U.S. patent application number 12/219294 was filed with the patent office on 2009-05-28 for hbm variants that modulate bone mass and lipid levels.
This patent application is currently assigned to Oscient Pharmaceuticals. Invention is credited to Kristina Allen, Anthony Anisowicz, James R. Graham, Wei Liu, Arturo Morales, Paul J. Yaworsky.
Application Number | 20090136507 12/219294 |
Document ID | / |
Family ID | 27501520 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090136507 |
Kind Code |
A1 |
Allen; Kristina ; et
al. |
May 28, 2009 |
HBM variants that modulate bone mass and lipid levels
Abstract
The present invention relates to methods and materials used to
express an HBM-like polypeptide derived from HBM, LRP5 or LRP6 in
animal cells and transgenic animals. The present invention also
relates to transgenic animals expressing the HBM-like polypeptides.
The invention provides nucleic acids, including coding sequences,
oligonucleotide primers and probes, proteins, cloning vectors,
expression vectors, transformed hosts, methods of developing
pharmaceutical compositions, methods of identifying molecules
involved in bone development, and methods of diagnosing and
treating diseases involved in bone development and lipid
modulation. In preferred embodiments, the present invention is
directed to methods for treating, diagnosing and preventing
osteoporosis.
Inventors: |
Allen; Kristina; (Hopkinton,
MA) ; Anisowicz; Anthony; (West Newton, MA) ;
Graham; James R.; (Arlington, MA) ; Morales;
Arturo; (Arlington, MA) ; Yaworsky; Paul J.;
(Rockland, MA) ; Liu; Wei; (Sudbury, MA) |
Correspondence
Address: |
DRINKER BIDDLE & REATH (DC)
1500 K STREET, N.W., SUITE 1100
WASHINGTON
DC
20005-1209
US
|
Assignee: |
Oscient Pharmaceuticals
Waltham
MA
Wyeth
Madison
NJ
|
Family ID: |
27501520 |
Appl. No.: |
12/219294 |
Filed: |
July 18, 2008 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10477173 |
Nov 4, 2004 |
7416849 |
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PCT/US02/14877 |
May 13, 2002 |
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12219294 |
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60290071 |
May 11, 2001 |
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60291311 |
May 17, 2001 |
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60353058 |
Feb 1, 2002 |
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60361293 |
Mar 4, 2002 |
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Current U.S.
Class: |
424/139.1 ;
530/387.3; 530/387.9 |
Current CPC
Class: |
C12N 2840/203 20130101;
A01K 2267/02 20130101; C12N 15/8509 20130101; C12N 2800/60
20130101; A61P 5/18 20180101; A61K 38/00 20130101; A01K 2227/105
20130101; A01K 67/0276 20130101; G01N 2500/00 20130101; A01K
2217/075 20130101; A01K 2267/03 20130101; A61P 9/00 20180101; A61P
19/10 20180101; A01K 2227/50 20130101; A01K 2267/0362 20130101;
A61P 5/14 20180101; A61P 3/14 20180101; A01K 2217/05 20130101; A61P
19/00 20180101; C07K 14/51 20130101; A01K 67/0278 20130101; A01K
2207/15 20130101; A61P 3/00 20180101; C12N 2830/30 20130101; C12N
2840/105 20130101; C12N 2830/008 20130101; G01N 2333/51 20130101;
C12N 2800/30 20130101; A01K 2217/00 20130101; A61P 19/08 20180101;
C12N 2830/00 20130101; C07K 16/18 20130101 |
Class at
Publication: |
424/139.1 ;
530/387.9; 530/387.3 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18 |
Claims
1-20. (canceled)
21. An antibody or immunogenic fragment thereof which binds to a
polypeptide or biologically active fragment thereof derived from
LRP5 comprising at least one amino acid change of Table 2, G171V,
A214V, A65V, M282V, G171K, G171F, G171I, G1710Q, L200V, T201V,
1202V, or S127V, or binds to a polypeptide or biologically active
fragment thereof derived from LRP6 comprising at least one amino
acid change of Table 2, G171V, A214V, A65V, M282V, G171K, G171F,
G1711, G1710Q, L200V, T201V, I1202V, or S127V when the amino acid
change is expressed in an equivalent position in LRP6.
22. The antibody or immunogenic fragment thereof of claim 21,
wherein the antibody is a monoclonal antibody, a chimeric antibody,
a bispecific antibody, a humanized antibody, a primatized antibody,
a human antibody, or a labeled antibody.
23. An antibody which binds to a polypeptide comprising
.sup.208KLYWADAKLSFIHRAN.sup.223, .sup.277ALYSPMDIQVLSQER.sup.291,
.sup.61GLEDAAAVDFQFSKGA.sup.73, .sup.234EGSLTHPFALTLSG.sup.247,
.sup.249TLYWTDWQTRSIHACN.sup.264, .sup.144VLFWQDLDQPRAI.sup.156,
.sup.194IYWPNGLTIDLEEQKLY.sup.210, .sup.34LLLFANRRDVRLVD.sup.47,
.sup.75GAVYWTDVSEEAIKQ.sup.89, .sup.121KLYWTDSETNRIEVA.sup.135 of
LRP5 or an equivalent domain on LRP6 or variants thereof.
24. An antibody which binds to a polypeptide comprising
.sup.969LILPLHGLRNVKAIDYDPLDKFIYW.sup.993,
.sup.989KFIYWVDGRQNIKRAKDDGTQPFVL.sup.1013,
.sup.1009QPFVLTSLSQGQNPDRQPHDLSIDI.sup.1033,
.sup.1029LSIDIYSRTLFWTCEATNTINVHRL.sup.1053,
.sup.1049NVHRLSGEAMGVVLRGDRDKPRAIV.sup.1073,
.sup.1253CGEPPTCSPDQFAC.sup.1266,
.sup.1278WRCDGFPECDDQSDEEGC.sup.1295,
.sup.1316RCDGEADCQDRSDEADC.sup.1332,
.sup.1370CEITKPPSDDSPAH.sup.1383 of LRP5 or an equivalent domain on
LRP6 or variants thereof, wherein said antibody modulates binding
of LRP5, HBM, LRP6 or variants thereof to Dkk.
25. (canceled)
26. A composition for modulating bone mass and/or lipid levels in a
subject comprising a therapeutically effective amount of an
antibody of claim 21 and a pharmaceutically acceptable carrier.
27. An antibody or an immunogenic fragment thereof of claim 21,
wherein the antibody or immunogenic fragment thereof can (1)
discriminate between LRP5 and an HBM-like protein, (2) discriminate
between LRP6 and an HBM-like protein, or (3) discriminate between
HBM and an HBM-like protein.
28-48. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the field of
genetics, genomics and molecular biology. The invention relates to
methods and materials used to isolate, detect and sequence a high
bone mass gene and corresponding wild-type gene, and mutants
thereof. The present invention also relates to the high bone mass
(HBM) gene, the corresponding wild-type gene, and mutants thereof.
The genes identified in the present invention are implicated in the
ontology and physiology of bone development. The invention also
provides nucleic acids, proteins, cloning vectors, expression
vectors, transformed hosts, methods of developing pharmaceutical
compositions, methods of identifying molecules involved in bbne
development, and methods of diagnosing and treating diseases
involved in bone development and lipid levels. The invention
further relates to transgenic animals for studying the HBM
phenotype and related variant phenotypes, the mechanism of action
of the HBM gene and its variants, and factors and treatments
affecting normal and abnormal bone conditions. In preferred
embodiments, the present invention is directed to methods for
treating, diagnosing, preventing and screening for normal and
abnormal conditions of bone, including metabolic bone diseases such
as osteoporosis.
BACKGROUND OF THE INVENTION
[0002] Two of the most common types of osteoporosis are
postmenopausal and senile osteoporosis. Osteoporosis affects men as
well as women, and, taken with other abnormalities of bone,
presents an ever-increasing health risk for an aging population.
The most common type of osteoporosis is that associated with
menopause. Most women lose between 20-60% of the bone mass in the
trabecular compartment of the bone within 3-6 years after the
cessation of menses. This rapid loss is generally associated with
an increase of bone resorption and formation. However, the
resorptive cycle is more dominant and the result is a net loss of
bone mass. Osteoporosis is a common and serious disease among
postmenopausal women. There are an estimated 25 million women in
the United States alone who are afflicted with this disease. The
results of osteoporosis are both personally harmful, and also
account for a large economic loss due to its chronicity and the
need for extensive and long-term support (hospitalization and
nursing home care) from the disease sequelae. This is especially
true in more elderly patients. Additionally, while osteoporosis is
generally not thought of as a life-threatening condition, a 20-30%
mortality rate is related to hip fractures in elderly women. A
large percentage of this mortality rate can be directly associated
with postmenopausal osteoporosis. The costs alone associated with
the treatment of osteoporotic fractures in the United States is $10
to $15 billion annually. Worldwide incidence of osteoporotic hip
fractures is estimated to exceed 1.7 million cases.
[0003] The most vulnerable tissue in the bone to the effects of
postmenopausal osteoporosis is the trabecular bone. This tissue is
often referred to as spongy bone and is particularly concentrated
near the ends of the bone near the joints and in the vertebrae of
the spine. The trabecular tissue is characterized by small
structures which inter-connect with each other as well as the more
solid and dense cortical tissue which makes up the outer surface
and central shaft of the bone. This crisscross network of
trabeculae gives lateral support to the outer cortical structure
and is critical to the biomechanical strength of the overall
structure. In postmenopausal osteoporosis, it is primarily the net
resorption and loss of the trabeculae which lead to the failure and
fracture of the bone. In light of the loss of the trabeculae in
postmenopausal women, it is not surprising that the most common
fractures are those associated with bones which are highly
dependent on trabecular support, e.g., the vertebrae, the neck of
the femur, and the forearm. Indeed, hip fracture, Colle's
fractures, and vertebral crush fractures are indicative of
postmenopausal osteoporosis.
[0004] One of the earliest generally accepted methods for treatment
of postmenopausal osteoporosis was estrogen replacement therapy.
Although this therapy frequently is successful, patient compliance
is low, primarily due to the undesirable side-effects of chronic
estrogen treatment. Frequently cited side-effects of estrogen
replacement therapy include reinitiation of menses, bloating,
depression, and fear of breast or uterine cancer. In order to limit
the known threat of uterine cancer in those women who have not
undergone a hysterectomy, a protocol of estrogen and progestin
cyclic therapy is often employed. This protocol is similar to that
which is used in birth control regimens, and often is not tolerated
by women because of the side-effects characteristic of progestin.
More recently, certain antiestrogens, originally developed for the
treatment of breast cancer, have been shown in experimental models
of postmenopausal osteoporosis to be efficacious. Among these
agents is raloxifene (See, U.S. Pat. No. 5,393,763, and Black et
al, J. Clin. Invest., 93:63-69 (1994)). In addition, tamoxifene, a
widely used clinical agent for the treatment of breast cancer, has
been shown to increase bone mineral density in post menopausal
women suffering from breast cancer (Love et al, N. Engl. J. Med.,
326:852-856 (1992)).
[0005] Another therapy for the treatment of postmenopausal
osteoporosis is the use of calcitonin. Calcitonin is a naturally
occurring peptide which inhibits bone resorption and has been
approved for this use in many countries (Overgaard et al, Br. Med.
J., 305:556-561 (1992)). The use of calcitonin has been somewhat
limited, however. Its effects are very modest in increasing bone
mineral density and the treatment is very expensive. Another
therapy for the treatment of postmenopausal osteoporosis is the use
of bis-phosphonates. These compounds were originally developed for
use in Paget's disease and malignant hypercalcemia. They have been
shown to inhibit bone resorption. Alendronate, one compound of this
class, has been approved for the treatment of postmenopausal
osteoporosis. These agents may be helpful in the treatment of
osteoporosis, but these agents also have potential liabilities
which include osteomalacia, extremely long half-life in bone
(greater than 2 years), and possible "frozen bone syndrome," e.g.,
the cessation of normal bone remodeling.
[0006] Senile osteoporosis is similar to postmenopausal
osteoporosis in that it is marked by the loss of bone mineral
density and resulting increase in fracture rate, morbidity, and
associated mortality. Generally, it occurs in later life, i.e.,
after 70 years of age. Historically, senile osteoporosis has been
more common in females, but with the advent of a more elderly male
population, this disease is becoming a major factor in the health
of both sexes. It is not clear what, if any, role hormones such as
testosterone or estrogen have in this disease, and its etiology
remains obscure. Treatment of this disease has not been very
satisfactory. Hormone therapy, estrogen in women and testosterone
in men, has shown equivocal results; calcitonin and
bis-phosphonates may be of some utility.
[0007] The peak mass of the skeleton at maturity is largely under
genetic control. Twin studies have shown that the variance in bone
mass between adult monozygotic twins is smaller than between
dizygotic twins (Slemenda et al, J. Bone Miner. Res., 6:561-567
(1991); Young et al, J. Bone Miner. Res., 6:561-567 (1995); Pocock
et al, J. Clin. Invest., 80:706-710 (1987); Kelly et al, J. Bone
Miner. Res., 8:11-17 (1993)), and it has been estimated that up to
60% or more of the variance in skeletal mass is inherited (Krall et
al, J. Bone Miner. Res., 10:S367 (1993)). Peak skeletal mass is the
most powerful determinant of bone mass in elderly years (Hui et al,
Ann. Int. Med., 111; 355-361 (1989)), even though the rate of
age-related bone loss in adult and later life is also a strong
determinant (Hui et al, Osteoporosis Int., 1:30-34 (1995)). Since
bone mass is the principal measurable determinant of fracture risk,
the inherited peak skeletal mass achieved at maturity is an
important determinant of an individual's risk of fracture later in
life. Thus, study of the genetic basis of bone mass is of
considerable interest in the etiology of fractures due to
osteoporosis.
[0008] Recently, a strong interest in the genetic control of peak
bone mass has developed in the field of osteoporosis. The interest
has focused mainly on candidate genes with suitable polymorphisms
to test for association with variation in bone mass within the
normal range, or has focused on examination of genes and gene loci
associated with low bone mass in the range found in patients with
osteoporosis. The vitamin D receptor locus (VDR) (Morrison et al,
Nature, 367:284-287 (1994)), PTH gene (Howard et al, J. Clin.
Endocrinol. Metab., 80:2800-2805 (1995); Johnson et al, J. Bone
Miner. Res., 8:11-17 (1995); Gong et al, J. Bone Miner. Res.,
10:S462 (1995)) and the estrogen receptor gene (Hosoi et al, J.
Bone Miner. Res., 10:S170 (1995); Morrison et al, Nature,
367:284-287 (1994)) have figured most prominently in this work.
These studies are difficult because bone mass (the phenotype) is a
continuous, quantitative, polygenic trait, and is confounded by
environmental factors such as nutrition, co-morbid disease, age,
physical activity, and other factors. Also, this type of study
design requires large numbers of subjects. In particular, the
results of VDR studies to date have been confusing and
contradictory (Garnero et al, J. Bone Miner. Res., 10:1283-1288
(1995); Eisman et al, J. Bone. Miner. Res., 10:1289-1293 (1995);
Peacock, J. Bone Miner. Res., 10:1294-1297 (1995)). Furthermore,
the work thus far has not shed much light on the mechanism(s)
whereby the genetic influences might exert their effect on bone
mass.
[0009] While it is well known that peak bone mass is largely
determined by genetic rather than environmental factors, studies to
determine the gene loci (and ultimately the genes) linked to
variation in bone mass are difficult and expensive. Study designs
which utilize the power of linkage analysis, e.g., sib-pair or
extended family, are generally more informative than simple
association studies, although the latter do have value. However,
genetic linkage studies involving bone mass are hampered by two
major problems. The first problem is the phenotype, as discussed
briefly above. Bone mass is a continuous, quantitative trait, and
establishing a discrete phenotype is difficult. Each anatomical
site for measurement may be influenced by several genes, many of
which may be different from site to site. The second problem is the
age component of the phenotype. By the time an individual can be
identified as having low bone mass, there is a high probability
that their parents or other members of prior generations will be
deceased and therefore unavailable for study, and younger
generations may not have even reached peak bone mass, making their
phenotyping uncertain for genetic analysis.
[0010] Regardless, linkage analysis can be used to find the
location of a gene causing a hereditary "disorder" and does not
require any knowledge of the biochemical nature of the disorder,
i.e., a mutated protein that is believed to cause the disorder does
not need to be known. Traditional approaches depend on assumptions
concerning the disease process that might implicate a known protein
as a candidate to be evaluated. The genetic localization approach
using linkage analysis can be used to first find the general
chromosomal region in which the defective gene is located and then
to gradually reduce the size of the region in order to determine
the location of the specific mutated gene as precisely as possible.
After the gene itself is discovered within the candidate region,
the messenger RNA and the protein are identified and, along with
the DNA, are checked for mutations.
[0011] The genetic localization approach has practical implications
since the location of the disease can be used for prenatal
diagnosis even before the altered gene that causes the disease is
found. Linkage analysis can enable families, even many of those
that do not have a sick child, to know whether they are carriers of
a disease gene and to evaluate the condition of an unborn child
through molecular diagnosis. The transmission of a disease within
families, then, can be used to find the defective gene. As used
herein, reference to "high bone mass" (HBM) is analogous to
reference to a disease state, although from a practical standpoint
high bone mass can actually help a subject avoid the disease known
as osteoporosis.
[0012] Linkage analysis is possible because of the nature of
inheritance of chromosomes from parents to offspring. During
meiosis, the two parental homologues pair to guide their proper
separation to daughter cells. While they are lined up and paired,
the two homologues exchange pieces of the chromosomes, in an event
called "crossing over" or "recombination." The resulting
chromosomes are chimeric, that is, they contain parts that
originate from both parental homologues. The closer together two
sequences are on the chromosome, the less likely that a
recombination event will occur between them, and the more closely
linked they are. In a linkage analysis experiment, two positions on
the chromosomes are followed from one generation to the next to
determine the frequency of recombination between them. In a study
of an inherited disease, one of the chromosomal positions is marked
by the disease gene or its normal counterpart, i.e., the
inheritance of the chromosomal region can be determined by
examining whether the individual displays symptoms of the disorder
or not. The other position is marked by a DNA sequence that shows
natural variation in the population such that the two homologues
can be distinguished based on the copy of the "marker" sequence
that they possess. In every family, the inheritance of the genetic
marker sequence is compared to the inheritance of the disease
state. If, within a family carrying an autosomal dominant disorder
such as high bone mass, every affected individual carries the same
form of the marker and all the unaffected individuals carry at
least one different form of the marker, there is a great
probability that the disease gene and the marker are located close
to each other. In this way, chromosomes may be systematically
checked with known markers and compared to the disease state. The
data obtained from the different families is combined, and analyzed
together by a computer using statistical methods. The result is
information indicating the probability of linkage between the
genetic marker and the disease allowing different distances between
them. A positive result can mean that the disease is very close to
the marker, while a negative result indicates that it is far away
on that chromosome, or on an entirely different chromosome.
[0013] Linkage analysis is performed by typing all members of the
affected family at a given marker locus and evaluating the
co-inheritance of a particular disease state with the marker probe,
thereby determining how often the two of them are co-inherited. The
recombination frequency can be used as a measure of the genetic
distance between two gene loci. A recombination frequency of 1% is
equivalent to 1 map unit, or 1 centiMorgan (cM), which is roughly
equivalent to 1,000 kb of DNA. This relationship holds up to
frequencies of about 20% or 20 cM.
[0014] The entire human genome is 3,300 cM long. In order to find
an unknown disease gene within 5-10 cM of a marker locus, the whole
human genome can be searched with roughly 330 informative marker
loci spaced at approximately 10 cM intervals (Botstein et al., Am.
J. Hum. Genet., 32:314-331 (1980)). The reliability of linkage
results is established by using a number of statistical methods.
The method most commonly used for the analysis of linkage in humans
is the LOD score method (Morton, Prog. Clin. Biol. Res.,
147:245-265 (1984), Morton et al., Am. J. Hun. Genet., 38:868-883
(1986)) which was incorporated into the computer program, LIPED, by
Ott, Am. J. Hum. Genet., 28:528-529 (1976). LOD scores are the
logarithm of the ratio of the likelihood that two loci are linked
at a given distance to that they are not linked (>50 cM apart).
The advantage of using logarithmic values is that they can be
summed among families with the same disease. This becomes necessary
given the relatively small size of human families.
[0015] By convention, a total LOD score greater than +3.0 (that is,
odds of linkage at the specified recombination frequency being 1000
times greater than odds of no linkage) is considered to be
significant evidence for linkage at that particular recombination
frequency. A total LOD score of less than -2.0 (that is, odds of no
linkage being 100 times greater than odds of linkage at the
specified frequency) is considered to be strong evidence that the
two loci under consideration are not linked at that particular
recombination frequency. Until recently, most linkage analyses have
been performed on the basis of two-point data, which is the
relationship between the disorder under consideration and a
particular genetic marker. However, as a result of the rapid
advances in mapping the human genome over the last few years, and
concomitant improvements in computer methodology, it has become
feasible to carry out linkage analyses using multi-point data.
Multi-point analysis provide a simultaneous analysis of linkage
between the disease and several linked genetic markers, when the
recombination distance among the markers is known.
[0016] Multi-point analysis is advantageous for two reasons. First,
the informativeness of the pedigree is usually increased. Each
pedigree has a certain amount of potential information, dependent
on the number of parents heterozygous for the marker loci and the
number of affected individuals in the family. However, few markers
are sufficiently polymorphic as to be informative in all those
individuals. If multiple markers are considered simultaneously,
then the probability of an individual being heterozygous for at
least one of the markers is greatly increased. Second, an
indication of the position of the disease gene among the markers
may be determined. This allows identification of flanking markers,
and thus eventually allows isolation of a small region in which the
disease gene resides. Lathrop et al., Proc. Natl. Acad. Sci. USA,
81:3443-3446 (1984) have written the most widely used computer
package, LINKAGE, for multi-point analysis.
[0017] There is a need in the art for identifying the gene
associated with a high bone mass phenotype. There is also a need
for tools for the study of the high bone mass gene and phenotype.
More generally there is need for the development of diagnostic
tools and treatments. The present invention is directed to these,
as well as other, important ends.
SUMMARY OF THE INVENTION
[0018] It is an object of the invention to provide a nucleic acid
comprising a mutation in LRP5 or LRP6 which results in a HBM
phenotype when expressed in a cell, wherein said HBM phenotype
results in bone mass modulation and/or lipid level modulation.
Another embodiment contemplates that the mutation is located in
propeller 1. In another embodiment, the nucleic acid encodes a
mutation comprising at least one mutation of Tables 2 or 3 or which
results in a mutation of one of the following G171 V, A214V, A65V,
M282V, G171K, G171F, G171I, G171Q, L200V, T201V, 1202V, or S127V
when expressed in a cell, and wherein expression of the nucleic
acid in a subject results in bone mass modulation and/or lipid
level modulation. In the instance of LRP6, the mutation is located
in a position equivalent to LRP5 such that expression of the
nucleic acid in a subject results in bone mass modulation and/or
lipid level modulation. In another embodiment, the preferred
mutation of LRP5 is G171V, A214V, A65V, M282V, G171K, G171F, G171I
or G171Q or in an equivalent location if dealing with LRP6.
[0019] Another embodiment contemplated herein is a polypeptide
encoded by any of the above nucleic acids, wherein said polypeptide
when expressed in a cell modulates Wnt signaling, LRP5 activity
and/or LRP6 activity. The polypeptide can additionally or
alternatively modulate bone mass and/or lipid levels when expressed
in a subject. These polypeptides or biologically active fragments
thereof may preferably contain any of the following mutations of
Table 2, G171V, A214V, A65V, M282V, G171K, G171F, G171I, G171Q,
L200V, T201V, 202V, or S127V in LRP5 or in a equivalent location in
LRP6. The most preferred mutations are G171V, A214V, A65V, M282V,
G171K, G171F, G171I or G171Q in LRP5 or an equivalent location in
LRP6.
[0020] Yet another embodiment contemplates antibodies an
immunogenic fragments thereof which bind to these proteins. The
contemplated antibodies include a monoclonal antibody, a chimeric
antibody, a bispecific antibody, a humanized antibody, a
Primatized.RTM. antibody, a human antibody, or a labeled antibody.
Preferably, some of the antibodies can discriminate between the
wild type and variant forms of LRP5 and LRP6.
[0021] Another embodiment contemplates antibodies which bind to
polypeptides comprising: .sup.208KLYWADAKLSFIHRAN.sup.223,
.sup.277ALYSPMDIQVLSQER.sup.291, .sup.61GLEDAAAVDFQFSKGA.sup.73,
.sup.234 EGSLTPFALTLSG.sup.247, .sup.249TLYWTDWQTRSIFIACN.sup.264,
.sup.144VLFWQDLDQPRAI.sup.156, .sup.194IYWPNGLTIDLEEQKLY.sup.210,
.sup.34LLLFANRRDVRLVD.sup.47, .sup.75GAVYWTDVSEEAIKQ.sup.89,
.sup.121KLYWTDSETNRIEVA.sup.35 of LRP5 or an equivalent domain on
LRP6 or variants thereof.
[0022] The above antibodies can also be used in a composition for
modulating bone mass and/or lipid levels in a subject comprising a
therapeutically effective amount of the antibody or immunogenic
fragment and a pharmaceutically acceptable carrier.
[0023] The invention further contemplates a method of diagnosing a
HBM like phenotype in a subject comprising: (A) obtaining a
biological sample from said subject; (B) exposing the sample to one
of the described antibodies or immunogenic fragments; and (C)
detecting whether the antibody bound a protein from the biological
sample from said subject to determine whether the subject has a
IBM-like phenotype.
[0024] Another embodiment contemplates a transgenic animal having
somatic and/or germ cells comprising a nucleic acid which comprises
a promoter region that directs protein expression in animal and/or
human cells operably linked to any of the herein described nucleic
acids, and wherein said transgenic animal has at least three bone
parameters modulated by the expression of said nucleic acid. The
promoter region can be selected from the group consisting of CMV,
RSV, SV40, and EF-1a, CMV.beta.bActin, histone, type I collagen,
TGF.beta.1, SX2, cfos/cjun, Cbfa1, Fra/Jun, Dlx5, osteocalcin,
osteopontin, bone sialoprotein, and collagenase promoter
regions.
[0025] A further embodiment of the invention contemplates an animal
model for the study of bone density modulation and/or lipid level
modulation comprising a first group of animals composed of any of
the described transgenic animals and a second group of control
animals.
[0026] Another embodiment provides for a method of identifying
agents which modulate the activity of an HBM-like nucleic acid
comprising: (A) transfecting a cell with a vector of claim 11; (B)
exposing the transfected cell of step (A) to a compound; and (C)
determining whether the compound modulates the activity of the
HBM-like nucleic acid. Such agents can include a hormone, a growth
factor, a peptide, RNA, siRNA, DNA, a mineral, a vitamin, a natural
product, or a synthetic organic compound.
[0027] Another aspect of the invention provides for a method for
identifying compounds which modulate the interaction of Dkk with
the Wnt signaling pathway comprising: (A) transfecting cells with
constructs containing any of the described nucleic acids; (B)
assessing changes in expression of a reporter element linked to a
Wnt-responsive promoter; and (C) identifying as a Dkk/Wnt
interaction modulating compound any compound which alters reporter
gene expression compared with cells transfected with a Dkk
construct alone. The cells are preferably cancer cells, liver cells
or bone cells. The reporter element used is TCF-luciferase,
tk-Renilla, or a combination thereof.
[0028] Yet another embodiment includes a method of diagnosing a
subject as expressing a nucleic acid comprising a nucleotide change
of Tables 2 or 3 or any other mutations, the method comprising the
steps of: (A) obtaining a biological sample from the subject; and
(B) assaying for the presence of the nucleotide change which
results in HEM phenotype.
[0029] The invention also provides agents identified by the above
methods which regulate Wnt activity, Dkk activity, bone mass and/or
lipid levels.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1 shows the pedigree of the individuals used in the
genetic linkage studies. Under each individual is an ID number, the
z-score for spinal BMD, and the allele calls for the critical
markers on chromosome 11. Solid symbols represent "affected"
individuals. Symbols containing "N" are "unaffected" individuals.
DNA from 37 individuals was genotyped. Question marks denote
unknown genotypes or individuals who were not genotyped.
[0031] FIG. 2 depicts the BAC/STS content physical map of the HBM
region in 11q13.3. STS markers derived from genes, ESTs,
microsatellites, random sequences, and BAC end sequences are
denoted above the long horizontal line. For markers that are
present in GDB the same nomenclature has been used. Locus names
(D11S####) are listed in parentheses after the primary name if
available. STSs derived from BAC end sequences are listed with the
BAC name first followed by L or R for the left and right end of the
clone, respectively. The two large arrows indicate the genetic
markers that define the HBM critical region. The horizontal lines
below the STSs indicate BAC clones identified by PCR-based
screening of a nine-fold coverage BAC library. Open circles
indicate that the marker did not amplify the corresponding BAC
library address during library screening. Clone names use the
following convention: B for BAC, the plate, row and column address,
followed by --H indicating the HBM project (i.e., B36F16-H).
[0032] FIGS. 3A-3F show the genomic structure of Zmax1 (LRP5) with
flanking intron sequences. Translation is initiated by the
underlined "ATG" in exon 1. The site of the polymorphism in the HBM
gene is in exon 3 and is represented by the underlined "G," whereby
this nucleotide is a "T" in the HBM gene. The 3' untranslated
region of the mRNA is underlined within exon 23 (exon 1, SEQ ID
NO:40; exon 2, SEQ ID NO:41; exon 3, SEQ ID NO:42; exon 4, SEQ ID
NO:43; exon 5, SEQ ID NO:44; exon 6, SEQ ID NO:45; exon 7, SEQ ID
NO:46; exon 8, SEQ ID NO:47; exon 9, SEQ ID NO:48; exon 10, SEQ ID
NO:49; exon 11, SEQ ID NO:50; exon 12, SEQ ID NO:51; exon 13, SEQ
ID NO:52; exon 14, SEQ ID NO:53; exon 15, SEQ ID NO:54; exon 16,
SEQ ID NO:55; exon 17, SEQ ID NO:56; exon 18, SEQ ID NO:57; exon
19, SEQ ID NO:58; exon 20, SEQ ID NO:59; exon 21, SEQ ID NO:60;
exon 22, SEQ ID NO:61; and exon 23; SEQ ID NO:62).
[0033] FIG. 4 shows the domain organization of Zmax1 (LRP5),
including the YWTD spacers, the extracellular attachment site, the
binding site for LDL and calcium, the cysteine-rich growth factor
repeats, the transmembrane region, the ideal PEST region with the
CK-II phosphorylation site and the internalization domain. FIG. 4
also shows the site of the glycine to valine change that occurs in
the HBM protein. The signal peptide is located at amino acids 1-31,
the extracellular domain is located at amino acids 32-1385, the
transmembrane segment is located at amino acids 1386-1413, and the
cytoplasmic domain is located at amino acids 1414-1615.
[0034] FIG. 5 is a schematic illustration of the BAC contigs
B527D12 and B200E21 in relation to the HBM gene.
[0035] FIGS. 6A-6J are the nucleotide (SEQ ID NO:1) and amino acid
(SEQ ID NO:3) sequences of the wild-type gene, Zmax1 (LRP5). The
location for the base pair substitution at nucleotide 582, a
guanine to thymine, (SEQ ID NO: 2 and 4) is underlined. This
allelic variant is the HBM gene. The HBM gene encodes for a protein
with an amino acid substitution of glycine to valine at position
171. The 5' untranslated region (UTR) boundaries bases 1 to 70, and
the 3' UTR boundaries bases 4916-5120.
[0036] FIGS. 7A and 7B are northern blot analyses showing the
expression of Zmax1 (LRP5) in various tissues.
[0037] FIG. 8 is a PCR product analysis.
[0038] FIG. 9 is allele specific oligonucleotide detection of the
Zmax1 (LRP5) exon 3 mutation.
[0039] FIG. 10 is the cellular localization of mouse Zmax1 (LRP5)
by in situ hybridization at 100.times. magnification using sense
and antisense probes.
[0040] FIG. 11 is the cellular localization of mouse Zmax1 (LRP5)
by in situ hybridization at 400.times. magnification using sense
and antisense probes.
[0041] FIG. 12 is the cellular localization of mouse Zmax1 (LRP5)
by in situ hybridization of osteoblasts in the endosteum at
400.times. magnification using sense and antisense probes.
[0042] FIG. 13 shows antisense inhibition of Zmax1 (LRP5)
expression in MC-3T3 cells.
[0043] FIG. 14 shows a Zmax1 (LRP5) exon 3 allele specific
oligonucleotide (ASO) assay which illustrates the rarity of the HBM
allele (right panels; T-specific oligo; 58.degree. C. Wash) as
compared to the wild-type Zmax1 (LRP5) allele (left panels,
G-specific oligo; 55.degree. C. Wash). The positive spots appearing
in the right panels were positive controls.
[0044] FIG. 15 depicts a model representing the potential role of
Zmax1 (LRP5) in focal adhesion signaling.
[0045] FIG. 16 depicts a schematic of two Zmax1 (LRP5) gene
targeting vectors for the knock-out of endogenous mouse Zmax or
conditional knock-in of the HBM polymorphism. B, X, and R indicate
BamHI, XbaI, and EcoRI sites in DNA BAC 4735P5 respectively. Exons
3, 4, and 5 are indicated by black rectangles. A G->T base
change is engineered at base 24 of exon 3 to produce the HBM
polymorphism. The location of a LoxP flanked cassette containing a
neomycin resistance gene and a synthetic pause sequence and probes
used for screening and characterizing of ES cell clones are also
indicated.
[0046] FIG. 17 confirms expression by the transgenic (i.e., HBMMCBA
and HBMMTIC) and wild-type (i.e., ZmaxWTCBA and ZmaxWTTIC) plasmid
constructs. These constructs were transiently transfected into
HOB-02-02 cells and the mRNA levels determined using TaqMan.RTM.
quantitative PCR. HBMMCBA and ZmaxWTCBA are shown in the left
column (i.e. CMV.beta.Actin) and HBMMTIC and ZmaxWTTIC are shown in
the right column (i.e. Type I collagen) of the Table.
[0047] FIG. 18 depicts a comparison between the human and mouse
TaqMan.RTM. Primer/Probe sets. HOB (HOB-03-C5) and mouse
(MC-3T3-E1) osteoblastic cell mRNA was analyzed using the probes
and primers.
[0048] FIG. 19 depicts the quantification of human Zmax1 (LRP5)
mRNA expressed in a mixed human and mouse RNA background using the
TaqMan.RTM. Primer/Probe sets. Results are presented in Human Zmax1
(LRP5) mRNA added (ng) versus Human Zmax1 (LRP5) mRNA measured
(ng).
[0049] FIG. 20 depicts expression of HBM in transgenic mice based
on mRNA expression analyzed by TaqMan.RTM..
[0050] FIG. 21A-F Panels A-C depict the analysis of various
transgenic mouse lines which express the HBMMCBA construct in spine
(FIG. 21A), femur (FIG. 21B) and total body (FIG. 21C). Panels D-F
depict the analysis of various transgenic mouse lines which express
the HBMMTIC construct in spine (FIG. 21D), femur (FIG. 21E) and
total body (FIG. 21F).
[0051] FIG. 22 depicts changes in BMD in HBM transgenic mice (i.e.,
HBMMCBA and HBMMTIC constructs) at 5 weeks using in vivo pDXA*
analysis. The BMD changes are presented as compared to wild-type
animals which were also only 5 weeks old.
[0052] FIG. 23 depicts changes in BMD in HBM transgenic mice (i.e.,
HBMMCBA and HBMMTIC constructs) at 9 weeks using in vivo pDXA*
analysis. The BMD changes are presented as compared to wild-type
animals which were also only 9 weeks old.
[0053] FIG. 24 (A-D) presents the sequence of the insert of the
gene, HBMGI.sub.--2AS, subcloned into the vector (SEQ ID
NO:759).
[0054] FIG. 25 (A-D) presents the sequence of the insert of the
gene, ZMAXGI.sub.--3AS, subcloned into the vector (SEQ ID
NO:760).
[0055] FIG. 26 (A-C) presents an alignment of human (SEQ ID NO:761)
and mouse (SEQ ID NO:762) Zmax1 (LRP5) amino acid sequences.
[0056] FIG. 27 (A-C) presents an alignment of human LRP5 (Zmax1)
(SEQ ID NO:763) and LRP6 (SEQ ID NO:764) amino acid sequences.
[0057] FIG. 28 shows a schematic of the components of the Wnt
signal transduction pathway. Schematic obtained from:
http://www.stanford.edu/.about.musse/pathways/cell2.html.
[0058] FIG. 29 shows that the mutation G171F in LRP5 produces a
greater activation of the Wnt pathway than LRP5 which is consistent
with HBM activity.
[0059] FIG. 30 shows that the mutation M282V in LRP5 produces an
activation of the Wnt pathway which is consistent with HBM activity
in U2OS cells.
[0060] FIG. 31 shows a table of proteins identified in a Y2H screen
using a Dkk1d-1 bait sequence. These proteins are identified by
both their nucleic acid and amino acid accession numbers.
[0061] FIG. 32 shows the differential binding of an antibody
generated to a sequence (a.a. 165-177) containing the HBM mutation
in LRP5 in LRP5 and HBM virus-infected cells.
[0062] FIG. 33 shows a diagram of the Xenopus Embryo Assay for Wnt
activity.
[0063] FIG. 34 shows the effects of Zmax/LRP5 and HBM on Wnt
signaling in the Xenopus embryo assay.
[0064] FIG. 35 shows the effects of Zmax/LRP5 and HBM on induction
of secondary axis formation in the Xenopus embryo assay.
[0065] FIG. 36 shows the effects of human Dkk-1 on the repression
of the canonical Wnt pathway.
[0066] FIG. 37 shows the effects of human Dkk-1 on Zmax/LRP5 and
HBM-mediated Wnt signaling.
[0067] FIG. 38 shows a table of peptide aptamer insert sequences
identified in a Y2H screen using a LRP5 ligand binding domain bait
sequence.
[0068] FIG. 39 shows pcDNA3.1 construct names with nucleotide
sequences for LRP5-binding peptide aptamers, Dkk-1 peptides and
control constructs.
[0069] FIG. 40 shows the results of a minimum interaction domain
mapping screen of Dkk-1 with LRP5. At the top, a map of Dkk-1
showing the location of the signal sequence, and cysteine rich
domains 1 and 2. Below, the extent of domains examined using LRP5
LBD baits, LBD1 and LBD4. To the right, scoring of the binding
results observed in the experiment.
[0070] FIG. 41 shows the effects of Dkk-1 and Dkk-2 on Wnt1
signaling with coreceptors LRP5, HBM, and LRP6 in HOB03CE6
cells.
[0071] FIG. 42 shows the effects of Dkk-1 and Dkk-2 on Wnt3a
signaling with coreceptors LRP5, HBM, and LRP6 in HOB03CE6
cells.
[0072] FIG. 43 demonstrates that the LRP5-LBD peptide aptamer 262
activates Wnt signaling in the presence of Wnt3a in U2OS cells.
[0073] FIG. 44 shows the amino acid sequences for the corresponding
LRP5-binding peptides, Dkk-1 peptide aptamers and control
constructs in FIG. 39.
[0074] FIG. 45 shows that Dkk-1 represses Wnt3a-mediated Wnt
signaling in U2OS bone cells using the cell-based reporter gene
assay for high throughput screening.
[0075] FIG. 46 demonstrates that Wnt1-HBM generated signaling is
not efficiently inhibited by Dkk-1 in U2OS bone cells while LRP5
and LRP6-mediated signaling are using the cell-based reporter gene
assay for high throughput screening.
[0076] FIG. 47 shows that the TCF signal in the cell-based reporter
gene assay for high throughput screening can be modulated by Dkk-1
and Dkk-1-AP without Wnt DNA transfection.
[0077] FIG. 48 shows the morphological results in the Xenopus assay
using aptamers 261 and 262 from the LRP5-LBD to activate Wnt
signaling.
[0078] FIG. 49 demonstrates that LRP5-LBD aptamers 261 and 262
induce Wnt signaling over other LRP5 aptamers.
[0079] FIG. 50 depicts the LRP5 domain structure. The symbols are
defined at the upper right. The structural domains are numbered
successively from top to bottom corresponding to the N-terminal to
C-terminal ends of the protein as Propeller 1, EGF-like Domain 1,
Propeller 2, EGF-like Domain 2, and so forth. The structure was
determined using the motifs and nomenclature described in Springer
et al., 1998 J. Mol. Biol. 283: 837-62.
[0080] FIG. 51 CLUSTALW (1.8) multiple sequence alignment of the
.beta.-propeller segments of LRP5 from human (af077820h) and mouse
(af064984m and af077847m), LRP6 from human (af074264h) and mouse
(af074265m), and the sequences from proteins with modeled 6-bladed
.beta.-propellers: chicken LRP1(1lpx) and human nidogen (1ndx). For
the LPRs, the final suffix letter designates which of the four
propeller domains the sequence comes from (a=>prop. #1,
b=>prop. #2, etc.). The final four lines give the secondary
structure assignments predicted by DSC for all sequences given in
this alignment (H=helix, E=strand and C=coil) and the weights
assigned to each structural type at each position on a scale of 0
(least probable) to 9 (most probable). Sequences corresponding to
Springer's models' strand positions are underlined and color-coded
according to which of the six blades they belong to. In the
propeller geometry, alternate loops fall on opposite faces of the
disc-shaped domain. The loops on the "top" face (i.e., opposite the
points of entry and exit of the chain from the structure) are
colored red. The position of the G171V mutation is marked with
"*".
[0081] FIG. 52 displays the functional effect of mutations on side
chain interactions in HBM protein as compared the wild-type Zmax1
(LRP5) protein. It also shows the side chain interactions of G171F,
an HBM like variant.
[0082] FIG. 53 displays the structural change due to the OPPG
mutation versus the wild-type, Zmax1 (LRP5). Panel A depicts the
homology model of LRP5's second propeller domain; Panel B depicts
the third propeller domain and Panel C depicts the second propeller
domain with two mutations.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0083] To aid in the understanding of the specification and claims,
the following definitions are provided.
[0084] "Gene" refers to a DNA sequence that encodes through its
template or messenger RNA a sequence of amino acids characteristic
of a specific peptide. The term "gene" includes intervening,
non-coding regions, as well as regulatory regions, and can include
5' and 3' ends.
[0085] By "nucleic acid" is meant to include single stranded and
double stranded nucleic acids including, but not limited to DNAs,
RNAs (e.g., mRNA, tRNAs, siRNAs), cDNAs, recombinant DNA (rDNA),
rRNAs, antisense nucleic acids, oligonucleotides, and oligomers,
and polynucleotides. The term may also include hybrids such as
triple stranded regions of RNA and/or DNA or double stranded
RNA:DNA hybrids. The term also is contemplated to include modified
nucleic acids such as, but not limited to biotinylated nucleic
acids, tritylated nucleic acids, fluorophor labeled nucleic acids,
inosine, and the like.
[0086] "Gene sequence" refers to a nucleic acid molecule, including
DNA which contains a non-transcribed or non-translated sequence,
which comprises a gene. The term is also intended to include any
combination of gene(s), gene fragment(s), non-transcribed
sequence(s) or non-translated sequence(s) which are present on the
same DNA molecule.
[0087] The nucleic acid sequences of the present invention may be
derived from a variety of sources including DNA, cDNA, synthetic
DNA, synthetic RNA or combinations thereof. Such sequences may
comprise genomic DNA which may or may not include naturally
occurring introns. Moreover, such genomic DNA may be obtained in
association with promoter regions and/or poly (A) sequences. The
sequences, genomic DNA or cDNA may be obtained in any of several
ways. Genomic DNA can be extracted and purified from suitable cells
by means well known in the art. Alternatively, mRNA can be isolated
from a cell and used to produce cDNA by reverse transcription or
other means.
[0088] "cDNA" refers to complementary or copy DNA produced from an
RNA template by the action of RNA-dependent DNA polymerase (reverse
transcriptase). Thus, a "cDNA clone" means a duplex DNA sequence
for which one strand is complementary to an RNA molecule of
interest, carried in a cloning vector or PCR amplified. cDNA can
also be single stranded after first strand synthesis by reverse
transcriptase. In this form, it is a useful PCR template and does
not need to be carried in a cloning vector. This term includes
genes from which the intervening sequences have been removed. Thus,
the term "gene", as sometimes used generically, can also include
nucleic acid molecules comprising cDNA and cDNA clones.
[0089] "Recombinant DNA" means a molecule that has been engineered
by splicing in vitro a cDNA or genomic DNA sequence or altering a
sequence by methods such as PCR mutagenesis.
[0090] "Cloning" refers to the use of in vitro recombination
techniques to insert a particular gene or other DNA sequence into a
vector molecule. In order to successfully clone a desired gene, it
is necessary to use methods for generating DNA fragments, for
joining the fragments to vector molecules, for introducing the
composite DNA molecule into a host cell in which it can replicate,
and for selecting the clone having the target gene from amongst the
recipient host cells.
[0091] "cDNA library" refers to a collection of recombinant DNA
molecules containing cDNA inserts which together comprise the
entire or a partial repertoire of genes expressed in a particular
tissue or cell source. Such a cDNA library can be prepared by
methods known to one skilled in the art and described by, for
example, Cowell and Austin, "cDNA Library Protocols," Methods in
Molecular Biology (1997).
[0092] "Cloning vehicle" refers to a plasmid or phage DNA or other
DNA sequence which is able to replicate in a host cell. This term
can also include artificial chromosomes such as BACs and YACs. The
cloning vehicle is characterized by one or more endonuclease
recognition sites at which such DNA sequences may be cut in a
determinable fashion without loss of an essential biological
function of the DNA, which may contain a marker suitable for use in
the identification of transformed cells.
[0093] "Expression" refers to the process comprising transcription
of a gene sequence and subsequent processing steps, such as
translation of a resultant mRNA to produce the final end product of
a gene. The end product may be a protein (such as an enzyme or
receptor) or a nucleic acid (such as a tRNA, antisense RNA, or
other regulatory factor). The term "expression control sequence"
refers to a sequence of nucleotides that control or regulate
expression of structural genes when operably linked to those genes.
These include, for example, the lac systems, the trp system, major
operator and promoter regions of the phage lambda, the control
region of fd coat protein and other sequences known to control the
expression of genes in prokaryotic or eukaryotic cells. Expression
control sequences will vary depending on whether the vector is
designed to express the operably linked gene in a prokaryotic or
eukaryotic host, and may contain transcriptional elements such as
enhancer elements, termination sequences, tissue-specificity
elements and/or translational initiation and termination sites.
[0094] "Expression vehicle" refers to a vehicle or vector similar
to a cloning vehicle but which is capable of expressing a gene
which has been cloned into it, after transformation into a host.
The cloned gene is usually placed under the control of (i.e.,
operably linked to) an expression control sequence.
[0095] "Operator" refers to a DNA sequence capable of interacting
with the specific repressor, thereby controlling the transcription
of adjacent gene(s).
[0096] "Promoter" refers to a DNA sequence that can be recognized
by an RNA polymerase. The presence of such a sequence permits the
RNA polymerase to bind and initiate transcription of operably
linked gene sequences.
[0097] "Promoter region" is intended to include the promoter as
well as other gene sequences which may be necessary for the
initiation of transcription. The presence of a promoter region is
sufficient to cause the expression of an operably linked gene
sequence. The term "promoter" is sometimes used in the art to
generically indicate a promoter region. Many different promoters
are known in the art which direct expression of a gene in a certain
cell types. Tissue-specific promoters can comprise nucleic acid
sequences which cause a greater (or decreased) level of expression
in cells of a certain tissue type.
[0098] "Operably linked" means that the promoter controls the
initiation of expression of the gene. A promoter is operably linked
to a sequence of proximal DNA if upon introduction into a host cell
the promoter determines the transcription of the proximal DNA
sequence(s) into one or more species of RNA. A promoter is operably
linked to a DNA sequence if the promoter is capable of initiating
transcription of that DNA sequence.
[0099] "Prokaryote" refers to all organisms without a true nucleus,
including bacteria.
[0100] "Eukaryote" refers to organisms and cells that have a true
nucleus, including mammalian cells.
[0101] "Host" includes prokaryotes and eukaryotes, such as yeast
and filamentous fungi, as well as plant and animal cells. The term
includes an organism or cell that is the recipient of a replicable
expression vehicle.
[0102] The term "animal" is used herein to include all vertebrate
animals, except humans. It also includes an individual animal in
all stages of development, including embryonic and fetal stages.
Preferred animals include higher eukaryotes such as avians, rodents
(e.g., mice, rabbits, rats, chinchillas, guinea pigs, hamsters and
the like), and mammals. Preferred mammals include bovine, equine,
feline, canine, ovine, caprine, porcine, buffalo, humans, and
primates.
[0103] A "transgenic animal" is an animal containing one or more
cells bearing genetic information received, directly or indirectly,
by deliberate genetic manipulation or by inheritance from a
manipulated progenitor at a subcellular level, such as by
microinjection or infection with a recombinant viral vector (e.g.,
adenovirus, retrovirus, herpes virus, adeno-associated virus,
lentivirus). This introduced DNA molecule may be integrated within
a chromosome, or it may be extra-chromosomally replicating DNA.
[0104] "Embryonic stem cells" or "ES cells" as used herein are
cells or cell lines usually derived from embryos which are
pluripotent meaning that they are un-differentiated cells. These
cells are also capable of incorporating exogenous DNA by homologous
recombination and subsequently developing into any tissue in the
body when incorporated into a host embryo. It is possible to
isolate pluripotent cells from sources other than embryonic tissue
by methods which are well understood in the art.
[0105] Embryonic stem cells in mice have enabled researchers to
select for transgenic cells and perform gene targeting. This allows
more genetic engineering than is possible with other transgenic
techniques. For example, mouse ES cells are relatively easy to grow
as colonies in vitro. The cells can be transfected by standard
procedures and transgenic cells clonally selected by antibiotic
resistance. See, for example, Doetschman et al., 1994, Gene
transfer in embryonic stem cells. In Pinkert (Ed.) Transgenic
Animal Technology A Laboratory Handbook. Academic Press Inc., New
York, pp. 115-146. Furthermore, the efficiency of this process is
such that sufficient transgenic colonies (hundreds to thousands)
can be produced to allow a second selection for homologous
recombinants. Mouse ES cells can then be combined with a normal
host embryo and, because they retain their potency, can develop
into all the tissues in the resulting chimeric animal, including
the germ cells. The transgenic modification can then be transmitted
to subsequent generations.
[0106] Methods for deriving embryonic stem (ES) cell lines in vitro
from early preimplantation mouse embryos are well known. See for
example, Evans et al., 1981 Nature 29: 154-6 and Martin, 1981,
Proc. Nat. Acad. Sci. USA, 78: 7634-8. ES cells can be passaged in
an undifferentiated state, provided that a feeder layer of
fibroblast cells or a differentiation inhibiting source is
present.
[0107] The term "somatic cell" indicates any animal or human cell
which is not a sperm or egg cell or is capable of becoming a sperm
or egg cell. The term "germ cell" or "germ-line cell" refers to any
cell which is either a sperm or egg cell or is capable of
developing into a sperm or egg cell and can, therefore pass its
genetic information to offspring. The term "germ cell-line
transgenic animal" refers to a transgenic animal in which the
genetic information was incorporated in a germ line cell, thereby
conferring the ability to transfer the information to offspring. If
such offspring in fact possess some or all of that information,
then they, too, are transgenic animals.
[0108] The genetic alteration of genetic information may be foreign
to the species of animal to which the recipient belongs, or foreign
only to the particular individual recipient. In the last case, the
altered or introduced gene may be expressed differently than the
native gene.
[0109] "Fragment" of a gene refers to any portion of a gene
sequence. A "biologically active fragment" refers to any portion of
the gene that retains at least one biological activity of that
gene. For example, the fragment can perhaps hybridize to its
cognate sequence or is capable of being translated into a
polypeptide fragment encoded by the gene from which it is
derived.
[0110] "Variant" refers to a gene that is substantially similar in
structure and biological activity or immunological characteristics
to either the entire gene or to a fragment of the gene. Provided
that the two genes possess a similar activity, they are considered
variant as that term is used herein even if the sequence of encoded
amino acid residues is not identical. Preferentially, as used
herein (unless otherwise defined) the variant is one of LRP5, HBM
or LRP6. The variant preferably is one that yields an HBM-like
phenotype (i.e., enhances bones mass and/or modulates lipid
levels). These variants include missense mutations, single
nucleotide polymorphisms (SNPs), mutations which result in changes
in the amino acid sequence of the protein encoded by the gene or
nucleic acid, and combinations thereof, as well as com in the exon
domains of the HBM gene and mutations in LRP5 or LRP6 which result
in an HBM like phenotype.
[0111] "Amplification of nucleic acids" refers to methods such as
polymerase chain reaction (PCR), ligation amplification (or ligase
chain reaction, LCR) and amplification methods based on the use of
Q-beta replicase. These methods are well known in the art and
described, for example, in U.S. Pat. Nos. 4,683,195 and 4,683,202.
Reagents and hardware for conducting PCR are commercially
available. Primers useful for amplifying sequences from the HBM
region are preferably complementary to, and hybridize specifically
to sequences in the HBM region or in regions that flank a target
region therein. HBM sequences generated by amplification may be
sequenced directly. Alternatively, the amplified sequence(s) may be
cloned prior to sequence analysis.
[0112] "Antibodies" may refer to polyclonal and/or monoclonal
antibodies and fragments thereof, and immunologic binding
equivalents thereof, that can bind to the HBM proteins and
fragments thereof or to nucleic acid sequences from the HBM region,
particularly from the HBM locus or a portion thereof. Preferred
antibodies also include those capable of binding to LRP5, LRP6 and
HBM variants. The term antibody is used both to refer to a
homogeneous molecular entity, or a mixture such as a serum product
made up of a plurality of different molecular entities. Proteins
may be prepared synthetically in a protein synthesizer and coupled
to a carrier molecule and injected over several months into
rabbits. Rabbit sera is tested for immunoreactivity to the HBM
protein or fragment. Monoclonal antibodies may be made by injecting
mice with the proteins, or fragments thereof. Monoclonal antibodies
will be screened by ELISA and tested for specific immunoreactivity
with HBM protein or fragments thereof. Harlow et al., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1988) and Using Antibodies: A Laboratory Manual,
Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999). These
antibodies will be useful in assays as well as pharmaceuticals. By
"antibody" is meant to include but not limited to polyclonal,
monoclonal, chimeric, human, humanized, bispecific, multispecific,
Primatized.TM.antibodies.
[0113] "HBM protein" refers to a protein that is identical to a
Zmax1 (LRP5) protein except that it contains an alteration of
glycine 171 to a valine. An HBM protein is defined for any organism
that encodes a Zmax1 (LRP5) true homolog. For example, a mouse HBM
protein refers to the mouse Zmax1 (LRP5) protein having the glycine
170 to valine substitution.
[0114] By "HBM-like" is meant a variant of LRP5, LRP6 or HBM which
when expressed in a cell is capable of modulating bone mass, lipid
levels, Dkk activity, and/or Wnt activity.
[0115] In one embodiment of the present invention, "HBM gene"
refers to the genomic DNA sequence found in individuals showing the
HBM characteristic or phenotype, where the sequence encodes the
protein indicated by SEQ ID NO: 4. The HBM gene and the Zmax1
(LRP5) gene are allelic. The protein encoded by the HBM gene has
the property of causing elevated bone mass, while the protein
encoded by the Zmax1 (LRP5) gene does not. The HBM gene and the
Zmax1 (LRP5) gene differ in that the HBM gene has a thymine at
position 582, while the Zmax1 gene has a guanine at position 582.
The HBM gene comprises the nucleic acid sequence shown as SEQ ID
NO: 2. The HBM gene may also be referred to as an "HBM
polymorphism." Other HBM genes may further have silent mutations,
such as those discussed in Section 3 below.
[0116] In alternative embodiments of the present invention, "HBM
gene" may also refer to any allelic variant of Zmax1 (LRP5) or LRP6
which results in the HBM phenotype. Such variants may include
alteration from the wild-type protein coding sequence as described
herein and/or alteration in expression control sequences of Zmax1
(LRP5) or contains an amino acid mutation in LRP5 or LRP6, such
that the resulting protein produces a phenotype which enhances bone
mass and/or modulates lipid levels. A preferred example of such a
variant is an alteration of the endogenous Zmax1 (LRP5) promoter
region resulting in increased expression of the Zmax1 (LRP5)
protein.
[0117] "Normal," "wild-type," "unaffected", "Zmax1", "Zmax1", "LR3"
and "LRP5" all refer to the genomic DNA sequence that encodes the
protein indicated by SEQ ID NO: 3. LRP5 has also been referred to
LRP7 in mouse. Zmax1, LRP5 and Zmax may be used interchangeably
throughout the specification and are meant to be the same gene,
perhaps only relating to the gene in a different organism. The
Zmax1 gene has a guanine at position 582 in the human sequence. The
Zmax1 gene of human comprises the nucleic acid sequence shown as
SEQ ID NO: 1. "Normal," "wild-type," "unaffected", "Zmax1" and
"LRP5" also refer to allelic variants of the genomic sequence that
encodes proteins that do not contribute to elevated bone mass. The
Zmax1 (LRP5) gene is common in the human population, while the HBM
gene is rare.
[0118] "5YWTD+EGF" refers to a repeat unit found in the Zmax1
(LRP5) protein, consisting of five YWTD repeats followed by an EGF
repeat.
[0119] "Bone development" generally refers to any process involved
in the change of bone over time, including, for example, normal
development, changes that occur during disease states, and changes
that occur during aging. This may refer to structural changes in
and dynamic rate changes such as growth rates, resorption rates,
bone repair rates, and etc. "Bone development disorder"
particularly refers to any disorders in bone development including,
for example, changes that occur during disease states and changes
that occur during aging. Bone development may be progressive or
cyclical in nature. Aspects of bone that may change during
development include, for example, mineralization, formation of
specific anatomical features, and relative or absolute numbers of
various cell types.
[0120] "Bone modulation" or "modulation of bone formation" refers
to the ability to affect any of the physiological processes
involved in bone remodeling, as will be appreciated by one skilled
in the art, including, for example, bone resorption and
appositional bone growth, by, inter alia, osteoclastic and
osteoblastic activity, and may comprise some or all of bone
formation and development as used herein.
[0121] Bone is a dynamic tissue that is continually adapting and
renewing itself through the removal of old or unnecessary bone by
osteoclasts and the rebuilding of new bone by osteoblasts. The
nature of the coupling between these processes is responsible both
for the modeling of bone during growth as well as the maintenance
of adult skeletal integrity through remodeling and repair to meet
the everyday needs of mechanical usage. There are a number of
diseases of bone that result from an uncoupling of the balance
between bone resorption and formation. With aging there is a
gradual "physiologic" imbalance in bone turnover, which is
particularly exacerbated in women due to menopausal loss of
estrogen support, that leads to a progressive loss of bone. The
reduction in bone mass and deterioration in bone architecture
results in an increase in bone fragility and susceptibility to
spontaneous fractures. For every percent of bone that is lost the
risk of fracture doubles. Individuals with bone mineral density
(BMD) in the spine or proximal femur 2.5 or more standard
deviations below normal peak bone mass are classified as
osteoporotic. However, osteopenic individuals with BMD between 1
and 2.5 standard deviations below the norm are clearly at risk of
suffering bone loss related disorders.
[0122] Bone modulation may be assessed by measuring parameters such
as bone mineral density (BMD) and bone mineral content (BMC) by
pDXA X-ray methods, bone size, thickness or volume as measured by
X-ray, bone formation rates as measured for example by calcien
labeling, total, trabecular, and mid-shaft density as measured by
pQCT and/or mCT methods, connectivity and other histological
parameters as measured by mCT methods, mechanical bending and
compressive strengths as preferably measured in femur and vertebrae
respectively. Due to the nature of these measurements, each may be
more or less appropriate for a given situation as the skilled
practitioner will appreciate. Furthermore, parameters and
methodologies such as a clinical history of freedom from fracture,
bone shape, bone morphology, connectivity, normal histology,
fracture repair rates, and other bone quality parameters are known
and used in the art. Most preferably, bone quality may be assessed
by the compressive strength of vertebra when such a measurement is
appropriate. Bone modulation may also be assessed by rates of
change in the various parameters. Most preferably, bone modulation
is assessed at more than one age.
[0123] "Normal bone density" refers to a bone density within two
standard deviations of a Z score of 0 in the context of the HBM
linkage study. In a general context, the range of normal bone
density parameters is determined by routine statistical methods. A
normal parameter is within about 1 or 2 standard deviations of the
age and sex normalized parameter, preferably about 2 standard
deviations. A statistical measure of meaningfulness is the P value
which can represent the likelihood that the associated measurement
is significantly different from the mean. Significant P values are
P<0.05, 0.01, 0.005, and 0.001, preferably at least
P<0.01.
[0124] "HBM" refers to "high bone mass" although this term may also
be expressed in terms of bone density, mineral content, and
size.
[0125] The "HBM phenotype" and "HBM-like phenotype" may be
characterized by an increase of about 2 or more standard
deviations, preferably 2, 2.5, 3, or more standard deviations in 1,
2, 3, 4, 5, or more quantitative parameters of bone modulation,
preferably bone-density and mineral content and bone strength
parameters, above the age and sex norm for that parameter. The HBM
phenotype and HBM-like phenotype are characterized by statistically
significant increases in at least one parameter, preferably at
least 2 parameters, and more preferably at least 3 or more
parameters. The HBM phenotype and the HBM-like phenotype may also
be characterized by an increase in one or more bone quality
parameters and most preferably increasing parameters are not
accompanied by a decrease in any bone quality parameters. Most
preferably, an increase in bone modulation parameters and/or bone
quality measurements is observed at more than one age. The IBM
phenotype and HBM-like phenotype also includes changes of lipid
levels, Wnt activity and/or Dkk activity.
[0126] A "Zmax1 system" or "LRP5 system" refers to a purified
protein, cell extract, cell, animal, human or any other composition
of matter in which Zmax1 (LRP5) is present in a normal or mutant
form.
[0127] The terms "isolated" and "purified" refer to a substance
altered by hand of man from the natural environment. An isolated
peptide may be for example in a substantially pure form or
otherwise displaced from its native environment such as by
expression in an isolated cell line or transgenic animal. An
isolated sequence may for example be a molecule in substantially
pure form or displaced from its native environment such that at
least one end of said isolated sequence is not contiguous with the
sequence it would be contiguous with in nature.
[0128] A "surrogate marker" refers to a diagnostic indication,
symptom, sign or other feature that can be observed in a cell,
tissue, human or animal that is correlated with the HBM gene or
elevated bone mass or both, but that is easier to measure than bone
density. The general concept of a surrogate marker is well accepted
in diagnostic medicine.
[0129] The present invention encompasses the Zmax1 (LRP5) gene and
Zmax1 (LRP5) protein in the forms indicated by SEQ ID NOS: 1 and 3,
respectively, and other closely related variants, as well as the
adjacent chromosomal regions of Zmax1 (LRP5) necessary for its
accurate expression. In a preferred embodiment, the present
invention is directed to at least 15 contiguous nucleotides of the
nucleic acid sequence of SEQ ID NO: 1.
[0130] The present invention further encompasses variants of the
LRP6 gene and its corresponding protein which result in an enhanced
bone mass and/or modulate lipids and/or modulate the Wnt signaling
pathway.
[0131] "Biologically active" refers to those forms of proteins and
polypeptides, including conservatively substituted variants,
alleles of genes encoding a protein or polypeptide fragments of
proteins which retain a biological and/or immunological activity of
the wild-type protein or polypeptide. Preferably the activity is
one which induces a change in LRP5, LRP6, or Dkk activity, such as
inhibiting the interaction of LRP5 or LRP6 or variants thereof with
Dkk, or Dkk with another ligand binding partner (e.g., Dkk-1 with a
Dkk-1 interacting protein such as those shown in FIG. 31). By
biologically active is also meant to include any form which
modulates Wnt signaling.
[0132] By "modulate" and "regulate" is meant methods, conditions,
or agents which increase or decrease the wild-type activity of an
enzyme, inhibitor, signal transducer, receptor, transcription
activator, co-factor, and the like. This change in activity can be
an increase or decrease of mRNA translation, mRNA or DNA
transcription, and/or mRNA or protein degradation, which may in
turn correspond to an increase or decrease in biological
activity.
[0133] By "modulated activity" and "regulated activity" is meant
any activity, condition, disease or phenotype which is modulated by
a biologically active form of a protein. Modulation may be effected
by affecting the concentration or subcellular localization of
biologically active protein, i.e., by regulating expression or
degradation, or by direct agonistic or antagonistic effect as, for
example, through inhibition, activation, binding, or release of
substrate, modification either chemically or structurally, or by
direct or indirect interaction which may involve additional
factors.
[0134] By "effective amount" or "dose effective amount" or
"therapeutically effective amount" is meant an amount of an agent
which modulates a biological activity of the polypeptide of the
invention.
[0135] By "immunologically active" is meant any immunoglobulin
protein or fragment thereof which recognizes and binds to an
antigen.
[0136] By "Dkk" is meant to refer to the nucleic acids and proteins
of members of the Dkk (Dickkopf) family. This includes, but is not
limited to, Dkk-1, Dkk-2, Dkk-3, Dkk-4, Soggy, and related Dkk
proteins. Dkk-1 is a preferred embodiment of the present invention.
However, the Dkk proteins have substantial homology and one skilled
in the art will appreciate that all of the embodiments of the
present invention utilizing Dkk-1 may also be utilized with the
other Dkk proteins.
[0137] By "Dkk-1" is meant to refer to the Dkk-1 protein and
nucleic acids which encode the Dkk-1 protein. Dkk-1 refers to
Dickkopf-1, and in Xenopus it is related to at least Dkk-2, Dkk-3,
and Dkkk-4 (see Krupnik et al., 1999 Gene 238: 301-313). Dkk-1 was
first identified in Xenopus (Glinka et al., 1998 Nature 391:
357-62). It was recognized as a factor capable of inducing ectopic
head formation in the presence of inhibition of the BMP pathway. It
was then also found to inhibit the axis-inducing activity of
several Xenopus Wnt molecules by acting as an extracellular
antagonist of Wnt signaling. Mammalian homologs have been found
including Dkk-1, Dkk-2, Dkk-3, Dkk-4 and soggy (Fedi et al., 1999
J. Biol. Chem. 274: 19465-72; and Krupnick et al. 1999). Human
Dkk-1 was also referred to as sk (Fedi et al. 1999). As used
herein, Dkk-1 is meant to include proteins from any species having
a Wnt pathway in which Dkk-1 interacts. Particularly preferred are
mammalian species (e.g., murine, caprine, canine, bovine, feline,
equine, primate, ovine, porcine and the like), with particularly
preferred mammals being humans. Nucleic acid sequences encoding
Dkk-1 include, but are not limited to human Dkk-1 (GenBank
Accession Nos. AH009834, XM.sub.--005730, AF261158, AF261157,
AF177394, AF127563 and NM.sub.--012242), Mus musculus dickkopf
homolog 1 (GenBank Accession No. NM.sub.--010051), and Danio rerio
dickkopf-1 (GenBank Accession Nos. AF116852 and AB023488). The
genomic sequences with exon annotation are GenBank Accession Nos.
AF261157 and AF261158. Also contemplated are homologs of these
sequences which have Dkk-1 activity in the Wnt pathway. Dkk-1 amino
acid sequences include, but are not limited to human dickkopf
homolog 1 (GenBank Accession Nos. AAG15544, BAA34651,
NP.sub.--036374, AAF02674, AAD21087, and XP.sub.--005730), Danio
rerio (zebrafish) dickkopfl (GenBank Accession Nos. BAA82135 and
AAD22461) and murine dickkopf-1 (GenBank Accession Nos. O54908 and
NP.sub.--034181). Variants and homologs of these sequences which
possess Dkk-1 activity are also included when referring to
Dkk-1.
[0138] By "LRP5 mediated", "LRP6 mediated", and "Dkk mediated"
disorder, condition or disease is any abnormal state that involves
LRP5, LRP6 and/or Dkk activity. The abnormal state can be induced
by environmental exposure or drug administration. Alternatively,
the disease or disorder can be due to a genetic defect. Dkk
mediated diseases, disorders and conditions include but are not
limited to bone mass disorders or conditions and lipid disorders
and conditions. For example, bone mass
disorders/conditions/diseases, which may be mediated by Dkk, LRP5
and/or LRP6 include but are not limited to age related loss of
bone, bone fractures (e.g., hip fracture, Colle's fracture,
vertebral crush fractures), chondrodystrophies, drug-induced
disorders (e.g., osteoporosis due to administration of
glucocorticoids or heparin and osteomalacia due to administration
of aluminum hydroxide, anticonvulsants, or glutethimide), high bone
turnover, hypercalcemia, hyperostosis, osteoarthritis, osteogenesis
imperfecta, osteomalacia, osteomyelitis, osteoporosis, Paget's
disease, and rickets.
[0139] Lipid disorders/diseases/conditions, which may be mediated
by Dkk, LRP5, and/or LRP6 include but are not limited to familial
lipoprotein lipase deficiency, familial apoprotein CII deficiency,
familial type 3 hyperlipoproteinemia, familial
hypercholesterolemia, familial hypertriglyceridemia, multiple
lipoprotein-type hyperlipidemia, elevated lipid levels due to
dialysis and/or diabetes, and elevated lipid levels of unknown
etiologies
[0140] The term "recognizes and binds," when used to define
interactions of antisense nucleotides or siRNA's (small inhibitory
RNA) with a target sequence, means that a particular antisense or
small inhibitory RNA (siRNA) sequence is substantially
complementary to the target sequence, and thus will specifically
bind to a portion of an mRNA encoding polypeptide. As such,
typically the sequences will be highly complementary to the mRNA
target sequence, and will have no more than 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 base mismatches throughout the sequence. In many
instances, it may be desirable for the sequences to be exact
matches, i.e. be completely complementary to the sequence to which
the oligonucleotide specifically binds, and therefore have zero
mismatches along the complementary stretch. As such, highly
complementary sequences will typically bind quite specifically to
the target sequence region of the mRNA and will therefore be highly
efficient in reducing, and/or even inhibiting the translation of
the target mRNA sequence into polypeptide product.
[0141] Substantially complementary oligonucleotide sequences will
be greater than about 80 percent complementary (or `% exact-match`)
to the corresponding mRNA target sequence to which the
oligonucleotide specifically binds, and will, more preferably be
greater than about 85 percent complementary to the corresponding
mRNA target sequence to which the oligonucleotide specifically
binds. In certain aspects, as described above, it will be desirable
to have even more substantially complementary oligonucleotide
sequences for use in the practice of the invention, and in such
instances, the oligonucleotide sequences will be greater than about
90 percent complementary to the corresponding mRNA target sequence
to which the oligonucleotide specifically binds, and may in certain
embodiments be greater than about 95 percent complementary to the
corresponding mRNA target sequence to which the oligonucleotide
specifically binds, and even up to and including 96%, 97%, 98%,
99%, and even 100% exact match complementary to the target mRNA to
which the designed oligonucleotide specifically binds.
[0142] Percent similarity or percent complementary of any of the
disclosed sequences may be determined, for example, by comparing
sequence information using the GAP computer program, version 6.0,
available from the University of Wisconsin Genetics Computer Group
(UWGCG). The GAP program utilizes the alignment method of Needleman
and Wunsch (1970). Briefly, the GAP program defines similarity as
the number of aligned symbols (i.e., nucleotides or amino acids)
which are similar, divided by the total number of symbols in the
shorter of the two sequences. The preferred default parameters for
the GAP program include: (1) a unary comparison matrix (containing
a value of 1 for identities and 0 for non-identities) for
nucleotides, and the weighted comparison matrix of Gribskov and
Burgess (1986), (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps.
[0143] By "mimetic" is meant a compound or molecule that performs
the same function or behaves similarly to the compound
mimicked.
[0144] By "reporter element" is meant a polynucleotide that encodes
a polypeptide capable of being detected in a screening assays.
Examples of polypeptides encoded by reporter elements include, but
are not limited to, lacZ, GFP, luciferase, and chloramphenicol
acetyltransferase.
2. Introduction
[0145] The present invention also encompasses the HBM gene and HBM
protein in the forms indicated by SEQ ID NO: 2 and 4, respectively,
and other closely related variants, as well as the adjacent
chromosomal regions of the HBM gene necessary for its accurate
expression. In a preferred embodiment, the present invention is
directed to at least 15 contiguous nucleotides of the nucleic acid
sequence of SEQ ID NO: 2. More preferably, the present invention is
directed to at least 15 contiguous nucleotides of the nucleic acid
sequence of SEQ ID NO: 2, wherein one of the 15 contiguous
nucleotides is the thymine at nucleotide 582.
[0146] The invention also relates to the nucleotide sequence of the
Zmax1 (LRP5) gene region, as well as the nucleotide sequence of the
HBM region. More particularly, a preferred embodiment are the BAC
clones containing segments of the Zmax1 (LRP5) gene region
B200E21-H and B527D12-H. A preferred embodiment is the nucleotide
sequence of the BAC clones consisting of SEQ ID NOS: 5-12.
[0147] The invention also concerns the use of the nucleotide
sequence to identify DNA probes for the Zmax1 (LRP5) gene and the
HBM gene, PCR primers to amplify the Zmax1 (LRP5) gene and the HBM
gene, nucleotide polymorphisms in the Zmax1 (LRP5) gene and the HBM
gene, and regulatory elements of the Zmax1 (LRP5) gene and the HBM
gene.
[0148] This invention describes the further localization of the
chromosomal location of the Zmax1 (LRP5) gene and HBM gene on
chromosome 11q13.3, between genetic markers D11S987 and
SNP_CONTIG033-6, as well as the DNA sequences of the Zmax1 (LRP5)
gene and the HBM gene. The chromosomal location was refined by the
addition of more genetic markers to the mapping panel used to map
the gene, and by the extension of the pedigree to include more
individuals. The pedigree extension was critical because the new
individuals that have been genotyped harbor critical recombination
events that narrow the region. To identify genes in the region on
11q13.3, a set of BAC clones containing this chromosomal region was
identified. The BAC clones served as a template for genomic DNA
sequencing, and also as a reagent for identifying coding sequences
by direct cDNA selection. Genomic sequencing and direct cDNA
selection were used to characterize more than 1.5 million base
pairs of DNA from 11q13.3. The Zmax1 (LRP5) gene was identified
within this region and the HBM gene was then discovered after
mutational analysis of affected and unaffected individuals.
[0149] When a gene has been genetically localized to a specific
chromosomal region, the genes in this region can be characterized
at the molecular level by a series of steps that include: cloning
of the entire region of DNA in a set of overlapping clones
(physical mapping), characterization of genes encoded by these
clones by a combination of direct cDNA selection, exon trapping and
DNA sequencing (gene identification), and identification of
mutations in these genes by comparative DNA sequencing of affected
and unaffected members of the HBM kindred (mutation analysis).
[0150] Physical mapping is accomplished by screening libraries of
human DNA cloned in vectors that are propagated in E. coli or S.
cereviseae using PCR assays designed to amplify unique molecular
landmarks in the chromosomal region of interest. To generate a
physical map of the HBM candidate region, a library of human DNA
cloned in Bacterial Artificial Chromosomes (BACs) was screened with
a set of Sequence Tagged Site (STS) markers that had been
previously mapped to chromosome 11q12-q13 by the efforts of the
Human Genome Project.
[0151] STSs are unique molecular landmarks in the human genome that
can be assayed by PCR. Through the combined efforts of the Human
Genome Project, the location of thousands of STSs on the twenty-two
autosomes and two sex chromosomes has been determined. For a
positional cloning effort, the physical map is tied to the genetic
map because the markers used for genetic mapping can also be used
as STSs for physical mapping. By screening a BAC library with a
combination of STSs derived from genetic markers, genes, and random
DNA fragments, a physical map comprised of overlapping clones
representing all of the DNA in a chromosomal region of interest can
be assembled.
[0152] BACs are cloning vectors for large (80 kilobase to 200
kilobase) segments of human or other DNA that are propagated in E.
coli. To construct a physical map using BACs, a library of BAC
clones is screened so that individual clones harboring the DNA
sequence corresponding to a given STS or set of STSs are
identified. Throughout most of the human genome, the STS markers
are spaced approximately 20 to 50 kilobases apart, so that an
individual BAC clone typically contains at least two STS markers.
In addition, the BAC libraries that were screened contain enough
cloned DNA to cover the human genome six times over. Therefore, an
individual STS typically identifies more than one BAC clone. By
screening a six-fold coverage BAC library with a series of STS
markers spaced approximately 50 kilobases apart, a physical map
consisting of a series of overlapping BAC clones, i.e. BAC contigs,
can be assembled for any region of the human genome. This map is
closely tied to the genetic map because many of the STS markers
used to prepare the physical map are also genetic markers.
[0153] When constructing a physical map, it often happens that
there are gaps in the STS map of the genome that result in the
inability to identify BAC clones that are overlapping in a given
location. Typically, the physical map is first constructed from a
set of STSs that have been identified through the publicly
available literature and World Wide Web resources. The initial map
consists of several separate BAC contigs that are separated by gaps
of unknown molecular distance. To identify BAC clones that fill
these gaps, it is necessary to develop new STS markers from the
ends of the clones on either side of the gap. This is done by
sequencing the terminal 200 to 300 base pairs of the BACs flanking
the gap, and developing a PCR assay to amplify a sequence of 100 or
more base pairs. If the terminal sequences are demonstrated to be
unique within the human genome, then the new STS can be used to
screen the BAC library to identify additional BACs that contain the
DNA from the gap in the physical map. To assemble a BAC contig that
covers a region the size of the HBM candidate region (2,000,000 or
more base pairs), it is often necessary to develop new STS markers
from the ends of several clones.
[0154] After building a BAC contig, this set of overlapping clones
serves as a template for identifying the genes encoded in the
chromosomal region. Gene identification can be accomplished by many
methods. Three methods are commonly used: (1) a set of BACs
selected from the BAC contig to represent the entire chromosomal
region can be sequenced, and computational methods can be used to
identify all of the genes, (2) the BACs from the BAC contig can be
used as a reagent to clone cDNAs corresponding to the genes encoded
in the region by a method termed direct cDNA selection, or (3) the
BACs from the BAC contig can be used to identify coding sequences
by selecting for specific DNA sequence motifs in a procedure called
exon trapping. The present invention includes genes identified by
the first two methods.
[0155] To sequence the entire BAC contig representing the HBM
candidate region, a set of BACs was chosen for subcloning into
plasmid vectors and subsequent DNA sequencing of these subclones.
Since the DNA cloned in the BACs represents genomic DNA, this
sequencing is referred to as genomic sequencing to distinguish it
from cDNA sequencing. To initiate the genomic sequencing for a
chromosomal region of interest, several non-overlapping BAC clones
are chosen. DNA for each BAC clone is prepared, and the clones are
sheared into random small fragments which are subsequently cloned
into standard plasmid vectors such as pUC18. The plasmid clones are
then grown to propagate the smaller fragments, and these are the
templates for sequencing. To ensure adequate coverage and sequence
quality for the BAC DNA sequence, sufficient plasmid clones are
sequenced to yield six-fold coverage of the BAC clone. For example,
if the BAC is 100 kilobases long, then phagemids are sequenced to
yield 600 kilobases of sequence. Since the BAC DNA was randomly
sheared prior to cloning in the phagemid vector, the 600 kilobases
of raw DNA sequence can be assembled by computational methods into
overlapping DNA sequences termed sequence contigs. For the purposes
of initial gene identification by computational methods, six-fold
coverage of each BAC is sufficient to yield ten to twenty sequence
contigs of 1000 base pairs to 20,000 base pairs.
[0156] The sequencing strategy employed in this invention was to
initially sequence "seed" BACs from the BAC contig in the HBM
candidate region. The sequence of the "seed" BACs was then used to
identify minimally overlapping BACs from the contig, and these were
subsequently sequenced. In this manner, the entire candidate region
was sequenced, with several small sequence gaps left in each BAC.
This sequence served as the template for computational gene
identification. One method for computational gene identification is
to compare the sequence of BAC contig to publicly available
databases of cDNA and genomic sequences, e.g., unigene, dbEST,
GenBank. These comparisons are typically done using the BLAST
family of computer algorithms and programs (Altschul et al., J.
Mol. Biol., 215:403-410 (1990)). The BAC sequence can also be
translated into protein sequence, and the protein sequence can be
used to search publicly available protein databases, using a
version of BLAST designed to analyze protein sequences (Altschul et
al., 1997 Nucl. Acids Res. 25: 3389-3402). Another method is to use
computer algorithms such as MZEF (Zhang, 1997 Proc. Natl. Acad.
Sci. USA, 94: 565-568) and GRAIL (Uberbacher et al., 1996 Methods
Enzymol. 266: 259-281), which predict the location of exons in the
sequence based on the presence of specific DNA sequence motifs that
are common to all exons, as well as the presence of codon usage
typical of human protein encoding sequences.
[0157] In addition to identifying genes by, computational methods,
genes were also identified by direct cDNA selection (Del Mastro et
al., 1995 Genome Res. 5(2): 185-194). In direct cDNA selection,
cDNA pools from tissues of interest are prepared, and the BACs from
the candidate region are used in a liquid hybridization assay to
capture cDNA which base-pairs to coding regions in the BAC. In the
methods described herein, the cDNA pools were created from several
different tissues by random priming the first strand cDNA from
poly-A RNA, synthesizing the second strand cDNA by standard
methods, and adding linkers to the ends of the cDNA fragments. The
linkers are used to amplify the cDNA pools. The BAC clones are used
as a template for in vitro DNA synthesis to create a biotin labeled
copy of the BAC DNA. The biotin labeled copy of the BAC DNA is then
denatured and incubated with an excess of the PCR amplified,
linkered cDNA pools which have also been denatured. The BAC DNA and
cDNA are allowed to anneal in solution, and heteroduplexes between
the BAC and the cDNA are isolated using streptavidin coated
magnetic beads. The cDNA which is captured by the BAC is then
amplified using primers complimentary to the linker sequences, and
the hybridization/selection process is repeated for a second round.
After two rounds of direct cDNA selection, the cDNA fragments are
cloned, and a library of these direct selected fragments is
created.
[0158] The cDNA clones isolated by direct selection are analyzed by
two methods. Since a pool of BACs from the HBM candidate region is
used to provide the genomic DNA sequence, the cDNAs must be mapped
to individual BACs. This is accomplished by arraying the BACs in
microtiter dishes, and replicating their DNA in high density grids.
Individual cDNA clones are then hybridized to the grid to confirm
that they have sequence identity to an individual BAC from the set
used for direct selection, and to determine the specific identity
of that BAC. cDNA clones that are confirmed to correspond to
individual BACs are sequenced. To determine whether the cDNA clones
isolated by direct selection share sequence identity or similarity
to previously identified genes, the DNA and protein coding
sequences are compared to publicly available databases using the
BLAST family of programs.
[0159] The combination of genomic DNA sequence and cDNA sequence
provided by BAC sequencing and by direct cDNA selection yields an
initial list of putative genes in the region. The genes in the
region were all candidates for the HBM locus. To further
characterize each gene, Northern blots were performed to determine
the size of the transcript corresponding to each gene, and to
determine which putative exons were transcribed together to make an
individual gene. For Northern blot analysis of each gene, probes
were prepared from direct selected cDNA clones or by PCR amplifying
specific fragments from genomic DNA or from the BAC encoding the
putative gene of interest. The Northern blots gave information on
the size of the transcript and the tissues in which it was
expressed. For transcripts which were not highly expressed, it was
sometimes necessary to perform a reverse transcription PCR assay
using RNA from the tissues of interest as a template for the
reaction.
[0160] Gene identification by computational methods and by direct
cDNA selection provides unique information about the genes in a
region of a chromosome. When genes are identified, then it is
possible to examine different individuals for mutations in each
gene.
[0161] The present invention also encompasses the HBM gene and HBM
protein in the forms indicated by SEQ ID NO: 2 and 4, respectively,
and other closely related variants, as well as the adjacent
chromosomal regions of the HBM gene necessary for its accurate
expression. In a preferred embodiment, the present invention is
directed to an isolated nucleic acid sequence of SEQ ID NO: 2, as
well as variants thereof. Variants include changes in SEQ ID NO:1
which result in a HBM like phenotype. Examples of such variants are
discussed further in Section 3 below and in the examples. These
variants preferably have at least about 90%, preferably at least
about 95%, or more preferably at least about 98% or more similarity
or identity to the nucleic acid sequence of SEQ ID NOS: 1 or 2 or
biologically active fragments thereof. Therefore, sequences which
are 96%, 97%, and 99% or more similar to SEQ ID NOS: 1 or 2 or
biologically active fragments thereof are also contemplated
herein.
[0162] Determination of the degree of variation between a high bone
mass (HBM) variant can be performed using BLAST or FASTA or other
suitable algorithm using standard default parameters. Preferably;
identity will be determined for coding regions of SEQ ID NOS: 1-2,
but can also include non-coding domains. Additionally, alignment
programs can be used to identify conserved sequences or potential
motifs across different animal species. Alignment programs can also
be used to align the nucleic acid and/or protein sequences of
related genes and the proteins that they encode. Preferred
alignment programs include CLUSTALW, PILEUP and GAP, and would
preferably be used with default parameters. For example, such
programs can be used to align the sequences of Zmax1 (LRP5), HBM,
and LDL receptor-related protein 6 (LRP6) and sequences related
thereto.
[0163] By a polynucleotide having a nucleotide sequence at least,
for example, 90% "similar" to a reference nucleotide sequence
encoding a polypeptide, is intended that the nucleotide sequence of
the polynucleotide is identical to the reference sequence except
that the polynucleotide sequence may include up to ten point
mutations per each 100 nucleotides of the reference nucleotide
sequence. These mutations of the reference sequence may occur at
any location in SEQ ID NO: 1 or 2 or in the LRP6 gene. The
mutations may be silent.
[0164] Another embodiment contemplates that such polynucleotide
variants of SEQ ID NO: 1 or 2 comprise nucleic acid sequences which
are at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,
150, 200, 300, 400, or 500 contiguous nucleotides of SEQ ID NO: 1
or 2. More preferably, such polynucleotide variants have a
contiguous nucleic acid sequence corresponding with the
polymorphism at nucleotide 582 (G-T substitution) of SEQ ID NO: 2
or other variants of SEQ ID NO: 1 or 2, which comprise a mutation
which modulates bone mass and/or lipid levels when the polypeptide
encoded thereby is administered to a subject. All variants of SEQ
ID NO: 1 or 2 contemplated possess the characteristic of encoding a
protein or polypeptide which when administered to a subject induces
bone modulation. Additional variants which may be responsible for
modulating bone mass when administered to a subject may lie within
the domain known to contain the HBM polymorphism and which encodes
the beta propeller domain (YWTD motifs). Alternatively, other
variants of Zmax1 (LRP5) which modulate high bone mass in a subject
may be due to mutations in the nucleic acid sequences encoding any
of the other conserved domains of Zmax1 (LRP5), such as those set
forth in FIG. 4 (e.g., the RGD extracellular attachment site, the
binding site for LDL and calcium, the cysteine rich growth factor
repeats, the ideal PEST region, and the internalization domain).
See Section 3 and the Examples below for additional mutations which
may confer enhance bone mass and/or lipid modulation.
[0165] HBM polynucleotides and HBM like variants contemplated
include those which hybridize under stringent conditions to SEQ ID
NO: 2. Hybridization methods are known in the art and include, but
are not limited to: (a) washing with 0.1.times.SSPE (0.62 MNaCl,
0.06 M NaH.sub.2 PO.sub.4.H.sub.2O, 0.075 M EDTA, pH 7.4) and 0.1%
sodium dodecyl sulfate (SDS) at 50.degree. C.; (b) washing with 50%
formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM
sodium phosphate (pH 6-8), 0.1% sodium pyrophosphate,
5.times.Denhardt's solution, sonicated salmon sperm DNA (50
.mu.g/ml), 0.1% SDS and 10% dextran sulfate at 42.degree. C.,
followed by washing at 42.degree. C. in 0.2.times.SSC and 0.1% SDS;
and (c) washing with of 0.5 M NaPO.sub.4, 7% SDS at 65.degree. C.
followed by washing at 60.degree. C. in 0.5.times.SSC and 0.1% SDS.
Additional conditions under which HBM variants can be isolated by
hybridization to SEQ ID NO: 2 or nucleic acid fragments thereof can
be performed by varying the hybridization temperature. High
stringency hybridization conditions are those performed at about
2.degree. C. below the melting temperature (T.sub.m) of SEQ ID NO:
2 or fragments thereof. Preferred stringency is performed at about
5-10.degree. C. below the T.sub.m of SEQ ID NO: 2 or fragments
thereof. Additional hybridization conditions can be prepared as
described in Chapter 11 of Sambrook et al., Molecular Cloning: A
Laboratory Manual (1989), or as would be known to the artisan of
ordinary skill.
[0166] Alternatively, mammalian libraries (e.g., equine, primate;
caprine, bovine, ovine, feline, porcine, and canine) can be probed
using degenerate primers and polymerase chain reaction (PCR)
techniques to identify variants of SEQ ID NO: 2 or fragments
thereof. Preferably primers are utilized which hybridize under
stringent conditions to the open reading frame of SEQ ID NO: 2, or
to non-coding portions of the sequence. More preferably, such
primers hybridize to conserved domains within SEQ ID NO: 2. For
example, conserved domains include those coding for the YWTD
beta-propeller domains or other domains, such as those listed in
FIG. 4. Preferred primers are typically 15 nucleotides in length,
but can vary to be at least, about 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 30, 35, 40 or 50 (and any range in between) nucleotides in
length. Heterologous hybridization is to amplify the target gene or
nucleic acid sequence using degenerate PCR primers. Probes for
variants of SEQ ID NO: 2 and the polypeptide encoded thereby can be
obtained by preparing mixed oligonucleotides of greater than 10,
preferably of 15 or more, nucleotides in length representing all
possible nucleotide sequences which could encode the corresponding
amino acid sequences (e.g., SEQ ID NO: 4 fragments thereof). This
method is clearly documented by Gould et al., 1989 Proc. Natl.
Acad. Sci. USA 86(6): 1934-8.
[0167] Another embodiment includes nucleic acids which encode an
HBM polypeptide or HBM like variant which is at least about 90%
similar to SEQ ID NO: 4 and fragments thereof, and which when
administered to a subject modulate bone mass in that subject. HBM
variants may have a valine corresponding to position 171 of SEQ ID
NO: 4 (Gly to Val substitution) or 170 of the mouse homolog, or an
amino acid change elsewhere in propeller 1 or in the protein which
results in enhanced bone mass and/or lipid modulation. Other
preferred embodiments include high bone mass polypeptides which
have at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,
90, 100, 150, 200, 250, 300, 500 or more contiguous amino acids of
SEQ ID NO: 3 or 4, with a mutation in the sequence resulting in
enhanced bone mass and/or modulated lipid levels. Such contemplated
contiguous sequences preferably overlap with a polymorphism
corresponding to high bone mass, such as valine-171 of SEQ ID NO:
4, other mutations in propeller 1 or is predicted from the model
provided in the Examples. Also contemplated are the polynucleotides
encoding polypeptides which are at least about 95%, 96%, 97%, 98%
and 99% or more similar to SEQ ID NO: 3 or 4 and fragments thereof,
wherein these polypeptide contain at least one mutation (e.g.,
valine-171 or like mutation).
[0168] In another embodiment, a synthetic nucleic acid encoding SEQ
ID NO: 4 is contemplated wherein the nucleic acid sequence has been
conservatively substituted based on the degeneracy of the code such
that no amino acids are altered in SEQ ID NO: 4, but perhaps
wherein the resulting synthetic polynucleotide encoding said SEQ ID
NO: 4 is one that is at least, about 50% similar to SEQ ID NO: 2.
Alternatively, SEQ ID NO: 4 may contain any of the silent mutations
identified in Section 3 below.
[0169] By a polypeptide having an amino acid sequence of at least,
for example, 95% "identity" to a reference amino acid sequence of
SEQ ID NO: 3 or 4 or fragment thereof is intended that the amino
acid sequence of the polypeptide is identical to the reference
sequence except that the polypeptide sequence may include up to
five amino acid alterations per each 100 amino acids of the
reference amino acid sequence of SEQ ID NO: 3 or 4 and has an HBM
like phenotype. In other words, to obtain a polypeptide having an
amino acid sequence 95% identical to a reference amino acid
sequence, up to 5% of the amino acid residues in the reference
sequence may be deleted or substituted with another amino acid, or
a number of amino acids up to 5% of the total amino acid residues
in the reference sequence may be inserted into the reference
sequence. These alterations of the reference sequence may occur at
the amino or carboxy terminal positions of the reference amino acid
sequence or anywhere between those terminal positions, interspersed
either individually among residues in the reference sequence or in
one or more contiguous groups within the reference sequence.
[0170] Additional HBM polypeptides and nucleic acids which encode
said HBM polypeptides are contemplated wherein amino acid residues
are conservatively substituted. For example, guidance concerning
how to make phenotypically silent amino acid substitutions is
provided in Bowie et al., "Deciphering the Message in Protein
Sequences: Tolerance to Amino Acid Substitutions," Science 247:
1306-10 (1990), wherein the authors indicate that there are two
main approaches for studying the tolerance of an amino acid
sequence to change. The first method relies on the process of
evolution, in which mutations are either accepted or rejected by
natural selection. The second approach uses genetic engineering to
introduce amino acid changes at specific positions of a cloned gene
and selections or screens to identify sequences that maintain
functionality. These studies have revealed that proteins are
surprisingly tolerant of amino acid substitutions. The authors
further indicate which amino acid changes are likely to be
permissive at a certain position of the protein. Numerous
phenotypic substitutions are described in Bowie et al., supra, and
the references cited therein, which are herein incorporated by
reference in their entirety. Preferred substitutions would be in
domains which are less conserved across species, and which do not
correspond to a structurally or functionally important domain
(e.g., a binding site, catalytic site, or beta propeller or other
domain described in FIG. 4).
[0171] Recent studies have indicated that LRP5 participates in the
Wnt signal transduction pathway. The Wnt pathway is critical in
limb early embryological development. A recently published sketch
of the components of Wnt signaling is shown in FIG. 28 (Nusse, 2001
http://www.stanford.edu/.about.musse/pathways/cell2.html) (see
also, Nusse, 2001 Nature 411: 255-6; and Mao et al., 2001 Nature
411: 321-5).
[0172] Briefly summarized, Wnt proteins are secreted proteins which
interact with the transmembrane protein Frizzled (Fz). LRP
proteins, such as LRP5 and LRP6, are believed to modulate the Wnt
signal in a complex with Fz (Tamai et al., 2000 Nature 407: 530-5).
The Wnt pathway acts intracellularly through the Disheveled protein
(Dsh) which in turn inhibits glycogen synthetase kinase-3 (GSK3)
from phosphorylating .beta.-catenin. Phosphorylated .beta.-catenin
is rapidly degraded following ubiquitination. However, the
stabilized .beta.-catenin accumulates and translocates to the
nucleus where it acts as a cofactor of the T-cell factor (TCF)
transcription activator complex.
[0173] The protein dickkopf-1 (Dkk-1) is reported to be an
antagonist of Wnt pathway. Dkk-1 is required for head formation in
early development (Glinka et al., 1998 Nature 391: 357-62). Dkk-1
and its function in the Wnt pathway are described in e.g., Krupnik,
et al., 1999 Gene 238: 301-13; Fedi et al., 1999 J. Biol. Chem.
274: 19465-72; see also for Dkk-1 and the Wnt pathway, Wu et al.,
2000 Curr. Biol. 10: 1611-4; Shinya et al., 2000 Mech. Dev. 98:
3-17; Mukhopadhyay et al., 2001 Dev. Cell. 1: 423-434; and in PCT
Patent Application No. WO 00/52047, and in references cited in
each. It has been known that Dkk-1 acts upstream of Dsh, however
the nature of the mechanism of inhibition by Dkk-1 is just
beginning to be elucidated. Dkk-1 is expressed in the mouse
embryonic limb bud and its disruption results in abnormal limb
morphogensis, among other developmental defects. (Gotewold et al.,
1999 Mech. Dev. 89:151-3; and Mukhopadhyay et al., 2001 Dev. Cell.
1: 423-34).
[0174] Related U.S. Ser. No. 60/291,311 (herein incorporated by
reference in its entirety) disclosed a novel interaction between
Dkk-1 (GenBank Accession No. XM 005730) and LRP5. The interaction
between Dkk-1 and LRP5 was discovered by a yeast two hybrid (Y2H)
screen for proteins which interact with the ligand binding domain
of LRP5. The two-hybrid screen is a common procedure in the art,
which is described, for example, by Gietz et al., 1997 Mol. Cell.
Biochem. 172: 67-79; Young, 1998 Biol. Reprod. 58: 302-11; Brent et
al., 1997 Ann. Rev. Genet. 31: 663-704; and Lu et al., eds., Yeast
Hybrid Technologies, Eaton Publishing, Natick Mass., (2000). More
recently, other studies confirm that Dkk-1 is a binding partner for
LRP and modulates the Wnt pathway via direct binding with LRP (R.
Nusse, 2001 Nature 411: 255-6; Bafico et al., 2001 Nat. Cell Biol.
3: 683-6; Semenov, 2001 Curr. Biol. 11: 951-61; Mao, 2001 Nature
411: 321-5 (2001); Zorn, 2001 Curr. Biol. 11: R592-5); and Li et
al., 2002 J. Biol. Chem. 277: 5977-81).
[0175] Mao and colleagues (2001) identified Dkk-1 as a ligand for
LRP6. Mao et al. suggest that Dkk-1 and LRP6 interact
antagonistically where Dkk proteins inhibit the Wnt coreceptor
functions of LRP6. Using co-immunoprecipitation, the group verified
that the Dkk-1/LRP6 interaction was direct. Dkk-2 was also found to
directly bind LRP6. Contrary to data contained in provisional
application 60/291,311, Mao et al. report that no interaction was
detected between any Dkk protein and LRP5, as well as no
interaction with LDLR, VLDLR, ApoER, or LRP). Additionally, Mao et
al. demonstrated that LRP6 can titrate Dkk1-1's effects of
inhibiting Wnt signaling using the commercial TCF-luciferase
reporter gene assay (TOPFLASH). A similar conclusion was drawn from
analogous studies in Xenopus embryos. Deletion analyses of LRP6
functional domains revealed that EGF repeats (beta-propellers) 3
and 4 were necessary for Dkk-1 binding and that the ligand binding
domains of LRP6 had no effect on Dkk-1 binding. The findings of Mao
et al. contrast with data obtained by the present inventors
indication that the ligand binding domains of LRP5 were necessary
and sufficient for Dkk-1 binding in yeast. Using classical
biochemical ligand-receptor studies, Mao et al. determined a
Kd=0.34 nM for Dkk-1/LRP6 and a Kd=0.73 nM for Dkk-2/LRP6. Semenov
et al. (2001) verified the Mao group's results and confirmed by
coimmunoprecipitation that Dkk-1 does not directly bind to Wnt or
Frizzled but rather interacts with LRP6. Their Scatchard analyses
found a Kd=0.5 nM for Dkk-1/LRP6.
[0176] Semenov et al. also demonstrated that Dkk-1 could abolish an
LRP5/Frizzled8 complex implying that Dkk-1 can also repress Wnt
signaling via interactions with LRP5. A Dkk-1 mutant where cysteine
220 was changed to alanine abolished LRP6 binding and was unable to
repress Wnt signaling. Studies in Xenopus embryos confirmed the
results and revealed a functional consequence of Dkk-1/LRP6:
repression of Wnt signaling. Their Xenopus work also suggested that
LRP6/Dkk-1 may be specific for the canonical,
.beta.-catenin-mediated, Wnt pathways as opposed to the Wnt Planar
Cell Polarity pathway.
[0177] Bafico et al. (2001) employed a .sup.125I-labeled Dkk-1
molecule to identify LRP6 as its sole membrane receptor with a
Kd=0.39 nM. Again, the functional consequences of the Dkk-1/LRP6
interaction was a repression of the canonical Wnt signaling even
when Dkk-1 was added at extremely low concentrations (30
.mu.M).
[0178] Not wishing to be bound by theory, it is believed that the
present invention provides an explanation for the mechanism of
Dkk-1 inhibition of the Wnt pathway and provides a mechanism
whereby the Wnt pathway may be modulated. The present application
and related U.S. Ser. No. 60/291,311 (which is herein incorporated
by reference in its entirety) describe Dkk-1/LRP5 interactions and
demonstrate that the interaction between LRP5/LRP6/HBM, and Dkk can
be used in a method as an intervention point in the Wnt pathway for
an anabolic bone therapeutic or a modulator of lipid
metabolism.
[0179] As detailed in the Examples, Dkk-1 is able to repress
LRP5-mediated Wnt signaling but not HBM-mediated Wnt signaling.
This observation is of particular interest because the HBM mutation
in LRP5 is a gain of function or activation mutation. That is, Wnt
signaling, via the canonical pathway, is enhanced with HBM versus
LRP5. Thus, other HBM-like mutations would also function similarly.
The present data suggest the mechanism of this functional
activation: the inability of Dkk-1 to repress HBM-mediated Wnt
signaling. Further investigations of other Wnt or Dkk family
members show differential activities in the canonical Wnt pathway
that demonstrate the complexity and variability in Wnt signaling
that can be achieved depending on the LRP/Dkk/Wnt/Frizzled
repertoire that is expressed in a particular cell or tissue. This
may attest to the apparent bone specificity of the HBM or HBM like
phenotypes in humans and in the HEM transgenic animals.
[0180] Furthermore, the present data reveal the importance and
functional consequence for the potential structural perturbation of
the first beta-propeller domain of LRP5. Our data identified the
ligand binding domain of LRP5 as the interacting region with Dkk-1
while the Mao et al. publication demonstrated the functional role
of propellers 3 and 4 in their LRP6/Dkk-1 studies. In the present
invention, we implicate the first beta propeller domain, via the
HBM mutation at residue 171, as having a functional consequence in
the Dkk-1-mediated Wnt pathway. The involvement of position 171 of
propeller 1 may be direct or indirect with Dkk-1. Direct
involvement could arise from perturbations of the 3-dimensional
structure of the HBM extracellular domain that render Dkk-1 unable
to bind. Alternatively, residue 171 of propeller 1 may directly
interact with Dkk-1; however, by itself, it is insufficient to bind
and requires other LRP5 domains. Potential indirect candidate
molecules may be among the proteins identified the Dkk-1
yeast-two-hybrid experiments.
[0181] It may be that the disruption of Dkk activity is not
necessarily mediated by enhancing or preventing the binding of Dkk
to LRP5/LRP6/HBM. More than one mechanism may be involved. Indeed,
the inventors have observed that Dkk-1 binds LRP5, LRP6, and HBM.
It is able to effectively inhibit LRP6, and to a slightly lesser
extent, LRP5 activity. Further, has been observed that different
members of the Dkk family differentially affect LRP5/LRP6/HBM
activity. For example, Dkk-1 inhibits LRP5/LRP6/HBM activity while
another Dkk may enhance LRP5/LRP6/HBM activity. An endpoint to
consider is the modulation of the LRP5/LRP6/HBM activity, not
simply binding.
[0182] The present disclosure shows that targeting the modulation
of the Dkk-1/LRP5 interaction is a therapeutic intervention point
for an HBM or HBM-like mimetic agent. A therapeutic agent of the
invention may be a small molecule, peptide or nucleic acid aptamer,
antibody, or other peptide/protein, etc. Methods of reducing Dkk-1
expression may also be therapeutic using methodologies such as: RNA
interference (i.e., siRNAs), small hairpin RNAs (shRNAs), antisense
oligonucleotides, morpholino oligonucleotides, PNAs, antibodies to
Dkk-1 or Dkk-1 interacting proteins, decoy or scavenger LRP5 or
LRP6 receptors, and knockdown of Dkk-1 or Dkk-1 interactor
transcription. For discussion of small hairpin RNAs, see Yu et al.,
2002 Proc. Natl. Acad. Sci. USA 99: 6047-52; Tuschl, 2002 Nature
Biotech. 20: 446-8; Lee et al., 2002 Nature Biotech. 19: 500-5;
Paddison et al., 2002 Genes & Devel. 16: 948-58; and
Brummelkamp et al., 2002 Science 296: 550-3.
[0183] In an embodiment of the present invention, the activity of
Dkk-1 or the activity of a Dkk-1 interacting protein may be
modulated for example by binding with a peptide aptamer of the
present invention. In another embodiment, LRP5 activity may be
modulated by a reagent provided by the present invention (e.g., a
peptide aptamer). In another embodiment, the Dkk-1/LRP5 interaction
may be modulated by a reagent of the present invention (e.g.; a
Dkk-1 interacting protein such as those identified in FIG. 31). In
another embodiment, the Wnt signal transduction pathway may be
modulated by use of one or more of the above methods. In a
preferred embodiment of the present invention, the Dkk-1 mediated
activity of the Wnt pathway may be specifically modulated by one or
more of the above methods. In another preferred embodiment of the
present invention, the Wnt signal transduction pathway may be
stimulated by down-regulating Dkk-1 interacting protein activity;
such down-regulation could, for example, yield-greater LRP5
activity. In a more preferred embodiment, by stimulating LRP5
activity, bone mass regulation may be stimulated to restore or
maintain a more optimal level. In another preferred embodiment, by
stimulating LRP5 activity, lipid metabolism may be stimulated to
restore or maintain a more optimal level. Alternative embodiments
provide methods for screening candidate drugs and therapies
directed to correction of bone mass disorders or lipid metabolism
disorders. And, preferred embodiments of the present invention
provide drugs and therapies developed by the use of the reagents
and/or methods of the present invention. One skilled in the art
will understand that the present invention provides important
research tools to develop an effective model of osteoporosis, to
increase understanding of bone mass and lipid modulation, and to
modulate bone mass and lipid metabolism. For a more detailed
description of Dkk-1 and Dkk-1 interacting protein modulation,
please refer to U.S. Ser. No. 60/361,293, which is herein
incorporated by reference in its entirety.
3. Alternative Variants of LTRP5/T, RP6 Having HBM Activity
[0184] A structural model of the LRP5/Zmax1 first beta-propeller
module was generated based on a model prediction in Springer et
al., 1998 J. Mol. Biol. 283: 837-862. Based on the model, certain
amino acid residues were identified as important variants of
LRP5/HBM/Zmax1. The model and modifications thereof are discussed
in more detail in the Examples. The following three categories
provide examples of such variants:
[0185] The shape of the beta-propeller resembles a disk with
inward-sloping sides and a hole down the middle. Residue 171 is in
a loop on the outer or top surface of the domain in blade 4 of
propeller module 1. Thus, variants comprising changed residues in
structurally equivalent positions in other blades; as well as
residues that are slightly more interior to the binding pocket, but
still accessible to the surface, are important embodiments of the
present invention for the study of bone mass modulation by
LRP5/HBM, for the development of pharmaceuticals and treatments of
bone mass disorders, and for other objectives of the present
invention. The following Table contains examples of such
variants:
TABLE-US-00001 TABLE 1 Variant Effect of Mutation A214V a position
equivalent to 171 in blade 5 of propeller 1; alanine is not
conserved in other propellers E128V a position equivalent to 171 in
blade 3 of propeller 1; glutamate is not conserved in other
propellers A65V a position equivalent to 171 in blade 2 of
propeller 1; alanine is conserved in propellers 1-3 but not 4 G199V
an accessible interior position in blade 5 of propeller 1; glycine
is conserved in propellers 1-3 but not 4 M282V accessible interior
position in blade 1 of propeller 1; methionine is conserved in
propellers 1-3 but not 4
These mutations were further analyzed based on a more sophisticated
model, as discussed and described in Example 11 below.
[0186] LRP5/Zmax1 has four beta-propeller structures; the first
three beta-propeller modules conserve a glycine in the position
corresponding to residue 171 in human LRP5/Zmax1. Therefore,
variants bearing a valine in the equivalent positions in the other
propellers are important embodiments of the present invention. The
following variants are useful for the study of bone mass modulation
by LRP5/HBM, for the development of pharmaceuticals and treatments
of bone mass disorders, and for other objectives of the present
invention: G479V, G781V, and Q1087V of SEQ ID NO: 3, which
demonstrate that propeller 1 is an important determinant of an HBM
or HBM-like effect.
[0187] The G171V HBM polymorphism (SEQ ID NO: 4) results in
"occupied space" of the beta-propeller 1, with the side-chain from
the valine residue sticking out into an open binding pocket and
potentially altering a ligand/protein interaction. The glycine
residue is conserved in LRP5/Zmax1 propellers 1, 2 and 3 but is a
glutamine in propeller 4. Therefore, the following variants of
LRP5/HBM are important embodiments of the present invention for the
study of bone mass modulation by LRP5/HBM, for the development of
pharmaceuticals and treatments of bone mass disorders, and for
other objectives of the present invention:
[0188] G171K: introduces a charged side-chain
[0189] G171F: introduces a ringed side-chain
[0190] G171I: introduces a branched side-chain
[0191] G171Q: introduces the propeller 4 residue
These substitutions along with substitutions in other regions of
propeller 1 of SEQ ID NO: 3 (i.e., A214V and M282V) have been shown
to produce an IBM-like effect by TCF assay (FIGS. 29 and 30). Thus
these substitutions in other propeller 1 domains would similarly
have an expectation of producing an HBM-like phenotype readily
assayable by TCF assay.
[0192] Furthermore, LRP6 is the closest homolog of LRP5/Zmax1. LRP6
has a beta-propeller structure predicted to be similar, if not
identical to Zmax1. The position corresponding to glycine 171 of
human LRP5/Zmax1 is glycine 158 of human LRP6. Thus, corresponding
variants of LRP6 are an important embodiment of the present
invention for the study of the specificity of LRP5/Zmax1 versus its
related family member, for the development of pharmaceuticals and
treatments of bone mass disorders, and for other objectives of the
present invention. Specifically, for example, a glycine to valine
substitution at the structurally equivalent position, residue 158,
of human LRP6 and similar variants of other species' LRP6 homologs
represent important research tools.
[0193] Site-directed mutants of LRP5 were generated in the
full-length human LRP5 cDNA using the QuikChange XL-Site-Directed
Mutagenesis Kit (catalog #200516, Stratagene, La Jolla, Calif.)
following the manufacturer's protocol. The mutant sequences were
introduced using complementary synthetic oligonucleotides:
TABLE-US-00002 Mutation Complementary oligos A65V:
5'-TGGTCAGCGGCCTGGAGGATGTGGCCGCAGTGGACTTCC-3'
5'-GGAAGTCCACTGCGGCCACATCCTCCAGGCCGCTGACCA-3' E128V
5'-AAGCTGTACTGGACGGACTCAGTGACCAACCGCATCGAGG-3'
5'-CCTCGATGCGGTTGGTCACTGAGTCCGTCCAGTACAGCTT-3' G171K
5'-ATGTACTGGACAGACTGGAAGGAGACGCCCCGGATTGAGCG-3'
5'-CGCTCAATCCGGGGCGTCTCCTTCCAGTCTGTCCAGTACAT-3' G171F
5'-ATGTACTGGACAGACTGGTTTGAGACGCCCCGGATTGAGCG-3'
5'-CGCTCAATCCGGGGCGTCTCAAACCAGTCTGTCCAGTACAT-3' G171I
5'-ATGTACTGGACAGACTGGATTGAGACGCCCCGGATTGAGCG-3'
5'-CGCTCAATCCGGGGCGTCTCAATCCAGTCTGTCCAGTACAT-3' G171Q
5'-ATGTACTGGACAGACTGGCAGGAGACGCCCCGGATTGAGCG-3'
5'-CGCTCAATCCGGGGCGTCTCCTGCCAGTCTGTCCAGTACAT-3' G199V
5'-CGGACATTTACTGGCCCAATGTACTGACCATCGACCTGGAGG-3'
5'-CCTCCAGGTCGATGGTCAGTACATTGGGCCAGTAAATGTCCG-3' A214V
5'-AGCTCTACTGGGCTGACGTCAAGCTCAGCTTCATCCACCG-3'
5'-CGGTGGATGAAGCTGAGCTTGACGTCAGCCCAGTAGAGCT-3' M282V
5'-GAGTGCCCTCTACTCACCCGTGGACATCCAGGTGCTGAGCC-3'
5'-GGCTCAGCACCTGGATGTCCACGGGTGAGTAGAGGGCACTC-3' G479V
5'-CATGTACTGGACAGACTGGGTAGAGAACCCTAAAATCGAGTGTGC-3'
5'-GCACACTCGATTTTAGGGTTCTCTACCCAGTCTGTCCAGTACATG-3' G781V
5'-CATCTACTGGACCGAGTGGGTCGGCAAGCCGAGGATCGTGCG-3'
5'-CGCACGATCCTCGGCTTGCCGACCCACTCGGTCCAGTAGATG-3' Q1087V
5'-GTACTTCACCAACATGGTGGACCGGGCAGCCAAGATCGAACG-3'
5'-CGTTCGATCTTGGCTGCCCGGTCCACCATGTTGGTGAAGTAC-3' G158V of
5'-GTACTGGACAGACTGGGTAGAAGTGCCAAAGATAGAACGTGC-3' LRP6
5'-GCACGTTCTATCTTTGGCACTTCTACCCAGTCTGTCCAGTAC-3'
[0194] All constructs were sequence verified to ensure that only
the engineered modification was present in the gene. Once verified,
each variant was functionally evaluated in the TCF-luciferase assay
in U2OS cells (essentially as described in Example 6. Other
functional evaluations could also be performed, such as the Xenopus
embryo assay (essentially as described in Example 5); or other
assays to evaluate Wnt signaling, Dkk modulation, or anabolic bone
effect. Binding of these mutants to Dkk, LRP-interacting proteins,
Dkk-interacting proteins, or peptide aptamers to any of the
preceding could also be investigated in a variety of ways such as
in a two-hybrid system (such as in yeast as described in this
application), or other methods.
[0195] FIG. 29 shows the effects of the G171F mutation in propeller
1 of LRP5. This mutation is at the same position as HBM's G171V
substitution. Expression of G171F results in an HBM effect. That
is, in the presence of Wnt, G171F is able to activate the
TCF-luciferase reporter construct. In fact, it may activate the
reporter to a greater extent than either LRP5 or HBM. Furthermore,
in the presence of Dkk1 and Wnt1, G171F is less susceptible than
LRP5 to modulation by Dkk. These data exemplify that the G171F
variant modulates Wnt signaling in a manner similar to IBM. In
addition, this data confirms that HBM's valine residue at 171 is
not the only modification at 171 that can result in an HBM effect.
Together these data support an important role for LRP5 propeller 1
in modulating Wnt pathway activity; in responding to Dkk
modulation; and, in the ability to generate an HBM effect.
[0196] FIG. 30 shows the effects of the M282V mutation in propeller
1 of LRP5. M282 expression results in an HBM-effect. That is, in
the presence of Wnt, M282 is able to activate the TCF-luciferase
reporter construct. Furthermore, in the presence of Dkk1 and Wnt1,
M282V is less susceptible than LRP5 to modulation by Dkk. These
data show that the M282V variant modulates Wnt signaling in a
manner similar to HBM. In addition, this data confirms that
modifications of other residues in propeller 1 of LRP5 can result
in an HBM effect.
[0197] These data support an "occupied space" model of the HBM
mutation in propeller 1 and show that multiple mutations of
propeller 1 are capable of generating an HBM effect; the original
G171V HBM mutation is not unique in this ability. Moreover, various
perturbations in propeller 1 can modulate Dkk activity.
[0198] These data illustrate the molecular mechanism of Dkk
modulation of LRP signaling. Using the methods disclosed herein and
in U.S. Application 60/290,071, generation of a comprehensive
mutant panel will reveal residues in LRP that function in Dkk
modulation of Wnt signaling. Such variants of LRP5 and LRP6 that
modulate Dkk activity and the residues which distinguish them from
LRP5 and LRP6 are points for therapeutic intervention by small
molecule compound, antibody, peptide aptamer, or other agents.
Furthermore, models of each HBM-effect mutation/polymorphism may be
used in rational drug design of an HBM mimetic agent.
[0199] These and the examples provided infra are only a few
illustrative examples presented to better describe the present
invention. Variants of LRP5 which have demonstrated HBM activity in
assays include A65V, G171I, G171V (HBM), G171F, M282V, G171K, G171Q
and A214V. Clearly, other variants may be contemplated within the
scope of the present invention. Furthermore, wherever HBM is
recited in the methods of the invention, it should be understood
that any such alternative variant of LRP5 or LRP6 which
demonstrates HBM like biological activity is also encompassed by
those claims.
[0200] Additional mutations may also result in conformational
changes such as those described above and in the Examples below.
These mutations may also result in a HBM-like phenotype when
expressed. The following mutations have been identified in Table 2
based on familial genetics in the exons of Zmax1 (LRP5).
TABLE-US-00003 TABLE 2 Mutations in the Exons of LRP5 Location Met
(ATG as as AA Exon nt 1) Reference Change AA1 Change 2 249
CGAGGAGGCCATC C/T S83 None 2 266 AGACCTACCT A/G Q89 G-R 3 512
GTGAGACGCC G/T G171 G-V 6 1199 CGTACCTGGACGGG C/T A400 A-V 8 1647
CGGGTTCACGCTGC C/T F549 None 9 1932 GGCCTTCTTGGTCT G/A E644 None 9
1999 GTGGCCATCCCGCT G/A V667 V-M 10 2220 CAGAATCGAAGTG C/T N740
None 14 3107 GGACACTGTT G/A R1036 R-Q 15 3297 CGGCACCGAGCG C/T
D1099 None 15 3357 AGACAACACACTG A/G V1119 None 16 3564 GACTCGCATC
G/A R1188 None 18 3989 CGGACTGTGA C/T A1330 A-V 20 4137
CAGCCCGGCCCAC C/T D1379 None 20 4248 GGGGGCCAACGG G/A A1416 None 20
4565 CGGCCACTGC C/T P1522 P-L 23 4635 CGACGTGTGTGACA C/T T1545
None
By, for example "C/T" is meant there is a C to T mutation.
[0201] Additional mutations have also been identified in the
introns, which may result in splice variants are provided in Table
3 below.
TABLE-US-00004 TABLE 3 Mutations in the Introns of LRP5 Nucleotide
Postion with respect Sequence with SNP Exon to Exon Location
Underlined Nucleotide 2 +53 CTCTCTCTCGAATT C/T 5 -4 TCAGTCCACACTCG
T/C 5 +8 GGGCGGGGGCTGGG G/A 7 -50 GCAGAGACCAGAC G/A 8 -118
TGCTCTTGGGCATT T/G 9 -131 TGGGGGTGAGTCCT T/C 10 +6 TGTTTGCCTGTCCC
T/C 11 -173 ATGTGTGTGGCAG A/G 11 -152 GGTCTCGCCCTTC G/A 11 -49
CGGTGAGAGCAGAC C/T 11 +37 CGGGGCAGCCGGG C/T 11 +78 GTACCCTGTGGCCT
G/A 12 +80 CTCATCTGGGGTTC C/G 12 +141 ATGATGCTACCTGG A/G 15 -166
CGGGAATTTGGAGA C/T 15 -149 TTGTTCAACTAGTA T/C 15 -52 TCCGAGGAGACGC
T/G 17 -213 TTGTTTCCGGCATC T/C 17 -82 CATTTGGCCCCTA C/T 18 -72
GCCCAGTCAC G/A 18 -63 CGCCATTGCC C/T 18 -30 GTGTGATGTT G/A 18 +23
TGATCTGGAGGAGG T/C 18 +47 GTCTGGGCAGCTTT G/C 18 +54 CACCGTCAGTGCT
C/A 22 -118 GGCACCTGCC G/A
[0202] These splice variants may also produce an altered phenotype
capable of conferring a HBM-like effect.
[0203] These mutations were identified using 80 ng of genomic DNA,
which was PCR amplified with the primers indicated in Table 4 below
with M13F (TGTAAAACGACGGCCAGT) attached to the 5' end of the
Forward primer or M13R (AGGAAACAGCTATGACCAT) attached to the 5' end
of the Reverse primer. 4 .mu.l of the PCR reaction was diluted to a
final volume of 1001 with water. Sequencing was performed using ET
and M13F and M13R primers or by the ABI-PRISM.RTM.
Big-Dye.TM.method of Applied Biosystems using the indicated nested
sequencing primers. The sequences are assembled on consed with the
appropriate reference sequence.
[0204] The PCR mixes used are as follows [0205] Promega: 50 mM KCl,
10 mM Tris-HCl, pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl.sub.2 [0206]
Invitrogen D: 60 mM Tris-HCl, pH 8.5, 15 mM
(NH.sub.4).sub.2SO.sub.4, 3.5 mM MgCl.sub.2 [0207] Invitrogen J: 60
mM Tris-HCL, pH 9.5, 15 mM (NH.sub.4).sub.2SO.sub.4, 2.0 mM
MgCl.sub.2 [0208] Invitrogen M: 60 mM Tris-HCl, pH 10.0, 15 mM
(NH.sub.4).sub.2SO.sub.4, 1.5 mM MgCl.sub.2 To all the PCT mixes,
120 .mu.M of all 4 dNTPs was added, 0.4 .mu.M of the forward and
reverse primers, 80 ng genomic DNA, 1 U of AmpliTaq.RTM. DNA
polymerase, and 1.1 U of TaqStart antibody (Clontech). The PCR
reaction is than run as follows: 94.degree. C., 2 min; (94.degree.
C., 30 sec; X-anneal-temp, 30 sec; 72.degree. C., 2 min) for 35
cycles and 72.degree. C. for 3 min.
TABLE-US-00005 [0208] TABLE 4 Forward and Reverse Primers anneal
Prod. PCR Exon F-primer (PCR) R-primer (PCR) temp Size buffer
F-primer (seq) R-primer (seq) 1 GAGACGCGGCGCGGCTTC
CGCCCCAACTCGCTCCCA 2- 429 dmso TCCCGCGCGCCCAGCT TCGCCAAGTCGCTTCCG
AC cycle- C 68 2 AAGGAACTGGAGGTCTTG CAGAGTCACACCCTTTTT 62 670
promega GGCATGGGCAGGGCAG TGAAAAACAACTTGGGCT C T C 3
CCAAGTTCTGAGAAGTCC AATACCTGAAACCATACC 58 523 promega
TGCATTCCTCAGGGCC TTGGTTATTTCCGATGGG TG C 4 GGCGTAGTGGTGGGCATCAG
CCCAGCCAGCCACACACC 62 680 dmso ACTGTCGGGGGACCCT ATTGCAGCAGGTACCCC
TC C 5 GGGAAATTGCAGGCCGTCTG CTGAGGACCAAGGCGGAG 58 633 promega
AGGCTGAGGGCCCCAT CAGGATTGACCTCCTGG AG G 6 CACCTAACATCACCAGCC
GATGCAAGACAGTGTCCC 62 672 promega CCTGGCTGAGTATTTC
TCAATCTCCCTCTCGCC C 7 ACACCGACATTTACGAGCAC AAATAGCAGAGCACAGGC 58
484 promega + CGACTTTACTGACACC CCATCGGTGCCTCGCCA AC 10% dmso A 8
TCTCAAACAAACAAACAAACAA TCCTTGGCCAGATACTGT 60 638 promega
ACAAACAAACAAAGCG CTTTCCTGTCCTGCCCT AAA CAC TCA 9 TGTGTGGCGGGAATAAAG
TTGAGGCAGGAACAGAGG 60 648 promega CTGTGCACATTGGAGC
CAAGGTTTTCCCATAAAG T G 10 ATGTCTACAAAACACGCTG CTAATCACTGAGGGCCAC 60
744 promega GTTCTGGCCTGGCGTG ACGGACAGCCTGCCACC G 11
TCACCTTACGAGTGAGCC AGCCTCTCCCGACATAAC 60 677 promega
ACTGTGGGAATTCAGG TGCAGCAAAGGCACCCA G 12-1 GAAAACCAGCAAAAGCCC
TGTCCATCACGAAGTCCA 60 677 promega ATGAGGGCGGCCATGT
GCGGTTCCGGCCGCTAG G G 12-2 ACAGCGATTATATCTACTGGAC
ATGGAAAGCACTCAGCAC 60 622 promega AGACTGGAATCTGCAC
TGTGCTGGCAGTATGAG AG 13 AGAATGAAATTCCCCATAGCG AGACAAAAGTCCTGTGGG 52
676 Invitro- TCCCGTGGACCTCCAG ACAACGGGGAAACCCAG GTC gen D C 14
GAGAGACCCCCACACCAATAC CAGGTGGAAAGTCTCCCC 63 571 promega
TGGGATTTGACTTTCA CCTGTGAGAGGCTGGCA AG GG 15 CAGTTGGATTTAGGGCCTACC
CAGCTGTCAGTGTGAGGA 60 656 promega CTCACCCATTGTGGTC
AACACGCTACACACAAAG CAA G 16 AACTGATGGCCTTCATCCC TCTTAAAACGCTGTTCGT
60 699 promega GAGCCCAGCCCAGGTG GATTTGTTCTGCGGCAAA GGT G AG 17
AGACTGATGGTATGGGCACAG ATTTGTGAGATGCAGGAA 60 662 promega
TAGAGACTGGTGCAGA CAGTACTTAGAGGAAAAT ACG CTC 18
ATTCTCCCAGCCTCTCTTCTG GCAGGAAGGTGAGATAGA 58 722 promega
GGTTGGGGCTGGAGGT TCACAGTCTGCCTTCAAG CCC G 19 GGGGTCTGTTACTCCTTGCAT
CAGCTCACAGTCTGTCCT 60 645 promega GGCGTAGACCTCCCCA
CCGCTGCCCTGGGAAAG CCT C 20 ACGTTACCCTGAGGTTGGC AGGCCTCTGTGTTGAAGG
60 640 promega AGGCTCCCCAGGCCTA CTGATGCCAAGCACAGG ATT G 21
TTGGAGGAGGTACCATGTGTC GGTGGATTTGGGTGAGAT 55 643 Invitro-
TCCCGTTTCACAGATG CAGATTATCCACAATCAA TTT gen J AG C 22
TTCTGCTGATTTCTGAACCC TTGCCTTTCACTGAGATG 58 558 promega +
AACATGCAGTGCCCGC CTTCGGGGCAGGTGGCT AC 10% dmso T 23
GGCCTGCATCTTCTGGAGC CATTCCCTCCACGGGGAC 62 683 promega
TGGCCAGTGGACAGGC ACACAACTCAAATGCACA C C
4. Genotyping of Microsatellite Markers
[0209] To narrow the genetic interval to a region smaller than that
originally reported by Johnson et al., 1997 Am. J. Hum. Genet.,
60:1326-1332, additional microsatellite markers on chromosome
11q12-13 were typed. The new markers included: D11S4191, D11S1883,
D11S1785, D11S4113, D11S4136, D11S4139, (Dib et al., 1996 Nature,
380:152-154), FGF3 (Polymeropolous et al., 1990 Nucl. Acid Res.,
18: 7468), as well as GTC_HBM_Marker.sub.--1,
GTC_HBM_Marker.sub.--2, GTC_HBM_Marker.sub.--3,
GTC_HBM_Marker.sub.--4, GTC_HBM_Marker.sub.--5,
GTC_HBM_Marker.sub.--6, and GTC_HBM_Marker.sub.--7 (See FIG.
2).
[0210] Blood (20 ml) was drawn into lavender cap (EDTA containing)
tubes by a certified phlebotomist. The blood was stored
refrigerated until DNA extraction. DNA has been extracted from
blood stored for up to 7 days in the refrigerator without reduction
in the quality or quantity of yield. For those subjects that have
blood drawn at distant sites, a shipping protocol was successfully
used on more than a dozen occasions. Blood samples were shipped by
overnight express in a styrofoam container with freezer packs to
provide cooling. Lavender cap tubes were placed on individual
plastic shipping tubes and then into "zip-lock" biohazard bags.
When the samples arrived the next day, they were immediately
processed to extract DNA.
[0211] The DNA extraction procedure used a kit purchased from
Gentra Systems, Inc. (Minneapolis, Minn.). Briefly, the procedure
involved adding 3 volumes of a red blood cell lysis buffer to the
whole blood. After incubations for 10 minutes at room temperature,
the solution was centrifuged in a Beckman tabletop centrifuge at
2,000.times.g for 10 minutes. The white blood cell pellet was
resuspended in Cell Lysis Buffer. Once the pellet was completely
resuspended and free of cell clumps, the solution was digested with
RNase A for minutes at 37.degree. C. Proteins were precipitated by
addition of the provided Protein Precipitation Solution and removed
by centrifugation. The DNA was precipitated out of the supernatant
by addition of isopropanol. This method was simple and fast,
requiring only 1-2 hours, and allowed for the processing of dozens
of samples simultaneously. The yield of DNA was routinely >8 mg
for a 20 ml sample of whole blood and had a MW of >50 kb. DNA
was archived by storing coded 50 .mu.g aliquots at -80.degree. C.
as an ethanol precipitate.
[0212] DNA was genotyped using one fluorescently labeled
oligonucleotide primer and one unlabeled oligonucleotide primer.
Labeled and unlabeled oligonucleotides were obtained from
Integrated DNA Technologies, Inc. (Coralville, Iowa). All other
reagents for microsatellite genotyping were purchased from Perkin
Elmer-Applied Biosystems, Inc. ("PE-ABI") (Norwalk, Conn.).
Individual PCR reactions were performed for each marker, as
described by PE-ABI using AmpliTaq.TM.DNA Polymerase. The reactions
were added to 3.5 .mu.l of loading buffer containing deionized
formamide, blue dextran and TAMRA 350 size standards (PE-ABI).
After heating at 95.degree. C. for 5 minutes to denature the DNA,
the samples were loaded and electrophoresed as described in the
operator's manual for the Model 377 DNA Sequencer (PE-ABI, Foster
City, Calif.). After gel electrophoresis, the data was analyzed
using PE-ABI GENESCAN.TM.and GENOTYPER.TM.software. First, within
the GENESCAN.TM.software, the lane tracking was manually optimized
prior to the first step of analysis. After the gel lane data was
extracted, the standard curve profiles of each lane were examined
and verified for linearity and size calling. Lanes, which had
problems with either of these parameters, were re-tracked and
verified. Once all lanes were tracked and the size standards were
correctly identified, the data were imported into GENOTYPER.TM.for
allele identification To expedite allele calling (binning), the
program Linkage Designer from the Internet web-site of Dr. Guy Van
Camp (http://alt.www.uia.ac.be/u/dnalab/ld.html) was used. This
program greatly facilitates the importing of data generated by
GENOTYPER.TM.into the pedigree drawing program Cyrillic (Version
2.0, Cherwell Scientific Publishing Limited, Oxford, Great Britain)
and subsequent linkage analysis using the program LINKAGE (Lathrop
et al., 1985 Am. J. Hum. Genet. 37: 482-98).
5. Linkage Analysis
[0213] FIG. 1 demonstrates the pedigree of the individuals used in
the genetic linkage studies for this invention. Specifically,
two-point linkage analysis was performed using the MLINK and
LINKMAP components of the program LINKAGE (Lathrop et al., 1985 Am.
J. Hun. Genet., 37: 482-98). Pedigree/marker data was exported from
Cyrillic as a pre-file into the Makeped program and converted into
a suitable ped-file for linkage analysis.
[0214] The original linkage analysis was performed using three
models: (i) an autosomal dominant, fully penetrant model, (ii) an
autosomal dominant model with reduced penetrance, and (iii) a
quantitative trait model. The HBM locus was mapped to chromosome
11q12-13 by analyzing DNA for linked markers from 22 members of a
large, extended kindred. A highly automated technology was used
with a panel of 345 fluorescent markers which spanned the 22
autosomes at a spacing interval ranging from 6-22 cM. Only markers
from this region of chromosome 11 showed evidence of linkage (LOD
score .about.3.0). The highest LOD score (5.74) obtained by
two-point and multipoint analysis was D11S987 (map position 55 in
FIG. 2). The 95% confidence interval placed the HBM locus between
markers D11S905 and D11S937 (map position 41-71 in FIG. 2).
Haplotype analysis also places the Zmax1 (LRP5) gene in this same
region. Further descriptions of the markers D11S987, D11S905, and
D11S937 can be found in Gyapay et al., 1994 Nature Genetics, Vol.
7.
[0215] In this invention, the inventors report the narrowing of the
HBM interval to the region between markers D11S987 and
GTC_HBM_Marker.sub.--5. These two markers lie between the
delimiting markers from the original analysis (D11S905 and D11S937)
and are approximately 3 cM from one another. The narrowing of the
interval was accomplished using genotypic data from the markers
D11S4191, D11S1883, D11S1785, D11S4113, D11S4136, D11S4139, (Dib et
al., 1996 Nature 380: 152-4), FGF3 (Polymeropolous et al., 1990
Nucl. Acid Res., 18: 7468) (information about the genetic markers
can be found at the Internet site of the Genome Database,
http://gdbwww.gdb.org/), as well as the markers
GTC_HBM_Marker.sub.--1, GTC_HBM_Marker.sub.--2,
GTC_HBM_Marker.sub.--3, GTC_HBM_Marker.sub.--4,
GTC_HBM_Marker.sub.--5, GTC_HBM_Marker.sub.--6, and
GTC_HBM_Marker.sub.--7.
[0216] As shown in FIG. 1, haplotype analysis with the above
genetic markers identifies recombination events (crossovers) in
individuals 9019 and 9020 that significantly refine the interval of
chromosome 11 to which the Zmax1 (LRP5) gene is localized.
Individual 9019 is an HBM-affected individual that inherits a
portion of chromosome 11 from the maternal chromosome with the HBM
gene, and a portion from the chromosome 11 homologue. The portion
inherited from the HBM gene-carrying chromosome includes markers
D11S935, D11S1313, GTC_HBM_Marker.sub.--4, D11S987, D11S1296,
GTC_HBM_Marker.sub.--6, GTC_HBM_Marker.sub.--2, D11S970,
GTC_HBM_Marker.sub.--3, D11S4113, GTC_HBM_Marker.sub.--1,
GTC_HBM_Marker.sub.--7 and GTC_HBM_Marker.sub.--5. The portion from
D11S4136 and continuing in the telomeric direction is derived from
the non-HBM chromosome. This data places the Zmax1 (LRP5) gene in a
location centromeric to the marker GTC_HBM_Marker_S5. Individual
9020 is an unaffected individual who also exhibits a critical
recombination event. This individual inherits a recombinant
paternal chromosome 11 that includes markers D11S935, D11S1313,
GTC_HBM_Marker.sub.--4, D11S987, D11S1296 and
GTC_HBM_Marker.sub.--6 from her father's (individual 0115)
chromosome 11 homologue that carries the HBM gene, and markers
GTC_HBM_Marker.sub.--2, D11S970, GTC_HBM_Marker.sub.--3,
GTC_HBM_Marker.sub.--1, GTC_HBM_Marker.sub.--7,
GTC_HBM_Marker.sub.--5, D11S4136, D11S4139, D11S1314, and D11S937
from her father's chromosome 11 that does not carry the HBM gene.
Marker D11S4113 is uninformative due to its homozygous nature in
individual 0115. This recombination event places the centromeric
boundary of the HBM region between markers D11S1296 and
D11S987.
[0217] Two-point linkage analysis was also used to confirm the
location of the Zmax1 (LRP5) gene on-chromosome 11. The linkage
results for two point linkage analysis under a model of full
penetrance are presented in Table 5 below. This table lists the
genetic markers in the first column and the recombination fractions
across the top of the table. Each cell of the column shows the LOD
score for an individual marker tested for linkage to the Zmax1
(LRP5) gene at the recombination fraction shown in the first row.
For example, the peak LOD score of 7.66 occurs at marker D11S970,
which is within the interval defined by haplotype analysis.
TABLE-US-00006 TABLE 5 Marker 0.0 0.05 0.1 0.15 0.2 0.25 0.3 0.35
0.4 D11S935 -infinity 0.39 0.49 0.47 0.41 0.33 0.25 0.17 0.10
D11S1313 -infinity 2.64 2.86 2.80 2.59 2.30 1.93 1.49 1.00 D11S987
-infinity 5.49 5.18 4.70 4.13 3.49 2.79 2.03 1.26 D11S4113 4.35
3.99 3.62 3.24 2.83 2.40 1.94 1.46 0.97 D11S1337 2.29 2.06 1.81
1.55 1.27 0.99 0.70 0.42 0.18 D11S970 7.66 6.99 6.29 5.56 4.79 3.99
3.15 2.30 1.44 D11S4136 6.34 5.79 5.22 4.61 3.98 3.30 2.59 1.85
1.11 D11S4139 6.80 6.28 5.73 5.13 4.50 3.84 3.13 2.38 1.59 FGF3
0.59 3.23 3.15 2.91 2.61 2.25 1.84 1.40 0.92 D11S1314 6.96 6.49
5.94 5.34 4.69 4.01 3.27 2.49 1.67 D11S937 -infinity 4.98 4.86 4.52
4.06 3.51 2.88 2.20 1.47
[0218] A single nucleotide polymorphism (SNP) further defines the
HBM region. This SNP is termed SNP_Contig033-6 and is located 25 kb
centromeric to the genetic marker GTC_HBM_Marker.sub.--5. This SNP
is telomeric to the genetic marker GTC_HBM_Marker.sub.--7.
SNP_Contig033-6 is present in HBM-affected individual 0113.
However, the HBM-affected individual 9019, who is the son of 0113,
does not carry this SNP. Therefore, this indicates that the
crossover is centromeric to this SNP. The primer sequence for the
genetic markers GTC_HBM_Marker.sub.--5 and GTC_HBM_Marker.sub.--7
is shown in Table 6 below.
TABLE-US-00007 TABLE 6 Marker Primer (Forward) Primer (Reverse)
GTC_HBM_Marker_5 TTTTGGGTACACAATTCAGTCG AAAACTGTGGGTGCTTCTGG (SEQ
ID NO:63) (SEQ ID NO:65) GTC_HBM_Marker_7 GTGATTGAGCCAATCCTGAGA
TGAGCCAAATAAACCCCTTCT (SEQ ID NO:64) (SEQ ID NO:66)
[0219] The kindred described have several features of great
interest, notably that their bones, while very dense, have an
absolutely normal shape. The outer dimensions of the skeletons of
the HBM-affected individuals are normal, and, while medullary
cavities are present, there is no interference with hematopoiesis.
The HBM-affected members seem to be resistant to fracture, and
there are no neurologic symptoms, and no symptoms of impairment of
any organ or system function in the members examined. HBM-affected
members of the kindred live to advanced age without undue illness
or disability. Furthermore, the HEM phenotype matches no other bone
disorders such as osteoporosis, osteoporosis pseudoglioma,
Engelmann's disease, Ribbing's disease, hyperphosphatasemia, Van
Buchem's disease, melorheostosis, osteopetrosis, pycnodysostosis,
sclerostenosis, osteopoikilosis, acromegaly, Paget's disease,
fibrous dysplasia, tubular stenosis, osteogenesis imperfecta,
hypoparathyroidism, pseudohypoparathyroidism,
pseudopseudohypoparathyroidism, primary and secondary
hyperparathyroidism and associated syndromes, hypercalciuria,
medullary carcinoma of the thyroid gland, osteomalacia and other
diseases. Clearly, the HBM locus in this family has a very powerful
and substantial role in regulating bone density, and its
identification is an important step in understanding the pathway(s)
that regulate bone density and the pathogenesis of diseases such as
osteoporosis.
[0220] In addition, older individuals carrying the HBM gene, and
therefore expression of the HEM protein, do not show loss of bone
mass characteristic of normal individuals. In other words, the HBM
gene is a suppressor of osteoporosis. In essence, individuals
carrying the HBM gene are dosed with the HBM protein, and, as a
result, do not develop osteoporosis. This in vivo observation is
strong evidence that treatment of normal individuals with the HBM
gene or protein, or a fragment thereof, will ameliorate
osteoporosis.
6. Physical Mapping
[0221] To provide reagents for the cloning and characterization of
the HBM locus, the genetic mapping data described above were used
to construct a physical map of the region containing Zmax1 (LRP5)
on chromosome 11q13.3. The physical map consists of an ordered set
of molecular landmarks, and a set of BAC clones that contain the
Zmax1 (LRP5) gene region from chromosome 11q13.3.
[0222] Various publicly available mapping resources were utilized
to identify existing STS markers (Olson et al., 1989 Science
245:1434-5) in the HBM region. Resources included the GDB, the
Whitehead Institute Genome Center, dbSTS and dbEST (NCBI), 11 db,
the University of Texas Southwestern GESTEC, the Stanford Human
Genome Center, and several literature references (Courseaux et al.,
1997 Genomics 40: 13-23; Courseaux et al., 1996 Genomics 37:
354-65; Guru et al., 1997 Genomics 42: 436-45; Hosoda et al., 1997
Genes Cells 2: 345-57; James et al., 1994 Nat. Genet. 8: 70-76;
Kitamura et al., 1997 DNA Research, 4: 281-9; Lemmens et al., 1997
Genomics 44: 94-100; and Smith et al., 1997 Genome Res. 7: 835-42).
Maps were integrated manually to identify markers mapping to the
region containing Zmax1 (LRP5).
[0223] Primers for existing STSs were obtained from the GDB or
literature references are listed in Table 6 below. Thus, Table 7
shows the STS markers used to prepare the physical map of the Zmax1
(LRP5) gene region.
TABLE-US-00008 TABLE 7 HBM STS Table Locus Size STS Name Name Type
GDB Access. # (kb) SEQ ID NO: Forward Primer ACTN3 Gene GDB: 197568
0.164 67: CTGGACTACGTGGCCTTCTC PC-B/PC-Y Gene GDB: 197884 0.125 69:
CTCAGTGCCATGAAGATGGA D11S2161E Gene 0.322 71: GTTTCAGGAGACTCAGAGTC
ADRBKI Gene GDB: 4590179 0.117 73: TTATTGTGATTTCCCGTGGC PSANK3 GENE
0.259 75: GAGAAAGAAATAAGGGGACC PP1 (1/2)/ Gene GDB: 197566 0.208
77: GAAGTACGGGCAGTTCAGTGGCCT PP1 (2/2) GSTP1.PCR1 Gene GDB: 270066
0.19 79: AGCCTGGGCCACAGCGTGAGACTACGT NDUFV1 Gene 0.521 81:
CATGTGCCCACCTCATTCAT PSANK2 GENE 0.157 83: CAGAGAAGTCAAGGGACTTG
PSANK1 EST 0.3 85: CAAGGCTAAAAGACGAAAAA UT5620 D11S1917 MSAT GDB:
314521 0.211 87: AAGTCGAGGCTGCAAGGAG AFM289ya9 D11S1337 MSAT GDB:
199805 0.287 89: AAGGTGTGAGGATCACTGG GALN Gene 0.322 91:
GCTTCTCCGAGTGTATCAAC pMS51 D11S97 VNTR GDB: 177850 93:
GATCAGCGAACTTCCTCTCGGCTC BCL1 (1)/ Gene 0.205 95:
GCTAATCACAGTCTAACCGA BCL1 (2) CCND1 Gene GDB: 4590141 0.248 97:
GCACAGCTGTAGTGGGGTTCTAGGC FGF4 Gene GDB: 5690113 0.549 99:
CACCGATGAGTGCACGTTCAAGGAG FGF3.PCR1 Gene GDB: 188627 0.161 101:
TTTCTGGGTGTGTCTGAAT AFM164ZF12 D11S913 MSAT GDB: 188151 0.22 103:
CATTTGGGAAATCCAGAAGA AFMA190YD5 MSAT GDB: 1222329 0.275 105:
GACATACCATGAACACTATAAGAGG SHGC-15295 D11S4689 STS GDB: 740600 0.147
107: GAACAAGAGGGGTAAGTTGGC SHGC 3084 D11S4540 STS GDB: 740102 0.167
109: GAAGTGTTCCCTCTTAAATTCTTTG SHGC-14407 D11S4664 STS GDB: 740516
0.158 111: CCTGTAACCCCCAGTCCC SHGC 10946 D11S4327 Gene GDB: 674522
0.311 113: ACTCCATCCACCTCATCACTG S515 D11S703 STS GDB: 196290 0.166
115: GTGGACAGGCATAGCTGAGG AFM147XD10 D11S1889 MSAT GDB: 307895
0.183 117: AGCTGGACTCTCACAGAATG AFMA131YES D11S987 MSAT GDB: 195002
0.082 119: GACTCCAGTCTGGGCAATAAAAGC AFMb358xa9 D11S4178 MSAT GDB:
611922 0.237 121: CAGGCCCAGTCTCTTG AFMa272yb5 D11S4113 MSAT GDB:
608115 0.218 123: ACCTCACGGTGTAATCCC WI-17803 EST GDB: 4581644 0.15
125: TATTTGCAAAGCTTGAGAGTTCT SGC31923 EST GDB: 4578606 0.126 127:
ACTTTATTGTCAGCGTGGGC WI-7741 D11S4364 GENE GDB: 677652 0.324 129:
GAGCAGGGGAGAGAAGGC SGC35223 EST GDB: 4582599 0.13 131:
AGCCACTTTATTGTTATTTTGATGC WI-16754 EST GDB: 4578377 0.15 133:
GTGGAGTGTGGGATTGGG WI-6315 D11S4418 EST GDB: 678804 0.224 135:
ATGCTTTTGCATGATTCTAATTATT WI-16915 EST GDB: 4584055 0.126 137:
CTGGTCTTCCTTGTGTGCTG SGC30608 EST 0.128 139:
TCAGAAGCAGAACTGTTTTTAACA WI-17663 EST GDB: 4583346 0.126 141:
CAAGCCGGGTTTTATTGAAA WI-6383 Gene GDB: 1222237 0.199 143:
GCATATAGAAACAATTTATTGCCG SGC31567 Gene GDB: 4578432 0.207 145:
CTACCACACCACACCAGGC SGC30658 EST GDB: 4584037 0.15 147:
GTTGTCTTGACTTCAGGTCTGTC SGC34590 EST 0.13 149: GCGTGGGGATATAGAGGTCA
SGC33927 EST GDB: 4582382 0.15 151: TAATATATCCCCAGTCTAAGGCAT
WI-8671 EST GDB: 1222235 0.124 153: TGGTTTTAAACCTTTAATGAGAAAA
WI-12334 EST GDB: 1222257 0.127 155: AATTATTTAAAAGAGAGGAAAGGCA
WI-18402 EST GDB: 4581874 0.113 157: GGTTACAGAAAAACATTTGAGAGAT
WI-18671 EST GDB: 4584947 0.131 159: TTGAAAAACCATTTATTTCACCG
WI-12856 EST GDB: 4576606 0.209 161: TTGAAAAAACCATTTATTTCACCG
SGC33767 EST GDB: 4581106 0.15 163: CTTTATTGAAAACATTGAGTGCA
AFM343YB5 MSAT GDB: 1222332 0.181 165: AAACCACGACCNCCAA SGC33744
EST GDB: 4575826 0.15 167: CTTTTGGTAGAGACAAGGTCTCA SGC32272 EST
GDB: 4581592 0.135 169: GACGAAGGTGATTCAGGGC SGC34148 EST GDB:
4583084 0.1 171: CAGATAAAAGAGTCACTATGGCTCA WI-18546 EST GDB:
4574598 0.133 173: TTATTGATAAGCATTAGTGAACCCC SGC31103 EST GDB:
4567265 0.1 175: CTATGCCCAGAGATGAACAGG SGC30028 Gene GDB: 4580505
0.128 177: GCCAGCTTTATTGAGTAAACTTCC WI-2875 D11S4407 STS GDB:
678546 0.125 179: CATCCCAACCATCACTCAGT SGC36985 Gene GDB: 4577182
0.223 181: AGACTACATTTTGGAACCAGTGG GCT16B07 D11S4270 STS GDB:
626245 0.137 183: GAAGGTTTTGTCCCTCGATC WI-6504 D11S3974 EST GDB:
588142 0.174 185: CCTTCATAGCCACACCCG SGC31049 EST GDB: 4580093 0.15
187: TCTTTACTGTGCTTACAACTTTCCT TIGR-A002J17 EST GDB: 1222193 0.199
189: AGATCAGCAAGCAGATAG WI-5996 D11S2382 EST GDB: 458683 0.1 191:
CATACCTATGAGGTGTGCTACAGG WI-16987 EST GDB: 4575848 0.15 193:
TTACAGCCACCAAGGTTTCC SGC31912 EST GDB: 4567868 0.101 195:
CACTGTTATCTCATTAACTGTGAGG WI-13500 EST GDB: 4577893 0.15 197:
CCCCACTCCCACTTTTATTT CHLC.GAAT1B01.P7 D11S971 MSAT GDB: 684255
0.103 199: AGGACACAGCCTGCATCTAG SGC35519 Gene GDB: 4577180 0.134
201: GATGGGTCACACTAACCTGTCA WI-11974 EST GDB: 1222255 0.108 203:
AGCATCTTTAATGTGTCAGGCA WI-15244 Gene GDB: 4574740 0.108 205:
TCACATTCAAAAATCGGCAA WI-17496 EST GDB: 4597336 0.131 207:
TGTTTTATTTCTCAGTACAAAGCCA WI-9159 D11S4381 EST GDB: 678144 0.111
209: CCACCAAATTATTTATAGTTCTGCG WI-4232 STS GDB: 1222250 0.175 211:
CCTATAATGGGCTGGACCAA SHGC-4167 EST GDB: 4566789 0.161 213:
CAGTGTGCACGTTTTCATTT WI-14303 EST GDB: 4576938 0.15 215:
CTGCATTTATTATGAGAATCAACAG WI-16597 EST GDB: 4585666 0.13 217:
CAGGGCACTGAGATACACTTACC RC29S1CATTFOR/R D11S970 MSAT GDB: 191084
0.15 219: ACACATCTCTTCTGTGCCCC UT979 D11S1296 MSAT GDB: 198525
0.362 221: CATTCCCCAGTTTGCAGAC 1281/1282 D11S1959E EST GDB: 335216
0.07 223: GCAGAGAAGTCCTGTTAGCC D11S468 D11S468 STS 0.096 225:
AGTGTGGGGCAGGACCTCTG D11S668 D11S668 STS GDB: 179349 0.143 227:
TCCCTCATCCCCTTGTCTGT RH18048 Gene GDB: 4572853 0.188 229:
GATGCTTACCTACCACGGC IGHMBP2 Gene GDB: 4590087 0.699 231:
TGGCAGACCATGCTCCGCCT NUMA Gene GDB: 4590244 0.277 233:
CTCCATCACAACCAGATTTGAGGCT KRN1 Gene GDB: 4590232 0.228 235:
AGTGGGAAACCTCAGGTAGCTCCCGA Cda1ff06 D11S2302E EST GDB: 445887 0.091
237: CATTAAGTAGTGGGGGGACAG RH10753 Gene GDB: 4563588 0.194 239:
GGAGTAGACCATGATTACTG EMS1 Gene GDB: 459016 0.64 241:
CGCCCTGGATCCTCACACTACA SHGC-11098 DXS9736 Gene GDB: 737674 0.137
243: GCTCCTATCTGTGTTTTGAATGG INPPL1 Gene GDB: 4590093 0.382 245:
CTTGGAGCGCTATGAGGAGGGC RH18051 EST GDB: 4572859 0.195 247:
TTGGAGTCACAGGGGC Cdalcc11 D11S2297E EST GDB: 445869 0.1 249:
AACAAAGCTGCTTAGCACCTG 1249/1250 D11S1957E EST GDB: 335210 0.247
251: TTTTCCAATAATGTGACTTC NDUFV1 D11S2245E EST GDB: 445695 253:
CTTGATCTCGCCCAGGAAC AFMb032zg5 D11S4136 MSAT GDB: 609546 0.19 255:
GAATCGCTTGAACCCAG AFMa059xg9 D11S4196 MSAT GDB: 614025 0.2 257:
GAACGTTNTTCATGTAGGCGT Cda17c12 D11S2288E EST GDB: 445842 0.158 259:
AGGGAAAATGGTATGTGGGGAG SHGC-1364 D11S951E EST GDB: 4562765 0.137
261: AGTGGACAAAATGAGGAAAACAGG RH17410 EST GDB; 4571587 0.126 263:
TGACATCTTTGCATTATGGC RH17414 EST GDB: 4571595 0.121 265:
AGCTCTTGCTTCTCAGTCCA RH17770 EST GDB: 4572301 0.267 267:
GCCTCTCAAAGTAGTTGGAACC SEA EST GDB: 4590169 0.13 269:
CTCAAGGCCAGGCATCACT RH10689 EST GDB: 4563460 0.107 271:
AATGATGATCTCAACTCTG TIGR-A006P20 EST GDB: 4587692 0.236 273:
GACATCTGTTAGTCTCATAATTC TIGR-A007D15 Gene GDB: 4588398 0.24 275:
CTATGTACAAAACAGGAAGAG TIGR-A008B14 EST GDB: 4588882 0.141 277:
GTAAATGAGAAACAGACAAATGA TIGR-A008K11 EST GDB: 4589094 0.203 279:
AAGTAGAAACAAAATGAGGGAC TIGR-A008P15 EST GDB: 4589662 0.182 281:
ACTTCCTATAAATGGAGGTGAG TIGR-A008T11 EST GDB: 4589278 0.138 283:
CATACTCCTAGACTCAAGGAATC TIGR-A008U48 EST GDB: 4589364 0.107 285:
GTGTTGAGGAGAAAAGCACT TIGR-A008X45 EST GDB: 4589838 0.242 287:
CAAGTTACAAATAACTTAAGCCG SHGC-11839 D11S4611 Gene GDB: 740339 0.151
289: TTTATTAGAAGTGACTCTTGGCCC N1B1242 D11S4929E EST GDB: 3888276
0.149 291: TTCTCATGTACAAAGCGGTC SHGC-13599 D22S1553 Gene GDB:
737558 0.147 293: CACCAGAAGGTTGGGGTG SHGC-11867 D11S4331 Gene GDB:
674684 0.14 295: CTCATGCTGGATGACCCC SHGC 15349 D12S2124 EST GDB:
740819 0.141 297: TCACAGCCTTCAGTCAGGG BdaB4a05 D11S2235E EST GDB:
445662 0.095 299: CCTGAGCTACTGCCACAG Bda99d07 D11S2238E EST GDB:
445674 0.09 301: TCAGAGTCACTCCTGCCC folr1 Gene GDB: 197840 0.3 303:
CGGCATTTCATCCAGGAC NIB1738 D11S4284 EST GDB: 626260 0.173 305:
TTCCATTTATTGAGCACCTG WI-7351 D11S4433 Gene GDB: 679143 0.324 307:
CCTCCTACACCTGCAAAAGC
WI-14325 EST GDB: 4578507 0.132 309: AAAGCACAAAAGTAACAGCAACA
WI-15192 EST GDB: 4575806 0.15 311: AGAGCACCTTTCCTCAGCAC WI-17872
EST GDB: 4577492 0.141 313: AAAAAGGACAGTGTCTAAAATTTGA SHGC-30732
EST GDB: 4567830 0.105 315: GATTTAGGGAGTACAAGTGCGG stSG4288 EST
GDB: 4566057 0.123 317: CCATCATCATATTGGTGTGACC WI-13814 EST GDB:
4579290 0.15 319: TTAAGATGCCATTAAACTCATGAC WI-14122 Gene GDB:
4576114 0.126 321: CCATCTCTTTTATCAGGGTTGG 2729/2730 D11S4057 EST
GDB: 596509 0.118 323: CGACTGTGTATTTTCCACAG SHGC-31329 EST GDB:
4567386 0.15 325: AGCTTAAAGTAGGACAACCATGG SGC33858 EST GDB: 4578600
0.127 327: TGTTGTTTATTTCCACCTGCC WI-12191 EST GDB: 1222208 0.15
329: TTTTTTTTTTTTACACGAATTTGAGG WI-13701 EST GDB: 4574892 0.15 331:
ATGAAATCTTAAGCAGAATCCCA WI-14069 EST GDB: 4584373 0.15 333:
AAAGGCCTTTATTTATCTCTCTCTG WI-14272 EST GDB: 4578525 0.125 335:
GCTTCTAAGTCTTAGAGTCAGCTGG WI-17347 EST GDB: 4578523 0.127 337:
TTGGTTAAATGATGCCCAGA stSG1561 EST GDB: 4584415 0.215 339:
ACACAGCATGCAGGGAGAG stSG1938 EST GDB: 4564568 0.137 341:
GATGGAAGTAGCTCCTCTCGG stSG2759 EST GDB: 4565137 0.141 343:
CCGGTGCTTGGAAAGATG RH97 EST GDB: 4559690 0.17 345:
TTACAGGCATGAGTCACTACGC 5tSG4794 EST GDB: 4573113 0.141 347:
CCCTCCCTCCACACACAC stSG4957 EST GDB: 4569051 0.171 349:
AGATACGGGCAAAACACTGG stSG4974 EST GDB: 4569063 0.166 351:
TTCTGAGGTCAGGGCTGTCT stSG8144 EST GDB: 4573137 0.17 353:
ACTCAGTCCCTCCCACCC stSG9275 EST GDB: 4569999 0.19 355:
GTGATCACGGCTCAACCTG SHGC-10667 D11S4583 Gene GDB: 740246 0.277 357:
CTGCAGCTGCCTCAGTTTC SHGC-11930 Gene GDB: 1231223 0.21 359:
ATTTCCAGAGCCAGCTCAAA SHGC-32786 EST GDB: 4567878 0.125 361:
GATCATGCACTGTTGACCAC FKBP2 Gene 0.064 363: AACTGAGCTGTAACCAGACTGGGA
WI-13116 EST GDB: 4585099 0.203 365: TTATCCCTTTATTGTTTCTCCTTTG MDU1
Gene GD8: 4590064 0.859 367: TCTTCAAAGCCTCTGCAGTACC S453 D11S579
STS GDB: 196276 0.108 369: GTGGCTGCAGCTAATGTAAGACAC
STS1-cSRL-112e11 D11S3866 STS GDB: 547681 0.135 371:
CTGATTGAGAACCAGAACAG STS1-cSRL-44a3 D11S3830 STS GTC: 547609 0.118
373: TAGTAAGGGACCTTCACCAG STS1-cSRL31b12 D11S2439 STS GDB: 459738
0.123 375: GATGATTAAACTCTCCTGGC cSRL-419 D11S1137 STS GDB: 197824
0.196 377: GAGGTGGTGGGCACCTGTA SHGC-10323 D11S4351 Gene GDB: 676135
0.141 379: GACCAGAGTCTGCCCAGAAG WI-9219 Gene GDB: 678179 0.1 381:
GGAGGGATGGACAAGTCTGA GTC_ZNF Gene 0.172 383: TCAAAACACAGTCATCTCCA
AFMa152yh1 D11S4087 MSAT GDB: 603797 0.158 385: GCTCAGCACCCCCATT
AFMb331zh5 D11S4162 MSAT GDB: 611241 0.263 387:
GTTCTCCAGAGAGACAGCAC AFMb038yb9 D11S4139 MSAT GDB: 609621 0.151
389: TATAGACTTCAGCCCTGCTGC AFM212xe3 D11S1314 MSAT GDB: 199292
0.209 391: TTGCTACGCACTCCTCTACT WI-18813 EST 0.13 393:
ATCCTAGACCAGAGGAGCCC WI-19549 EST 0.252 395: AACTTTCATTTGCCAAGGGA
WI-20154 EST 0.25 397: bACAGTTGTCATCGGTAGGCA WI-22393 EST GDB:
4583084 0.142 399: GTGCAGGTGGCGTTTATTTT WI-7587 EST GDB: 1223732
0.274 401: GCTCTAGTGGGAAACCTCAGG EST455579 EST 0.273 403:
GGTTTGGTCTCAAAGGCAAA WI-21134 EST 0.293 405: GCTGCCTTGGAATTTCTGTT
WI-21698 EST 0.25 407: ATTCAAGCTCATCCAGACCC SHGC-7373 D11S4567 STS
GDB: 740192 0.225 409: ATATTGACCGTGCACAAATACG SHGC-36533 STS 0.125
411: ATTGGCAGTGGAAAATGCTT ARIX Gene 0.242 413:
tctgtcctcctttcaccggaagc CLCI.PCR Gene GDB: 6362613 415:
TCAGGGCCTGTGTTGCCGCACTCTG B188N21-HL STS 417: AGGCATGCAAGCTTCTTA
B234C17-HR STS 419: TGGTAAGCACAGAAAATGC B235G10-HR STS 421:
CTGGACGTTATGTCTGCC B247F23-HR STS 423: ATCACTCTGAACTGCCACT
B337H24-HL STS 425: CAAGCTTTGAAGGAAGAG B337L5-HL STS 427:
GCTCTGCAGTGGGTAAAA B382N10-HR STS 429: CCCTTTCTGAGGCAAGAT B1211-HR
STS 431: CGCTATGAGTCCCATCTG B180D17-HR STS 433: TTGAGTACACGGGGTGAC
B236E6-HR STS 435: ACCTGTCTCCTCTCCTGG B278E22-HR STS 437:
ATGACCAGCAAGCATTGT B312F21-HR STS 439: GCAGAAGGTCCTTTGGAT
B337H24-HR STS 441: CGACATTCTTTTCTGGAGG B358H9-HR STS 443:
GCACTTTTCCTTCCTTCC B148N18-HL STS 445: ACAGCTCCAGAGAGAAGGA
B172N12-HL STS 447: AGGCATCAAGCTTTCCTT B172N12-HR STS 449:
GTGGTGCTGCAAGTTACC B215J11-HR STS 451: GACCATTTGTTACGCAGC B223E5-HR
STS 453: CTCAAGCTTCTGTTCATGC B312B3-HR STS 455: TACAGAAAACCGCAGCTC
B328G19-HL STS 457: AAAAGGAGGGAATCATGG B328G19-HR STS 459:
CTGAGCATCCGATGAGAC B329110-HL STS 461: TCTAACCCTTACTGGGC B329110-HR
STS 463: TTTACACAGGACCAGGGA B368G19-HL STS 465: GTCCACGGGCTTTATTCT
B368G19-HR STS 467: GGAAGAGCAAAATAAATCCA B39F19-HL STS 469:
AGCACGCTTATTTCATGG B250K11-HR STS 471: TCCTGCTGCATTATGGAT
B338D17-HR STS 473: ATGGGGATTAAATACGGG B268I23-HL STS 475:
CTGAGGAGAAGAGGCTGG B268I23-HR STS 477: AGGATGCTTGCTAGGGTT
B371E15-HR STS 479: GGTCTCAGGAGCCCTTTA B312F21-HL STS 481:
ACTTAACCAAGGATGGGG B338D17-HL STS 483: TAGGCTCTGCACTCTTGG
B369H19-HL STS 485: TAAAGGCGGTGAAGTGAG B369H19-HR STS 487:
TGGGGCCAGATAATTCTT B444M11-HR STS 489: AAGGAAGAGGTCACCAGG
B269L23-HL STS 491: TCAATAGGTGATCCAACATTT B250K11-HL STS 493:
GGGTAGGGGGATCTTTTT B269L23-HR STS 495: GTCCTGGGAAAGATGGAA B364H4-HL
STS 497: TCTTTCGCTGTACTTGGC B364H4-HR STS 499: GGACAGTGTATGTGTTGGG
B473O3-HR STS 501: CTTCTTGAGTCCCGTGTG B180D17-HL STS 503:
GCTGGGAGAGAATCACAA B200E21-HL STS 505: ACGCTGTCAGGTCACACT
B200E21-HR STS 507: TAGGGGGATCTTTTTCCA B14L15-HR STS 509:
ATGGTCCAGCTCCTCTGT B442P6-HR STS 511: AACATTGCTGTTAGCCCA B188N21-HR
STS 513: ATGAGTTGGCAGCTGAAG GTC-ARRB1 Gene 0.097 515:
GAGGAGAAGATCCACAAGCG B508A5-HL STS 517: CTGAGCTTTTGGCACTGT
B36F16-HR STS 519: TGTATGAGTCTGGAGGGTGT B117N18-HL STS 521:
GCAGGGGACGTGATAATA B14L15-HL STS 523: AAAATTGTGAGCACCTCC B21K22-HL
STS 525: GTGCAAAGCCCACAGTAT B21K22-HR STS 527: CCACTGAATTGCATACTTTG
B223E5-HL STS 529: AGATTTTGGGGAGTCAGG B278E22-HL STS 531:
CAAGCCCCAAAGTAGTCA B444M11-HL STS 533: AGCCTCCAGGTGACTACC
B543O19-HR STS 535: ATGCTTTCAGCATTTTCG B117N18-HR STS 537:
GTCGGATTGGTTTCACAA B543O19-HL STS 539: TTTGGAAAAGAACAGAAATGT
B442P6-HL STS 541: CCTTAATGCCCCTGATTC B367H4-HR STS 543:
TCAAGCTTGCTTTCTCAA B250E21-HR STS 545: CCTGGCTGGAGATAGGAT
B250E21-HL STS 547: GGCACGTACTTCCTACCA B248C16-HR STS 549:
ACCCAGGCTGGTGTGT B248C16-HL STS 551: GATGCATTTTGCTTCACC B160D8-HR
STS 553: TCAAGCTTCAAAGAGCAGA B539L7-HR STS 555: TGGTGCTTTTAAATCCAGA
B473O3-HL STS 557: TCTTCTCCCAGGGAATCT
AFMa190xd9 D11S4095 STS GDB: 606064 0.193 559: TCCCTGGCTATCTTGAATC
ARRB1 (2) STS 561: CGAGACGCCAGTAGATACCA ARRB1 (1) STS 563:
AGTTCCAGAGAACGAGACGC P102F3S STS GDB: 6054145 565:
GAGCGTGAGAGGTTGAGGAG N172A STS GDB: 6054146 0.208 567:
CTGAACCACTACCTGTATGACCTG N60A STS GDB: 6054147 0.23 569:
GAAGCATTTCAATACTTTAACTG cCI11-44A STS GDB: 6054148 0.239 571:
CTTCTCCTGGCCACTCTGAC CN1677-2A STS GDB: 6054149 0.271 573:
TGAGGATGAATGAGCACATAGG cCI11-524B STS GDB: 6054150 0.221 575:
AGGGGAAGGAATGTGCTTGG P117F3T STS GDB: 6054151 0.168 577:
ATTGAAGGTCCTCCAAAAGAATGCTGCAG ARRB1 (3) Gene 579:
TTGTATTTGAGGACTTTGCTCG B215J11-HL STS 0.122 581:
TTTTTGCCTCATCTATGCCC B317G1-HR STS 583: TTGCTCAAGTTCTCCTGG
B317G1-HL STS 585: CTTGGCTATTTGGACAGC B292J18-HR STS 587:
CTTGTGTCAGTTGTCAGGG B10A18-HL STS 589: CCAGTTCCACTGGATGTT B10A18-HR
STS 591: CTGCCTATCCCTGGACTT B527D12-HL STS 593: CAACACGTCTGACATCCAT
B372J11-HR STS 595: TGGGTGGTACTATTGTTCCAT B372J11-HL STS 597:
GGCCACTATCATCCCTGTGT B37E17-HR (GS) STS 599: ACAGTGACACTAGGGACGGG
B37317-HL (GS) STS 601: CCTGTGGCACACATATCACC B34F22-HR (GS) STS
603: TGCTGTGTAACAAGTCCCCA B34F22-HL (GS) STS 605:
GCAGGGTCCGACTCACTAAG B648P22-HR1 STS 607: ACAGTGGGGACAAAGACAGG
B82A4-HR2 STS 609: TCTTCTGTTAAGGTTTCCCCC B648P22-HL STS 611:
AACATATTTCCTCCCCAGCC B82L11-HL (GS) STS 613: CTCCTCTGCATGGGAGAATC
B86J13-HL (GS) STS 615: GGGAGACGACGTCACAAGAT 144A24HL STS 617:
CAGGCATCTTCTATGTGCCA B82L11-HR (GS) STS 619: ACTTCGTGGCACTGAGTGTG
B86J13-HR (GS) STS 621: GGCTGCTGAGCTCTTCTGAT B82L11-HL2 (GS) STS
623: TCACCTACTTCCAGCTTCCG B82L11-HL3 (GS) STS 625:
CTCCTCTGCATGGGAGAATC STS Name SEQ ID NO: Reverse Primer Gene Name
ACTN3 68: TTCAGAAGCACTTGGCTGG Actinin, alpha 3-skeletal muscle
PC-B/PC-Y 70: CAAGATCACTCGATCTCCAGG Pyruvate Carboxyiase 72:
TTCTGCAGGTTGCTGTTGAG Adenosine Receptor (A2) Gene ADRBKI 74:
GCCCTCTGTCCTGACTTCAGG Beta-adrenergic receptor kinase PSANK3 76:
TGCTTTGTAAAGCACTGAGA sim. to Human endogenous retrovirus mRNA long
terminal repeat PP1 (1/2)/ 78: ATACACCAAGGTCCATGTTCCCCGT Protein
phosphatase 1, catalytic subunit, alpha PP1 (2/2) isoform
GSTP1.PCR1 80: TCCCGGAGCTTGCACACCCGCTTCACA Glutathione
S-transferase pi NDUFV1 82: CAAGATTCTGTAGCTTCTGG NADH dehydrogenase
(ubiquinone) flavoprotein 1 (51 kD) PSANK2 84: ATCCTCTCACATCCCACACT
Aldehyde Dehydrogenase 8 (ALDH8) PSANK1 86: TCAGGAGCATTTCATCTTTT
Human ribosomal protein L37 (PSANK1) pseudogene UT5620 88:
GCCCTGTGTTCCTTTCAGTA AFM289ya9 90: ACGTCATGGGGGCTATT GALN 92:
ATGGCAGAGGACTTAGAACA Preprogalanin (GAL1) pMS51 94:
TCCACATTGAGGACTGTGGGAACG BCL1 (1)/ 96: TTGCACTGTCTTGGATGCA B-cell
CLL/lymphoma 1-cyclin D1 (PRAD1 gene) BCL1 (2) CCND1 98:
CAGGCGCAAAGGACATGCACACGGC Cyclin D1 FGF4 100:
CAGACAGAGATGCTCCACGCCATAC Fbroblast growth factor 4 FGF3.PCR1 102:
ACACAGTTGCTCTAAAGGGT Fibroblast growth factor 3 AFM164ZF12 104:
TAGGTGTCTTATTTTTTGTTGCTTC AFMA190YD5 106: CAACCCATACCAGGGATAAG
SHGC-15295 108: TGAGGACACAGATACTGATGGG SHGC 3084 110:
GAACTATATTGTAGTTAGTGAGGAG SHGC-14407 112: TCTTGCTTCCTAAGTTTCTCGG
SHGC 10946 114: TGCTGTTTGCCTCATCTGAC Choline Kinase S515 116:
TGTTCACTCTTCTGCCTGCAG AFM147XD10 118: CAAGAGGCTGGTAGAAGGTG
AFMA131YES 120: GGTGGCAGCATGACCTCTAAAG AFMb358xa9 122:
CGTGTCCAGATGAAAGTG AFMa272yb5 124: CTTGAAGCCCATCTTTGC WI-17803 126:
AATCACTGTGCTTTGTTGCC SGC31923 128: ACTCCCTCGATGGCTTCC WI-7741 130:
CCCAACTGGCTTGTTTTATTG Transformation-sensistive protein IEF SSP
3521 SGC35223 132: AAGAGTGAACAAAAGCAAACATACC ZNF162-spicing factor
1 WI-16754 134: TACTGTTCTTGATAAGTATGTCGGC WI-6315 136:
TCCCCCAAAAGAATGTAAAGG WI-16915 138: ATCACCCAGGCCAGGGAT Mitogen
inducible gene (MIG-2) SGC30608 140: CCTGCTTGAAAGTTCTAGAGCC
WI-17663 142: GATGCCAGGACCATGGAC WI-6383 144: CTCTGAAGCAGGGACCAGAG
Human tat interactive protein (TIP60) SGC31567 146:
CAAGCGAAAGCTGCCTTC Calcium activated neutral protease large
subunit, muCANP, calpain SGC30658 148: TTTTCCTTCAACAATCACTACTCC
SGC34590 150: TACGTGGCCAAGAAGCTAGG SGC33927 152: AGCTTGCAGATGGAGCCC
WI-8671 154: TGTTGATCTATACCCTGTTTCCG WI-12334 156:
TGGCTGTGAACTTCCTCTGA WI-18402 158: TGAGCTTTAGTTCCCTTCTCTG Hlark
WI-18671 160: TCTGCGGCTGTTGGATTT WI-12856 162: TGTTCTCTTCTCCCAGCAGG
Hlark SGC33767 164: TTGTCAAATTCCCCCCAAAA AFM343YB5 166:
CCCTGGAAAGGTAAGATGCT SGC33744 168: TATCTGTCTGTAGTGCTTCAAATGT
SGC32272 170: ACTGAAGAACTCTTGTCCT SGC34148 172:
CACTTCTCCCACTTTGTCCC WI-18546 174: TGGCAAGTTAGGCACAGTCA Human 1.1
kb mRNA upregulated in retinoic acid treated HL-60 neutrophitic
cells SGC31103 176: TCCACTAAGGGCTATGTCGC SGC30028 178:
CACTGGAGACTACAAGTGGTGG Human pyruvate carboxylase precursor WI-2875
180: GGGGACTAGCTTACAGATTTGA SGC36985 182: TGAAAGGATATTTATAGCCTGGA
LAR-interacting protein 1b GCT16B07 184: TGAGGGTTGGGAAGATCATA
WI-6504 186: CAGCTAACTGTTGACATGCCA SGC31049 188:
CAACAGTGCAGTCGGTATCG TIGR-A002J17 190: CATTCCACATGGATAGAC NDUFV1
WI-5996 192: GCATTTTCTCATCATCCTTGC amplaxin (EMS1) WI-16987 194:
AGGTGTGTGTGCCAGGTTGA Nuclear mitotic apparatus protein 1, NUMA
SGC31912 196: TTTGATTTTGTGTCTCCCAAA WI-13500 198:
CCAGTCACCTTTACTAGTCCTTTG CHLC.GAAT1B01.P7 200: ACCAGGCATTGCACTAAAAG
SGC35519 202: ACATTTATATTTGGACATGCAACC LAR-interacting protein 1a
mRNA WI-11974 204: ATGTGCTGGGCTGGAAAG Carniline palmitoyl
transferase 1 WI-15244 206: CTGCCTGTGTGGTGTCGC Beta-adrenergic
receptor kinase 1, ADRB1 WI-17496 208: GACCTCCTGTGACACCACG WI-9159
210: GTAAGATTCTCCACTGTTGCACC FGF4 WI-4232 212:
ACTCCTCATGTGAAGTCACCG SHGC-4167 214: CAGCATCTTCAGCACTTACC Human DNA
helicase gen (SMBP2) WI-14303 216: TGCTGCTGGGAGTCAGAGTC WI-16597
218: AAGGATCAAGCAGGCATTTG RC29S1CATTFOR/R 220: TGAACCCTGGAGGCAGAG
UT979 222: GTGCTGGGATTACAGGTGT 1281/1282 224: CCATGCTAGAGAAGCACAAC
D11S468 226: CAGACAGATAGCCCTGGGTTC D11S668 228: AGCCCCCCTGGGGATAATC
RH18048 230: AGGATTCCTATCTGGGCTATG Aldehyde dehydrogenase (ALDH8)
IGHMBP2 232: GAGAAGGCCGGGAGGCTCTG Human DNA helicasae gen
(SMBP2)
NUMA 234: GGGTGTGAGCTGCTGCTGAAGG Nuclear mitotic apparatus protein
1, NUMA KRN1 236: CAGTTTGGCTCAGACATATGGGGGCA High sulphur keratin,
KRN Cda1ff06 238: CAAAGCGACAGTGAGTTAGGG RH10753 240:
CATGGTCTATTTATTCTCG protein phosphatase 2A, PP2A EMS1 242:
GGGCATCAGGGGATGGGTAGA Amplaxin SHGC-11098 244:
CCGTGGCATAAGATAAGTAAACG Androgen Receptor INPPL1 246:
ATGGCAACTGACCTTCCGTCCTG 51C protein, inositol polyphosphate
phosphatase- like 1 RH18051 248: CAGCACTATCCTTGGGG NOF1 Cdalcc11
250: GATGAGGACCAACTGGTGAC 1249/1250 252: CAATCCCAACCGTAACAGGC
NDUFV1 254: GCTCGCTGAAGGATGAAGAC NDUFV1 AFMb032zg5 256:
CCAGGTGGTCTTAACGG AFMa059xg9 258: TAATGGTCGCTGTCCC Cda17c12 260:
GCAGTGTGTGAAGGCAGG SHGC-1364 262: CCAACACAGTTTGCTCACATGCC RH17410
264: AGTTATCCCACCTGATACCG RH17414 266: CAAAAGTTGTTTCTGTGTTTGTTC
RH17770 268: TGTGTATCCATAGTGCAAAACAG SEA 270: GGACTCTTCCATGCCAGTG
S13 avian eryghroblastosis oncongene homolog RH10689 272:
ACTGAAGAACTCTTGTCCT TIGR-A006P20 274: GGTAACAGTGTCTTGCTT
TIGR-A007D15 276: ATCCTAGTTTCCTCTCCTT Menin gene (MENI)
TIGR-A008B14 278: CTATTGGATGTGATATGTTATGG TIGR-A008K11 280:
CCTACCCCAAGGTAACAG TIGR-A008P15 282: GAGGAGCTTCAAGAGGAA
TIGR-A008T11 284: GAATGATGTACATGAATTCTTTG TIGR-A008U48 286:
CTCCCAGTAGTCACATTCC TIGR-A008X45 288: CAAGACCCTATCTCTACAAAAAC
SHGC-11839 290: GACTACCTGCCCTCAGCTTG Folate receptor 2 (FBP2)
N1B1242 292: CCACTGGCTTCTCTCTTTTT cGMP-stimulated 3',5'-cyclic
nucleotide phospho- diesterase PDE2A3 (PDE2A) SHGC-13599 294:
ACTATTACGACATGAACGCGG Macrophage Migration Inhibitory factor
SHGC-11867 296: TTGCCTTTCTTGAAACTTAATTCC P2U Purinoceptor SHGC
15349 298: ACATGCTGTGGCACCATG BdaB4a05 300: CCCTGACTTGGACAGTGTCC
Bda99d07 302: CAAATTCAAGCTCATCCAGACC folr1 304:
GGTGTAGGAGGTGCGACAAT Folate receptor2 (FBP2) NIB1738 306:
CTTAAGCCACTGTGTTTTGG WI-7351 308: TGGAAGAACCCCAGAGGAC Folate
receptor3 (FBP3) WI-14325 310: GTGTGTGGGCCACAATATTG WI-15192 312:
AGAATCTCATCACAGGGGCG WI-17872 314: AATTGTTTTTGTTTGTTTTTTGAGT
SHGC-30732 316: GGGGACAAATTATACTTTATTCAGG stSG4288 318:
TGGCTGCCCAAGAAGAAG WI-13814 320: CCAAGGAGATGACCAAGTGG (DRES9
WI-14122 322: CTCTGTGCAAGTAAGCATCTTACA Human VEGF related factor
isoform VRF 186 pre- cursor (VRF) 2729/2730 324:
AGAAGCCCATATCAATGCAC SHGC-31329 326: GGATGCTTCACTCCAGAAAG SGC33858
328: AGAGTGGCTGCAGGCCAG WI-12191 330: TGAGGAAGTAAAAACAGGTCATC
WI-13701 332: CACAGAGTCCCAGGGTCTGT WI-14069 334: GCCTCAGAGCTGGTGGGT
WI-14272 336: AGCCCACAGTCAGCCTACC WI-17347 338: TGGTCCCACTCACATCCC
stSG1561 340: ATCCCTGGTGCTTAGGTGG stSG1938 342:
GGAAGGCCAGCAAGTACTACC stSG2759 344: GAAGTGTCTCTGTTGGGGGA RH97 346:
ACCACTCTCACAGCCCTTACA 5tSG4794 348: GCTCACTGAACTTTCAGGGC stSG4957
350: GTTGAATATAGAGCAGGGCCC stSG4974 352: AGCTTGGAAAATCTCGTGTCA
stSG8144 354: TCCTCTCACTCCTTCCCAGA stSG9275 356: TGGAGGACTGCTTGAGCC
SHGC-10667 358: TCAAAAGTGCTGGTGACAGC Human protein kinase (MLK 3)
SHGC-11930 360: CTTTAATGTTGTGATGACACAAAGC FGF3 SHGC-32786 362:
TACATTTGAAACATTTAAAACCTGA FKBP2 364: TGGAACAGTCTGGTCCTGATGG
FK506-Binding Protein Precursor (FKBP-13) WI-13116 366:
TGGTCACCTGTATTTATTGCTAGG MDU1 368: CTCATCTCCAACCTGTCTAACC 4F2
CellL-Surface Antigen Heavy Chain (4F2HC) S453 370:
CAGCAGAGACAATGGCGTAAGTCC STS1-cSRL-112e11 372: TAAAGCCCTATAACCTCTCC
STS1-cSRL-44a3 374: AGATGTTTGGTATGACTTGG STS1-cSRL31b12 376:
GAGACAGCTAAGCACTCATG cSRL-419 378: AGAGGGGAGGAACACACCTT Folate
receptor2 (FBP2) SHGC-10323 380: TCCCCAGCTCTATCCCAAC Collagen
binding protein 2, colligin-2 gene (CBP2) WI-9219 382:
GTCCAGCTCGCTGACTATCC Retinal outer segment membrane protein 1, ROM1
GTC_ZNF 384: GCAAAGGCTTTACCATATTG ZNF126 AFMa152yh1 386:
TCCCTGCTCGGGAAAC AFMb331zh5 388: GAGAGCAACACTATTGCCC AFMb038yb9
390: CCTCTGTAGGATGCAGTTGG AFM212xe3 392: GTGAAGGCAGGAAATGTGAC
WI-18813 394: CTCCCCCTGGTCCAGTTATT Serine/threonine kinase WI-19549
396: AGCAGATCTGCTCTTGCGAT WI-20154 398: AAAAGTATGAATGGGATGGAGC
WI-22393 400: CCCTATATCTCCGTGTGCTCC DRES9 WI-7587 402:
GAATTCCAGGCTCTTGCTTG Ultra high-sulphur keratin protein (KRN1)
EST455579 404: CCAGTACATGGTGGTCACCA WI-21134 406:
GTGCTGTGGTGGGGAAAG Fas-associating death domain-containing protein,
FADD WI-21698 408: GGACTGGCCCTTTGAAACTC SHGC-7373 410:
AGACCTGGGAAAAGTGGAGAA SHGC-36533 412: TTAATCTTTTGTCAACTTCCTGATT
ARIX 414: ggataaagaaactccgctctgctggtaga Arix homeodomain protein,
neuroendocrine specific, 1x factor CLCI.PCR 416:
AGCGATGTAAAGGGTACCAGTGCCGAGG Chloride channel current inducer, ICLN
gene B188N21-HL 418: CCGGGAGGAGACATCTAT B234C17-HR 420:
AATGGATGGGGGATTATT B235G10-HR 422: AGAGGCCCAGTCACAGAT B247F23-HR
424: CCCTTCTGTTTTTCTGTTTT B337H24-HL 426: TAGGACGTTAAGTGAGGAC
B337L5-HL 428: ACTCTCCAAGACTGTGCG B382N10-HR 430:
GACCACCTGGGAGAGAAC B1211-HR 432: GATCAGCTGCAATGAAGG B180D17-HR 434:
CGCAGGACTGAAAGATGA B236E6-HR 436: TGCTTTTCTTCTGTGGGA B278E22-HR
438: GTACTGGGATTACAGGCG B312F21-HR 440: TTTGCAGGATTCATGCTT
B337H24-HR 442: ACCTTTGCATGTTGGTTTT B358H9-HR 444:
TGCTTTGCTTTCTTCTGG B148N18-HL 446: GCAGTCACTTGAAACCAGA B172N12-HL
448: GGTTTAGAGAACCGAGCC B172N12-HR 450: GGAATCCCTTTCTTTCCA
B215J11-HR 452: GATGGGTGTGAATGAACAA B223E5-HR 454:
GCTGTGAGTGTCTTGGCT B312B3-HR 456: GCCACCAAAGGAAAGATT B328G19-HL
458: TCACTTAGCAGGAGGCAG B328G19-HR 460: GTGCAAAATGAGCAGCTT
B329110-HL 462: TCCTCAAACTGGGAATGA B329110-HR 464:
ATCTCCCCCACTCAGAAG
B368G19-HL 466: TGAGCATAAATTTCATTAGCTG B368G19-HR 468:
GGTGCACAGAATTGTTCAT B39F19-HL 470: GTAACACCAGCAGGGACA B250K11-HR
472: GGGGGTGAGAAGTAGGAA B338D17-HR 474: AGCTAGCATTGGGCTCTT
B268I23-HL 476: CGCCTTACAAGGCAAGTA B268I23-HR 478:
CACAAGTGTCTGGAAGGC B371E15-HR 480: ACATGCCACTCTTCTCACTAA B312F21-HL
482: CAACCCACGAGCATAAGA B338D17-HL 484: ACCCACGGAGTCTCTCTC
B369H19-HL 486: CTACCGCTCTCCTAGGCT B369H19-HR 488:
CTGGTGTTTGGTGGTGTT B444M11-HR 490: CACAAATTCCATTTCCCA B269L23-HL
492: AAAGTCCCACAAAGGGTC B250K11-HL 494: TGTGGAACATTCATTGGC
B269L23-HR 496: TCAAAGCGTCTCCCATAA B364H4-HL 498:
TGGGAGGTCAGAGTGATG B364H4-HR 500: AGGCAGCTGTTTTTGTGA B473O3-HR 502:
CAACCGAGAATCCTCTAGC B180D17-HL 504: GCTTTGCAGAAGAGACCA B200E21-HL
506: GGAGGATGCTCAGGTGAT B200E21-HR 508: GAGCAATTTGAAAAGCCA
B14L15-HR 510: ATAGAGCACCCCATCTCC B442P6-HR 512: GCAATCGAAACAGCATTC
B188N21-HR 514: AATGAAGGTCTTGCCTCC GTC-ARRB1 516:
TCTCTGGGGCATACTGAACC Beta-arrestin-1 B508A5-HL 518:
CTGCTAGGTGACAGCAGG B36F16-HR 520: ACACCTGGCTGAGGAAAT B117N18-HL
522: TTTTGCTTCCTACCATGC B14L15-HL 524: TTTATATTTAAAGTGGCTTTGTT
B21K22-HL 526: AGGAAAATGCAAGAGCAG B21K22-HR 528: TCTGGGTCCAGTCTGCTA
B223E5-HL 530: GCGCTCAAGCAATTCTC B278E22-HL 532: GAATCATCCAATCCACGA
B444M11-HL 534: GAAGGACATGGTCAGCAG B543O19-HR 536:
TGATCCGTGGTAGGGTTA B117N18-HR 538: TTTTATGGGAATTTCAGCC B543O19-HL
540: GGCTAGTCTTTCCTGAACC B442P6-HL 542: GCGTTTACAAGCTGAGGA
B367H4-HR 544: GTAGCCCAGCAAGTGTCT B250E21-HR 546:
CTTCCCCTCTGCCTATGT B250E21-HL 548: GGTGCTTCTTACAGGCAA B248C16-HR
550: ACTGAGTTAATTATCACTCCCCT B248C16-HL 552: TCTGCTTTTAGAGCTGTTAGC
B160D8-HR 554: GGAGTACATCCCAGGACC B539L7-HR 556:
CTCCCTTACTTACTTGCATTG B473O3-HL 558: TTTATGTCCCCTGAGCAC AFMa190xd9
560: CTTGACTGGGTCCACG ARRB1 (2) 562: CATCCTCCATGCCTTTCAGT ARRB1 (1)
564: CTTGTCATCCTCCATGCCTT P102F3S 566: AAACAAACTCCAGACGCACC N172A
568: CTAACTACTTACTCCTACAGGGCCC N60A 570: CCACTCCAGTGCACCCAATC
cCI11-44A 572: GGTTTACCTTTGAATCCCAGC CN1677-2A 574:
TTTGTGGTCCATTGAGTAGGC cCI11-524B 576: TTCGGCTGAGCGGGCAGTGT P117F3T
578: AGAACGTCAACATATCTTTTTGGGGGACAC ARRB1 (3) 580:
CGGTACCATCCTCCTCTTCC B215J11-HL 582: GGGTGACAGAGCAAGACTCC B317G1-HR
584: ACCTTGTTTTGAGGGGAG B317G1-HL 586: GGGCATTTACTCACTTGC
B292J18-HR 588: TGGAATTGTTGTGTCTTGG B10A18-HL 590:
ATGGGCTGTGTTTCTCAA B10A18-HR 592: AGTTTGTCCCTAGTGCCC B527D12-HL
594: GGATAGTGCACACCCA B372J11-HR 596: AGTTCCAGCCCCCTTACCAG
B372J11-HL 598: TTTCACATGGGAAGAACACG B37E17-HR (GS) 600:
TGCCAGGATGGAGATAACAA B37317-HL (GS) 602: ACAACCAAGAATGGAGCCAC
B34F22-HR (GS) 604: TGAACGGAGGACCTACCAAG B34F22-HL (GS) 606:
GCTGTGAGTTCCCTTTACGC B648P22-HR1 608: TACAGGGCACCTCCCAGTAG
B82A4-HR2 610: TGTCTCAAACCTCCCTCTGC B648P22-HL 612:
CAGTCCCAGCCAATGAGAAC B82L11-HL (GS) 614: AGACCTGGGACCAGTCTGTG
B86J13-HL (GS) 616: TGATGTTGGGAAGATGGTGA 144A24HL 618:
GGGAGGCACAAGTTCTTTCA B82L11-HR (GS) 620: CCTTTCTTACGGATGAGGCA
B86J13-HR (GS) 622: TGGGTCTCTCTGCCTGACTT B82L11-HL2 (GS) 624:
AGACCTGGGACCAGTCTGTG B82L11-HL3 (GS) 626: AATTCAGGAGACCTGGGACC
[0224] Novel STSs were developed either from publicly available
genomic sequence or from sequence-derived BAC insert ends. Primers
were chosen using a script which automatically performs vector and
repetitive sequence masking using Cross_match (P. Green, Univ. of
Washington) and subsequent primer picking using Primer3 (Rozen,
Skaletsky (1996, 1997). Primer3 is available at:
www.genome.wi.mit.edu/genome_software/other/primer3.html.
[0225] Polymerase chain reaction (PCR) conditions for each primer
pair were initially optimized with respect to MgCl.sub.2
concentration. The standard buffer was 10 mM Tris-HCl (pH 8.3), 50
mM KCl, MgCl.sub.2, 0.2 mM each dNTP, 0.2 .mu.M each primer, 2.7
ng/.mu.l human DNA, 0.25 units of AmpliTaq (Perkin Elmer) and
MgCl.sub.2 concentrations of 1.0 mM, 1.5 mM, 2.0 mM or 2.4 mM.
Cycling conditions included an initial denaturation at 94.degree.
C. for 2 minutes followed by 40 cycles at 94.degree. C. for 15
seconds, 55.degree. C. for 25 seconds, and 72.degree. C. for 25
seconds followed by a final extension at 72.degree. C. for 3
minutes. Depending on the results from the initial round of
optimization the conditions were further optimized if necessary.
Variables included increasing the annealing temperature to
58.degree. C. or 60.degree. C., increasing the cycle number to 42
and the annealing and extension times to 30 seconds, and using
AmpliTaq.TM.Gold (Perkin Elmer).
[0226] BAC clones (Kim et al., 1996 Genomics 32: 213-8; and Shizuya
et al., 1992 Proc. Natl. Acad. Sci. USA 89: 8794-7) containing STS
markers of interest were obtained by PCR-based screening of DNA
pools from a total human BAC library purchased from Research
Genetics. DNA pools derived from library plates 1-596 were used
corresponding to nine genomic equivalents of human DNA. The initial
screening process involved PCR reactions of individual markers
against superpools, i.e.; a mixture of DNA derived from all BAC
clones from eight 384-well library plates. For each positive
superpool, plate (8), row (16) and column (24), pools were screened
to identify a unique library address. PCR products were
electrophoresed in 2%-agarose gels (Sigma) containing 0.5 .mu.g/ml
ethidium bromide in 1.times.TBE at 150 volts for 45 min. The
electrophoresis units used were the Model A3-1 systems from Owl
Scientific Products. Typically, gels contained 10 tiers of lanes
with 50 wells/tier. Molecular weight markers (100 bp ladder, Life
Technologies, Bethesda, Md.) were loaded at both ends of the gel.
Images of the gels were captured with a Kodak DC40 CCD camera and
processed with Kodak 1D software. The gel data were exported as tab
delimited text files; names of the files included information about
the library screened, the gel image files and the marker screened.
These data were automatically imported using a customized Perl
script into Filemaker.TM. PRO (Claris Corp.) databases for data
storage and analysis. In cases where incomplete or ambiguous clone
address information was obtained, additional experiments were
performed to recover a unique, complete library address.
[0227] Recovery of clonal BAC cultures from the library involved
streaking out a sample from the library well onto LB agar (Maniatis
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1982)) containing 12.5
.mu.g/ml chloramphenicol (Sigma). Two individual colonies and a
portion of the initial streak quadrant were tested with appropriate
STS markers by colony PCR for verification. Positive clones were
stored in LB broth containing 12.5 .mu.g/ml chloramphenicol and 15%
glycerol at -70.degree. C.
[0228] Several different types of DNA preparation methods were used
for isolation of BAC DNA. The manual alkaline lysis miniprep
protocol listed below (Maniatis et al., 1982) was successfully used
for most applications, i.e., restriction mapping, CHEF gel
analysis, FISH mapping, but was not successfully reproducible by
endsequencing. The Autogen and Qiagen protocols were used
specifically for BAC DNA preparation for end sequencing
purposes.
[0229] Bacteria were grown in 15 ml Terrific Broth containing 12.5
.mu.g/ml chloramphenicol in a 50 ml conical tube at 37.degree. C.
for 20 hrs with shaking at 300 rpm. The cultures were centrifuged
in a Sorvall RT 6000 D at 3000 rpm (.about.1800.times.g) at
4.degree. C. for 15 min. The supernatant was then aspirated as
completely as possible. In some cases, cell pellets were frozen at
-20.degree. C. at this step for up to 2 weeks. The pellet was then
vortexed to homogenize the cells and minimize clumping. 250 .mu.l
of P1 solution (50 mM glucose, 15 mM Tris-HCl, pH 8, 10 mM EDTA,
and 100 g/ml RNase A) was added and the mixture pipetted up and
down to mix. The mixture was then transferred to a 2 ml Eppendorf
tube. 350 .mu.l of P2 solution (0.2 N NaOH, 1% SDS) was then added,
the mixture mixed gently and incubated for min at room temperature.
350 .mu.l of P3 solution (3 M KOAc, pH 5.5) was added and the
mixture mixed gently until a white precipitate formed. The solution
was incubated on ice for min and then centrifuged at 4.degree. C.
in a microfuge for 10 min. The supernatant was transferred
carefully (avoiding the white precipitate) to a fresh 2 ml
Eppendorf tube, and 0.9 ml of isopropanol was added, the solution
mixed and left on ice for 5 min. The samples were centrifuged for
10 min, and the supernatant removed carefully. Pellets were washed
in 70% ethanol and air dried for 5 min. Pellets were resuspended in
200 .mu.l of TE8 (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA), and RNase A
(Boehringer Mannheim) added to 100 .mu.g/ml. Samples were incubated
at 37.degree. C. for 30 min, then precipitated by addition of
C.sub.2H.sub.3O.sub.2Na.3H.sub.2O to 0.5 M and 2 volumes of
ethanol. Samples were centrifuged for 10 min, and the pellets
washed with 70% ethanol followed by air drying and dissolving in 50
.mu.l TE8. Typical yields for this DNA prep were 3-5 .mu.g/15 ml
bacterial culture. Ten to 15 .mu.l were used for HindIII
restriction analysis; 5 .mu.l was used for NotI digestion and clone
insert sizing by CHEF gel electrophoresis.
[0230] BACs were inoculated into 15 ml of 2.times.LB Broth
containing 12.5 g/ml chloramphenicol in a 50 ml conical tube. Four
tubes were inoculated for each clone. Cultures were grown overnight
(.about.16 hr) at 37.degree. C. with vigorous shaking (>300
rpm). Standard conditions for BAC DNA isolation were followed as
recommended by the Autogen 740 manufacturer. 3 ml samples of
culture were placed into Autogen tubes for a total of 60 ml or 20
tubes per clone. Samples were dissolved finally in 100 .mu.l TE8
with 15 seconds of shaking as part of the Autogen protocol. After
the Autogen protocol was finished DNA solutions were transferred
from each individual tube and pooled into a 2 ml Eppendorf tube.
Tubes with large amounts of debris (carry over from the pelleting
debris step) were avoided. The tubes were then rinsed with 0.5 ml
of TE8 successively and this solution added to the pooled material.
DNA solutions were stored at 4.degree. C.; clumping tended to occur
upon freezing at -20.degree. C. This DNA was either used directly
for restriction mapping, CHEF gel analysis or FISH mapping or was
further purified as described below for use in endsequencing
reactions.
[0231] The volume of DNA solutions was adjusted to 2 ml with TE8,
samples were then mixed gently and heated at 65.degree. C. for 1.0
min. The DNA solutions were then centrifuged at 4.degree. C. for 5
min and the supernatants transferred to a 15 ml conical tube. The
NaCl concentration was then adjusted to 0.75 M (.about.0.3 ml of 5
M NaCl to the 2 ml sample). The total volume was then adjusted to 6
ml with Qiagen column equilibration buffer (Buffer QBT). The
supernatant containing the DNA was then applied to the column and
allowed to enter by gravity flow. Columns were washed twice with 10
ml of Qiagen Buffer QC. Bound DNA was then eluted with four
separate 1 ml aliquots of Buffer QF kept at 65.degree. C. DNA was
precipitated with 0.7 volumes of isopropanol (.about.2.8 ml). Each
sample was then transferred to 4 individual 2.2 ml Eppendorf tubes
and incubated at room temperature for 2 hr or overnight. Samples
were centrifuged in a microfuge for 10 min. at 4.degree. C. The
supernatant was removed carefully and 1 ml of 70% ethanol was
added. Samples were centrifuged again and because the DNA pellets
were often loose at this stage, the supernatant removed carefully.
Samples were centrifuged again to concentrate remaining liquid
which was removed with a micropipet tip. DNA pellets were then
dried in a desiccator for 10 min. 20 .mu.l of sterile distilled and
deionized H.sub.2O was added to each tube which was then placed at
4.degree. C. overnight. The four 20 .mu.l samples for each clone
were pooled and the tubes rinsed with another 20 .mu.l of sterile
distilled and deionized H.sub.2O for a final volume of 100 .mu.l.
Samples were then heated at 65.degree. C. for 5 min. and then mixed
gently. Typical yields were 2-5 .mu.g/60 ml culture as assessed by
NotI digestion and comparison with uncut lambda DNA.
[0232] 3 ml of LB Broth containing 12.5 .mu.g/ml of chloramphenicol
was dispensed into autoclaved Autogen tubes. A single tube was used
for each clone. For inoculation, glycerol stocks were removed from
-70.degree. C. storage and placed on dry ice. A small portion of
the glycerol stock was removed from the original tube with a
sterile toothpick and transferred into the Autogen tube; the
toothpick was left in the Autogen tube for at least two minutes
before discarding. After inoculation the tubes were covered with
tape making sure the seal was tight. When all samples were
inoculated, the tube units were transferred into an Autogen rack
holder and placed into a rotary shaker at 37.degree. C. for 16-17
hours at 250 rpm. Following growth, standard conditions for BAC DNA
preparation, as defined by the manufacturer, were used to program
the Autogen. Samples were not dissolved in TE8 as part of the
program and DNA pellets were left dry. When the program was
complete, the tubes were removed from the output tray and 30 .mu.l
of sterile distilled and deionized H.sub.2O was added directly to
the bottom of the tube. The tubes were then gently shaken for 2-5
seconds and then covered with parafilm and incubated at room
temperature for 1-3 hours. DNA samples were then transferred to an
Eppendorf tube and used either directly for sequencing or stored at
4.degree. C. for later use.
7. BAC Clone Characterization for Physical Mapping
[0233] DNA samples prepared either by manual alkaline lysis or the
Autogen protocol were digested with HindIII for analysis of
restriction fragment sizes. This data were used to compare the
extent of overlap among clones. Typically 1-2 .mu.g were used for
each reaction. Reaction mixtures included: 1.times. Buffer 2 (New
England Biolabs), 0.1 mg/ml bovine serum albumin (New England
Biolabs), 50 .mu.g/ml RNase A (Boehringer Mannheim), and 20 units
of HindIII (New England Biolabs) in a final volume of 25 .mu.l.
Digestions were incubated at 37.degree. C. for 4-6 hours. BAC DNA
was also digested with NotI for estimation of insert size by CHEF
gel analysis (see below). Reaction conditions were identical to
those for HindIII except that 20 units of NotI were used. Six .mu.l
of 6.times. Ficoll loading buffer containing bromphenol blue and
xylene cyanol was added prior to electrophoresis.
[0234] HindIII digests were analyzed on 0.6% agarose (Seakem, FMC
Bioproducts) in 1.times.TBE containing 0.5 .mu.g/ml ethidium
bromide. Gels (20 cm.times.25 cm) were electrophoresed in a Model
A4 electrophoresis unit (Owl Scientific) at 50 volts for 20-24 hrs.
Molecular weight size markers included undigested lambda DNA,
HindIII digested lambda DNA, and HaeIII digested X174 DNA.
Molecular weight markers were heated at 65.degree. C. for 2 min
prior to loading the gel. Images were captured with a Kodak DC40
CCD camera and analyzed with Kodak 1D software.
[0235] NotI digests were analyzed on a CHEF DRII (BioRad)
electrophoresis unit according to the manufacturer's
recommendations. Briefly, 1% agarose gels (BioRad pulsed field
grade) were prepared in 0.5.times.TBE, equilibrated for 30 minutes
in the electrophoresis unit at 14.degree. C., and electrophoresed
at 6 volts/cm for 14 hrs with circulation. Switching times were
ramped from 10 sec to 20 sec. Gels were stained after
electrophoresis in 0.5 .mu.g/ml ethidium bromide. Molecular weight
markers included undigested lambda DNA, HindIII digested lambda
DNA, lambda ladder PFG ladder, and low range PFG marker (all from
New England Biolabs).
[0236] BAC DNA prepared either by the manual alkaline lysis or
Autogen protocols were labeled for FISH analysis using a Bioprime
labeling kit (BioRad) according to the manufacturer's
recommendation with minor modifications. Approximately 200 ng of
DNA was used for each 50 .mu.l reaction. 3 .mu.l were analyzed on a
2% agarose gel to determine the extent of labeling. Reactions were
purified using a Sephadex G50 spin column prior to in situ
hybridization. Metaphase FISH was performed as described (Ma et
al., 1996 Cytogenet. Cell Genet., 74: 266-71).
8. BAC Endseaencing
[0237] The sequencing of BAC insert ends utilized DNA prepared by
either of the two methods described above. The DYEnamic energy
transfer primers and Dynamic Direct cycle sequencing kits from
Amersham were used for sequencing reactions. Ready made sequencing
mix including the M13-40 forward sequencing primer was used
(Catalog #US79730) for the T7 BAC vector terminus; ready made
sequencing mix (Catalog #US79530) was mixed with the M13-28 reverse
sequencing primer (Catalog #US79339) for the SP6 BAC vector
terminus. The sequencing reaction mixes included one of the four
fluorescently labeled dye-primers, one of the four dideoxy
termination mixes, dNTPs, reaction buffer, and Thermosequenase. For
each BAC DNA sample, 3 .mu.l of the BAC DNA sample was aliquoted to
4 PCR strip tubes. 2 .mu.l of one of the four dye
primer/termination mix combinations was then added to each of the
four tubes. The tubes were then sealed and centrifuged briefly
prior to PCR. Thermocycling conditions involved a 1 minute
denaturation at 95.degree. C., 15 second annealing at 45.degree.
C., and extension for 1 minute at 70.degree. C. for 35 total
cycles. After cycling the plates were centrifuged briefly to
collect all the liquid to the bottom of the tubes. 5 .mu.l of
sterile distilled and deionized H.sub.2O was then added into each
tube, the plates sealed and centrifuged briefly again. The four
samples for each BAC were then pooled together. DNA was then
precipitated by adding 1.5 .mu.l of 7.5 M NH.sub.4OAc and 100 .mu.l
of -20.degree. C. 100% ethanol to each tube. Samples were mixed by
pipetting up and down once. The plates were then sealed and
incubated on ice for 10 minutes. Plates were centrifuged in a table
topHaraeus centrifuge at 4000 rpm (3,290.times.g) for 30 minutes at
4.degree. C. to recover the DNA. The supernatant was removed and
excess liquid blotted onto paper towels. Pellets were washed by
adding 100 .mu.l of -20.degree. C. 70% ethanol into each tube and
re-centrifuging at 4000 rpm (3,290.times.g) for 10 minutes at
4.degree. C. The supernatant was removed and excess liquid again
removed by blotting on a paper towel. Remaining traces of liquid
were removed by placing the plates upside down over a paper towel
and centrifuging only until the centrifuge reached 800 rpm. Samples
were then air dried at room temperature for 30 min. Tubes were
capped and stored dry at -20.degree. C. until electrophoresis.
Immediately prior to electrophoresis the DNA was dissolved in 1.5
.mu.l of Amersham loading dye. Plates were then sealed and
centrifuged at 2000 rpm (825.times.g). The plates were then
vortexed on a plate shaker for 1-2 minutes. Samples were then
recentrifuged at 2000 rpm (825.times.g) briefly. Samples were then
heated at 65.degree. C. for 2 min and immediately placed on ice.
Standard gel electrophoresis was performed on ABI 377 fluorescent
sequencers according to the manufacturer's recommmendation.
9. Sub-Cloning and Sequencing of HBM BAC DNA
[0238] The physical map of the Zmax1 (LRP5) gene region provides a
set of BAC clones that contain within them the Zmax1 (LRP5) gene
and the HBM gene. DNA sequencing of several of the BACs from the
region has been completed. The DNA sequence data is a unique
reagent that includes data that one skilled in the art can use to
identify the Zmax1 (LRP5) gene and the HBM gene, or to prepare
probes to identify the gene(s), or to identify DNA sequence
polymorphisms that identify the gene(s).
[0239] BAC DNA was isolated according to one of two protocols,
either a Qiagen purification of BAC DNA (Qiagen, Inc. as described
in the product literature) or a manual purification which is a
modification of the standard alkaline lysis/Cesium Chloride
preparation of plasmid DNA (see e.g., Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons (1997)).
Briefly for the manual protocol, cells were pelleted, resuspended
in GTE (50 mM glucose, 25 mM Tris-Cl (pH 8), 10 mM EDTA) and
lysozyme (50 mg/ml solution), followed by NaOH/SDS (1% SDS/0.2 N
NaOH) and then an ice-cold solution of 3 M KOAc (pH 4.5-4.8).
RNaseA was added to the filtered supernatant, followed by
Proteinase K and 20% SDS. The DNA was then precipitated with
isopropanol, dried and resuspended in TE (10 mM Tris, 1 mM EDTA (pH
8.0)). The BAC DNA was further purified by Cesium Chloride density
gradient centrifugation (Ausubel et al., 1997).
[0240] Following isolation, the BAC DNA was sheared
hydrodynamically using an HPLC (Hengen, 1997 Trends in Biochem.
Sci. 22: 273-4) to an insert size of 2000-3000 bp. After shearing,
the DNA was concentrated and separated on a standard 1% agarose
gel. A single fraction, corresponding to the approximate size, was
excised from the gel and purified by electroelution (Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring, N.Y. (1989)).
[0241] The purified DNA fragments were then blunt-ended using T4
DNA polymerase. The blunt-ended DNA was then ligated to unique
BstXI-linker adapters (5'-GTCTTCACCACG GGG-3' and 5'-GTGGTGAAGAC-3'
in 100-1000 fold molar excess; SEQ ID NOS: 627 and 628
respectively). These linkers were complimentary to the BstXI-cut
pMPX vectors (constructed by the inventors), while the overhang was
not self-complimentary. Therefore, the linkers would not
concatemerize nor would the cut-vector religate itself easily. The
linker-adapted inserts were separated from the unincorporated
linkers on a 1% agarose gel and purified using GeneClean (BIO 101,
Inc.). The linker-adapted insert was then ligated to a modified
pBlueScript vector to construct a "shotgun" subclone library. The
vector contained an out-of-frame lacZ gene at the cloning site
which became in-frame in the event that an adapter-dimer is cloned,
allowing these to be avoided by their blue-color.
[0242] All subsequent steps were based on sequencing by ABI377
automated DNA sequencing methods. Only major modifications to the
protocols are highlighted. Briefly, the library was then
transformed into DH5.alpha. competent cells (Life Technologies,
Bethesda, Md., DH5.alpha. transformation protocol). It was assessed
by plating onto antibiotic plates containing ampicillin and
IPTG/Xgal. The plates were incubated overnight at 37.degree. C.
Successful transformants were then used for plating of clones and
picking for sequencing. The cultures were grown overnight at
37.degree.. DNA was purified using a silica bead DNA preparation
(Ng et al., 1996 Nucl. Acids Res. 24: 5045-7) method. In this
manner, 25 .mu.g of DNA was obtained per clone.
[0243] These purified DNA samples were then sequenced using ABI
dye-terminator chemistry. The ABI dye terminator sequence reads
were run on ABI377 machines and the data was directly transferred
to UNIX machines following lane tracking of the gels. All reads
were assembled using PHRAP (P. Green, Abstracts of DOE-Human Genome
Program Contractor-Grantee Workshop V, January 1996, p. 157) with
default parameters and quality scores. The initial assembly was
done at 6-fold coverage and yielded an average of 8-15 contigs.
Following the initial assembly, missing mates (sequences from
clones that only gave one strand reads) were identified and
sequenced with ABI technology to allow the identification of
additional overlapping contigs. Primers for walking were selected
using a Genome Therapeutics program Pick_primer near the ends of
the clones to facilitate gap closure. These walks were sequenced
using the selected clones and primers. Data were reassembled with
PHRAP into sequence contigs.
10. Gene Identification by Computational Methods
[0244] Following assembly of the BAC sequences into contigs, the
contigs were subjected to computational analyses to identify coding
regions and regions bearing DNA sequence similarity to known genes.
This protocol included the following steps. [0245] 1. Degap the
contigs: the sequence contigs often contain symbols (denoted by a
period symbol) that represent locations where the individual ABI
sequence reads have insertions or deletions. Prior to automated
computational analysis of the contigs, the periods were removed.
The original data was maintained for future reference. [0246] 2.
BAC vector sequences were "masked" within the sequence by using the
program cross match (Phil Green,
http:\\chimera.biotech.washington.edu\UVGC). Since the shotgun
libraries construction detailed above leaves some BAC vector in the
shotgun libraries, this program was used to compare the sequence of
the BAC contigs to the BAC vector and to mask any vector sequence
prior to subsequent steps. Masked sequences were marked by an "X"
in the sequence files, and remained inert during subsequent
analyses. [0247] 3. E. coli sequences contaminating the BAC
sequences were masked by comparing the BAC contigs to the entire E.
coli DNA sequence. [0248] 4. Repetitive elements known to be common
in the human genome were masked using cross match. In this
implementation of crossmatch, the BAC sequence was compared to a
database of human repetitive elements (Jerzy Jerka, Genetic
Information Research Institute, Palo Alto, Calif.). The masked
repeats were marked by X and remained inert during subsequent
analyses. [0249] 5. The location of exons within the sequence was
predicted using the MZEF computer program (Zhang, 1997 Proc. Natl.
Acad. Sci. USA. 94: 565-8). [0250] 6. The sequence was compared to
the publicly available unigene database (National Center for
Biotechnology Information, National Library of Medicine, 38A,
8N905, 8600 Rockville Pike, Bethesda, Md. 20894;
www.ncbi.nlm.nih.gov) using the blastn2 algorithm (Altschul et al.,
1997 Nucl. Acids Res. 25: 3389-3402). The parameters for this
search were: E=0.05, v=50, B=50 (where E is the expected
probability score cutoff, V is the number of database entries
returned in the reporting of the results, and B is the number of
sequence alignments returned in the reporting of the results
(Altschul et al., J. Mol. Biol., 215:403-410 (1990)). [0251] 7. The
sequence was translated into protein for all six reading frames,
and the protein sequences were compared to a non-redundant protein
database compiled from Genpept Swissprot PIR (National Center for
Biotechnology Information, National Library of Medicine, 38A,
8N905, 8600 Rockville Pike, Bethesda, Md. 20894;
www.ncbi.nlm.nih.gov). The parameters for this search were E=0.05,
V=50, B=50, where E, V, and B are defined as above. [0252] 8. The
BAC DNA sequence was compared to the database of the cDNA clones
derived from direct selection experiments (described below) using
blastn2 (Altschul et al., 1997). The parameters for this search
were E=0.05, V=250, B=250, where E, V, and B are defined as above.
[0253] 9. The BAC sequence was compared to the sequences of all
other BACs from the IBM region on chromosome 11q12-13 using blastn2
(Altschul et al., 1997). The parameters for this search were
E=0.05, V=50, B=50, where E, V, and B are defined as above. [0254]
10. The BAC sequence was compared to the sequences derived from the
ends of BACs from the HBM region on chromosome 11q12-13 using
blastn2 (Altschul et al., 1997). The parameters for this search
were E=0.05, V=50, B=50, where E, V, and B are defined as above.
[0255] 11. The BAC sequence was compared to the GenBank database
(National Center for Biotechnology Information, National Library of
Medicine, 38A, 8N905, 8600 Rockville Pike, Bethesda, Md. 20894;
www.ncbi.nlm.nih.gov) using blastn2 (Altschul et al., 1997). The
parameters for this search were E=0.05, V=50, B=50, where E, V, and
B are defined as above. [0256] 12. The BAC sequence was compared to
the STS division of GenBank database (National Center for
Biotechnology Information, National Library of Medicine, 38A,
8N905, 8600 Rockville Pike, Bethesda, Md. 20894;
www.ncbi.nlm.nih.gov) using blastn2 (Altschul et al., 1997). The
parameters for this search were E=0.05, V=50, B=50, where E, V, and
B are defined as above. [0257] 13. The BAC sequence was compared to
the Expressed Sequence (EST) Tag GenBank database (National Center
for Biotechnology Information, National Library of Medicine, 38A,
8N905, 8600 Rockville Pike, Bethesda, Md. 20894;
www.ncbi.nhn.nih.gov) using blastn2 (Altschul et al., 1997). The
parameters for this search were E=0.05, V=250, B=250, where E, V,
and B are defined as above.
11. Gene Identification by Direct cDNA Selection
[0258] Primary linkered cDNA pools were prepared from bone marrow,
calvarial bone, femoral bone, kidney, skeletal muscle, testis and
total brain. Poly (A)+RNA was prepared from calvarial and femoral
bone tissue (Chomczynski et al., 1987 Anal. Biochem. 162: 156-9;
and D'Alessio et al., 1987 Focus 9: 1-4) and the remainder of the
mRNA was purchased from Clontech (Palo Alto, Calif.). In order to
generate oligo(dT) and random primed cDNA pools from the same
tissue, 2.5 .mu.g mRNA was mixed with oligo(dT) primer in one
reaction and 2.5 .mu.g mRNA was mixed with random hexamers in
another reaction, and both were converted to first and second
strand cDNA according to manufacturers recommendations (Life
Technologies, Bethesda, Md.). Paired phosphorylated cDNA linkers
(see sequence below) were annealed together by mixing in a 1:1
ratio (10 .mu.g each) incubated at 65.degree. C. for five minutes
and allowed to cool to room temperature.
Paired Linkers Oligo 1/2
TABLE-US-00009 [0259] OLIGO 1: 5'-CTG AGC GGA ATT CGT GAG ACC-3'
(SEQ D NO: 12) OLIGO 2: 5'-TTG GTC TCA CGT ATT CCG CTC GA-3' (SEQ
ID NO:13)
Paired Linkers Oligo3/4
TABLE-US-00010 [0260] OLIGO 3: 5'-CTC GAG AAT TCT GGA TCC TC-3'
(SEQ ID NO:14) OLIGO 4: 5'-TTG AGG ATC CAG AAT TCT CGA G-3' (SEQ ID
NO:15)
Paired Linkers Oligo5/6
TABLE-US-00011 [0261] OLIGO 5: 5'-TGT ATG CGA ATT CGC TGC GCG-3'
(SEQ ID NO:16) OLIGO 6: 5'-TTC GCG CAG CGA ATT CGC ATA CA-3' (SEQ
ID NO:17)
Paired Linkers Oligo7/8
TABLE-US-00012 [0262] OLIGO 7: 5'-GTC CAC TGA ATT CTC AGT GAG-3'
(SEQ ID NO:18) OLIGO 8: 5'-TTG TCA CTG AGA ATT CAG TGG AC-3' (SEQ
ID NO:19)
Paired Linkers Oligo11/12
TABLE-US-00013 [0263] OLIGO 11: 5'-GAA TCC GAA TTC CTG GTC AGC-3'
(SEQ ID NO:20) OLIGO 12: 5'-TTG CTG ACC AGG AAT TCG GAT TC-3' (SEQ
ID NO:21)
[0264] Linkers were ligated to all oligo(dT) and random primed cDNA
pools (see below) according to manufacturers instructions (Life
Technologies, Bethesda, Md.).
[0265] Oligo 1/2 was ligated to oligo(dT) and random primed cDNA
pools prepared from bone marrow. Oligo 3/4 was ligated to oligo(dT)
and random primed cDNA pools prepared from calvarial bone. Oligo
5/6 was ligated to oligo(dT) and random primed cDNA pools prepared
from brain and skeletal muscle. Oligo 7/8 was ligated to oligo(dT)
and random primed cDNA pools prepared from kidney. Oligo 11/12 was
ligated to oligo(dT) and random primed cDNA pools prepared from
femoral bone.
[0266] The cDNA pools were evaluated for length distribution by PCR
amplification using 1 .mu.l of a 1:1, 1:10, and 1:100 dilution of
the ligation reaction, respectively. PCR reactions were performed
in a Perkin Elmer 9600, each 25 .mu.l volume reaction contained 1
.mu.l of DNA, mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2,
0.001% gelatin, 200 mM each dNTPs, 10 .mu.M primer and 1 unit Taq
DNA polymerase (Perkin Elmer) and was amplified under the following
conditions: 30 seconds at 94.degree. C., 30 seconds at 60.degree.
C. and 2 minutes at 72.degree. C. for 30 cycles. The length
distribution of the amplified cDNA pools were evaluated by
electrophoresis on a 1% agarose gel. The PCR reaction that gave the
best representation of the random primed and oligo(dT) primed cDNA
pools was scaled up so that .about.2-3 .mu.g of each cDNA pool was
produced. The starting cDNA for the direct selection reaction
comprised of 0.5 .mu.g of random primed cDNAs mixed with 0.5 .mu.g
of oligo(dT) primed cDNAs.
[0267] The DNA from the 54 BACs that were used in the direct cDNA
selection procedure was isolated using Nucleobond AX columns as
described by the manufacturer (The Nest Group, Inc.).
[0268] The BACs were pooled in equimolar amounts and 1 .mu.g of the
isolated genomic DNA was labeled with biotin 16-UTP by nick
translation in accordance with the manufacturers instructions
(Boehringer Mannheim). The incorporation of the biotin was
monitored by methods that could be practiced by one skilled in the
art (Del Mastro et al., Methods in Molecular Biology, Humana Press
Inc., NJ (1996)).
[0269] Direct cDNA selection was performed using methods that could
be practiced by one skilled in the art (Del Mastro et al., 1996).
Briefly, the cDNA pools were multiplexed in two separate reactions:
In one reaction cDNA pools from bone marrow, calvarial bone, brain
and testis were mixed, and in the other cDNA pools from skeletal
muscle, kidney and femoral bone were mixed. Suppression of the
repeats, yeast sequences and plasmid in the cDNA pools was
performed to a Cot of 20. 100 ng of biotinylated BAC DNA was mixed
with the suppressed cDNAs and hybridized in solution to a Cot of
200. The biotinylated DNA and the cognate cDNAs was captured on
streptavidin-coated paramagnetic beads. The beads were washed and
the primary selected cDNAs were eluted. These cDNAs were PCR
amplified and a second round of direct selection was performed. The
product of the second round of direct selection is referred to as
the secondary selected material. A Galanin cDNA clone, previously
shown to map to 11q12-13 (Evans, 1993 Genomics 18: 473-7), was used
to monitor enrichment during the two rounds of selection.
[0270] The secondary selected material from bone marrow, calvarial
bone, femoral bone, kidney, skeletal muscle, testis and total brain
was PCR amplified using modified primers of oligos 1, 3, 5, 7 and
11, shown below, and cloned into the UDG vector pAMP10 (Life
Technologies, Bethesda, Md.), in accordance with the manufacturer's
recommendations.
[0271] Modified Primer Sequences:
TABLE-US-00014 SEQ ID Primer NO. Sequence Oligo 1 22 5'-CUA CUA CUA
CUA CTG AGC GGA ATT CGT GAG ACC-3' Oligo 3 23 5'-CUA CUA CUA CUA
CTC GAG AAT TCT GGA TCC TC-3' Oligo 5 24 5'-CUA CUA CUA CUA TGT ATG
CGA ATT CGC TGC GCG-3' Oligo 7 25 5'-CUA CUA CUA CUA GTC CAC TGA
ATT CTC AGT GAG-3' Oligo 11 26 5'-CUA CUA CUA CUA GAA TCC GAA TTC
CTG GTC AGC-3'
[0272] The cloned secondary selected material, from each tissue
source, was transformed into MAX Efficiency DH5a Competent Cells
(Life Technologies, Bethesda, Md.) as recommended by the
manufacturer. 384 colonies were picked from each transformed source
and arrayed into four 96 well microtiter plates.
[0273] All secondarily selected cDNA clones were sequenced using
M13 dye primer terminator cycle sequencing kit (Applied
Biosystems), and the data collected by the ABI 377 automated
fluorescence sequencer (Applied Biosystems).
[0274] All sequences were analyzed using the BLASTN, BLASTX and
FASTA programs (Altschul et al., 1990 J. Mol. Biol. 215: 403-410;
and Altschul et al., Nucl. Acids. Res. 25: 3389-3402). The cDNA
sequences were compared to a database containing sequences derived
from human repeats, mitochondrial DNA, ribosomal RNA, E. coli DNA
to remove background clones from the dataset using the program
cross_match. A further round of comparison was also performed using
the program BLASTN2 against known genes (GenBank) and the BAC
sequences from the HBM region. Those cDNAs that were >90%
homologous to these sequences were filed according to the result
and the data stored in a database for further analysis. cDNA
sequences that were identified but did not have significant
similarity to the BAC sequences from the HBM region or were
eliminated by cross_match were hybridized to nylon membranes which
contained the BACs from the HBM region, to ascertain whether they
hybridized to the target.
[0275] Hybridization analysis was used to map the cDNA clones to
the BAC target that selected them. The BACs that were identified
from the HBM region were arrayed and grown into a 96 well
microtiter plate. LB agar containing 25 .mu.g/ml kanamycin was
poured into 96 well microtiter plate lids. Once the agar had
solidified, pre-cut Hybond N+ nylon membranes (Amersham) were laid
on top of the agar and the BACs were stamped onto the membranes in
duplicate using a hand held 96 well replica plater (V&P
Scientific, Inc.). The plates were incubated overnight at
37.degree. C. The membranes were processed according to the
manufacturers recommendations.
[0276] The cDNAs that needed to be mapped by hybridization were PCR
amplified using the relevant primer (oligos 1, 3, 5, 7 and 11) that
would amplify that clone. For this PCR amplification, the primers
were modified to contain a linkered digoxigenin molecule at the 5'
of the oligonucleotide. The PCR amplification was performed under
the same conditions as described in Preparation of cDNA Pools
(above). The PCR products were evaluated for quality and quantity
by electrophoresis on a 1% agarose gel by loading 5 .mu.l of the
PCR reaction. The nylon membranes containing the stamped BACs were
individually pre-hybridized in 50 ml conical tubes containing 10 ml
of hybridization solution (5.times.SSPE, 0.5.times. Blotto, 2.5%
SDS and 1 mM EDTA (pH 8.0)). The 50 ml conical tubes were placed in
a rotisserie oven (Robbins Scientific) for 2 hours at 65.degree. C.
Twenty-five ng of each cDNA probe was denatured and added into
individual 50 ml conical tubes containing the nylon membrane and
hybridization solution. The hybridization was performed overnight
at 65.degree. C. The filters were washed for 20 minutes at
65.degree. C. in each of the following solutions: 3.times.SSPE,
0.1% SDS; 1.times.SSPE, 0.1% SDS and 0.1.times.SSPE, 0.1% SDS.
[0277] The membranes were removed from the 50 ml conical tubes and
placed in a dish. Acetate sheets were placed between each membrane
to prevent them from sticking to each other. The incubation of the
membranes with the Anti-DIG-AP and CDP-Star was performed according
to manufacturers recommendations (Boehringer Mannheim). The
membranes were wrapped in Saran wrap and exposed to Kodak Bio-Max
X-ray film for 1 hour.
12. cDNA Cloning and Expression Analysis
[0278] To characterize the expression of the genes identified by
direct cDNA selection and genomic DNA sequencing in comparison to
the publicly available databases, a series of experiments were
performed to further characterize the genes in the HBM region.
First, oligonucleotide primers were designed for use in the
polymerase chain reaction (PCR) so that portions of a cDNA, EST, or
genomic DNA could be amplified from a pool of DNA molecules (a cDNA
library) or RNA population (RT-PCR and RACE). The PCR primers were
used in a reaction containing genomic DNA to verify that they
generated a product of the size predicted based on the genomic
(BAC) sequence. A number of cDNA libraries were then examined for
the presence of the specific cDNA or EST. The presence of a
fragment of a transcription unit in a particular cDNA library
indicates a high probability that additional portions of the same
transcription unit will be present as well.
[0279] A critical piece of data that is required when
characterizing novel genes is the length, in nucleotides, of the
processed transcript or messenger RNA (mRNA). One skilled in the
art primarily determines the length of an mRNA by Northern blot
hybridization (Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y.
(1989)). Groups of ESTs and direct-selected cDNA clones that
displayed significant sequence similarity to sequenced BACs in the
critical region were grouped for convenience into approximately 30
kilobase units. Within each 30 kilobase unit there were from one up
to fifty ESTs and direct-selected cDNA clones which comprised one
or more independent transcription units. One or more ESTs or
direct-selected cDNAs were used as hybridization probes to
determine the length of the mRNA in a variety of tissues, using
commercially available reagents (Multiple Tissue Northern blot;
Clontech, Palo Alto, Calif.) under conditions recommended by the
manufacturer.
[0280] Directionally cloned cDNA libraries from femoral bone, and
calvarial bone tissue were constructed by methods familiar to one
skilled in the art (for example, Soares in Automated DNA Sequencing
and Analysis, Adams, Fields and Venter, Eds., Academic Press, NY,
pages 110-114 (1994)). Bones were initially broken into fragments
with a hammer, and the small pieces were frozen in liquid nitrogen
and reduced to a powder in a tissue pulverizer (Spectrum Laboratory
Products). RNA was extracted from the powdered bone by homogenizing
the powdered bone with a standard Acid Guanidinium
Thiocyanate-Phenol-Chloroform extraction buffer (e.g., Chomczynski
et al., 1987 Anal. Biochem. 162: 156-9) using a polytron
homogenizer (Brinkman Instruments). Additionally, human brain and
lung total RNA was purchased from Clontech. PolyA RNA was isolated
from total RNA using dynabeads-dT according to the manufacturer's
recommendations (Dynal, Inc.). First strand cDNA synthesis was
initiated using an oligonucleotide primer with the sequence:
5'-AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTT TT-3' (SEQ ID
NO:27). This primer introduces a NotI restriction site (underlined)
at the 3', end of the cDNA. First and second strand synthesis were
performed using the "one-tube" cDNA synthesis kit as described by
the manufacturer (Life Technologies, Bethesda, Md.). Double
stranded cDNAs were treated with T4 polynucleotide kinase to ensure
that the ends of the molecules were blunt (Soares in Automated DNA
Sequencing and Analysis, Adams, Fields and Venter, Eds., Academic
Press, NY, pages 110-114 (1994)), and the blunt ended cDNAs were
then size selected by a Biogel column (Huynh et al. in DNA Cloning,
Vol. 1, Glover, Ed., IRL Press, Oxford, pages 49-78 (1985)) or with
a size-sep 400 Sepharose column (Pharmacia, catalog #27-5105-01).
Only cDNAs of 400 base pairs or longer were used in subsequent
steps. EcoRI adapters (sequence: 5' OH-AATTCGGCACGAG-OH 3' (SEQ ID
NO:28), and 5' p-CTCGTGCCG-OH 3' (SEQ ID NO:29)) were then ligated
to the double stranded cDNAs by methods familiar to one skilled in
the art (Soares, 1994). The EcoRI adapters were then removed from
the 3' end of the cDNA by digestion with NotI (Soares, 1994). The
cDNA was then ligated into the plasmid vector pBluescript.RTM. II
KS+ (Stratagene, La Jolla, Calif.), and the ligated material was
transformed into E. coli host DH10B or DH12S by electroporation
methods familiar to one skilled in the art (Soares, 1994). After
growth overnight at 37.degree. C., DNA was recovered from the E.
coli colonies after scraping the plates by processing as directed
for the Mega-prep kit (Qiagen, Chatsworth, Calif.). The quality of
the cDNA libraries was estimated by counting a portion of the total
numbers of primary transformants and determining the average insert
size and the percentage of plasmids with no cDNA insert. Additional
cDNA libraries (human total brain, heart, kidney, leukocyte, and
fetal brain) were purchased from Life Technologies, Bethesda,
Md.
[0281] cDNA libraries, both oligo (dT) and random hexamer (N.sub.6)
primed, were used for isolating cDNA clones transcribed within the
HBM region: human bone, human brain, human kidney and human
skeletal muscle (all cDNA libraries were made by the inventors,
except for skeletal muscle (dT) and kidney (dT) cDNA libraries).
Four 10.times.10 arrays of each of the cDNA libraries were prepared
as follows: the cDNA libraries were titered to 2.5.times.10.sup.6
using primary transformants. The appropriate volume of frozen stock
was used to inoculate 2 L of LB/ampicillin (100 mg/ml). This
inoculated liquid culture was aliquoted into 400 tubes of 4 ml
each. Each tube contained approximately 5000 cfu. The tubes were
incubated at 30.degree. C. overnight with gentle agitation. The
cultures were grown to an OD of 0.7-0.9. Frozen stocks were
prepared for each of the cultures by allocating 100 .mu.l of
culture and 300 .mu.l of 80% glycerol. Stocks were frozen in a dry
ice/ethanol bath and stored at -70.degree. C. The remaining culture
was DNA prepared using the Qiagen (Chatsworth, Calif.) spin
miniprep kit according to the manufacturer's instructions. The DNAs
from the 400 cultures were pooled to make 80 column and row pools.
The cDNA libraries were determined to contain HBM cDNA clones of
interest by PCR. Markers were designed to amplify putative exons.
Once a standard PCR optimization was performed and specific cDNA
libraries were determined to contain cDNA clones of interest, the
markers were used to screen the arrayed library. Positive addresses
indicating the presence of cDNA clones were confirmed by a second
PCR using the same markers.
[0282] Once a cDNA library was identified as likely to contain cDNA
clones corresponding to a specific transcript of interest from the
HBM region, it was manipulated to isolate the clone or clones
containing cDNA inserts identical to the EST or direct-selected
cDNA of interest. This was accomplished by a modification of the
standard "colony screening" method (Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor N.Y. (1989)). Specifically, twenty 150 mm
LB+ampicillin agar plates were spread with 20,000 colony forming
units (cfu) of cDNA library and the colonies allowed to grow
overnight at 37.degree. C. Colonies were transferred to nylon
filters (Hybond from Amersham, or equivalent) and duplicates
prepared by pressing two filters together essentially as described
(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor N.Y. (1989)). The
"master" plate was then incubated an additional 6-8 hours to allow
the colonies to grow back. The DNA from the bacterial colonies was
then affixed to the nylon filters by treating the filters
sequentially with denaturing solution (0.5 N NaOH, 1.5 M NaCl) for
two minutes, neutralization solution (0.5 M Tris-Cl pH 8.0, 1.5 M
NaCl) for two minutes (twice). The bacterial colonies were removed
from the filters by washing in a solution of 2.times.SSC/0.1% SDS
for one minute while rubbing with tissue paper. The filters were
air dried and baked under vacuum at 80.degree. C. for 1-2
hours.
[0283] A cDNA hybridization probe was prepared by random hexamer
labeling (Fineberg and Vogelstein, Anal. Biochem., 132:6-13 (1983))
or by including gene-specific primers and no random hexamers in the
reaction (for small fragments). Specific activity was calculated
and was >5.times.10.sup.8 cpm/10.sup.8 .mu.g of cDNA. The colony
membranes were then prewashed in 10 mM Tris-Cl pH 8.0, 1 M NaCl, 1
mM EDTA, 0.1% SDS for 30 minutes at 55.degree. C. Following the
prewash, the filters were prehybridized in >2 ml/filter of
6.times.SSC, 50% deionized formamide, 2% SDS, 5.times.Denhardt's
solution, and 100 mg/ml denatured salmon sperm DNA, at 42.degree.
C. for 30 minutes. The filters were then transferred to
hybridization solution (6.times.SSC, 2% SDS, 5.times.Denhardt's,
100 mg/ml denatured salmon sperm DNA) containing denatured
.alpha..sup.32P-dCTP-labeled cDNA probe and incubated at 42.degree.
C. for 16-18 hours.
[0284] After the 16-18 hour incubation, the filters were washed
under constant agitation in 2.times.SSC, 2% SDS at room temperature
for 20 minutes, followed by two washes at 65.degree. C. for minutes
each. A second wash was performed in 0.5.times.SSC, 0.5% SDS for 15
minutes at 65.degree. C. Filters were then wrapped in plastic wrap
and exposed to radiographic film for several hours to overnight.
After film development, individual colonies on plates were aligned
with the autoradiograph so that they could be picked into a 1 ml
solution of LB Broth containing ampicillin. After shaking at
37.degree. C. for 1-2 hours, aliquots of the solution were plated
on 150 mm plates for secondary screening. Secondary screening was
identical to primary screening (above) except that it was performed
on plates containing .about.250 colonies so that individual
colonies could be clearly identified for picking.
[0285] After colony screening with radiolabeled probes yielded cDNA
clones, the clones were characterized by restriction endonuclease
cleavage, PCR, and direct sequencing to confirm the sequence
identity between the original probe and the isolated clone. To
obtain the full-length cDNA, the novel sequence from the end of the
clone identified was used to probe the library again. This process
was repeated until the length of the cDNA cloned matches that
estimated to be full-length by the northern blot analysis.
[0286] RT-PCR was used as another method to isolate full length
clones. The cDNA was synthesized and amplified using a "Superscript
One Step RT-PCR" kit (Life Technologies, Gaithersburg, Md.). The
procedure involved adding 1.5 .mu.g of RNA to the following: 25
.mu.l of reaction mix provided which is a proprietary buffer mix
with MgSO.sub.4 and dNTP's, 1 .mu.l sense primer (10 .mu.M) and 1
.mu.l anti-sense primer (10 .mu.M), 1 .mu.l reverse transcriptase
and Taq DNA polymerase mix provided and autoclaved water to a total
reaction mix of 50 .mu.l. The reaction was then placed in a
thermocycler for 1 cycle at 50.degree. C. for 15 to 30 minutes,
then 94.degree. C. for 15 seconds, 55-60.degree. C. for 30 seconds
and 68-72.degree. C. for 1 minute per kilobase of anticipated
product and finally 1 cycle of 72.degree. C. for 5-10 minutes. The
sample was analyzed on an agarose gel. The product was excised from
the gel and purified from the gel (GeneClean, Bio 101). The
purified product was cloned in pCTNR (General Contractor DNA
Cloning System, 5 Prime-3 Prime, Inc.) and sequenced to verify that
the clone was specific to the gene of interest.
[0287] Rapid Amplification of cDNA ends (RACE) was performed
following the manufacturer's instructions using a Marathon cDNA
Amplification Kit (Clontech, Palo Alto, Calif.) as a method for
cloning the 5' and 3' ends of candidate genes. cDNA pools were
prepared from total RNA by performing first strand synthesis, where
a sample of total RNA sample was mixed with a modified oligo (dT)
primer, heated to 70.degree. C., cooled on ice and followed by the
addition of: 5.times. first strand buffer, 10 mM dNTP mix, and AMV
Reverse Transcriptase (20 U/.mu.l). The tube was incubated at
42.degree. C. for one hour and then the reaction tube was placed on
ice. For second strand synthesis, the following components were
added directly to the reaction tube: 5.times. second strand buffer,
10 mM dNTP mix, sterile water, 20.times. second strand enzyme
cocktail and the reaction tube was incubated at 16.degree. C. for
1.5 hours. T4 DNA Polymerase was added to the reaction tube and
incubated at 16.degree. C. for 45 minutes. The second-strand
synthesis was terminated with the addition of an EDTA/Glycogen mix.
The sample was subjected to a phenol/chloroform extraction and an
ammonium acetate precipitation. The cDNA pools were checked for
quality by analyzing on an agarose gel for size distribution.
Marathon cDNA adapters (Clontech) were then ligated onto the cDNA
ends. The specific adapters contained priming sites that allowed
for amplification of either 5' or 3' ends, depending on the
orientation of the gene specific primer (GSP) that was chosen. An
aliquot of the double stranded cDNA was added to the following
reagents: 10 .mu.M Marathon cDNA adapter, 5.times.DNA ligation
buffer, T4 DNA ligase. The reaction was incubated at 16.degree. C.
overnight. The reaction was heat inactivated to terminate the
reaction. PCR was performed by the addition of the following to the
diluted double stranded cDNA pool: 10.times.cDNA PCR reaction
buffer, 10 .mu.M dNTP mix, 10 .mu.M GSP, 10 .mu.M API primer (kit),
50.times. Advantage cDNA Polymerase Mix. Thermal Cycling conditions
were 94.degree. C. for 30 seconds, 5 cycles of 94.degree. C. for 5
seconds, 72.degree. C. for 4 minutes, 5 cycles of 94.degree. C. for
5 seconds, 70.degree. C. for 4 minutes, 23 cycles of 94.degree. C.
for 5 seconds, 68.degree. C. for 4 minutes. After the first round
of PCR was performed using the GSP to extend to the end of the
adapter to create the adapter primer binding site, exponential
amplification of the specific cDNA of interest was observed.
Usually a second nested PCR is performed to confirm the specific
cDNA. The RACE product was analyzed on an agarose gel and then
excised and purified from the gel (GeneClean, BIO 101). The RACE
product was then cloned into pCTNR (General Contractor DNA Cloning
System, 5'-3', Inc.) and the DNA sequence determined to verify that
the clone is specific to the gene of interest.
13. Mutation Analysis
[0288] Comparative genes were identified using the above procedures
and the exons from each gene were subjected to mutation detection
analysis. Comparative DNA sequencing was used to identify
polymorphisms in HBM candidate genes from chromosome 11q12-13. DNA
sequences for candidate genes were amplified from patient
lymphoblastoid cell lines.
[0289] The inventors developed a method based on analysis of direct
DNA sequencing of PCR products amplified from candidate regions to
search for the causative polymorphism. The procedure consisted of
three stages that used different subsets of HBM family to find
segregating polymorphisms and a population panel to assess the
frequency of the polymorphisms. The family resources result from a
single founder leading to the assumption that all affected
individuals will share the same causative polymorphism.
[0290] Candidate regions were first screened in a subset of the IBM
family consisting of the proband, daughter, and her mother, father
and brother. Monochromosomal reference sequences were produced
concurrently and used for comparison. The mother and daughter
carried the HBM polymorphism in this nuclear family, providing the
ability to monitor polymorphism transmission. The net result is
that two HBM chromosomes and six non-HBM chromosomes were screened.
This allowed exclusion of numerous frequent alleles. Only alleles
exclusively present in the affected individuals passed to the next
level of analysis.
[0291] Polymorphisms that segregated exclusively with the HBM
phenotype in this original family were then re-examined in an
extended portion of the HBM pedigree consisting of two additional
nuclear families. These families consisted of five HBM and three
unaffected individuals. The HBM individuals in this group included
the two critical crossover individuals, providing the centromeric
and telomeric boundaries of the critical region. Tracking the
heredity of polymorphisms between these individuals and their
affected parents allowed for further refining of the critical
region. This group brought the total of HBM chromosomes screened to
seven and the total of non-HBM chromosomes to seventeen.
[0292] When a given polymorphism continued to segregate exclusively
with the HBM phenotype in the extended group, a population panel
was then examined. This panel of 84 persons consisted of 42
individuals known to have normal bone mineral density and 42
individuals known to be unrelated but with untyped bone mineral
density. For this purpose, normal bone mineral density is within
two standard-deviations of a BMD Z score of 0. The second group was
from the widely used CEPH panel of individuals. Any segregating
polymorphisms found to be rare in this population were subsequently
examined on the entire HBM pedigree and a larger population.
[0293] Polymerase chain reaction (PCR) was used to generate
sequencing templates from the HBM family's DNA and monochromosomal
controls. Enzymatic amplification of genes within the HBM region on
11q12-13 was accomplished using the PCR with oligonucleotides
flanking each exon as well as the putative 5' regulatory elements
of each gene. The primers were chosen to amplify each exon as well
as 15 or more base pairs within each intron on either side of the
splice. All PCR primers were made as chimeras to facilitate dye
primer sequencing. The M13-21F (5'-GTA A CGA CGG CCA GT-3') (SEQ ID
NO:30) and -28REV (5'-AAC AGC TAT GAC CAT G-3') (SEQ ID NO:31)
primer binding sites were built on to the 5' end of each forward
and reverse PCR primer, respectively, during synthesis. 150 ng of
genomic DNA was used in a 50 .mu.l PCR with 2 U AmpliTaq, 500 nM
primer and 125 .mu.M dNTP. Buffer and cycling conditions were
specific to each primer set. TaqStart antibody (Clontech) was used
for hot start PCR to minimize primer dimer formation. 10% of the
product was examined on an agarose gel. The appropriate samples
were diluted 1:25 with deionized water before sequencing.
[0294] Each PCR product was sequenced according to the standard
Energy Transfer primer (Amersham) protocol. All reactions took
place in 96 well trays. 4 separate reactions, one each for A, C, G
and T were performed for each template. Each reaction included 2
.mu.l of the sequencing reaction mix and 3 .mu.l of diluted
template. The plates were then heat sealed with foil tape and
placed in a thermal cycler and cycled according to the
manufacturer's recommendation. After cycling, the 4 reactions were
pooled. 3 .mu.l of the pooled product was transferred to a new 96
well plate and 1 .mu.l of the manufacturer's loading dye was added
to each well. All 96 well pipetting procedures occurred on a Hydra
96 pipetting station (Robbins Scientific, USA). 1 .mu.l of pooled
material was directly loaded onto a 48 lane gel running on an ABI
377 DNA sequencer for a 10 hour, 2.4 kV run.
[0295] Polyphred (University of Washington) was used to assemble
sequence sets for viewing with Consed (University of Washington).
Sequences were assembled in groups representing all relevant family
members and controls for a specified target region. This was done
separately for each of the three stages. Forward and reverse reads
were included for each individual along with reads from the
monochromosomal templates and a color annotated reference sequence.
Polyphred indicated potential polymorphic sites with a purple flag.
Two readers independently viewed each assembly and assessed the
validity of the purple-flagged sites.
[0296] A total of 23 exons present in the mature mRNA and several
other portions of the primary transcript were evaluated for
heterozygosity in the nuclear family of two HBM-affected and two
unaffected individuals. 25 SNPs were identified, as shown in the
table below.
TABLE-US-00015 TABLE 8 Single Nucleotide Polymorphisms in the Zmax1
(LRP5) Gene and Environs Exon Name Location Base Change
b200e21-h_Contig1_1.nt 69169 (309G) C/A b200e21-h_Contig4_12.nt
27402 (309G) A/G b200e21-h_Contig4_13.nt 27841 (309G) T/C
b200e21-h_Contig4_16.nt 35600 (309G) A/G b200e21-h_Contig4_21.nt
45619 (309G) G/A b200e21-h_Contig4_22.nt-a 46018 (309G) T/G
b200e21-h_Contig4_22.nt-b 46093 (309G) T/G
b200e21-h_Contig4_22.nt-c 46190 (309G) A/G
b200e21-h_Contig4_24.nt-a 50993 (309G) T/C
b200e21-h_Contig4_24.nt-b 51124 (309G) C/T b200e21-h_Contig4_25.nt
55461 (309G) C/T b200e21-h_Contig4_33.nt-a 63645 (309G) C/A
b200e21-h_Contig4_33.nt-b 63646 (309G) A/C b200e21-h_Contig4_61.nt
24809 (309G) T/G b200e21-h_Contig4_62.nt 27837 (309G) T/C
b200e21-h_Contig4_63.nt-a 31485 (309G) C/T
b200e21-h_Contig4_63.nt-b 31683 (309G) A/G b200e21-h_Contig4_9.nt
24808 (309G) T/G b527d12-h_Contig030g_1.nt-a 31340 (308G) T/C
b527d12-h_Contig030g_1.nt-b 32538 (308G) A/G
b527d12-h_Contig080C_2.nt 13224 (308G) A/G
b527d12-h_Contig087C_1.nt 21119 (308G) C/A
b527d12-h_Contig087C_4.nt 30497 (308G) G/A
b527d12-h_Contig088C_4.nt 24811 (309G) A/C
b527d12-h_Contig089_1HP.nt 68280 (309G) G/A
In addition to the polymorphisms presented in Table 8, two
additional polymorphisms can also be present in SEQ ID NO:2. These
is a change at position 2002 of SEQ ID NO:2. Either a guanine or an
adenine can appear at this position. This polymorphism is silent
and is not associated with any change in the amino acid sequence.
The second change is at position 4059 of SEQ ID NO:2 corresponding
in a cytosine (C) to thymine (T) change. This polymorphism results
in a corresponding amino acid change from a valine (V) to an
alanine (A). Other polymorphisms were found in the candidate gene
exons and adjacent intron sequences as displayed in Tables 2 and 3.
Any one or combination of the polymorphisms listed in Tables 2, 3
or S or the two discussed above could also have a minor effect oh
bone mass when present in SEQ ID NO:2. These could also be in
combination with any of the other mutations discussed in Section 3
and in the Examples below.
[0297] The present invention encompasses the nucleic acid sequences
having the nucleic acid sequence of SEQ ID NO: 1 with any one or
more of the above-identified point mutations.
[0298] Preferably, the present invention encompasses the nucleic
acid of SEQ ID NO: 2 or is a mutation in SEQ ID NO:1 that produces
a phenotype like that observed with the protein encoded by SEQ ID
NO:2 (HBM phenotype). Specifically, a base-pair substitution
changing G to T at position 582 in the coding sequence of Zmax1
(the HBM gene) was identified as heterozygous in all HBM
individuals, and not found in the unaffected individuals (i.e.,
b527d12-h Contig087C.sub.--1.nt). FIG. 6 shows the order of the
contigs in B527D12. The direction of transcription for the HBM gene
is from left to right. The sequence of contig308G of B527D12 is the
reverse complement of the coding region to the HBM gene. Therefore,
the relative polymorphism in contig 308G shown in Table 8 as a base
change substitution of C to A is the complement to the G to T
substitution in the HBM gene. This mutation causes a substitution
of glycine 171 with valine (G171V).
[0299] The HBM polymorphism was confirmed by examining the DNA
sequence of different groups of individuals. In all members of the
HBM pedigree (38 individuals), the HBM polymorphism was observed in
the heterozygous form in affected (i.e., elevated bone mass)
individuals only (N=18). In unaffected relatives (N=20)
(BMDZ<2.0), the HBM polymorphism was never observed. To
determine whether this polymorphism was ever observed in
individuals outside of the HBM pedigree, 297 phenotyped individuals
were characterized at the site of the HBM gene. None were
heterozygous at the site of the HBM polymorphism. In an
unphenotyped control group, none of 64 individuals were observed to
be heterozygous at position 582. Taken together, these data prove
that the polymorphism observed in the kindred displaying the high
bone mass phenotype is strongly correlated with the G-T
polymorphism at position 582 of Zmax1 (LRP5). Furthermore, these
results coupled with the ASO results described below, establish
that the HBM polymorphism genetically segregates with the HEM
phenotype, and that both the HEM polymorphism and phenotype are
rare in the general population.
13. Allele Specific Oligonucleotide (ASO) Analysis
[0300] The amplicon containing the HBM polymorphism was PCR
amplified using primers specific for the exon of interest. The
appropriate population of individuals was PCR amplified in 96 well
microtiter plates as follows. PCR reactions (201) containing
1.times. Promega PCR buffer (Cat. #M1883 containing 1.5 mM
MgCl.sub.2), 100 mM dNTP, 200 nM PCR primers (1863F:
5'-CCAAGTTCTGAGAAGTCC-3' and 1864R: 5'-AATACCTGAAACCATACCTG-3'; SEQ
ID NOS: 629 and 630 respectively), 1 U AmpliTaq.TM., and 20 ng of
genomic DNA were prepared and amplified under the following PCR
conditions: 94.degree. C., 1 minute, (94.degree. C., 30 sec.;
58.degree. C., 30 sec.; 72.degree. C., 1 min X35 cycles),
72.degree. C., 5 min, 4.degree. C., hold. Loading dye was then
added and 10 .mu.l of the products was electrophoresed on 1.5%
agarose gels containing 1 .mu.g/ml ethidium bromide at 100-150 V
for 5-10 minutes. Gels were treated 20 minutes in denaturing
solution (1.5 M NaCl, 0.5 N NaOH), and rinsed briefly with water.
Gels were then neutralized in 1 M Tris-HCl, pH 7.5, 1.5 M NaCl, for
20 minutes and rinsed with water. Gels were soaked in 10.times.SSC
for 20 minutes and blotted onto nylon transfer membrane (Hybond
N+-Amersham) in 10.times.SSC overnight. Filters were the rinsed in
6.times.SSC for 10 minutes and UV crosslinked.
[0301] The allele specific oligonucleotides (ASO) were designed
with the polymorphism approximately in the middle. Oligonucleotides
were phosphate free at the 5' end and were purchased from Gibco
BRL. Sequences of the oligonucleotides are:
TABLE-US-00016 2326 Zmax1.ASO.g: 5'-AGACTGGGGTGAGACGC-3' (SEQ ID
NO:631) 2327 Zmax1.ASO.t: 5'-CAGACTGGGTTGAGACGCC-3' (SEQ ID
NO:632)
[0302] The polymorphic nucleotides are underlined. To label the
oligos, 1.5 .mu.l of 1 g/.mu.l ASO oligo (2326.Zmax1.ASO.g or
2327.Zmax1.ASO.t), 11 .mu.l ddH.sub.2O, 2 .mu.l 10.times. kinase
forward buffer, 5 .mu.l .gamma..sup.32P-ATP (6000 Ci/mMole), and 1
.mu.l T4 polynucleotide kinase (10 U/.mu.l) were mixed, and the
reaction incubated at 37.degree. C. for 30-60 minutes. Reactions
were then placed at 95.degree. C. for 2 minutes and 30 ml H.sub.2O
was added. The probes were purified using a G25 microspin column
(Pharmacia).
[0303] Blots were prehybridized in 10 ml 5.times.SSPE,
5.times.Denhardt's, 2% SDS, and 100 .mu.g/ml, denatured, sonicated
salmon sperm DNA at 40.degree. C. for 2 hr. The entire reaction mix
of kinased oligo was then added to 10 ml fresh hybridization buffer
(5.times.SSPE, 5.times.Denhardt's, 2% SDS) and hybridized at
40.degree. C. for at least 4 hours to overnight.
[0304] All washes done in 5.times.SSPE, 0.1% SDS. The first wash
was at 45.degree. C. for 15 minutes; the solution was then changed
and the filters washed 50.degree. C. for 15 minutes. Filters were
then exposed to Kodak biomax film with 2 intensifying screens at
-70.degree. C. for 15 minutes to 1 hr. If necessary the filters
were washed at 55.degree. C. for 15 minutes and exposed to film
again. Filters were stripped by washing in boiling 0.1.times.SSC,
0.1% SDS for 10 minutes at least 3 times.
[0305] The two films that best captured the allele specific assay
with the 2 ASOs were converted into digital images by scanning them
into Adobe PhotoShop. These images were overlaid against each other
in Graphic Converter and then scored and stored in FileMaker Pro
4.0 (see FIG. 9).
[0306] In order to determine the HBM allele frequency in ethnically
diverse populations, 672 random individuals from various ethnic
groups were typed by the allele specific oligonucleotide (ASO)
method. This population included 96 CEPH grandparents (primarily
Caucasian), 192 Caucasian, 192 African-American, 96 Hispanic, and
96 Asian individuals. No evidence was obtained for the presence of
the HBM polymorphism in any of these individuals. Overall, a total
of 911 individuals were typed either by direct sequencing or ASO
hybridization; all were homozygous GG at the site of the HBM
polymorphism (FIG. 14). This information illustrates that the HBM
allele is rare in various ethnic populations.
[0307] Thus this invention provides a rapid method of identifying
individuals with the HBM allele. This method could be used in the
area of diagnostics and screening of an individual for
susceptibility to osteoporosis or other bone disorder. The assay
could also be used to identify additional individuals with the HBM
allele or the additional polymorphisms described herein.
14. Cellular Localization of Zmax1 ARP5)
[0308] 14.1 Gene Expression in Rat Tibia by Non Isotopic In Situ
Hybridization
[0309] In situ hybridization was conducted by Pathology Associates
International (PAI), Frederick, Md. This study was undertaken to
determine the specific cell types that express the Zmax1 (LRP5)
gene in rat bone with particular emphasis on areas of bone growth
and remodeling. Zmax1 (LRP5) probes used in this study were
generated from both human (HuZmax1) and mouse (MsZmax1) cDNAs,
which share an 87% sequence identity. The homology of human and
mouse Zmax1 (LRP5) with rat Zmax1 (LRP5) is unknown.
[0310] For example, gene expression by non-isotopic in situ
hybridization was performed as follows, but other methods would be
known to the skilled artisan. Tibias were collected from two 6 to 8
week old female Sprague Dawley rats euthanized by carbon dioxide
asphyxiation. Distal ends were removed and proximal tibias were
snap frozen in OCT embedding medium with liquid nitrogen
immediately following death. Tissues were stored in a -80.degree.
C. freezer.
[0311] Probes for amplifying PCR products from cDNA were prepared
as follows. The primers to amplify PCR products from a cDNA clone
were chosen using published sequences of both human LRP5 (GenBank
Accession No. ABO17498) and mouse LRP5 (GenBank Accession No.
AF064984). In order to minimize cross reactivity with other genes
in the LDL receptor family, the PCR products were derived from an
intracellular portion of the protein coding region. PCR was
performed in a 501 reaction volume using cDNA clone as template.
PCR reactions contained 1.5 mM MgCl.sub.2, 1 unit AmpliTaq, 200
.mu.M dNTPs and 2 .mu.M each primer. PCR cycling conditions were
94.degree. C. for 1 min., followed by 35 cycles of 94.degree. C.
for 30 seconds, 55.degree. C. for 30 seconds, 72.degree. C. for 30
seconds; followed by a 5 minute extension at 72.degree. C. The
reactions were then run on a 1.5% agarose Tris-Acetate gel. DNA was
eluted from the agarose, ethanol precipitated and resuspended in 10
mM Tris, pH 8.0. Gel purified PCR products were prepared for both
mouse and human cDNAs and supplied to Pathology Associates
International for in situ hybridizations.
[0312] The sequence of the human and mouse PCR primers and products
were as follows:
Human Zmax1 (LRP5) Sense Primer (HBM25)
TABLE-US-00017 [0313] 5'-CCCGTGTGCTCCGCCGCCCAGTTC-3' (SEQ ID
NO:633)
Human Zmax1 (LRP5) Antisense Primer (HBM465)
TABLE-US-00018 [0314] 5'-GGCTCACGGAGCTCATCATGGACTT-3' (SEQ ID
NO:634)
Human Zmax1 PCR Product
TABLE-US-00019 [0315] (SEQ ID NO:635)
5'CCCGTGTGCTCCGCCGCCCAGTTCCCCTGCGCGCGGGGTCAGTGTGTG
GACCTGCGCCTGCGCTGCGACGGCGAGGCAGACTGTCAGGACCGCTCAGA
CGAGGTGGACTGTGACGCCATCTGCCTGCCCAACCAGTTCCGGTGTGCGA
GCGGCCAGTGTGTCCTCATCAAACAGCAGTGCGACTCCTTCCCCGACTGT
ATCGACGGCTCCGACGAGCTCATGTGTGAAATCACCAAGCCGCCCTCAGA
CGACAGCCCGGCCCACAGCAGTGCCATCGGGCCCGTCATTGGCATCATCC
TCTCTCTCTTCGTCATGGGTGGTGTCTATTTTGTGTGCCAGCGCGTGGTG
TGCCAGCGCTATGCGGGGGCCAACGGGCCCTTCCCGCACGAGTATGTCAG
CGGGACCCCGCACGTGCCCCTCAATTTCATAGCCCCGGGCGGTTCCCAGC
ATGGCCCCTTCACAGGCATCGCATGCGGAAAGTCCATGATGAGCTCCGTG AGCC-3'
Mouse Zmax1 (LRP5) Sense Primer (HBM655)
TABLE-US-00020 [0316] 5'-AGCGAGGCCACCATCCACAGG-3' (SEQ ID
NO:636)
Mouse Zmax1 (LRP5) Antisense Primer (HBM656)
TABLE-US-00021 [0317] 5'-TCGCTGGTCGGCATAATCAAT-3' (SEQ ID
NO:637)
Mouse (LRP5) 1 PCR Product
TABLE-US-00022 [0318] (SEQ ID NO:638)
5'-AGCAGAGCCACCATCCACAGGATCTCCCTGGAGACTAACAACAACGA
TGTGGCTATCCCACTCACGGGTGTCAAAGAGGCCTCTGCACTGGACTTTG
ATGTGTCCAACAATCACATCTACTGGACTGATGTTAGCCTCAAGACGATC
AGCCGAGCCTTCATGAATGGGAGCTCAGTGGAGCACGTGATTGAGTTTGG
CCTCGACTACCCTGAAGGAATGGCTGTGGACTGGATGGGCAAGAACCTCT
ATTGGGCGGACACAGGGACCAACAGGATTGAGGTGGCCCGGCTGGATGGG
CAGTTCCGGCAGGTGCTTGTGTGGAGAGACCTTGACAACCCCAGGTCTCT
GGCTCTGGATCCTACTAAAGGCTACATCTACTGGACTGAGTGGGGTGGCA
AGCCAAGGATTGTGCGGGCCTTCATGGATGGGACCAATTGTATGACACTG
GTAGACAAGGTGGGCCGGGCCAACGACCTCACCATTGATTATGCCGACCA GCGA-3'
[0319] Riboprobes were synthesized as follows. The PCR products
were reamplified with chimeric primers designed to incorporate
either a T3 promoter upstream, or a T7 promoter downstream of the
reamplification products. The resulting PCR products were used as
template to synthesize digoxigenin-labeled riboprobes by in vitro
transcription (IVT). Antisense and sense riboprobes were
synthesized using T7 and T3 RNA polymerases, respectively, in the
presence of digoxigenin-11-UTP (Boehringer-Mannheim) using a
MAXIscript IVT kit (Ambion) according to the manufacturer. The DNA
was then degraded with Dnase-1, and unincorporated digoxigenin was
removed by ultrafiltration. Riboprobe integrity was assessed by
electrophoresis through a denaturing polyacrylamide gel. Molecular
size was compared with the electrophoretic mobility of a 100-1000
base pair (bp) RNA ladder (Ambion). Probe yield and labeling was
evaluated by blot immunochemistry. Riboprobes were stored in 5
.mu.l aliquots at -80.degree. C.
[0320] The in situ hybridization was performed as follows. Frozen
rat bone was cut into 5 .mu.M sections on a Jung CM3000 cryostat
(Leica) and mounted on adhesive slides (Instrumedics). Sections
were kept in the cryostat at -20.degree. C. until all the slides
were prepared in order to prevent mRNA degradation prior to
post-fixation for 15 minutes in 4% paraformaldehyde. Following
post-fixation, sections were incubated with 1 ng/.mu.l of either
antisense or sense riboprobe in Pathology Associates International
(PAI) customized hybridization buffer for approximately 40 hours at
58.degree. C. Following hybridization, slides were subjected to a
series of post-hybridization stringency washes to reduce
nonspecific probe binding. Hybridization was visualized by
immunohistochemistry with an anti-digoxigenin antibody (FAB
fragment) conjugated to alkaline phosphatase. Nitroblue tetrazolium
chloride/bromochloroindolyl phosphate (Boehringer-Mannheim), a
precipitating alkaline phosphatase substrate, was used as the
chromogen to stain hybridizing cells purple to nearly black,
depending on the degree of staining. Tissue sections were
counter-stained with nuclear fast red. Assay controls included
omission of the probe, omission of probe and anti-digoxigenin
antibody.
[0321] Specific cell types were assessed for demonstration of
hybridization with antisense probes by visualizing a purple to
black cytoplasmic and/or peri-nuclear staining indicating a
positive hybridization signal for mRNA. Each cell type was compared
to the replicate sections, which were hybridized with the
respective sense probe. Results were considered positive if
staining was observed with the antisense probe and no staining or
weak background with the sense probe.
[0322] The cellular localization of the hybridization signal for
each of the study probes is summarized in Table 9. Hybridization
for Zmax1 (LRP5) was primarily detected in areas of bone involved
in remodeling, including the endosteum and trabecular bone within
the metaphysis. Hybridization in selected bone lining cells of the
periosteum and epiphysis were also observed. Positive signal was
also noted in chondrocytes within the growth plate, particularly in
the proliferating chondrocytes. See FIGS. 10, 11 and 12 for
representative photomicrographs of in situ hybridization
results.
TABLE-US-00023 TABLE 9 Summary of Zmax1 (LRP5) in situ
hybridization in rat tibia PROBE SITE ISH SIGNAL Hu Zmax1 Epiphysis
Osteoblasts + Osteoclasts - Growth Plate resting chondrocytes -
proliferating chondrocytes + hypertrophic chondrocytes - Metaphysis
osteoblasts + osteoclasts + Diaphysis - Endosteum osteoblasts +
osteoclasts + Periosteum - MsZmax1 Epiphysis Osteoblasts +
Osteoclasts - Growth Plate resting chondrocytes - proliferating
chondrocytes + hypertrophic chondrocytes + Metaphysis osteoblasts +
osteoclasts + Diaphysis - Endosteum osteoblasts + osteoclasts +
Periosteum + Legend: "+" = hybridization signal detected "-" = no
hybridization signal detected "ISH"--In situ hybridization
[0323] These studies confirm the positional expression of Zmax1
(LRP5) in cells involved in bone remodeling and bone formation.
Zmax1 (LRP5) expression in the zone of proliferation and in the
osteoblasts and osteoclasts of the proximal metaphysis, suggests
that the Zmax1 (LRP5) gene is involved in the process of bone
growth and mineralization. The activity and differentiation of
osteoblasts and osteoclasts are closely coordinated during
development as bone is formed and during growth as well as in adult
life as bone undergoes continuous remodeling. The formation of
internal bone structures and bone remodeling result from the
coupling of bone resorption by activated osteoclasts with
subsequent deposition of new material by osteoblasts. Zmax1 (LRP5)
is related to the LDL receptor gene, and thus may be a receptor
involved in mechanosensation and subsequent signaling in the
process of bone remodeling. Therefore, changes in the level of
expression of this gene could impact on the rate of remodeling and
degree of mineralization of bone.
15. Antisense
[0324] Antisense oligonucleotides are short synthetic nucleic acids
that contain complementary base sequences to a targeted RNA.
Hybridization of the RNA in living cells with the antisense
oligonucleotide interferes with RNA function and ultimately blocks
protein expression. Therefore, any gene for which the partial
sequence is known can be targeted by an antisense
oligonucleotide.
[0325] Antisense technology is becoming a widely used research tool
and will play an increasingly important role in the validation and
elucidation of therapeutic targets identified by genomic sequencing
efforts.
[0326] Antisense technology was developed to inhibit gene
expression by utilizing an oligonucleotide complementary to the
mRNA that encodes the target gene. There are several possible
mechanisms for the inhibitory effects of antisense
oligonucleotides. Among them, degradation of mRNA by RNase H is
considered to be the major mechanism of inhibition of protein
function. This technique was originally used to elucidate the
function of a target gene, but may also have therapeutic
applications, provided it is designed carefully and properly.
[0327] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An
antisense nucleic acid of the invention can be constructed using
chemical synthesis and enzymatic ligation reactions using
procedures known in the art. For example, an antisense nucleic acid
(e.g., an antisense oligonucleotide) can be chemically synthesized
using naturally occurring nucleotides or variously modified
nucleotides designed to increase the biological stability of the
molecules or to increase the physical stability of the duplex
formed between the antisense and sense nucleic acids, e.g.,
phosphorothioate derivatives and acridine substituted nucleotides
can be used. Examples of modified nucleotides which can be used to
generate the antisense nucleic acid include 5-fluorouracil,
5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,
xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethlaminomethyluracil, dihydrouracil,
.beta.-D-galactosylqueosine, inosine, N6-isopentenyladenine,
I-methylguanine, I-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-n-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), t-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine.
[0328] In addition, the use of morpholino oligonucleotides could be
employed. Morpholinos are oligomers with modification of the ribose
moiety to a morpholino group. This technology is covered by U.S.
Pat. No. 5,185,444 and is described in Summerton et al., 1997
Antisense Nucleic Acid Drug Dev. 7(3): 187-95. The antisense
nucleic acid molecules of the invention are typically administered
to a subject or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding an HBM or Zmax1
(LRP5) protein or a protein which interacts with Zmax1 (LRP5)
and/or HBM to thereby inhibit expression of the protein, e.g., by
inhibiting transcription and/or translation. The hybridization can
be by conventional nucleotide complementarity to form a stable
duplex, or, for example, in the case of an antisense nucleic acid
molecule which binds to DNA duplexes, through specific interactions
in the major groove of the double helix. An example of a route of
administration of an antisense nucleic acid molecule of the
invention includes direct injection at a tissue site.
Alternatively, an antisense nucleic acid molecule can be modified
to target selected cells and then administered systemically. For
example, for systemic administration, an antisense molecule can be
modified such that it specifically binds to a receptor or an
antigen expressed on a selected cell surface, e.g., by linking the
antisense nucleic acid molecule to a peptide or an antibody which
binds to a cell surface receptor or antigen. The antisense nucleic
acid molecule can also be delivered to cells using the vectors
described herein.
[0329] In yet another embodiment, the antisense nucleic acid
molecule of the invention is an .alpha.-anomeric nucleic acid
molecule. An .mu.-anomeric nucleic acid molecule forms specific
double-stranded hybrids with complementary RNA in which, contrary
to the usual .gamma.-units, the strands run parallel to each other
(Gaultier et al., 1987 Nucl. Acids Res. 15:6625-41). The antisense
nucleic acid molecule can also comprise a 2'-o-methylribonucleotide
(Inoue et al., 1987 Nucl. Acids Res. 15: 6131-6148) or a chimeric
RNA-DNA analogue (Inoue et al., 1987 FEBS Lett. 215: 327-330). In
still another embodiment, an antisense nucleic acid of the
invention is a ribozyme. Ribozymes are catalytic RNA molecules with
ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an in RNA, to which they have
a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes
(described in Haselhoff et al., 1988 Nature 334: 585-591) can be
used to catalytically cleave Zmax1 (LRP5) or HBM mRNA transcripts
to thereby inhibit translation of Zmax1 (LRP5) or HBM mRNA. A
ribozyme having specificity for a Zmax1- or HBM-encoding nucleic
acid can be designed based upon the nucleotide sequence of a Zmax1
(LRP5) or HBM cDNA disclosed herein (i.e., SEQ ID NOS:1 or 3). For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in an HBM or
Zmax1-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071
and Cech et al. U.S. Pat. No. 5,116,742 both incorporated herein by
reference. Alternatively, Zmax1 (LRP5) or HBM mRNA can be used to
select a catalytic RNA having a specific ribonuclease activity from
a pool of RNA molecules. See, e.g., Bartel et al., 1993 Science
261: 1411-1418. Alternatively, Zmax1 (LRP5) or HBM gene expression
can be inhibited by targeting nucleotide sequences complementary to
the regulatory region of the Zmax1 (LRP5) or HBM gene (e.g., the
Zmax1 or HBM gene promoter and/or enhancers) to form triple helical
structures that prevent transcription of the Zmax1 (LRP5) or HBM
genes in target cells. See generally, Helene, 1991 Anticancer Drug
Des. 6(6):569-84; Helene et al., 1992 Ann. N.Y. Acad. Sci.
660:27-36; and Maher, 1992 Bioassays 14(12):807-15.
[0330] Zmax1 (LRP5), LRP6, HBM-like and HBM gene expression can
also be inhibited using RNA interference (RNAi) caused by small
inhibitory RNAs (siRNAs). This is a technique for
post-transcriptional gene silencing (PTGS), in which target gene
activity is specifically abolished with cognate double-stranded RNA
(dsRNA). siRNAs resemble in many aspects PTGS in plants and has
been detected in many invertebrates including trypanosome, hydra,
planaria, nematode and fruit fly (Drosophila melanogaster). It may
be involved in the modulation of transposable element mobilization
and antiviral state formation. RNAi in mammalian systems is
disclosed in PCT application WO 00/63364 which is incorporated by
reference herein in its entirety. See also, Elbashir et al., 2001
Nature 411: 494-98. Basically, dsRNA, homologous to the target
(Zmax1 or HBM) is introduced into the cell and a sequence specific
reduction in gene activity is observed.
[0331] Another embodiment contemplates the use of small hairpin
RNAs (shRNAs). These compounds are described further in Yu et al.,
2002 Proc. Natl. Acad. Sci. USA 99: 6047-52; and Paddison et al.,
2002 Genes & Devel. 16: 948-58.
[0332] As an example, preparing antisense oligonucleotides can be
performed as follows. Studies have been undertaken using antisense
technology in the osteoblast-like murine cell line, MC3T3. These
cells can be triggered to develop along the bone differentiation
sequence. An initial proliferation period is characterized by
minimal expression of differentiation markers and initial synthesis
of collagenous extracellular matrix. Collagen matrix synthesis is
required for subsequent induction of differentiation markers. Once
the matrix synthesis begins, osteoblast marker genes are activated
in a clear temporal sequence: alkaline phosphatase is induced at
early times while bone sialoprotein and osteocalcin appear later in
the differentiation process. This temporal sequence of gene
expression is useful in monitoring the maturation and
mineralization process. Matrix mineralization, which does not begin
until several days after maturation has started, involves
deposition of mineral on and within collagen fibrils deep within
the matrix near the cell layer-culture plate interface. The
collagen fibril-associated mineral formed by cultured osteoblasts
resembles that found in woven bone in vivo and therefore is used
frequently as a study reagent.
[0333] MC3T3 cells were transfected with antisense oligonucleotides
for the first week of the differentiation, according to the
manufacturer's specifications (U.S. Pat. No. 5,849,902).
[0334] The oligonucleotides designed for Zmax1 (LRP5) are given
below:
TABLE-US-00024 10875: 5'-AGUACAGCUUCUUGCCAACCCAGUC-3' (SEQ ID
NO:639) 10876: 5'-UCCUCCAGGUCGAUGGUCAGCCCAU-3' (SEQ ID NO:640)
10877: 5'-GUCUGAGUCCGAGUUCAAAUCCAGG-3' (SEQ ID NO:641)
[0335] FIG. 13 shows the results of antisense inhibition of (LRP5)
1 in MC3T3 cells. The three oligonucleotides shown above were
transfected into MC3T3 and RNA was isolated according to standard
procedures. Northern analysis clearly shows markedly lower steady
state levels of the Zmax1 (LRP5) transcript while the control gene
GAPDH remained unchanged. Thus, antisense technology using the
primers described above allows for the study of the role of Zmax1
(LRP5) expression on bone biology.
16. Yeast Two Hybrid
[0336] In order to identify the signaling pathway that Zmax1 (LRP5)
participates in to modulate bone density, the yeast two hybrid
protein interaction technology was utilized. This technique
facilitates the identification of proteins that interact with one
another by coupling tester proteins to components of a yeast
transcription system (Fields et al., 1989 Nature 340: 245-246; U.S.
Pat. No. 5,283,173 by Fields et al.; Johnston, 1987 Microbiol. Rev.
51: 458-476; Keegan et al., 1986 Science 231: 699-704; Durfee et
al., 1993, Genes Dev. 7: 555-569; Chien et al., 1991 Proc. Natl.
Acad. Sci. USA 88: 9578-9582; Fields et al., 1994 Trends in
Genetics 10: 286-292; and Gyuris et al., 1993 Cell 75: 791-803).
First a "bait" protein, the protein for which one seeks interacting
proteins, is fused to the DNA binding domain of a yeast
transcription factor. Second, a cDNA library is constructed that
contains cDNAs fused to the transcriptional activation domain of
the same yeast transcription factor; this is termed the prey
library. The bait construct and prey library are transformed into
yeast cells and then mated to produce diploid cells. If the bait
interacts with a specific prey from the cDNA library, the
activation domain is brought into the vicinity of the promoter via
this interaction. Transcription is then driven through selectable
marker genes and growth on selective media indicates the presence
of interacting proteins.
[0337] The amino acid sequence used in the yeast two hybrid
experiments discussed herein consisted of the entire cytoplasmic
domain and a portion of the transmembrane domain and is shown below
(amino to carboxy orientation):
TABLE-US-00025 (SEQ ID NO: 765) RVVCQRYAGA NGPFPHEYVS GTPHVPLNFI
APGGSQHGPF TGIACGKSMM SSVSLMGGRG GVPLYDRNHV TGASSSSSSS TKATLYPPIL
NPPPSPATDP SLYNMDMFYS SNIPATVRPY RPYIIRGMAP PTTPCSTDVC DSDYSASRWK
ASKYYLDLNS DSDPYPPPPT PHSQYLSAED SCPPSPATER SYFHLFPPPP SPCTDSS
[0338] The last 6 amino acids of the putative transmembrane domain
are indicated in bold. Putative SH3 domains are underlined.
Additional amino acid sequences of 50 amino acids or greater in
either the proteins encoded by the Zmax1 (LRP5) or HBM alleles can
also be used as bait. The upper size of the polypeptide used as
bait is limited only by the presence of a complete transmembrane
domain (see FIG. 4), which will render the bait to be nonfunctional
in a yeast two hybrid system. These additional bait proteins can be
used to identify additional proteins which interact with the
proteins encoded by HBM or Zmax1 (LRP5) in the focal adhesion
signaling pathway or in other pathways in which these HBM or Zmax1
(LRP5) proteins may act. Once identified, methods of identifying
agents which regulate the proteins in the focal adhesion signaling
pathway or other pathways in which HBM acts can be performed as
described herein for the HBM and Zmax1 (LRP5) proteins.
[0339] In order to identify cytoplasmic Zmax1 (LRP5) signaling
pathways, the cytoplasmic domain of Zmax1 (LRP5) was subcloned into
two bait vectors. The first vector was pDBleu, which was used to
screen a brain, and Hela prey cDNA library cloned into the vector
pPC86 (Clontech). The second bait vector used was pDBtrp, which was
used to screen a cDNA prey library derived from the TE85
osteosarcoma cell line in vector pOP46. Another suitable vector
which is widely available, is p86 (Gibco, Iest.TM. System).
Standard techniques known to those skilled in the art were used as
described in Fields and Song, 1989 Nature 340: 245-246; U.S. Pat.
No. 5,283,173 by Fields and Song; Johnston, 1987 Microbiol. Rev.
51: 458-476; Keegan et al., 1986 Science 231: 699-704; Durfee et
al., 1993 Genes Dev. 7: 555-569; Chien et al., 1991 Proc. Natl.
Acad. Sci. USA 88: 9578-9582; Fields et al., 1994 Trends in
Genetics 10: 286-292; and Gyuris et al., 1993 Cell 75: 791-803. The
bait construct and prey cDNA libraries were transformed into yeast
using standard procedures.
[0340] To perform the protein interaction screen, an overnight
culture of the bait yeast strain was grown in 20 ml SD selective
medium with 2% glucose (pDBLeu, SD-Leu medium, pDBtrp, SD-trp
medium). The cultures were shaken vigorously at 30.degree. C.
overnight. The cultures were diluted 1:10 with complete medium
(YEPD with 2% glucose) and the cultures then incubated with shaking
for 2 hrs at 30.degree. C.
[0341] The frozen prey library was thawed, and the yeast cells
reactivated by growing them in 150 ml YEPD medium with 2% glucose
for 2 hrs at 30.degree. C. A filter unit was sterilized with 70%
ethanol and washed with sterile water to remove the ethanol. The
cell densities of both bait and prey cultures were measured by
determining the OD at 600 nm. An appropriate volume of yeast cells
that corresponded to a cell number of 1 ml of OD 600=4 of each
yeast strain, bait and prey (library) was placed in a 50 ml Falcon
tube. The mixture was then filtered through the sterilized filter
unit. The filter was then transferred onto a prewarmed YEPD agar
plate with the cell side up, removing all air bubbles underneath
the filter. Plates were then incubated at 30.degree. C. for 6 hrs.
One filter was transferred into a 50 ml Falcon tube, and 10 ml of
SD with 2% Glucose was added; cells were resuspended by vortexing
for 10 sec.
[0342] The number of primary diploid cells (growth on SD-Leu, -Trp
plates) versus the numbers of colony forming units growing on
SD-Trp and SD-Leu plates only was then titered. Different dilutions
were plated and incubated at 30.degree. C. for two days. The number
of colony forming units was then counted. The number of diploid
colonies (colonies on SD-Leu-Trp plates) permits the calculation of
whether or not the whole library of prey constructs was mated to
the yeast expressing the bait. This information is important to
judge the quality of the screen.
[0343] 16.1 Indirect Selection
[0344] Resuspended cells from 5 filter-matings were then pooled and
the cells sedimented by centrifugation in a 50 ml Falcon tube.
Cells were then resuspended in 16 ml SD medium with 2% Glc. Two ml
of this cell suspension was plated onto 8 square plates each
(SD-Leu, -Trp) with sterile glass beads and selected for diploid
cells by incubating at 30.degree. C. for 18-20 hrs.
[0345] Cells were then scraped off the square plates, the cells
centrifuged and combined into one 50 ml Falcon tube. The cell
pellet was then resuspended in 25 ml of SD medium with 2% glucose.
The cell number was then determined by counting of an appropriate
dilution (usually 1:100 to 1:1000) with a Neugebauer chamber.
Approximately 5.times.10.sup.7 diploid cells were plated onto the
selective medium. The observations about the growth of the bait
strain together with irrelevant prey vectors helps to determine
which selective plates will have to be chosen for the library
screen. Generally, all screens were plated on one square plate each
with SD-Leu, -Trp, -His; SD-Leu, -Trp, His, 5 mM 3AT, and SD-Leu,
-Trp, -His, -Ade is recommended.
[0346] The yeast cells were then spread homogeneously with sterile
glass beads and incubated at 30.degree. C. for 4 days. The number
of colony forming yeast cells was titered by plating different
dilutions of the scraped cell suspension onto SD-Leu, -Trp plates.
Usually, plating of 100 .mu.l of a 10.sup.-3 and 10.sup.-4 dilution
gave 100-1000 colonies per plate.
[0347] 16.2 Direct selection
[0348] Five filters with the mated yeast cells were each
transferred into separate 50 ml Falcon tubes and the cells
resuspended with 10 ml SD medium with 2% Glc, each, followed by
vortexing for 10 sec. The resuspended cells were combined and
centrifuged in a Beckman centrifuge at 3000 rpm. The supernatant
was discarded and the cells resuspended in 6 ml of SD medium with
2% Glc. Two ml of the suspension was spread onto each selective
square plate and incubated at 30.degree. C. for 4-5 days.
[0349] 16.3 Isolation of Single Colonies
[0350] Yeast cells from an isolated colony were picked with a
sterile tooth pick and transferred into individual wells of a 96
well plate. The cells were resuspended in 50 .mu.l of SD-Leu, -Trp,
-His medium and incubated at 30.degree. C. for one day. The yeast
cells were then stamped onto a SD-Leu, -Trp, -His plate in 96 well
format and incubated at 30.degree. C. for 2 days.
[0351] Yeast cells were also stamped onto a Nylon filter covering a
YEPD plate and incubated at 30.degree. C. for one day. The cells on
the Nylon filter were used for the analysis of the .beta.-Gal
reporter activity.
[0352] Yeast colonies were scraped from the SD-Leu, -Trp, -His
plate with a sterile tooth pick, and reconfigured, if necessary,
according to the 13-Gal activity and then resuspended in 20%
glycerol. This served as a master plate for storage at -80.degree.
C.
[0353] For DNA preparation, yeast cells from the glycerol stock
were stamped onto a SD-Trp plate and incubated at 30.degree. C. for
2 days. After two days of incubation, the yeast colonies were ready
for colony PCR and sequencing. Standard colony PCR conditions were
used to amplify inserts from preys recovered from the interaction
screen. Sequencing was done using standard sequencing reactions and
ABI377 (Perkin Elmer) fluorescent sequencing machines.
[0354] 16.4 Verification of bait/prey interaction
[0355] Glycerol stocks of the prey of interest were thawed and
inoculated in a 10 ml overnight culture of SD with glucose-Trp.
After overnight growth, plasmid DNA preparation was performed using
the BIO 101 RPM Yeast Plasmid Isolation Kit with 10 ml of culture.
The culture was centrifuged and transferred to a 1.5 ml
microcentrifuge tube.
[0356] Yeast Lysis Matrix was then added to the pellet followed by
250 .mu.l of Alkaline Lysis Solution. Samples were then vortexed
for 5 minutes. 250 .mu.l Neutralizing Solution was added and the
sample mixed briefly. Samples were centrifuged for 2 minutes at
room temperature in a microcentrifuge. The supernatant was
transferred to a Spin Filter avoiding debris and Lysis Matrix. 250
.mu.l of Glassmilk Spin Buffer was added, and the tubes inverted to
mix. Samples were centrifuged for 1 min and the liquid in the Catch
Tube was discarded. 500 .mu.l of Wash Solution was added, the
samples were centrifuged for 1 min, and the wash solution was
discarded. The wash step was repeated once followed by a 1 min dry
centrifugation to drive the remaining liquid out of the Spin
Filter. The filter was transferred to a new Catch Tube and 100
.mu.l of sterile H.sub.2O was added; samples were then vortexed
briefly to resuspend and centrifuged for 30 seconds to collect the
DNA in the bottom of the Catch Tube.
[0357] Five .mu.l of DNA was then transformed into DH10B Electromax
cells using standard procedures and glycerol stocks prepared.
Miniprep DNA was prepared using the Qiagen QIAprep Spin Miniprep
Kit. DNA was finally eluted with 30 .mu.l of Qiagen EB buffer. One
.mu.l of the plasmid DNA samples was then used to transform yeast
cells using standard procedures. After 2 days of growth on SD-trp
media, colonies were picked and patched onto fresh media.
Similarly, bait colonies were patched onto SD-Leu media. Both were
grown overnight at 30.degree. C.
[0358] For mating, cells from bait and prey patches were spread
together on YAPD media and incubated at 30.degree. C. for 12 hr.
This plate was then replica plated onto an SD Agar-Leu-Trp plate
and grown for 2 days at 30.degree. C. To test the strength of
interaction these plates were replica plated onto SD
Agar-Leu-Trp-His, SD Agar-Leu-Trp-His with 5 mM 3AT and 10 mM 3AT,
SD Agar-Leu-Trp-His-Ade, and SD Agar-Leu-Trp-Ura media and grown
for 2 days at 30.degree. C.
[0359] 16.5 Galacton Star .beta.-Galactosidase Activity Assay
[0360] After streaking and replica plating positive interactors on
selection plates, colonies were placed in a 96 well dish with 200
.mu.l of SD-medium, leaving wells 1 and 96 blank. Ten microliters
from the first 96 well dish was plated into another flat bottom 96
well dish containing 100 .mu.l of SD-medium. Controls consisted of
a negative control and a very weak positive control. The cell
density was measured at OD.sub.600 (a value of 1 corresponds to
1.times.10.sup.7 cells utilizing a 96 well spectrophotometer). The
OD was usually between 0.03 and 0.10. Using microplates
specifically for the luminometer, 50 .mu.l of reaction mixture were
pipetted into each well. Fifty microliters of culture were then
added and mixed by pipetting up and down twice. The reaction was
incubated for 30 minutes at room temperature followed by
measurement of Relative Light Units using a luminometer.
[0361] Table 9 lists the genes identified in the yeast two hybrid
screens from the 3 prey libraries tested. Two genes, zyxin and
axin, were found to interact with the cytoplasmic domain of (LRP5)
1 in all three screens. Three genes, alpha-actinin, TCB and S1-5
interacted in two of the three screens.
[0362] A variety of proteins found at sites of cell-cell and
cell-matrix contact (focal contacts/adhesion plaques) were shown to
interact with the cytoplasmic domain of Zmax1 (LRP5). These include
.alpha.-actinin, Trio, Pinch-like protein, and Zyxin. PINCH is a
LIM domain-containing protein that is known to interact with
integrin-linked kinase, an early signaler in integrin and growth
factor signaling pathways. The finding of a closely related gene in
the yeast two hybrid screen raises the possibility of a novel
pathway linked to integrin signaling from extracellular matrix
signals. Trio, also known to localize to focal adhesions, is
thought to play a key role in coordinating cell-matrix interactions
and cytoskeletal rearrangements involved in cell movement. Zyxin,
another LIM domain-containing protein, is also localized to
adhesion plaques and is thought to be involved in reorganization of
the cytoskeleton when triggers are transmitted via integrin
signaling pathways. Zyxin also interacts with alpha actinin, which
we identified as interacting with Zmax1 (LRP5). Other LIM domain
containing proteins identified include the human homologue of mouse
ajuba, LIMD1, and a novel LIMD1-like protein.
[0363] Axin was also identified from the two hybrid experiments.
This protein is involved in inhibition of the Wnt signaling pathway
and interacts with the tumor suppressor APC. There is a link here
with the focal adhesion signaling described above: one common step
in the two pathways involves inhibition of glycogen synthase kinase
3, which in turn results in the activation of .beta.-catenin/Lef-1
and AP-1 transcription factors. Axin/APC are involved in this as
well as integrin linked kinase. The Wnt pathway has a role in
determining cell fates during embryogenesis. If inappropriately
activated, the Wnt pathway may also lead to cancer. The Wnt pathway
also seems to have a role in cytoskeletal rearrangements. In a
Xenopus embryo assay, the combination of HBM and Wnt5a proteins
stimulated the Wnt pathway to a much greater extent than the
combination of Zmax1 (LRP5) and Wnt5a, which was modestly above the
control and Wnt5a alone scores. The HBM and Zmax1 (LRP5)
extracellular domains (ECD) caused a modest stimulation of Wnt
signaling in the absence of Wnt5a which was slightly increased by
the presence of Wnt5a in the presence of HBM ECD. A model depicting
Zmax1 (LRP5) involvement in focal adhesion signaling is depicted in
FIG. 15.
[0364] This data coupled with other studies suggest that integrin
signaling pathways have a role in cellular responses to mechanical
stress and adhesion. This provides an attractive model for the
mechanism of action of Zmax1 (LRP5) in bone biology. It is possible
that Zmax1 (LRP5) is involved in sensing either mechanical stress
directly or binding a molecule in the extracellular matrix that is
related to mechanical sensation. Signaling through subsequent
pathways may be involved in bone remodeling due to effects on cell
morphology, cell adhesion, migration, proliferation,
differentiation, and apoptosis in bone cells.
TABLE-US-00026 TABLE 10 Yeast Two Hybrid Results NT AA Gene Genbank
SEQ ID SEQ ID Symbol Gene Accession # NO: NO: ACTN1 alpha-actinin
NM_001102 642 AES amino-terminal enhancer of NM_001130.3 643 AIP4
atrophin-1 interacting protein AF038564.1 644 Novel Ajuba 645 AXIN
Wnt signaling AF009674.1 646 CDC23 cell division cycle 23, yeast,
NM_004661.1 647 homolog HSM800944 Similar to TRIO AL117435.1 748
HSM800936 AL117427.1 649 Novel Similar to LIM domains 650
containing protein 1 DEEPEST mitotic spindle coiled-coil
NM_006461.1 651 related protein ECM1 extracellular matrix protein 1
U65932.1 652 EF1A elongation factor 1-alpha X16869.1 653 FN
fibronectin X02761.1 654 HOXB13 homeodomain protein U81599.1 655
Novel Glu-Lys Rich protein 656 LIMD1 LIM domains containing 1
NM_014240.1 657 Novel PINCH-like 658 RANBPM centrosomal protein
NM_005493.1 659 S1-5 extracellular protein U03877.1 660 TCB gene
encoding cytosolic M26252.1 661 thyroid hormone-binding TID
tumorous imaginal discs NM_005147.1 662 ZYX Zyxin NM_003461.1 663
TRIO GTPase U42390.1 664 HUMPITPB phosphatidylinositol transfer
D30037.1 665 protein ACTN1 alpha-actinin NP_001093.1 666 AES
amino-terminal enhancer of NP_001121.2 667 AIP4 atrophin-1
interacting protein AAC04845.1 668 Novel Ajuba 669 AXIN Wnt
signaling AAC51624.1 670 CDC23 cell division cycle 23, yeast
NP_004652.1 671 homolog Novel Similar to TRIO CAB55923.1 672 Novel
Similar to LIM domains 673 containing protein 1 DEEPEST mitotic
spindle coiled-coil NP_006452.1 674 related protein ECM1
extracellular matrix protein 1 AAB05933.1 675 EF1A elongation
factor 1-alpha CAA34756.1 676 FN fibronectin CAA26536.1 677 Novel
Glu-Lys rich protein 678 HOXB13 homeodomain protein B13 AAB39863.1
679 LIMD1 LIM domains containing 1 NP_055055.1 680 Novel PINCH-like
681 RANBPM centrosomal protein NP_005484.1 682 S1-5 extracellular
protein AAA65590.1 683 TCB cytosolic thyroid hormone- AAA36672.1
684 binding protein TID tumorous imaginal discs NP_005138.1 685 ZYX
Zyxin NP_003452.1 686 TRIO GTPase AAC34245.1 687 PTDINSTP
phosphatidylinositol transfer P48739 688 protein beta isoform
[0365] In light of the model depicted in FIG. 15 and the results
shown in Table 10, another aspect contemplated by the invention
would be to regulate bone density and bone mass disorders by the
regulating focal adhesion signaling. The regulation can occur by
regulating the DNA, mRNA transcript or protein encoded by any of
the members involved in the focal adhesion signaling pathway as
identified by the yeast two hybrid system.
[0366] Also contemplated are the novel nucleic acids and proteins
identified by the HBM yeast two hybrid system. These include but
are not limited to SEQ ID NO: 645 (Ajuba), SEQ ID NO: 651 (a gene
similar to a gene encoding LIM domains containing protein 1), SEQ
ID NO: 656 (Glu-Lys Rich protein), SEQ ID NO: 658 (PINCH-like
gene), SEQ ID NO: 669 (Ajuba protein), SEQ ID NO: 672 (protein
similar to TRIO), SEQ ID NO: 673, SEQ ID NO: 678 (Glu-Lys rich
protein) and SEQ ID NO: 681 (PINCH-like protein).
17. Potential Function
[0367] The protein encoded by Zmax1 (LRP5) and LRP6 are related to
the Low Density Lipoprotein receptor (LDL receptor). See, Goldstein
et al., 1985 Ann. Rev. Cell Biology, 1: 1-39; Brown et al., 1986
Science, 232:34-47. The LDL receptor is responsible for uptake of
low density lipoprotein, a lipid-protein aggregate that includes
cholesterol. Individuals with a defect in the LDL receptor are
deficient in cholesterol removal and tend to develop
artherosclerosis. In addition, cells with a defective LDL receptor
show increased production of cholesterol, in part because of
altered feedback regulation of cholesterol synthetic enzymes and in
part because of increased transcription of the genes for these
enzymes. In some cell types, cholesterol is a precursor for the
formation of steroid hormones.
[0368] Thus, the LDL receptor may, directly or indirectly, function
as a signal transduction protein and may regulate gene expression.
Because Zmax1 (LRP5) and LRP6 are related to the LDL receptor, this
protein may also be involved in signaling between cells in a way
that affects bone remodeling.
[0369] The glycine 171 amino acid is likely to be important for the
function of Zmax1 (LRP5) because this amino acid is also found in
the mouse homologue of Zmax1 (LRP5).
[0370] The closely related LRP6 (Genbank Accession No. JE0272)
protein also contains glycine at the corresponding position (Brown
et al., 1988 Biochem. Biophys. Res. Comm. 248: 879-88). Amino acids
that are important in a protein's structure or function tend to be
conserved between species, because natural selection prevents
mutations with altered amino acids at important positions from
arising.
[0371] In addition, the extracellular domain of Zmax1 (LRP5)
contains four repeats consisting of five YWTD motifs followed by an
EFG motif. This 5YWTD+EGF repeat is likely to form a distinct
folded protein domain, as this repeat is also found in the LDL
receptor and other LDL receptor-related proteins. The first three
5YWTD+EGF repeats are very similar in their structure, while the
fourth is highly divergent. Glycine 171 occurs in the central YWTD
motif of the first 5YWTD+EGF repeat in Zmax1 (LRP5). The other two
similar 5YWTD+EGF repeats of Zmax1 (LRP5) also contain glycine at
the corresponding position, as does the 5YWTD+EGF repeat in the LDL
receptor protein. However, only 17.6% of the amino acids are
identical among the first three 5YWTD+EGF repeats in Zmax 1 (LRP5)
and the single repeat in the LDL receptor. These observations
indicate that glycine 171 is essential to the function of this
repeat, and mutation of glycine 171 causes a functional alteration
of Zmax1 (LRP5). The cDNA and peptide sequences are shown in FIGS.
6A-6J. The critical base at nucleotide position 582 is indicated in
bold and is underlined.
[0372] Northern blot analysis (FIGS. 7A-B) reveals that Zmax1
(LRP5) is expressed in human bone tissue as well as numerous other
tissues. A multiple-tissue Northern blot (Clontech, Palo Alto,
Calif.) was probed with exons from Zmax1 (LRP5). As shown in FIG.
7A, the 5.5 kb Zmax1 (LRP5) transcript was highly expressed in
heart, kidney, lung, liver and pancreas and is expressed at lower
levels in skeletal muscle and brain. A second northern blot, shown
in FIG. 7B, confirmed the transcript size at 5.5 kb, and indicated
that Zmax1 (LRP5) is expressed in bone, bone marrow, calvaria and
human osteoblastic cell lines.
[0373] Taken together, these results coupled with the yeast two
hybrid results indicate that the HBM polymorphism in the Zmax1
(LRP5) gene is responsible for the HBM phenotype, and that the
Zmax1 gene is important in bone development. In addition, because
mutation of Zmax1 can alter bone mineralization and development, it
is likely that molecules that bind to Zmax1 may usefully alter bone
development. Such molecules may include, for example, small
molecules, proteins, RNA aptamers, peptide aptamers, and the
like.
18. Preparation of Nucleic Acids, Vectors, Transformations and Host
Cells
[0374] Large amounts of the nucleic acids of the present invention
may be produced by replication in a suitable host cell. Natural or
synthetic nucleic acid fragments coding for a desired fragment will
be incorporated into recombinant nucleic acid constructs, usually
DNA constructs, capable of introduction into and replication in a
prokaryotic or eukaryotic cell. Usually the nucleic acid constructs
will be suitable for replication in a unicellular host, such as
yeast or bacteria, but may also be intended for introduction to
(with and without integration within the genome) cultured mammalian
or plant or other eukaryotic cell lines. The purification of
nucleic acids produced by the methods of the present invention is
described, for example, in Sambrook et al., Molecular Cloning, A
Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1989) or Ausubel et al., Current Protocols in
Molecular Biology, J. Wiley and Sons, NY (1992).
[0375] The nucleic acids of the present invention may also be
produced by chemical synthesis, e.g., by the phosphoramidite method
described by Beaucage et al., 1981 Tetra. Lett. 22: 1859-62 or the
triester method according to Matteucci, et al., 1981 J. Am. Chem.
Soc. 103: 3185, and may be performed on commercial, automated
oligonucleotide synthesizers. A double-stranded fragment may be
obtained from the single-stranded product of chemical synthesis
either by synthesizing the complementary strand and annealing the
strands together under appropriate conditions or by adding the
complementary strand using DNA polymerase with an appropriate
primer sequence.
[0376] Nucleic acid constructs prepared for introduction into a
prokaryotic or eukaryotic host may comprise a replication system
recognized by the host, including the intended nucleic acid
fragment encoding the desired protein, and will preferably also
include transcription and translational initiation regulatory
sequences operably linked to the protein encoding segment.
Expression vectors may include, for example, an origin of
replication or autonomously replicating sequence (ARS) and
expression control sequences, a promoter, an enhancer and necessary
processing information sites, such as ribosome-binding sites, RNA
splice sites, polyadenylation sites, transcriptional terminator
sequences, and mRNA stabilizing sequences. Secretion signals may
also be included where appropriate, whether from a native IBM or
Zmax1 (LRP5) protein or from other receptors or from secreted
proteins of the same or related species, which allow the protein to
cross and/or lodge in cell membranes, and thus attain its
functional topology, or be secreted from the cell. Such vectors may
be prepared by means of standard recombinant techniques well known
in the art and discussed, for example, in Sambrook et al., (1989)
or Ausubel et al., (1992).
[0377] An appropriate promoter and other necessary vector sequences
will be selected so as to be functional in the host, and may
include, when appropriate, those naturally associated with Zmax1
(LRP5) or HBM genes. Examples of workable combinations of cell
lines and expression vectors are described in Sambrook et al.,
(1989) or Ausubel et al., (1992). Many useful vectors are known in
the art and may be obtained from such vendors as Stratagene, New
England BioLabs, Promega Biotech, and others. Promoters such as the
trp, lac and phage promoters, tRNA promoters and glycolytic enzyme
promoters may be used in prokaryotic hosts. Useful yeast promoters
include promoter regions for metallothionein, 3-phosphoglycerate
kinase or other glycolytic enzymes such as enolase or
glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for
maltose and galactose utilization, and others. Vectors and
promoters suitable for use in yeast expression are further
described in EP 73,675A. Appropriate non-native mammalian promoters
might include the early and late promoters from SV40 (Fiers et al.,
1978 Nature 273: 113) or promoters derived from murine Moloney
leukemia virus, mouse tumor virus, avian sarcoma viruses,
adenovirus II, bovine papilloma virus or polyoma. In addition, the
construct may be joined to an amplifiable gene (e.g., DHFR) so that
multiple copies of the gene may be made. For appropriate enhancer
and other expression control sequences, see also Enhancers and
Eukaryotic Gene Expression, Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. (1983).
[0378] While such expression vectors may replicate autonomously,
they may also replicate by being inserted into the genome of the
host cell, by methods well known in the art.
[0379] Expression and cloning vectors will likely contain a
selectable marker, a gene encoding a protein necessary for survival
or growth of a host cell transformed with the vector. The presence
of this gene ensures growth of only those host cells which express
the inserts. Typical selection genes encode proteins that a) confer
resistance to antibiotics or other toxic substances, e.g.
ampicillin, neomycin, methotrexate, etc.; b) complement auxotrophic
deficiencies, or c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for
Bacilli. The choice of the proper selectable marker will depend on
the host cell, and appropriate markers for different hosts are well
known in the art.
[0380] The vectors containing the nucleic acids of interest can be
transcribed in vitro, and the resulting RNA introduced into the
host cell by well-known methods, e.g., by injection (see, Kubo et
al., FEBS Letts. 241: 119 (1988)), or the vectors can be introduced
directly into host cells by methods well known in the art, which
vary depending on the type of cellular host, including
electroporation; transfection employing calcium chloride, rubidium
chloride, calcium phosphate, DEAE-dextran, or other substances;
microprojectile bombardment; lipofection; infection (where the
vector is an infectious agent, such as a retroviral genome); and
other methods. See generally, Sambrook et al., 1989 and Ausubel et
al., 1992. The introduction of the nucleic acids into the host cell
by any method known in the art, including those described above,
will be referred to herein as "transformation." The cells into
which have been introduced nucleic acids described above are meant
to also include the progeny of such cells.
[0381] Large quantities of the nucleic acids and proteins of the
present invention may be prepared by expressing the Zmax1 (LRP5),
LRP6, HBM or HBM-like nucleic acids or portions thereof in vectors
or other expression vehicles in compatible prokaryotic or
eukaryotic host cells. The most commonly used prokaryotic hosts are
strains of Escherichia coli, although other prokaryotes, such as
Bacillus subtilis or Pseudomonas may also be used.
[0382] Mammalian or other eukaryotic host cells, such as those of
yeast, filamentous fungi, plant, insect, or amphibian or avian
species, may also be useful for production of the proteins of the
present invention. Propagation of mammalian cells in culture is per
se well known. See, Jakoby and Pastan (eds.), Cell Culture, Methods
in Enzymology, volume 58, Academic Press, Inc., Harcourt Brace
Jovanovich, N.Y., (1979). Examples of commonly used mammalian host
cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)
cells, and WI38, BHK, and COS cell lines, although it will be
appreciated by the skilled practitioner that other cell lines may
be appropriate, e.g., to provide higher expression desirable
glycosylation patterns, or other features.
[0383] Clones are selected by using markers depending on the mode
of the vector construction. The marker may be on the same or a
different DNA molecule, preferably the same DNA molecule. In
prokaryotic hosts, the transformant may be selected, e.g., by
resistance to ampicillin, tetracycline or other antibiotics.
Production of a particular product based on temperature sensitivity
may also serve as an appropriate marker.
[0384] Prokaryotic or eukaryotic cells transformed with the nucleic
acids of the present invention will be useful not only for the
production of the nucleic acids and proteins of the present
invention, but also, for example, in studying the characteristics
of Zmax1 (LRP5), LRP6, IBM or HBM-like proteins.
[0385] Antisense nucleic acid-sequences are useful in preventing or
diminishing the expression of Zmax1 (LRP5), LRP6, HBM or HBM-like
molecules, as will be appreciated by one skilled in the art. For
example, nucleic acid vectors containing all or a portion of the
Zmax1 (LRP5), LRP6, HBM or HBM-like nucleic acid may be placed
under the control of a promoter in an antisense orientation and
introduced into a cell. Expression of such an antisense construct
within a cell will interfere with Zmax1(LRP5), LRP6, HBM or
HBM-like transcription and/or translation and/or replication.
[0386] The probes and primers based on the Zmax1(LRP5), LRP6, HBM
or HBM-like gene sequences disclosed herein are used to identify
homologous gene sequences and proteins in other species. The gene
sequences and proteins can also be used in the
diagnostic/prognostic, therapeutic and drug screening methods
described herein for the species from which they have been
isolated.
19. Protein Expression and Purification
[0387] Expression and purification of the Zmax1 (LRP5), LRP6, HBM
or HBM-like proteins of the invention can be performed essentially
as outlined below (LRP5, LRP6 and HBM-like proteins are also
included when referring only to HBM). To facilitate the cloning,
expression and purification of membrane and secreted protein from
the HBM gene, a gene expression system, such as the pET System
(Novagen), for cloning and expression of recombinant proteins in E.
coli was selected. Also, a DNA sequence encoding a peptide tag, the
His-Tap, was fused to the 3' end of DNA sequences of interest to
facilitate purification of the recombinant protein products. The 3'
end was selected for fusion to avoid alteration of any 5' terminal
signal sequence.
[0388] Nucleic acids chosen, for example, from the nucleic acids
set forth in SEQ ID NOS: 1, 3 and 5-12 for cloning HBM were
prepared by polymerase chain reaction (PCR). Synthetic
oligonucleotide primers specific for the 5' and 3' ends of the HBM
nucleotide sequence were designed and purchased from Life
Technologies (Gaithersburg, Md.). All forward primers (specific for
the 5' end of the sequence) were designed to include an NcoI
cloning site at the 5' terminus. These primers were designed to
permit initiation of protein translation at the methionine residue
encoded within the NcoI site followed by a valine residue and the
protein encoded by the HBM DNA sequence. All reverse primers
(specific for the 3' end of the sequence) included an EcoRI site at
the 5' terminus to permit cloning of the HBM sequence into the
reading frame of the pET-28b. The pET-28b vector provided a
sequence encoding an additional 20 carboxyl-terminal amino acids
including six histidine residues (at the C-terminus), which
comprised the histidine affinity tag.
[0389] Genomic DNA prepared from the HBM gene was used as the
source of template DNA for PCR amplification (Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley & Sons
(1994)). To amplify a DNA sequence containing the HBM nucleotide
sequence, genomic DNA (50 ng) was introduced into a reaction vial
containing 2 mM MgCl.sub.2, 1 .mu.M synthetic oligonucleotide
primers (forward and reverse primers) complementary to and flanking
a defined HBM, 0.2 mM of each of deoxynucleotide triphosphate,
dATP, dGTP, dCTP, dTTP and 2.5 units of heat stable DNA polymerase
(AmpliTaq.TM., Roche Molecular Systems, Inc., Branchburg, N.J.) in
a final volume of 100 microliters.
[0390] Upon completion of thermal cycling reactions, each sample of
amplified DNA was purified-using the Qiaquick Spin PCR purification
kit (Qiagen, Gaithersburg, Md.). All amplified DNA samples were
subjected to digestion with the restriction endonucleases, e.g.,
NcoI and EcoRI (New England BioLabs, Beverly, Mass.) (Ausubel et
al., Current Protocols in Molecular Biology, John Wiley & Sons,
Inc. (1994)). DNA samples were then subjected to electrophoresis on
1.0% NuSeive (FMC BioProducts, Rockland, Me.) agarose gels. DNA was
visualized by exposure to ethidium bromide and long wave UV
irradiation. DNA contained in slices isolated from the agarose gel
was purified using the Bio 101 GeneClean Kit protocol (Bio 101,
Vista, Calif.).
[0391] The pET-28b vector was prepared for cloning by digestion
with restriction endonucleases, e.g., NcoI and EcoRI (New England
BioLabs, Beverly, Mass.) (Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons, Inc. (1994)). The pET-28a
vector, which encodes the histidine affinity tag that can be fused
to the 5' end of an inserted gene, was prepared by digestion with
appropriate restriction endonucleases.
[0392] Following digestion, DNA inserts were cloned (Ausubel et
al., Current Protocols in Molecular Biology, John Wiley & Sons,
Inc. (1994)) into the previously digested pET-28b expression
vector. Products of the ligation reaction were then used to
transform the BL21 strain of E. coli (Ausubel et al., 1994) as
described below.
[0393] Competent bacteria, E. coli strain BL21 or E. coli strain
BL21 (DE3), were transformed with recombinant pET expression
plasmids carrying the cloned HBM sequence according to standard
methods (Ausubel et al., 1994). Briefly, 1 .mu.l of ligation
reaction was mixed with 50 .mu.l of electrocompetent cells and
subjected to a high voltage pulse, after which samples were
incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone,
10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20
mM glucose) at 37.degree. C. with shaking for 1 hour. Samples were
then spread on LB agar plates containing 25 .mu.g/ml kanamycin
sulfate for growth overnight. Transformed colonies of BL21 were
then picked and analyzed to evaluate cloned inserts, as described
below.
[0394] Individual BL21 clones transformed with recombinant pET-28b
HBM nucleotide sequences were analyzed by PCR amplification of the
cloned inserts using the same forward and reverse primers specific
for the HBM sequences that were used in the original PCR
amplification cloning reactions. Successful amplification verifies
the integration of the HBM sequence in the expression vector
(Ausubel et al., 1994).
[0395] Individual clones of recombinant pET-28b vectors carrying
properly cloned HBM nucleotide sequences were picked and incubated
in 5 ml of LB broth plus 25 .mu.g/ml kanamycin sulfate overnight.
The following day plasmid DNA was isolated and purified using the
Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth,
Calif.).
[0396] The pET vector can be propagated in any E. coli K-12 strain,
e.g., HMS174, HB101, JM109, DH5 and the like, for purposes of
cloning or plasmid preparation. Hosts for expression include E.
coli strains containing a chromosomal copy of the gene for T7 RNA
polymerase. These hosts were lysogens of bacteriophage DE3, a
lambda derivative that carries the lacI gene; the lacUV5 promoter
and the gene for T7 RNA polymerase. T7 RNA polymerase was induced
by addition of isopropyl-.beta.-D-thiogalactoside (IPTG), and the
T7 RNA polymerase transcribes any target plasmid containing a
functional T7 promoter, such as pET-28b, carrying its gene of
interest. Strains include, for example, BL21(DE3) (Studier et al.,
1990 Meth. Enzymol., 185: 60-89).
[0397] To express the recombinant HBM sequence, 50 ng of plasmid
DNA are isolated as described above to transform competent
BL21(DE3) bacteria as described above (provided by Novagen as part
of the pET expression kit). The lacZ gene (.beta.-galactosidase) is
expressed in the pET-System as described for the HBM recombinant
constructions-Transformed cells were cultured in SOC medium for 1
hour, and the culture was then plated on LB plates containing 25
.mu.g/ml kanamycin sulfate. The following day, the bacterial
colonies were pooled and grown in LB medium containing kanamycin
sulfate (25 .mu.g/ml) to an optical density at 600 nM of 0.5 to 1.0
O.D. units, at which point 1 mM IPTG was added to the culture for 3
hours to induce gene expression of the HBM recombinant DNA
constructions.
[0398] After induction of gene expression with IPTG, bacteria were
collected by centrifugation in a Sorvall RC-3B centrifuge at
3500.times.g for 15 minutes at 4.degree. C. Pellets were
resuspended in 50 ml of cold mM Tris-HCl, pH 8.0, 0.1 M NaCl and
0.1 mM EDTA (STE buffer). Cells were then centrifuged at
2000.times.g for 20 minutes at 4.degree. C. Wet pellets were
weighed and frozen at -80.degree. C. until ready for protein
purification.
[0399] 19.1 Chinese Hamster Ovary (CHO) Expression System
[0400] Alternatively, HBM and Zmax1 (LRP5) may be expressed in
eukaryotic cells. Eukaryotic cells, such as mammalian derived cell
lines, are more capable of expressing properly folded proteins
containing cystine rich domains such as the EGF and LDLR
modules.
[0401] 19.2 Development of Constructs
[0402] HBM and Zmax1 (LRP5) extracellular domain fusions (ECD) to
IgG-Fc were prepared. These ECD fusions to the IgG-Fc domain remove
the endogenous transmembrane and cytoplasmic portion of the
Zmax1/HBM receptor and should produce a secreted fusion protein.
The Fc region is separated from the Zmax1/HBM ECD by an
enterokinase recognition site so that purified Zmax1 or HBM ECD
protein can be obtained without the Fc domain present. The vector
used for this construct was pHTop, a derivative of pED (Kaufman et
al., 1991 Nuc. Acids Res. 19: 4485-90) in which the majority of the
adenomajor late promoter was replaced by six repeats of the tet
operator (Gossen et al., 1992 Proc. Natl. Acad. Sci. USA 89:
5547-51). This vector contains the dihydrofolate reductase (dhfr)
gene, and when introduced in the cell line CHO/A2 (see description
below), functions very efficiently. Clones with high expression can
be selected by isolating cells which survive in high methotrexate
(MTX) concentrations.
[0403] The CHO expression vector pHTOP-Fc was digested with SalI
and NotI. The intervening sequence was purified away from the rest
of the vector by electroelution from an acrylamide gel slice. SalI
cuts 5' to the intrinsic honey bee mellitin signal sequence in
pHTOP-Fc, and NotI cuts just 5' to the coding sequence IgG1-Fc. The
resulting SalI-NotI pHTOP-Fc vector has the signal sequence removed
and the NotI cloning site is amenable to creating a 5' fusion to
IgG-Fc. Full-length Zmax1 (LRP5) in pCMVSPORT6 and full-length HBM
in pCMVSPORT6 were digested individually with Xma1 which cuts
within the region of the ORF that encodes the signal sequence) and
BamHI (that cuts internally in the ORF) to generate a 2286 bp 5'
fragment of Zmax1 and HBM. The mutation which distinguishes Zmax1
from HBM lies on this fragment. Separately, the Zmax1 DNA was
digested with BamHI and SacI to isolate an 1800 bp 3' fragment
which is common to both the Zmax1 and the HBM genes. Together,
these two fragments constitute the coding sequence for the HBM and
Zmax1 extracellular domains, less the coding sequence for the first
6 amino, acids of the signal sequence and ending 18 amino acids
prior to the end of the extracellular domain, which we estimated
from Kyte-Doolittle plots to end at the sequence "SPAHSS" (SEQ ID
NO:698).
[0404] A synthetic duplex was designed to recreate the coding
sequence of the Zmax1/HBM signal sequence 5' of the native Xma1
site, which included the initiator methionine and Kozak sequence.
This duplex was designed to contain SalI (5') and XmaI (3')
cohesive ends to adapt ends to adapt the gene fragments described
above to the pHTOP-Fc vector. This synthetic duplex was constructed
from two partially complementary oligonucleotides as given
below:
TABLE-US-00027 5'-TCGACCACCATGGAGGCAGCGCCGC-3' (SEQ ID NO:699)
3'-GGTGGTACCTCCGTCGCGGCGGGCC-5' (SEQ ID NO:700)
A second synthetic duplex was designed to recreate the 3' coding
sequence from a native SacI site to the estimated end of the
extracellular domain following the serine in the sequence " . . .
SPAHSS", and to also encode a cloning site to allow in-frame fusion
to the downstream IgG-Fc. This duplex was designed to contain SacI
(5') and NotI (3') cohesive ends to adapt the gene fragments
described above to the pHTOP-Fc vector. This synthetic duplex was
constructed from two partially complementary oligonucleotides whose
sequences are given below:
TABLE-US-00028 (SEQ ID NO:701)
5'-CATGTGTGAAATCACCAAGCCGCCCTCAGACGACAGCCCGGCCCACA GCAGTGGC-3' (SEQ
ID NO:702) 3'-TCGAGTACACACTTTAGTGGTTCGGCGGGAGTCTGCTGTC GGGCCG
GGTGTCGTCACCGCCGG-5'
[0405] The fragments, synthetic duplexes, and vector were ligated
together in a single reaction. Separate reactions were performed
for Zmax (LRP5) 1 and IBM. The ligation mixtures were used to
transform electrocompetent E. coli DH10B cells, and the resulting
colonies were screened by radioactive colony hybridization using
the common SacI-BamHI 3' fragment as a probe. Colonies containing
plasmids with the Zmax1 or HBM fragment inserted were identified,
and plasmids were isolated from multiple candidates and their
sequences were verified by DNA sequencing. Verified constructs were
then used for transfection into CHO cells.
[0406] 19.2.1 Establishment of CHO Stable Cell Lines
[0407] The CHO/A2 cell line is derived from CHO DUKX B11 (Urlaub et
al., 1980 Proc. Natl. Acad. Sci. USA 77: 4216-20) by stably
integrating a transcriptional activator (tTA), a fusion protein,
between the Tet repressor and the herpes virus VP16 transcriptional
domain (Gossen et al.) CHO cell lines expressing extracellular
HBM-1.Fc and Zmax1.Fc were established by transfecting (using
lipofection) pHTopHBM-1.Fc into CHO/A2 cells and pHTopZmax1.Fc into
CHO/A2 cells. Clones were selected using by culturing the cells in
0.02 .mu.M methotrexate. Clones were later amplified step-wise to a
final concentration of 0.5 .mu.M methotrexate:
[0408] 19.2.2 Screening of CHO Stable Cell Lines
[0409] Multiple clones were screened by a variety of techniques.
Clones were screened by Western blot assay using a (mouse)
anti-human IgG.Fc horseradish peroxidase (1-IRP) antibody. The same
clones were also metabolically labeled with .sup.35 S-Met/Cys) for
a 6 hour pulse, or a 15 minute pulse, followed by a 1 hour, 4 hour,
or 24 hour chase in media without radiolabeled Met/Cys.
Immunoprecipitations were performed on proteins obtained from
conditioned media, as well as from cell extracts. Purification is
then performed followed by sequencing of the proteins using
N-terminal sequencing as known in the art.
[0410] 19.2.3 Fusion Protein Purification
[0411] Zmax1-IgG or HBM-IgG fusion protein can be purified from
conditioned media or cell extracts of CHO stable cells. The fusion
protein is isolated by affinity binding to protein A (for example
using protein A coated beads or columns). The IgG-FC domain can
then subsequently be cleaved from the Zmax/HBM1 ECD protein by
enterokinase digestion.
[0412] 19.2.4 Potential Uses for Cell Lines and Protein
[0413] Stable cell lines may be used for generation of purified
protein for use in ligand hunting, antibody generation,
determination of crystal structure, and competitive binding
assays.
[0414] A variety of methodologies known in the art can be used to
purify the isolated proteins (Coligan et al., Current Protocols in
Protein Science, John Wiley & Sons (1995)). For example, the
frozen cells can be thawed, resuspended in buffer and ruptured by
several passages through a small volume microfluidizer (Model
M-110S, Microfluidics International Corp., Newton, Mass.). The
resultant homogenate is centrifuged to yield a clear supernatant
(crude extract) and, following filtration, the crude extract is
fractioned over columns. Fractions are monitored by absorbance at
OD.sub.280 nm and peak fractions may be analyzed by SDS-PAGE.
[0415] The concentrations of purified protein preparations are
quantified spectrophotometrically using absorbance coefficients
calculated from amino acid content (Perkins, 1986 Eur. J. Biochem.
157:169-180). Protein concentrations are also measured by the
method of Bradford, 1976 Anal. Biochem. 72: 248-54 and Lowry et
al., 1951 J. Biol. Chem. 193: 265-275 using bovine serum albumin as
a standard.
[0416] SDS-polyacrylamide gels of various concentrations were
purchased from BioRad (Hercules, Calif.), and stained with
Coomassie blue. Molecular weight markers may include rabbit
skeletal muscle myosin (200 kDa), E. coli-galactosidase (116 kDa),
rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin
(66.2 kDa), ovalbumin (45 kDa), bovine carbonic anhydrase (31 kDa),
soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa)
and bovine aprotinin (6.5 kDa).
[0417] Once a sufficient quantity of the desired protein has been
obtained, it may be used for various purposes. A typical use is the
production of antibodies specific for binding. These antibodies may
be either polyclonal or monoclonal, and may be produced by in vitro
or in vivo techniques well known in the art. Monoclonal antibodies
to epitopes of any of the peptides identified and isolated as
described can be prepared from murine hybridomas (Kohler, 1975
Nature, 256: 495). In summary, a mouse is inoculated with a few
micrograms of HBM protein over a period of two weeks. The mouse is
then sacrificed. The cells that produce antibodies are then removed
from the mouse's spleen. The spleen cells are then fused with
polyethylene glycol with mouse myeloma cells. The successfully
fused cells are diluted in a microtiter plate and growth of the
culture is continued. The amount of antibody per well is measured
by immunoassay methods such as ELISA (Engvall, 1980 Meth. Enzymol.
70: 419). Clones producing antibody can be expanded and further
propagated to produce HBM antibodies. Other suitable techniques
involve in vitro exposure of lymphocytes to the antigenic
polypeptides, or alternatively, to selection of libraries of
antibodies in phage or similar vectors. See Huse et al., 1989
Science 246: 1275-81. For additional information on antibody
production see Davis et al., Basic Methods in Molecular Biology,
Elsevier, N.Y., Section 21-2 (1989).
[0418] Additional uses for purified or isolated protein includes
use in X-ray crystallography, binding assays, and so forth as
described in greater detail infra.
[0419] 19.3 Zmax1, LRP6, and Variant Antibodies
[0420] Polyclonal antibodies were developed to both human Zmax1
(LRP5) (SEQ ID NO:3) and LRP6 (GenBank Accession No. AF074264).
Antibodies can similarly be prepared against HBM and HBM like
proteins and polypeptides. Peptides from the Zmax1 amino acid
sequence were selected as immunogens based on five goals. 1)
Maximize differences between Zmax1 and LRP6 amino acid sequences
(71% amino acid identity). See FIG. 27. For sequence comparison,
typically one sequence acts as a reference sequence, to which test
sequences are compared. When using a sequence comparison algorithm,
test and reference sequences are input into a computer, subsequence
coordinates are designated, if necessary, and sequence algorithm
program parameters are designated. The sequence comparison
algorithm then calculates the percent sequence identity for the
test sequence(s), relative to the reference sequence, based on the
designated program parameters. 2) Minimize potential cross
reactivity with other known genes by performing sequence alignment
and similarity searches. Optimal alignment of sequences for
comparison can be conducted, e.g., by the local homology algorithm
of Smith et al, 1981 Adv. Appl. Math. 2: 482, by the homology
alignment algorithm of Needleman et al., 1970 J. Mol. Biol. 48:
443, by the search for similarity method of Pearson et al., 1988
Proc. Natl. Acad. Sci. USA 85: 2444, by computerized
implementations of these algorithms and others in programs
contained in the Wisconsin genetics software package, Genetics
Computer Group, 585 Science Dr., Madison, Wis., or by visual
inspection (see generally Ausubel et al., Current Protocols in
Molecular Biology, John Wiley & Sons (1997). Another example of
algorithm that is suitable for determining percent sequence
identity and sequence similarity is the BLAST algorithm, which is
described in Altschul et al., 1990 J. Mol. Biol. 215: 403-10.
Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information. 3)
Obtain peptides with the highest antigenicity index as possible as
determined by Peptide Structure protein analysis using software
programs contained in the Wisconsin Genetics software package,
Genetics Computer Group, 575 Science Dr., Madison, Wis. 4) Locating
peptides relative to highly homologous domains (e.g., EGF-like
domains and LDL receptor repeats) within the gene family and the
location relative to the extracellular and cytoplasmic regions of
the gene. 5) And, for human Zmax1 specific antibodies, the human
amino acid sequence (SEQ ID NO: 3) was compared to the mouse Zmax1
sequence (GenBank Accession No. AF064984) and peptides were
selected based on the above criteria in addition to minimizing the
sequence similarity between the two species (See FIG. 26).
[0421] Using the same criteria above, LRP6 specific peptides were
selected for polyclonal antibody production. Table 11 lists the
amino acid sequences that were chosen, the amino acid differences
within the peptide between the human and mouse sequences. All the
peptide sequences (ranging from 12-18 amino acids) were provided to
Sigma/Genosys (St. Louis, Mo.) for peptide synthesis and subsequent
polyclonal antibody production in New Zealand White Rabbits. The
IgG fraction from the serum of each immunized rabbit was isolated
using Protein G Sepharose (Amersham). Polyclonal antibody
generation using these peptides may be done in other species as
well, for example, chickens. This is often advantageous when there
is a high degree of similarity between the human (reference) and
murine/rodent sequence.
TABLE-US-00029 TABLE 11 SEQ H/M* Amino ID Differ- Acids Amino Acids
NO ences Comments 171-187 VETPRIERAGMDGSTRK 703 5 Contains HBM
poly- morphism 264-278 NKRTGGKRKEILSAL 704 3 Extra- cellular
290-301 ERQPFFHTRCEE 705 2 Adjacent to EGF-I, extra- cellular
532-546 VDGTKRRTLLEDKLP 706 5 Extra- cellular 901-915
DGLNDCMHNNGQCGQ 707 2 In EGF-III, extra- cellular 1010-1021
PFVLTSLSQGQN 708 6 Extra- cellular, human specific 1415-1429
YAGANGPFPHEYVSG 709 3 Cyto- plasmic 1452-1464 ACGKSMMSSVSLM 710 5
Cyto- plasmic, human specific 1556-1573 RWKASKYYLDLNSDSDPY 711 1
Cyto- plasmic 888-902 SGWNECASSNGHCSH 712 LRP6 specific 1308-1321
NGDANCQDKSDEKN 713 LRP6 specific *H/M-differences between human and
mouse sequences
[0422] Antibodies towards variants of LRP6. HBM and LRP5 are also
contemplated. Based on the analysis of the structural model of the
LRP5 beta propeller 1 (discussed in more detail in the Examples),
interior regions of the propeller were analysed. Since the
site-directed mutagenesis experiments had confirmed that modulation
of propeller 1, particularly in the interior regions of beta
propeller 1, could result in an HBM effect, a strategy was employed
to generate antibodies with epitopes specific to these regions.
Such antibodies to the wild type LRP5 receptor could serve, for
examples, as an HBM mimetic, by altering ligand/receptor
interactions, protein-protein interactions, or by modulating Wnt or
Dkk activity. Such antibodies could be used as therapies to treat,
for example osteoporosis.
[0423] One set of preferred antibodies includes:
TABLE-US-00030 LRP5 Amino Acids Sequence 208-223 KLYWADALKLSFIHRAN
277-291 ALYSPMDIQVLSQER 61-73 GLEDAAAVDFQFSKGA 234-247
EGSLTHPFALTLSG 249-264 TLYWTDWQTRSIHACN 144-156 VLFWQDLDQPPAI
194-210 IYWPNGLTIDLEEQKLY 34-47 LLLFANRRDVRLVD 75-89
GAVYWTDVSEEAIYQ 121-135 KLYWTDSETNRIEVA
Similar peptides can be used to prepare antibodies based on the
LRP6 or HBM structures and the proposed mutations in their
propellers. Other antibodies could easily be prepared based on the
structural model discussed in the Examples, as would be readily
appreciated by the skilled artisan.
[0424] Another series of antibodies could be prepared which target
the domains on HBM, LRP5, LRP6 and their variants which interact
Dkk. Such antibodies could again serve as HBM mimetics, for example
by displacing Dkk binding and thereby could be used as an
osteoporosis therapeutic. Preferred peptides for preparing
antibodies include but are not limited to:
TABLE-US-00031 LRP5 Amino Acids Sequence 969-993
LILPLHGLRNVKAIDYDPLDKFIYW 989-1013 KFIYWVDGRQNIKRAKDDGTQPFVL
1009-1033 QPFVLTSLSQGQNPDRQPHDLSIDI 1029-1053
LSIDIYSRTLFWTCEATNTINVHRL 10491073 NVHRLSGEAMGVVLRGDRDKPRAIV
1253-1266 CGEPPTCSPDQFAC 1278-1295 WRCDGFPECDDQSDEEGC 1316-1332
RCDGEADCQDRSDEADC 1370-1383 CEITKPPSDDSPAH
Additional peptides would be readily apparent to the artisan of
ordinary skill.
[0425] 19.3.1 Single Chain Fv Molecules Developed by Phage
Display
[0426] Peptides were chosen from the Zmax1 (LRP5) sequence (SEQ ID
NO: 3) to screen for single chain Fv (scFv) molecules by phage
display. Similar peptides can be chosen for LRP6, HBM and HBM-like
proteins to screen for scFv molecules. As discussed below, all
mention of LRP5 is also meant to include these other proteins.
[0427] A total of 17 peptides from the Zmax1 sequence were selected
for synthesis and subsequent phage display screen for scFv
molecules. All peptide synthesis and phage display work was
performed at Cambridge Antibody Technology (CaT) in Cambridge, UK.
Peptides were selected based on criteria as described above.
TABLE-US-00032 TABLE 12 Protein Domain Zmax1 Residues LRP6 Residues
% Identity Spacer 1 (+G171V) 161-181 148-168 76% Spacer 1 (-G171V)
161-181 148-168 76% EGF 1 301-321 288-308 76% Spacer 2 401-421
388-408 52% EGF 2 611-631 598-618 62% Spacer 3 781-801 768-788 62%
EGF 3 921-941 908-928 10% Spacer 4 1000-1021 988-1008 26% EGF 4
1229-1249 1219-1239 76% LDLR 1 1261-1282 1252-1272 81% LDLR 2
1300-1320 1290-1310 57% LDLR 3 1338-1358 1328-1348 48% Cytosolic 1
1418-1438 1405-1425 14% Cytosolic 1 1516-1536 1503-1525 52%
Cytosolic 1 1535-1555 1524-1544 81% Cytosolic 1 1595-1615 1592-1613
82% Spacer 2 (cross reactive) 421-441 408-428 100%
[0428] Note that a number of these regions (e.g., 401-421, 421-441,
781-801, and 1229-1249) share 100% identity with mouse LRP5 (see
FIG. 26). Therefore, these may be used against both mouse and human
forms of the protein. The peptide 421-441 was included to
facilitate the generation of an antibody that would recognize both
Zmax1 (LRP5) and LRP6 (see FIG. 27). Two peptides were synthesized
spanning the HBM mutation site (Zmax1 residues 161-181), one with
the Zmax1 sequence and the other containing the HBM sequence.
[0429] Once scFv molecules were isolated, they were used as
reagents in immunochemistry to detect Zmax1 (LRP5) protein
expression in a variety of human normal and diseased tissues. The
details of the scFV antibody immunohistochemical analysis of three
phage clones against peptide 1000-1021 (i.e.,
IKRAKDDGTQPFVLTSLSQGQN; SEQ ID NO: 714) of the extracellular domain
of Zmax1 showed positive staining with cardiac muscle, kidney, lung
and liver. Expression was also detected in prostate carcinoma.
These results are consistent with mRNA tissue distribution profiles
as well as with the published reports of LRP5 mRNA localization
(Kim et al., 1998 J. Biochem. 124: 1072-6). The resulting phage
clones arise from pools and will be sequenced to identify potential
variants in the Fv region of the molecules. Once identified, the
suitable scFVs can then be subcloned into variable heavy chain and
variable light chain DNA constructs for cotransfection into COS
cells for final assembly of an intact and functional immunoglobulin
gamma (IgG) molecule. The IgG that is expressed by the cells can
then be further characterized for specificity and reactivity as
would be known in the art.
[0430] 19.3.2 Monoclonal Antibody Development
[0431] Monoclonal antibodies can be developed to Zmax1 (LRP5),
LRP6, HBM and HBM-like proteins/polypeptides by complete cell and
adenovirus immunization of, for example, Balb/c mice (antibodies to
all forms are contemplated even when only a few examples are
discussed in detail). Dendritic cells can be isolated from spleens
of Balb/c mice, for example, and the cells expanded in vitro in the
presence of growth factors IL-4 and GM-CSF. The dendritic cells can
then be infected with HBM or Zmax1 adenovirus particles. The cells
are then cultured for 24 hours prior to intravenous injection into
Balb/c mice. Dendritic cells (1.times.10.sup.6 cells/mouse) are
injected 2-3 times every 3-4 weeks over a three month period.
[0432] Alternatively, purified HBM and Zmax DNAs in, for example,
the pcDNA3.1 expression vector, can be coated on colloidal gold
particles. These particles can then be injected subcutaneously into
the desired mouse using gene gun technology. Approximately, 5 .mu.g
cDNA/mouse can be injected. Injections are performed 4-6 times
every 2 weeks over approximately a 3 month period.
[0433] Another option is that cells (any species of animal, but
preferably Balb/c mouse strain or the same species as the mouse
strain being used which is related to limit antigen response to
non-specific protein) over expressing HBM and Zmax1 (LRP5) and
their respective adenovirus will be injected into the mice every
2-3 weeks for a period of about 1.5 to 3 months, as necessary. The
bleeds from the mice can be tested for reactivity with the native
and denatured protein by ELISA (using purified protein or
protein-fusions), cell based ELISA, immunohistochemical staining
and Western blotting. Serum samples from the animals can be
screened by FACS (fluorescent activated cell sorting) using cells
infected with Zmax1 or HBM adenovirus. The spleen cells (antibody
producing cells) from the mice with the strongest reactivity can
then be fused with a myeloma to generate the hybridoma cells. The
conditioned media from the hybridoma is then screened for the
positive cell colonies for subsequent cloning. These cloned cells
can then be injected into the intraperitoneal space in mice for
ascites production.
[0434] 19.3.3 Polyclonal Antibody Applications
[0435] Polyclonal antibodies directed against Zmax1 (LRP5) and LRP6
were developed to determine the function of these proteins, analyze
the expressed pattern and levels in various tissues, cells or any
biological sample. Similar antibodies could be prepared which
distinguish the wild type forms from the HBM and HBM-like
variants.
[0436] Uses for polyclonal antibodies against Zmax1, HBM, HBM-like
polypeptides, and LRP6 include but are not limited to: analysis of
bone cross-sectional mounts, tissue distribution, evaluation of
expression of the protein from bone biopsy samples of
affected/non-affected family members (e.g., bone cell digests,
explants of bone marrow stromal cell cultures), evaluation of
protein expression levels in transiently or stably transfected
cells, evaluating protein concentration in tissues, serum or body
fluid, purification of full length or fragments of these proteins
for ligand hunting and functional assay development, identification
of ligands or proteins which interact with these proteins, and
elucidations of the signaling pathways of LRP6, Zmax1 (LRP5) and
HBM, and related variants.
[0437] For example, Zmax1 cloned in pcDNA3.1 (Invitrogen, Carlsbad,
Calif.). This was used to generate .sup.35S-labeled in vitro
translated (Promega, Madison, Wis.) Zmax1. Antibody (10 .mu.g/ml)
3109 and 3110, which are directed against peptide immunogen,
RWKASKYYLDLNSDSDPY (SEQ ID NO:711), was combined with 20 .mu.l of
the in vitro translated product in the presence of either 10
.mu.g/ml specific peptide (i.e., RWKASKYYLDLNSDSDPY; --SEQ ID NO:
711) or non-specific peptide (i.e., SGWNECASSNGHCSH; SEQ ID NO:
712) or no peptide and incubated for 1.5 hr at 4.degree. C. Protein
A Sepharose was then added to the samples (previously blocked for
about 1.5 hr with reticulocyte lysate), and the samples were shaken
for 1 hour at 4.degree. C. The protein A Sepharose was washed 3
times with 0.5 ml of PBS. The bound protein was subsequently
separated on a 4-12% gradient NuPAGE gel (Invitrogen) according to
manufacturer's instructions. The gel was dried at 80.degree. C. for
30 min and then exposed to Kodak X-OMAT-AR film for 24 to 48 hr.
The specific peptide was observed to significantly compete for the
.sup.35 S-labeled Zmax1 immunoprecipitated protein with either
antibody. The competition was not observed with a non-specific
peptide.
[0438] These antibodies can also be used for immunohistochemistry.
For example, HBM transgenic and wild-type mice were sacrificed
using CO.sub.2 narcosis. Mouse calvariae were removed intact, and
the soft tissues gently dissected. The bones were fixed in 10%
phosphate buffered formalin for 24 hours for further processing and
analysis. After fixation, calvariae were decalcified in TBD-2
decalcifying agent (Shandon, Pittsburgh, Pa.) for about 7-8 hours
and then dehydrated in graded alcohol. Calvariae were then bisected
perpendicular to the sagittal suture through the central portion of
the parietal bones parallel to the lambdoidal and coronal sutures
and embedded in paraffin. Four to six 5 .mu.m thick representative
sections were cut.
[0439] For example, the rabbit polyclonal antibody, Zmax1/HBM
(i.e., antibody 3109 and 3110) recognize Zmax1 (LRP5) in both HBM
transgenic and wild-type mouse calvariae. An anti-Zmax1 or anti-HBM
antibody can be used to detect Zmax1 or HBM protein in order to
evaluate its abundance and pattern of protein expression. Detection
can be facilitated by coupling (i.e., physically linking) the
antibody to a detectable substance. Examples of detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials, bioluminescent materials, and
radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, P-galactosidase, or
acetylcholinesterase; example of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; and example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin and acquorien; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S, and .sup.3H.
Alternatively, a secondary antibody can be employed that detects
the presence of the primary Zmax1 polyclonal antibody. An example
would be an antibody that recognized all rabbit immunoglobulins.
This secondary antibody could be coupled in an identical manner as
described above to facilitate detection. Controls comprised samples
with the avidin peroxidase, but without antibody. Intensive
positive staining of stroma cells and mesenchymal cells was
observed in the suture area. Pre-osteoblasts and osteoblasts were
observed to stain within the periosteum and some osteocytes with
antibody 3109 and 3110 in the calvariae of the HBM compared to
wild-type mice. High magnification of tissue calvaria sections of
the HBM transgenic mice showed a pronounced cell membrane staining
of the osteocytes and the cells within the suture area.
[0440] Similar antibodies could be prepared to HBM-like proteins
and polypeptides, as would be readily appreciated by the artisan of
ordinary skill.
20. Methods of Use: Gene Therapy
[0441] In recent years, significant technological advances have
been made in the area of gene therapy for both genetic and acquired
diseases (Kay et al., 1997 Proc. Natl. Acad. Sci. USA, 94:
12744-6). Gene therapy can be characterized as the deliberate
transfer of DNA for therapeutic purposes. Improvement in gene
transfer methods has allowed for development of gene therapy
protocols for the treatment of diverse types of diseases. Gene
therapy has also taken advantage of recent advances in the
identification of new therapeutic genes, improvement in both viral
and nonviral gene delivery systems, better understanding of gene
regulation, and improvement in cell isolation and
transplantation.
[0442] The preceding experiments identify the HBM gene as a
dominant mutation conferring elevated bone mass. Additional
HBM-like genes are identifiable based on the model and data
presented herein regarding the propellers and structure of the HBM
protein. The fact that this mutation is dominant indicates that
expression of the HBM protein causes elevated bone mass. Older
individuals carrying the HBM gene, and, therefore expressing the
HBM protein, do not suffer from osteoporosis. These individuals are
equivalent to individuals being treated with the HBM protein. These
observations are a strong experimental indication that therapeutic
treatment with the HBM protein prevents osteoporosis. The bone mass
elevating activity of the HBM gene is termed "HBM function" or "HBM
like phenotype" in the instance of other HBM variants.
[0443] Therefore, according to the present invention, a method is
also provided of supplying HBM function to mesenchymal stem cells
(Onyia et al., 1998 J. Bone Miner. Res. 13: 20-30; Ko et al., 1996
Cancer Res. 56: 4614-9). Supplying such a function provides
protection against osteoporosis. The HBM gene or a part of the gene
or other HBM-like gene may be introduced into the cell in a vector
such that the gene remains extrachromosomal. In such a situation,
the gene will be expressed by the cell from the extrachromosomal
location.
[0444] Vectors for introduction of genes both for recombination and
for extrachromosomal maintenance are known in the art, and any
suitable vector may be used. Methods for introducing DNA into cells
such as electroporation, calcium phosphate co-precipitation, and
viral transduction are known in the art, and the choice of method
is within the competence of one skilled in the art (Robbins, ed.,
Gene Therapy Protocols, Human Press, NJ (1997)). Cells transformed
with the HBM gene (or HBM-like gene) can be used as model systems
to study osteoporosis and drug treatments that promote bone
growth.
[0445] As generally discussed above, the HBM or HBM-like gene or
biologically active fragment thereof; where applicable, may be used
in gene therapy methods in order to increase the amount of the
expression products of such genes in mesenchymal stem cells (in all
instances where discussing HBM gene and its cognate product, the
HBM-like gene and cognate protein is also contemplated). It may be
useful also to increase the level of expression of a given HBM
protein, or a fragment thereof, even in those cells in which the
wild type gene is expressed normally. Gene therapy would be carried
out according to generally accepted methods as described by, for
example, Friedman, Therapy for Genetic Diseases, Friedman, Ed.,
Oxford University press, pages 105-121 (1991).
[0446] A virus or plasmid vector containing a copy of the HBM gene
linked to expression control elements and capable of replicating
inside mesenchymal stern cells, is prepared. Suitable vectors are
known and described, for example, in U.S. Pat. No. 5,252,479 and WO
93/07282, the disclosures of which are incorporated by reference
herein in their entirety. The vector is then injected into the
patient, either locally into the bone marrow or systemically (in
order to reach any mesenchymal stem cells located at other sites,
i.e., in the blood). If the transfected gene is not permanently
incorporated into the genome of each of the targeted cells, the
treatment may have to be repeated periodically.
[0447] Gene transfer systems known in the art may be useful in the
practice of the gene therapy methods of the present invention.
These include viral and non-viral transfer methods. A number of
viruses have been used as gene transfer vectors, including polyoma,
i.e., SV40 (Madzak et al., 1992 J. Gen. Virol. 73: 1533-6),
adenovirus (Berkner, 1992 Curr. Top. Microbiol. Immunol. 158:
39-61; Berkner et al., 1988 BioTechniques, 6: 616-629; Gorziglia et
al., 1992 J. Virol. 66: 4407-12; Quantin et al., 1992 Proc. Natl.
Acad. Sci. USA 89: 2581-2584; Rosenfeld et al., 1992 Cell 68:
143-155; Wilkinson et al., 1992 Nucl. Acids Res. 20: 2233-39;
Stratford-Perricaudet et al., 1990 Hum. Gene Ther. 1: 241-256),
vaccinia virus (Mackett et al., 1992 Biotechnology 24: 495-499),
adeno-associated virus (Muzyczka, 1992 Curr. Top. Microbiol.
Immunol. 158: 91-123; Ohi et al., 1990 Gene 89: 279-282), herpes
viruses including HSV and EBV (Margolskee, 1992 Curr. Top.
Microbiol. Immunol. 158: 67-90; Johnson et-al., 1992 J. Virol., 66:
2952-65; Fink et al., 1992 Hum. Gene Ther. 3: 11-9; Breakfield et
al., 1987 Mol. Neurobiol. 1: 337-371; Fresse et al., 1990 Biochem.
Pharmacol., 40: 2189-2199), and retroviruses of avian
(Brandyopadhyay et al., 1984 Mol. Cell Biol., 4: 749-754;
Petropouplos et al., 1992 J. Virol. 66: 3391-3397), murine (Miller,
1992 Curr. Top. Microbiol. Immunol. 158: 1-24; Miller et al., 1985
Mol. Cell. Biol. 5: 431-437; Sorge et al., 1984 Mol. Cell. Biol. 4:
1730-7; Mann et al., 1985 J. Virol. 54: 401-7), and human origin
(Page et al., 1990 J. Virol. 64: 5370-6; Buchschalcher et al., 1992
J. Virol. 66: 2731-9). Most human gene therapy protocols have been
based on disabled murine retroviruses.
[0448] Non-viral gene transfer methods known in the art include
chemical techniques such as calcium phosphate coprecipitation
(Graham et al., 1973 Virology 52: 456-67; Pellicer et al., 1980
Science 209: 1414-22), mechanical techniques, for example
microinjection (Anderson et al., 1980 Proc. Natl. Acad. Sci. USA,
77: 5399-5403; Gordon et al., 1980 Proc. Natl. Acad. Sci: USA, 77:
7380-4; Brinster et al., 1981 Cell 27: 223-231; Constantini et al.,
1981 Nature 294: 92-94), membrane fusion-mediated transfer via
liposomes (Felgner et al., 1987 Proc. Natl. Acad. Sci. USA 84:
7413-7; Wang et al., 1989 Biochemistry 28: 9508-14; Kaneda et al.,
1989 J. Biol. Chem. 264: 12126-9; Stewart et al., 1992 Hum. Gene
Ther. 3: 267-275; Nabel et al., 1990 Science 249: 1285-8; Lim et
al., 1992 Circulation 83: 2007-11), and direct DNA uptake and
receptor-mediated DNA transfer (Wolff et al., 1990 Science 247:
1465-8; Wu et al., 1991 BioTechniques 11: 474-85; Zenke et al.,
1990 Proc. Natl. Acad. Sci. USA 87: 3655-9; Wu et al., 1989 J.
Biol. Chem. 264: 16985-7; Wolff et al., 1991 BioTechniques 11:
474-45; Wagner et al., 1990; Wagner et al., 1991 Proc. Natl. Acad.
Sci. USA, 88: 4255-9; Cotten et al., 1990 Proc. Natl. Acad. Sci.
USA, 87: 4033-7; Curiel et al., 1991 Proc. Natl. Acad. Sci. USA,
88: 8850-4; Curiel et al., 1991 Hum. Gene Ther. 3: 147-54).
Viral-mediated gene transfer can be combined with direct in vivo
vectors to the mesenchymal stem cells and not into the surrounding
cells (Romano et al., 1998 In Vivo, 12: 59-67; Gonez et al., 1998
Hum. Mol. Genetics. 7:1913-9). Alternatively, the retroviral vector
producer cell line can be injected into the bone marrow (Culver et
al., 1992 Science 256: 1550-2). Injection of producer cells would
then provide a continuous source of vector particles. This
technique has been approved for use in humans with inoperable brain
tumors.
[0449] In an approach which combines biological and physical gene
transfer methods, plasmid DNA of any size is combined with a
polylysine-conjugated antibody specific to the adenovirus hexon
protein, and the resulting complex is bound to an adenovirus
vector. The trimolecular complex is then used to infect cells. The
adenovirus vector permits efficient binding, internalization, and
degradation of the endosome before the coupled DNA is damaged.
[0450] Liposome/DNA complexes have been shown to be capable of
mediating direct in vivo gene transfer. While in standard liposome
preparations the gene transfer process is non-specific, localized
in vivo uptake and expression have been reported in tumor deposits,
for example, following direct in situ administration (Nabel, Hum.
Gene Ther., 3:399-410 (1992)).
21. Methods of Use: Transformed Hosts and Transgenic Animals as
Research Tools and for the Development of Pharmaceuticals
[0451] Cells and animals that carry the HBM, HBM-like, Zmax1
(LRP5), or LRP6 genes, used as model systems, are valuable research
tools to study and test for substances that have potential as
therapeutic agents (Onyia et al., 1998 J. Bone Miner. Res., 13:
20-30; Broder et al., 1997 Bone, 21: 225-35). Discussion of one of
these is meant to include the others, e.g., discussion of HBM is
meant to include HBM-like variants, and so forth.
[0452] Cells for this purpose are typically cultured mesenchymal
stem cells. These may be isolated from individuals with somatic or
germline HBM genes. Alternatively, the cell line can be engineered
to carry the HBM or HBM-like gene, as described above. After a test
substance is applied to the cells, the transformed phenotype of the
cell is determined. Any trait of transformed cells can be assessed,
including, for example, formation of bone matrix in culture (Broder
et al., 1997 Bone, 21: 225-235), mechanical properties (Kizer et
al., 1997 Proc. Natl. Acad. Sci. USA 94: 1013-8), expression of
marker genes and response to application of putative therapeutic
agents.
[0453] Transgenic modified animals and cell lines may be used to
test therapeutic agents. Transgenic modifications include, for
example, insertion of the Zmax1 (LRP5) gene, LRP6, HBM gene or
HBM-like gene and disrupted homologous genes. Alternatively, the
inserted Zmax1, LRP6, HBM, and/or HBM-like gene of the animals may
be disrupted by insertion or deletion mutation of other genetic
alterations using conventional techniques, such as those described
by, for example, Capecchi, 1989 Science 244: 1288; Valancuis et
al., 1991 Mol. Cell Biol. 11: 1402; Hasty et al., 1991 Nature 350:
243; Shinkai et al., Cell, 68:855 (1992); Mombaerts et al., Cell,
68:869 (1992); Philpott et al., 1992 Science 256: 1448; Snouwaert
et al., 1992 Science 257: 1083; Donehower et al., 1992 Nature 356:
215. After test substances have been administered to the animals,
the growth of bone must be assessed. If the test substance
modulates (e.g., enhances) the growth of bone, then the test
substance is a candidate therapeutic agent. These animal models
provide an extremely important vehicle for potential therapeutic
products.
[0454] The present invention also provides animals and cell lines
wherein the expression of endogenous genes are activated, and may
be further amplified, which does not require in vitro manipulation
and transfection of exogenous DNA encoding Zmax1 (LRP5) or --IBM
proteins. These methods are described for example in PCT
Application WO 94/12650 and U.S. Pat. No. 5,968,502, both of which
are herein incorporated in their entirety by reference. In
addition, the present invention includes methods wherein endogenous
activation or over-expression is achieved by in situ homologous
recombination, non-homologous recombination, or illegitimate
recombination methods. These methods are described for example in
PCT Applications WO 99/15659 and WO 00/49162, both of which are
incorporated herein in their entirety.
[0455] 21.1 Creating Transgenic and Gene-Targeted Animals
[0456] The present invention provides genetically modified animals
that recapitulate the human HBM or a HBM-like phenotype. The
approaches taken involve the creation of both transgenic and
gene-targeted animals that have the human G to T nucleotide
substitution incorporated into the genome, express human Zmax1
(LRP5) or express a variant with a bone mass altering phenotype.
These approaches can be used with any gene, such as HBM, HBM-like,
LRP5 and LRP6 systems.
[0457] 21.1.1 Transgenic Mice Over-Expressing the HBM
Polymorphism
[0458] Plasmid constructs were prepared that utilized either the
CMVbActin or type I collagen promoters to drive expression of the
human HBM cDNA. The most commonly-used promoters for mammalian
expression are from cytomegalovirus (CMV), Rous sarcoma virus
(RSV), Simian virus 40 (SV40), and EF-1a (human elongation factor
1a-subunit). CMV is derived from the human cytomegalovirus
immediate-early viral promoter. CMV is a stronger promoter in most
cell lines than either RSV or SV40. The RSV promoter is derived
from an avian virus and tends to be a strong promoter in avian cell
lines. The SV40 promoter expresses well in cell lines that carry
the large. T antigen, such as COS-1. In these cell lines, the
plasmid is replicated to higher copy numbers. EF-1a is beginning to
be more widely used for recombinant protein expression. EF-1a is
the promoter from the human elongation factor 1a-subunit, a gene
that is highly expressed and conserved in mammalian cell lines.
[0459] The chimeric CMVbActin promoter is a strong promoter that
has been shown to produce ubiquitous gene expression in transgenic
mice including bone. This promoter was chosen to drive expression
of HBM in a manner consistent with the reported widespread
expression of the endogenous mouse Zmax1 (LRP5) gene. Although the
HBM phenotype is observed in bone, the HBM gene may have direct or
indirect effects in other tissues. Therefore, other strong
ubiquitous promoters may be utilized as would be known to those
skilled in the art.
[0460] Type I collagen promoters provide tissue-restricted gene
expression wherein expression is primarily limited to bone. Other
bone-specific promoters are available that could result in
expression of IBM in bone. For example, promoters associated with
proliferation of osteoblasts include histone, type I collagen,
TGF.beta.1, MSX2, cfos/cJun and Cbfa1 may be used. Promoters
associated with bone matrix maturation including alkaline
phosphatase, MGP, Cbfa1, Fra/Jun and Dlx5 also can be used.
Promoters associated with bone mineralization such as osteocalcin,
osteopontin, bone sialoprotein and collagenase also can be used.
The promoter chosen would be determined by, for example, the tissue
expression, the degree of regulatable control and the like as would
be known to the skilled artisan. For example, the type I collagen
promoters were chosen to insure that HBM would be expressed in bone
in a temporal, spatial and bone cell-specific pattern resembling
the endogenous pattern of Zmax1 (LRP5) expression in bone.
[0461] 21.1.2 Transgenic Mice Over-Expressing the Wild-Type (LRP5)
Zmax1 Gene
[0462] Plasmid constructs were prepared using the CMVbActin and
type I collagen promoters driving expression of Zmax1 (LRP5). These
animals can serve as a control animal model for the HBM, HBM-like
and LRP6 transgenic mice (all of which are contemplated when
discussing LRP5/Zmax1). Additional controls include non-transgenic
littermates and wild-type animals of an identical genetic
background. Methods for preparing these animals would be similar to
what is discussed for mice which over express the HBM
polymorphism.
[0463] 21.1.3 TLRP6 Gene Targeted Knock Out Mice
[0464] LRP6 knock-out mice were generated using Omnibank embryonic
stem (ES) cells carrying a gene trap vector which inserted into the
first intron of the LRP6 gene. The insert location was determined
to be the LRP6 gene by an Omnibank Sequence Tag (OST) generated by
reverse transcription PCR (RT-PCR) of a fusion transcript comprised
of 5' gene trap vector sequence spliced to the host gene transcript
3' of the insertion site. The gene trap vector functionally knocks
out the mouse LRP6 gene by forced spicing of LRP6 exon 1 to the
IRES-LacZ-PolyA element of the gene trap, preventing transcription
of LRP.
[0465] Chimeric mice were generated with ES cells, identified as
OST38808, by injection into C57BL/6 albino host blastocysts which
were then transferred to pseudopregnant females and allowed to
develop through birth. Germ line chimeras were backcrossed to
129SvEVBrd strain mice to maintain the knockout allele of LRP6 on
an inbred 1298SvEvBrd genetic background. Germ line transmission of
the LRP6-KO allele was identified by PCR amplification of a gene
trap specific sequence. Heterozygous LRP6-KO mating pairs were used
for continued breeding. The genotype of wt and LRP6-KO progeny is
determined by tail DNA PCR.
[0466] Measurements of bone density at 9 weeks of age in female
heterozygous knock-out mice has shown significant (p<0.05)
decreases in bone volume, trabecular number, and trabecular
thickness as measured by uCT. These results are consistent with the
hypothesis that LRP6 is also involved in modulating bone density
and is a target for development of therapies and drugs.
Accordingly, LRP6 transgenic animals and transgenic animals
expressing bone modulating variants of LRP6 are contemplated within
the scope of the invention.
[0467] 21.1.4. Gene-Targeted Mice Expressing the HBM
Polymorphism
[0468] A gene-targeting construct was prepared that could be used
to create animals containing a HBM or HBM-like knock-in (KI) allele
and a Zmax1 (LRP5) knock-out (KO) allele. The gene-targeting
construct contained the HBM polymorphism in exon 3 and included a
neomycin selection cassette that was linked to a transcriptional
stop sequence and was flanked with lox P sites. The HBM
polymorphism in mouse Zmax1 results in a glycine to valine change
in the amino acid sequence at position 170 of the mouse LRP5
homolog (Genbank Accession No. AF064984). Homologous recombination
is used to stably introduce the construct into the mouse genome. If
the transcriptional stop sequence functioned to completely block
transcription of the modified Zmax1 allele, then a functional Zmax1
knock-out allele would be generated. This would facilitate
production of a homozygous knock-out animal for the Zmax1 gene.
[0469] To create the knock-in allele, Cre recombinase could be used
to excise the neomycin selection cassette leaving behind the
modified exon 3 and one copy of the loxP site. Cre could be
introduced into single-cell fertilized embryos to facilitate
ubiquitous expression of HBM or by crossing animals with transgenic
mice to obtain bone-specific HBM expression.
[0470] Homozygous animals could be made for the HBM knock-in
allele. Alternatively, animals could be created by nuclear transfer
techniques, wherein nuclei from homozygous animals is transferred
into a prepared oocyte (e.g., enucleated) as is known in the art.
See, e.g., Campbell et. al., Nature 380: 64-68 (1996). Additional
methods of creating knock out mice include engineering a homologous
recombination vector wherein the ATG start codon is deleted or
mutated, engineering a frame-shift mutation into the vector,
engineering deletions of critical portions of the promoter region,
and/or engineering a vector to delete critical regions of the
gene.
[0471] The methods used for the LRP5, LRP6 and HBM mice can
similarly be used for other variants of these genes.
[0472] 21.2 Materials and Methods
[0473] 21.2.1 Construction of the Zmax1 (LRP5) Plasmid Zmax1
GI.sub.--3AS
[0474] The full-length Zmax1 cDNA construct has been engineered
into the XbaI-NotI sites of the pCMVSPORT6.0 vector from Life
Technologies (part of the Gateway cloning system) to create
Zmax1GI.sub.--3AS. The insert (5,278 bp) can be released from the
vector by digestion with either HindIII or XbaI on the 5' side
together with either EcoRV or EcoRI on the 3' end. A similar
construct can be prepared for LRP6 and HBM-like nucleic acids as
would be apparent to one of ordinary skill and is contemplated as
well in the discussion of this section.
[0475] The Zmax1 (LRP5) construct was generated from four
independent partial clones. These clones were isolated from a Zmax1
specific primed cDNA library. A partial Zmax1 cDNA clone existed in
the internal survey sequencing clone set as L236B_P0049E08 isolated
from an oligo-dT primed HeLa cell cDNA library. This clone was
truncated at the 5-primed end. In order to isolate more 5-prime
containing fragments necessary to generate a full length cDNA, a
Zmax1 gene-specific cDNA library was generated from Clontech human
liver poly-A mRNA (catalog #6510-1, lot# 9060032) and Life
Technologies SuperScript.RTM. Plasmid System for cDNA Synthesis and
Plasmid Cloning kit (catalog no. 18248-013). This library was
designated as L401. The manufacturer's protocol was carried out
with the following modifications. 1) In both first and second
strand synthesis reactions, DEPC-treated water was substituted for
.alpha.-.sup.32P-dCTP. 2) Reverse transcription was primed using
oligonucleotides that were selected to be specific for the Zmax1
gene at approximately 1 kb intervals. These sequences were checked
using the program BLAST against the public databases to ensure
Zmax1 specificity. 3) Two separate reverse transcription reactions
were performed. The first reaction, (A), was primed with
oligonucleotides which annealed to the more 3' regions of Zmax1
(LRP5) as follows:
TABLE-US-00033 47114: (SEQ ID NO: 715)
5'-CGTACGTAAAGCGGCCGCTTGGCAATACAGATGTGGGA-3' 47116: (SEQ ID NO:
716) 5'-CGTACGTAAAGCGGCCGCAGTAGCTCCTCTCGGTGGC-3' 47118: (SEQ ID NO:
717) 5'-CGTACGTAAAGCGGCCGCGCTCATCATGGACTTTCCG-3' 47120: (SEQ ID NO:
718) 5'-CGTACGTAAAGCGGCCGCGCACTGCTGTTTGATGAGG-3'
The second reaction, (B), used the previously mentioned four
oligonucleotides, as well as
TABLE-US-00034 47108: (SEQ ID NO: 719)
5'-CGTACGTAAAGCGGCCGCGAGTGTGGAAGAAAGGCTGC-3' 47110: (SEQ ID NO:
720) 5'-CGTACGTAAAGCGGCCGCAGTAGAGCTTCCCCTCCTGC-3' 47112: (SEQ ID
NO: 721) 5'-CGTACGTAAAGCGGCCGCGTCCATCACGAAGTCCAGGT-3'
All oligonucleotides contained a NotI linker sequence and were used
at a concentration of 0.02 ug/ul. 4) The SalI-adapted cDNA from
both reverse transcription reactions was size-fractionated by
electrophoresis on 1% agarose, 0.1 ug/ml ethidium bromide,
1.times.TAE gels. The ethidium bromide-stained cDNA between 0.6 and
8.0 kb was excised from the gel. The cDNA was purified from the
agarose gel by electroelution (ISCO Little Blue Tank
Electroelutor.RTM.) using the manufacturer's protocol. The purified
cDNA from reactions A and B were then pooled together. 5) The
size-fractionated SalI-adapted cDNA was ligated to NotI-SalI
digested pBluescript.RTM. (Stratagene, La Jolla, Calif.).
[0476] Ligated library cDNA (3 ml) was used to transform
electrocompetent E. coli cells (ElectroMAX.RTM. DH10B cells and
protocol, Life Technologies catalog no. 18290-015, BioRad E. coli
pulser, voltage 1.8 KV, 3-5 msec pulse). The transformed cells were
plated on LB-ampicillin (100 .mu.g/ml) agar plates and incubated
overnight at 37.degree. C. Approximately 10.sup.6 colony forming
units (cfu) were plated at a density of 50,000 cfu/150 mm plate.
Cells were washed off the plates with LB media, and collected by
centrifugation. Plasmid DNA was purified from the cells using the
QIAGEN Plasmid Giga Kit and protocol (catalog no. 12191) at a final
concentration of 2.05 .mu.g/.mu.l.
[0477] Two probes for use in library screening were generated by
the polymerase chain reaction (PCR) using 100 ng of library L401 as
template. Standard PCR techniques were used. A reaction mixture
contained 10 .mu.mol of each oligo primer; 0.2 mM each dATP, dTTP,
dCTP and dGTP (PE Applied Biosystems catalog no. N.sub.808-0260);
1.5 units Expand.TM. High Fidelity Taq DNA polymerase and
1.times.PCR reaction buffer (Roche Molecular Biochemicals, catalog
no. 732-641; 10 mM Tris-HCl, 1.5 mM MgCl.sup.2, 50 mM KCl, pH 8.3).
The mixture was incubated at 99.degree. C. for one minute, followed
by 30 cycles of 96.degree. C. for 15 seconds, 50.degree. C. for 30
seconds, 72.degree. C. for 1 minute with a final incubation at
72.degree. C. for 7 minutes (MJResearch DNA Engine.RTM. Tetrat
PTC-225). The first was generated using oligos
TABLE-US-00035 107335: 5'-CAGCGGCCTGGAGGATGC-3' (SEQ ID NO: 722)
107338: 5'-CGGTCCAGTAGAGGTTTCG-3' (SEQ ID NO: 723)
which amplify a NotI-SalI fragment of the Zmax1 gene. The second
was generated using oligos
TABLE-US-00036 107341: 5'-CATCAGCCGCGCCTTCATG-3' (SEQ ID NO: 724)
107342: 5'-CCTGCATGTTGGTGAAGTAC-3' (SEQ ID NO: 725)
which amplify a SacI-KpnI fragment of the Zmax1 gene. Both PCR
products were purified using the Qiaquick.RTM. kit and the
manufacturer's protocol (Qiagen catalog 28106) then subcloned into
the vector pCRII-TOPO.RTM. (Invitrogen catalog no. K4600) following
the manufacturer's protocol. Positive subclones were identified by
restriction digestion of purified plasmid DNA (using standard
molecular biology techniques) and subsequent DNA sequence analysis
(ABI Prism BigDye Terminator Cycle.RTM.sequencing, catalog no.
4303154, ABI 377 instruments). Probe DNA was isolated by EcoRI
restriction digestion (New England Biolabs, catalog no. R0101L) of
the respective sequence-verified pCRII-TOPO.RTM. clone. Restriction
fragments were size-fractionated by gel electrophoresis on 1%
agarose, 0.1 ug/ml ethidium bromide, 1.times.TAE gels. Insert DNA
was excised from the gel and purified using the Clontech
NucleoSpin.RTM. Nucleic Acid Purification Kit (catalog no. K3051-2)
following the manufacturer's protocol. The purified DNA fragment
(25-50 ng) was labeled with Redivue.RTM. (.alpha..sup.32P)-dCTP
(Amersham Pharmacia, catalog no. AA0005) using the Prime-It II.RTM.
Random Primer labeling Kit and protocol (Stratagene, catalog no.
300385). Unincorporated dCTP was removed with Amersham's NICK.RTM.
column and protocol (catalog no. 17-0855-02).
[0478] Two rounds of screening library L401 were initiated to
isolate fragments of the Zmax1 (LRP5) gene. In the first,
forty-three 150 cm LB-100 .mu.g/ml ampicillin agar plates were
plated with primary transformants from L401 at a density of about
3,000-4,000 colonies per plate. This library was screened using the
.sup.32P-labeled probe (NotI-SalI fragment) as described above at
500,000-1,000,000 cpm/m hybridization buffer, using standard
molecular biology protocols. From this primary screen, 13 single
colonies were identified based on positive hybridization to the
Zmax1 probe. Plasmid DNA, prepared using the QIAprep Spin Miniprep
Kit and protocol (Qiagen Inc., catalog no. 27106), was analyzed by
restriction digestion and sequence analysis as described above.
cDNA clone #44 was isolated from this screen and sequence verified
to contain a partial Zmax1 clone.
[0479] In the second library screen, one hundred-and-four 150 cm LB
ampicillin 100 ug/ml agar plates were plated with primary
transformants from L401 at a density of 3000-4000 colonies per
plate. This library was screened using the .sup.32P-labeled probe
(SacI-KpnI) exactly as previously described. From this primary
screen, 48 colonies were identified based on positive hybridization
to the Zmax1 (LRP5) probe. Since these colonies were not single
colony isolates, a secondary screen was initiated where each of the
48 primary isolates was plated at a density of approximately 500
colonies per plate. These colonies were then screened exactly as
the primaries using the labeled SacI-KpnI fragment as probe.
Thirty-four of the 48 primary clones resulted in positive
hybridization to the Zmax1 probe and were isolated as single
colonies. Plasmid DNA was prepared and analyzed as described above.
cDNA isolate #71.sub.--2 was isolated from this screen and sequence
verified to contain a partial Zmax1 clone.
[0480] In all cases, the sequence of any Zmax1 (LRP5) isolate was
compared to a reference sequence (i.e., the sequence of the
wild-type Zmax1 allele from an affected member of the HBM kindred).
This analysis was important since DNA polymorphisms had been
reported for this gene in the literature. This reference sequence
is predicted to encode a polypeptide of Genbank Accession No.
AF077820.
[0481] The four independent partial clones used to prepare
ZmaxIGI.sub.--3AS are as follows: [0482] 1). Bases 1-1366: A
XbaI-SalI fragment was obtained from a Zmax1 cDNA construct,
GTC.Zmax1.sub.--13. GTC.Zmax1.sub.--13 contains a 5075 BP insert
containing the entire ORF of LRP5. The clone was blunt end cloned
in the EcoRV site of pSTBlue-1. This clone was generated by fusing
a 5' clone derived from screening a bone random primed cDNA library
in a pBluescript.TM. II derivative with a 3' clone derived from a
PCR product from a bone dT primed cDNA library in pBluescript.TM.
II. PCR was performed using LRP5 specific forward primer
5'-GCCCGAAACCTCTACTGG ACCGAC-3' (SEQ ID NO: 726) and reverse primer
5'-GCCCACCCCATCACAGTTCA CATT-3' (SEQ ID NO: 727) using DNAzyme
polymerase. The resultant 3.7 kb PCR product was cloned into
PCR-XL-TOPO. To generate the full length clone the 5' and 3'
plasmids were transformed into DM1 (dam-) from Gibco/BRL. The 5'
plasmid was digested with XbaI and the 3' plasmid was digested with
HindIII. The digested plasmids were filled in with T4 polymerase to
generate blunt ends and cut with BclI the 1.7 kb 5' fragment and
3.5 kb 3' fragments were gel purified, ligated together, and cloned
in the EcoRV site of pSTBlue-1. It provides a short 5'UTR, with
coding sequence beginning at base 100. Furthermore, it carries some
additional restriction sites at the 5' multiple cloning site. This
fragment also contains a DNA polymorphism relative to the Genbank
Accession No. AF077820 sequence at position 558 resulting in an A
(AF077820) to a G change; this mutation does not result in an amino
acid difference (Pro). [0483] 2). Bases 1367-2403: This clone was
obtained from a Zmax1-gene primed cDNA library made from commercial
human liver RNA described above. This fragment is a SalI-BamHI
piece of DNA obtained from isolate #44. The sequence is identical
to Genbank Accession No. AF077820. [0484] 3). Bases 2404-4013: This
BamHI-BssHII fragment was obtained from isolate #71-2 from the same
library as described above. At position 3456, there is a DNA
polymorphism resulting in a G (AF077820) to an A. This nucleotide
difference does not change the encoded amino acid (Val). [0485] 4).
Bases 4014-5278: This BssHII-NotI fragment came from an internal
clone, L236B_P0049E08. It was obtained from an oligo-dT primed HeLa
cell cDNA library. The stop codon occurs at base 4947. The clone
contains 331 bp of 3' UTR sequence, including a 120 bp poly-A tail
followed by the NotI site. The 3' NotI site used in this subcloning
step is a result of an added linker that was introduced at the end
of the poly-A tail during library construction. A DNA polymorphism
is present at base 4515 resulting in a G (AF077820) to C change
that is silent at the amino acid level (Leu).
[0486] To generate the 5' section of the Zmax1 (LRP5) gene, the
XbaI-SalI fragment and SalI-BamHI fragment were ligated into the
XbaI-BamHI sites of pBluescript.RTM. (Stratagene). The 3' section
of the gene was obtained by ligating the 1.61 kb BamHI-BssHII
fragment from Zmax1 isolate #71.sub.--2 to the 1.26 kb BssHII-NotI
fragment from L236B_P0049E08. These two fragments were ligated into
the BamHI-NotI sites of pBluescript.RTM.. The full length Zmax1
cDNA was engineered into the XbaI-NotI sites of the vector
pCMVSPORT6.0 (Life Technologies) by ligation of the XbaI-BamHI 5'
section and the BamHI-NotI 3' section. The resulting plasmid,
ZmaxGI.sub.--3AS, contains an insert of 5278 bp from the XbaI site
to the NotI site of the vector's multiple cloning site. This clone
is in the antisense orientation with respect to the CMV promoter
present in the vector. Zmax1 coding sequence begins at base 100 and
ends at base 4947, followed by 331 bp of 3-primed UTR sequence
including a 120 bp poly-A tail. This full length cDNA contains
three DNA polymorphisms from the reference sequence (GenBank
Accession No. AF077820) that do not alter the predicted amino acid
sequence. These polymorphisms great position 558 resulting in an A
to G that maintains the proline residue; at position 3456 resulting
in a G to A that maintains the valine residue; and, at position
4515 resulting in a G to C that maintains the leucine residue.
[0487] The sequence of Zmax1GI.sub.--3AS (FIG. 25) also contains a
DNA polymorphism relative to SEQ ID NO. 1 at base 4088 resulting in
a C (SEQ ID NO: 1 and Genbank Accession No: AB017498) to T change
that results in an amino acid change at position 1330 of alanine to
valine. This is consistent with the sequence determined in the
wild-type allele from an affected member of the HBM kindred as well
as with the published sequence of Genbank Accession No. AF077820.
Zmax1GI 3AS also has 29 additional bases at the 5' end relative to
SEQ ID NO: 1, as well as 129 bases at the 3' end consisting of an
extra G, 120 bases of poly-A tract, and the NotI site.
[0488] 21.2.2 Creation of the HBM Mutation G171V
[0489] The HBM mutation that results in a predicted amino acid
change from glycine to valine at amino acid 171 was introduced into
the full length human Zmax1 (LRP5) cDNA (plasmid Zmax1GI.sub.--3AS)
using PCR to change the G at position 611 to a T. Introduction of
the HBM mutation was done using oligos 107335: (5'-CAGCGGCCTGGA
GGATGC-3'; SEQ ID NO: 728) and 49513: (5'-CGGGTACATGTACTGGACAGC
TGATTAGC-3'; SEQ ID NO: 729), which flank the endogenous NotI site
of the Zmax1 (LRP5) gene. This method creates a new PvuII site at
the 3' end of the PCR product. A second PCR reaction was completed
using oligos which introduce a ScaI site at the 5' end of the
product and contains the endogenous SalI site of Zmax1 in the
3-primed end. PCR products were purified using the QiaQuick.RTM.
procedure (Qiagen Inc.); subcloned into the vector pCRII-TOPO
(Invitrogen) as described above. Plasmid DNA was purified from
single bacterial colonies and analyzed by restriction digest and
subsequent sequence analysis, all as described above. The
sequence-verified pCRII-TOPO clones were restriction digested with
NotI-PvuII and ScaI-SalI, respectively. The resulting DNA fragments
were size fractionated and purified as described above. These two
fragments were then subcloned into the vector, pBluescript.RTM.
that had been prepared by NotI-SalI digestion. Both PvuII and ScaI
produce blunt ends when used to digest double stranded nucleic
acids. Thus, the resulting ligated fragment fails to recreate
either the PvuII or ScaI site and contains only the consensus Zmax1
sequence, with the exception of the newly introduced HBM mutation.
To introduce the mutation into the full length Zmax1 gene, this
resulting plasmid was digested with MscI and SalI, while the 5'
region of Zmax1 was obtained by XbaI-MscI digestion of Zmax1
plasmid GTC.Zmax1.sub.--13. These two fragments were ligated
together into XbaI-SalI digested pBluescript.RTM., in effect
creating a similar 1.366 kb XbaI-SalI fragment. The only difference
being that this construct contains the HBM mutation described
above. The full length HBM cDNA then was assembled into
pCMVSPORT6.0 exactly as described above for the Zmax1 gene, with
the substitution of this newly created XbaI-SalI fragment
containing the HBM mutation. The entire cDNA insert was verified by
DNA sequence analysis and the introduction of the HBM mutation was
confirmed.
[0490] The resulting plasmid, HBMGI.sub.--2AS (FIG. 24), contains
an insert of 5,278 bp from the XbaI site to the NotI site of the
vector's multiple cloning site. This clone is in the antisense
orientation with respect to the CMV promoter in the vector. HBM
coding sequence begins at base 100 and ends at base 4947, followed
by 331 bp of 3-primed UTR sequence which includes a 120 bp poly-A
tail. This full length cDNA contains three DNA polymorphisms from
the reference sequence, which do not alter the amino acid sequence.
These polymorphisms occur at position 558, resulting in an A to G
change that maintains the proline residue; at position 3456
resulting in a G to A change that maintains the valine residue;
and, at position 4515 resulting in a G to C change that maintains
the leucine residue. Additionally, the HBM mutation is present at
position 611 (G in Zmax1 to T in HBM) which results in a predicted
amino acid change of glycine to valine at amino acid position 171,
as found in affected members of the HBM kindred. This insert
sequence was used to generate the construct used for HEM
over-expressing transgenic mice.
[0491] 21.3 Transgene Preparation
[0492] The examples provided herein are illustrations of how
transgenic animals can be prepared. Additional transgenic animals
can be prepared as would be known in the art. See, for example,
Glenn Monastersley et al., ed, Strategies in Transgenic Animal
Studies (Amer. Soc. Microbiology 1995) and the references cited
therein.
[0493] 21.3.1 CMVbActin Promoter-HRM cDNA (HBMMCBA)
[0494] To prepare the CMVbactin-HBM construct, pCX-EGFP, a plasmid
containing the chimeric CMVbactin promoter, was purified as a 4778
bp EcoRI fragment. Subsequently, the HBM cDNA was excised from
HBMGI.sub.--2AS as a 4994 bp XbaI/DraI fragment, treated with
Klenow fragment of DNA polymerase, ligated to EcoRI linkers, and
digested with EcoRI. This fragment was then inserted into the EcoRI
site of pCX-EGFP. A SpeI/HindIII 7265 bp CMVbactin-HBM fragment was
purified for microinjection into mouse embryos.
[0495] 21.3.2 Type I Collagen Promoter-HBM cDNA (HBMMTIC)
[0496] The rat type I collagen promoter-HBM construct was created
by first replacing the pBS(SK-) (Stratagene) polylinker with
another polylinker (i.e., comprising
Kpn1-SpeI-HindIII-BglII-NdeI-SalI-SmalI-EcoRI-PstI-BamHI-XbaI-ScaI-NcoI-C-
laI-NotI-SacII-SacI), that is referred to as BS(SK-)A/D. The SV40
splice and poly (A), XbaI-NcoI region (750 bp) from pcDNA I
(Invitrogen, Inc.) was directionally cloned into BS(SK-)A/D. Next,
a 4994 bp EcoRI HBM cDNA fragment (above) was cloned into the EcoRI
site. A 3640 bp XbaI, type I collagen promoter fragment was
subcloned into the XbaI site of BS(SK-) (Stratagene). The promoter
fragment was then excised from BS(SK-) with SacII, blunt-ended with
T4 DNA polymerase, digested with SpeI, and ligated into the HBM
BS(SK-) A/D construct, which was digested with Nde I, blunted with
T4 DNA polymerase, and digested with SpeI. A SpeI/ClaI 9435 bp type
I collagen-HBM fragment was purified for microinjection into mouse
embryos.
[0497] 21.3.3 CMVbActin promoter-Zmax1 cDNA (Zmax1WTCBA)
[0498] The CMVbActin promoter-HBM cDNA construct from above was
used to generate the final plasmid. The following three fragments
were ligated together: 1) a 6.34 kb XbaI-KpnI backbone fragment
from HBMMCBA; 2) a 0.64 kb XbaI-SapI fragment from HBMMCBA
containing the 3' end of the bActin promoter and the 5' end of the
HBM cDNA; and 3) a 2.8 kb SapI-KpnI fragment derived from the Zmax1
cDNA that contains the wild-type sequence. A 7.2 kb SpeI-HindIII
CMVbActin-Zmax1 fragment was purified for micro-injection into
mouse embryos.
[0499] 21.3.4 Type I Collagen Promoter-Zmax1 cDNA (Zmax1WTTIC)
[0500] The type I collagen --HBM cDNA construct from above was used
to generate the final plasmid. The HBMMTIC plasmid was linearized
with HindIII and cut with either SalI to yield a 8.52 kb
HindIII-SalI fragment or SapI to yield a 2.98 kb SapI-HindIII
fragment.
[0501] A 2.8 kb SapI-SalI fragment from the Zmax1 (LRP5) cDNA
containing the wild-type sequence was then ligated to the above two
fragments to yield the final plasmid. A 9.4 kb SpeI-ClaI type I
collagen-Zmax1 fragment was purified for micro-injection into mouse
embryos.
[0502] 21.4 Confirmation of Transgene Expression In Vitro
[0503] Plasmid constructs for HBMMCBA, HBMMTIC, Zmax1WTCBA and
Zmax1WTTIC were transiently transfected into human osteoblast (HOB)
cells to measure mRNA expression as a test for functionality.
[0504] 21.4.1 Transient Transfections
[0505] HOB-02-02 cells are a clonal, post-senescent, cell line
derived from the HOB-02-C1 cells (Bodine et. al, 1996, J. Bone
Miner. Res. 11: 806-819). Like the parental cell line, the
HOB-02-02 cells express the temperature-sensitive SV40 large
T-antigen mutant, tsA209. Consequently, these cells proliferate at
the permissive temperature of 34.degree. C., but stop dividing at
non-permissive temperatures of 37.degree. C. or above. Also like
the parental cell line, the HOB-02-02 cells are cultured with
Growth Medium (D-MEM/F-12 containing 10% heat inactivated fetal
bovine serum, 1% penicillin-streptomycin and 2 mM GlutaMAX-1) at
34.degree. C. in a 5% CO.sub.2/95% humidified air incubator (Form a
Scientific, Marietta, Ohio).
[0506] For the transient transfections, the HOB-02-02 cells were
seeded with Growth Medium at 400,000 cells/well into 6-well plates
and incubated overnight at 34.degree. C. The cells were transfected
with 0.3 mg/well of either the CMV.beta.Actin-HBM expression
plasmid, the Type I Collagen-HBM expression plasmid or the
corresponding empty vectors using LipofectAMINE 2000 transfection
reagent according to the manufacturer's instructions (Life
Technologies, Rockville, Md.). After a 24 hr incubation at
34.degree. C., the medium was changed, and the cells were incubated
for an additional 24 hr at 39.degree. C. At the end of this last
incubation, the cells were rinsed with Hank's buffered salt
solution. Total cellular RNA was then isolated using TRIzol.RTM.
according to the manufacturer's instructions (GibcoBRL, Grand
Island, N.Y.). The RNA was treated with RNase-free DNase in order
to remove contaminating DNA as previously described (Bodine et al.,
1997, J. Cell. Biochem. 65: 368-387).
[0507] 21.4.2 TaqMan.RTM. Assay for mRNA Expression
[0508] TaqMan.RTM. primers and probes were chosen based on human
and mouse Zmax1 (LRP5) cDNA sequences. The selected sequences were
designed to be gene-specific by analysis of an alignment of human
and mouse Zmax1 (LRP5) sequences as illustrated in FIG. 26.
[0509] TaqMan.RTM. quantitative reverse transcriptase-polymerase
chain reaction (RT-PCR) analysis of RNA isolated from human cells
was performed as described by the manufacturer (PE Applied
Biosystems, Foster City, Calif.) using the following primers and
probe set:
Human Zmax1-1/HBM-1:
TABLE-US-00037 [0510] Forward Primer: 5'-GTCAGCCTGGAGGAGTTCTCA-3'
(SEQ ID NO: 730) Reverse Primer: 5'-TCACCCTTGGCAATACAGATGT-3' (SEQ
ID NO: 731) Probe: 6-FAM-5'-CCCACCCATGTGCCCGTGACA-3' (SEQ ID NO:
732)
[0511] Results from the experimental primers/probe set were
normalized to human GAPDH levels using the multiplex protocol with
the human GAPDH control kit from PE Applied Biosystems.
Species-specific TaqMan.RTM. quantitative RT-PCR analysis of RNA
isolated from murine cells and tissues was performed as described
by the manufacturer (PE Applied Biosystems, Foster City, Calif.)
using the following primers and probes sets:
Human Zmax-1/HBM-1:
TABLE-US-00038 [0512] Forward Primer: (SEQ ID NO:733)
5'-CGTGATTGCCGACGATCTC-3' Reverse Primer: (SEQ ID NO:734)
5'-TTCCGGCCGCTAGTCTTGT-3' Probe: (SEQ ID NO:735)
6-FAM-5'-CGCACCCGTTCGGTCTGACGCAGTAC-3'
Mouse Zmax-1/HBM-1:
TABLE-US-00039 [0513] Forward Primer: (SEQ ID NO:736)
5'-CTTTCCCCACGAGTATGTTGGT-3' Reverse Primer: (SEQ ID NO:737)
5'-AAGGGACCGTGCTGTGAGC-3' Probe: (SEQ ID NO:738)
6-FAM-5'-AGCCCCTCATGTGCCTCTCAACTTCATAG-3'
[0514] Results from the experimental primers/probe sets were
normalized to 18S ribosomal RNA levels using the multiplex protocol
with the 18S ribosomal RNA control kit from PE Applied Biosystems.
A summary of these results is presented in FIGS. 17-20.
[0515] 21.5 Production of Transgenic Mice
[0516] 21.5.1 DNA Microinjection
[0517] Transgene fragments for micro-injection were first purified
on 1% agarose gels according to the GELase protocol from Epicentre
Technologies. Fragments were then further purified on cesium
chloride density gradients and extensively dialyzed against 5 mM
Tris (pH 7.4), and 0.1 mM EDTA.
[0518] Linearized DNA was microinjected into mouse embryos
according to standard procedures. DNA was injected into primarily
the male pronucleus of fertilized C57BL/6T mouse embryos. Injected
embryos (n=20-35) were transferred to the oviduct (unilaterally) of
day 0.5 post coitum pseudopregnant Swiss Webster embryo recipients.
Offspring were tail-biopsied and genotyped at age 10-14 days.
[0519] 21.6 Production of Gene-Targeted Transgenic Mice
[0520] 21.6.1 Gene Targeting Vectors and Probes
[0521] Two gene-targeting vectors were constructed for modification
of the Zmax1 (LRP5) gene in embryonic stem (ES) cells. The two
constructs, illustrated in FIG. 16, designated as Zmax1-KI/KO
A&B were designed to generate two types of mutations, a
knock-out (KO) of the Zmax1 gene and a Cre recombinase dependent
knock-in (KI) of a nucleotide substitution in order to create a
mouse model (i.e., glycine 170 to valine amino acid substitution in
mouse Zmax1, of the HBM kindred.
[0522] Both gene-targeting vectors were constructed using genomic
DNA of the mouse genomic DNA BAC clone 473P5 (Genbank Accession No.
AZ095413) containing the first five exons of the mouse Zmax1 gene.
This clone was isolated by Research Genetics (Huntsville, Ala.)
from their mouse 129SvJ genomic BAC library using a polymerase
chain reaction (PCR) screen for exon 3. A forward and reverse
primers of
TABLE-US-00040 Forward: 5'-GAGCGGGCAGGGATGGATGG-3' (SEQ ID NO: 739)
Reverse: 5'-AGGTTGGCACGGTGGATGAAGC-3' (SEQ ID NO: 740)
were used to amplify exon 3 by PCR; the following thermal cycling
conditions were employed: for thirty cycles, 95.degree. C. for 0.5
minute, 55.degree. C. for 1 minute and 72.degree. C. for 1 minute.
Identity of this clone with mouse Zmax1 was confirmed by sequencing
exon 3 using the BAC clone DNA as template. PCR products were
cloned using the pGEM-T-easy T/A cloning kit.
[0523] 21.6.2 Zmax1 (LRP5) Knock-In/Knock-Out Vector
[0524] The organization of the genomic BAC clone 473P5 was
characterized by Southern blot analysis using subcloned exon 1,
exon 2 and exon 3 as probes and by sequencing the region spanning
exon 1 through exon 5. Two different constructs were prepared for
the Zmax1 KI/KO targeting. These constructs (A and B) differ only
in flanking arms of homology. Construct (A) contains a 6.5 kb
BstEII-XbaI 5' arm of homology and a 1 kb XbaI-EcoRI 3' arm of
homology; whereas, construct (B) contains a 1 kb 5' arm of homology
and a 6.0 kb 3' arm of homology. The constructs were prepared by
ligating short and long arms of homology to a LoxP flanked cassette
containing the neomycin resistance gene (MC1-Neo, Stratagene) and a
synthetic transcriptional pause sequence (Promega).
[0525] Both Zmax1-KI/KO-targeting vectors (A and B) were modified
to a G-to-T nucleotide substitution, encoding the G170V amino acid
substitution, in exon 3. These modifications were introduced into
by overlapping PCR mutagenesis using the wild type sequence of the
short arm of homology as template. In addition, the 1 kb short arm
of the Zmax1-KI/KO (B) targeting vector was modified to include a
5' terminal PmeI restriction recognition site. The 5' overlapping
fragment was made using the following forward primer and reverse
mutagenic primer:
TABLE-US-00041 Forward: (SEQ ID NO: 741)
5'-AAGCTTGTTTAAACTGGGCATGGTGGCACATGGTTGTAAT-3' Reverse: (SEQ ID NO:
742) 5'-GGGCTTCCACCCAGTCAGTCCAGTACATGTACCT-3'.
The thermal cycling conditions utilized for thirty cycles are
95.degree. C. for 0.5 minute, 55.degree. C. for 1 minute and
72.degree. C. for 1 minute. The 3' fragment was made using the
following forward and reverse primers:
TABLE-US-00042 Forward: (SEQ ID NO: 743)
5'-CTGACTGGGTGGAAGCACCCCGGATCGAGC-3' Reverse (mutagenic): (SEQ ID
NO: 744) 5'-GAATTCATCGGTACCTGTGCGGCCGCTTCATTG-3'.
The thermal cycling conditions utilized for thirty cycles are
95.degree. C. for 0.5 minute, 55.degree. C. for 1 minute and
72.degree. C. for 1 minute. The final overlapping PCR used 1 ml
each of the 5' fragment and 3' fragment PCR reactions as template
and amplification was performed using the forward and reverse
primers of the 5' and 3' fragments respectively and the same
thermal cycling parameters. The final PCR product was cloned using
the pGEM-T-easy T/A cloning kit. The mutagenized exon 3 was excised
from Zmax1-KI/KO (B) and transferred to Zmax1-KI/KO (A) as a 600 bp
BsmBI-XbaI fragment.
[0526] Probes for screening for and characterization of Zmax1-KI/KO
(A) gene targeted ES cell clones are prepared by subcloning
restriction fragments of BAC clone 473P5. The 5' outside probe is a
400 bp Nde-BstEII fragment, and the 3' outside probe is a 500 bp
EcoRI-BstXI fragment.
[0527] The outside probes for Zmax1-KI/KO (B) are prepared by PCR
cloning genomic fragments flanking and immediately adjacent to the
targeting vector region of homology. The 5' outside probe used for
Zmax-KI/KO (B) is a 498 bp fragment generated using the forward
primer of the sequence (5'-TGAGATGTCCTGTCTGTGGC-3'; SEQ ID NO: 745)
and a reverse primer of the sequence (5'-TCCTTCCTTCCCTACAGTTG-3';
SEQ ID NO: 746). The thermal cycling conditions utilized with these
probes for thirty cycles are: 95.degree. C. for 0.5 minute,
55.degree. C. for 1 minute and 72.degree. C. for 1 minute. The 3'
outside probe is a 600 bp fragment generated using the forward
primer of the sequence (5'-CCTAAGGATCTCCTTGT GTCTGTGG-3'; SEQ ID
NO: 747) and a reverse primer of the sequence (5'-CTGCAGCAG
GTCAGTAGCCTGC-3'; SEQ ID NO: 748). The thermal cycling conditions
utilized with these probes for thirty cycles were: 95.degree. C.
for 0.5 minute, 55.degree. C. for 1 minute and 72.degree. C. for 1
minute. Both probes are specific for the Zmax1 gene in genomic
southern analysis. PCR products are cloned using the pGEM-T-easy
T/A cloning kit.
[0528] A probe for ribonuclease protection analysis of Zmax1 mRNA
structure and transcription levels was prepared by PCR cloning a
cDNA fragment containing exon 3 through exon 4. The PCR reaction
used a complete cDNA as template, a forward primer of the sequence
(5'-TGAGATGTCCTGTCTGTGGC-3'; SEQ ID NO: 749), a reverse primer of
the sequence (5'-TCCTTCCTTCCCTACAGTTG-3'; SEQ ID NO: 750) and the
following thermal cycling conditions for thirty cycles; 95.degree.
C. for 0.5 minute, 55.degree. C. for 1 minute and 72.degree. C. for
1 minute. The PCR product is cloned using the pGEM-T-easy T/A
cloning kit.
[0529] One skilled in the art could use similar protocols to
generate other engineered KI-alleles to produce transgenic animals
with an HBM-like phenotype.
[0530] 21.6.3 Gene Targeting in ES Cells
[0531] For gene targeting, embryonic stem (ES) cells are
electroporated with 50 mg of linearized targeting vector and
selected in 200 mg/ml G418 for 7-10 days beginning the day after
electroporation. G418 resistant clones are picked, expanded and
cryopreserved. Resistant clones were screened for homologous
recombination by an EcoRI genomic Southern-restriction fragment
length analysis using the 5' outside probe, which detects the wild
type and targeted alleles of Zmax1 as 4 kb and 5 kb fragments,
respectively. Gene targeted ES cell clones are thawed, expanded,
and characterized by ScaI genomic restriction fragment length
analysis using the 3' outside probe, which detects the wild type
and targeted alleles of Zmax1 as 9 kb and 8 kb fragments,
respectively. Gene targeted clones are also characterized by
sequence analysis of Zmax1 exon 3 to ensure that the G to T
substitution was included in homologous recombination.
[0532] 21.6.4 Production of Gene Targeted Mice by Blastocyst
Injection
[0533] To generate chimeric mice, gene targeted ES cell clones are
thawed, expanded and 9-14 ES cells injected into the blastocoel of
3.5 post coitum (p.c.) host C57BL/6 blastocysts. Injected
blastocysts (12-17) are then transferred unilaterally into the
uterus of 2.5 p.c. pseudopregnant Swiss Webster embryo recipients
and allowed to develop to term. Chimeric males are back-crossed to
129SvEv females and tested for transmission of the targeted allele
by PCR geneotyping with primers specific to the neomycin resistance
gene.
[0534] 21.6.5 In Vitro Deletion of the Neomycin Resistance Cassette
Via Cre Recombinase
[0535] To generate Zmax1 (LRP5) KI mice from the Zmax1 KI/KO mice
the Neomycin resistance (KO) cassette was deleted by micro
injection of a Cre expressing plasmid (2 mg/ml) into the male
pronucleus of Zmax1 KI/KO pre-fusion zygotes. Deletion of the KO
cassette was confirmed by PCR analysis of the cassette insertion
site.
[0536] 21.6.6 Genotyping Transgenic Mice
[0537] Genomic DNA was isolated from mouse tail snips by digestion
in 500 ul buffer containing 50 mM Tris-HCl, (pH 7.2), 50 mM EDTA,
(pH 8.0), 0.5% SDS and 0.8 mg/ml proteinase K. Samples are
incubated at 55.degree. C. with shaking overnight. A 10 .mu.l
aliquot was heat-inactivated at 99.degree. C. for 5 minutes and
diluted 1:20 in water. For PCR, 1 .mu.l of the diluted DNA was
amplified under the following conditions: Denature: 96.degree. C.
for 4.5 min; 45 cycles: 96.degree. C. for 30 sec; 63.degree. C. for
1 min; 72.degree. C. for 1 min; Extension: 72.degree. C. for 5 min;
4.degree. C. hold.
[0538] The following primer sets are used for genotyping:
HBMMCBA:
5' Primers: 296 bp Fragment (SEQ ID NOS: 751-752 Respectively)
TABLE-US-00043 [0539] Forward: 5'-GCT TCT GGC GTG TGA CCG GCG-3'
Reverse: 5'-GCC GCA CAG CGC CAG CAG CAG C-3'
3' Primers: 400 hp Fragment (SEQ ID NOS: 753-754 Respectively)
TABLE-US-00044 [0540] Forward: 5'-CAC CCA CGC CCC ACA GCC AGT A-3'
Reverse: 5'-ATT TGC CCT CCC ATA TGT CCT TCC-3'
HBMMTIC:
5' Primers: 382 bp Fragment (SEQ ID NOS: 755-756 Respectively)
TABLE-US-00045 [0541] Forward: 5'-TTC CTC CCA GCC CTC CTC CAT
CAG-3' Reverse: 5'-GCC GCA CAG CGC CAG CAG CAG C-3'
3' Primers: 524 bp Fragment (SEQ ID NOS: 757-758 Respectively)
TABLE-US-00046 [0542] Forward: 5'-GAA TGG CGC CCC CGA CGA C-3'
Reverse: 5'-GCT CCC ATT CAT CAG TTC CAT AGG-3'
[0543] 21.6.7. Confirmation of Genotype by Southern Analysis
[0544] Mouse genomic DNA was digested with EcoRI and probed with a
1.0 kb SalI-BamHI restriction fragment from the Zmax1 cDNA. The
probe hybridizes to a 5 kb fragment in transgene positive
animals.
[0545] 21.7 Phenotyping
[0546] Both in vivo and ex vivo assays are used to evaluate the
phenotype in transgenic mice. Two strains of wild-type mice, namely
C57BL/6 and 129 SvEv, are studied to provide control data for
phenotypic evaluation in transgenic and gene-targeted mice. In
addition, non-transgenic littermate animals are used as
controls.
[0547] 21.7.1 In Vivo Analysis
[0548] Spinal bone mineral content (BMC) and bone mineral density
(BMD) measurements performed at Creighton University (Omaha, Nebr.)
were made by DXA using a Norland Instruments densitometer (Norland
XR2600Densitometer, Dual Energy X-ray Absorptiometry, DXA). Spinal
BMC and BMD at other locations used the machinery available. There
are estimated to be 800 DXA machines currently operating in the
U.S. Most larger cities have offices or imaging centers which have
DXA capabilities, usually a Lunar or Hologic machine. Each location
that provided spine BMC and BMD data included copies of the
printouts from their machines to provide verification that the
regions of interest for measurement of BMD have been chosen
appropriately. Complete clinical histories and skeletal radiographs
were obtained.
[0549] The HBM phenotype (and HBM like phenotype which is also
included when discussing the HBM phenotype) in human and animal
subjects, preferably humans, can be described using criteria such
as: very high spinal BMD; a clinical history devoid of any known
high bone mass syndrome; and skeletal radiographs showing a normal
shape of the appendicular skeleton.
[0550] pDXA: Wild-type and transgenic mice are anesthetized,
weighed and whole-body X-ray scans of the skeleton generated using
the LUNAR small animal PIXImus device. Scans are begun when the
mice are weaned (i.e., at 3 weeks of age) and repeated at 2 week
intervals. Wild-type animals are scanned at 3, 5, 7, 9, 11, 13, 15,
17, 19, 21, 23, 27, and 29 weeks. Scanning of transgenic animals
would be performed for periods up to 17 weeks. Scans are analysed
for BMD (bone mineral density), BMC (bone mineral content), TTM
(total tissue mass), and % fat for various body regions.
[0551] Faxitron radiographs: Following pDXA scanning of
anesthetized animals, an additional X-ray was taken using a
Faxitron device allowing measurement of bone size.
[0552] Calcein labeling: Animals are dosed with 15 mg/kg calcein
intraperitoneally on two consecutive occasions. The first dose was
given 9 days before euthanasia and the second given 2 days before
euthanasia allowing measurement of bone formation rate.
[0553] 21.7.2 Ex Vivo Analysis
[0554] RNA isolation: Total RNA was isolated from tibia and other
tissues using TRIzol.RTM. to determine mRNA expression.
[0555] pQCT: The right femur was cleaned of soft tissue and stored
in 70% ethanol for determination of total and trabecular density of
the distal metaphysis and cortical density of the mid-shaft.
[0556] MicroCT. The right femur was used to determine trabecular
indices of the distal metaphysis.
[0557] Histology: The right femur was used to determine bone area
and static and dynamic parameters of the distal metaphysis.
[0558] Bending strength: The left femur was cleaned of soft tissue
and stored at -20.degree. C. prior to analysis of 3-point bending
strength of the mid-shaft.
[0559] Compressive strength of vertebra: The entire spine was
removed from T10-L6-7. Soft tissue was left on and the spine frozen
at -20.degree. C. until analysis. Compressive strength was measured
at the L5 vertebra.
[0560] Serum: Animals are euthanized and serum prepared from blood
to measure total cholesterol, triglycerides, osteocalcin and other
biochemical markers.
[0561] Lysis: Examples include immunocytochemistry, such as in situ
hybridization of osteogenic markers and TUNEL staining of cells
undergoing apoptosis.
[0562] 21.8 Results
[0563] 21.8.1 Confirmation of Expression from Transgenic Plasmid
Constructs
[0564] The HBM (HBMMCBA and HBMMTIC) and wild-type (Zmax1WTCBA and
Zmax1WTTIC) plasmid constructs were transiently transfected into
HOB-02-02 cells, which have a very low endogenous level of Zmax1
(LRP5) expression. Two days after transfection, RNA was isolated
and TaqMan.RTM. quantitative RT-PCR was performed to determine the
mRNA levels of Zmax1/HBM in the cells. To control for contaminating
plasmid DNA, PCR was performed with or without the prior RT step,
in the absence of the RT step, only very low levels of Zmax1/HBM
mRNA were detected. However; with the RT step, a 1000-fold increase
in HBM and Zmax1 mRNA was observed in cells transfected with
CMVbActin-promoter constructs as compared to those transfected with
the CMV-b-Galactosidase control. The type I collagen promoter
constructs showed approximately 10-fold increases in HBM and Zmax1
mRNAs, which is consistent with the weaker nature of this promoter
compared to the CMV.beta.Actin promoter. See FIG. 17.
[0565] 21.8.2 Species Specific TagMan.RTM. Reagents for HBM/Zmax1
Expression
[0566] Species specific TaqMan.RTM. primer and probe sets for
Zmax1/HBM were developed. In a series of experiments using HOB
cells and mouse MC-3T3-E1 osteoblastic cells, Zmax1/HBM mRNA was
measured in a mouse background, and vise versa. These reagents
useful for the detection and quantization of species-specific
expression. As demonstrated in FIG. 18, the primer sets are species
specific in the mouse and human cell lines. Further, FIG. 19
demonstrates the quantitative measurement of human Zmax1 RNA in a
background of mouse RNA. These TaqMan.RTM. sets can be used to
determine the levels of human or mouse HEM or Zmax1 (or other
HBM-like variant) message that are being expressed in the mouse
transgenic lines.
[0567] The species-specific TaqMan.RTM. reagents are novel tools
for the characterization of both endogenous Zmax1 mRNA levels and
human Zmax1/HBM mRNA levels in the transgenic mouse tissues. These
tools have several advantages over other conventional methods, such
as Northern hybridization and standard RT-PCR. Some of these
advantages are as follows: (1) specificity, since only a small
region (<100 bps) is amplified primers and probes are chosen to
sequence regions predicted to have no or minimal cross-reactivity;
(2) speed, since the procedure is less labor intensive; (3)
accuracy, since it is truly quantitative; and (4) sensitivity,
since it requires only small amounts of starting material (i.e.,
RNA) and the signal-to-noise ratio is high. These advantages are
especially important for analyzing mRNA levels in bone, because it
is difficult to obtain large amounts of RNA from bone. Thus, the
primer sets developed for TaqMan.RTM.analysis of a HBM and Zmax1
expression are important embodiments of the present invention. One
skilled in the art will recognize that the primers described here
are preferred embodiments; modifications such as extension or
truncation of a primer or base substitution are encompassed by the
present invention so long as the resultant nucleic acid continues
to perform substantially the same function.
[0568] 21.8.3 HBM Expression in Transgenic Mice
[0569] Eight transgenic founder animals were produced for the
CMVbActin (HBMMCBA) construct and a breeding program initiated to
establish lines. Expression of mRNA determined by TaqMan.RTM.
analysis, shown in FIG. 20, showed variable levels of bone
expression in 4 lines. In tibia, expression levels (relative to
endogenous Zmax1 (LRP5) in HOB-03-C5 cells) showed the following
range: line 18 (x10-11 fold); line 2 (x7-10 fold); line 13 (x1-2
fold) and line 28 (x1 fold). Expression was also detected in other
tissues as expected based on the known activity of the promoter.
For lines 2 and 13, the highest levels of HBM expression were found
in the heart. A TaqMan.RTM. genotyping assay will screen for
potential homozygous animals.
[0570] Six transgenic founder animals were produced for the type I
collagen (HBMMTIC) construct, and a breeding program initiated to
establish lines. Expression of mRNA was found in two lines
initially tested. In line 19, expression was 7-8 fold and 19-20
fold greater than Zmax1 in HOB-03-C5 cells in tibia and femur,
respectively. In line 35, a low level of expression was detected in
tibia and femur.
[0571] 21.9 In Vivo pDXA in HBM Transgenic Mice
[0572] 21.9.1 HBMMCBA Construct
[0573] Initial analysis of limited numbers of mice, illustrated in
FIG. 21 (A-C), at the 5 week and 9 week time-points showed that one
line tested to date had greater BMD values compared to control. At
5 weeks, HBMMCBA line 2 (n=11) BMD in femur, spine and total body
was 21%, 24% and 10% greater respectively, than wild-type control.
At 9 weeks (n=3), these increases in BMD amounted to 19%, 32% and
12%, respectively.
[0574] 21.9.2 HBMMTIC Construct
[0575] Analysis of limited numbers of mice, illustrated in FIG. 21
(D-F), at the 5 week and 9 week time points showed that two lines
tested to date had greater BMD values as compared to control. At 5
weeks, HBMMTIC line 19 (n=5) BMD in femur, spine and total body was
63%, 70% and 41% greater respectively than the wild-type control.
At 9 weeks (n=2), these increases in BMD amounted to 52%, 64% and
37% respectively.
[0576] At 5 weeks, HBMMTIC line 35 (n=1) BMD in femur, spine and
total body was 4%, 47% and 6% greater, respectively than the
wild-type control. At 9 weeks (n=3), these increases in BMD
amounted to 32%, 43% and 18% respectively.
[0577] These results indicate that three lines analyzed to date
show evidence for a high bone mass phenotype. There appeared to be
no obvious correlation between levels of mRNA expression and BMD
phenotype from the limited numbers of lines and animals studied to
date. For example, HBMMCBA line 2 and HBMMTIC line 19 have similar
levels of HBM mRNA in tibia, but the phenotype is more evident in
line 19. Also, HBMMTIC line 35 shows a very low level of expression
when compared to HBMMCBA line 2, but HBMMTIC line 35 appears to
have a stronger phenotype. These observations point to possible
differences in cellular expression that may impact the
phenotype.
[0578] Overall, the BMD results from the transgenic mice show
similarities in magnitude to the phenotype observed in the HBM
affected kindred (Johnson et al., 1997, Am. J. Hum. Genetics,
60:1326-1332). For example, spinal BMD measured in affected
individuals is approximately 34-63% greater than non-affected
family members. The data for spinal BMD from the transgenic animals
ranges from .about.30-70% greater than normal at 9 weeks of
age.
[0579] 21.9.3 Ex Vivo Analysis of Transgenic Mice
[0580] In order to further examine increases in bone density that
were detected in select transgenic lines through monitoring of the
animals by non-invasive bone imaging, necropsies were performed on
animals of these lines at 5 and 9 weeks of age for direct bone
densitometric and histologic analysis. The left femur was isolated,
cleaned and positioned in an XCT Research peripheral Quantitative
Computed Tomograph (pQCT; Stradtec Medizintechnik, Pforzheim,
Germany). The distal end of the femur was located and pQCT scanning
was initiated 2.5 mm proximal from this point for total and
trabecular measurements. The pQCT scan for cortical measurements
was initiated 3.5 mm proximal from the first scan (i.e., 6 mm
proximal from the distal end). The pQCT scans were 0.5 mm thick,
had a voxel (i.e., three dimensional pixel) size of 0.07 mm, and
consisted of 360 projections through the slice. After the scans
were completed, the images were displayed on the monitor and a
region of interest, including the entire femur for each scan, was
outlined. The soft tissue was automatically removed using an
iterative algorithm, and the density of the remaining bone (total
density) in the first slice was determined. The outer 55% of the
bone was then peeled away in a concentric spiral and the density of
the remaining bone (trabecular density) of the first slice was
reported in mg/cm.sup.3. In the second slice, the boundary between
cortical and trabecular bone was determined using an iterative
algorithm, and the density of the cortical bone was determined.
[0581] Analysis of Line 2 F1 CMVbActin-HBM 5 week old transgenic
animals revealed that total density, trabecular density and
cortical density, were 20%, 37% and 4% higher, respectively, in the
transgenic male mice versus the non-transgenic males. In 5 week old
Line 19 type I collagen-HBM transgenic males, an even more dramatic
increase in bone density over their non-transgenic littermates was
evident. Total density, trabecular density and cortical density
were 53%, 104% and 5% higher, respectively. In the Line 19 animals,
the phenotype was found to be maintained at 9 weeks of age with
total, trabecular and cortical bone density being 44%, 101% and 6%
higher than the non-transgenic littermates. At 17 weeks, total and
trabecular density were increased 46%, 202%, respectively. The
effects on the total trabecular parameters in line 19 at all three
time points were statistically significantly higher (p<0.001). A
somewhat different pattern of bone phenotypic expression was
evident from males of type I collagen-HBM transgenic Line 35. At 5
weeks of age total, trabecular and cortical density were only
marginally higher (7%, 4% and 4%, respectively). However, at 9
weeks of age a clear and statistically significant (P<0.05,
0.005, and 0.001 respectively) increase in these parameters became
evident (i.e., 25%, 37% and 4%, respectively). The occurrence of
different patterns of age-related expression of the phenotype is
not unexpected, particularly with the "bone specific" type I
collagen transgene, which is influenced by stage of bone cell
differentiation. Both Line 2 and Line 19 animals at 5 weeks of age
express comparable levels of HBM mRNA in tibia samples, and these
levels are significantly greater (>7-8-fold) than other lines
that show no apparent bone phenotype at this age. Line 19, which is
driven by the type I collagen promoter, unlike line 2, shows very
low expression of the transgene in tissues other than bone. At 5
weeks of age, Line 35 animals show low level expression in bone and
none in other tissues. Immunohistochemistry of calvarial bone
sections using an IBM/Zmax1 specific antibody reveals much more
intense staining in bone cells of transgenic animals from Lines 2
and 19 at 5 weeks of age and from Line 35 at 9 weeks of age versus
their non-transgenic littermates.
[0582] The findings revealed by pQCT analysis were further examined
under greater resolution using .mu.CT instrumentation (Scanco). The
femur was positioned such that the region being imaged includes the
distal end of the femur extending approximately 4 mm proximally
with the view being perpendicular to the axis of the articulating
cartilage. The reference line for beginning the .mu.CT measurement
was placed to minimally overlap the growth plate and extends
proximally for 200 scan slices (9 mm thickness). After completing
the .mu.CT measurement, the first slice in which the condyles have
fully merged was identified. A region of interest was outlined to
include a maximum amount of the trabecular space, while excluding
the cortex. For the first thirty slices, regions of interest were
drawn every five slices and merged. For the remaining 105 slices,
regions of interest were drawn every 10-20 slices. The more regular
the trabecular space, the less frequently a region of interest
needed to be drawn. Each region of interest was merged with its
predecessor after it was drawn. After regions of interest had been
established for all 135 slices, three dimensional evaluation was
performed using a threshold setting of 350.
[0583] The increased bone densities identified by pQCT were
confirmed and extended by .mu.CT to include elements of bone
architecture. In the Line 2 transgenic animals, .mu.CT bone
volume/total volume, connectivity density and trabecular thickness
were 50%, 83% and 12% higher, respectively. Both the connectivity
density and trabecular thickness indices suggest that the increased
density is also associated with increased structural strength. Bone
surface/bone volume was lower by 17% in the transgenic males, which
may suggest that there may be fewer resorptive surfaces and pits.
The trabecular bone response was further confirmed by histological
evaluation of non-decalcified, Goldner's stained sections, which
revealed 36% greater bone mineral area in the distal femoral
metaphysis of the transgenic males. Dynamic histomorphometric
analysis revealed that a substantial increase in bone mineral
apposition rate (+100%), as determined by calcein double labeling,
may be partially responsible for the increased bone in the
transgenics. The dramatic effects evident by pQCT on trabecular
bone in Line 19 were supported by .mu.CT evaluation where bone
volume/total volume, trabecular number, trabecular thickness and
connectivity density were found to be 130%, 45%, 30% and 121%
higher, respectively, in the transgenic males. As seen in Line 2,
the bone surface/bone volume was lower in the transgenic males
(-36%). All of these effects were statistically significant with
p<0.01. The bone phenotype seen at 5 weeks of age in Line 19 was
maintained in 9 week-old animals where bone volume/total volume,
trabecular number and connectivity density were 125%, 38% and 110%
higher than in the non-transgenic littermates.
[0584] .mu.CT analysis of the Line 35 transgenics revealed a
somewhat different pattern than the other two lines. In contrast to
only modestly increased density indicated by pQCT in 5 week-old
females from Line 35, a statistically significant effect
(p<0.01) was seen with mCT, which has greater image resolution
and encompasses a larger volumetric sample. Bone volume/total
volume, trabecular thickness and connectivity density were 35%, 9%
and 27% higher. A similar result was seen in 5 week-old males from
Line 35 where bone volume/total volume and connectivity density
increases of 37% and 45%, respectively, were evident by .mu.CT.
analysis, where only slight increases were revealed by pQCT. The
differences between the Line 35 transgenic males and their
non-transgenic littermates appeared to increase with age such that
statistically significant increases in total density (25%) and
trabecular density (37%) were evident by pQCT at 9 weeks of age.
The .mu.CT results support an age-related divergence in bone
phenotype in this line and show that differences between transgenic
and non-transgenic animals, in terms of bone volume/total volume
and connectivity density, had nearly doubled those seen at 5 weeks
to 70% and 83%, respectively. The bone volume increases seen in the
transgenic animals is in agreement with a significant increase in
this parameter that was detected in a bone biopsy sample from an
adult male affected member of the HBM kindred. The other parameters
that were found to be affected in the transgenic lines may reflect
changes that lead to an increased peak/adult bone mass, which in
this strain of mice occurs between the ages of 17-20 weeks
[0585] The bone density and bone architectural changes seen in the
over-expressing transgenic lines would suggest potentially greater
bio-mechanical strength. This was tested directly by evaluating
3-point bending strength of femurs from 5 week old Line 19 males.
The femora were cleaned of soft tissue and the femoral length
measured using a digital caliper. Periosteal and endosteal
circumferences, as well as cortical thickness, were measured 6 mm
from the distal end of the bone using pQCT. The femur was then
placed on a fixture so that the center of mid-shaft was at an equal
distance from fixed supports located 5 mm apart. The cross bar of
an Instron 5543 load device was placed over the mid-shaft and a
force applied at a speed of 1 mm/minute until fracture occurred. A
force vs. displacement curve was generated and peak load determined
using Instron Merlin software. There was a 75% increase (p<0.01)
in strength that appears to be due to an increase in periosteal
circumference leading to an increase in cortical thickness. Thus,
it appears that the changes in bone density and bone geometry, as
seen in the HBM transgenic animals, do translate into increases in
biomechanical strength.
[0586] In view of the association of HBM/Zmax1 within the class of
LDL related receptor proteins, it was of interest to determine
whether the mutation might affect lipid profiles. Indeed, lipid
studies in the HBM kindred (i.e., 8 affected and 7 unaffected
members) have revealed that triglyceride and VLDL levels are
statistically lower in the affected members. Serum samples from the
transgenic lines were analyzed on a Hitachi 911 instrument using
Boehringer Mannheim (for Cholesterol) and Roche (for triglycerides)
reagents. The cholesterol was measured via o-quinone imine dye
(which is formed following enzymatic reactions with cholesterol)
photometrically at 505 nm at 37.degree. C. Enzymatic methods for
triglyceride measurements are based on determination of the
glycerol part of triglyceride after hydrolysis of triglycerides and
fatty acids. The end dye product of enzymatic reaction was measured
at 505 nm. In 5 week old male Line 2 transgenics, although serum
cholesterol was only slightly reduced, serum triglyceride levels
were reduced by 26% in the transgenics versus their non-transgenic
littermates. In a limited sample of Line 2 animals at 9 weeks of
age, triglyceride levels remained 20% lower. Similarly, at 5 weeks
of age triglyceride levels in male transgenics from Line 19 were
32% lower. In contrast, at 5 weeks of age both male and female
transgenics of Line 35 did not have lower triglyceride levels. The
fact that the 5 week old Line 35 animals did have statistically
greater bone volume/total volume suggests that the lipid change may
not be directly related to the skeletal phenotype. This would
appear to be supported by the fact that the Line 35 animals at 9
weeks of age had only slightly reduced triglyceride levels (11%)
but exhibited substantially higher bone density than at 5 weeks of
age. Due to the different levels and sites of expression of the
transgene in these lines we can not rule out the possibility that
serum lipid levels could serve as a surrogate marker for agents
favorably affecting a bone phenotype through HBM/Zmax1.
[0587] These and other transgenic lines based on HBM or HBM-like
genes will serve as valuable models for exploring the nature, of
bone homeostasis. Bone density in all species accommodates to its
customary loading conditions. In the HBM kindred and in the
transgenic lines, the sensor/effector systems of the skeleton
appear to perceive greater load signals resulting in greater bone
density. Experimental models have been established showing that
increased bone loading can lead to increased bone density and that
unloading or disuse leads to a loss of bone density. Evaluating the
histological, biochemical and genetic responses of the skeleton of
the transgenic animals in these experimental paradigms will yield
much information regarding the sensor/effector system responsible
for bone homeostasis. The application of the transgenic animals in
other established models of altered bone turnover, including but
not limited to steroid deficiency-induced osteopenia and
aging-related osteopenia will provide further insight into the role
of Zmax1 in bone homeostasis and the nature of the favorable
changes induced by the HBM mutation.
[0588] 21.9.4 HBM Gene-Targeting
[0589] The Zmax1 KI/KO gene-targeting vector is electroporated into
129 SvEv, C57BL/6 ES and 129 ES cells. Restriction fragment length
analysis of genomic DNA and sequencing of PCR amplified fragments
can be used to identify gene targeted clones. The knock-in version
of the gene-targeting vector allows for the introduction of the HBM
mutation into the endogenous Zmax (LRP5) genomic locus with minimal
impact on the mouse genome. It permits the production of the HBM
protein in a more natural environment, i.e. not in an
over-expression model such as the transgenic mice or transfected
cell lines. The knock-out version of the gene-targeting vector was
engineered to contain a transcriptional stop sequence that has the
potential to result in loss of one functional LRP5 allele. Breeding
heterozygous animals with this mutation would lead to the
production of embryos homozygous for the null allele. In a
different design of the gene-targeting vector, lox P sites could be
positioned in such a way that would facilitate production of a
conditional knock-out of the endogenous LRP5 gene. In the presence
of Cre recombinase, a critical region of the LRP5 gene would be
deleted in between the lox P sites, thus resulting in the potential
loss of one functional allele. Animal breeding would then be used
to create homozygotes with a null allele. Other recombinase enzyme
systems, such as flp recombinase in combination with cognate frt
sites, could be used to create the deletion. The recombinase could
be administered in a number of ways as described earlier, including
plasmid injection into embryos and use of transgenic animals
expressing Cre. The promoter used to drive expression of Cre could
be chosen in a manner that would result in ubiquitous or
tissue-specific deletion of the LRP5 gene thus resulting in a
conditional knockout. In a further embodiment expression of the Cre
enzyme itself could be made conditional using inducible systems
such as GeneSwitch and Tetracycline paradigms.
[0590] 21.9.5 Uses of Transgenic Animals and Cells
[0591] The transgenic animals and cells of the present invention
are useful tools in methods for identifying surrogate markers for
the HBM phenotype. The surrogate markers provided by the present
invention are also useful tools for the assessment and screening of
prospective treatments. Individuals carrying the HBM gene have
elevated bone mass. The HBM gene causes this phenotype by altering
the activities, levels, expression patterns, and modification
states of other molecules involved in bone development. Using a
variety of established techniques, it is possible to identify
molecules, preferably proteins or mRNAs, whose activities, levels,
expression patterns, and modification states are different between
systems containing the Zmax1 gene and systems containing the HBM
gene. Such systems can be, for example, cell-free extracts, cells,
tissues or living organisms, such as mice or humans. For a mutant
form of Zmax1, a complete deletion of Zmax1, mutations lacking the
extracellular or intracellular portion of the protein, or any other
mutation in the Zmax1 gene may be used. It is also possible to use
expression of antisense Zmax1 RNA or oligonucleotides to inhibit
production of the Zmax1 protein. For a mutant form of HBM, a
complete deletion of HBM, mutations lacking the extracellular or
intracellular portion of the HBM protein, or any other mutation in
the HBM gene may be used. It is also possible to use expression of
antisense HBM RNA or oligonucleotides to inhibit production of the
HBM protein.
[0592] Molecules identified by comparison of Zmax1 systems and HBM
systems can be used as surrogate markers in pharmaceutical
development or in diagnosis of human or animal bone disease.
Alternatively, such molecules may be used in treatment of bone
disease. See, Schena et al., 1995 Science, 270: 467-70.
[0593] For example, a transgenic mouse carrying the HBM gene or
HBM-like variant in the mouse homologue locus is constructed. A
mouse of the genotype HBM/+ is viable, healthy and has elevated
bone mass. To identify surrogate markers for elevated bone mass,
HBM/+ (i.e., heterozygous) and isogenic +/+ (i.e., wild-type) mice
are sacrificed. Bone tissue mRNA is extracted from each animal, and
a "gene chip" corresponding to mRNAs expressed in the +/+
individual is constructed. mRNA from different tissues is isolated
from animals of each genotype, reverse-transcribed, fluorescently
labeled, and then hybridized to gene fragments affixed to a solid
support. The ratio of fluorescent intensity between the two
populations is indicative of the relative abundance of the specific
mRNAs in the +/+ and HBM/+ animals. Alternatively, mRNA may be
isolated from wild-type and transgenic animals. cDNA prepared from
these samples is transcribed in vitro to obtain labeled mRNA for
use on custom made or commercially available gene array chips such
as are manufactured by Affymetrix. Sets of genes with altered
expression as a function of phenotype may be identified be a
variety of routine computational analyses. Genes encoding mRNA
over- and under-expressed relative to the wild-type control are
candidates for genes coordinately regulated by the HBM gene or
HBA-like gene.
[0594] One standard procedure for identification of new proteins
that are part of the same signaling cascade as an
already-discovered protein is as follows. Cells are treated with
radioactive phosphorous, and the already-discovered protein is
manipulated to be more or less active. The phosphorylation state of
other proteins in the cell is then monitored by polyacrylamide gel
electrophoresis and autoradiography, or similar techniques. Levels
of activity of the known protein may be manipulated by many
methods, including, for example, comparing wild-type mutant
proteins using specific inhibitors such as drugs or antibodies,
simply adding or not adding a known extracellular protein, or using
antisense inhibition of the expression of the known protein (Tamura
et al., 1998 Science, 280: 1614-7; Meng, 1998 EMBO J., 17:
4391-403; Cooper et al., 1982 Cell 1: 263-73).
[0595] In another example, proteins with different levels of
phosphorylation are identified in TE85 osteosarcoma cells
expressing either a sense or antisense cDNA for Zmax1. TE85 cells
normally express high levels of Zmax1 (Dong et al., 1998 Biochem.
& Biophys. Res. Comm., 251: 784-90). Cells containing the sense
construct express even higher levels of Zmax1, while cells
expressing the antisense construct express lower levels. Cells are
grown in the presence of .sup.32P, harvested, lysed, and the
lysates run on SDS polyacrylamide gels to separate proteins, and
the gels subjected to autoradiography (Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons (1997)).
Bands that differ in intensity between the sense and antisense cell
lines represent phosphoproteins whose phosphorylation state or
absolute level varies in response to levels of Zmax1. As an
alternative to the .sup.32P-labeling, unlabeled proteins may be
separated by SDS-PAGE and subjected to immunoblotting, using the
commercially available anti-phosphotyrosine antibody as a probe
(Thomas et al., 1995 Nature, 376: 267-71). As an alternative to the
expression of antisense RNA, transfection with chemically modified
antisense oligonucleotides can be used (Woolf et al., 1990 Nucleic
Acids Res., 18: 1763-9).
[0596] Many bone disorders, such as osteoporosis, have a slow onset
and a slow response to treatment. It is therefore useful to develop
surrogate markers for bone development and mineralization. Such
markers can be useful in developing treatments for bone disorders,
and for diagnosing patients who may be at risk for later
development of bone disorders. Examples of preferred markers are N-
and C-terminal telopeptide markers described, for example, in U.S.
Pat. Nos. 5,455,179, 5,641,837 and 5,652,112, the disclosures of
which are incorporated by reference herein in their entirety. In
the area of HIV disease, CD4 counts and viral load are useful
surrogate markers for disease progression (Vlahov et al., 1998
JAMA, 279(1): 35-40). There is a need for analogous surrogate
markers in the area of bone disease.
[0597] A surrogate marker can be any characteristic that is easily
tested and relatively insensitive to non-specific influences. For
example, a surrogate marker can be a molecule such as a protein or
mRNA in a tissue or in blood serum. Alternatively, a surrogate
marker may be a diagnostic sign such as sensitivity to pain, a
reflex response or the like.
[0598] In yet another example, surrogate markers for elevated bone
mass are identified using a pedigree of humans carrying the HBM
gene or HBM-like gene. Blood samples are withdrawn from three
individuals that carry the HBM gene or HBM-like gene, and from
three closely related individuals that do not. Proteins in the
serum from these individuals are electrophoresed on a two
dimensional gel system, in which one dimension separates proteins
by size, and another dimension separates proteins by isoelectric
point (Epstein et al., 1996 Electrophoresis 17: 1655-70). Spots
corresponding to proteins are identified. A few spots are expected
to be present in different amounts or in slightly different
positions for the HBM individuals compared to their normal
relatives. These spots correspond to proteins that are candidate
surrogate markers. The identities of the proteins are determined by
microsequencing, and antibodies to the proteins can be produced by
standard methods for use in diagnostic testing procedures.
Diagnostic assays for HBM proteins or other candidate surrogate
markers include using antibodies described in this invention and a
reporter molecule to detect HBM in human body fluids, membranes,
bones, cells, tissues or extracts thereof. The antibodies can be
labeled by joining them covalently or noncovalently with a
substance that provides a detectable signal. In many scientific and
patent literature, a variety of reporter molecules or labels are
described including radionuclides, enzymes, fluorescent,
chemiluminescent or chromogenic agents (U.S. Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and
4,366,241). The transgenic or genetically modified animals can also
serve in a method for surrogate marker identification.
[0599] Using these antibodies, the levels of candidate surrogate
markers are measured in normal individuals and in patients
suffering from a bone disorder, such as osteoporosis, osteoporosis
pseudoglioma, Engelmann's disease, Ribbing's disease,
hyperphosphatasemia, Van Buchem's disease, melorheostosis,
osteopetrosis, pychodysostosis, sclerosteosis, osteopoilkilosis,
acromegaly, Paget's disease, fibrous dysplasia, tubular stenosis,
osteogenesis imperfecta, hypoparathyroidism,
pseudohypoparathyroidism, pseudopseudohypoparathyroidism, primary
and secondary hyperparathyroidism and associated syndromes,
hypercalciuria, medullary carcinoma of the thyroid gland,
osteomalacia and other diseases. Techniques for measuring levels of
protein in serum in a clinical setting using antibodies are well
established. A protein that is consistently present in higher or
lower levels in individuals carrying a particular disease or type
of disease is a useful surrogate marker.
[0600] A surrogate marker can be used in diagnosis of a bone
disorder. For example, consider a child that presents to a
physician with a high frequency of bone fracture. The underlying
cause may be child abuse, inappropriate behavior by the child, or a
bone disorder. To rapidly test for a bone disorder, the levels of
the surrogate marker protein are measured using the antibody
described above.
[0601] Levels of modification states of surrogate markers can be
measured as indicators of the likely effectiveness of a drug that
is being developed. It is especially convenient to use surrogate
markers in creating treatments for bone disorders, because
alterations in bone development or mineralization may require a
long time to be observed. For example, a set of bone mRNAs, termed
the "HBM-inducible mRNA set" is found to be over-expressed in HBM/+
mice as compared to +/+ mice, as described above. Expression of
this set can be used as a surrogate marker. Specifically, if
treatment of +/+ mice with a compound results in over-expression of
the HBM-inducible mRNA set, then that compound is considered a
promising candidate for further development.
[0602] This invention is particularly useful for screening
compounds by using the Zmax1 or HBM protein or HBM-like proteins or
binding fragments thereof in any of a variety of drug screening
techniques.
[0603] The Zmax1 or HBM protein or fragment employed in such a test
may either be free in solution, affixed to a solid support, or
borne on a cell surface. One method of drug screening utilizes
eukaryotic or prokaryotic host cells which are stably transformed
with recombinant nucleic acids expressing the protein or fragment,
preferably in competitive binding assays. Such cells, either in
viable or fixed form, can be used for standard binding assays. One
may measure, for example, for the formation of complexes between a
Zmax1 or HBM protein or fragment and the agent being tested, or
examine the degree to which the formation of a complex between a
Zmax1 or HBM protein or fragment and a known ligand is interfered
with by the agent being tested.
[0604] Thus, the present invention provides methods of screening
for drugs comprising contacting such an agent with a Zmax1 (LRP5)
or HBM protein or fragment thereof and assaying (i) for the
presence of a complex between the agent and the Zmax1 or HBM
protein or fragment, or (ii) for the presence of a complex between
the Zmax1 or HBM protein or fragment and a ligand, by methods well
known in the art. In such competitive binding assays the Zmax1 or
HBM protein or fragment is typically labeled. Free Zmax1 or HBM
protein or fragment is separated from that present in a
protein:protein complex, and the amount of free (i.e., uncomplexed)
label is a measure of the binding of the agent being tested to
Zmax1 or HBM or its interference with Zmax1 or IBM: ligand binding,
respectively.
[0605] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to the Zmax1 or HBM proteins and is described in detail in WO
84/03564. Briefly stated, large numbers of different small peptide
test compounds are synthesized on a solid substrate, such as
plastic pins or some other surface.
[0606] The peptide test compounds are reacted with Zmax1 or HBM
proteins and washed. Bound Zmax1 or HBM protein is then detected by
methods well known in the art. Purified Zmax1 or HBM can be coated
directly onto plates for use in the aforementioned drug screening
techniques. However, non-neutralizing antibodies to the protein can
be used to capture antibodies to immobilize the Zmax1 or HBM
protein on the solid phase.
[0607] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
specifically binding the Zmax1 or HBM protein compete with a test
compound for binding to the Zmax1 or HBM protein or fragments
thereof. In this manner, the antibodies can be used to detect the
presence of any peptide that shares one or more antigenic
determinants of the Zmax1 or HBM protein.
[0608] A further technique for drug screening involves the use of
host eukaryotic cell lines or cells (such as described above) that
have a nonfunctional Zmax1 or HBM gene. These host cell lines or
cells are defective at the Zmax1 or HBM protein level. The host
cell lines or cells are grown in the presence of drug compound. The
rate of growth of the host cells is measured to determine if the
compound is capable of regulating the growth of Zmax1 or HBM
defective cells.
[0609] The goal of rational drug design is to produce structural
analogs of biologically active proteins of interest or of small
molecules with which they interact (e.g., agonists, antagonists,
inhibitors) in order to fashion drugs which are, for example, more
active or stable forms of the protein, or which, e.g., enhance or
interfere with the function of a protein in vivo. See, e.g.,
Hodgson, 1991 Bio/Technology, 9: 19-21. In one approach, one first
determines the three-dimensional structure of a protein of interest
(e.g., Zmax1 or HBM protein) or, for example, of the Zmax1- or
HBM-receptor or ligand complex, by x-ray crystallography, by
computer modeling or most typically, by a combination of
approaches. Less often, useful information regarding the structure
of a protein may be gained by modeling based on the structure of
homologous proteins. An example of rational drug design is the
development of HIV protease inhibitors (Erickson et al., 1990
Science, 249: 527-33). In addition, peptides (e.g., Zmax1 or HBM
protein) are analyzed by an alanine scan (Wells, 1991 Methods in
Enzymol., 202: 390-411). In this technique, an amino acid residue
is replaced by Ala, and its effect on the peptide's activity is
determined. Each of the amino acid residues of the peptide is
analyzed in this manner to determine the important regions of the
peptide.
[0610] It is also possible to isolate a target-specific antibody,
selected by a functional assay, and then to solve its crystal
structure. In principle, this approach yields a pharmacore upon
which subsequent drug design can be based. It is possible to bypass
protein crystallography altogether by generating anti-idiotypic
antibodies (anti-ids) to a functional, pharmacologically active
antibody. As a mirror image of a mirror image, the binding site of
the anti-ids would be expected to be an analog of the original
receptor. The anti-id could then be used to identify and isolate
peptides from banks of chemically or biologically produced banks of
peptides. Selected peptides would then act as the pharmacore.
[0611] Thus, one may design drugs which have, e.g., improved Zmax1
or HBM protein activity or stability or which act as inhibitors;
agonists, antagonists, etc. of Zmax1 or HBM protein activity. By
virtue of the availability of cloned Zmax1 or HEM sequences,
sufficient amounts of the Zmax1 or HBM protein may be made
available to perform such analytical studies as X-ray
crystallography. In addition, the knowledge of the Zmax1 or HBM
protein sequence provided herein will guide those employing
computer modeling techniques in place of, or in addition to X-ray
crystallography.
[0612] Identified drug candidates (known as "leads") may be further
studied by use of transgenic animals. The transgenic animals of the
present invention are useful for creating an animal model of bone
density modulation which may be used to test and refine drug leads.
The transgenic animals described above represent a single example
of the Zmax1 and HBM or HBM-like transgenic animals contemplated
herein. One skilled in the art is aware of variations and the
considerations which will be routinely applied in modifying the
present invention to a specific purpose. Examples of the
development of transgenic animal models are given for example in
Strategies in Transgenic Animal Science (1995, Monastersky and Robl
Eds., Washington, D.C.: American Society for Microbiology) and
references therein which are all incorporated herein by reference
in their entirety.
[0613] As an example, at least two groups of transgenic animals can
be created as described, so that one group expresses HBM and
another group expresses Zmax1. These animals can be treated with
the candidate drug for some time spanning from a few days to the
remainder of the animal's life-span. The animals are monitored for
changes in bone mass and/or surrogate markers for the HEM
phenotype. The transgenic animals used in such a study may express
human HBM protein and Zmax1 protein or the homologous HBM and Zmax1
proteins defined for each species or variants thereof. Expression
may be driven by a ubiquitous promoter or a bone specific promoter
as would be known. It will be informative to compare groups of
animals utilizing different promoters.
[0614] The transgenic animals of the method according to the
present inventions may also comprise knock-in (KI) and/or knock-out
(KO) animals, such as mice, which express HBM, Zmax1, or neither
under the control of the animal's native promoter. Such animals may
be created by homologous recombination in ES cells as described
above (and elsewhere in the literature of the art such as, for
example, U.S. Pat. Nos. 6,187,991 and 6,187,992 and references
cited therein which are incorporated herein in their entirety). The
experimental groups of transgenic animals treated with candidate
drugs may be monitored by non-invasive means, by the monitoring of
surrogate markers as described above, and/or by ex vivo analysis of
bones from sacrificed animals at given time-points.
[0615] Likewise the effect of such treatments as dietary control
(e.g. varying intake of vitamins, minerals, proteins, lipids,
etc.), ovariectomy, direct administration of all or part of
purified HBM or Zmax1 proteins, administration of antisense
nucleotides, antibodies against Zmax1 or gene therapy in adults may
be investigated by systematic administration of the treatment to
transgenic animals according to the invention. Such treatments may
include, administration of estrogens, tamoxifen, raloxifene, (or
other selective estrogen modulators, SERMs), vitamin D analogs,
calcitonin, cathepsin K inhibitors, statins (e.g. simvastatin,
pravastatin, and lovastatin), bis-phosphonates, parathyroid hormone
(PTH), bone morphogenetic proteins (BMP) as described in U.S. Pat.
Nos. 6,190,880 and 5,866,364, and combinations of the above
compounds.
[0616] In view of the homology between Zmax1 and LRP6 and to the
LDL receptor and the further observations of markers for cardiac
health being modulated in HBM subjects, it is an aspect of the
present invention to use the novel research methods disclosed
herein, to screen known cardio-protective treatments for bone
modulating effects. Thereby, the present invention provides
therapeutic methods which are both cardio-protective and which
improve bone quality. The models are useful for testing drugs and
researching lipid modulation effects related to Zmax1, LRP6, HBM
and HBM like proteins and nucleic acids.
[0617] The effect of various mutations of Zmax1, HBM and HBM-like
genes may be investigated by creation of additional lines of
transgenic animals according to the invention, wherein these
animals comprise such mutations. By comparison of direct measures
of bone development or surrogate markers, an embodiment of the
invention provides a useful research tool for screening gene
therapy reagents, candidate drug therapies, and elucidating
molecular mechanisms of bone development modulation. One skilled in
the art knows how to use the methods of the present invention to
achieve these goals.
[0618] The present invention provides a method and useful research
tools for testing prospective gene therapies. Transgenic knock-out
mice are useful for testing prospective gene therapies. A
transgenic knock-out animal such as a mouse is created as described
above which does not express endogenous Zmax1 or HBM. A prospective
gene therapy, such as intravenous injection of a recombinant
replication-defective adenovirus encoding the human HBM protein
driven by the CMV.beta.Actin promoter, is administered. Parameters
of bone density and/or surrogate markers are monitored over time
following therapy (Ishibashi et al., 1993 J. Clin. Invest. 92:
883-93) A TaqMan.RTM. primer set such as that described above may
be used to measure expression of transgenic HBM. One skilled in the
art knows alternative methods such as the Northern blot-method.
Comparison of treated and untreated animals both within and between
groups of germ-line transgenic animals, knock-out (null allele)
background, and wild-type endogenous Zmax1 background animals
provides complementary controls for assessing the relative
effectiveness of various modalities of gene therapy.
[0619] Uses for the transgenic animals models contemplated herein
also include, but are not limited to: (1) sources for generating
bone cell cultures from the calvaria of the transgenic animals to
study bone cell (e.g., osteoblast and osteoclast) function and
number; (2) models for studying the effects of estrogen loss by
ovariectomizing (ovx) the transgenic animals; (3) models for
testing mechanical loading on the bones and other stress/strength
tests; (4) breeding models with which to breed to other genetically
modified or naturally occurring mutant animals that display bone
abnormalities; (Chipman et al., 1993 PNAS, 90: 1701-05; Phillips et
al., 2000 Bone 27: 219-26; Kajkenova et al., 1997 J. Bone Min. Res.
12: 1772-79; Jilka et al., 1996 J. Clin. Invest. 97: 1732-40;
Takahashi et al., 1994 Bone and Mineral 24: 245-55); (5) bone
mis/disuse models to test the effects of weight bearing or gravity;
(6) models for identifying and screening reagents which may or are
known to modulate bone metabolism (e.g., --PTH, estrogen, vitamin D
analogs, bisphosphonates, statins, leptin, BMP, apoE); (7) models
for investigating prospective treatments to improve fracture
repair.
[0620] The transgenic animal models can be analyzed using, but not
limited to, such methods as bone densitometry by pDEXA, pQCT and
microCT; histology, molecular marker analysis, apoptosis, cell
proliferation, cell cycle, mineralization, serum biochemistry,
transcriptional profiling, and the like.
22. Methods of Use: Avian and Mammalian Animal Husbandry
[0621] The Zmax1 (LRP5) DNA and Zmax1 (LRP5) protein and/or the HBM
DNA and HBM protein (or LRP6 or an HBM-like nucleic acid or protein
as also contemplated herein) can be used for vertebrate and
preferably human therapeutic agents and for avian and mammalian
veterinary agents, including for livestock breeding. Birds,
including, for example, chickens, roosters, hens, turkeys,
ostriches, ducks, pheasants and quails, can benefit from the
identification of the gene and pathway for high bone mass. In many
examples cited in literature (for example, McCoy et al., 1996 Res.
Vet. Sci., 60: 185-6), weakened bones due to husbandry conditions
cause cage layer fatigue, osteoporosis and high mortality rates.
Additional therapeutic agents to treat osteoporosis or other bone
disorders in birds can have considerable beneficial effects on
avian welfare and the economic conditions of the livestock
industry, including, for example, meat and egg production.
23. Methods of Use: Diagnostic Assays Using Zmax1-Specific
Oligonucleotides for Detection of Genetic Alterations Affecting
Bone Development
[0622] In cases where an alteration or disease of bone development
is suspected to involve an alteration of the Zmax1, LRP6, HBM or
HBM-like gene, specific oligonucleotides may be constructed and
used to assess the level of Zmax1 mRNA or HBM mRNA, respectively,
in bone tissue or in another tissue that affects bone
development.
[0623] For example, to test whether a person has the HBM gene,
which affects bone density, polymerase chain reaction can be used.
Two oligonucleotides are synthesized by standard methods or are
obtained from a commercial supplier of custom-made
oligonucleotides. The length and base composition are determined by
standard criteria using the Oligo 4.0 primer Picking program
(Wojchich Rychlik, 1992) or any suitable alternative. One of the
oligonucleotides is designed so that it will hybridize only to HBM
DNA under the PCR conditions used. The other oligonucleotide is
designed to hybridize a segment of Zmax1 genomic DNA such that
amplification of DNA using these oligonucleotide primers produces a
conveniently identified DNA fragment. For example, the pair of
primers CCAAGTTCTGAGAAGTCC (SEQ ID NO:32) and AATACCTGAAACCATACCTG
(SEQ ID NO:33) will amplify a 530 base pair DNA fragment from a DNA
sample when the following conditions are used: step 1 at 95.degree.
C. for 120 seconds; step 2 at 95.degree. C. for 30 seconds; step 3
at 58.degree. C. for 30 seconds; step 4 at 72.degree. C. for 120
seconds; where steps 2-4 are repeated times. Tissue samples may be
obtained from hair follicles, whole blood, or the buccal
cavity.
[0624] The fragment generated by the above procedure is sequenced
by standard techniques. Individuals heterozygous for the HBM gene
will show an equal amount of G and T at the Second position in the
codon for glycine 171. Normal or homozygous wild-type individuals
will show only G at this position. Similar routine procedures may
be used to develop assays for other polymorphisms and variants
according to the invention.
[0625] Other amplification techniques besides PCR may be used as
alternatives, such as ligation-mediated PCR or techniques involving
Q-beta replicase (Cahill et al., Clin. Chem., 37(9):1482-5 (1991)).
For example, the oligonucleotides 5'-AGCTGCTCGTAGCTG
TCTCTCCCTGGATCACGGGTACATGTACTGGACAGACTGGGT-3'(SEQ ID NO:34) and
T5'-GAGACGCCCCGGATTGAGCGGGCAGGGATAGCTTATTCCCTGTGCCGCA TTACGGC-3'
(SEQ ID NO: 35) can be hybridized to a denatured human DNA sample,
treated with a DNA ligase, and then subjected to PCR amplification
using the primer oligonucleotides 5'-AGCTGCTCGTAGCTGTCTCTCCCTGGA-3'
(SEQ ID NO:36) and 5'-GCCGTAATGCGGCACAGGGAATAAGCT-3' (SEQ ID
NO:37). In the first two oligonucleotides, the outer 27 bases are
random sequence corresponding to primer binding sites, and the
inner 30 bases correspond to sequences in the Zmax1 gene. The T at
the end of the first oligonucleotide corresponds to the HBM gene.
The first two oligonucleotides are ligated only when hybridized to
human DNA carrying the HBM gene, which results in the formation of
an amplifiable 114 bp DNA fragment.
[0626] Products of amplification can be detected by agarose gel
electrophoresis, quantitative hybridization, or equivalent
techniques for nucleic acid detection known to one skilled in the
art of molecular biology (Sambrook et al., Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring, N.Y.
(1989)).
[0627] Other alterations in the Zmax1 gene or the HBM gene (or LRP6
or HBM-like nucleic acids, which are also contemplated herein) may
be diagnosed by the same type of amplification-detection
procedures, by using oligonucleotides designed to identify those
alterations. These procedures can be used in animals as well as
humans to identify alterations in Zmax1 or HBM that affect bone
development.
[0628] Expression of Zmax1 or HBM in bone tissue may be
accomplished by fusing the cDNA of Zmax1 or HBM, respectively, to a
bone-specific promoter in the context of a vector for genetically
engineering vertebrate cells. DNA constructs are introduced into
cells by packaging the DNA into virus capsids, by the use of
cationic liposomes, electroporation, or by calcium phosphate
transfection. Transfected cells, preferably osteoblasts, may be
studied in culture or may be introduced into bone tissue in animals
by direct injection into bone or by intravenous injection of
osteoblasts, followed by incorporation into bone tissue (Ko et al.,
1996 Cancer Res. 56: 4614-9). For example, the osteocalcin
promoter, which is specifically active in osteoblasts, maybe used
to direct transcription of the Zmax1 gene or the HBM gene. Any of
several vectors and transfection methods may be used, such as
retroviral vectors, adenovirus vectors, or vectors that are
maintained after transfection using cationic liposomes, or other
methods and vectors described herein.
[0629] Alteration of the level of functional Zmax1 protein or HBM
protein affects the level of bone mineralization. By manipulating
levels of functional Zmax1 protein or HBM protein, it is possible
to affect bone development and to increase or decrease levels of
bone mineralization. For example, it may be useful to increase bone
mineralization in patients with osteoporosis. Alternatively, it may
be useful to decrease bone mineralization in patients with
osteopetrosis or Paget's disease. Alteration of Zmax1 levels or HBM
levels can also be used as a research tool. Specifically, it is
possible to identify proteins, mRNA and other molecules whose level
or modification status is altered in response to changes in
functional levels of Zmax1 or HBM. The pathology and pathogenesis
of bone disorders is known and described, for example, in Rubin and
Farber (Eds.), Pathology, 2nd Ed., S. B. Lippincott Co.,
Philadelphia, Pa. (1994).
[0630] A variety of techniques can be used to alter the levels of
functional Zmax1 or HBM. For example, intravenous or intraosseous
injection of the extracellular portion of Zmax1 or mutations
thereof, or IBM or mutations thereof, will alter the level of Zmax1
activity or HBM activity, respectively, in the body of the treated
human, animal or bird. Truncated versions of the Zmax1 protein or
HBM protein can also be injected to alter the levels of functional
Zmax1 protein or HBM protein, respectively. Certain forms of Zmax1
or HEM enhance the activity of endogenous protein, while other
forms are inhibitory.
[0631] In a preferred embodiment, the HBM protein is used to treat
osteoporosis, fracture, or other bone disorder. In a further
preferred embodiment, the extracellular portion of HBM or fragment
thereof (e.g., the Dkk binding domain) protein is used. This HBM
protein may be optionally modified by the addition of a moiety that
causes the protein to adhere to the surface of cells. The protein
is prepared in a pharmaceutically acceptable solution and is
administered by injection or another method that achieves
acceptable pharmacokinetics and distribution.
[0632] In a second embodiment of this method, Zmax1, HBM, HEM
variant, and/or LRP6 levels are increased or decreased by gene
therapy techniques. To increase Zmax1 or HBM levels, osteoblasts or
another useful cell type are genetically engineered to express high
levels of Zmax1 or HBM as described above. Alternatively, to
decrease Zmax1 or HBM levels, antisense constructs that
specifically reduce the level of translatable Zmax1 or HEM mRNA can
be used. In general, a tissue-nonspecific promoter may be used,
such as the CMV promoter or another commercially available promoter
found in expression vectors (Wu et al., 1996 Toxicol. Appl.
Pharmacol. 141: 330-9). In a preferred embodiment, a Zmax1 cDNA or
its antisense is transcribed by a bone-specific promoter, such as
the osteocalcin or another promoter, to achieve specific expression
in bone tissue. In this way, if a Zmax1-expressing DNA construct or
HBM-expressing construct is introduced into non-bone tissue, it
will not be expressed.
[0633] In a third embodiment of this method, antibodies against
Zmax1, LRP6, IBM-like or HBM are used to modulate its function.
Such antibodies are identified herein.
[0634] In a fourth embodiment of this method, drugs that are
agonists or antagonists of Zmax1 function or HBM function are used.
Such drugs are described herein and optimized according to
techniques of medicinal chemistry well known to one skilled in the
art of pharmaceutical development.
[0635] Zmax 1 and HBM interact with several proteins, such as ApoE.
Molecules that inhibit the interaction between Zmax1 or IBM and
ApoE or another binding partner are expected to alter bone
development and mineralization. Such inhibitors may be useful as
drugs in the treatment of osteoporosis, osteopetrosis, or other
diseases of bone mineralization. Such inhibitors may be low
molecular weight compounds, proteins or other types of molecules.
See, Kim et al., 1998 J. Biochem. (Tokyo) 124: 1072-1076.
[0636] Inhibitors of the interaction between Zmax1 or HBM and
interacting proteins may be isolated by standard drug-screening
techniques. For example, Zmax1 protein, (or a fragment thereof) or
HBM protein (or a fragment thereof) can be immobilized on a solid
support such as the base of microtiter well. A second protein or
protein fragment, such as ApoE is derivatized to aid in detection,
for example with fluorescein. Iodine, or biotin, then added to the
Zmax1 or HBM in the presence of candidate compounds that may
specifically inhibit this protein-protein domain of Zmax1 or HBM,
respectively, and thus avoid problems associated with its
transmembrane segment. Drug screens of this type are well known to
one skilled in the art of pharmaceutical development.
[0637] Because Zmax1 and HBM are involved in bone development,
proteins that bind to Zmax1 and HBM are also expected to be
involved in bone development. Such binding proteins can be
identified by standard methods, such as co-immunoprecipitation,
co-fractionation, or the two-hybrid screen (Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons (1997)). For
example, to identify Zmax1-interacting proteins or HBM-interacting
proteins using the two-hybrid system, the extracellular domain of
Zmax1 or HBM is fused to LexA and expressed for the yeast vector
pEG202 (the "bait") and expressed in the yeast strain EGY48. The
yeast strain is transformed with a "prey" library in the
appropriate vector, which encodes a galactose-inducible
transcription-activation sequence fused to candidate interacting
proteins. The techniques for initially selecting and subsequently
verifying interacting proteins by this method are well known to one
skilled in the art of molecular biology (Ausubel et al., 1997).
[0638] In a preferred embodiment, proteins that interact with
--IBM, but not Zmax1, are identified using a variation of the above
procedure (Xu et al., 1997 Proc. Natl. Acad. Sci. USA, 94:
12473-8). This variation of the two-hybrid system uses two baits,
and Zmax1 and HBM are each fused to LexA and TetR, respectively.
Alternatively, proteins that interact with the HBM but not Zmax1
are also isolated. These procedures are well known to one skilled
in the art of molecular biology, and are a simple variation of
standard two-hybrid procedures.
[0639] As an alternative method of isolating substances interacting
with Zmax1 or HBM, a biochemical approach is used. The Zmax1
protein or a fragment thereof, such as the extracellular domain, or
the HBM protein or a fragment thereof, such as the extracellular
domain, is chemically coupled to Sepharose beads. The Zmax1- or
HBM-coupled beads are poured into a column. A biological extract,
such as a lipid fraction, serum proteins, proteins in the
supernatant of a bone biopsy, or cellular contents from gently
lysed osteoblast cells, is added to the column. Non-specifically
bound compounds are eluted, the column is washed several times with
a low-salt buffer, and then tightly binding compounds may be eluted
with a high-salt buffer. These are candidate compounds that bind to
Zmax1 or HBM, and can be tested for specific binding by standard
tests and control experiments. Sepharose beads used for coupling
proteins and the methods for performing the coupling are
commercially available (Sigma), and the procedures described here
are well known to one skilled in the art of protein
biochemistry.
[0640] As a variation of the above procedure, proteins that are
eluted by high salt from the Zmax1- or HBM-sepharose column are
then added to an HBM-Zmax1-sepharose column. Proteins that flow
through without sticking are proteins that bind to Zmax1 but not to
HBM. Alternatively, proteins that bind to the HBM protein and not
to the Zmax1 protein can be isolated by reversing the order in
which the columns are used.
[0641] Isolated compounds may be identified by standard methods
such as 2D gel electrophoresis, chromatography, and mass
spectroscopy.
24. Method of Use: Transformation-Associated Recombination (TAR)
Cloning
[0642] Essential for the identification of novel allelic variants
of Zmax1 (LRP5) and LRP6 is the ability to examine the sequence of
both copies of the gene in an individual. To accomplish this, two
"hooks," or regions of significant similarity, are identified
within the genomic sequence such that they flank the portion of DNA
that is to be cloned. Most preferably, the first of these hooks is
derived from sequences 5' to the first exon of interest and the
second is derived from sequences 3' to the last exon of interest.
These two "hooks" are cloned into a bacterial/yeast shuttle vector
such as that described by Larionov et al., 1997 Proc. Natl. Acad.
Sci. USA, 94: 7384-7. Other similar vector systems may also be
used. To recover the entire genomic copy of the Zmax1 gene, the
plasmid containing the two "hooks" is linearized with a restriction
endonuclease or is produced by another method such as PCR. This
linear DNA fragment is introduced into yeast cells along with human
genomic DNA. Typically, the yeast Saccharomyces cerevisiae is used
as a host cell, although Kourina et al., 1998 Genome Res. 8: 666-72
have reported using chicken host cells as well. During and after
the process of transformation, the endogenous host cell converts
the linear plasmid to a circle by a recombination event whereby the
region of the human genomic DNA homologous to the "hooks" is
inserted into the plasmid. This plasmid can be recovered and
analyzed by methods well known to one skilled in the art.
Obviously, the specificity for this reaction requires the host cell
machinery to recognize sequences similar to the "hooks" present in
the linear fragment. However, 1.00% sequence identity is not
required, as shown by Kouprina et al., 1998 Genomics 53: 21-8,
where the author describes using degenerate repeated sequences
common in the human genome to recover fragments of human DNA from a
rodent/human hybrid cell line.
[0643] In another example, only one "hook" is required, as
described by Larionov et al., 1998 Proc. Natl. Acad. Sci. USA, 95:
4469-74. For this type of experiment, termed "radial TAR cloning,"
the other region of sequence similarity to drive the recombination
is derived from a repeated sequence from the genome. In this way,
regions of DNA adjacent to the Zmax1 gene coding region can be
recovered and examined for alterations that may affect
function.
25. Methods of Use: Genomic Screening
[0644] The use of polymorphic genetic markers linked to the HBM
gene, or to Zmax1 (LRP5) or to LRP6 is very useful in predicting
susceptibility to osteoporosis or other bone diseases. Koller et
al., 1998 Amer. J. Bone Min. Res. 13: 1903-8 have demonstrated that
the use of polymorphic genetic markers is useful for linkage
analysis. Similarly; the identification of polymorphic genetic
markers within the high bone mass gene will allow the
identification of specific allelic variants that are in linkage
disequilibrium with other genetic lesions that affect bone
development. Using the DNA sequence from the BACs, a dinucleotide
CAn repeat was identified and two unique PCR primers that will
amplify the genomic DNA containing this repeat were designed, as
shown below:
TABLE-US-00047 B200E21C16_L: GAGAGGCTATATCCCTGGGC (SEQ ID NO: 38)
B200E21C16_R: ACAGCACGTGTTTAAAGGGG (SEQ ID NO: 39)
and used in the genetic mapping study.
[0645] This method has been used successfully by others skilled in
the art (e.g., Sheffield et al., 1995 Genet., 4:1837-44;
LeBlanc-Straceski et al., 1994 Genomics, 19: 341-9; Chen et al.,
1995 Genomics, 25:1-8). Use of these reagents with populations or
individuals will predict their risk for osteoporosis. Similarly,
single nucleotide polymorphisms (SNPs), such as those shown in
Table 4 above, can be used as well to predict risk for developing
bone diseases or resistance to osteoporosis in the case of the HBM
gene.
26. Methods of Use: Modulators of Tissue Calcification
[0646] The calcification of tissues in the human body is well
documented. Towler et al. (1998 J. Biol. Chem. 273: 30427-34)
demonstrated that several proteins known to regulate calcification
of the developing skull in a model system are expressed in
calcified aorta. The expression of Msx2, a gene transcribed in
osteoprogenitor cells, in calcified vascular tissue indicates that
genes which are important in bone development are involved in
calcification of other tissues.
[0647] Treatment with HBM protein, HBM-like proteins and
polypeptides, agonists of LRP5 and LRP6 and HBM or antagonists of
Dkk-1 are likely to ameliorate calcification (such as the
vasculature, dentin and bone of the skull viscera) due to its
demonstrated effect on bone mineral density. In experimental
systems where tissue calcification is demonstrated, the
over-expression or repression of Zmax1(LRP5) activity permits the
identification of molecules that are directly regulated by the
Zmax1 gene. These genes are potential targets for therapeutics
aimed at modulating tissue calcification. For example, an animal,
such as the LDLR -/-, mouse is fed a high fat diet and is observed
to demonstrate expression of markers of tissue calcification,
including Zmax1. These animals are then treated with antibodies to
Zmax1 or HBM protein, antisense oligonucleotides directed against
Zmax1 or HBM cDNA, or with compounds known to bind the Zmax1 or HBM
protein or its binding partner or ligand. RNA or proteins are
extracted from the vascular tissue and the relative expression
levels of the genes expressed in the tissue are determined by
methods well known in the art. Genes that are regulated in the
tissue are potential therapeutic targets for pharmaceutical
development as modulators of tissue calcification.
[0648] The nucleic acids, proteins, peptides, amino acids, small
molecules or other pharmaceutically useful compounds of the present
invention that are to be given to an individual may be administered
in the form of a composition with a pharmaceutically acceptable
carrier, excipient or diluent, which are well known in the art. The
individual may be a mammal or a bird, preferably a human, a rat, a
mouse or bird. Such compositions may be administered to an
individual in a pharmaceutically effective amount. The amount
administered will vary depending on the condition being treated and
the patient being treated. The compositions may be administered
alone or in combination with other treatments.
27. Methods to Identify Agents that Modulate the Expression of a
Nucleic Acid Encoding the Dkk and/or LRP5 Proteins and/or Dkk
Interacting Proteins
[0649] Another embodiment of the present invention provides methods
for identifying agents that modulate the expression of a nucleic
acid encoding Dkk, which is part of the Wnt pathway and that
interacts with LRP5, LRP6 and to much lesser extent to HBM and its
variants. Such assays may utilize any available means of monitoring
for changes in the expression level of the nucleic acids of the
invention. As used herein, an agent is said to modulate the
expression of Dkk, if it is capable of up- or down-regulating
expression of the nucleic acid in a cell (e.g., mRNA).
[0650] In one assay format, cell lines that contain reporter gene
fusions between the nucleic acid encoding Dkk (or proteins which
modulate the activity of Dkk) and any assayable fusion partner may
be prepared. Numerous assayable fusion partners are known and
readily available, including but not limited to the firefly
luciferase gene and the gene encoding chloramphenicol
acetyltransferase (Alam et al., 1990 Anal. Biochem. 188: 245-54).
Cell lines containing the reporter gene fusions are then exposed to
the agent to be tested under appropriate conditions and time.
Differential expression of the reporter gene between samples
exposed to the agent and control samples identifies agents which
modulate the expression of a nucleic acid encoding Dkk or other
protein which modulates Dkk activity. Such assays can similarly be
used to determine whether LRP5 and even LRP6 activity is modulated
by regulating Dkk activity. This can also be performed with the HBM
variants.
[0651] Additional assay formats may be used to monitor the ability
of the agent(s) to modulate the expression of a nucleic acid
encoding Dkk, alone or Dkk and LRP5, and/or Dkk interacting
proteins such as those identified in FIG. 31. For instance, mRNA
expression may be monitored directly by hybridization to the
nucleic acids of the invention. Cell lines are exposed to the agent
to be tested under appropriate conditions and time and total RNA or
mRNA is isolated by standard procedures such those disclosed in
Sambrook et al. (1989); Ausubel et al., Current Protocols in
Molecular Biology (Greene Publishing Co., NY, 1995); Maniatis et
al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1982); and Short Protocols in
Molecular Biology: A Compendium of Methods from Current Protocols
in Molecular Biology (Frederick M. Ausubel et al., April 1999).
[0652] Probes to detect differences in RNA expression levels
between cells exposed to the agent and control cells may be
prepared from the nucleic acids of the invention. It is preferable,
but not necessary, to design probes which hybridize only with
target nucleic acids under conditions of high stringency. Only
highly complementary nucleic acid hybrids form under conditions of
high stringency. Accordingly, the stringency of the assay
conditions determines the amount of complementarity which should
exist between two nucleic acid strands in order to form a hybrid.
Stringency should be chosen to maximize the difference in stability
between the probe:target hybrid and potential probe:non-target
hybrids.
[0653] Probes may be designed from the nucleic acids of the
invention through methods known in the art. For instance, the G+C
content of the probe and the probe length can affect probe binding
to its target sequence. Methods to optimize probe specificity are
commonly available. See for example, Sambrook et al. (1989) or
Ausubel et al. (Current Protocols in Molecular Biology, Greene
Publishing Co., NY, 1995).
[0654] Hybridization conditions are modified using known methods,
such as those described by Sambrook et al. (1989) and Ausubel et
al. (1995), as suitable for each probe. Hybridization of total
cellular RNA or RNA enriched for polyA RNA can be accomplished in
any available format. For instance, total cellular RNA or RNA
enriched for polyA RNA can be affixed to a solid support and the
solid support exposed to at least one probe comprising at least
one, or part of one of the nucleic acid sequences of the invention
under conditions in which the probe will specifically hybridize.
Alternatively, nucleic acid fragments comprising at least one, or
part of one of the sequences of the invention can be affixed to a
solid support, such as a porous glass wafer. The glass or silica
wafer can then be exposed to total cellular RNA or polyA RNA from a
sample under conditions in which the affixed sequences will
specifically hybridize. Such glass wafers and hybridization methods
are widely available, for example, those disclosed by Beattie (WO
95/11755). By examining for the ability of a given probe to
specifically hybridize to an RNA sample from an untreated cell
population and from a cell population exposed to the agent, agents
which up- or down-regulate the expression of a nucleic acid
encoding Dkk, a Dkk interacting protein, and/or LRP5 can be
identified.
[0655] Microarray technology and transcriptional profiling are
examples of methods which can be used to analyze the impact of
putative Dkk or Dkk interacting protein modulating compounds. For
transcriptional profiling, mRNA from cells exposed in vivo to a
potential Dkk modulating agent, such as the Dkk interacting
proteins identified in the present invention (e.g., those
identified in FIG. 31), agents which modulate Dkk interacting
proteins, and mRNA from the same type of cells that were not
exposed to the agent could be reverse transcribed and hybridized to
a chip containing DNA from numerous genes, to thereby compare the
expression of genes in cells treated and not treated with the
agent. If, for example a putative Dkk modulating agent
down-regulates the expression of Dkk in the cells, then use of the
agent may be undesirable in certain patient populations. For
additional methods of transcriptional profiling and the use of
microarrays, refer to, for example, U.S. Pat. No. 6,124,120 issued
to Lizardi (2000).
[0656] Additional methods for screening the impact of Dkk and Dkk
interacting protein modulating compounds or the impact of Dkk or
Dkk interacting proteins on modulation of LRP5, LRP6, HBM, HBM
variants or the Wnt pathway include the use of TaqMan.RTM. PCR,
conventional reverse transcriptase PCR (RT-PCR), changes in
downstream surrogate markers (i.e., Wnt responsive genes), and
anti-Dkk Western blots for protein detection. Other methods would
be readily apparent to the artisan of ordinary skill.
28. Methods to Identify Agents that Modulate at Least One Activity
of Dkk, a Dkk Interacting Protein, or LRP5/LRP6/HBM/HBM-Like
[0657] Another embodiment of the present invention provides methods
for identifying agents that modulate, at least one activity of Dkk,
Dkk interacting proteins, and/or LRP5/LRP6/HBM/HBM-like proteins or
preferably which specifically modulate an activity of a Dkk/Dkk
interacting protein complex or an LRP5 (or LRP6/HBM/HBM-like)/Dkk
complex, or a biologically active fragment of Dkk (e.g., comprising
the domain which binds LRP5/LRP6/HBM/HBM-like) or a Dkk interacting
protein complex. Such methods or assays may utilize any means of
monitoring or detecting the desired activity as would be known in
the art (See, e.g., Wu et al., 2000 Curr. Biol. 10:1611-4; Fedi et
al., 199 J. Biol. Chem. 274:19465-72; Grotewold et al., 1999 Mech.
Dev. 89:151-3; Shibata et al., 2000 Mech. Dev. 96: 243-6; Wang et
al., 2000 Oncogene 19: 1843-8; and Glinka et al., 1998 Nature 391:
357-62). Potential agents which modulate Dkk include, for example,
p 53, the tumor suppressor protein, which can induce Dkk-1. Damage
to DNA has also been observed to up-regulate Dkk-1 expression via a
stabilization and activation of p 53 (Wang et al., 2000 Oncogene
19: 1843-48); and, Shou et al., 2002 Oncogene 21: 878-89).
Additionally, Fedi et al. (1999) purportedly showed that Dkk-1 can
block the Wnt2-induced oncogenic transformation of NIH-3T3 cells.
Furthermore, it has been suggested that Dkk expression may be
modulated by BMP signaling in the developing skeleton (Mukhopadhyay
et al., 2001 Dev. Cell. 1: 423-34; and Grotewold et al., 2002 EMBO
J. 21: 966-75). Grotewald et al. additionally describe altered Dkk
expression levels in response to stress signals including UV
irradiation and other genotoxic stimuli. They propose that Dkk
expression is pro-apoptotic. In the HBMMTIC animals described
herein, a reduced osteoblast apoptosis effect was observed. Thus,
HBM and HBM like variants may control/alter Dkk's role in
programmed cell death. Other agents which potentially modulate Dkk
activity include the Dkk interacting proteins identified in FIG.
31.
[0658] In one embodiment, the relative amounts of Dkk or a Dkk
interacting protein of a cell population that has been exposed to
the agent to be tested is compared to an un-exposed control cell
population. Antibodies can be used to monitor the differential
expression of the protein in the different cell populations. Cell
lines or populations are exposed to the agent to be tested under
appropriate conditions and time. Cellular lysates may be prepared
from the exposed cell line or population and a control, unexposed
cell line or population. The cellular lysates are then analyzed
with the probe, as would be known in the art. See, e.g., Ed Harlow
and David Lane, Antibodies: A Laboratory Manual (Cold Spring
Harbor, N.Y., 1988) and Ed Harlow and David Lane, Using Antibodies:
A Laboratory Manual (Cold Spring Harbor, N.Y. 1998).
[0659] For example, N- and C-terminal fragments of Dkk can be
expressed in bacteria and used to search for proteins which bind to
these fragments. Fusion proteins, such as His-tag or GST fusion to
the N- or C-terminal regions of Dkk (or to biologically active
domains of Dkk-1) or a whole Dkk protein can be prepared. These
fusion proteins can be coupled to, for example, Talon or
Glutathione-Sepharose beads and then probed with cell lysates to
identify molecules which bind to Dkk. Prior to lysis, the cells may
be treated with purified Wnt proteins, RNA, or drugs which may
modulate Wnt signaling or proteins that interact with downstream
elements of the Wnt pathway. Lysate proteins binding to the fusion
proteins can be resolved by SDS-PAGE, isolated and identified by,
for example protein sequencing or mass spectroscopy, as is known in
the art. See, e.g., Protein Purification Applications: A Practical
Approach (Simon Roe, ed., 2.sup.nd ed. Oxford Univ. Press, 2001)
and "Guide to Protein Purification" in Meth. Enzymology vol. 182
(Academic Press, 1997).
[0660] The activity of Dkk, a Dkk interacting protein, or a complex
of Dkk with LRP5/LRP6/HBM/HBM-like may be affected by compounds
which modulate the interaction between Dkk and a Dkk interacting
protein (such as those shown in FIG. 31) and/or Dkk and
LRP5/LRP6/HBM/HBM-like. The present invention provides methods and
research tools for the discovery and characterization of these
compounds. The interaction between Dkk and a Dkk interacting
protein and/or Dkk and LRP5/6/HBM/HBM-like may be monitored in vivo
and in vitro. Compounds which modulate the stability of a
Dkk-LRP5/LRP6/HBM/HBM-like complex are potential therapeutic
compounds. Example in vitro methods include: Binding
LRP5/6/HBM/HBM-like, Dkk, or a Dkk interacting protein to a sensor
chip designed for an instrument such are made by Biacore (Uppsala,
Sweden) for the performance of an plasmon resonance spectroscopy
observation. In this method, the chip with one of Dkk, a Dkk
interacting protein, or LRP5/6 is first exposed to the other under
conditions which permit them to form the complex. A test compound
is then introduced and the output signal of the instrument provides
an indication of any effect exerted by the test compound. By this
method, compounds may be rapidly screened. Another, in vitro,
method is exemplified by the SAR-by-NMR methods (Shuker et al.,
Science. 274:1531-4 (1996)). Briefly, a Dkk-1 binding domain and/or
LRP 5 or 6 LBD are expressed and purified as .sup.15N labeled
protein by expression in labeled media. The labeled protein(s) are
allowed to form the complex in solution in an NMR sample tube. The
heteronuclear correlation spectrum in the presence and absence of a
test compound provides data at the level of individual residues
with regard to interactions with the test compound and changes at
the protein-protein interface of the complex. One of skill in the
art knows of many other protocols, e.g. affinity capillary
electrophoresis (Okun et al., 2001 J. Biol. Chem. 276: 1057-62;
Vergnon et al., 1999 Methods, 19: 270-7), fluorescence
spectroscopy, electron paramagnetic resonance, etc. which can
monitor the modulation of a complex and/or measure binding
affinities for complex formation.
[0661] In vitro protocols for monitoring the modulation of a
Dkkk/LRP5/LRP6/HBM/HBM-like complex include the yeast two hybrid
protocol. The yeast two hybrid method may be used to monitor the
modulation of a complex in vivo by monitoring the expression of
genes activated by the formation of a complex of fusion proteins of
Dkk and LRP ligand binding domains. Nucleic acids according to the
invention which encode the interacting Dkk and LRP LBD domains are
incorporated into bait and prey plasmids. The Y2H protocol is
performed in the presence of one or more test compounds. The
modulation of the complex is observed by a change in expression of
the complex activated gene. It will be appreciated by one skilled
in the art that test compounds can be added to the assay directly
or, in the case of proteins, can be coexpressed in the yeast with
the bait and prey compounds. Similarly, fusion proteins of Dkk and
Dkk interacting proteins can also be used in a Y2H screen to
identify other proteins which modulate the Dkk/Dkk interacting
protein complex.
[0662] Assay protocols such as these may be used in methods to
screen for compounds, drugs, treatments which modulate the Dkk/Dkk
interacting protein and/or Dkk/LRP5/6 complex, whether such
modulation occurs by competitive binding, or by altering the
structure of either LRP 5/6 or Dkk at the binding site, or by
stabilizing or destablizing the protein-protein interface. It may
be anticipated that peptide aptamers may competitively bind,
although-induction of an altered binding site structure by steric
effects is also possible.
[0663] 28.1 Antibodies and Antibody Fragments
[0664] Polyclonal and monoclonal antibodies and fragments of these
antibodies which bind to Dkk or LRP5/LRP6/HBM/HBM-like can be
prepared as would be known in the art. For example, suitable host
animals can be immunized using appropriate immunization protocols
and the peptides, polypeptides or proteins of the invention.
Peptides for use in immunization are typically about 8-40 residues
long. If necessary or desired, the polypeptide immunogens can be
conjugated to suitable carriers. Methods for preparing immunogenic
conjugates with carriers such as bovine serum albumin (BSA),
keyhole limpet hemocyanin (KLH), or other carrier proteins are well
known in the art (See, Harlow et al., 1988). In some circumstances,
direct conjugation using, for example, carbodiimide reagents, may
be effective; in other instances linking reagents such as those
supplied by Pierce Chemical Co., Rockford, Ill., may be desirable
to provide accessibility to the polypeptide or hapten. The hapten
peptides can be extended at either the amino or carboxy terminus
with a cysteine residue or interspersed with cysteine residues, for
example, to facilitate linking to a carrier. Administration of the
immunogens is conducted generally by injection over a suitable time
period and with use of suitable adjuvants, as is generally
understood in the art. During the immunization schedule, titers of
antibodies are taken to determine adequacy of antibody
formation.
[0665] Anti-peptide antibodies can be generated using synthetic
peptides, for example, the peptides derived from the sequence of
any Dkk, including Dkk-1, or LRP5/LRP6/HBM/HBM-like. Synthetic
peptides can be as small as 2-3 amino acids in length, but are
preferably at least 3, 5, 10, or 15 or more amino acid residues
long. Such peptides can be determined using programs such as
DNAStar. The peptides are coupled to KLH using standard methods and
can be immunized into animals such as rabbits. Polyclonal anti-Dkk
or anti-LRP5/LRP6/HBM/HBM-like peptide antibodies can then be
purified, for example using Actigel beads containing the covalently
bound peptide.
[0666] While the polyclonal antisera produced in this way may be
satisfactory for some applications, for pharmaceutical
compositions, use of monoclonal preparations is preferred.
Immortalized cell lines which secrete the desired monoclonal
antibodies may be prepared using the standard method of Kohler and
Milstein or modifications which effect immortalization of
lymphocytes or spleen cells, as is generally known (See, e.g.,
Harlow et al., 1988 and 1998). The immortalized cell lines
secreting the desired antibodies can be screened by immunoassay in
which the antigen is the peptide hapten, polypeptide or protein.
When the appropriate immortalized cell culture secreting the
desired antibody is identified, the cells can be cultured either in
vitro or by production in ascites fluid.
[0667] The desired monoclonal antibodies are then recovered from
the culture supernatant or from the ascites supernatant. Fragments
of the monoclonal antibodies which contain the immunologically
significant portion can be used as agonists or antagonists of Dkk
activity. Use of immunologically reactive fragments, such as the
Fab, scFV, Fab', of F(ab').sub.2 fragments are often preferable,
especially in a therapeutic context, as these fragments are
generally less immunogenic than the whole immunoglobulin.
[0668] The antibodies or fragments may also be produced, using
current technology, by recombinant means. Regions that bind
specifically to the desired regions of Dkk or
LRP5/LRP6/HBM/HBM-like can also be produced in the context of
chimeras with multiple species origin. Antibody reagents so created
are contemplated for use diagnostically or as stimulants or
inhibitors of Dkk activity.
[0669] In one embodiment, antibodies against Dkk, bind Dkk with
high affinity, i.e., ranging from 10.sup.-5 to 10.sup.-9 M.
Preferably, the anti-Dkk antibody will comprise a chimeric,
primate, Primatized.RTM., human or humanized antibody. Also, the
invention embraces the use of antibody fragments, e.g., Fab's,
Fv's, Fab's, F(ab).sub.2, and aggregates thereof.
[0670] Another embodiment contemplates chimeric antibodies which
recognize Dkk or LRP5/LRP6/HBM/HBM-like. A chimeric antibody is
intended to refer to an antibody with non-human variable regions
and human constant regions, most typically rodent variable regions
and human constant regions.
[0671] A "Primatized.RTM. antibody" refers to an antibody with
primate variable regions, e.g., CDR's, and human constant regions.
Preferably, such primate variable regions are derived from an Old
World monkey.
[0672] A "humanized antibody" refers to an antibody with
substantially human framework and constant regions, and non-human
complementarity-determining regions (CDRs). "Substantially" refers
to the fact that humanized antibodies typically retain at least
several donor framework residues (i.e., of non-human parent
antibody from which CDRs are derived).
[0673] Methods for producing chimeric, primate, Primatized.RTM.,
humanized and human antibodies are well known in the art. See,
e.g., U.S. Pat. No. 5,530,101, issued to Queen et al.; U.S. Pat.
No. 5,225,539, issued to Winter et al.; U.S. Pat. Nos. 4,816,397
and 4,816,567, issued to Boss et al. and Cabilly et al.
respectively, all of which are incorporated by reference in their
entirety.
[0674] The selection of human constant regions may be significant
to the therapeutic efficacy of the subject anti-Dkk or
LRP5/LRP6/HBM/HBM-like antibody. In a preferred embodiment, the
subject anti-Dkk or LRP5/LRP6/HBM/HBM-like antibody will comprise
human, gamma 1, or gamma 3 constant regions and, more preferably,
human gamma 1 constant regions.
[0675] Methods for making human antibodies are also known and
include, by way of example, production in SCID mice, and in vitro
immunization.
[0676] The subject anti-Dkk or LRP5/LRP6/HBM/HBM-like antibodies
can be administered by various routes of administration, typically
parenteral. This is intended to include intravenous, intramuscular,
subcutaneous, rectal, vaginal, and administration with intravenous
infusion being preferred.
[0677] The anti-Dkk or LRP5/LRP6/HBM/HBM-like antibody will be
formulated for therapeutic usage by standard methods, e.g., by
addition of pharmaceutically acceptable buffers, e.g., sterile
saline, sterile buffered water, propylene glycol, and combinations
thereof.
[0678] Effective dosages will depend on the specific antibody,
condition of the patient, age, weight, or any other treatments,
among other factors. Typically effective dosages will range from
about 0.001 to about 30 mg/kg body weight, more preferably from
about 0.01 to 25 mg/kg body weight, and most preferably from about
0.1 to about 20 mg/kg body weight.
[0679] Such administration may be effected by various protocols,
e.g., weekly, bi-weekly, or monthly, depending on the dosage
administered and patient response. Also, it may be desirable to
combine such administration with other treatments.
[0680] Antibodies to Dkk-1 interacting proteins, such as those
identified in FIG. 31, are also contemplated according to the
present invention, and can be used similarly to the Dkk-1
antibodies mentioned in the above methodology.
[0681] The antibodies of the present invention can be utilized in
experimental screening, as diagnostic reagents, and in therapeutic
compositions.
[0682] 28.2 Chemical Libraries
[0683] Agents that are assayed by these methods can be randomly
selected or rationally selected or designed. As used herein, an
agent is said to be randomly selected when the agent is chosen
randomly without considering the specific sequences involved in the
association of Dkk-1 alone, Dkk-1 interacting proteins alone, or
with their associated substrates, binding partners, etc. An example
of randomly selected agents is the use of a chemical library or a
peptide combinatorial library, or a growth broth of an
organism.
[0684] The agents of the present invention can be, as examples,
peptides, small molecules, vitamin derivatives, as well as
carbohydrates. A skilled artisan can readily recognize that there
is no limit as to the structural nature of the agents of the
present invention.
[0685] 28.3 Peptide Synthesis
[0686] The peptide agents of the invention can be prepared using
standard solid phase (or solution phase) peptide synthesis methods,
as is known in the art. In addition, the DNA encoding these
peptides may be synthesized using commercially available
oligonucleotide synthesis instrumentation and produced
recombinantly using standard recombinant production systems. The
production of polypeptides using solid phase peptide synthesis is
necessitated if non-nucleic acid-encoded amino acids are to be
included.
29. Uses for Agents that Modulate at Least One Activity of Dkk, a
Dkk Interacting Protein, a Dkk/Dkk Interacting Protein Complex, or
a Dkk/LRP5 or Dkk LRP6 Complex
[0687] The proteins and nucleic acids of the invention, such as the
proteins or polypeptides containing an amino acid sequence of LRP5,
Dkk, and Dkk interacting proteins are involved in bone mass
modulation and lipid modulation of other Wnt pathway mediated
activity. Agents that modulate (i.e., up and down-regulate) the
expression of Dkk or Dkk interacting proteins, or agents, such as
agonists and antagonists respectively, of at least one activity of
Dkk or a Dkk interacting protein may be used to modulate biological
and pathologic processes associated with the function and activity
of Dkk or a Dkk interacting protein.
[0688] As used herein, a subject can be preferably any mammal, so
long as the mammal is in need of modulation of a pathological or
biological process modulated by a protein of the invention. The
term "mammal" means an individual belonging to the class Mammalia.
The invention is particularly useful in the treatment of human
subjects.
[0689] As used herein, a biological or pathological process
modulated by Dkk or a Dkk interacting protein may include binding
of Dkk to a Dkk interacting protein, Dkk to LRP5 or LRP6 or release
therefrom, inhibiting or activating Dkk or a Dkk interacting
protein mRNA synthesis or inhibiting Dkk or Dkk interacting protein
modulated inhibition of LRP5 or LRP6 mediated Wnt signaling.
Further bone-related markers may be observed such as alkaline
phosphatase activity, osteocalcin production, or
mineralization.
[0690] Pathological processes refer to a category of biological
processes which produce a deleterious effect. For example,
expression or up-regulation of expression of LRP5 or LRP6 and/or
Dkk and/or a Dkk interacting protein may be associated with certain
diseases or pathological conditions. As used herein, an agent is
said to modulate a pathological process when the agent
statistically significantly (p<0.05) alters the process from its
base level in the subject. For example, the agent may reduce the
degree or severity of the process mediated by that protein in the
subject to which the agent was administered. For instance, a
disease or pathological condition may be prevented, or disease
progression modulated by the administration of agents which reduce
or modulate in some way the expression or at least one activity of
a protein of the invention.
[0691] As LRP5/6 and Dkk are involved both directly and indirectly
in bone mass modulation, one embodiment of this invention is to use
Dkk or Dkk interacting protein expression as a method of diagnosing
a bone condition or disease. Certain markers are associated with
specific Wnt signaling conditions (e.g., TCF/LEF activation).
Diagnostic tests for bone conditions may include the steps of
testing a sample or an extract thereof for the presence of Dkk or
Dkk interacting protein nucleic acids (i.e., DNA or RNA), oligomers
or fragments thereof or protein products of TCF/LEF regulated
expression. For example, standard in situ hybridization or other
imaging techniques can be utilized to observe products of Wnt
signaling. Other diagnostic techniques, as described herein, would
also be useful as would be apparent to the skilled artisan (e.g., a
serum marker).
[0692] This invention also relates to methods of modulating bone
development or bone loss conditions. Inhibition of bone loss may be
achieved by inhibiting or modulating changes in the LRP5/6 mediated
Wnt signaling pathway. For example, absence of LRP5 activity may be
associated with low bone mass. Increased activity LRP5 may be
associated with high bone mass. Therefore, modulation of LRP5
activity will in turn modulate bone development. Modulation of the
Dkk/LRP5/6 or Dkk/Dkk interacting protein complex via agonists and
antagonists is one embodiment of a method to regulate bone
development. Such modulation of bone development can result from
inhibition of the activity of, for example, a Dkk/LRP(5/6) protein
complex, a Dkk/Dkk interacting protein complex, upregulated
transcription of the LRP5 gene or inhibited transcription or
translation of Dkk or Dkk interacting protein mRNA.
[0693] The agents of the present invention can be provided alone,
or in combination with other agents that modulate a particular
pathological process. As used herein, two agents are said to be
administered in combination when the two agents are administered
simultaneously or are administered independently in a fashion such
that the agents will act at the same time.
[0694] The agents of the present invention can be administered via
parenteral, subcutaneous (sc), intravenous (iv), intramuscular
(im), intraperitoneal (ip), transdermal or buccal routes.
Alternatively, or concurrently, administration may be by the oral
route. The dosage administered will be dependent upon the age,
health, and weight of the recipient, kind of concurrent treatment,
if any, frequency of treatment, and the nature of the effect
desired.
[0695] The present invention further provides compositions
containing one or more agents which modulate expression or at least
one activity of a protein of the invention. While individual needs
vary, determination of optimal ranges of effective amounts of each
component is within the skill of the art. Typical dosages of the
active agent which mediate Dkk or Dkk interacting protein activity
comprise from about 0.0001 to about 50 mg/kg body weight. The
preferred dosages comprise from about 0.001 to about 50 mg/kg body
weight. The most preferred dosages comprise from about 0.1 to about
1 mg/kg body weight. In an average human of 70 kg, the range-would
be from about 7 .mu.g to about 3.5 g, with a preferred range of
about 0.5 mg to about 5 mg.
[0696] In addition to the pharmacologically active agent, the
compositions of the present invention may contain suitable
pharmaceutically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically for delivery
to the site of action. Suitable formulations for parenteral
administration include aqueous solutions of the active compounds in
water-soluble form, for example, water-soluble salts. In addition,
suspensions of the active compounds as appropriate oily injection
suspensions may be administered. Suitable lipophilic solvents or
vehicles include fatty oils, for example, sesame oil, or synthetic
fatty acid esters, (e.g., ethyl oleate or triglycerides). Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol and/or dextran. Optionally, the
suspension may also contain stabilizers.
[0697] Liposomes and other non-viral vectors can also be used to
encapsulate the agent for delivery into the cell.
[0698] The pharmaceutical formulation for systemic administration
according to the invention may be formulated for enteral,
parenteral, or topical (top) administration. Indeed; all three
types of formulations may be used simultaneously to achieve
systemic administration of the active ingredient.
[0699] Suitable formulations for oral administration include hard
or soft gelatin capsules, pills, tablets, including coated tablets,
elixirs, suspensions, syrups or inhalations and controlled release
forms thereof.
[0700] Potentially, any compound which binds Dkk or a Dkk
interacting protein or modulates the Dkk/LRP5 or Dkk/LRP6 or
Dkk/Dkk interacting protein complex may be a therapeutic compound.
In one embodiment of the invention, a peptide or nucleic acid
aptamer according to the invention is used in a therapeutic
composition. Such compositions may comprise an aptamer, or a LRP5
or LRP6 fragment unmodified or modified. In another embodiment, the
therapeutic compound comprises a-Dkk-1 interacting protein, or
biologically active fragment thereof.
[0701] Nucleic acid aptamers have been used in compositions for
example by chemical bonding to a carrier molecule such as
polyethylene glycol (PEG) which may facilitate uptake or stabilize
the aptamer. A di-alkylgylcerol moiety attached to an RNA will
embed the aptamer in liposomes, thus stabilizing the compound.
Incorporating chemical substitutions (i.e. changing the 2'OH group
of ribose to a 2'NH in RNA confers ribonuclease resistance) and
capping, etc. can prevent breakdown. Several such techniques are
discussed for RNA aptamers in Brody and Gold (Rev. Mol. Biol.
74:3-13, 2000).
[0702] Peptide aptamers may by used in therapeutic applications by
the introduction of an expression vector directing aptamer
expression into the affected tissue such as for example by
retroviral delivery, by encapsulating the DNA in a delivery complex
or simple by naked DNA injection. Or, the aptamer itself or a
synthetic analog may be used directly as a drug. Encapsulation in
polymers and lipids may assist in delivery. The use of peptide
aptamers as therapeutic and diagnostic agents is reviewed by
Hoppe-Syler and Butz (J. Mol. Med. 78:426-430 (2000)).
[0703] In another aspect of the invention. The structure of a
constrained peptide aptamer of the invention may be determined such
as by NMR or X-ray crystallography. (Cavanagh et al., Protein NMR
Spectroscopy: Principles and Practice, Academic Press, 1996;
Drenth, Principles of Protein X-Ray Crystallography, Springer
Verlag, 1999) Preferably the structure is determined in complex
with the target protein. A small molecule analog is then designed
according to the positions of functional elements of the 3D
structure of the aptamer. (Guidebook on Molecular Modeling in Drug
Design, Cohen, Ed., Academic Press, 1996; Molecular Modeling and
Drug Design (Topics in Molecular and Structural Biology), Vinter
and Gardner Eds., CRC Press, 1994) Thus the present invention
provides a method for the design of effective and specific drugs
which modulate the activity of Dkk, Dkk interacting proteins,
Dkk/Dkk interacting protein complex and the Dkk/LRP complex. Small
molecule mimetics of the peptide aptamers of the present invention
are encompassed within the scope of the invention.
[0704] In practicing the methods of this invention, the compounds
of this invention may be used alone or in combination, or in
combination with other therapeutic or diagnostic agents. In certain
preferred embodiments, the compounds of this invention may be
co-administered along with other compounds typically prescribed for
these conditions according to generally accepted medical practice.
For example, the compounds of this invention can be administered in
combination with other therapeutic agents for the treatment of bone
loss. Bone loss mediating agents include bone resorption inhibitors
such as bisphosphonates (e.g., alendronic acid, clodronic acid,
etidronic acid, pamidronic acid, risedronic acid and tiludronic
acid), vitamin D and vitamin D analogs, cathepsin K inhibitors,
hormonal agents (e.g., calcitonin and estrogen), and selective
estrogen receptor modulators or SERMs (e.g., raloxifene). And bone
forming agents such as parathyroid hormone (PTH) and bone
morphogenetic proteins (BMP).
[0705] Additionally contemplated are combinations of agents which
regulate Dkk-1 and agents which regulate lipid levels such as
HMG-CoA reductase inhibitors (i.e., statins such as Mevacor.RTM.,
Lipitor.RTM. and other inhibitors such as Baycol.RTM., Lescol.RTM.,
Pravachol.RTM. and Zocor.RTM.), bile acid sequestrants (e.g.,
Colestid.RTM. and Welchol.RTM.), fibric acid derivatives
(Atromid-S.RTM., Lopid.RTM., Tricor.RTM.), and nicotinic acid.
[0706] The compounds of this invention can be utilized in vivo,
ordinarily in vertebrates and preferably in mammals, such as
humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or
in vitro.
30. Peptide and Nucleotide Aptamers and Peptide Aptamer
Mimetics
[0707] Another embodiment contemplates the use of peptide and
nucleotide aptamer technology to screen for agents which interact
with Dkk, which block Dkk from interacting with LRP5, LRP6, HBM, or
HBM-like molecules, or which block any other Dkk ligand
interaction, or which interact with Dkk interacting proteins, such
as those shown in FIG. 5. Peptide aptamers are molecules in which a
variable peptide domain is displayed from a scaffold protein.
Thioredoxin A (trxA) is commonly used for a scaffold. The peptide
insert destroys the catalytic site of trxA. It is recognized that
numerous proteins may also be used as scaffolding proteins to
constrain and/or present a peptide aptamer. Other scaffold proteins
that could display a constrained peptide aptamer could include
staphylococcal nuclease, the protease inhibitor eglin C, the
Streptomyces tendea alpha-amylase inhibitor Tendamistat, Sp1, green
fluorescent protein (GFP) (reviewed in Hoppe-Seyler et al., 2001 J.
Steroid Biochem Mol. Biol. 78: 105-11), and the S1 nucleas from
Staphyloccus or M13 for phage display. Any molecule to which the
aptamer could be anchored and presented in its bioactive
conformation would be suitable.
[0708] Aptamers can then specifically bind to a given target
protein in vitro and in vivo and have the potential to selectively
block the function of their target protein. Peptide aptamers are
selected from randomized expression libraries on the basis of their
in vivo binding capacity to the desired target protein. Briefly, a
target protein (e.g., Dkk, a Dkk interacting protein, or LRP5/6) is
linked to a heterologous DNA binding domain (BD) and expressed as
bait in a yeast test strain. Concomitantly, a library coding for
different peptides (e.g., 16-mers) of randomized sequence inserted
in a scaffold protein sequence, which are linked to a heterologous
transcriptional activation domain (AD) is expressed as prey. If a
peptide binds to a target protein, a functional transcription
factor is reconstituted, in which the BD and AD are bridged
together by interacting proteins. This transcription factor is then
able to activate the promoter of a marker gene which can be
monitored by colorimetric enzymatic assays or by growth selection.
Additional variation, methods of preparing and screening
methodologies are described in, for example, Hoppe-Seyler et al.,
2000 J. Mol. Med. 78: 426-430.
[0709] Nucleotide aptamers are described for example in Brody et
al., 2000 Trends Mol. Biotechnol. 74: 5-13. Additional methods of
making and using nucleotide aptamers include SELEX, i.e.,
Systematic Evolution of Ligands by Exponential Enrichment. SELEX is
a process of isolating oligonucleotide ligands of a chosen target
molecule (see Tuerk and Gold, Science 249:505-510 (1990); U.S. Pat.
Nos. 5,475,096, 5,595,877, and 5,660,985). SELEX, as described in
Tuerk and Gold, involves admixing the target molecule with a pool
of oligonucleotides (e.g., RNA) of diverse sequences; retaining
complexes formed between the target and oligonucleotides;
recovering the oligonucleotides bound to the target;
reverse-transcribing the RNA into DNA; amplifying the DNA with
polymerase chain reactions (PCR); transcribing the amplified DNA
into RNA; and repeating the cycle with ever increasing binding
stringency. Three enzymatic reactions are required for each cycle.
It usually takes 12-15 cycles to isolate aptamers of high affinity
and specificity to the target. An aptamer is an oligonucleotide
that is capable of binding to an intended target substance but not
other molecules under the same conditions.
[0710] In another reference, Bock et al., 1990 Nature 355: 564-6,
describe a different process from the SELEX method of Tuerk and
Gold in that only one enzymatic reaction is required for each cycle
(i.e., PCR) because the nucleic acid library in Bock's method is
comprised of DNA instead of RNA. The identification and isolation
of aptamers of high specificity and affinity with the method of
Bock et al. still requires repeated cycles in a chromatographic
column.
[0711] Other nucleotide aptamer methods include those described by
Conrad et al., 1996 Meth. Enzymol. 267: 336-367. Conrad et al.
describe a variety of methods for isolating aptamers, all of which
employ repeated cycles to enrich target-bound ligands and require a
large amount of purified target molecules. More recently described
methods of making and using nucleotide aptamers include, but are
not limited to those described in U.S. Pat. Nos. 6,180,348;
6,051,388; 5,840,867; 5,780,610, 5,756,291 and 5,582,981.
[0712] Potentially, any compound which binds Dkk or a Dkk
interacting protein or modulates the Dkk/Dkk interacting protein or
Dkk/LRP5, Dkk/LRP6, Dkk/HBM, or Dkk/HBM-like complex may be a
therapeutic compound. In one embodiment of the invention, a peptide
or nucleic acid aptamer according to the invention is used in a
therapeutic composition. Such compositions may comprise an aptamer,
or a LRP5 or LRP6 fragment unmodified or modified.
[0713] Nucleic acid aptamers have been used in compositions for
example by chemical bonding to a carrier molecule such as
polyethylene glycol (PEG) which may facilitate uptake or stabilize
the aptamer. A di-alkylglycerol moiety attached to an RNA will
embed the aptamer in liposomes, thus stabilizing the compound.
Incorporating chemical substitutions (i.e., changing the 2'-OH
group of ribose to a 2'--NH in RNA confers ribonuclease resistance)
and capping, etc. can prevent breakdown. Several such techniques
are discussed for RNA aptamers in Brody et al., 2000 Rev. Mol.
Biol. 74: 3-13.
[0714] Peptide aptamers may by used in therapeutic applications by
the introduction of an expression vector directing aptamer
expression into the affected tissue such as for example by
retroviral delivery, by encapsulating the DNA in a delivery complex
or simple by naked DNA injection. Or, the aptamer itself or a
synthetic analog may be used directly as a drug.
[0715] Encapsulation in polymers and lipids may assist in delivery.
The use of peptide aptamers as therapeutic and diagnostic agents is
reviewed by Hoppe-Syler et al., 2000 J. Mol. Med. 78: 426-430.
[0716] In another aspect of the invention, the structure of a
constrained peptide aptamer of the invention may be determined such
as by NMR or X-ray crystallography. (Cavanagh et al., Protein NMR
Spectroscopy: Principles and Practice, Academic Press, 1996;
Drenth, Principles of Protein X-Ray Crystallography, Springer
Verlag, 1999) Preferably the structure is determined in complex
with the target protein. A small molecule analog is then designed
according to the positions of functional elements of the 3D
structure of the aptamer. (Guidebook on Molecular Modeling in Drug
Design, Cohen, Ed., Academic Press, 1996; Molecular Modeling and
Drug Design (Topics in Molecular and Structural Biology), Vinter
and Gardner Eds., CRC Press, 1994) Thus, a method is provided for
the design of effective and specific drugs which modulate the
activity of Dkk, Dkk interacting proteins, Dkk/Dkk interacting
protein complex, and the Dkk/LRP complex. Small molecule mimics of
the peptide aptamers of the present invention are also encompassed
within the scope of the invention.
EXAMPLES
[0717] The present invention is described by reference to the
following Examples, which are offered by way of illustration and
are not intended to limit the invention in any manner. Standard
techniques well known in the art or the techniques specifically
described below were utilized.
Example 1
[0718] The propositus was referred by her physicians to the
Creighton Osteoporosis Center for evaluation of what appeared to be
unusually dense bones. She was 18 years old and came to medical
attention two years previous because of back pain, which was
precipitated by an auto accident in which the car in which she was
riding as a passenger was struck from behind. Her only injury was
soft tissue injury to her lower back that was manifested by pain
and muscle tenderness. There was no evidence of fracture or
subluxation on radiographs. The pain lasted for two years, although
she was able to attend school full time. By the time she was seen
in the Center, the pain was nearly resolved and she was back to her
usual activities as a high school student. Physical exam revealed a
normal healthy young woman standing 66 inches and weighing 128
pounds. Radiographs of the entire skeleton revealed dense looking
bones with thick cortices. All bones of the skeleton were involved.
Most importantly, the shapes of all the bones were entirely normal.
The spinal BMC was 94.48 grams in L1-4, and the spinal BMD was
1.667 gm/cm.sup.2 in L1-4. BMD was 5.62 standard deviations (SD)
above peak skeletal mass for women. These were measured by DXA
using a Hologic 2000.about.. Her mother was then scanned and a
lumbar spinal BMC of 58.05 grams and BMD of 1.500 gm/cm.sup.2 were
found. Her mother's values place her 4.12 SD above peak mass and
4.98 SD above her peers. Her mother was 51 years old, stood 65
inches and weighed 140 pounds. Her mother was in excellent health
with no history of musculoskeletal or other symptoms. Her father's
lumbar BMC was 75.33 grams and his BMD was 1.118 gm/cm.sup.2. These
values place him 0.25 SD above peak bone mass for males. He was in
good health, stood 72 inches tall, and weighed 187 pounds.
[0719] These clinical data suggested that the propositus inherited
a trait from her mother, which resulted in very high bone mass, but
an otherwise normal skeleton, and attention was focused on the
maternal kindred. In U.S. Pat. No. 5,691,153, twenty-two of these
members had measurement of bone mass by DXA. In one case, the
maternal grandfather of the propositus, was deceased, however,
medical records, antemortem skeletal radiographs and a gall bladder
specimen embedded in paraffin for DNA genotyping were obtained. His
radiographs showed obvious extreme density of all of the bones
available for examination including the femur and the spine, and he
was included among the affected members. In this invention, the
pedigree has been expanded to include 37 informative individuals.
These additions are a significant improvement over the original
kinship (Johnson et al., Am. J. Hum. Genet., 60:1326-1332 (1997))
because, among the fourteen individuals added since the original
study, two individuals harbor key crossovers. X-linkage is ruled
out by the presence of male-to-male transmission from individual 12
to 14 and 15.
Example 2
[0720] The present invention describes DNA sequences derived from
two BAC clones from the HBM gene region, as evident in Table 7
below, which is an assembly of these clones. Clone b200e21-h (ATCC
No. 980812; SEQ ID NOS: 10-11) was deposited at the American Type
Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.
20110-2209 U.S.A., on Dec. 30, 1997. Clone b527d12-h (ATCC No.
980720; SEQ ID NOS: 5-9) was deposited at the American Type Culture
Collection (ATCC), 10801 University Blvd:, Manassas, Va. 20110-2209
U.S.A., on Oct. 2, 1998. These sequences are unique reagents that
can be used by one skilled in the art to identify DNA probes for
the Zmax1 gene, PCR primers to amplify the gene, nucleotide
polymorphisms in the Zmax1 gene, or regulatory elements of the
Zmax1 gene.
Example 3
Yeast-2 Hybrid Screen for Peptide Aptamer Sequences to Dkk-1
[0721] Peptide aptamer library construction. A peptide aptamer
library, Tpep, was constructed, which provides a means to identify
chimeric proteins that bind to a protein target (or bait) of
interest using classic yeast two hybrid (Y2H) assays. The Tpep
library is a combinatorial aptamer library composed of constrained
random peptides, expressed within the context of the disulfide loop
of E. coli thioredoxin (trxA), and as C-termini fusion to the S.
cerevisiae Gal4 activation domain. The Tpep library was generated
using a restriction enzyme modified recombinant Y2H prey vector,
pPC86 (Gibco), which contains the trxA scaffold protein.
[0722] Generation of aptamer-encoding sequences. Aptamer-encoding
sequences were produced as follows. DNA encoding random stretches
of approximately sixteen amino acids surrounded by appropriate
restriction sites were generated by semi-random oligonucleotide
synthesis. The synthetic oligonucleotides were PCR-amplified,
restriction digested, and cloned into the permissive sites within
the trxA scaffold protein. The cloning strategy was to insert the
random oligonucleotide sequence is in-frame with the scaffold
protein coding sequence, resulting in expression of a scaffold
protein-aptamer chimera. The scaffold protein is itself in-frame
with the activation domain of Gal4, within the pPC86 vector that is
appropriate for the aptamer to be expressed and functional in a
regular Y2H assay. Additional methods of preparing aptamers would
be apparent to the skilled artisan.
[0723] Generation of a permissive recombinant pPC86 vector
containing the trxA coding sequence. First the RsrII restriction
site located within the Gal4 activation domain of pPC86 (Gibco) was
eliminated by site-directed mutagenesis (Quickchange.TM.kit,
Stratagene). The amino acid sequence, of the Gal4 activation domain
was unchanged by this modification. The strength of different
control interactions was verified to be unchanged by the
modification.
[0724] Second, the E. coli trxA coding sequence was cloned into the
Sail and NotI sites of the RsrII-modified pPC86. EcoRI and SpeI
sites were then introduced within the trxA RsrII site. The
oligonucleotides encoding the peptideaptamers were cloned into the
EcoRI and SpeI sites of the resulting vector.
TABLE-US-00048 TABLE 13 SEQ ID Length Contig ATCC No. NO. (base
pairs) b527d12-h_contig302G 980720 5 3096 b527d12-h_contig306G
980720 6 26928 b527d12-h_contig307G 980720 7 29430
b527d12-h_contig308G 980720 8 33769 b527d12-h_contig309G 980720 9
72049 b200e21-h_contig1 980812 10 8705 b200e21-h_contig4 980812 11
66933
Example 4
Generation of Antibodies
[0725] In each of the following antibody-generating examples, the
synthesis of these linear peptides is followed by injection into
two New Zealand Rabbits. Subsequent boosts and bleeds are taken
according to a standard ten-week protocol. The end-user receives
back 5 mgs of peptide, aliquots of pre-bleeds, roughly 80 ml of
crude sera from each of the two rabbits and, and ELISA titration
data is obtained.
[0726] Generation of LRP5 Polymorphism-specific antibodies.
Antibodies were generated to the following peptides to obtain
antibodies which distinguish the HBM polymorphism versus wild-type
LRP5/Zmax: MYWTDWVETPRIE (SEQ ID NO: ) (mutant peptide) and
MYWTDWGETPRIE (SEQ ID NO: ) (wild-type peptide for negative
selection). Immunofluorescence data confirmed that the antibody,
after affinity purification, is specific for HBM and does not
recognize LRP5 (FIG. 32).
[0727] Generation of LRP5 Monospecific antibodies. LRP5
monospecific polyclonal antibodies were generated to the following
amino acid sequences of LRP5: Peptide 1 (a.a.
265-277)-KRTGGKRKEILSA (SEQ ID NO: ), Peptide 2 (a.a.
1178-1194)-ERVEKTTGDKRTRIQGR (SEQ ID NO: ), and Peptide 3 (a.a.
1352-1375)-KQQCDSFPDCIDGSDE (SEQ ID NO: ). Immunofluorescence
confirmed that the antibody generated detects LRP5.
[0728] Generation of Dkk-1 monospecific polyclonal antibodies.
Dkk-1 monospecific polyclonal antibodies were generated to the
following amino acid sequences of Dkk-1:
TABLE-US-00049 Peptide 1 GNKYQTIDNYQPYPC, (SEQ ID NO: ) Peptide 2
LDGYSRRTTLSSKMYHTKGQEG, (SEQ ID NO: ) Peptide 3
RIQKDHHQASNSSRLHTCQRH, (SEQ ID NO: ) Peptide 4 RGEIEETITESFGND,
(SEQ ID NO: ) and Peptide 5 EIFQRCGEGLSCRIQKD (SEQ ID NO: ) of
human Dkk-1.
Western Blots demonstrated that the antibodies generated against
peptides 2 and 4 are specific toward Dkk-1.
Example 5
Effects of Exogenous Dkk-1 on Wnt-Mediated Signaling in the Xenopus
Embryo Assay
[0729] Xenopus embryos are an informative and well-established in
vivo assay system to evaluate the modulation of Wnt signaling
(McMahon et al., 1989 Cell 58: 1075-84; Smith et al., 1991 reviewed
in Wodarz and Nusse, 1998).
[0730] Modification of the Wnt signaling pathway can be visualized
by examining the embryos for a dorsalization phenotype (duplicated
body axis) after RNA injection into the ventral blastomere at the
4- or 8-cell stage. On the molecular level, phenotypes can be
analyzed by looking for expression of various marker-genes in stage
10.5 embryos. Such markers would include general endoderm,
mesoderm, and ectoderm markers as well as a variety of
tissue-specific transcripts.
[0731] Analysis can be done by RT-PCR/TaqMan.RTM. and can be done
on whole embryo tissue or in a more restricted fashion
(microdissection). Because this system is very flexible and rapid,
by injecting combinations of transcripts, such as HBM and different
Wnts or Wnt antagonists, the mechanism of HBM in the Wnt pathway
can thereby be dissected. Furthermore, investigations are conducted
to determine whether Zmax/LRP5 and HBM differentially modulate Wnt
signaling either alone, or in combination with other components.
Previous studies have demonstrated that LRP6 alone or LRP5+Wnt5a
were able to induce axis duplication (dorsalization) in this system
(Tamai et al., 2000 Nature 407: 530-35).
[0732] Constructs for Xenopus Expression (Vector pCS2.sup.+).
Constructs were prepared using the vector pCS2.sup.+. DNA inserts
were subcloned in the sense orientation with respect to the vector
SP6 promoter. The pCS2.sup.+ vector contains an SV40 virus
polyadenylation signal and T3 promoter sequence (for generation of
antisense mRNA) downstream of the insert.
[0733] Full length Zmax/LRP5 and HBM ORF cDNA: Insert cDNA was
isolated from the full length cDNA retrovirus constructs (with
optimized Kozak sequences) by BglII-EcoRI digestion and subcloned
into the BamHI-EcoRI sites of the pCS2.sup.+ vector. CDNAs encoding
a HBM-like molecule could be subcloned into pCS2.sup.+ vectors and
processed similarly by one of ordinary skill.
[0734] Full length XWnt8: This cDNA was PCR amplified from a
Xenopus embryo cDNA library using oligos 114484 (SEQ ID NO: )
(5'-CAGTGAATTCACCATGCAAAACACC ACTTTGTTC-3') and 114487 (SEQ ID NO:
) (5'-CAGTTGCGGCCGCTCATCTCCGGTG GCCTCTG-3'). The oligos were
designed to amplify the ORF with a consensus Kozak sequence at the
5' end as determined from GenBank #X57234. PCR was carried out
using the following conditions: 96.degree. C., 45 sec.; 63.degree.
C., 45 sec.; 72.degree. C., 2 min. for 30 cycles. The resulting PCR
product was purified, subcloned into pCRII-TOPO (Invitrogen Corp.),
sequence verified, and digested with BamH/XhoI. This insert was
subcloned into the vector at the BamHI-XhoI sites.
[0735] Full length Wnt5a: A murine Wnt5a cDNA clone was purchased
from Upstate Biotechnology (Lake Placid, N.Y.) and subcloned into
the EcoRI site of the vector. Sequencing confirmed insert
orientation.
[0736] Full length human Dkk-1: A human cDNA with GenBank accession
number AF127563 was available in the public database. Using this
sequence, PCR primers were designed to amplify the open reading
frame with a consensus Kozak sequence immediately upstream of the
initiating ATG. Oligos 117162 (SEQ ID NO: )
(5'-CAATAGTCGACGAATTCACCATGGCTCTGGGCGCAGCGG-3') and 117163 (SEQ ID
NO: ) (5'-GTATTGCGGCCGCTCTAGATTAGTGTCTCTGACAAGTGTGAA-3') were used
to screen a human uterus cDNA library by PCR. The resulting PCR
product was purified, subcloned into pCRII-TOPO (Invitrogen Corp.),
sequence verified, and digested with EcoRI/XhoI. This insert was
subcloned into the pCS2.sup.+ vector at the EcoRI-XhoI sites.
[0737] Full length human Dkk-2: A full length cDNA encoding human
Dkk-2 was isolated to investigate the specificity of the
Zmax/LRP5/HBM interaction with the Dkk family of molecules. Dkk-1
was identified in yeast as a potential binding partner of
Zmax/LRP5/HBM. Dkk-1 has also been shown in the literature to be an
antagonist of the Wnt signaling pathway, while Dkk-2 is not
(Krupnik et al., 1999). The Dkk-2 full length cDNA serves as a tool
to discriminate the specificity and biological significance of
Zmax/LRP5/HBM interactions with the Dkk family (e.g., Dkk-1, Dkk-2,
Dkk-3, Dkk-4, Soggy, their homologs and variant, etc.). A human
cDNA sequence for Dkk-2 (GenBank Accession No. NM.sub.--014421) was
available in the public database. Using this sequence, PCR primers
were designed to amplify the open reading frame with a consensus
Kozak sequence immediately upstream of the initiating ATG. Oligos
51409 (SEQ ID NO: ) (5'-CTAACGGATCCACCATGGCCGCGTTGATGCGG-3') and
51411 (SEQ ID NO: ) (5'-GATTCGAATTCTCAAATTTTCTGACACACATGG-3') were
used to screen human embryo and brain cDNA libraries by PCR. The
resulting PCR product was purified, subcloned into pCRII-TOPO,
sequence verified, and digested with BamHI/EcoRI. This insert was
subcloned into the pCS2.sup.+ vector at the BamHI-EcoRI sites.
[0738] Full length LRP6 was isolated from the pED6dpc4 vector by
XhoI-XbaI digestion. The full length cDNA was reassembled into the
XhoI-XbaI sites of pCS2.sup.+. Insert orientation was confirmed by
DNA sequencing.
[0739] mRNA Synthesis and Microinjection Protocol. mRNA for
microinjection into Xenopus embryos is generated by in vitro
transcription using the cDNA constructs in the pCS2.sup.+ vector
described above as template. RNA is synthesized using the Ambion
mMessage mMachine high yield capped RNA transcription kit (Cat.
#1340) following the manufacturer's specifications for the Sp6
polymerase reactions. RNA products were brought up to a final
volume of 50 .mu.l in sterile, glass-distilled water and purified
over Quick Spin Columns for Radiolabeled RNA Purification
G50-Sephadex (Roche, Cat. #1274015) following the manufacturer's
specifications. The resulting eluate was finally extracted with
phenol:chloroform:isoamyl alcohol and isopropanol precipitated
using standard protocols (Sambrook et al., 1989). Final RNA volumes
were approximately 50 .mu.l. RNA concentration was determined by
absorbance values at 260 nm and 280 nm. RNA integrity was
visualized by ethidium bromide staining of denaturing
(formaldehyde) agarose gel electrophoresis (Sambrook et al., 1989).
Various amounts of RNA (2 pg to 1 ng) are injected into the ventral
blastomere of the 4- or 8-cell Xenopus embryo. These protocols are
described in Moon et al., 1989 Technique-J. of Methods in Cell
& Mol. Biol. 1: 76-89; and Peng, 1991 Meth. Cell. Biol. 36:
657-62.
[0740] Screening for Duplicated Body Axis. In vitro transcribed RNA
is purified and injected into a ventral blasomere of the 4- or
8-cell Xenopus embryo (approx. 2 hours post-fertilization). At
stage 10.5 (approx. 11 hours post-fertilization), the injected
embryos are cultured for a total of 72 hours and then screened for
the presence of a duplicated body axis (dorsalization) (FIG. 33).
Using XWnt8-injected (2-10 pg) as a positive control (Christian et
al., 1991) and water-injected or non-injected embryos as negative
controls, we replicated the published observation that
Zmax(LRP5)+Wnt5a (500 and 20 pg, respectively) could induce axis
duplication. Wnt5a (20 pg) alone could not induce axis duplication
(as previously reported by Moon et al., 1993). We have also
injected GFP RNA (100-770 pg) as a negative control to show that
the amount of RNA injected is not perturbing embryo development
(not shown). Strikingly, HBM+Wnt5a (500 and 20 pg, respectively)
yielded an approximately 3.5 fold more robust response of the
phenotype (p=0.043 by Fisher's exact test) compared to
Zmax(LRP5)+Wnt5a, suggesting that the HBM mutation is activating
the Wnt pathway (FIGS. 34 and 35). The HBM/Wnt5a embryos also
appear to be more "anteriorized" than the Zmax(LRP5)/Wnt5a embryos,
again suggestive of a gain-of-function mutation.
[0741] The role of Dkk-1 as a modulator of Zmax/LRP5- and
HBM-mediated Wnt signaling was investigated. Literature reports
have previously characterized Xenopus and murine Dkkk-1 as
antagonists of the canonical Wnt pathway in the Xenopus system
(Glinka et al., 1998 Nature 391: 357-62). Using the human Dkk-1
construct, a dose-response assay was performed to confirm that our
construct was functional and to identify the optimal amount of RNA
for microinjection. Using 250 pg/embryo of hDkk-1 RNA, over 90%
(p<0.001) of the embryos were observed to display enlarged
ariterior structures (big heads) as anticipated from the published
reports (FIG. 36).
[0742] The mechanism of hDkk-1 modulation of Wnt signaling in the
presence of Zmax/LRP5 or HBM was also investigated. Without any
hDkk-1 present, it was confirmed that HBM+Wnt5a was a more potent
activator of Wnt signaling than Zmax/LRP5+Wnt5a (p<0.05).
Interestingly, in the presence of hDkk-1 (250 pg),
Zmax/LRP5-mediated Wnt signaling was repressed (p<0.05) but
hDkk-1 was unable to repress HBM-mediated Wnt signaling (p<0.01)
(FIG. 37). The specificity of this observation can be further
addressed by investigating other members of the Dkk family, other
Wnt genes, LRP6, additional Zmax/LRP5 mutants, and the peptide
aptamers.
Example 6
Effects of exogenous Dkk and LRP5 on Wnt signaling in the
TCF-luciferase Assay
[0743] Wnt activity can be antagonized by many proteins including
secreted Frizzled related proteins (SFRPs), Cerberus, Wrt
Inhibitory Factor-1 and Dkk-1 (Krupnik et al., 1999). The Dkk
family of proteins consists of Dkk-1-4 and Soggy, a Dkk-3-like
protein. Dkk-1 and Dkk-4 have been shown to antagonize Wnt mediated
Xenopus embryo development, whereas Dkk-2, Dkk-3, and Soggy do not.
Unlike many of these proteins that antagonize Wnt activity by
directly interacting with Wnt proteins, Dkk-1 acts by binding to
two recently identified Wnt coreceptors, LRP5 and LRP6 (Mao et al.,
2001; Bafico et al., 2001). The details of this interaction have
been examined by the present inventors and Mao et al. using
deletion constructs of LRP6, which demonstrated that EGF repeats 3
and 4 are important for Dkk-1 interaction. Accordingly, the
activity of two Dkk proteins, Dkk-1 and Dkk-2, were investigated
with various Wnt members, LRP5, LRP6, and the mutant form of LRP5,
designated HBM. The present invention explores whether there is any
functional difference between LRP5 and HBM with regard to Dkk
action on Wnt mediated signaling. Various reagents were developed,
including Dkk-1 peptides, constrained LRP5 peptide aptamers,
constrained Dkk-1 peptide aptamers and polyclonal antibodies to
Dkk-1 (in Example 4 above) to identify factors that mimic HBM
mediated Wnt signaling.
[0744] Methods. Various LRP5 constrained peptides were developed.
Specifically, four peptides that interact with the LBD of LRP5
(FIG. 38, constructs OST259-262 in FIG. 39) and three peptides that
interact with the cytoplasmic domain of LRP5 (constructs
OST266-OST268 in FIG. 39). In addition two Dkk-1 peptides were
developed: constructs OST264 and OST265 in FIG. 39, corresponding
to Dkk-1 amino acids 139-266 and 96-245, containing the smallest
region of Dkk-1 that interacts with LRP5 (FIG. 40). The cDNA clones
encoding the LRP5 LBD interacting peptides and the Dkk-1 peptides
were subcloned into pcDNA3.1 with the addition of a Kozak and
signal sequence to target the peptide for secretion. The constructs
encoding the three peptides interacting with the cytoplasmic domain
of LRP5 were also subcloned into pcDNA3.1. However, these latter
constructs do not contain a signal sequence.
[0745] HOB-03-CE6 osteoblastic cells developed by Wyeth Ayerst
(Philadelphia, Pa.) were seeded into 24-well plates at 150,000
cells per well in 1 ml of the growth media (D-MEM/F12 phenol
red-free) containing 10% (v/v) heat-inactivated FBS, 1.times.
penicillin streptomycin, and 1.times. Glutamax-1, and incubated
overnight at 34.degree. C. The following day, the cells were
transfected using Lipofectamine 2000.RTM. (as described by the
manufacturer, Invitrogen) in OptiMEM (Invitrogen) with 0.35
.mu.g/well of LRP5, HBM, or control plasmid DNA (empty vector
pcDNA3.1) and either Wnt1 or Wnt3a plasmid DNA. Similar experiments
were performed with LRP6 plasmid DNA (0.35 g/well) or a control
pEDdpc4 empty vector. Furthermore, each of these groups were then
divided into three groups, those receiving 0.35 .mu.g/well Dkk-1,
Dkk-2, or pcDNA3.1 control DNA. All wells were transfected with
0.025 .mu.g/well of CMV beta-galactosidase plasmid DNA and 0.35
.mu.g/well 16.times.TCF(AS)-luciferase reporter DNA (developed by
Ramesh Bhat, Wyeth-Ayerst (Philadelphia, Pa.)). After 4 hours of
incubation, the cells were rinsed and 1 ml of fresh growth media
was added to each well. The cells were cultured overnight at
34.degree. C., followed by a wash and a change of media. Cells were
cultured for an additional 18-24 hours at 37.degree. C. Cells were
then lysed with 50 .mu.l/well of 1.times. lysis buffer. The
extracts were assayed for beta-galactosidase activity (Galacto
Reaction Buffer Diluent & Light Emission Accelerator, Tropix)
using 5 .mu.l extract+50 .mu.l beta-galactosidase diluerit and
luciferase activity (Luciferase Assay Reagent, Promega) using 20
.mu.l extract.
[0746] U2OS human osteosarcoma cells. were also utilized. U2OS
cells (ATCC) were seeded into 96-well plates at 30,000 cells per
well in 200 .mu.l of the growth media (McCoy's 5A) containing 10%
(v/v) heat-inactivated FBS, 1.times. penicillin streptomycin, and
1.times. Glutamax-1, and incubated overnight at 37.degree. C. The
following day, the media was replaced with OptiMEM (Invitrogen) and
cells were transfected using Lipofectamine 2000.RTM. (as described
by the manufacturer, Invitrogen) with 0.005 .mu.g/well of LRP5,
HBM, LRP6 or control plasmid DNA (empty vector pcDNA3.1) and either
Wnt1 (0.0025 .mu.g/well) or Wnt3a (0.0025 .mu.g/well) plasmid DNA.
In addition, the 16.times.-(AS) TCF-TK-firefly-luciferase and
control TK-renilla luciferase (Promega Corp.) were co-transfected
at 0.3 .mu.g/well and 0.06 .mu.g/well respectively in all
experiments. Furthermore, each of these groups was then divided
into different groups, those receiving 0.05 .mu.g/well Dkk-1,
Dkk-2, Dkk3, Dkk1-Alkaline Phosphatase (AP), mutant Dkk-1 (C220A),
Soggy or pcDNA3.1 control DNA. In other experiments, cells were
co-transfected with 0.005 .mu.g/well of LRP5, 0.0025 .mu.g/well of
Wnt1 or Wnt3a (using 0.0025 .mu.g/well of a control pcDNA3.1) with
LRP5-interacting aptamers (0.05 .mu.g/well). Cells were cultured
for an additional 18-20 hours at 37.degree. C. Culture medium was
removed. Cells were cultured for an additional 18-20 hours at
37.degree. C. Culture medium was removed. Cells were then lysed
with 100 .mu.l/well of 1.times. Passive Lysis Buffer (PLB) of Dual
Luciferase Reagent kit (DLR-kit-Promega Corp.) 20/1 of the lysates
were combined with LARII reagent of DLR-kit and assayed for
TCF-firefly luciferase signal in Top Count (Packard) instrument.
After measuring the Firefly readings, 100 .mu.l of the "Stop and
Glo" reagent of DLR kit that contains a quencher and a substrate
for renilla luciferase was added into each well. Immediately the
renilla luciferase reading was measured using the Top Count
(Packard) Instrument. The ratios of the TCF-firefly luciferase to
control renilla readings were calculated for each well and the mean
ratio of triplicate or more wells was expressed in all data.
[0747] Results. The results of these experiments demonstrate that
Dkk-1, in the presence of Wnt1 and LRP5, significantly antagonized
TCF-luciferase activity (FIG. 41). In marked contrast, Dkk-1 had no
effect on HBM/Wnt1 mediated TCF-luciferase activity (FIG. 41). In
similar experiments, Dkk-1 was also able to antagonize LRP5/Wnt3a
but not HBM/Wnt3a mediated TCF-luciferase activity (FIG. 42). These
results indicate that the HBM mutation renders Dkk-1 inactive as an
antagonist of Wnt1 and Wnt3a signaling in HOB03CE6 osteoblastic
cells. In other experiments with Wnt1, Dkk-1 had no effect on LRP5
or HBM mediated TCF-luciferase activity (FIG. 41). In contrast,
with either LRP5 or HBM in the presence of Wnt3a, Dkk-2 was able to
antagonize the TCF-luciferase activity (FIG. 42). These latter
results indicate that the HBM mutation has no effect on Dkk-2
action in the presence of Wnt3a. Experiments were also performed
using the closely related LRP6 cDNA in HOB-03-CE6 cells: In these
experiments, LRP6/Wnt1 and LRP6/Wnt3a mediated TCF-luciferase were
regulated in the same manner as LRP5. Specifically, Dkk-1
antagonized LRP6/Wnt1 mediated TCF-luciferase activity, whereas
Dkk-2 had no effect (FIG. 41). However, similar to the action of
Dkk-2 with LRP5/Wnt3a, Dkk-2 was able to antagonize LRP6/Wnt3a
mediated TCF-luciferase activity (FIG. 42).
[0748] The results in the U2OS cells show a robust effect of the
OST262 LRP5 peptide aptamer activation of Wnt signaling in the
presence of Wnt3a (FIG. 43). These functional results are confirmed
by the results shown below in Example 7 using LRP5 peptide aptamers
in the Xenopus assay. Such results affirmatively demonstrate that
the effects of small molecules on LRP5/LRP6/HBM signaling can be
detected using the TCF-luciferase assay.
[0749] These data demonstrate that there is a functional difference
between LRP5 and HBM regarding the ability of Dkk-1 to antagonize
Wnt1 and Wnt3a signaling. These data and previous data showing that
Dkk-1 directly interacts with LRP5 suggests that the inability of
Dkk-1 to antagonize HBM/Wnt signaling may in part contribute to the
HBM phenotype. These experiments further demonstrate the ability to
test various molecules (e.g., small molecules, aptamers, peptides,
antibodies, LRP5 interacting proteins or Dkk-1 interacting
proteins, and the like) for a LRP5 ligand that mimics HBM mediated
Wnt signaling or factors that block Dkk-1 interaction with
LRP5.
[0750] This was the assay that was used to show the responsiveness
of two HBM-like variants. See FIGS. 29 and 30. The data also
demonstrates that these variants are less susceptible to modulation
by Dkk.
Example 7
Cell-Based Functional High-Throughput Assay
[0751] To develop a high throughput assay, the TCF-luciferase assay
described in Example 6 was modified utilizing low level expression
of endogenous LRP5/6 in U2OS and HEK293 cells. However, HOB-03-CE6
cells and any other cells which show a differential response to Dkk
depending on whether LRP5, LRP6 or HBM are expressed. Using U2OS
(human osteosarcoma) and HEK293 (ATCC) cells, the TCF-luciferase
and tk-Renilla reporter element constructs were co-transfected
along with Wnt3a/1 and Dkk. Wnt3a alone, by using endogenous
LRP5/6, was able to stimulate TCF reporter gene activation. When
Dkk, is co-transfected with Wnt3a/Wnt 1 and reporters (TCF-luci and
tk-Renilla), Dkk represses reporter element activity. In addition,
the TCF-luci signal is activated by Wnt3a/Wnt1 can be repressed by
the addition of Dkk-enriched conditioned media to the cells
containing Wnt3a/Wnt1 and reporters. The assay is further validated
by the lack of TCF-reporter inhibition by a point mutant construct
(C220A) of Dkk1.
[0752] The Dkk-mediated repression of the reporter is dependent
upon the concentration of transfected Dkk cDNA or on the amount of
Dkk-conditioned media added. In addition, the Dkk-mediated reporter
suppression can be altered by the co-transfection of LRP5, LRP6,
and HBM cDNAs in the U2OS or HEK293 cells. In general, U2OS cells
show greater sensitivity to Dkk-mediated reporter suppression than
that in HEK-293 cells. In U2OS cells, the transfection of
LRP5/LRP6/HMHBMBM-like cDNA leads to moderate activation of
TCF-luci in the absence of Wnt3a/Wnt1 transfection. This activation
presumably utilizes the endogenous Wnts present in U2OS cells.
Under this condition, Dkk1 can repress TCF-luci and shows a
differential signal between LRP5 and HBM. By co-transfecting
Wnt3a/Wnt1, there is a generalized increase in the TCF-luci signal
in the assay. Further, one can detect Dkk-mediated differential
repression of the reporter due to LRP5 and HBM cDNA expression as
well as between LRP5 and LRP6 cDNA. The repression is maximal with
LRP6, moderate with LRP5, and least with HBM cDNA expression. In
addition, the assay can detect the functional impact of the LRP5
interacting peptide aptamers (FIG. 38), Dkk1 interacting aptamers
and binding domains of Dkk-1 (FIG. 40; OST264 and OST265 of FIGS.
39 and 44).
[0753] Using this system with a suppressed Wnt-TCF signal due to
the presence of both Dkk and Wnt3a, one can screen for compounds
that could alter Dkk modulation of Wnt signaling, by looking for
compounds that activate or the TCF-luciferase reporter, and thereby
relieve the Dkk-mediated repression of the Wnt pathway. Such
compounds identified may potentially serve as HBM-mimetics and be
useful, for example, as osteogenic therapeutics. Data generated
from this high throughput screen are demonstrated in FIGS. 45-47.
FIG. 45 shows that Dkk1 represses Wnt3a-mediated signaling in U2OS
bone cells. FIG. 46 demonstrates the functional differences between
LRP5, LRP6, and HBM. Dkk-1 represses LRP6 and LRP5 but has little
or no effect on HBM-generated Wnt1 signaling in U2OS cells. FIG. 47
demonstrates the differential effects of various Dkk family members
and modified Dkks, including Dkk-1, a mutated Dkk-1 (C220A),
Dkk-1-AP (modified with alkaline phosphatase), Dkk-3, and
Soggy.
Example 8
DKK/LRP5/6/HBM/FARM-like ELISA Assay
[0754] A further method to investigate Dkk binding to LRP or HBM
and HBM like polypeptides is via ELISA assay. Two possible
permutations of this assay are exemplified. LRP5 is immobilized to
a solid surface, such as a tissue culture plate well. One skilled
in the art will recognize that other supports such as a nylon or
nitrocellulose membrane, a silicon chip, a glass slide, beads, etc.
can be utilized. In this example, the form of LRP5 used is actually
a fusion protein where the extracellular domain of LRP5 is fused to
the Fc portion of human IgG. The LRP5-Fc fusion protein is produced
in CHO cell extracts from stable cell lines. The LRP5-Fc fusion
protein is immobilized on the solid surface via anti-human Fc
antibody or by Protein-A or Protein G-coated plates, for example.
The plate is then washed to remove any non-bound protein.
Conditioned media containing secreted Dkk protein or secreted
Dkk-epitope tagged protein (or purified Dkk or purified Dkk-epitope
tagged protein) is incubated in the wells and binding of Dkk to LRP
is investigated using antibodies to either Dkk or to an epitope
tag. Dkk-V5 epitope tagged protein would be detected using an
alkaline phosphatase tagged anti-V5 antibody.
[0755] Alternatively, the Dkk protein could be directly fused to a
detection marker, such as alkaline phosphatase. Here the detection
of the Dkk-LRP interaction can be directly investigated without
subsequent antibody-based experiments. The bound Dkk is detected in
an alkaline phosphatase assay. If the Dkk-alkaline phosphatase
fusion protein is bound to the immobilized LRP5, alkaline
phosphatase activity would be detected in a colorimetric readout.
As a result, one can assay the ability of small molecule compounds
to alter the binding of Dkk to LRP using this system. Compounds,
when added with Dkk (or epitope-tagged Dkk) to each well of the
plate, can be scored for their ability to modulate the interaction
between Dkk and LRP based on the signal intensity of bound Dkk
present in the well after a suitable incubation time and washing.
The assay can be calibrated by doing cold competition experiments
with unlabeled Dkk or with a second type of epitope-tagged Dkk. Any
small molecule that is able to modulate the Dkk-LRP interaction may
be a suitable therapeutic candidate, more preferably an osteogenic
therapeutic candidate.
Example 9
Functional Evaluation of Peptide Aptamers in Xenopus
[0756] The constrained peptide aptamers constructs OST258-263
(where 258 contains the signal sequence by itself and 263 contains
an irrelevant constrained peptide) (FIGS. 39 and 44) were used to
generate RNA substantially as described in Example 6, except the
vector was linearized by restriction endonuclease digestion and RNA
was generated using T7 RNA polymerase.
[0757] Aptamer RNA was injected at 250 pg per blastomere using the
protocol of Example 6. Wnt signaling was activated, as visualized
by embryo dorsalization (duplicated body axis) with aptamers 261
and, more strongly, 262. The results of this assay are shown in
FIGS. 48 and 49. These results suggest that aptamers 261 and 262
are able to activate Wnt signaling possibly by binding to the LBD
of LRP, thereby preventing the modulation of LRP-mediated signaling
by Dkk.
[0758] The aptamers of the present invention can serve as
HBM-mimetics. In the Xenopus system they are able to induce Wnt
signaling all by themselves. They may also serve as tools for
rational drug design by enhancing the understanding of how peptides
are able to interact with LRP and modulate Wnt signaling at the
specific amino acid level. Thus, one would be able to design small
molecules to mimic their effects as therapeutics. In addition, the
aptamers identified as positives in this assay may be used as
therapeutic molecules themselves.
Example 10
Homogenous Assay
[0759] An excellent method to investigate perturbations in
protein-protein interactions is via Fluorescence Resonance Energy
Transfer (FRET). FRET is a quantum mechanical process where a
fluorescent molecule, the donor, transfers energy to an acceptor
chromophore molecule which is in close proximity. This system has
been successfully used in the literature to characterize the
intermolecular interactions between LRP5 and Axin (Mao et al.,
Molec. Cell Biol. 7: 801-9). There are many different fluorescent
tags available for such studies and there are several ways to
fluorescently tag the proteins of interest. For example, CFP (cyan
fluorescent protein) and YFP (yellow fluorescent protein) can be
used as donor and acceptor, respectively. Fusion proteins, with a
donor and an acceptor, can be engineered, expressed, and
purified.
[0760] For instance, purified LRP protein, or portions or domains
thereof, fused to CFP and purified Dkk protein, or portions or
domains thereof that interact with Dkk or LRP respectively, fused
to YFP can be generated and purified using standard approaches. If
LRP-CFP and Dkk-YFP are in close proximity, the transfer of energy
from CFP to YFP will result in a reduction of CFP emission and an
increase in YFP emission. Energy is supplied with an excitation
wavelength of 450 nm and the energy transfer is recorded at
emission wavelengths of 480 nm and 570 nm. The ratio of YFP
emission to CFP emission provides a gauge for changes in the
interaction between LRP and Dkk. This system is amenable for
screening small molecule compounds that may alter the Dkk-LRP
protein-protein interaction. Compounds that disrupt the interaction
would be identified by a decrease in the ratio of YFP emission to
CFP emission. Such compounds that modulate the LRP-Dde interaction
would then be considered candidate --IBM mimetic molecules. Further
characterization of the compounds can be done using the
TCF-luciferase or Xenopus embryo assays to elucidate the effects of
the compounds on Wnt signaling.
[0761] While the above example describes a cell-fee, solution-phase
assay using purified components, a similar cell-based assay could
also be performed. For example, LRP-CFP fusion protein can be
expressed in cells. The Dkk-YFP fusion protein then could be added
to the cells either as purified protein or as conditioned media.
The interaction of LRP and Dkk is then monitored as described
above.
Example 11
Identification of Variants of LRP/Zmax1/HBM
[0762] Because the YWTD repeats constitute the major part of domain
which interacts with Dkk-1, a search was begun from the repeats to
look for protein folds that contain such repeats. Springer, 1998 J.
Mol. Biol. 283: 837-62 proposed that these YWTD repeats, which had
been previously described as "spacers" and considered to have no
defined structure, are in fact the propeller-blade subdomains of
highly structured six-bladed .beta.-propellers. Springer (1998)
therefore proposed two theoretically modeled protein structures
with such YWTD repeats.
[0763] The Model. A set of LRP5 propeller domain sequences from
mouse and human were assembled and aligned using CLUSTALW, together
with the sequences corresponding to two of the modeled
YWTD-propeller structures of Springer (1998) [1lpx, based on the
LRP1 .beta.-propeller domain no. 7 from chicken (SwissProt
LRP1_CH7), and 1ndx, based on human nidogen (Swiss-Prot NIDO_HU1)].
The alignment was manually edited based on the rationale and
knowledge of protein structures. FIG. 50 depicts a schematic model
of LRP5. The secondary structures consist exclusively of
.beta.-strands and turns. The secondary structure assignments were
verified by the predictive program, DSC (Discrimination of Protein
Secondary Structure Class available at
http://bioweb.pasteur.fr/seqanal/interfaces/dsc.html). Preliminary
checks of the exon-intron boundaries were performed manually. A
tertiary-structure homology model for human LRP5 using the 1lpx
model as a template was built with InsightII (Accelrys Inc., San
Diego, Calif.) and examined using the graphics display programs
from Insight II and Rasmol (freely available at
http://www.uuass.edu/microbio/rasmol/index2.html).
[0764] Results. Based on the homology alignments and modeling, the
domain diagram of LRP5 in FIG. 50 was obtained. FIG. 51 shows the
complete alignment and secondary structure assignments for the
propeller domains of mouse LRP5 and LRP6, human LRP5 and human
LRP6, and the sequences corresponding to the two theoretical models
constructed by Springer (1998).
[0765] To a good first approximation, the sequence alignments
support the proposal that there are four 6-bladed .beta.-propeller
domains in human LRP5. This model nicely accommodates the sequence
(as should be expected since it derives from a carefully
constructed model of a chicken LRP propeller domain). The overall
shape of this domain resembles a disk with inward-sloping sides and
a hole down the middle. The polypeptide chain enters and leaves
from the same (comparatively flat) "bottom" surface. As indicated
in the alignment (FIG. 51) and in several of the structure
illustrations, the G171V mutation falls into a loop on the outer or
"top" face of the domain. This immediately suggests that--contrary
to what one might expect, given the role of highly conserved
glycine residues in determining protein folds--the mutation should
not have any significant effect on the domain's tertiary structural
stability. It is rather more likely that the mutation interferes
with binding of some ligand, possibly a macromolecule (Smith et
al., 1999 Trends Biochem. Sci. 24: 181-5) or alters a
protein-protein interaction.
[0766] Closer scrutiny of the protein in the vicinity of G171 shows
that this residue sits at the bottom of a pocket on the outer edge
of the top surface of the domain. Moreover, one side of this pocket
consists of a cluster of very hydrophobic side chains, while the
other side has more polar groups, most notably glutamate 172. In
the mutant form of the protein, with V171 in place of the glycine,
the pocket largely disappears as the isopropyl group of valine
replaces the hydrogen side chain of glycine. It is easy to
understand how such a substitution could seriously disrupt ligand
binding if the ligand normally protrudes into the pocket. By the
same rationale, any other mutations that block such pockets in any
of the four propellers could also result in impaired ligand
binding.
[0767] The reasonableness of the proposed model is based on
Springer's results as well as additional data. For example, data
derived from the typical features of such protein domains
(including the well-studied YWTD repeats), e.g., that are robust
and rigid tertiary structures. Loop conformations may vary
somewhat, especially for the longer loops, but the basic features
of the protein scaffold are almost certainly well predicted. Of
course we would be on somewhat more secure ground if there were a
crystal structure for one of these domains. That naturally implies
that some of the more interesting features, such as the identity of
residues exposed on the outer, putative ligand-binding surface,
will also be well-predicted. Note that in FIG. 51 these residues
are marked in red on the sequences corresponding to Springer's
(1998) models. Even though different models could possibly be built
that are not dependent on Springer's assumptions and lead to a
different topology of the propeller, this would not alter the
conclusion that the G171V mutation lies in a surface loop.
[0768] At the very least, the model affords an opportunity to think
about this protein in molecular terms and that should facilitate
both experimental design and evaluation of results, i.e., candidate
epitopes might be selected more rationally. One interesting aspect,
evoked forcefully by the diagram in FIG. 50, is the question of how
the different modular propeller domains interact. The EGF-like
domains are well-studied (dozens of crystal and NMR structures
exist, see for example Bork et al., 1996 Quart. Rev. Biophys. 29:
119-67). The EGF-like domains consist mainly of a couple of
.beta.-hairpins, cross-linked by disulfide bonds, and in some cases
they have tightly bound calcium ions, which must further stabilize
their structure. Interestingly, in contrast to the YWTD propellers,
the EGF-like domains have the polypeptide chain entering at one end
and exiting at the other. Thus, mechanically, these domains could
act as spacers, even swivels (since rotation can occur around the
tails that extend from either end). Given the size of the
propellers, there are almost certainly inter-domain interactions
between them as they assemble in some higher-order structure,
connected by the EGF-like domains. Because of the topology of the
propellers, their "top" faces would have to face outward since the
EGF-like domains will tie them together by their protruding N- and
C-terminal extensions.
[0769] A more sophisticated analysis of the .beta.-propeller
structure was modeled using X-ray crystallographic data from the
LDL receptor. The primary amino acid sequences of the various beta
propeller domains of LRP5 (and other domains) were used to develop
homology models of their 3-dimensional structures according to the
following method: [0770] (A) Search for suitable structural
templates and check sequence identity with target. The ExNRL-3D
database (derived from the Protein Data Bank
(http://pdb.ccdc.cam.ac.uk/pdb/) was searched using BLASTP2
(Altschul et al., 1990 J. Mol. Biol. 215: 403-10; Huang et al.,
1991 Adv. Appl. Math. 12: 337-67; and Peitsch, 1995 PDB Quart.
Newsletter 72: 4) to find all similarities of the target sequence
with sequences of known structure. This program selects all
templates with sequence identities above 25% and projected model
size greater than 20 residues. This step also detects domains that
can be modelled based on unrelated templates. Use of this step
resulted in the selection of Protein Data Bank (PDB) structure,
1IJQ ("Crystal Structure Of The LDL Receptor YWTD-EGF Domain Pair")
(Jeon et al., 2001 Nat. Struct. Biol. 8: 499). [0771] (B) Create
ProModII (Altschul et al., 1990; Huang et al., 1991; and Peitsch,
1995) jobs and generate models with ProModII. [0772] (C)
Superimpose related 3D structures. This method is based upon the
diagonals of sequence similarity (the Dynamic sequence alignment
algorithm (SIM) (Altschul et al., 1990; Huang et al., 1991; and
Peitsch, 1995). Specifically, the following steps are performed:
[0773] (i) Primary match: Regions with sequence similarity are
selected automatically (manual selection is also possible) and the
corresponding residues matched in three-space. (ii) The primary
match is further refined using expanding context spheres. [0774]
(D) Generate a multiple alignment comprising the sequences to be
modeled. [0775] (E) Generate a framework for the new sequence.
[0776] (F) Based on the topological arrangement of corresponding
atoms: (a) atoms which occupy a similar portion of space and are
expected to have a structural counterpart in the new structure are
used to compute the framework coordinates (averaged positions); (b)
Side chains with fully incorrect geometries are removed. [0777] (G)
Rebuild the lacking loops based on the geometry of the loop stems:
(a) the stems of the loops to rebuild are used to scan a database
of structural fragments derived from the Brookhaven Data Bank.
Either the best fitting fragment or a framework derived from the
five best fragments, is used as the new loop, or (b) the
conformational space is searched using a CSP approach (seven
allowed .PHI.-.PSI. angle combinations, space allocation for the
loop, space allocation for each .alpha.-carbon in the loop). Both
methods will place only .alpha.-carbons when loops are in steric
conflict with the surrounding context. [0778] (H) Rebuilt
incomplete backbones based on the position of the .alpha.-carbons,
the backbone was rebuilt using a set of seven allowed .PHI.-.PSI.
angle combinations and a database of backbone fragments (a sliding
window of five residues was run through the protein sequence. The
best matching backbone fragment for each overlapping pentapeptide
was then stored. A framework for the main chain atoms is derived
from these peptides, using only the coordinates of the three
central residues of each pentapeptide. [0779] (I) The model's
structural quality is verified and packing is checked as follows:
[0780] (i) Verification of structural quality is based on the
method described by R. Luthy et al., 1992 Nature 356: 83-85. This
method analyzes the 3D context of each residue and allows the
identification of mis-folded regions. [0781] (ii). Packing is
checked based on a probe accessible surface (Connoly surface)
computation and a cubic grid passed through the structure. Inside
and outside surfaces are detected and the center and size of each
cavity is computed. The algorithm then compares the size and
distribution of these cavities between the model and the structure
in order to detect possibly mis-folded regions. [0782] (J) Refine
structure by energy minimization and molecular dynamics based on
force field computations following Gromos96 (Methods for the
evaluation of long-range electrostatic forces in computer
simulations of molecular systems, IN COMPUTER SIMULATION OF
BIOMOLECULAR SYSTEMS, THEORETICAL AND EXPERIMENTAL APPLICATIONS,
Vol. 2, 182-212 (W. F. van Gunsteren et al., eds., Escorn Science
Publishers, Leiden, The Netherlands, 1993); van Gunsteren et al.,
1990 Angew. Chem. Int. Ed. Engl. 29: 992-1023; van Gunsteren et
al., 1992 Eur. J. Biochem. 204: 947-961; Torda and W. F. van
Gunsteren. Molecular Modeling Using Nuclear Magnetic Resonance
Data, IN REVIEWS IN COMPUTATIONAL CHEMISTRY, Vol. II, 143-172 (K.
B. Lipkowitz et al., eds., VCH Publishers, Inc. New York, 1992);
van Gunsteren, Molecular dynamics and stochastic dynamics
simulation: A primer. in COMPUTER SIMULATION OF BIOMOLECULAR
SYSTEMS, THEORETICAL AND EXPERIMENTAL APPLICATIONS 3-36; van
Gunsteren et al., Computation of free energy in practice: choice of
approximations and accuracy limiting factors. IN COMPUTER
SIMULATION OF BIOMOLECULAR SYSTEMS, THEORETICAL AND EXPERIMENTAL
APPLICATIONS 315-348; Smith et. al., Methods for the evaluation of
long-range electrostatic forces in computer simulations of
molecular systems, IN COMPUTER SIMULATION OF BIOMOLECULAR SYSTEMS,
THEORETICAL AND EXPERIMENTAL APPLICATIONS 182-212; van Gunsteren et
al., Accounting for Molecular Mobility in Structure Determination
Based on Nuclear Magnetic Resonance Spectroscopic and X-Ray
Diffraction Data, in Methods IN ENZYMOLOGY: NUCLEAR MAGNETIC
RESONANCE, Vol. 239 619-654 (T. L. James et al., eds., Academic
Press, New York, 1994); van Gunsteren et al., 1994 Quart. Rev.
Biophysics 27: 435-481; van Gunsteren et al., 1995 Biomolecular
Modelling: Overview of Types of Methods to Search and Sample
Conformational Space, IN PROCEEDINGS OF THE 1ST EUROPEAN CONFERENCE
ON COMPUTATIONAL CHEMISTRY, AMERICAN INSTITUTE OF PHYSICS CONF.
PROC. 330: 253-268; and van Gunsteren et al., 1995 Computer Phys.
Communications 91: 305-319). [0783] (K) The structure is then
manually refined based on sequence alignment and structural
elements using Deep View, the SWISS-PDB Viewer (Guex et al., 1997
Electrophoresis 18: 2714-23).
[0784] Uses of structural model. Having a three-dimensional model
of the protein domain allows one to appreciate the context of the
HBM mutation, or other HBM-like mutations, in three dimensional
space. This facilitates the prediction of possible mechanisms of
action in a manner impossible in any other way because one cannot
predict from primary sequence alone the proximity of one distant
(in sequence space) amino acid to another. This also allows one to
make functional predictions that can then be tested using
molecular/cellular biology techniques to further refine the model
and validate the target. For example, the structural model of the
G171V mutation predicted an alteration in side chain-side chain
interactions, so we were able to use this information to
successfully predict similar mutations in other amino acids that
would have analogous structural effects. Overlaying other
functional data on this model would also able highlight other
potentially important regions of the protein domain. Additionally,
such models show the accessible residues, which would be useful for
developing small compounds, antibodies and the like which recognize
and bind to this region.
[0785] Results. Based on the space filling hypothesis set forth in
Section 3 above, two residues from blade 6 of propeller 1 were
identified as being in structurally equivalent locations to residue
171 in blade 4. Accordingly, substitutions with valine were
engineered: F241V and A242V.
[0786] Residues that were predicted to be accessible to the
interior surface of the propeller were identified in both blades 3
and 5 of Zmax1 (LRP5). Original predictions from the Springer model
identified G199 on blade 5 and E128 on blade 3 as the equivalent
position to residue 171 of blade 4 (HBM). Using the more
sophisticated model described above, G199 and E128 are not
predicted to be in the equivalent position to residue 171 of blade
4. Based on the new, more sophisticated model, the following
residues were chosen for valine substitutions:
TABLE-US-00050 Blade No. Mutations 5 L200V, T201V and I202V 3
S127V
Another residue T125 was selected for a conservative substitution
with T125S as well as a less conservative substitution of
T125G.
[0787] The role of the YWTD repetitive motifs in the
.beta.-propeller was examined by carrying out an alanine
replacement scan over this region in Blade 4. These repeats are
predicted to create the .beta.-sheet blades of the propeller. Based
on this model data, the following additional mutations were
prepared in propeller 1: Y164A, M165A, Y166A, W167A, T168A, and
D169A. These substitutions would be predicted to be detrimental to
the .beta.-propeller structure.
[0788] The spacing filling or occupied space model was further
examined by mutating a residue predicted by the above methods to
reside on the exterior surface of propellar 1, blade 4. This
mutation, K215V (K.fwdarw.V at position 215), was selected because
it was believed to not produce an HBM-like effect.
[0789] Based on this information, the following model of the HBM
G171V mutation was obtained. Specifically, substitutions of valine
at specific locations within propeller 1 could result in the
creation of hydrophobic patches on the surface of the propeller.
For example, the V at residue 171 of the HBM mutant comes in close
proximity to L150 on the adjacent strand, potentially creating a
hydrophobic patch that was not present with the wild type G171. An
example of this prediction, and others are presented in a separate
file (FIG. 52).
[0790] Based on this model, a mutation of L150G which would remove
the hydrophobic side chain at this site may not produce an HBM
effect. In addition, a double mutant of G171V (HBM) and L150G may
not produce an HBM effect due to the elimination of the hydrophobic
side chain interactions. The model can also be used to test this
prediction on a different propeller 1 blade. The substitution A214V
was shown to result in an HBM effect. I194 is the residue that is
in the equivalent position to A214 on blade 5 as L150 is to G171 on
blade 4. While the mutation of I194G alone may not be predicted to
result an HBM effect, the double mutant of I194G and A214V may no
longer permit a hydrophobic patch to form and as a result not
generate the HBM effect observed with A214V. Additional analyses of
the residues in proximity to A214 that may contribute to the
formation of hydrophobic patches identified P240 and F241.
Mutations of either P240G or F241G together with A214V may no
longer permit the formation of hydrophobic patches and may
eliminate the HBM effect observed with A214V alone.
[0791] Lastly, the new model was used to investigate specific
aspects of the other three propellers. The relevance of residues
R494, R570 and V667 in the three other propellars was investigated.
These three residues were chosen since they have been reported to
be the site of osteoporosis pseudoglioma disease causing missense
mutations: R494Q, R570 W and V667M (Gong et al., 2001 Cell 107:
513-523). Based on the structural location of these residues on the
outer surface of the propeller, the model would not predict that
these substitutions are either gain- or loss-of-function
substitutions (FIG. 53A-C). For example, the mutations related to
osteoporosis-pseudoglioma syndrome (OPPG) result in patients having
very low bone mass and who are prone to developing fractures and
deformation. This genetic condition is known now to result from
mutations in LRP5 (Gong et al., 2001 Cell 107: 513-23). Based on
this information, this mutation would be categorized as a loss in
function mutation.
[0792] As a result, we have engineered these receptor variant to
functionally test. These new receptor variants were generated
exactly as described above with the following oligonucleotides:
TABLE-US-00051 L150GF GAAGGTGCTCTTCTGGCAGGACGGTGACCAGCCGAGGGCC
L150GR GGCCCTCGGCTGGTCACCGTCCTGCCAGAAGAGCACCTTC I194GF
GATCATTGTGGACTCGGACGGTTACTGGCCCAATGGACTG II94GR
CAGTCCATTGGGCCAGTAACCGTCCGAGTCCACAATGATC L200VF
CATTTACTGGCCCAATGGAGTGACCATCGACCTGGAGGAGCAGA AGC L200VR
GCTTCTGCTCCTCCAGGTCGATGGTCACTCCATTGGGCGAGTAA ATG T201VF
CATTTACTGGCCCAATGGACTGGTCATCGACCTGGAGGAGCAGA AGC T201VR
GCTTCTGCTCCTCCAGGTCGATGACCAGTCCATTGGGCCAGTAA ATG I202VF
CATTTACTGGCCCAATGGACTGACCGTCGACCTGGAGGAGCAGA AGC I202VR
GCTTCTGCTCCTCCAGGTCGACGGTCAGTCCATTGGGCCAGTAA ATG T125SF
GGCAAGAAGCTGTACTGGTCGGACTCAGAGACCAACCGCATC T125SR
GATGCGGTTGGTCTCTGAGTCCGACCAGTACAGCTTCTTGCC T125GF
GGCAAGAAGCTGTACTGGGGGGACTCAGAGACCAACCGCATC T125GR
GATGCGGTTGGTCTCTGAGTCCCCGGAGTACAGCTTCTTGCC S127VF
GAAGCTGTACTGGACGGACGTAGAGACCAACCGCATCGAGGTG S127VR
GACCTCGATGCGGTTGGTCTCTACGTCCGTCCAGTACAGCTTC F241VF
CCTGACGCACCCCGTCGCCCTGACGCTCTCCGGGGACACTC F241VR
GAGTGTCCCCGGAGAGCGTCAGGGCGACGGGGTGCGTCAGG A242VF
CCTGACGCACCCCTTCGTCCTGACGCTCTCCGGGGACACTC A242VR
GAGTGTCCCGGGAGAGCGTCAGGACGAAGGGGTGCGTCAGG K215VF
CTACTGGGCTGACGCCGTGCTCAGCTTCATCCACCGTGCC K215VR
GGCACGGTGGATGAAGCTGAGCACGGCGTCAGCCGAGTAG Y164AF
CTTGGACCCCGCTCACGGGGGCATGTACTGGACAGACTGG Y164AR
CCAGTCTGTCCAGTACATGGCCCCGTGAGCGGGGTCCAAG M165AF
GGACCCCGCTCACGGGTACGCGTACTGGACAGACTGGGGTG M165AR
CACCCCAGTCTGTCCAGTACGCGTACCCGTGAGCGGGGTCC Y166AF
CCCGCTCACGGGTACATGGCCTGGACAGACTGGGGTGAGAC Y166AR
GTCTCACCCCAGTCTGTCCAGGCCATGTACCCGTGAGCGGG W167AF
CGCTCACGGGTACATGTACGCGACAGACTGGGGTGAGACGC W167AR
GCGTCTGACCCCAGTCTGTCGCGTACATGTACCCGTGAGCG T168AF
GCTCACGGGTACATGTACTGGGCAGACTGGGGTGAGACGCC T168AR
GGCGTCTCACCCCAGTCTGCCCAGTACATGTACCCGTGAGC D169AF
CACGGGTACATGTACTGGACAGCCTGGGGTGAGACGCCCCG D169AR
CGGGGCGTCTCACCCCAGGCTGTCCAGTACATGTACCCGTG R494QF
CAACTTGGATGGGGAGGAGCAGCGTGTGCTGGTCAATGCCTC R490QR
GAGGCATTGACCAGCACACGCTGCTCCTGCCCATCCAAGTTG R570WF
GCAGCGCCGCAGCATCGAGTGGGTGCACAAGGTCAAGGCCAG R570WR
CTGGCCTTGACCTTGTGCACCCACTCGATGCTGCGGCGCTGC V667MF
CGAGACCAATAACAACGACATGGCCATCCCGCTCACGGGCG V667MR
CGCCCGTGACGGGGATGGCCATGTCGTTGTTATTGGTCTCG
[0793] Point mutations in other molecules with beta-propellers have
been described in the literature. For example, alpha-4-integrin
(Guerrero-Esteo et al., 1998 FEBS Letter 429: 123-8) has had two
mutations introduced, which resulted in altered protein function
(i.e., G130R and G190S). Both of these glycines are in structurally
equivalent locations to G171 of LRP5, just located on different
blades of the beta-propeller at the upper surface. Using the model
discussed above, we mapped these residues of the alpha-4-integrin
and confirmed their placement. The result of these substitutions in
the alpha4 integrin was loss of ligand binding and reduced affinity
to its heterodimer binding partner: integrin-beta1, such that the
alpha4-beta1 (.alpha.4.beta.1) heterodimer cannot be formed.
Examples of modulation of other residues in integrin-alpha4 with
functional consequences are referenced within Guerrero-Esteo et al.
(1998). Thus, these data support the notion that disruption of
beta-propeller structure can result in significant and dramatic
effects on receptor function.
[0794] All references cited are herein incorporated by reference in
their entirety for all purposes. The following applications are
also incorporated by reference in their entirety herein: U.S.
application Ser. Nos. 09/543,771 and 09/544,398 filed on Apr. 5,
2000, which are a continuation-in-part of application Ser. No.
09/229,319, filed Jan. 13, 1999, which claims benefit of U.S.
Provisional Application No. 60/071,449, filed Jan. 13, 1998, and
U.S. Provisional Application No. 60/105,511, filed Oct. 23, 1998.
Additionally This application claims priority of Application Nos.
60/290,071 filed May 11, 2001; 60/291,311 filed May 17, 2001;
60/353,058 filed Feb. 1, 2002, and 60/361,293 filed Mar. 4, 2002
the texts of which are herein incorporated by reference in their
entirety for all purposes.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090136507A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090136507A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References