U.S. patent application number 10/823197 was filed with the patent office on 2004-11-18 for diagnostics and therapeutics for restenosis.
This patent application is currently assigned to Interleukin Genetics, Inc.. Invention is credited to Crossman, David C., Duff, Gordon W., Francis, Sheila E., Kornman, Kenneth S., Stephenson, Katherine.
Application Number | 20040229264 10/823197 |
Document ID | / |
Family ID | 32043474 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229264 |
Kind Code |
A1 |
Crossman, David C. ; et
al. |
November 18, 2004 |
Diagnostics and therapeutics for restenosis
Abstract
Methods and kits for determining whether a subject has or is
predisposed to developing restenosis are provided.
Inventors: |
Crossman, David C.;
(Sheffield, GB) ; Duff, Gordon W.; (South
Yorkshire, GB) ; Francis, Sheila E.; (Sheffield,
GB) ; Kornman, Kenneth S.; (San Antonio, TX) ;
Stephenson, Katherine; (San Antonio, TX) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Assignee: |
Interleukin Genetics, Inc.
|
Family ID: |
32043474 |
Appl. No.: |
10/823197 |
Filed: |
April 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10823197 |
Apr 12, 2004 |
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09578534 |
May 24, 2000 |
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6720141 |
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09578534 |
May 24, 2000 |
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09431352 |
Nov 1, 1999 |
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6524795 |
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09431352 |
Nov 1, 1999 |
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09320395 |
May 26, 1999 |
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09320395 |
May 26, 1999 |
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08813456 |
Mar 10, 1997 |
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6210877 |
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Current U.S.
Class: |
435/6.11 ;
435/6.1; 514/44R |
Current CPC
Class: |
G01N 2800/323 20130101;
C12Q 2600/156 20130101; C12Q 1/6883 20130101; C12Q 1/683 20130101;
C07K 14/545 20130101; G01N 33/6893 20130101; G01N 2800/324
20130101; C07K 14/54 20130101; G01N 2800/32 20130101 |
Class at
Publication: |
435/006 ;
514/044 |
International
Class: |
C12Q 001/68 |
Claims
1-7. (Cancelled)
8. A kit for determining the existence of or a susceptibility to
developing a restenosis in a subject, said kit comprising a first
primer oligonucleotide that hybridizes 5' or 3' to an allele
selected from the group consisting of allele 1 of any of the
following markers: IL-1A (+4845), IL-1B (-511), IL-1B (+3954),
IL-1RN (VNTR) and IL-1RN (+2018) or an allele in linkage
disequilibrium therewith.
9. The kit of claim 8, which additionally comprises a second primer
oligonucleotide that hybridizes either 3' or 5' respectively to the
allele so that the allele can be amplified.
10. The kit of claim 9, wherein said first primer and said second
primer hybridize to a region in the range of between about 50 and
about 1000 base pairs.
11. The kit of claim 8, wherein said primer is selected from the
group consisting of any of SEQ ID Nos. 1-14.
12. The kit of claim 8, which additionally comprises a detection
means.
13. The kit of claim 12, wherein the detection means is selected
from the group consisting of: a) allele specific oligonucleotide
hybridization; b) size analysis; c) sequencing; d) hybridization;
e) 5' nuclease digestion; f) single-stranded conformation
polymorphism; g) allele specific hybridization; h) primer specific
extension; and j) oligonucleotide ligation assay.
14. The kit of claim 8, which additionally comprises an
amplification means.
15. The kit of claim 8, which further comprises a control.
16-79. (Cancelled)
Description
1. BACKGROUND OF THE INVENTION
[0001] Restenosis
[0002] Percutaneous transluminal coronary angioplasty (PTCA) is
used to treat obstructive coronary artery disease by compressing
atheromatous plaque to the sides of the vessel wall. PTCA is widely
used with an initial success rate of over 90%. Approximately
666,000 angioplasties were conducted in the United States alone in
1996, and more of these procedures were performed on men (452,000)
than women (214,000). Of this total, 482,000 were percutaneous
transluminal coronary angioplasty (P.T.C.A. (American Heart
Association; www.amhrt.org). Despite the frequent application of
this procedure and its high initial success rate, the long-term
success of PTCA is limited by intraluminal renarrowing or
restenosis at the site of the procedure. This occurs within 6
months following the procedure in approximately 30% to 40% of
patients who undergo a single vessel procedure and in more than 50%
of those who undergo multivessel angioplasty.
[0003] Stent placement has largely supplanted balloon angioplasty
because it is able to more widely restore intraluminal dimensions
which has the effect of reducing restenosis by approximately 50%.
Ironically, stent placement actually increases neointimal growth at
the treatment site, but because a larger lumen can be achieved with
stent placement, the tissue growth is more readily accommodate, and
sufficient luminal dimensions are maintained, so that the
restenosis rate is nearly halved by stent placement compared with
balloon angioplasty alone.
[0004] The pathophysiological mechanisms involved in restenosis are
not fully understood. While a number of clinical, anatomical and
technical factors have been linked to the development of
restenosis, at least 50% of the process has yet to be explained.
However, it is known that following endothelial injury, a series of
repair mechanisms are initiated. Within minutes of the injury, a
layer of platelets and fibrin is deposited over the damaged
endothelium. Within hours to days, inflammatory cells begin to
infiltrate the injured area. Within 24 hours after an injury,
vascular smooth muscle cells (SMCs) located in the vessel media
commence DNA synthesis. A few days later, these activated,
synthetic SMCs migrate through the internal elastic lamina towards
the luminal surface. A neointima is formed by these cells by their
continued replication and their production of extracellular matrix.
An increase in the intimal thickness occurs with ongoing cellular
proliferation matrix deposition. When these processes of vascular
healing progress excessively, the pathological condition is termed
intimal hyperplasia or neointimial hyperplasia. Histological
studies in animal models have identified neointimal hyperplasia as
the central element in restenosis.
[0005] Neointimal hyperplasia is understood to figure prominently
in peripheral vascular restenosis following reconstructive
procedures. One series of 5,000 arterial reconstructions reports
50% of late failures to be due to neointimal hyperplasia (Imparato
et al. (1972) Surg. 72:1107-1117). Restenosis following stenting is
similarly thought to involve an important component of neointimal
hyperplasia (Dussaillant et al. (1995) J. Am. Coll. Cardiol
26:720-724). In the coronary system, by contrast, restenosis
following balloon angioplasty involves vascular remodeling as well
as neointimal hyperplasia. The importance of vascular remodeling in
this setting may be attributable to the nature of the injury to the
vessel wall following balloon angioplasty. Commonly, the injury to
the vessel wall with this procedure involves dissection planes
extending through the atherosclerotic plaque into the vessel media
(Mintz et al. (1996) Circ. 94:35043). Furthermore, plaque fracture,
medial stretch, focal medial rupture and adventitial stretch all
may occur following angioplasty. Repair of the deeper layers of the
vessel wall takes place by the general processes of wound healing,
including inflammation, neovascularization, fibroblast
proliferation and eventual collagen deposition. Cumulatively, these
processes lead to remodeling of the coronary vessel wall that may
culminate in restenosis.
[0006] The biology of vascular wall healing implicated in
restenosis therefore includes the general processes of wound
healing and the specific processes of neointimal hyperplasia.
Inflammation is generally regarded as an important component in
both these processes. (Munro and Cotran (1993) Lab. Investig.
58:249-261; and Badimon et al. (1993), Supp II 87:3-6).
Understanding the effects of acute and chronic inflammation in the
blood vessel wall can thus suggest methods for diagnosing and
treating restenosis and related conditions.
[0007] In its initial phase, inflammation is characterized by the
adherence of leukocytes to the vessel wall. Leukocyte adhesion to
the surface of damaged endothelium is mediated by several complex
glycoproteins on the endothelial and neutrophil surfaces. Two of
these binding molecules have been well-characterized: the
endothelial leukocyte adhesion molecule-1 (ELAM-1) and the
intercellular adhesion molecule-1 (ICAM-1). During inflammatory
states, the attachment of neutrophils to the involved cell surfaces
is greatly increased, primarily due to the upregulation and
enhanced expression of these binding molecules. Substances thought
to be primary mediators of the inflammatory response to tissue
injury, including interleukin-1 (IL-1), tumor necrosis factor alpha
(TNF-.alpha.), lymphotoxin and bacterial endotoxins, all increase
the production of these binding substances.
[0008] After binding to the damaged vessel wall, leukocytes migrate
into it. Once in place within the vessel wall, the leukocytes, in
particular activated macrophages, then release additional
inflammatory mediators, including IL-1, TNF, prostaglandin E.sub.2,
(PGE.sub.2), bFGF, and transforming growth factors .alpha. and
.beta. (TGF.alpha., TNF.beta.). All of these inflammatory mediators
recruit more inflammatory cells to the damaged area, and regulate
the further proliferation and migration of smooth muscle. A
well-known growth factor elaborated by the monocyte-macrophage is
monocyte- and macrophage-derived growth factor (MDGF), a stimulant
of smooth muscle cell and fibroblast proliferation. MDGF is
understood to be similar to platelet-derived growth factor (PDGF);
in fact, the two substances may be identical. By stimulating smooth
muscle cell proliferation, inflammation can contribute to the
development and the progression of neointimal hyperplasia.
[0009] Leukocytes, attracted to the vessel wall by the
abovementioned chemical mediators of inflammation, produce
substances that have direct effects on the vessel wall that may
exacerbate the local injury and prolong the healing response.
First, leukocytes activated by the processes of inflammation
secrete lysosomal enzymes that can digest collagen and other
structural proteins. Releasing these enzymes within the vessel wall
can affect the integrity of its extracellular matrix, permitting
SMCs and other migratory cells to pass through the wall more
readily. Hence, the release of these lysosomal proteases can
enhance the processes leading to neointimal hyperplasia. Second,
activated leukocytes produce free radicals by the action of the
NADPH system on their cell membranes. These free radicals can
damage cellular elements directly, leading to an extension of a
local injury or a prolongation of the cycle of
injury-inflammation-healing.
[0010] The responses to vascular injury that lead to restenosis
have certain features in common with the processes leading to the
development of the vascular lesions of atherosclerosis. Currently,
it is understood that the lesions of atherosclerosis are initiated
by some form of injury to arterial endothelium, whether due to
hemodynarnic factors, endothelial dysfunction or a combination of
these or other factors (Schoen, "Blood vessels," pp. 467-516 in
Pathological Basis of Disease (Philadelphia: Saunders, 1994)).
Inflammation has been implicated in the formation and progression
of atherosclerotic lesions. Several inflammatory products,
including IL-1.beta., have been identified in atherosclerotic
lesions or in the endothelium of diseased coronary arteries (Galea,
et al. (1996) Arterioscler Thromb Vasc Biol. 16:1000-6). Also,
serum concentrations of IL-1.beta. are elevated in patients with
coronary disease (Hasdai, et al. (1996) Heart, 76:24-8). Realizing
the importance of inflammatory processes in the final common
pathways of vascular response to injury allows analogies to be
drawn between the lesions seen in restenosis and those seen in
atherosclerosis.
[0011] Currently, approximately 500,000 patients per year undergo
vascular reconstructive procedures, with half involving the
coronary vessels and the other half involving the periphery.
Restenosis and progressive atherosclerosis are the most common
mechanisms for late failure in these reconstructions. It would be
desirable to determine which patients would respond well to
invasive treatments for occlusive vascular disease such as
angioplasty and intravascular stent placement. It would be further
desirable to identify those patients at increased risk for stenosis
so that they could be targeted with appropriate therapies to
prevent, modulate or reverse the condition. It would be desirable,
moreover, to identify those individuals for whom PTCA and stent
placement is a suboptimal therapeutic choice because of the risk of
restenosis. Those patients might become candidates at earlier
stages for vascular reconstructive procedures, possibly combined
with other pharmacological interventions.
[0012] Genetics of the IL-1 Gene Cluster
[0013] The IL-1 gene cluster is on the long arm of chromosome 2
(2q13) and contains at least the genes for IL-1.alpha. (IL-1A),
IL-1.beta. (IL-1B), and the IL-1 receptor antagonist (IL-1RN),
within a region of 430 Kb (Nicklin, et al. (1994) Genomics, 19:
382-4). The agonist molecules, IL-1.alpha. and IL-1.beta., have
potent pro-inflammatory activity and are at the head of many
inflammatory cascades. Their actions, often via the induction of
other cytokines such as IL-6 and IL-8, lead to activation and
recruitment of leukocytes into damaged tissue, local production of
vasoactive agents, fever response in the brain and hepatic acute
phase response. All three IL-1 molecules bind to type I and to type
II IL-1 receptors, but only the type I receptor transduces a signal
to the interior of the cell. In contrast, the type II receptor is
shed from the cell membrane and acts as a decoy receptor. The
receptor antagonist and the type II receptor, therefore, are both
anti-inflammatory in their actions.
[0014] Inappropriate production of IL-1 plays a central role in the
pathology of many autoimmune and inflammatory diseases, including
rheumatoid arthritis, inflammatory bowel disorder, psoriasis, and
the like. In addition, there are stable inter-individual
differences in the rates of production of IL-1, and some of this
variation may be accounted for by genetic differences at IL-1 gene
loci. Thus, the IL-1 genes are reasonable candidates for
determining part of the genetic susceptibility to inflammatory
diseases, most of which have a multifactorial etiology with a
polygenic component. Indeed, there is increasing evidence that
certain alleles of the IL-1 genes are over-represented in these
diseases.
[0015] Certain alleles from the IL-1 gene cluster are already known
to be associated with particular disease states. For example,
IL-1RN allele 2 has been shown to be associated with coronary
artery disease (PCT/US/98/04725, and U.S. Ser. No. 08/813456),
osteoporosis (U.S. Pat. No. 5,698,399), nephropathy in diabetes
mellitus (Blakemore, et al. (1996) Hum. Genet. 97(3): 369-74),
alopecia areata (Cork, et al., (1995) J. Invest. Dermatol. 104(5
Supp.): 15S-16S; Cork et al. (1996) Dermatol Clin 14: 671-8),
Graves disease (Blakemore, et al. (1995) J. Clin. Endocrinol.
80(1): 111-5), systemic lupus erythematosus (Blakemore, et al.
(1994) Arthritis Rheum. 37: 1380-85), lichen sclerosis (Clay, et
al. (1994) Hum. Genet. 94: 407-10), and ulcerative colitis
(Mansfield, et al. (1994) Gastoenterol. 106(3): 637-42).
[0016] In addition, the IL-1A allele 2 from marker -889 and IL-1B
(TaqI) allele 2 from marker +3954 have been found to be associated
with periodontal disease (U.S. Pat. No. 5,686,246; Kornman and
diGiovine (1998) Ann Periodont 3: 327-38; Hart and Kornman (1997)
Periodontol 2000 14: 202-15; Newman (1997) Compend Contin Educ Dent
18: 881-4; Komman et al. (1997) J. Clin Periodontol 24: 72-77). The
IL-1A allele 2 from marker -889 has also been found to be
associated with juvenile chronic arthritis, particularly chronic
iridocyclitis (McDowell, et al. (1995) Arthritis Rheum. 38:
221-28). The IL-1B (TaqI) allele 2 from marker +3954 of IL-1B has
also been found to be associated with psoriasis and insulin
dependent diabetes in DR3/4 patients (di Giovine, et al. (1995)
Cytokine 7: 606; Pociot, et al. (1992) Eur J. Clin. Invest. 22:
396-402). Additionally, the IL-1RN (VNTR) allele 1 has been found
to be associated with diabetic retinopathy (see U.S. Ser. No.
09/037472, and PCT/GB97/02790). Furthermore allele 2 of IL-1RN
(VNTR) has been found to be associated with ulcerative colitis in
Caucasian populations from North America and Europe (Mansfield, J.
et al., (1994) Gastroenterology 106: 637-42). Interestingly, this
association is particularly strong within populations of ethnically
related Ashkenazi Jews (PCT WO97/25445).
[0017] Genotype Screening
[0018] Traditional methods for the screening of heritable diseases
have depended on either the identification of abnormal gene
products (e.g., sickle cell anemia) or an abnormal phenotype (e.g.,
mental retardation). These methods are of limited utility for
heritable diseases with late onset and no easily identifiable
phenotypes such as, for example, a predisposition to restenosis.
With the development of simple and inexpensive genetic screening
methodology, it is now possible to identify polymorphisms that
indicate a propensity to develop disease, even when the disease is
of polygenic origin. The number of diseases that can be screened by
molecular biological methods continues to grow with increased
understanding of the genetic basis of multifactorial disorders.
[0019] Genetic screening (also called genotyping or molecular
screening), can be broadly defined as testing to determine if a
patient has mutations (or alleles or polymorphisms) that either
cause a disease state or are "linked" to the mutation causing a
disease state. Linkage refers to the phenomenon that DNA sequences
which are close together in the genome have a tendency to be
inherited together. Two sequences may be linked because of some
selective advantage of co-inheritance. More typically, however, two
polymorphic sequences are co-inherited because of the relative
infrequency with which meiotic recombination events occur within
the region between the two polymorphisms. The co-inherited
polymorphic alleles are said to be in linkage disequilibrium with
one another because, in a given human population, they tend to
either both occur together or else not occur at all in any
particular member of the population. Indeed, where multiple
polymorphisms in a given chromosomal region are found to be in
linkage disequilibrium with one another, they define a quasi-stable
genetic "haplotype." In contrast, recombination events occurring
between two polymorphic loci cause them to become separated onto
distinct homologous chromosomes. If meiotic recombination between
two physically linked polymorphisms occurs frequently enough, the
two polymorphisms will appear to segregate independently and are
said to be in linkage equilibrium.
[0020] While the frequency of meiotic recombination between two
markers is generally proportional to the physical distance between
them on the chromosome, the occurrence of "hot spots" as well as
regions of repressed chromosomal recombination can result in
discrepancies between the physical and recombinational distance
between two markers. Thus, in certain chromosomal regions, multiple
polymorphic loci spanning a broad chromosomal domain may be in
linkage disequilibrium with one another, and thereby define a
broad-spanning genetic haplotype. Furthermore, where a
disease-causing mutation is found within or in linkage with this
haplotype, one or more polymorphic alleles of the haplotype can be
used as a diagnostic or prognostic indicator of the likelihood of
developing the disease. This association between otherwise benign
polymorphisms and a disease-causing polymorphism occurs if the
disease mutation arose in the recent past, so that sufficient time
has not elapsed for equilibrium to be achieved through
recombination events. Therefore identification of a human haplotype
which spans or is linked to a disease-causing mutational change,
serves as a predictive measure of an individual's likelihood of
having inherited that disease-causing mutation. Importantly, such
prognostic or diagnostic procedures can be utilized without
necessitating the identification and isolation of the actual
disease-causing lesion. This is significant because the precise
determination of the molecular defect involved in a disease process
can be difficult and laborious, especially in the case of
multifactorial diseases such as inflammatory disorders.
[0021] Indeed, the statistical correlation between an inflammatory
disorder and an IL-1 polymorphism does not necessarily indicate
that the polymorphism directly causes the disorder. Rather the
correlated polymorphism may be a benign allelic variant which is
linked to (i.e. in linkage disequilibrium with) a disorder-causing
mutation which has occurred in the recent human evolutionary past,
so that sufficient time has not elapsed for equilibrium to be
achieved through recombination events in the intervening
chromosomal segment. Thus, for the purposes of diagnostic and
prognostic assays for a particular disease, detection of a
polymorphic allele associated with that disease can be utilized
without consideration of whether the polymorphism is directly
involved in the etiology of the disease. Furthermore, where a given
benign polymorphic locus is in linkage disequilibrium with an
apparent disease-causing polymorphic locus, still other polymorphic
loci which are in linkage disequilibrium with the benign
polymorphic locus are also likely to be in linkage disequilibrium
with the disease-causing polymorphic locus. Thus these other
polymorphic loci will also be prognostic or diagnostic of the
likelihood of having inherited the disease-causing polymorphic
locus. Indeed, a broad-spanning human haplotype (describing the
typical pattern of co-inheritance of alleles of a set of linked
polymorphic markers) can be targeted for diagnostic purposes once
an association has been drawn between a particular disease or
condition and a corresponding human haplotype. Thus, the
determination of an individual's likelihood for developing a
particular disease of condition can be made by characterizing one
or more disease-associated polymorphic alleles (or even one or more
disease-associated haplotypes) without necessarily determining or
characterizing the causative genetic variation.
2. SUMMARY OF THE INVENTION
[0022] In one aspect, the present invention provides novel methods
and kits for determining whether a subject has or is predisposed to
developing restenosis. Diagnosis of the presence of a restenosis
disorder identifies those patients predisposed to the development
of a restenosis disease, characterized by clinical events related
to the recurrence of the initial vascular stenosis that is being
treated by the stent. Determining which patients are at risk for
developing the disease because they have the disorder thus opens
the possibility of selecting therapies for the initial vascular
stenosis most likely to avoid subsequent stenoses. Such patients
might be preferred candidates for surgical revascularization rather
than percutaneous transluminal angioplasty, for example, or such
patients may benefit from pharmacological or topical interventions
at an early stage that could affect the progression of the
restenosis disorder.
[0023] In one embodiment, the method comprises determining whether
a restenosis associated allele is present in a nucleic acid sample
obtained from the subject. In a preferred embodiment, the
restenosis associated allele is selected from the group consisting
of allele 1 of each of the following markers: IL-1A (+4845), IL-1B
(+3954), IL-1B (-511), IL-1RN (+2018) and IL-1RN (VNTR) or an
allele that is in linkage disequilibrium with one of the
aforementioned alleles. In preferred embodiments, the presence of a
particular allelic pattern of one or more of the abovementioned
IL-1 polymorphic loci is used to predict the susceptibility of an
individual to developing restenosis. In particular, there are three
patterns of alleles at four polymorphic loci in the IL-1 gene
cluster that show various associations with particular
cardiovascular disorders. These patterns are referred to herein as
patterns 1, 2 and 3. Pattern 1 comprises an allelic pattern
including allele 2 of IL-1A (+4845) or IL-1B (+3954) and allele 1
of IL-1B (-511) or IL-1RN (+2018), or an allele that is in linkage
disequilibrium with one of the aforementioned allele. In a
preferred embodiment, this allelic pattern permits the diagnosis of
occlusive cardiovascular disorder. Pattern 2 comprises an allelic
pattern including allele 2 of IL-1B (-511) or IL-1RN (+2018) and
allele 1 of IL-1A (+4845) or IL-1B (+3954), or an allele that is in
linkage disequilibrium with one of the aforementioned alleles. In a
preferred embodiment, this allelic pattern permits the diagnosis of
occlusive cardiovascular disorder. Pattern 3 comprises an allelic
pattern including allele 1 of IL-1A (+4845) or allele 1 of IL-1B
(+3954), and allele 1 of IL-1B (-511) or allele 1 of IL-1RN
(+2018), or an allele that is in linkage disequilibrium with one of
the aforementioned alleles. In a preferred embodiment, this allelic
pattern permits the diagnosis of a restenosis disorder.
[0024] In another embodiment, the method of the invention may be
employed by detecting the presence of an IL-1 associated
polymorphism that is in linkage disequilibrium with one or more of
the aforementioned restenosis-predictive alleles. For example, the
following alleles of the IL-1 (44112332) haplotype are known to be
in linkage disequilibrium:
1 allele 4 of the 222/223 marker of IL-1A allele 4 of the gz5/gz6
marker of IL-1A allele 1 of the -889 marker of IL-1A allele 1 of
the +3954 marker of IL-1B allele 2 of the -511 marker of IL-1B
allele 3 of the gaat.p33330 marker allele 3 of the Y31 marker
allele 2 of the VNTR or (+2018) marker of IL-1RN
[0025] Also, the following alleles of the IL-1 (33221461) haplotype
are in linkage disequilibrium:
2 allele 3 of the 222/223 marker of IL-1A allele 3 of the gz5/gz6
marker of IL-1A allele 2 of the -889 marker of IL-1A allele 2 of
the +3954 marker of IL-1B allele 1 of the -511 marker of IL-1B
allele 4 of the gaat.p33330 marker allele 6 of the Y31 marker
allele 1 of the VNTR or (+2018) marker of IL-1RN
[0026] A restenosis associated allele can be detected by any of a
variety of available techniques, including: 1) performing a
hybridization reaction between a nucleic acid sample and a probe
that is capable of hybridizing to the allele; 2) sequencing at
least a portion of the allele; or 3) determining the
electrophoretic mobility of the allele or fragments thereof (e.g.,
fragments generated by endonuclease digestion). The allele can
optionally be subjected to an amplification step prior to
performance of the detection step. Preferred amplification methods
are selected from the group consisting of: the polymerase chain
reaction (PCR), the ligase chain reaction (LCR), strand
displacement amplification (SDA), cloning, and variations of the
above (e.g. RT-PCR and allele specific amplification).
Oligonucleotides necessary for amplification may be selected for
example, from within the IL-1 gene loci, either flanking the marker
of interest (as required for PCR amplification) or directly
overlapping the marker (as in ASO hybridization). In a particularly
preferred embodiment, the sample is hybridized with a set of
primers, which hybridize 5' and 3' in a sense or antisense sequence
to the restenosis associated allele, and is subjected to a PCR
amplification.
[0027] A restenosis associated allele may also be detected
indirectly, e.g. by analyzing the protein product encoded by the
DNA. For example, where the marker in question results in the
translation of a mutant protein, the protein can be detected by any
of a variety of protein detection methods. Such methods include
immunodetection and biochemical tests, such as size fractionation,
where the protein has a change in apparent molecular weight either
through truncation, elongation, altered folding or altered
post-translational modifications.
[0028] In another aspect, the invention features kits for
performing the above-described assays. The kit can include a
nucleic acid sample collection means and a means for determining
whether a subject carries a restenosis associated allele. The kit
may also contain a control sample either positive or negative or a
standard and/or an algorithmic device for assessing the results and
additional reagents and components including: DNA amplification
reagents, DNA polymerase, nucleic acid amplification reagents,
restrictive enzymes, buffers, a nucleic acid sampling device, DNA
purification device, deoxynucleotides, oligonucleotides (e.g.
probes and primers) etc.
[0029] As described above, the control samples may be positive or
negative controls. Further, the control sample may contain the
positive (or negative) products of the allele detection technique
employed. For example, where the allele detection technique is PCR
amplification, followed by size fractionation, the control sample
may comprise DNA fragments of the appropriate size. Likewise, where
the allele detection technique involves detection of a mutated
protein, the control sample may comprise a sample of mutated
protein. However, it is preferred that the control sample comprises
the material to be tested. For example, the controls may be a
sample of genomic DNA or a cloned portion of the IL-1 gene cluster.
Preferably, however, the control sample is a highly purified sample
of genomic DNA where the sample to be tested is genomic DNA.
[0030] The oligonucleotides present in said kit may be used for PCR
amplification of the region of interest or for direct allele
specific oligonucleotide (ASO) hybridization to the markers in
question. Thus, the oligonucleotides may either flank the marker of
interest (as required for PCR amplification) or directly overlap
the marker (as in ASO hybridization).
[0031] Such oligonucleotides can include, but are not limited
to:
[0032] 5' ATG GTT TTA GAA ATC ATC AAG CCT AGG GCA 3' (SEQ ID No. 1)
and
[0033] 5' AAT GAA AGG AGG GGA GGA TGA CAG AAA TGT 3' (SEQ ID No.
2)
[0034] which can be used to amplify the human IL-1A (+4845)
polymorphic locus;
[0035] 5' TGG CAT TGA TCT GGT TCA TC 3' (SEQ ID No. 3) and
[0036] 5' GTT TAG GAA TCT TCC CAC TT-3' (SEQ ID No. 4)
[0037] which can be used to amplify the human IL-1B (-511)
polymorphic locus;
[0038] 5'-CTC AGG TGT CCT CGA AGA AAT CAA A-3' (SEQ ID No. 5)
and
[0039] 5' GCT TTT TTG CTG TGA GTC CCG-3' (SEQ ID No. 6)
[0040] which can be used to amplify the human IL-1B (+3954)
polymorphic locus;
[0041] 5'-CTC.AGC.AAC.ACT.CCT.AT-3' (SEQ ID NO. 7) and
[0042] 5'-TCC.TGG.TCT.GCA.GCT.AA-3' (SEQ ID NO. 8)
[0043] which can be used to amplify the human IL-1RN (VNTR)
polymorphic locus;
[0044] 5'-CTA TCT GAG GAA CAA CCA ACT AGT AGC-3' (SEQ ID NO. 9)
and
[0045] 5'-TAG GAC ATT GCA CCT AGG GTT TGT-3' (SEQ ID NO. 10)
[0046] which can be used to amplify the human IL-1RN (+2018)
polymorphic locus;
[0047] 5' ATT TTT TTA TAA ATC ATC AAG CCT AGG GCA 3' (SEQ. ID No.
11) and
[0048] 5' AAT TAA AGG AGG GAA GAA TGA CAG AAA TGT 3' (SEQ. ID No.
12)
[0049] which can also be used to amplify the human IL-1A (+4845)
polymorphic locus;
[0050] 5'-AAG CTT GTT CTA CCA CCT GAA CTA GGC.-3' (SEQ. ID NO. 13)
and
[0051] 5'-TTA CAT ATG AGC CTT CCA TG.-3' (SEQ. ID NO. 14)
[0052] which can be used to amplify the human IL-1A (-889)
polymorphic locus;
[0053] Information obtained using the assays and kits described
herein (alone or in conjunction with information on another genetic
defect or environmental factor, which contributes to restenosis) is
useful for determining whether a non-symptomatic subject has or is
likely to develop restenosis. In addition, the information can
allow a more customized approach to preventing the onset or
progression of restenosis. For example, this information can enable
a clinician to more effectively prescribe a therapy that will
address the molecular basis of restenosis. In yet a further aspect,
the invention features methods for treating or preventing the
development of restenosis in a subject by administering to the
subject an appropriate restenosis therapeutic of the invention. In
still another aspect, the invention provides in vitro or in vivo
assays for screening test compounds to identify restenosis
therapeutics. In one embodiment, the assay comprises contacting a
cell transfected with a restenosis causative mutation that is
operably linked to an appropriate promoter with a test compound and
determining the level of expression of a protein in the cell in the
presence and in the absence of the test compound. In a preferred
embodiment, the restenosis causative mutation results in decreased
production of IL-1 receptor antagonist, and increased production of
the IL-1 receptor antagonist in the presence of the test compound
indicates that the compound is an agonist of IL-1 receptor
antagonist activity. In another preferred embodiment, the
restenosis causative mutation results in increased production of
IL-1.alpha. or IL-1.beta., and decreased production of IL-1.alpha.
or IL-1.beta. in the presence of the test compound indicates that
the compound is an antagonist of IL-1.alpha. or IL-1.beta.
activity. In another embodiment, the invention features transgenic
non-human animals and their use in identifying antagonists of
IL-1.alpha. or IL-1.beta. activity or agonists of IL-1Ra
activity.
[0054] Other embodiments and advantages of the invention are set
forth in part in the description which follows, and will be obvious
from this description.
3. BRIEF DESCRIPTION OF THE FIGURES
[0055] FIG. 1 shows the nucleic acid sequence for IL-1A (GEN
X03833; SEQ ID No. 15).
[0056] FIG. 2 shows the nucleic acid sequence for IL-1B (GEN
X04500; SEQ ID No. 16).
[0057] FIG. 3 shows the nucleic acid sequence for the secreted
IL-1RN (GEN X64532;
[0058] SEQ ID No. 17).
[0059] FIG. 4 depicts the organization of the IL-1 genes, and
associated polymorphic loci, on human chromosome 2.
[0060] FIG. 5 shows linkage disequilibrium values for the IL-1
polymorphic loci in a Caucasian population.
[0061] FIG. 6 is a bar graph illustrating the frequency of
particular IL-1 polymorphic allelic patterns in a Caucasian
population.
[0062] FIG. 7 indicates the relative risk for restenosis associated
with each of the IL-1 polymorphic patterns.
[0063] FIG. 8 indicates the association between homozygous and
heterozygous allelic patterns at the IL-1RN(+2018) locus and the
occurrence of restenosis and target vessel revascularization.
[0064] FIG. 9 is a graph showing the odds ratios for clinical
events and angiographic restenosis associated with the presence of
the IL-1RN*2 allele for the whole population (left panel) and
patients <60 years (right panel)
[0065] FIG. 10 is a bar graph showing the decrease in the incidence
of restenosis and target vessel revascularization (TVR) in patients
<60 years with the increase in the number of IL-1RN*2
alleles.
4. DETAILED DESCRIPTION OF THE INVENTION
4.1 Definitions
[0066] For convenience, the meaning of certain terms and phrases
employed in the specification, examples, and appended claims are
provided below.
[0067] The term "allele" refers to the different sequence variants
found at different polymorphic regions. For example, IL-1RN (VNTR)
has at least five different alleles. The sequence variants may be
single or multiple base changes, including without limitation
insertions, deletions, or substitutions, or may be a variable
number of sequence repeats.
[0068] The term "allelic pattern" refers to the identity of an
allele or alleles at one or more polymorphic regions. For example,
an allelic pattern may consist of a single allele at a polymorphic
site, as for IL-1RN (VNTR) allele 1, which is an allelic pattern
having at least one copy of IL-1RN allele 1 at the VNTR of the
IL-1RN gene loci. Alternatively, an allelic pattern may consist of
either a homozygous or heterozygous state at a single polymorphic
site. For example, IL1-RN (VNTR) allele 2,2 is an allelic pattern
in which there are two copies of the second allele at the VNTR
marker of IL-1RN and that corresponds to the homozygous IL-RN
(VNTR) allele 2 state. Alternatively, an allelic pattern may
consist of the identity of alleles at more than one polymorphic
site.
[0069] The term "antibody" as used herein is intended to refer to a
binding agent including a whole antibody or a binding fragment
thereof which is specifically reactive with an IL-1B polypeptide.
Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described
above for whole antibodies. For example, F(ab).sub.2 fragments can
be generated by treating an antibody with pepsin. The resulting
F(ab).sub.2 fragment can be treated to reduce disulfide bridges to
produce Fab fragments. The antibody of the present invention is
further intended to include bispecific, single-chain, and chimeric
and humanized molecules having affinity for an IL-1B polypeptide
conferred by at least one CDR region of the antibody.
[0070] "Biological activity" or "bioactivity" or "activity" or
"biological function", which are used interchangeably, for the
purposes herein means an effector or antigenic function that is
directly or indirectly performed by an IL-1 polypeptide (whether in
its native or denatured conformation), or by any subsequence
thereof. Biological activities include binding to a target peptide,
e.g., an IL-1 receptor. An IL-1 bioactivity can be modulated by
directly affecting an IL-1 polypeptide. Alternatively, an IL-1
bioactivity can be modulated by modulating the level of an IL-1
polypeptide, such as by modulating expression of an IL-1 gene.
[0071] As used herein the term "bioactive fragment of an IL-1
polypeptide" refers to a fragment of a full-length IL-1
polypeptide, wherein the fragment specifically mimics or
antagonizes the activity of a wild-type IL-1 polypeptide. The
bioactive fragment preferably is a fragment capable of interacting
with an interleukin receptor.
[0072] The term "an aberrant activity", as applied to an activity
of a polypeptide such as IL-1, refers to an activity which differs
from the activity of the wild-type or native polypeptide or which
differs from the activity of the polypeptide in a healthy subject.
An activity of a polypeptide can be aberrant because it is stronger
than the activity of its native counterpart. Alternatively, an
activity can be aberrant because it is weaker or absent relative to
the activity of its native counterpart. An aberrant activity can
also be a change in an activity. For example an aberrant
polypeptide can interact with a different target peptide. A cell
can have an aberrant IL-1 activity due to overexpression or
underexpression of an IL-1 locus gene encoding an IL-1 locus
polypeptide.
[0073] "Cells", "host cells" or "recombinant host cells" are terms
used interchangeably herein to refer not only to the particular
subject cell, but to the progeny or potential progeny of such a
cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact be identical to the parent cell, but
is still included within the scope of the term as used herein.
[0074] A "chimera," "mosaic," "chimeric mammal" and the like,
refers to a transgenic mammal with a knock-out or knock-in
construct in at least some of its genome-containing cells.
[0075] The terms "control" or "control sample" refer to any sample
appropriate to the detection technique employed. The control sample
may contain the products of the allele detection technique employed
or the material to be tested. Further, the controls may be positive
or negative controls. By way of example, where the allele detection
technique is PCR amplification, followed by size fractionation, the
control sample may comprise DNA fragments of an appropriate size.
Likewise, where the allele detection technique involves detection
of a mutated protein, the control sample may comprise a sample of a
mutant protein. However, it is preferred that the control sample
comprises the material to be tested. For example, the controls may
be a sample of genomic DNA or a cloned portion of the IL-1 gene
cluster. However, where the sample to be tested is genomic DNA, the
control sample is preferably a highly purified sample of genomic
DNA.
[0076] A "cardiovascular disease" is a cardiovascular disorder, as
defined herein, characterized by clinical events including clinical
symptoms and clinical signs. Clinical symptoms are those
experiences reported by a patient that indicate to the clinician
the presence of pathology. Clinical signs are those objective
findings on physical or laboratory examination that indicate to the
clinician the presence of pathology. "Cardiovascular disease"
includes both "coronary artery disease" and "peripheral vascular
disease," both terms being defined below. Clinical symptoms in
cardiovascular disease include chest pain, shortness of breath,
weakness, fainting spells, alterations in consciousness, extremity
pain, paroxysmal nocturnal dyspnea, transient ischemic attacks and
other such phenomena experienced by the patient. Clinical signs in
cardiovascular disease include such findings as EKG abnormalities,
altered peripheral pulses, arterial bruits, abnormal heart sounds,
rales and wheezes, jugular venous distention, neurological
alterations and other such findings discerned by the clinician.
Clinical symptoms and clinical signs can combine in a
cardiovascular disease such as a myocardial infarction (MI) or a
stroke (also termed a "cerebrovascular accident" or "CVA"), where
the patient will report certain phenomena (symptoms) and the
clinician will perceive other phenomena (signs) all indicative of
an underlying pathology. "Cardiovascular disease" includes those
diseases related to the cardiovascular disorders of fragile plaque
disorder, occlusive disorder and stenosis. For example, a
cardiovascular disease resulting from a fragile plaque disorder, as
that term is defined below, can be termed a "fragile plaque
disease." Clinical events associated with fragile plaque disease
include those signs and symptoms where the rupture of a fragile
plaque with subsequent acute thrombosis or with distal embolization
are hallmarks. Examples of fragile plaque disease include certain
strokes and myocardial infarctions. As another example, a
cardiovascular disease resulting from an occlusive disorder can be
termed an "occlusive disease." Clinical events associated with
occlusive disease include those signs and symptoms where the
progressive occlusion of an artery affects the amount of
circulation that reaches a target tissue. Progressive arterial
occlusion may result in progressive ischemia that may ultimately
progress to tissue death if the amount of circulation is
insufficient to maintain the tissues. Signs and symptoms of
occlusive disease include claudication, rest pain, angina, and
gangrene, as well as physical and laboratory findings indicative of
vessel stenosis and decreased distal perfusion. As yet another
example, a cardiovascular disease resulting from restenosis can be
termed an in-stent stenosis disease. In-stent stenosis disease
includes the signs and symptoms resulting from the progressive
blockage of an arterial stent that has been positioned as part of a
procedure like a percutaneous transluminal angioplasty, where the
presence of the stent is intended to help hold the vessel in its
newly expanded configuration. The clinical events that accompany
in-stent stenosis disease are those attributable to the restenosis
of the reconstructed artery.
[0077] A "cardiovascular disorder" refers broadly to both to
coronary artery disorders and peripheral arterial disorders. The
term "cardiovascular disorder" can apply to any abnormality of an
artery, whether structural, histological, biochemical or any other
abnormality. This term includes those disorders characterized by
fragile plaque (termed herein "fragile plaque disorders"), those
disorders characterized by vaso-occlusion (termed herein "occlusive
disorders"), and those disorders characterized by restenosis. A
"cardiovascular disorder" can occur in an artery primarily, that
is, prior to any medical or surgical intervention. Primary
cardiovascular disorders include, among others, atherosclerosis,
arterial occlusion, aneurysm formation and thrombosis. A
"cardiovascular disorder" can occur in an artery secondarily, that
is, following a medical or surgical intervention. Secondary
cardiovascular disorders include, among others, post-traumatic
aneurysm formation, restenosis, and post-operative graft
occlusion.
[0078] A "cardiovascular disorder causative functional mutation"
refers to a mutation which causes or contributes to the development
of a cardiovascular disorder in a subject. Preferred mutations
occur within the IL-1 complex. A cardiovascular disorder causative
functional mutation occurring within an IL-1 gene (e.g. IL-1A,
IL-1B or IL-1RN) or a gene locus, which is linked thereto, may
alter, for example, the open reading frame or splicing pattern of
the gene, thereby resulting in the formation of an inactive or
hypoactive gene product. For example, a mutation which occurs in
intron 6 of the IL-1A locus corresponds to a variable number of
tandem repeat 46 bp sequences corresponding to from five to 18
repeat units (Bailly, et al. (1993) Eur. J. immunol. 23: 1240-45).
These repeat sequences contain three potential binding sites for
transcriptional factors: an SPI site, a viral enhancer element, and
a glucocorticoid-responsive element; therefore individuals carrying
IL-1A intron 6 VNTR alleles with large numbers of repeat units may
be subject to altered transcriptional regulation of the IL-1A gene
and consequent perturbations of inflammatory cytokine production.
Indeed, there is evidence that increased repeat number at this
polymorphic IL-1A locus leads to decreased IL-1.alpha. synthesis
(Bailly et al. (1996) Mol Immunol 33: 999-1006). Alternatively, a
mutation can result in a hyperactive gene product. For example,
allele 2 of the IL-1B (G at +6912) polymorphism occurs in the 3'
UTR (untranslated region) of the IL-1B MRNA and is associated with
an approximately four-fold increase in the steady state levels of
both IL-1B MRNA and IL-1B protein compared to those levels
associated with allele 1 of the IL-1B gene at +6912). Further, an
IL-1B (-511) mutation occurs near a promoter binding site for a
negative glucocorticoid response element (Zhang et al. (1997) DNA
Cell Biol 16: 145-52). This element potentiates a four-fold
repression of IL-1B expression by dexamethosone and a deletion of
this negative response elements causes a 2.5-fold increase in IL-1B
promoter activity. The IL-1B (-511) polymorphism may thus directly
affect cytokine production and inflammatory responses. These
examples demonstrate that genetic variants occurring in the IL-1A
or IL-1B gene can directly lead to the altered production or
regulation of IL-1 cytokine activity.
[0079] A "cardiovascular disorder therapeutic" refers to any agent
or therapeutic regimen (including pharmaceuticals, nutraceuticals
and surgical means) that prevents or postpones the development of
or reduces the extent of an abnormality constitutive of a
cardiovascular disorder in a subject. Cardiovascular disorder
therapeutics can be directed to the treatment of any cardiovascular
disorder, including fragile plaque disorder, occlusive disorder and
restenosis. Examples of therapeutic agents directed to each
category of cardiovascular disorder are provided herein. It is
understood that a therapeutic agent may be useful for more than one
category of cardiovascular disorder. The therapeutic can be a
polypeptide, peptidomimetic, nucleic acid or other inorganic or
organic molecule, preferably a "small molecule" including vitamins,
minerals and other nutrients. Preferably the therapeutic can
modulate at least one activity of an IL-1 polypeptide, e.g.,
interaction with a receptor, by mimicking or potentiating
(agonizing) or inhibiting (antagonizing) the effects of a
naturally-occurring polypeptide. An IL-1 agonist can be a wild-type
protein or derivative thereof having at least one bioactivity of
the wild-type, e.g., receptor binding activity. An IL-1 agonist can
also be a compound that upregulates expression of a gene or which
increases at least one bioactivity of a protein. An IL-1 agonist
can also be a compound which increases the interaction of a
polypeptide with another molecule, e.g., a receptor. An IL-1
antagonist can be a compound which inhibits or decreases the
interaction between a protein and another molecule, e.g., a
receptor or an agent that blocks signal transduction or
post-translation processing (e.g., IL-1 converting enzyme (ICE)
inhibitor). Accordingly, a preferred antagonist is a compound which
inhibits or decreases binding to a receptor and thereby blocks
subsequent activation of the receptor. An IL-1 antagonist can also
be a compound that downregulates expression of a gene or which
reduces the amount of a protein present. The antagonist can be a
dominant negative form of a polypeptide, e.g., a form of a
polypeptide which is capable of interacting with a target peptide,
e.g., a receptor, but which does not promote the activation of the
receptor. The antagonist can also be a nucleic acid encoding a
dominant negative form of a polypeptide, an antisense nucleic acid,
or a ribozyme capable of interacting specifically with an RNA. Yet
other antagonists are molecules which bind to a polypeptide and
inhibit its action. Such molecules include peptides, e.g., forms of
target peptides which do not have biological activity, and which
inhibit binding to receptors. Thus, such peptides will bind to the
active site of a protein and prevent it from interacting with
target peptides. Yet other antagonists include antibodies that
specifically interact with an epitope of a molecule, such that
binding interferes with the biological function of the polypeptide.
In yet another preferred embodiment, the antagonist is a small
molecule, such as a molecule capable of inhibiting the interaction
between a polypeptide and a target receptor. Alternatively, the
small molecule can function as an antagonist by interacting with
sites other than the receptor binding site. Preferred therapeutics
include lipid lowering drugs, antiplatelet agents,
anti-inflammatory agents and antihypertensive agents.
[0080] "Cerebrovascular disease," as used herein, is a type of
peripheral vascular disease (as defined below) where the peripheral
vessel blocked is part of the cerebral circulation. The cerebral
circulation includes the carotid and the vertebral arterial
systems. This definition of cerebrovascular disease is intended
specifically to include intracranial hemorrhage that does not occur
as a manifestation of an arterial blockage. Blockage can occur
suddenly, by mechanisms such as plaque rupture or embolization.
Blockage can occur progressively, with narrowing of the artery via
myointimal hyperplasia and plaque formation. Blockage can be
complete or partial. Certain degrees and durations of blockage
result in cerebral ischemia, a reduction of blood flow that lasts
for several seconds to minutes. The prolongation of cerebral
ischemia can result in cerebral infarction. Ischemia and infarction
can be focal or widespread. Cerebral ischemia or infarction can
result in the abrupt onset of a non-convulsive focal neurological
defect, a clinical event termed a "stroke" or a "cerebrovascular
accident (CVA)". Cerebrovascular disease has two broad categories
of pathologies: thrombosis and embolism. Thrombotic strokes occur
without warning symptoms in 80-90% of patients; between 10 and 20%
of thrombotic strokes are heralded by transient ischemic attacks. A
cerebrovascular disease can be associated with a fragile plaque
disorder. The signs and symptoms of this type of cerebrovascular
disease are those associated with fragile plaque, including stroke
due to sudden arterial blockage with thrombus or embolus formation.
A cerebrovascular disease can be associated with occlusive
disorder. The signs and symptoms of this type of cerebrovascular
disease relate to progressive blockage of blood flow with global or
local cerebral ischemia. In this setting, neurological changes can
be seen, including stroke.
[0081] A "clinical event" is an occurrence of clinically
discernible signs of a disease or of clinically reportable symptoms
of a disease. "Clinically discernible" indicates that the sign can
be appreciated by a health care provider. "Clinically reportable"
indicates that the symptom is the type of phenomenon that can be
described to a health care provider. A clinical event may comprise
clinically reportable symptoms even if the particular patient
cannot himself or herself report them, as long as these are the
types of phenomena that are generally capable of description by a
patient to a health care provider.
[0082] A "coronary artery disease" ("CAD") refers to a vascular
disorder relating to the blockage of arteries serving the heart.
Blockage can occur suddenly, by mechanisms such as plaque rupture
or embolization. Blockage can occur progressively, with narrowing
of the artery via myointimal hyperplasia and plaque formation.
Those clinical signs and symptoms resulting from the blockage of
arteries serving the heart are manifestations of coronary artery
disease. Manifestations of coronary artery disease include angina,
ischemia, myocardial infarction, cardiomyopathy, congestive heart
failure, arrhythmias and aneurysm formation. It is understood that
fragile plaque disease in the coronary circulation is associated
with arterial thrombosis or distal embolization that manifests
itself as a myocardial infarction. It is understood that occlusive
disease in the coronary circulation is associated with arterial
stenosis accompanied by anginal symptoms, a condition commonly
treated with pharmacological interventions and with
angioplasty.
[0083] A "disease" is a disorder characterized by clinical events
including clinical signs and clinical symptoms. The diseases
discussed herein include cardiovascular disease, peripheral
vascular disease, CAD, cerebrovascular disease, and those diseases
in any anatomic location associated with fragile plaque disorder,
with occlusive disorder or with restenosis.
[0084] A "disorder associated allele" or "an allele associated with
a disorder" refers to an allele whose presence in a subject
indicates that the subject has or is susceptible to developing a
particular disorder. One type of disorder associated allele is a
"cardiovascular disorder associated allele," the presence of which
in a subject indicates that the subject has or is susceptible to
developing a cardiovascular disorder. These include broadly within
their scope alleles which are associated with "fragile plaque
disorders," alleles associated with "occlusive disorders," and
alleles associated with restenosis. Examples of alleles associated
with "fragile plaque disorders" include those alleles comprising
the IL-1 pattern 1--i.e. allele 2 of the IL-1A +4825; allele 2 of
the +3954 marker of IL-1B; and allele 1 of the +2018 marker of
IL-1RN; and allele 1 of the (-511) marker of the IL-1B gene or an
allele that is in linkage disequilibrium with one of the
aforementioned alleles. Examples of alleles associated with
"occlusive disorders" include those comprising the IL-1 pattern
2--i.e. allele 1 of the IL-1A +4825; allele 1 of the +3954 marker
of IL-1B; and allele 2 of the +2018 marker of IL-1RN; and allele 2
of the (-511) marker of the IL-1B gene or an allele that is in
linkage disequilibrium with one of the aforementioned alleles.
Examples of alleles associated with restenosis include the
combination of either allele 1 of the +4825 marker of IL-1A or
allele 1 of the +3954 marker as combined with either allele 1 of
the -511 marker of IL-1B or allele 1 of the +2018 marker of IL-1RN,
or an allele that is in linkage disequilibrium with one of the
aforementioned alleles. A "periodontal disorder associated allele"
refers to an allele whose presence in a subject indicates that the
subject has or is susceptible to developing a periodontal
disorders.
[0085] The phrases "disruption of the gene" and "targeted
disruption" or any similar phrase refers to the site specific
interruption of a native DNA sequence so as to prevent expression
of that gene in the cell as compared to the wild-type copy of the
gene. The interruption may be caused by deletions, insertions or
modifications to the gene, or any combination thereof.
[0086] The term "haplotype" as used herein is intended to refer to
a set of alleles that are inherited together as a group (are in
linkage disequilibrium) at statistically significant levels
(p.sub.corr<0.05). As used herein, the phrase "an IL-1
haplotype" refers to a haplotype in the IL-1 loci.
[0087] The term "hyperplasia" as used herein is intended to refer
to an abnormal or unusual increase in growth or division of the
cells composing a tissue or organ. It is understood that the term
"hyperplasia," as used herein, encompasses a wide variety of
specific proliferative states including "neointimal hyperplasia" or
"neointimal growth," which refers to hyperplasia of the of cells in
the endothelial layer of a blood vessel and "myointimal
hyperplasia" or "myointimal growth," which refers to an abnormal
proliferation of smooth muscle cells of the vascular wall. The
terms myointimal and neointimal are used interchangeably
herein.
[0088] An "IL-1 agonist" as used herein refers to an agent that
mimics, upregulates (potentiates or supplements) or otherwise
increases an IL-1 bioactivity or a bioactivity of a gene in an IL-1
biological pathway. IL-1 agonists may act on any of a variety of
different levels, including regulation of IL-1 gene expression at
the promoter region, regulation of mRNA splicing mechanisms,
stabilization of mRNA, phosphorylation of proteins for translation,
conversion of proIL-1 to mature IL-1 and secretion of IL-1.
Agonists that increase IL-1 synthesis include: lipopolysaccharides,
IL-1B, cAMP inducing agents, Nf.kappa.B activating agents, AP-1
activating agents, TNF-.alpha., oxidized LDL, advanced
glycosylation end products (AGE), sheer stress, hypoxia, hyperoxia,
ischemia reperfusion injury, histamine, prostaglandin E 2 (PGE2),
IL-2, IL-3, IL-12, granulocyte macrophage-colony stimulating factor
(GM-CSF), monocyte colony stimulating factor (M-CSF), stem cell
factor, platelet derived growth factor (PDGF), complement C5A,
complement C5b9, fibrin degradation products, plasmin, thrombin,
9-hydroxyoctadecaenoic acid, 13-hydroxyoctadecaenoic acid, platelet
activating factor (PAF), factor H, retinoic acid, uric acid,
calcium pyrophosphate, polynucleosides, c-reactive protein,
a-antitrypsin, tobacco antigen, collagen, P-1 integrins, LFA-3,
anti-HLA-DR, anti-IgM, anti-CD3, phytohemagglutinin (CD2), sCD23,
ultraviolet B radiation, gamma radiation, substance P,
isoproterenol, methamphetamine and melatonin. Agonists that
stabilize IL-1 MRNA include bacterial endotoxin and IL-1. Other
agonists, that function by increasing the number of IL-1 type 1
receptors available, include IL-1, PKC activators, dexamethasone,
IL-2, IL-4 and PGE2. Other preferred antagonists interfere or
inhibit signal transduction factors activated by IL-1 or utilized
in an IL-1 signal transduction pathway (e.g NFKB and AP-1, P13
kinase, phospholipase A2, protein kinase C, JNK-1, 5-lipoxygenase,
cyclooxygenase 2, tyrosine phosphorylation, iNOS pathway, Rac, Ras,
TRAF). Still other agonists increase the bioactivity of genes whose
expression is induced by IL-1, including: IL-1, IL-1Ra, TNF, IL-2,
IL-3, IL-6, IL-12, GM-CSF, G-CSF, TGF-.beta., fibrinogen, urokinase
plasminogen inhibitor, Type 1 and type 2 plasminogen activator
inhibitor, p-selectin (CD62), fibrinogen receptor, CD-11/CD18,
protease nexin-1, CD44, Matrix metalloproteinase-1 (MMP-1),MMP-3,
Elastase, Collagenases, Tissue inhibitor of metalloproteinases-1
(TIMP-1),Collagen, Triglyceride increasing Apo CIII,
Apolipoprotein, ICAM-1, ELAM-1, VCAM-1, L-selectin, Decorin, stem
cell factor, Leukemia inhibiting factor, IFN.alpha.,.beta.,.gamma.,
L-8, IL-2 receptor, IL-3 receptor, IL-5 receptor, c-kit receptor,
GM-CSF receptor, Cyclooxygenase-2 (COX-2), Type 2 phospholipase A2,
Inducible nitric oxide synthase (iNOS), Endothelin-1,3, Gamma
glutamyl transferase, Mn superoxide dismutase, C-reactive protein,
Fibrinogen, Serum amyloid A, Metallothioneins, Ceruloplasmin,
Lysozyme, Xanthine dehydrogenase, Xanthine oxidase, Platelet
derived growth factor A chain (PDGF), Melanoma growth stimulatory
activity (gro-.alpha., .beta., .gamma., Insulin-like growth
factor-1 (IGF-1), Activin A, Pro-opiomelanocortiotropin,
corticotropin releasing factor, B amyloid precursor, Basement
membrane protein-40, Laminin B1 and B2, Constitutive heat shock
protein p70, P42 mitogen, activating protein kinase, ornithine
decarboxylase, heme oxygenase and G-protein .alpha. subunit).
[0089] An "IL-1 antagonist" as used herein refers to an agent that
downregulates or otherwise decreases an IL-1 bioactivity. IL-1
antagonists may act on any of a variety of different levels,
including regulation of IL-1 gene expression at the promoter
region, regulation of mRNA splicing mechanisms, stabilization of
mRNA, phosphorylation of proteins for translation, conversion of
proIL-1 to mature IL-1 and secretion of IL-1. Antagonists of
IL-iproduction include: corticosteroids, lipoxygenase inhibitors,
cyclooxygenase inhibitors, .gamma.-interferon, IL-4, IL-10, IL-13,
transforming growth factor .beta. (TGF-.beta.), ACE inhibitors, n-3
polyunsaturated fatty acids, antioxidants and lipid reducing
agents. Antagonists that destabilize IL-1mRNA include agents that
promote deadenylation. Antagonists that inhibit or prevent
phosphorylation of IL-1 proteins for translation include
pyridinyl-imadazole compounds, such as tebufelone and compounds
that inhibit microtubule formation (e.g. colchicine, vinblastine
and vincristine). Antagonists that inhibit or prevent the
conversion of proIL-1 to mature IL-1 include interleukin converting
enzyme (ICE) inhibitors, such as .epsilon.ICE isoforms, ICE
.alpha., .beta., and .gamma. isoform antibodies, CXrm-A, transcript
X, endogenous tetrapeptide competitive substrate inhibitor,
trypsin, elastase, chyrnotrypsin, chymase, and other nonspecific
proteases. Antagonists that prevent or inhibit the scretion of IL-1
include agents that block anion transport. Antagonists that
interefere with IL-1 receptor interactions, include: agents that
inhibit glycosylation of the type I IL-1 receptor, antisense
oligonucleotides against IL-1 RI, antibodies to IL-1RI and
antisense oligonucleotides against IL-1RacP. Other antagonists,
that function by decreasing the number of IL-1 type 1 receptors
available, include TGF-.beta., COX inhibitors, factors that
increase IL-1 type II receptors, dexamethasone, PGE2, IL-1 and
IL-4. Other preferred antagonists interfere or inhibit signal
transduction factors activated by IL-1 or utilized in an IL-1
signal transduction pathway (e.g NFKB and AP-1, PI3 kinase,
phospholipase A2, protein kinase C, JNK-1, 5-lipoxygenase,
cyclooxygenase 2, tyrosine phosphorylation, iNOS pathway, Rac, Ras,
TRAF). Still other antagonists interfere with the bioactivity of
genes whose expression is induced by IL-1, including: IL-1, IL-1Ra,
TNF, IL-2, IL-3, IL-6, IL-12, GM-CSF, G-CSF, TGF-.beta.,
fibrinogen, urokinase plasminogen inhibitor, Type 1 and type 2
plasminogen activator inhibitor, p-selectin (CD62), fibrinogen
receptor, CD-11/CD18, protease nexin-1, CD44, Matrix
metalloproteinase-1(MMP-1),MMP-3, Elastase, Collagenases, Tissue
inhibitor of metalloproteinases-1(TIMP-1),Collagen, Triglyceride
increasing Apo CIII, Apolipoprotein, ICAM-1, ELAM-1, VCAM-1,
L-selectin, Decorin, stem cell factor, Leukemia inhibiting factor,
IFN.alpha.,.beta.,.gamma., L-8, IL-2 receptor, IL-3 receptor, IL-5
receptor, c-kit receptor, GM-CSF receptor, Cyclooxygenase-2
(COX-2), Type 2 phospholipase A2, Inducible nitric oxide synthase
(iNOS), Endothelin-1,3, Gamma glutamyl transferase, Mn superoxide
dismutase, C-reactive protein, Fibrinogen, Serum amyloid A,
Metallothioneins, Ceruloplasmin, Lysozyme, Xanthine dehydrogenase,
Xanthine oxidase, Platelet derived growth factor A chain (PDGF),
Melanoma growth stimulatory activity (gro-.alpha.,.beta.,.gamma.),
Insulin-like growth factor-1 (IGF-1), Activin A,
Pro-opiomelanocortiotropin, corticotropin releasing factor, B
amyloid precursor, Basement membrane protein-40, Laminin B1 and B2,
Constitutive heat shock protein p70, P42 mitogen, activating
protein kinase, ornithine decarboxylase, heme oxygenase and
G-protein .alpha. subunit). Other preferred antagonists include:
hymenialdisine, herbimycines (e.g. herbamycin A), CK-103A and its
derivatives (e.g. 4,6-dihydropyridazino[4,5-c]pyridazin-5
(1H)-one), CK-119, CK-122, iodomethacin, aflatoxin B1, leptin,
heparin, bicyclic imidazoles (e.g SB203580), PD15306 HCl,
podocarpic acid derivatives, M-20, Human [Gly2] Glucagon-like
peptide-2, FR167653, Steroid derivatives, glucocorticoids,
Quercetin, Theophylline, NO-synthetase inhibitors, RWJ 68354,
Euclyptol (1.8-cineole), Magnosalin, N-Acetylcysteine,
Alpha-Melatonin-Stimulating Hormone (.alpha.-MSH), Triclosan
(2,4,4'-trichloro-2'-hydroxyldiphenyl ether), Prostaglandin E2 and
4-aminopyridine Ethacrynic acid and
4,4'-diisothiocyanatostilbene-2,2- '-disulfonic acid (DIDS),
Glucose, Lipophosphoglycan, aspirin, Catabolism-blocking agents,
Diacerhein, Thiol-modulating agents, Zinc, Morphine, Leukotriene
biosynthesis inhibitors (e.g. MK886), Platelet-activating factor
receptor antagonists (e.g. WEB 2086), Amiodarone, Tranilast,
S-methyl-L-thiocitrulline, Beta-adrenoreceptor agonists
(e.g.Procaterol, Clenbuterol, Fenoterol, Terbutaline, Hyaluronic
acid, anti-TNF-.alpha. antibodies, anti-IL-1.alpha. autoantibodies,
IL-1 receptor antagonist, IL-1R-associated kinase, soluble TNF
receptors and antiinflammatory cytokines (e.g IL-4, IL-13, IL-10,
IL-6, TGF-.alpha., angiotensin II, Soluble IL-1 type II receptor,
Soluble IL-1 type I receptor, Tissue plasminogen activator, Zinc
finger protein A20 IL-1 Peptides (e.g (Thr-Lys-Pro-Arg) (Tuftsin),
(Ile-Thr-Gly-Ser-Glu) IL-1-alpha, Val-Thr-Lys-Phe-Tyr-Phe,
Val-Thr-Asp-Phe-Tyr-Phe, Interferon alpha2b, Interferon beta,
IL-1-beta analogues (e.g. IL-1-beta tripeptide: Lys-D-Pro-Thr),
glycosylated IL-1-alpha, and IL-1ra peptides.
[0090] The terms "IL-1 gene cluster" and "IL-1 loci" as used herein
include all the nucleic acid at or near the 2q13 region of
chromosome 2, including at least the IL-1A, IL-1B and IL-1RN genes
and any other linked sequences. (Nicklin et al., Genomics 19:
382-84, 1994). The terms "IL-1A", "IL-1B", and "IL-1RN" as used
herein refer to the genes coding for IL-1, IL-1, and IL-1 receptor
antagonist, respectively. The gene accession number for IL-1A,
IL-1B, and IL-1RN are X03833, X04500, and X64532, respectively.
[0091] "IL-1 functional mutation" refers to a mutation within the
IL-1 gene cluster that results in an altered phenotype (i.e.
effects the function of an IL-1 gene or protein). Examples include:
IL-1A(+4845) allele 2, IL-1B (+3954) allele 2, IL-1B (+6912) allele
2 and IL-1RN (+2018) allele 2.
[0092] "IL-1X (Z) allele Y"refers to a particular allelic form,
designated Y, occurring at an IL-1 locus polymorphic site in gene
X, wherein X is IL-1A, B, or RN or some other gene in the IL-1 gene
loci, and positioned at or near nucleotide Z, wherein nucleotide Z
is numbered relative to the major transcriptional start site, which
is nucleotide +1, of the particular IL-1 gene X. As further used
herein, the term "IL-1X allele (Z)" refers to all alleles of an
IL-1 polymorphic site in gene X positioned at or near nucleotide Z.
For example, the term "IL-1RN (+2018) allele" refers to alternative
forms of the IL-1RN gene at marker+2018. "IL-1RN (+2018) allele
1"refers to a form of the IL-1RN gene which contains a cytosine (C)
at position +2018 of the sense strand. Clay et al., Hum. Genet.
97:723-26, 1996. "IL-1RN (+2018) allele 2" refers to a form of the
IL-1RN gene which contains a thymine (T) at position +2018 of the
plus strand. When a subject has two identical IL-1RN alleles, the
subject is said to be homozygous, or to have the homozygous state.
When a subject has two different IL-1RN alleles, the subject is
said to be heterozygous, or to have the heterozygous state. The
term "IL-1RN (+2018) allele 2,2" refers to the homozygous IL-1RN
(+2018) allele 2 state. Conversely, the term "IL-1RN (+2018) allele
1,1" refers to the homozygous IL-1 RN (+2018) allele 1 state. The
term "IL-1RN (+2018) allele 1,2" refers to the heterozygous allele
1 and 2 state.
[0093] "IL-1 related" as used herein is meant to include all genes
related to the human IL-1 locus genes on human chromosome 2 (2q
12-14). These include IL-1 genes of the human IL-1 gene cluster
located at chromosome 2 (2q 13-14) which include: the IL-1A gene
which encodes interleukin-1.alpha., the IL-1B gene which encodes
interleukin-1, and the IL-1RN (or IL-1ra) gene which encodes the
interleukin-1 receptor antagonist. Furthermore these IL-1 related
genes include the type I and type II human IL-1 receptor genes
located on human chromosome 2 (2q12) and their mouse homologs
located on mouse chromosome 1 at position 19.5 cM.
Interleukin-1.alpha., interleukin-1, and interleukin-1RN are
related in so much as they all bind to IL-1 type I receptors,
however only interleukin-1.alpha. and interleukin-1.beta. are
agonist ligands which activate IL-1 type I receptors, while
interleukin-1RN is a naturally occurring antagonist ligand. Where
the term "IL-1" is used in reference to a gene product or
polypeptide, it is meant to refer to all gene products encoded by
the interleukin-1 locus on human chromosome 2 (2q 12-14) and their
corresponding homologs from other species or functional variants
thereof. The term IL-1 thus includes secreted polypeptides which
promote an inflammatory response, such as IL-1.alpha. and
IL-1.beta., as well as a secreted polypeptide which antagonize
inflammatory responses, such as IL-1 receptor antagonist and the
IL-1 type II (decoy) receptor.
[0094] An "IL-1 receptor" or "IL-IR" refers to various cell
membrane bound protein receptors capable of binding to and/or
transducing a signal from IL-1 locus-encoded ligand. The term
applies to any of the proteins which are capable of binding
interleukin-1 (IL-1) molecules and, in their native configuration
as mammalian plasma membrane proteins, presumably play a role in
transducing the signal provided by IL-1 to a cell. As used herein,
the term includes analogs of native proteins with IL-1-binding or
signal transducing activity. Examples include the human and murine
IL-1 receptors described in U.S. Pat. No. 4,968,607. The term "IL-1
nucleic acid" refers to a nucleic acid encoding an IL-1
protein.
[0095] An "IL-1 polypeptide" and "IL-1 protein" are intended to
encompass polypeptides comprising the amino acid sequence encoded
by the IL-1 genomic DNA sequences shown in
[0096] FIGS. 1, 2, and 3, or fragments thereof, and homologs
thereof and include agonist and antagonist polypeptides.
[0097] "In-stent stenosis" refers to the progressive occlusion
within a stent that has been placed during angioplasty. In-stent
stenosis is a form of restenosis that takes place within an
arterial stent.
[0098] "Increased risk" refers to a statistically higher frequency
of occurrence of the disease or condition in an individual carrying
a particular polymorphic allele in comparison to the frequency of
occurrence of the disease or condition in a member of a population
that does not carry the particular polymorphic allele.
[0099] The term "interact" as used herein is meant to include
detectable relationships or associations (e.g. biochemical
interactions) between molecules, such as interactions between
protein-protein, protein-nucleic acid, nucleic acid-nucleic acid
and protein-small molecule or nucleic acid-small molecule in
nature.
[0100] The term "isolated" as used herein with respect to nucleic
acids, such as DNA or RNA, refers to molecules separated from other
DNAs, or RNAs, respectively, that are present in the natural source
of the macromolecule. For example, an isolated nucleic acid
encoding one of the subject IL-1 polypeptides preferably includes
no more than 10 kilobases (kb) of nucleic acid sequence which
naturally immediately flanks the IL-1 gene in genomic DNA, more
preferably no more than 5 kb of such naturally occurring flanking
sequences, and most preferably less than 1.5 kb of such naturally
occurring flanking sequence. The term isolated as used herein also
refers to a nucleic acid or peptide that is substantially free of
cellular material, viral material, or culture medium when produced
by recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. Moreover, an "isolated
nucleic acid" is meant to include nucleic acid fragments which are
not naturally occurring as fragments and would not be found in the
natural state. The term "isolated" is also used herein to refer to
polypeptides which are isolated from other cellular proteins and is
meant to encompass both purified and recombinant polypeptides.
[0101] A "knock-in" transgenic animal refers to an animal that has
had a modified gene introduced into its genome and the modified
gene can be of exogenous or endogenous origin.
[0102] A "knock-out" transgenic animal refers to an animal in which
there is partial or complete suppression of the expression of an
endogenous gene (e.g, based on deletion of at least a portion of
the gene, replacement of at least a portion of the gene with a
second sequence, introduction of stop codons, the mutation of bases
encoding critical amino acids, or the removal of an intron
junction, etc.).
[0103] A "knock-out construct" refers to a nucleic acid sequence
that can be used to decrease or suppress expression of a protein
encoded by endogenous DNA sequences in a cell. In a simple example,
the knock-out construct is comprised of a gene, such as the IL-1RN
gene, with a deletion in a critical portion of the gene so that
active protein cannot be expressed therefrom. Alternatively, a
number of termination codons can be added to the native gene to
cause early termination of the protein or an intron junction can be
inactivated. In a typical knock-out construct, some portion of the
gene is replaced with a selectable marker (such as the neo gene) so
that the gene can be represented as follows: IL-1RN 5'/neo/IL-1RN
3', where IL-1RN5' and IL-1RN 3', refer to genomic or cDNA
sequences which are, respectively, upstream and downstream relative
to a portion of the IL-1RN gene and where neo refers to a neomycin
resistance gene. In another knock-out construct, a second
selectable marker is added in a flanking position so that the gene
can be represented as: IL-1RN/neo/IL-1RN/TK, where TK is a
thymidine kinase gene which can be added to either the IL-1RN5' or
the IL-1RN3' sequence of the preceding construct and which further
can be selected against (i.e. is a negative selectable marker) in
appropriate media. This two-marker construct allows the selection
of homologous recombination events, which removes the flanking TK
marker, from non-homologous recombination events which typically
retain the TK sequences. The gene deletion and/or replacement can
be from the exons, introns, especially intron junctions, and/or the
regulatory regions such as promoters.
[0104] "Linkage disequilibrium" refers to co-inheritance of two
alleles at frequencies greater than would be expected from the
separate frequencies of occurrence of each allele in a given
control population. The expected frequency of occurrence of two
alleles that are inherited independently is the frequency of the
first allele multiplied by the frequency of the second allele.
Alleles that co-occur at expected frequencies are said to be in
"linkage equilibrium". The cause of linkage disequilibrium is often
unclear. It can be due to selection for certain allele combinations
or to recent admixture of genetically heterogeneous populations. In
addition, in the case of markers that are very tightly linked to a
disease gene, an association of an allele (or group of linked
alleles) with the disease gene is expected if the disease mutation
occurred in the recent past, so that sufficient time has not
elapsed for equilibrium to be achieved through recombination events
in the specific chromosomal region. When referring to allelic
patterns that are comprised of more than one allele, a first
allelic pattern is in linkage disequilibrium with a second allelic
pattern if all the alleles that comprise the first allelic pattern
are in linkage disequilibrium with at least one of the alleles of
the second allelic pattern. An example of linkage disequilibrium is
that which occurs between the alleles at the IL-1RN (+2018) and
IL-1RN (VNTR) polymorphic sites. The two alleles at IL-1RN (+2018)
are 100% in linkage disequilibrium with the two most frequent
alleles of IL-1RN (VNTR), which are allele 1 and allele 2.
[0105] The term "marker" refers to a sequence in the genome that is
known to vary among individuals. For example, the IL-1RN gene has a
marker that consists of a variable number of tandem repeats
(VNTR).
[0106] "Modulate" refers to the ability of a substance to regulate
bioactivity. When applied to an IL-1 bioactivity, an agonist or
antagonist can modulate bioactivity for example by agonizing or
antagonizing an IL-1 synthesis, receptor interaction, or IL-1
mediated signal transduction mechanism.
[0107] A "mutated gene" or "mutation" or "functional mutation"
refers to an allelic form of a gene, which is capable of altering
the phenotype of a subject having the mutated gene relative to a
subject which does not have the mutated gene. The altered phenotype
caused by a mutation can be corrected or compensated for by certain
agents. If a subject must be homozygous for this mutation to have
an altered phenotype, the mutation is said to be recessive. If one
copy of the mutated gene is sufficient to alter the phenotype of
the subject, the mutation is said to be dominant. If a subject has
one copy of the mutated gene and has a phenotype that is
intermediate between that of a homozygous and that of a
heterozygous subject (for that gene), the mutation is said to be
co-dominant.
[0108] A "non-human animal" of the invention includes mammals such
as rodents, non-human primates, sheep, dogs, cows, goats, etc.
Preferred non-human animals are selected from the rodent family
including rat and mouse, most preferably mouse, though transgenic
amphibians, such as members of the Xenopus genus, and transgenic
chickens can also provide important tools for understanding and
identifying agents which can affect, for example, embryogenesis and
tissue formation. The term "chimeric animal" is used herein to
refer to animals in which the recombinant gene is found, or in
which the recombinant gene is expressed in some but not all cells
of the animal. The term "tissue-specific chimeric animal" indicates
that one of the recombinant IL-1 genes is present and/or expressed
or disrupted in some tissues but not others. The term "non-human
mammal" refers to any members of the class Mammalia, except for
humans.
[0109] As used herein, the term "nucleic acid" refers to
polynucleotides or oligonucleotides such as deoxyribonucleic acid
(DNA), and, where appropriate, ribonucleic acid (RNA). The term
should also be understood to include, as equivalents, analogs of
either RNA or DNA made from nucleotide analogs (e.g. peptide
nucleic acids) and as applicable to the embodiment being described,
single (sense or antisense) and double-stranded
polynucleotides.
[0110] "Occlusive disorder" refers to that cardiovascular disorder
characterized by the progressive thickening of an arterial wall,
associated with the presence of an atherosclerotic intimal lesion
within an artery. Occlusive disorder leads to progressive blockage
of the artery. With sufficient progression, the occlusive disorder
can reduce flow in the artery to the point that clinical signs and
symptoms are produced in the tissues perfused by the artery. These
clinical events relate to ischemia of the perfused tissues. When
severe, ischemia is accompanied by tissue death, called infarction
or gangrene. Occlusive disorder is associated with the allele
pattern 2s at the IL-1 locus.
[0111] An "occlusive disorder therapeutic" refers to any agent or
therapeutic regimen (including pharmaceuticals, nutraceuticals and
surgical means) that prevents or postpones the development of or
reduces the extent of an abnormality constitutive of an occlusive
disorder in a subject. Examples of occlusive disorder therapeutics
include those agents that are anti-oxidants, those that lower serum
lipids, those that block the action of oxidized lipids and other
agents that influence lipid metabolism or otherwise have
lipid-active effects.
[0112] A "peripheral vascular disease" ("PVD") is a cardiovascular
disease resulting from the blockage of the peripheral (i.e.,
non-coronary) arteries. Blockage can occur suddenly, by mechanisms
such as plaque rupture or embolization, as occurs in fragile plaque
disease. Blockage can occur progressively, with narrowing of the
artery via myointimal hyperplasia and plaque formation, as in
occlusive disease. Blockage can be complete or partial. Those
clinical signs and symptoms resulting from the blockage of
peripheral arteries are manifestations of peripheral vascular
disease. Manifestations of peripheral vascular diseases include,
inter alia, claudication, ischemia, intestinal angina,
vascular-based renal insufficiency, transient ischemic attacks,
aneurysm formation, peripheral embolization and stroke. Ischemic
cerebrovascular disease is a type of peripheral vascular disease.
The term "polymorphism" refers to the coexistence of more than one
form of a gene or portion (e.g., allelic variant) thereof. A
portion of a gene of which there are at least two different forms,
i.e., two different nucleotide sequences, is referred to as a
"polymorphic region of a gene". A specific genetic sequence at a
polymorphic region of a gene is an allele. A polymorphic region can
be a single nucleotide, the identity of which differs in different
alleles. A polymorphic region can also be several nucleotides
long.
[0113] The term "propensity to disease," also "predisposition" or
"susceptibility" to disease or any similar phrase, means that
certain alleles are hereby discovered to be associated with or
predictive of ILD. The alleles are thus over-represented in
frequency in individuals with disease as compared to healthy
individuals. Thus, these alleles can be used to predict disease
even in pre-symptomatic or pre-diseased individuals.
[0114] The term "restenosis" refers to any preocclusive lesion that
develops following a reconstructive procedure in a diseased blood
vessel. The term is not only applied to the recurrence of a
pre-existing stenosis, but also to previously normal vessels such
as vein grafts that become partially occluded following vascular
bypass. Restenosis refers to any luminal narrowing that occurs
following an injury to the vessel wall. Injuries resulting in
restenosis can therefore include trauma to an atherosclerotic
lesion (as seen with angioplasty), a resection of a lesion (as seen
with endarterectomy), an external trauma (e.g., a cross-clamping
injury), or a surgical anastomosis. Restenosis typically results
from a hyperplasia.
[0115] Restenosis can occur as the result of any kind of vascular
reconstruction, whether in the coronary vasculature or in the
periphery (Colburn and Moore (1998) Myointimal Hyperplasia
pp.690-709 in Vascular Surgery: A Comprehensive Review
(Philadelphia: Saunders, 1998)). For example, studies have reported
symptomatic restenosis rates of 30-50% following coronary
angioplasties (see Berk and Harris (1995) Adv. Intern. Med.
40:455-501). After carotid endarterectomies, as a further example,
20% of patients studied had a luminal narrowing greater than 50%
(Clagett et al. (1986) J. Vasc. Surg. 3:10-23). Yet another example
of restenosis is seen in infrainguinal vascular bypasses, where
40-60% of prosthetic grafts and 20-40% of the vein grafts are
occluded at three years (Dalman and Taylor (1990) Ann. Vasc. Surg.
3:109-312, Szilagyi et al. (1973) Ann. Surg. 178:232-246).
Different degrees of symptomatology accompany preocclusive lesions
in different anatomical locations, due to a combination of factors
including the different calibers of the vessels involved, the
extent of residual disease and local hemodynamics.
[0116] A "restenosis associated allele" refers to an allele whose
presence in a subject indicates that the subject has or is
susceptible to developing a restenosis. Examples of restenosis
associated alleles include allele 1 of the +4845 marker of IL-1A;
allele 1 of the +3954 marker of IL-1B; allele 1 of the -511 marker
of IL-1B; and allele 1 of the +2018 marker of IL-1RN. Still other
linked polymorphic loci associated with restenosis include: the
IL-1RN(VNTR) polymorphism, the IL-1RN gene +1731 polymorphism; the
IL-1RN gene +1812 polymorphism; the IL-1RN gene +1868 polymorphism;
the IL-1RN gene +1887 polymorphism; the IL-1RN +8006 polymorphism,
the IL-1RN +8061 polymorphism, the IL-1B -31 polymorphism and the
IL-1B -511 polymorphism. Other restenosis associated alleles that
have been described in the art include certain alleles in
angiotensin converting enzymes (See e.g. Kasi et al., (1996) Am. J
Cardiol. 77: 875-77).
[0117] A "restenosis causative functional mutation" refers to a
mutation which causes or contributes to the development of
restenosis in a subject. Preferred mutations occur within the IL-1
complex. A restenosis causative functional mutation occurring
within an IL-1 gene (e.g. IL-1A, IL-1B or IL-1RN) or a gene locus,
which is linked thereto, may alter, for example, the open reading
frame or splicing pattern of the gene, thereby resulting in the
formation of an inactive or hypoactive gene product. For example, a
mutation which occurs in intron 6 of the IL-1A locus corresponds to
a variable number of tandem repeat 46 bp sequences corresponding to
from five to 18 repeat units (Bailly, et al. (1993) Eur. J.
Immunol. 23: 1240-45). These repeat sequences contain three
potential binding sites for transcriptional factors: an SP1 site, a
viral enhancer element, and a glucocorticoid-responsive element;
therefore individuals carrying IL-1A intron 6 VNTR alleles with
large numbers of repeat units may be subject to altered
transcriptional regulation of the IL-1A gene and consequent
perturbations of inflammatory cytokine production. Indeed, there is
evidence that increased repeat number at this polymorphic IL-1A
locus leads to decreased IL-1.alpha. synthesis (Bailly et al.
(1996) Mol Immunol 33: 999-1006). Alternatively, a mutation can
result in a hyperactive gene product. For example, allele 2 of the
IL-1B (G at +6912) polymorphism occurs in the 3' UTR (untranslated
region) of the IL-1B mRNA and is associated with an approximately
four-fold increase in the steady state levels of both IL-1B mRNA
and IL-1B protein compared to those levels associated with allele 1
of the IL-1B gene.COPYRGT. at +6912). Further, an IL-1B (-511)
mutation occurs near a promoter binding site for a negative
glucocorticoid response element (Zhang et al. (1997) DNA Cell Biol
16: 145-52). This element potentiates a four-fold repression of
IL-1B expression by dexamethosone and a deletion of this negative
response elements causes a 2.5-fold increase in IL-1B promoter
activity. The IL-1B (-511) polymorphism may thus directly affect
cytokine production and inflammatory responses. These examples
demonstrate that genetic variants occurring in the IL-1A or IL-1B
gene can directly lead to the altered production or regulation of
IL-1 cytokine activity.
[0118] A "restenosis therapeutic" refers to any agent or
therapeutic regimen (including pharmaceuticals, nutraceuticals and
surgical means) that prevents or postpones the development of or
alleviates the symptoms of a restenosis in a subject. A restenosis
therapeutic can be a polypeptide, peptidomimetic, nucleic acid or
other inorganic or organic molecule, preferably a "small molecule"
including vitamins, minerals and other nutrients. Preferably a
restenosis therapeutic can modulate at least one activity of an
IL-1 polypeptide, e.g., interaction with a receptor, by mimicking
or potentiating (agonizing) or inhibiting (antagonizing) the
effects of a naturally-occurring polypeptide. An agonist can be a
wild-type protein or derivative thereof having at least one
bioactivity of the wild-type, e.g., receptor binding activity. An
agonist can also be a compound that upregulates expression of a
gene or which increases at least one bioactivity of a protein. An
agonist can also be a compound which increases the interaction of a
polypeptide with another molecule, e.g., a receptor. An antagonist
can be a compound which inhibits or decreases the interaction
between a protein and another molecule, e.g., a receptor or an
agent that blocks signal transduction or post-translation
processing (e.g., IL-1 converting enzyme (ICE) inhibitor).
Accordingly, a preferred antagonist is a compound which inhibits or
decreases binding to a receptor and thereby blocks subsequent
activation of the receptor. An antagonist can also be a compound
that downregulates expression of a gene or which reduces the amount
of a protein present. The antagonist can be a dominant negative
form of a polypeptide, e.g., a form of a polypeptide which is
capable of interacting with a target peptide, e.g., a receptor, but
which does not promote the activation of the receptor. The
antagonist can also be a nucleic acid encoding a dominant negative
form of a polypeptide, an antisense nucleic acid, or a ribozyme
capable of interacting specifically with an RNA. Yet other
antagonists are molecules which bind to a polypeptide and inhibit
its action. Such molecules include peptides, e.g., forms of target
peptides which do not have biological activity, and which inhibit
binding to receptors. Thus, such peptides will bind to the active
site of a protein and prevent it from interacting with target
peptides. Yet other antagonists include antibodies that
specifically interact with an epitope of a molecule, such that
binding interferes with the biological function of the polypeptide.
In yet another preferred embodiment, the antagonist is a small
molecule, such as a molecule capable of inhibiting the interaction
between a polypeptide and a target receptor. Alternatively, the
small molecule can function as an antagonist by interacting with
sites other than the receptor binding site. Preferred restenosis
therapeutics include agents that suppress the development of
neointimal hyperplasia, including lipid lowering drugs,
antiplatelet agents, anti-inflammatory agents, antihypertensive
agents and anticoagulants; and agents that directly inhibit
cellular growth. Furthermore, surgical decisions at the time of the
primary procedure or at the time of a secondary surgical operation
could differ depending on whether the patient was at higher risk
for a more prolific inflammation-mediated injury response. The
decision to employ a stent as part of an endovascular procedure
could be governed, for example, by an awareness of a patient's
higher risk for more aggressive vascular response to injury.
[0119] A "risk factor" is a factor identified to be associated with
an increased risk. A risk factor for a cardiovascular disorder or a
cardiovascular disease is any factor identified to be associated
with an increased risk of developing those conditions or of
worsening those conditions. A risk factor can also be associated
with an increased risk of an adverse clinical event or an adverse
clinical outcome in a patient with a cardiovascular disorder. Risk
factors for cardiovascular disease include smoking, adverse lipid
profiles, elevated lipids or cholesterol, diabetes, hypertension,
hypercoagulable states, elevated homocysteine levels, and lack of
exercise. Carrying a particular polymorphic allele is a risk factor
for a particular cardiovascular disorder, and is associated with an
increased risk of the particular disorder.
[0120] "Small molecule" as used herein, is meant to refer to a
composition, which has a molecular weight of less than about 5 kD
and most preferably less than about 4 kD. Small molecules can be
nucleic acids, peptides, peptidomimetics, carbohydrates, lipids or
other organic or inorganic molecules.
[0121] As used herein, the term "specifically hybridizes" or
"specifically detects" refers to the ability of a nucleic acid
molecule to hybridize to at least approximately 6 consecutive
nucleotides of a sample nucleic acid.
[0122] "Transcriptional regulatory sequence" is a generic term used
throughout the specification to refer to DNA sequences, such as
initiation signals, enhancers, and promoters, which induce or
control transcription of protein coding sequences with which they
are operably linked.
[0123] As used herein, the term "transgene" means a nucleic acid
sequence (encoding, e.g., one of the IL-1 polypeptides, or an
antisense transcript thereto) which has been introduced into a
cell. A transgene could be partly or entirely heterologous, i.e.,
foreign, to the transgenic animal or cell into which it is
introduced, or, is homologous to an endogenous gene of the
transgenic animal or cell into which it is introduced, but which is
designed to be inserted, or is inserted, into the animal's genome
in such a way as to alter the genome of the cell into which it is
inserted (e.g., it is inserted at a location which differs from
that of the natural gene or its insertion results in a knockout). A
transgene can also be present in a cell in the form of an episome.
A transgene can include one or more transcriptional regulatory
sequences and any other nucleic acid, such as introns, that may be
necessary for optimal expression of a selected nucleic acid.
[0124] A "transgenic animal" refers to any animal, preferably a
non-human mammal, bird or an amphibian, in which one or more of the
cells of the animal contain heterologous nucleic acid introduced by
way of human intervention, such as by transgenic techniques well
known in the art. The nucleic acid is introduced into the cell,
directly or indirectly by introduction into a precursor of the
cell, by way of deliberate genetic manipulation, such as by
microinjection or by infection with a recombinant virus. The term
genetic manipulation does not include classical cross-breeding, or
in vitro fertilization, but rather is directed to the introduction
of a recombinant DNA molecule. This molecule may be integrated
within a chromosome, or it may be extrachromosomally replicating
DNA. In the typical transgenic animals described herein, the
transgene causes cells to express a recombinant form of one of an
IL-1 polypeptide, e.g. either agonistic or antagonistic forms.
However, transgenic animals in which the recombinant gene is silent
are also contemplated, as for example, the FLP or CRE recombinase
dependent constructs described below. Moreover, "transgenic animal"
also includes those recombinant animals in which gene disruption of
one or more genes is caused by human intervention, including both
recombination and antisense techniques. The term is intended to
include all progeny generations. Thus, the founder animal and all
F1, F2, F3, and so on, progeny thereof are included.
[0125] The term "treating" as used herein is intended to encompass
curing as well as ameliorating at least one symptom of a disease or
at least one abnormality associated with a disorder. Treating a
cardiovascular disorder can take place by administering a
cardiovascular disorder therapeutic. Treating a cardiovascular
disorder can also take place by modifying risk factors that are
related to the cardiovascular disorder.
[0126] A "treatment plan" refers to at least one intervention
undertaken to modify the effect of a risk factor upon a patient. A
treatment plan for a cardiovascular disorder or disease can address
those risk factors that pertain to cardiovascular disorders or
diseases. A treatment plan can include an intervention that focuses
on changing patient behavior, such as stopping smoking. A treatment
plan can include an intervention whereby a therapeutic agent is
administered to a patient. As examples, cholesterol levels can be
lowered with proper medication, and diabetes can be controlled with
insulin. Nicotine addiction can be treated by withdrawal
medications. A treatment plan can include an intervention that is
diagnostic. The presence of the risk factor of hypertension, for
example, can give rise to a diagnostic intervention whereby the
etiology of the hypertension is determined. After the reason for
the hypertension is identified, further treatments may be
administered.
[0127] The term "vector" refers to a nucleic acid molecule, which
is capable of transporting another nucleic acid to which it has
been linked. One type of preferred vector is an episome, i.e., a
nucleic acid capable of extra-chromosomal replication. Preferred
vectors are those capable of autonomous replication and/or
expression of nucleic acids to which they are linked. Vectors
capable of directing the expression of genes to which they are
operatively linked are referred to herein as "expression vectors".
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of "plasmids" which refer
generally to circular double stranded DNA loops which, in their
vector form are not bound to the chromosome. In the present
specification, "plasmid" and "vector" are used interchangeably as
the plasmid is the most commonly used form of vector. However, the
invention is intended to include such other forms of expression
vectors which serve equivalent functions and which become known in
the art subsequently hereto.
[0128] The term "wild-type allele" refers to an allele of a gene
which, when present in two copies in a subject results in a
wild-type phenotype. There can be several different wild-type
alleles of a specific gene, since certain nucleotide changes in a
gene may not affect the phenotype of a subject having two copies of
the gene with the nucleotide changes.
4.2 Predictive Medicine
[0129] 4.2.1. Polymorphisms Associated with Restenosis
[0130] The present invention is based at least in part, on the
identification of alleles that are associated (to a statistically
significant extent) with the development of a restenosis in
subjects. Therefore, detection of these alleles, alone or in
conjunction with another means in a subject indicate that the
subject has or is predisposed to the development of a restenosis.
For example, IL-1 polymorphic alleles which are associated with a
propensity for developing restenosis include allele 1 of each of
the following markers: IL-1A (+4845), IL-1B (+3954), IL-1B (-511),
IL-1RN (+2018) and IL-1RN (VNTR) or an allele that is in linkage
disequilibrium with one of the aforementioned alleles. In
particularly preferred embodiments, the presence of a particular
allelic pattern of one or more of the abovementioned IL-1
polymorphic loci is used to predict the susceptibility of an
individual to developing restenosis. In particular, there are three
patterns of alleles at four polymorphic loci in the IL-1 gene
cluster that show various associations with particular
cardiovascular disorders. These patterns are referred to herein as
patterns 1, 2 and 3. Pattern 1 comprises an allelic pattern
including allele 2 of IL-1A (+4845) or IL-1B (+3954) and allele 1
of IL-1B (-511) or IL-1RN (+2018), oran allele that is in linkage
disequilibrium with one of the aforementioned allele. In a
preferred embodiment, this allelic pattern permits the diagnosis of
fragile plaque disorder. Pattern 2 comprises an allelic pattern
including allele 2 of IL-1B (-511) or IL-1RN (+2018) and allele 1
of IL-1A (+4845) or IL-1B (+3954), or an allele that is in linkage
disequilibrium with one of the aforementioned alleles. In a
preferred embodiment, this allelic pattern permits the diagnosis of
occlusive cardiovascular disorder. Pattern 3 comprises an allelic
pattern including allele 1 of IL-1A (+4845) or allele 1 of IL-1B
(+3954), and allele 1 of IL-1B (-511) or allele 1 of IL-1RN
(+2018), or an allele that is in linkage disequilibrium with one of
the aforementioned alleles. In a preferred embodiment, this allelic
pattern permits the diagnosis of a restenosis disorder
[0131] These IL-1 locus polymorphisms represent single base
variations within the IL-1A/IL-1B/IL-1RN gene cluster (see FIG. 4).
The IL-1A (+4845) polymorphism is a single base variation (allele 1
is G, allele 2 is T) at position +4845 within Exon V of the IL-1A
gene which encodes the inflammatory cytokine IL-1.alpha. (Gubler,
et al.(1989) Interleukin, inflammation and disease (Bomford and
Henderson, eds.) p.31-45, Elsevier publishers; and Van den velden
and Reitsma (1993) Hum Mol Genetics 2:1753-50). The IL-1A (+4845)
polymorphism occurs in the coding region of the gene and results in
a single amino acid variation in the encoded protein (Van den
Velden and Reitsma (1993) Hum Mol Genet 2: 1753). The IL-1B (-511)
polymorphism is a single base pair variation (allele 1 is C, allele
2 is T) which occurs 511 base pairs upstream of the site of IL-1B
gene transcription initiation (Di Giovine et al. (1992) Hum Mol
Genet 1: 450). The IL-1B (+3954) polymorphism was first described
as a Taq I restriction fragment length polymorphism (RFLP) (Pociot
et al. (1992) Eur J Clin Invest 22: 396-402) and has subsequently
been characterized as a single base variation (allele 1 is C,
allele 2 is T) at position +3954 in Exon V of the IL-1B gene (di
Giovine et al. (1995) Cytokine 7: 600-606). This single nucleotide
change in the open reading frame of IL-1B does not appear to
qualitatively affect the sequence of the encoded IL-1 beta
polypeptide because it occurs at the third position of a TTC
phenylalanine codon (F) of allele 1 and therefore allele 2 merely
substitutes a TTT phenylalanine codon at this position which
encodes amino acid 105 of the IL-1B gene product. Finally, the
IL-RN variable number of tandem repeats (VNTR) polymorphism occurs
within the second intron the IL-1 receptor antagonist encoding gene
(Steinkasserer (1991) Nucleic Acids Res 19: 5090-5). Allele 2 of
the of the IL-1RN (VNTR) polymorphism corresponds to two repeats of
an 86-base pair sequence, while allele 1 corresponds to four
repeats, allele 3 to three repeats, allele 4 to five repeats, and
allele 5 to six repeats (Tarlow et al. (1993) Hum Genet 91: 403-4).
Detection of any one of these IL-1 allelic variants in an
individual suggests an increased likelihood of developing
restenosis in comparison to a control individual who does not carry
the allele 2 variant at the same locus.
[0132] However, because these alleles are in linkage disequilibrium
with other alleles, the detection of such other linked alleles can
also indicate that the subject has or is predisposed to the
development of a restenosis. For example, the following alleles of
the IL-1 (33221461) haplotype are in linkage disequilibrium:
3 allele 3 of the 222/223 marker of IL-1A allele 3 of the gz5/gz6
marker of IL-1A allele 2 of the -889 marker of IL-1A allele 2 of
the +3954 marker of IL-1B allele 1 of the -511 marker of IL-1B
allele 4 of the gaat.p33330 marker allele 6 of the Y31 marker
allele 1 of the VNTR or (+2018) marker of IL-1RN
[0133] Therefore, allele 1 of IL-1B (-511) and allele 1 of IL-1RN
(VNTR) are in strong linkage disequilibrium with one another and
each of these is in linkage disequilibrium with allele 1 of the
-511 marker of IL-1B. Furthermore, in alternative embodiments of
the present invention, genotyping analysis at the 222/223 marker of
IL-1A, the gzs/gz6 marker of IL-1A, the -889 marker of IL-1A, the
+3954 marker of IL-1B, the gaat.p33330 marker of the IL-1B/IL-1RN
intergenic region, or the Y31 marker of the IL-1B/IL-1RN intergenic
region is determined, and the presence of a polymorphic allele
which is linked to one or more of the preferred
restenosis-predictive alleles is detected.
[0134] In addition, allele 1 of the IL-1RN (+2018) polymorphism
(Clay et al. (1996) Hum Genet 97: 723-26), also referred to as exon
2 (8006) (GenBank:X64532 at 8006) is known to be in linkage
disequilibrium with allele 1 of the IL-1RN (VNTR) polymorphic
locus, which in turn is a part of the 33221461 human haplotype. In
contrast, allele 2 of the IL-1RN (+2018) locus (i.e. C at+2018), is
an allelic variant associated with the 44112332 haplotype and
allele 2 of the IL-1RN (VNTR) polymorphic locus. The IL-1RN (VNTR)
therefore provides an alternative target for prognostic genotyping
analysis to determine an individual's likelihood of developing
restenosis. Similarly, three other polymorphisms in an IL-1RN
alternative exon (Exon 1ic, which produces an intracellular form of
the gene product) are also in linkage disequilibrium with allele 2
of IL-1RN (VNTR) (Clay et al. (1996) Hum Genet 97: 723-26). These
include: the IL-1RN exon lic (1812) polymorphism (GenBank:X77090 at
1812); the IL-1RN exon lic (1868) polymorphism (GenBank:X77090 at
1868); and the IL-1RN exon lic (1887) polymorphism (GenBank:X77090
at 1887). Furthermore yet another polymorphism in the promoter for
the alternatively spliced intracellular form of the gene, the Pic
(1731) polymorphism (GenBank:X77090 at 1731), is also in linkage
disequilibrium with allele 2 of the IL-1RN (VNTR) polymorphic locus
(Clay et al. (1996) Hum Genet 97: 723-26). The corresponding
sequence alterations for each of these IL-1RN polymorphic loci is
shown below.
4 Exon 2 Exon lic-1 Exon lic-2 Exon lic-3 Pic (+2018 of (1812 of
(1868 of (1887 of (1731 of Allele # IL-1RN) GB: X77090) GB: X77090
GB: X77090) GB: X77090) 1 T G A G G 2 C A G C A
[0135] For each of these polymorphic loci, the allele 1 sequence
variant has been determined to be in linkage disequilibrium with
allele 1 of the IL-1RN (VNTR) locus (Clay et al. (1996) Hum Genet
97: 723-26).
[0136] Further, allele 1 of IL-1B (+3954), which has been pointed
out as a prognostic indicator of an increased propensity for
developing restenosis is a component of a second haplotype, the
44112332 haplotype of co-inherited IL-1 locus polymorphic alleles
(Cox, et al. (1998) Am. J. Hum. Genet. 62: 1180-88). Specifically,
the 44112332 haplotype comprises the following genotype:
5 allele 4 of the 222/223 marker of IL-1A allele 4 of the gz5/gz6
marker of IL-1A allele 1 of the -889 marker of IL-1A allele 1 of
the +3954 marker of IL-1B allele 2 of the -511 marker of IL-1B
allele 3 of the gaat.p33330 marker allele 3 of the Y31 marker
allele 2 of the VNTR marker of IL-1RN
[0137] In addition to the allelic patterns described above, as
described herein, one of skill in the art can readily identify
other alleles (including polymorphisms and mutations) that are in
linkage disequilibrium with an allele associated with restenosis.
For example, a nucleic acid sample from a first group of subjects
without restenosis can be collected, as well as DNA from a second
group of subjects with restenosis. The nucleic acid sample can then
be compared to identify those alleles that are over-represented in
the second group as compared with the first group, wherein such
alleles are presumably associated with restenosis. Alternatively,
alleles that are in linkage disequilibrium with a restenosis
associated allele can be identified, for example, by genotyping a
large population and performing statistical analysis to determine
which alleles appear more commonly together than expected.
Preferably the group is chosen to be comprised of genetically
related individuals. Genetically related individuals include
individuals from the same race, the same ethnic group, or even the
same family. As the degree of genetic relatedness between a control
group and a test group increases, so does the predictive value of
polymorphic alleles which are ever more distantly linked to a
disease-causing allele. This is because less evolutionary time has
passed to allow polymorphisms which are linked along a chromosome
in a founder population to redistribute through genetic cross-over
events. Thus race-specific, ethnic-specific, and even
family-specific diagnostic genotyping assays can be developed to
allow for the detection of disease alleles which arose at ever more
recent times in human evolution, e.g., after divergence of the
major human races, after the separation of human populations into
distinct ethnic groups, and even within the recent history of a
particular family line.
[0138] Linkage disequilibrium between two polymorphic markers or
between one polymorphic marker and a disease-causing mutation is a
meta-stable state. Absent selective pressure or the sporadic linked
reoccurrence of the underlying mutational events, the polymorphisms
will eventually become disassociated by chromosomal recombination
events and will thereby reach linkage equilibrium through the
course of human evolution. Thus, the likelihood of finding a
polymorphic allele in linkage disequilibrium with a disease or
condition may increase with changes in at least two factors:
decreasing physical distance between the polymorphic marker and the
disease-causing mutation, and decreasing number of meiotic
generations available for the dissociation of the linked pair.
Consideration of the latter factor suggests that, the more closely
related two individuals are, the more likely they will share a
common parental chromosome or chromosomal region containing the
linked polymorphisms and the less likely that this linked pair will
have become unlinked through meiotic cross-over events occurring
each generation. As a result, the more closely related two
individuals are, the more likely it is that widely spaced
polymorphisms may be co-inherited. Thus, for individuals related by
common race, ethnicity or family, the reliability of ever more
distantly spaced polymorphic loci can be relied upon as an
indicator of inheritance of a linked disease-causing mutation.
[0139] Appropriate probes may be designed to hybridize to a
specific gene of the IL-1 locus, such as IL-1A, IL-1B or IL-1RN or
a related gene. These genomic DNA sequences are shown in FIGS. 1, 2
and 3, respectively, and further correspond to formal SEQ ID Nos.
15, 16 and 17, respectively. Alternatively, these probes may
incorporate other regions of the relevant genomic locus, including
intergenic sequences. Indeed the IL-1 region of human chromosome 2
spans some 400,000 base pairs and, assuming an average of one
single nucleotide polymorphism every 1,000 base pairs, includes
some 400 SNPs loci alone. Yet other polymorphisms available for use
with the immediate invention are obtainable from various public
sources. For example, the human genome database collects intragenic
SNPs, is searchable by sequence and currently contains
approximately 2,700 entries (http://hgbase.interactiva.de). Also
available is a human polymorphism database maintained by the
Massachusetts Institute of Technology (MIT SNP database
(http://www.genome.wi.mit.edu/SNP/human/index.html)). From such
sources SNPs as well as other human polymorphisms may be found.
[0140] For example, examination of the IL-1 region of the human
genome in any one of these databases reveals that the IL-1 locus
genes are flanked by a centromere proximal polymorphic marker
designated microsatellite marker AFM220ze3 at 127.4 cM
(centiMorgans) (see GenBank Acc. No. Z17008) and a distal
polymorphic marker designated microsatellite anchor marker
AFM087xa1 at 127.9 cM (see GenBank Acc. No. Z16545). These human
polymorphic loci are both CA dinucleotide repeat microsatellite
polymorphisms, and, as such, show a high degree of heterozygosity
in human populations. For example, one allele of AFM220ze3
generates a 211 bp PCR amplification product with a 5' primer of
the sequence TGTACCTAAGCCCACCCTT-TAGAGC (SEQ ID No. 18) and a 3'
primer of the sequence TGGCCTCCAGAAACCTCCAA (SEQ ID No. 19).
Furthermore, one allele of AFM087xa1 generates a 177 bp PCR
amplification product with a 5' primer of the sequence
GCTGATATTCTGGTGGGAAA (SEQ ID No.20) and a 3' primer of the sequence
GGCAAGAGCAAAACTCTGTC (SEQ ID No.21). Equivalent primers
corresponding to unique sequences occurring 5' and 3' to these
human chromosome 2 CA dinucleotide repeat polymorphisms will be
apparent to one of skill in the art. Reasonable equivalent primers
include those which hybridize within about I kb of the designated
primer, and which further are anywhere from about 17 bp to about 27
bp in length. A general guideline for designing primers for
amplification of unique human chromosomal genomic sequences is that
they possess a melting temperature of at least about 50.degree. C.,
wherein an approximate melting temperature can be estimated using
the formula T.sub.melt=[2 x (# of A or T)+4 x (# of G or C)].
[0141] A number of other human polymorphic loci occur between these
two CA dinucleotide repeat polymorphisms and provide additional
targets for determination of a restenosis prognostic allele in a
family or other group of genetically related individuals. For
example, the National Center for Biotechnology Information web site
(www.ncbi.nlm.nih.gov/genem- ap/) lists a number of polymorphism
markers in the region of the IL-1 locus and provides guidance in
designing appropriate primers for amplification and analysis of
these markers.
[0142] Accordingly, the nucleotide segments of the invention may be
used for their ability to selectively form duplex molecules with
complementary stretches of human chromosome 2 q 12-13 or cDNAs from
that region or to provide primers for amplification of DNA or cDNA
from this region. The design of appropriate probes for this purpose
requires consideration of a number of factors. For example,
fragments having a length of between 10, 15, or 18 nucleotides to
about 20, or to about 30 nucleotides, will find particular utility.
Longer sequences, e.g., 40, 50, 80, 90, 100, even up to full
length, are even more preferred for certain embodiments. Lengths of
oligonucleotides of at least about 18 to 20 nucleotides are well
accepted by those of skill in the art as sufficient to allow
sufficiently specific hybridization so as to be useful as a
molecular probe. Furthermore, depending on the application
envisioned, one will desire to employ varying conditions of
hybridization to achieve varying degrees of selectivity of probe
towards target sequence. For applications requiring high
selectivity, one will typically desire to employ relatively
stringent conditions to form the hybrids. For example, relatively
low salt and/or high temperature conditions, such as provided by
0.02 M-0.15M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. Such selective conditions may tolerate little, if
any, mismatch between the probe and the template or target
strand.
[0143] Other alleles or other indicia of restenosis can be detected
or monitored in a subject in conjunction with detection of the
alleles described above. For example, echocardiography may be
performed during exercise, since studies have found an association
between the occurrence of clinical restenosis and both a positive
post-percutaneous transluminal coronary angioplasty exercise echo
as well as high values of the pre-surgical wall-motion score index
and duration of wall-motion abnormalities (Peters et al. (1997)
Circulation 95: 2254-61; Dagianti et al. (1997) Circulation 95:
1176-84; Gentile (1994) Cardiologia 39: 651-6). Furthermore,
angioscopic studies have shown that the color (yellow versus white)
of a patient's arterial plaque is highly predictive of the
occurrence of restenosis following balloon angioplasty in
individuals with stable angina (Itoh et al. (1995) Circulation 91:
1389-96). In addition, certain polymorphisms in the gene encoding
angiotensin converting enzyme have been associated with the
occurrence of restenosis after coronary angioplasty in unstable
angina pectoris (See e.g. Kasi et al., (1996) Am J Cardiol 77:
875-77).
[0144] In addition, behavioral studies have shown an association
between hostility and other aspects of a type A behavior pattern
and an increased risk for restenosis following percutaneous
transluminal coronary angioplasty (Goodman et al. (1996) Mayo Clin
Proc 71: 729-34). Still other studies have demonstrated an
association between various serum proteins and an increased
likelihood of restenosis. For example a drop in the level of
antibodies against heat shock protein-65 after percutaneous
transluminal coronary angioplasty is associated with a decreased
risk of developing restenosis relative to individuals in which no
decrease in the level of these antibodies occurred (Mukherjee et
al. (1996) Throm Haemost 75: 258-60). Another study has
demonstrated an association between an elevation of serum amyloid A
and the occurrence of restenosis following angioplasty (Blum et al.
(1998) Clin Cardiol 21: 655-58). Relatively high levels of
plasminogen activator inhibitor type-1 and relatively low levels of
plasmin-plasmin inhibitor complex are also associated with
restenosis (Ishiwata et al. (1997) Am Heart J 133: 387-92), as are
high levels of serum lipoprotein A (Hearn et al. (1992) Am J
Cardiol 69: 736-39) and elevated levels of monounsaturated fatty
acids (Foley et al. (1992) Cathet Cardiovasc Diagn 25: 25-30).
[0145] 4.2.2 Detection of Alleles
[0146] Many methods are available for detecting specific alleles at
human polymorphic loci. The preferred method for detecting a
specific polymorphic allele will depend, in part, upon the
molecular nature of the polymorphism. For example, the various
allelic forms of the polymorphic locus may differ by a single
base-pair of the DNA. Such single nucleotide polymorphisms (or
SNPs) are major contributors to genetic variation, comprising some
80% of all known polymorphisms, and their density in the human
genome is estimated to be on average 1 per 1,000 base pairs. SNPs
are most frequently biallelic-occurring in only two different forms
(although up to four different forms of an SNP, corresponding to
the four different nucleotide bases occurring in DNA, are
theoretically possible). Nevertheless, SNPs are mutationally more
stable than other polymorphisms, making them suitable for
association studies in which linkage disequilibrium between markers
and an unknown variant is used to map disease-causing mutations. In
addition, because SNPs typically have only two alleles, they can be
genotyped by a simple plus/minus assay rather than a length
measurement, making them more amenable to automation.
[0147] A variety of methods are available for detecting the
presence of a particular single nucleotide polymorphic allele in an
individual. Advancements in this field have provided accurate,
easy, and inexpensive large-scale SNP genotyping. Most recently,
for example, several new techniques have been described including
dynamic allele-specific hybridization (DASH), microplate array
diagonal gel electrophoresis (MADGE), pyrosequencing,
oligonucleotide-specific ligation, the TaqMan system as well as
various DNA "chip" technologies such as the Affymetrix SNP chips.
These methods require amplification of the target genetic region,
typically by PCR. Still other newly developed methods, based on the
generation of small signal molecules by invasive cleavage followed
by mass spectrometry or immobilized padlock probes and
rolling-circle amplification, might eventually eliminate the need
for PCR. Several of the methods known in the art for detecting
specific single nucleotide polymorphisms are summarized below. The
method of the present invention is understood to include all
available methods.
[0148] Several methods have been developed to facilitate analysis
of single nucleotide polymorphisms. In one embodiment, the single
base polymorphism can be detected by using a specialized
exonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C.
R. (U.S. Pat. No.4,656,127). According to the method, a primer
complementary to the allelic sequence immediately 3' to the
polymorphic site is permitted to hybridize to a target molecule
obtained from a particular animal or human. If the polymorphic site
on the target molecule contains a nucleotide that is complementary
to the particular exonuclease-resistant nucleotide derivative
present, then that derivative will be incorporated onto the end of
the hybridized primer. Such incorporation renders the primer
resistant to exonuclease, and thereby permits its detection. Since
the identity of the exonuclease-resistant derivative of the sample
is known, a finding that the primer has become resistant to
exonucleases reveals that the nucleotide present in the polymorphic
site of the target molecule was complementary to that of the
nucleotide derivative used in the reaction. This method has the
advantage that it does not require the determination of large
amounts of extraneous sequence data.
[0149] In another embodiment of the invention, a solution-based
method is used for determining the identity of the nucleotide of a
polymorphic site. Cohen, D. et al. (French Patent 2,650,840; PCT
Appln. No. WO91/02087). As in the Mundy method of U.S. Pat. No.
4,656,127, a primer is employed that is complementary to allelic
sequences immediately 3' to a polymorphic site. The method
determines the identity of the nucleotide of that site using
labeled dideoxynucleotide derivatives, which, if complementary to
the nucleotide of the polymorphic site will become incorporated
onto the terminus of the primer.
[0150] An alternative method, known as Genetic Bit Analysis or
GBA.TM. is described by Goelet, P. et al. (PCT Appln. No.
92/15712). The method of Goelet, P. et al. uses mixtures of labeled
terminators and a primer that is complementary to the sequence 3'
to a polymorphic site. The labeled terminator that is incorporated
is thus determined by, and complementary to, the nucleotide present
in the polymorphic site of the target molecule being evaluated. In
contrast to the method of Cohen et al. (French Patent 2,650,840;
PCT Appln. No. WO91/02087) the method of Goelet, P. et al. is
preferably a heterogeneous phase assay, in which the primer or the
target molecule is immobilized to a solid phase.
[0151] Recently, several primer-guided nucleotide incorporation
procedures for assaying polymorphic sites in DNA have been
described (Komher, J. S. et al., Nucl. Acids. Res. 17:7779-7784
(1989); Sokolov, B. P., Nucl. Acids Res. 18:3671 (1990); Syvanen,
A. -C., et al., Genomics 8:684-692 (1990); Kuppuswamy, M. N. et
al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147 (1991); Prezant,
T. R. et al., Hum. Mutat. 1: 159-164 (1992); Ugozzoli, L. et al.,
GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem. 208:171-175
(1993)). These methods differ from GBA.TM. in that they all rely on
the incorporation of labeled deoxynucleotides to discriminate
between bases at a polymorphic site. In such a format, since the
signal is proportional to the number of deoxynucleotides
incorporated, polymorphisms that occur in runs of the same
nucleotide can result in signals that are proportional to the
length of the run (Syvanen, A. -C., et al., Amer. J. Hum. Genet.
52:46-59 (1993)).
[0152] For mutations that produce premature termination of protein
translation, the protein truncation test (PTT) offers an efficient
diagnostic approach (Roest, et. al., (1993) Hum. Mol. Genet.
2:1719-21; van der Luijt, et. al., (1994) Genomics 20:1-4). For
PTT, RNA is initially isolated from available tissue and
reverse-transcribed, and the segment of interest is amplified by
PCR. The products of reverse transcription PCR are then used as a
template for nested PCR amplification with a primer that contains
an RNA polymerase promoter and a sequence for initiating eukaryotic
translation. After amplification of the region of interest, the
unique motifs incorporated into the primer permit sequential in
vitro transcription and translation of the PCR products. Upon
sodium dodecyl sulfate-polyacrylamide gel electrophoresis of
translation products, the appearance of truncated polypeptides
signals the presence of a mutation that causes premature
termination of translation. In a variation of this technique, DNA
(as opposed to RNA) is used as a PCR template when the target
region of interest is derived from a single exon.
[0153] Any cell type or tissue may be utilized to obtain nucleic
acid samples for use in the diagnostics described herein. In a
preferred embodiment, the DNA sample is obtained from a bodily
fluid, e.g, blood, obtained by known techniques (e.g. venipuncture)
or saliva. Alternatively, nucleic acid tests can be performed on
dry samples (e.g. hair or skin). When using RNA or protein, the
cells or tissues that may be utilized must express an IL-1
gene.
[0154] Diagnostic procedures may also be performed in situ directly
upon tissue sections (fixed and/or frozen) of patient tissue
obtained from biopsies or resections, such that no nucleic acid
purification is necessary. Nucleic acid reagents may be used as
probes and/or primers for such in situ procedures (see, for
example, Nuovo, G. J., 1992, PCR in situ hybridization: protocols
and applications, Raven Press, NY).
[0155] In addition to methods which focus primarily on the
detection of one nucleic acid sequence, profiles may also be
assessed in such detection schemes. Fingerprint profiles may be
generated, for example, by utilizing a differential display
procedure, Northern analysis and/or RT-PCR.
[0156] A preferred detection method is allele specific
hybridization using probes overlapping a region of at least one
allele of an IL-1 proinflammatory haplotype and having about 5, 10,
20, 25, or 30 nucleotides around the mutation or polymorphic
region. In a preferred embodiment of the invention, several probes
capable of hybridizing specifically to other allelic variants
involved in a restenosis are attached to a solid phase support,
e.g., a "chip" (which can hold up to about 250,000
oligonucleotides). Oligonucleotides can be bound to a solid support
by a variety of processes, including lithography. Mutation
detection analysis using these chips comprising oligonucleotides,
also termed "DNA probe arrays" is described e.g., in Cronin et al.
(1996) Human Mutation 7:244. In one embodiment, a chip comprises
all the allelic variants of at least one polymorphic region of a
gene. The solid phase support is then contacted with a test nucleic
acid and hybridization to the specific probes is detected.
Accordingly, the identity of numerous allelic variants of one or
more genes can be identified in a simple hybridization
experiment.
[0157] These techniques may also comprise the step of amplifying
the nucleic acid before analysis. Amplification techniques are
known to those of skill in the art and include, but are not limited
to cloning, polymerase chain reaction (PCR), polymerase chain
reaction of specific alleles (ASA), ligase chain reaction (LCR),
nested polymerase chain reaction, self sustained sequence
replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh, D.
Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), and
Q-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology
6:1197).
[0158] Amplification products may be assayed in a variety of ways,
including size analysis, restriction digestion followed by size
analysis, detecting specific tagged oligonucleotide primers in the
reaction products, allele-specific oligonucleotide (ASO)
hybridization, allele specific 5' exonuclease detection,
sequencing, hybridization, and the like.
[0159] PCR based detection means can include multiplex
amplification of a plurality of markers simultaneously. For
example, it is well known in the art to select PCR primers to
generate PCR products that do not overlap in size and can be
analyzed simultaneously. Alternatively, it is possible to amplify
different markers with primers that are differentially labeled and
thus can each be differentially detected. Of course, hybridization
based detection means allow the differential detection of multiple
PCR products in a sample. Other techniques are known in the art to
allow multiplex analyses of a plurality of markers.
[0160] In a merely illustrative embodiment, the method includes the
steps of (i) collecting a sample of cells from a patient, (ii)
isolating nucleic acid (e.g., genomic, mRNA or both) from the cells
of the sample, (iii) contacting the nucleic acid sample with one or
more primers which specifically hybridize 5' and 3' to at least one
allele of an IL-1 proinflammatory haplotype under conditions such
that hybridization and amplification of the allele occurs, and (iv)
detecting the amplification product. These detection schemes are
especially useful for the detection of nucleic acid molecules if
such molecules are present in very low numbers.
[0161] In a preferred embodiment of the subject assay, the allele
of an IL-1 proinflammatory haplotype is identified by alterations
in restriction enzyme cleavage patterns. For example, sample and
control DNA is isolated, amplified (optionally), digested with one
or more restriction endonucleases, and fragment length sizes are
determined by gel electrophoresis.
[0162] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
allele. Exemplary sequencing reactions include those based on
techniques developed by Maxim and Gilbert ((1977) Proc. Natl Acad
Sci USA 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci
USA 74:5463). It is also contemplated that any of a variety of
automated sequencing procedures may be utilized when performing the
subject assays (see, for example Biotechniques (1995) 19:448),
including sequencing by mass spectrometry (see, for example PCT
publication WO 94/16101; Cohen et al. (1996) Adv Chromatogr
36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol
38:147-159). It will be evident to one of skill in the art that,
for certain embodiments, the occurrence of only one, two or three
of the nucleic acid bases need be determined in the sequencing
reaction. For instance, A-track or the like, e.g., where only one
nucleic acid is detected, can be carried out.
[0163] In a further embodiment, protection from cleavage agents
(such as a nuclease, hydroxylamine or osmium tetroxide and with
piperidine) can be used to detect mismatched bases in RNA/RNA or
RNA/DNA or DNA/DNA heteroduplexes (Myers, et al. (1985) Science
230:1242). In general, the art technique of "mismatch cleavage"
starts by providing heteroduplexes formed by hybridizing (labeled)
RNA or DNA containing the wild-type allele with the sample. The
double-stranded duplexes are treated with an agent which cleaves
single-stranded regions of the duplex such as which will exist due
to base pair mismatches between the control and sample strands. For
instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA
hybrids treated with S1 nuclease to enzymatically digest the
mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA
duplexes can be treated with hydroxylamine or osmium tetroxide and
with piperidine in order to digest mismatched regions. After
digestion of the mismatched regions, the resulting material is then
separated by size on denaturing polyacrylamide gels to determine
the site of mutation. See, for example, Cotton et al (1988) Proc.
Natl Acad Sci USA 85:4397; and Saleeba et al (1992) Methods
Enzymol. 217:286-295. In a preferred embodiment, the control DNA or
RNA can be labeled for detection.
[0164] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes).
For example, the mutY enzyme of E. coli cleaves A at G/A mismatches
and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T
mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
According to an exemplary embodiment, a probe based on an allele of
an IL-1 locus haplotype is hybridized to a cDNA or other DNA
product from a test cell(s). The duplex is treated with a DNA
mismatch repair enzyme, and the cleavage products, if any, can be
detected from electrophoresis protocols or the like. See, for
example, U.S. Pat. No. 5,459,039.
[0165] In other embodiments, alterations in electrophoretic
mobility will be used to identify an IL-1 locus allele. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and
Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded DNA
fragments of sample and control IL-1 locus alleles are denatured
and allowed to renature. The secondary structure of single-stranded
nucleic acids varies according to sequence, the resulting
alteration in electrophoretic mobility enables the detection of
even a single base change. The DNA fragments may be labeled or
detected with labeled probes. The sensitivity of the assay may be
enhanced by using RNA (rather than DNA), in which the secondary
structure is more sensitive to a change in sequence. In a preferred
embodiment, the subject method utilizes heteroduplex analysis to
separate double stranded heteroduplex molecules on the basis of
changes in electrophoretic mobility (Keen et al. (1991) Trends
Genet 7:5).
[0166] In yet another embodiment, the movement of alleles in
polyacrylamide gels containing a gradient of denaturant is assayed
using denaturing gradient gel electrophoresis (DGGE) (Myers et al.
(1985) Nature 313:495). When DGGE is used as the method of
analysis, DNA will be modified to insure that it does not
completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing agent gradient to identify differences in the mobility
of control and sample DNA (Rosenbaum and Reissner (1987) Biophys
Chem 265:12753).
[0167] Examples of other techniques for detecting alleles include,
but are not limited to, selective oligonucleotide hybridization,
selective amplification, or selective primer extension. For
example, oligonucleotide primers may be prepared in which the known
mutation or nucleotide difference (e.g., in allelic variants) is
placed centrally and then hybridized to target DNA under conditions
which permit hybridization only if a perfect match is found (Saiki
et al. (1986) Nature 324:163); Saiki et al (1989) Proc. Natl Acad.
Sci USA 86:6230). Such allele specific oligonucleotide
hybridization techniques may be used to test one mutation or
polymorphic region per reaction when oligonucleotides are
hybridized to PCR amplified target DNA or a number of different
mutations or polymorphic regions when the oligonucleotides are
attached to the hybridizing membrane and hybridized with labelled
target DNA.
[0168] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation or
polymorphic region of interest in the center of the molecule (so
that amplification depends on differential hybridization) (Gibbs et
al (1989), Nucleic Acids Res. 17:2437-2448) or at the extreme 3'
end of one primer where, under appropriate conditions, mismatch can
prevent, or reduce polymerase extension (Prossner (1993) Tibtech
11:238. In addition it may be desirable to introduce a novel
restriction site in the region of the mutation to create
cleavage-based detection (Gasparini et al (1992) Mol. Cell Probes
6:1). It is anticipated that in certain embodiments amplification
may also be performed using Taq ligase for amplification (Barany
(1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation
will occur only if there is a perfect match at the 3' end of the 5'
sequence making it possible to detect the presence of a known
mutation at a specific site by looking for the presence or absence
of amplification.
[0169] In another embodiment, identification of the allelic variant
is carried out using an oligonucleotide ligation assay (OLA), as
described, e.g., in U.S. Pat. No. 4,998,617 and in Landegren, U. et
al. ((1988) Science 241:1077-1080). The OLA protocol uses two
oligonucleotides which are designed to be capable of hybridizing to
abutting sequences of a single strand of a target. One of the
oligonucleotides is linked to a separation marker, e.g,.
biotinylated, and the other is detectably labeled. If the precise
complementary sequence is found in a target molecule, the
oligonucleotides will hybridize such that their termini abut, and
create a ligation substrate. Ligation then permits the labeled
oligonucleotide to be recovered using avidin, or another biotin
ligand. Nickerson, D. A. et al. have described a nucleic acid
detection assay that combines attributes of PCR and OLA (Nickerson,
D. A. et al. (1990) Proc. Natl. Acad. Sci. USA 87:8923-27). In this
method, PCR is used to achieve the exponential amplification of
target DNA, which is then detected using OLA.
[0170] Several techniques based on this OLA method have been
developed and can be used to detect alleles of an IL-1 locus
haplotype. For example, U.S. Pat. No. 5,593,826 discloses an OLA
using an oligonucleotide having 3'-amino group and a
5'-phosphorylated oligonucleotide to form a conjugate having a
phosphoramidate linkage. In another variation of OLA described in
Tobe et al. ((1996) Nucleic Acids Res 24: 3728), OLA combined with
PCR permits typing of two alleles in a single microtiter well. By
marking each of the allele-specific primers with a unique hapten,
i.e. digoxigenin and fluorescein, each OLA reaction can be detected
by using hapten specific antibodies that are labeled with different
enzyme reporters, alkaline phosphatase or horseradish peroxidase.
This system permits the detection of the two alleles using a high
throughput format that leads to the production of two different
colors.
[0171] Another embodiment of the invention is directed to kits for
detecting a predisposition for developing a restenosis. This kit
may contain one or more oligonucleotides, including 5' and 3'
oligonucleotides that hybridize 5' and 3' to at least one allele of
an IL-1 locus haplotype. PCR amplification oligonucleotides should
hybridize between 25 and 2500 base pairs apart, preferably between
about 100 and about 500 bases apart, in order to produce a PCR
product of convenient size for subsequent analysis.
[0172] Particularly preferred primers for use in the diagnostic
method of the invention include the following:
6 5' ATG GTT TTA GAA ATC ATC (SEQ ID No. 1) AAG CCT AGG GCA 3' and
5' AAT GAA AGG AGG GGA GGA (SEQ ID No. 2) TGA CAG AAA TGT 3' 5' TGG
CAT TGA TCT GGT TCA (SEQ ID No. 3) TC-3' and 5' GTT TAG GAA TCT TCC
CAC (SEQ ID No. 4) TT-3'; 5' CTC AGG TGT CCT CGA AGA (SEQ ID No. 5)
AAT CAA A 3' and 5' GCT TTT TTG CTG TGA GTC (SEQ ID No. 6) CCG 3';
5'-CTC.AGC.AAC.ACT.CCT.AT-3' (SEQ ID NO. 7) and
5'-TCC.TGG.TCT.GCA.GCT.AA-3'; (SEQ ID NO. 8) 5'-CTA TCT GAG GAA CAA
ACT (SEQ ID NO. 9) AGT AGC-3' and 5'-TAG GAC ATT GCA CCT AGG (SEQ
ID NO. 10) GTT TGT-3'; 5' ATT TTT TTA TAA ATC ATC (SEQ. ID No. 11)
AAG CCT AGG GCA 3' 5' AAT TAA AGG AGG GAA GAA (SEQ. ID No. 12) TGA
CAG AAA TGT 3' 5'-AAG CTT GTT CTA CCA CCT (SEQ ID NO. 13) GAA CTA
GGC.-3' and 5'-TTA CAT ATG AGC CTT CCA (SEQ ID NO. 14) TG.-3';
[0173] The design of additional oligonucleotides for use in the
amplification and detection of IL-1 polymorphic alleles by the
method of the invention is facilitated by the availability of both
updated sequence information from human chromosome 2q13 --which
contains the human IL-1 locus, and updated human polymorphism
information available for this locus. For example, the DNA sequence
for the IL-1A, IL-1B and IL-1RN is shown in FIGS. 1 (GenBank
Accession No. X03833), 2 (GenBank Accession No. X04500) and 3
(GenBank Accession No. X64532) respectively. Suitable primers for
the detection of a human polymorphism in these genes can be readily
designed using this sequence information and standard techniques
known in the art for the design and optimization of primers
sequences. Optimal design of such primer sequences can be achieved,
for example, by the use of commercially available primer selection
programs such as Primer 2.1, Primer 3 or GeneFisher (See also,
Nicklin M. H. J., Weith A. Duff G. W., "A Physical Map of the
Region Encompassing the Human Interleukin-1.alpha.,
interleukin-1.beta., and Interleukin-1 Receptor Antagonist Genes"
Genomics 19: 382 (1995); Nothwang H. G., et al. "Molecular Cloning
of the Interleukin-1 gene Cluster: Construction of an Integrated
YAC/PAC Contig and a partial transcriptional Map in the Region of
Chromosome 2q13" Genomics 41: 370 (1997); Clark, et al. (1986)
Nucl. Acids. Res., 14:7897-7914 [published erratum appears in
Nucleic Acids Res., 15:868 (1987) and the Genome Database (GDB)
project at the URL http://www.gdb.org).
[0174] For use in a kit, oligonucleotides may be any of a variety
of natural and/or synthetic compositions such as synthetic
oligonucleotides, restriction fragments, cDNAs, synthetic peptide
nucleic acids (PNAs), and the like. The assay kit and method may
also employ labeled oligonucleotides to allow ease of
identification in the assays. Examples of labels which may be
employed include radio-labels, enzymes, fluorescent compounds,
streptavidin, avidin, biotin, magnetic moieties, metal binding
moieties, antigen or antibody moieties, and the like.
[0175] The kit may, optionally, also include DNA sampling means.
DNA sampling means are well known to one of skill in the art and
can include, but not be limited to substrates, such as filter
papers, the AmpliCard.TM. (University of Sheffield, Sheffield,
England S10 2JF; Tarlow, JW, et al., J. of Invest. Dermatol.
103:387-389 (1994)) and the like; DNA purification reagents such as
Nucleon.TM. kits, lysis buffers, proteinase solutions and the like;
PCR reagents, such as 10.times. reaction buffers, thermostable
polymerase, dNTPs, and the like; and allele detection means such as
the HinfI restriction enzyme, allele specific oligonucleotides,
degenerate oligonucleotide primers for nested PCR from dried
blood.
[0176] 4.2.3. Pharmacogenomics
[0177] Knowledge of the particular alleles associated with
restenosis, alone or in conjunction with information on other
genetic defects contributing to restenosis, such as the PL(A1/A2)
polymorphism in a platelet glycoprotein (See Abbate et al. (1998)
Am J Cardiol 82: 524-5), allows a customization of the restenosis
therapy to the individual's genetic profile, the goal of
"pharmacogenomics". For example, subjects having an allele 2 of any
of the following markers: IL-1A (+4845), IL-1B (-511), IL-1B
(+3954) or IL-1RN (VNTR) or any nucleic acid sequence in linkage
disequilibrium with any of these alleles may have or be predisposed
to developing restenosis and may respond better to particular
therapeutics that address the particular molecular basis of the
disease in the subject. Thus, comparison of an individual's IL-1
profile to the population profile for restenosis, permits the
selection or design of drugs or other therapeutic regimens that are
expected to be safe and efficacious for a particular patient or
patient population (i.e., a group of patients having the same
genetic alteration).
[0178] In addition, the ability to target populations expected to
show the highest clinical benefit, based on genetic profile can
enable: 1) the repositioning of marketed drugs with disappointing
market results; 2) the rescue of drug candidates whose clinical
development has been discontinued as a result of safety or efficacy
limitations, which are patient subgroup-specific; and 3) an
accelerated and less costly development for drug candidates and
more optimal drug labeling (e.g. since measuring the effect of
various doses of an agent on a restenosis causative mutation is
useful for optimizing effective dose).
[0179] The treatment of an individual with a particular therapeutic
can be monitored by determining protein (e.g. IL-1.alpha.,
IL-1.beta., or IL-1Ra), mRNA and/or transcriptional level.
Depending on the level detected, the therapeutic regimen can then
be maintained or adjusted (increased or decreased in dose). In a
preferred embodiment, the effectiveness of treating a subject with
an agent comprises the steps of: (i) obtaining a preadministration
sample from a subject prior to administration of the agent; (ii)
detecting the level or amount of a protein, mRNA or genomic DNA in
the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the protein, mRNA or genomic DNA
in the post-administration sample; (v) comparing the level of
expression or activity of the protein, mRNA or genomic DNA in the
preadministration sample with the corresponding protein, mRNA or
genomic DNA in the postadministration sample, respectively; and
(vi) altering the administration of the agent to the subject
accordingly.
[0180] Cells of a subject may also be obtained before and after
administration of a therapeutic to detect the level of expression
of genes other than an IL-1 gene to verify that the therapeutic
does not increase or decrease the expression of genes which could
be deleterious. This can be done, e.g., by using the method of
transcriptional profiling. Thus, mRNA from cells exposed in vivo to
a therapeutic and mRNA from the same type of cells that were not
exposed to the therapeutic 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 therapeutic.
4.3 Restenosis Therapeutics
[0181] Modulators of IL-1 (e.g. IL-1.alpha., IL-1p or IL-1 receptor
antagonist) or a protein encoded by a gene that is in linkage
disequilibrium with an IL-1 gene can comprise any type of compound,
including a protein, peptide, peptidomimetic, small molecule, or
nucleic acid. Preferred agonists include nucleic acids (e.g.
encoding an IL-1 protein or a gene that is up- or down-regulated by
an IL-1 protein), proteins (e.g. IL-1 proteins or a protein that is
up- or down-regulated thereby) or a small molecule (e.g. that
regulates expression or binding of an IL-1 protein). Preferred
antagonists, which can be identified, for example, using the assays
described herein, include nucleic acids (e.g. single (antisense) or
double stranded (triplex) DNA or PNA and ribozymes), protein (e.g.
antibodies) and small molecules that act to suppress or inhibit
IL-1 transcription and/or protein activity.
[0182] 4.3.1. Effective Dose
[0183] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining The LD.sub.50 (the
dose lethal to 50% of the population) and the E.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. Compounds
which exhibit large therapeutic indices are preferred. While
compounds that exhibit toxic side effects may be used, care should
be taken to design a delivery system that targets such compounds to
the site of affected tissues in order to minimize potential damage
to uninfected cells and, thereby, reduce side effects.
[0184] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound which achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0185] 4.3.2. Formulation and Use
[0186] Compositions for use in accordance with the present
invention may be formulated in a conventional manner using one or
more physiologically acceptable carriers or excipients. Thus, the
compounds and their physiologically acceptable salts and solvates
may be formulated for administration by, for example, injection,
inhalation or insufflation (either through the mouth or the nose)
or oral, buccal, parenteral or rectal administration.
[0187] For such therapy, the compounds of the invention can be
formulated for a variety of loads of administration, including
systemic and topical or localized administration. Techniques and
formulations generally may be found in Remmington's Pharmaceutical
Sciences, Meade Publishing Co., Easton, Pa. For systemic
administration, injection is preferred, including intramuscular,
intravenous, intraperitoneal, and subcutaneous. For injection, the
compounds of the invention can be formulated in liquid solutions,
preferably in physiologically compatible buffers such as Hank's
solution or Ringer's solution. In addition, the compounds may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included.
[0188] For oral administration, the compositions may take the form
of, for example, tablets or capsules prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulfate). The tablets may be
coated by methods well known in the art. Liquid preparations for
oral administration may take the form of, for example, solutions,
syrups or suspensions, or they may be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., ationd oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations may
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0189] Preparations for oral administration may be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions may take the form of tablets
or lozenges formulated in conventional manner. For administration
by inhalation, the compounds for use according to the present
invention are conveniently delivered in the form of an aerosol
spray presentation from pressurized packs or a nebuliser, with the
use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of e.g., gelatin for use in
an inhaler or insufflator may be formulated containing a powder mix
of the compound and a suitable powder base such as lactose or
starch.
[0190] The compounds may be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection may be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulating agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient may
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use.
[0191] The compounds may also be formulated in rectal compositions
such as suppositories or retention enemas, e.g., containing
conventional suppository bases such as cocoa butter or other
glycerides.
[0192] In addition to the formulations described previously, the
compounds may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt. Other suitable delivery systems include microspheres which
offer the possibility of local noninvasive delivery of drugs over
an extended period of time. This technology utilizes microspheres
of precapillary size which can be injected via a coronary catheter
into any selected part of the e.g. heart or other organs without
causing inflammation or ischemia. The administered therapeutic is
slowly released from these microspheres and taken up by surrounding
tissue cells (e.g. endothelial cells).
[0193] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration bile
salts and fusidic acid derivatives. In addition, detergents may be
used to facilitate permeation. Transmucosal administration may be
through nasal sprays or using suppositories. For topical
administration, the oligomers of the invention are formulated into
ointments, salves, gels, or creams as generally known in the art. A
wash solution can be used locally to treat an injury or
inflammation to accelerate healing.
[0194] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration.
4.4 Assays to Identify Restenosis Therapeutics
[0195] Based on the identification of mutations that cause or
contribute to the development of restenosis, the invention further
features cell-based or cell free assays, e.g., for identifying
restenosis therapeutics. In one embodiment, a cell expressing an
IL-1 receptor, or a receptor for a protein that is encoded by a
gene which is in linkage disequilibrium with an IL-1 gene, on the
outer surface of its cellular membrane is incubated in the presence
of a test compound alone or in the presence of a test compound and
another protein and the interaction between the test compound and
the receptor or between the protein (preferably a tagged protein)
and the receptor is detected, e.g., by using a microphysiometer
(McConnell et al. (1992) Science 257:1906). An interaction between
the receptor and either the test compound or the protein is
detected by the microphysiometer as a change in the acidification
of the medium. This assay system thus provides a means of
identifying molecular antagonists which, for example, function by
interfering with protein-receptor interactions, as well as
molecular agonist which, for example, function by activating a
receptor.
[0196] Cellular or cell-free assays can also be used to identify
compounds which modulate expression of an IL-1 gene or a gene in
linkage disequilibrium therewith, modulate translation of an mRNA,
or which modulate the stability of an mRNA or protein. Accordingly,
in one embodiment, a cell which is capable of producing an IL-1, or
other protein is incubated with a test compound and the amount of
protein produced in the cell medium is measured and compared to
that produced from a cell which has not been contacted with the
test compound. The specificity of the compound vis a vis the
protein can be confirmed by various control analysis, e.g.,
measuring the expression of one or more control genes. In
particular, this assay can be used to determine the efficacy of
antisense, ribozyme and triplex compounds.
[0197] Cell-free assays can also be used to identify compounds
which are capable of interacting with a protein, to thereby modify
the activity of the protein. Such a compound can, e.g., modify the
structure of a protein thereby effecting its ability to bind to a
receptor. In a preferred embodiment, cell-free assays for
identifying such compounds consist essentially in a reaction
mixture containing a protein and a test compound or a library of
test compounds in the presence or absence of a binding partner. A
test compound can be, e.g., a derivative of a binding partner,
e.g., a biologically inactive target peptide, or a small
molecule.
[0198] Accordingly, one exemplary screening assay of the present
invention includes the steps of contacting a protein or functional
fragment thereof with a test compound or library of test compounds
and detecting the formation of complexes. For detection purposes,
the molecule can be labeled with a specific marker and the test
compound or library of test compounds labeled with a different
marker. Interaction of a test compound with a protein or fragment
thereof can then be detected by determining the level of the two
labels after an incubation step and a washing step. The presence of
two labels after the washing step is indicative of an
interaction.
[0199] An interaction between molecules can also be identified by
using real-time BIA (Biomolecular Interaction Analysis, Pharmacia
Biosensor AB) which detects surface plasmon resonance (SPR), an
optical phenomenon. Detection depends on changes in the mass
concentration of macromolecules at the biospecific interface, and
does not require any labeling of interactants. In one embodiment, a
library of test compounds can be immobilized on a sensor surface,
e.g., which forms one wall of a micro-flow cell. A solution
containing the protein or functional fragment thereof is then flown
continuously over the sensor surface. A change in the resonance
angle as shown on a signal recording, indicates that an interaction
has occurred. This technique is further described, e.g., in
BIAtechnology Handbook by Pharmacia.
[0200] Another exemplary screening assay of the present invention
includes the steps of (a) forming a reaction mixture including: (i)
an IL-1 or other protein, (ii) an appropriate receptor, and (iii) a
test compound; and (b) detecting interaction of the protein and
receptor. A statistically significant change (potentiation or
inhibition) in the interaction of the protein and receptor in the
presence of the test compound, relative to the interaction in the
absence of the test compound, indicates a potential antagonist
(inhibitor). The compounds of this assay can be contacted
simultaneously. Alternatively, a protein can first be contacted
with a test compound for an appropriate amount of time, following
which the receptor is added to the reaction mixture. The efficacy
of the compound can be assessed by generating dose response curves
from data obtained using various concentrations of the test
compound. Moreover, a control assay can also be performed to
provide a baseline for comparison.
[0201] Complex formation between a protein and receptor may be
detected by a variety of techniques. Modulation of the formation of
complexes can be quantitated using, for example, detectably labeled
proteins such as radiolabeled, fluorescently labeled, or
enzymatically labeled proteins or receptors, by immunoassay, or by
chromatographic detection.
[0202] Typically, it will be desirable to immobilize either the
protein or the receptor to facilitate separation of complexes from
uncomplexed forms of one or both of the proteins, as well as to
accommodate automation of the assay. Binding of protein and
receptor can be accomplished in any vessel suitable for containing
the reactants. Examples include microtitre plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows the protein to be bound to
a matrix. For example, glutathione-S-transferase fusion proteins
can be adsorbed onto glutathione sepharose beads (Sigma Chemical,
St. Louis, Miss.) or glutathione derivatized microtitre plates,
which are then combined with the receptor, e.g. an .sup.35S-labeled
receptor, and the test compound, and the mixture incubated under
conditions conducive to complex formation, e.g. at physiological
conditions for salt and pH, though slightly more stringent
conditions may be desired. Following incubation, the beads are
washed to remove any unbound label, and the matrix immobilized and
radiolabel determined directly (e.g. beads placed in scintillant),
or in the supernatant after the complexes are subsequently
dissociated. Alternatively, the complexes can be dissociated from
the matrix, separated by SDS-PAGE, and the level of protein or
receptor found in the bead fraction quantitated from the gel using
standard electrophoretic techniques such as described in the
appended examples. Other techniques for immobilizing proteins on
matrices are also available for use in the subject assay. For
instance, either protein or receptor can be immobilized utilizing
conjugation of biotin and streptavidin. Transgenic animals can also
be made to identify agonists and antagonists or to confirm the
safety and efficacy of a candidate therapeutic. Transgenic animals
of the invention can include non-human animals containing a
restenosis causative mutation under the control of an appropriate
endogenous promoter or under the control of a heterologous
promoter.
[0203] The transgenic animals can also be animals containing a
transgene, such as reporter gene, under the control of an
appropriate promoter or fragment thereof. These animals are useful,
e.g., for identifying drugs that modulate production of an IL-1
protein, such as by modulating gene expression. Methods for
obtaining transgenic non-human animals are well known in the art.
In preferred embodiments, the expression of the restenosis
causative mutation is restricted to specific subsets of cells,
tissues or developmental stages utilizing, for example, cis-acting
sequences that control expression in the desired pattern. In the
present invention, such mosaic expression of a protein can be
essential for many forms of lineage analysis and can additionally
provide a means to assess the effects of, for example, expression
level which might grossly alter development in small patches of
tissue within an otherwise normal embryo. Toward this end,
tissue-specific regulatory sequences and conditional regulatory
sequences can be used to control expression of the mutation in
certain spatial patterns. Moreover, temporal patterns of expression
can be provided by, for example, conditional recombination systems
or prokaryotic transcriptional regulatory sequences. Genetic
techniques, which allow for the expression of a mutation can be
regulated via site-specific genetic manipulation in vivo, are known
to those skilled in the art.
[0204] The transgenic animals of the present invention all include
within a plurality of their cells a restenosis causative mutation
transgene of the present invention, which transgene alters the
phenotype of the "host cell". In an illustrative embodiment, either
the cre/loxP recombinase system of bacteriophage P1 (Lakso et al.
(1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or
the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et
al. (1991) Science 251:1351-1355; PCT publication WO 92/15694) can
be used to generate in vivo site-specific genetic recombination
systems. Cre recombinase catalyzes the site-specific recombination
of an intervening target sequence located between loxP sequences
loxP sequences are 34 base pair nucleotide repeat sequences to
which the Cre recombinase binds and are required for Cre
recombinase mediated genetic recombination. The orientation of loxP
sequences determines whether the intervening target sequence is
excised or inverted when Cre recombinase is present (Abremski et
al. (1984) J. Biol. Chem. 259:1509-1514); catalyzing the excision
of the target sequence when the loxP sequences are oriented as
direct repeats and catalyzes inversion of the target sequence when
loxP sequences are oriented as inverted repeats.
[0205] Accordingly, genetic recombination of the target sequence is
dependent on expression of the Cre recombinase. Expression of the
recombinase can be regulated by promoter elements which are subject
to regulatory control, e.g., tissue-specific, developmental
stage-specific, inducible or repressible by externally added
agents. This regulated control will result in genetic recombination
of the target sequence only in cells where recombinase expression
is mediated by the promoter element. Thus, the activation of
expression of the causative mutation transgene can be regulated via
control of recombinase expression.
[0206] Use of the cre/loxP recombinase system to regulate
expression of a causative mutation transgene requires the
construction of a transgenic animal containing transgenes encoding
both the Cre recombinase and the subject protein. Animals
containing both the Cre recombinase and the restenosis causative
mutation transgene can be provided through the construction of
"double" transgenic animals. A convenient method for providing such
animals is to mate two transgenic animals each containing a
transgene.
[0207] Similar conditional transgenes can be provided using
prokaryotic promoter sequences which require prokaryotic proteins
to be simultaneous expressed in order to facilitate expression of
the transgene. Exemplary promoters and the corresponding
trans-activating prokaryotic proteins are given in U.S. Pat. No.
4,833,080.
[0208] Moreover, expression of the conditional transgenes can be
induced by gene therapy-like methods wherein a gene encoding the
transactivating protein, e.g. a recombinase or a prokaryotic
protein, is delivered to the tissue and caused to be expressed,
such as in a cell-type specific manner. By this method, the
transgene could remain silent into adulthood until "turned on" by
the introduction of the transactivator.
[0209] In an exemplary embodiment, the "transgenic non-human
animals" of the invention are produced by introducing transgenes
into the germline of the non-human animal. Embryonal target cells
at various developmental stages can be used to introduce
transgenes. Different methods are used depending on the stage of
development of the embryonal target cell. The specific line(s) of
any animal used to practice this invention are selected for general
good health, good embryo yields, good pronuclear visibility in the
embryo, and good reproductive fitness. In addition, the haplotype
is a significant factor. For example, when transgenic mice are to
be produced, strains such as C57BL/6 or FVB lines are often used
(Jackson Laboratory, Bar Harbor, Me.). Preferred strains are those
with H-2.sup.b, H-2.sup.d or H-2.sup.q haplotypes such as C57BL/6
or DBA/1. The line(s) used to practice this invention may
themselves be transgenics, and/or may be knockouts (i.e., obtained
from animals which have one or more genes partially or completely
suppressed).
[0210] In one embodiment, the transgene construct is introduced
into a single stage embryo. The zygote is the best target for
microinjection. In the mouse, the male pronucleus reaches the size
of approximately 20 micrometers in diameter which allows
reproducible injection of 1-2 pl of DNA solution. The use of
zygotes as a target for gene transfer has a major advantage in that
in most cases the injected DNA will be incorporated into the host
gene before the first cleavage (Brinster et al. (1985) PNAS
82:4438-4442). As a consequence, all cells of the transgenic animal
will carry the incorporated transgene. This will in general also be
reflected in the efficient transmission of the transgene to
offspring of the founder since 50% of the germ cells will harbor
the transgene.
[0211] Normally, fertilized embryos are incubated in suitable media
until the pronuclei appear. At about this time, the nucleotide
sequence comprising the transgene is introduced into the female or
male pronucleus as described below. In some species such as mice,
the male pronucleus is preferred. It is most preferred that the
exogenous genetic material be added to the male DNA complement of
the zygote prior to its being processed by the ovum nucleus or the
zygote female pronucleus. It is thought that the ovum nucleus or
female pronucleus release molecules which affect the male DNA
complement, perhaps by replacing the protamines of the male DNA
with histones, thereby facilitating the combination of the female
and male DNA complements to form the diploid zygote. Thus, it is
preferred that the exogenous genetic material be added to the male
complement of DNA or any other complement of DNA prior to its being
affected by the female pronucleus. For example, the exogenous
genetic material is added to the early male pronucleus, as soon as
possible after the formation of the male pronucleus, which is when
the male and female pronuclei are well separated and both are
located close to the cell membrane. Alternatively, the exogenous
genetic material could be added to the nucleus of the sperm after
it has been induced to undergo decondensation. Sperm containing the
exogenous genetic material can then be added to the ovum or the
decondensed sperm could be added to the ovum with the transgene
constructs being added as soon as possible thereafter.
[0212] Introduction of the transgene nucleotide sequence into the
embryo may be accomplished by any means known in the art such as,
for example, microinjection, electroporation, or lipofection.
Following introduction of the transgene nucleotide sequence into
the embryo, the embryo may be incubated in vitro for varying
amounts of time, or reimplanted into the surrogate host, or both.
In vitro incubation to maturity is within the scope of this
invention. One common method in to incubate the embryos in vitro
for about 1-7 days, depending on the species, and then reimplant
them into the surrogate host.
[0213] For the purposes of this invention a zygote is essentially
the formation of a diploid cell which is capable of developing into
a complete organism. Generally, the zygote will be comprised of an
egg containing a nucleus formed, either naturally or artificially,
by the fusion of two haploid nuclei from a gamete or gametes. Thus,
the gamete nuclei must be ones which are naturally compatible,
i.e., ones which result in a viable zygote capable of undergoing
differentiation and developing into a functioning organism.
Generally, a euploid zygote is preferred. If an aneuploid zygote is
obtained, then the number of chromosomes should not vary by more
than one with respect to the euploid number of the organism from
which either gamete originated.
[0214] In addition to similar biological considerations, physical
ones also govern the amount (e.g., volume) of exogenous genetic
material which can be added to the nucleus of the zygote or to the
genetic material which forms a part of the zygote nucleus. If no
genetic material is removed, then the amount of exogenous genetic
material which can be added is limited by the amount which will be
absorbed without being physically disruptive. Generally, the volume
of exogenous genetic material inserted will not exceed about 10
picoliters. The physical effects of addition must not be so great
as to physically destroy the viability of the zygote. The
biological limit of the number and variety of DNA sequences will
vary depending upon the particular zygote and functions of the
exogenous genetic material and will be readily apparent to one
skilled in the art, because the genetic material, including the
exogenous genetic material, of the resulting zygote must be
biologically capable of initiating and maintaining the
differentiation and development of the zygote into a functional
organism.
[0215] The number of copies of the transgene constructs which are
added to the zygote is dependent upon the total amount of exogenous
genetic material added and will be the amount which enables the
genetic transformation to occur. Theoretically only one copy is
required; however, generally, numerous copies are utilized, for
example, 1,000-20,000 copies of the transgene construct, in order
to insure that one copy is functional. As regards the present
invention, there will often be an advantage to having more than one
functioning copy of each of the inserted exogenous DNA sequences to
enhance the phenotypic expression of the exogenous DNA
sequences.
[0216] Any technique which allows for the addition of the exogenous
genetic material into nucleic genetic material can be utilized so
long as it is not destructive to the cell, nuclear membrane or
other existing cellular or genetic structures. The exogenous
genetic material is preferentially inserted into the nucleic
genetic material by microinjection. Microinjection of cells and
cellular structures is known and is used in the art.
[0217] Reimplantation is accomplished using standard methods.
Usually, the surrogate host is anesthetized, and the embryos are
inserted into the oviduct. The number of embryos implanted into a
particular host will vary by species, but will usually be
comparable to the number of off spring the species naturally
produces.
[0218] Transgenic offspring of the surrogate host may be screened
for the presence and/or expression of the transgene by any suitable
method. Screening is often accomplished by Southern blot or
Northern blot analysis, using a probe that is complementary to at
least a portion of the transgene. Western blot analysis using an
antibody against the protein encoded by the transgene may be
employed as an alternative or additional method for screening for
the presence of the transgene product. Typically, DNA is prepared
from tail tissue and analyzed by Southern analysis or PCR for the
transgene. Alternatively, the tissues or cells believed to express
the transgene at the highest levels are tested for the presence and
expression of the transgene using Southern analysis or PCR,
although any tissues or cell types may be used for this
analysis.
[0219] Alternative or additional methods for evaluating the
presence of the transgene include, without limitation, suitable
biochemical assays such as enzyme and/or immunological assays,
histological stains for particular marker or enzyme activities,
flow cytometric analysis, and the like. Analysis of the blood may
also be useful to detect the presence of the transgene product in
the blood, as well as to evaluate the effect of the transgene on
the levels of various types of blood cells and other blood
constituents.
[0220] Progeny of the transgenic animals may be obtained by mating
the transgenic animal with a suitable partner, or by in vitro
fertilization of eggs and/or sperm obtained from the transgenic
animal. Where mating with a partner is to be performed, the partner
may or may not be transgenic and/or a knockout; where it is
transgenic, it may contain the same or a different transgene, or
both. Alternatively, the partner may be a parental line. Where in
vitro fertilization is used, the fertilized embryo may be implanted
into a surrogate host or incubated in vitro, or both. Using either
method, the progeny may be evaluated for the presence of the
transgene using methods described above, or other appropriate
methods.
[0221] The transgenic animals produced in accordance with the
present invention will include exogenous genetic material. Further,
in such embodiments the sequence will be attached to a
transcriptional control element, e.g., a promoter, which preferably
allows the expression of the transgene product in a specific type
of cell.
[0222] Retroviral infection can also be used to introduce the
transgene into a non-human animal. The developing non-human embryo
can be cultured in vitro to the blastocyst stage. During this time,
the blastomeres can be targets for retroviral infection (Jaenich,
R. (1976) PNAS 73:1260-1264). Efficient infection of the
blastomeres is obtained by enzymatic treatment to remove the zona
pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, 1986). The viral
vector system used to introduce the transgene is typically a
replication-defective retrovirus carrying the transgene (Jahner et
al. (1985) PNAS 82:6927-6931; Van der Putten et al. (1985) PNAS
82:6148-6152). Transfection is easily and efficiently obtained by
culturing the blastomeres on a monolayer of virus-producing cells
(Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
Alternatively, infection can be performed at a later stage. Virus
or virus-producing cells can be injected into the blastocoele
(Jahner et al. (1982) Nature 298:623-628). Most of the founders
will be mosaic for the transgene since incorporation occurs only in
a subset of the cells which formed the transgenic non-human animal.
Further, the founder may contain various retroviral insertions of
the transgene at different positions in the genome which generally
will segregate in the offspring. In addition, it is also possible
to introduce transgenes into the germ line by intrauterine
retroviral infection of the midgestation embryo (Jahner et al.
(1982) supra).
[0223] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984)
Nature 309:255-258; Gossler et al. (1986) PNAS 83: 9065-9069; and
Robertson et al. (1986) Nature 322:445-448). Transgenes can be
efficiently introduced into the ES cells by DNA transfection or by
retrovirus-mediated transduction. Such transformed ES cells can
thereafter be combined with blastocysts from a non-human animal.
The ES cells thereafter colonize the embryo and contribute to the
germ line of the resulting chimeric animal. For review see
Jaenisch, R. (1988) Science 240:1468-1474.
[0224] The present invention is further illustrated by the
following examples which should not be construed as limiting in any
way. The contents of all cited references (including literature
references, issued patents, published patent applications as cited
throughout this application) are hereby expressly incorporated by
reference. The practice of the present invention will employ,
unless otherwise indicated, conventional techniques that are within
the skill of the art. Such techniques are explained fully in the
literature. See, for example, Molecular Cloning A Laboratory
Manual, (2nd ed., Sambrook, Fritsch and Maniatis, eds., Cold Spring
Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D.
N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed.,
1984); U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; and
Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.,
1984).
5. EXAMPLES
5.1. IL-1RN*2 Allele Association with Decreased Risk of
Restenosis
[0225] In this example, DNA samples collected form 171 patients
attending for elective percutaneous transluminal coronary
angioplasty were studied at 4 and 6 months post-surgery using
angiography. At follow-up angiography, the patients were separated
into restenosers (>50% luminal narrowing) and non-restenosers
(<50% luminal narrowing), and were further assessed for their
genotype at the following IL-1 polymorphisms: IL-1A (-889), IL-1B
(-511), IL-1B (+3954), IL-1RN (intron 2 VNTR).
Methods
[0226] Patients
[0227] 171 patients who were scheduled to undergo follow-up
angiography after elective PTCA without stenting as part of other
protocols were studied. Quantitive coronary angiography was
performed on-line (Philips Integris HM 3000, (S); Siemens Micor
(L)). Patients were electively recruited in Sheffield where
follow-up angiography was performed at 6 months. 117 patients were
recruited from Leicester. These patients had been part of the SHARP
study (Subcutaneous Heparin and Angioplasty Restenosis Prevention)
where follow-up had been performed at 4 months .+-.2 weeks (Samani
NJ, et al., Lancet. 1995;345:1013-1016), and 67% of the original
cohort were electively recalled for the current study. The SHARP
study did not show any effect of subcutaneous heparin upon rates of
restenosis.
[0228] A dichotomous definition of restenosis was used setting
restenosis as a luminal narrowing >50% and non-restenosis
<50%, at follow-up angiography. Using this definition, the
cohort comprised 39% restenosers and 61% non-restenosers.
[0229] These studies were approved by the North Sheffield Ethics
Committee and by the Leicester Ethics Committee, and patients gavie
their written informed consent.
[0230] Analysis of genetic polymorphisms testing for significant
difference between the medians of the different genotype (or
carriage) groups. This may be done via a Mann-Whitney test (for 2
groups), or a Kruskall-Wallis (for>2 groups), although several
other tests also exist. In exactly the same way, this type of
analysis may also be performed solely within the disease group.
[0231] For use of qualitative traits in studies employing more than
one IL-1 polymorphic locus, the simple one locus case-control
analysis can be extended to one involving several loci (given a
sufficient sample size). In a similar way, a larger contingency
table can be calculated, with groups corresponding now to composite
genotypes. As before, a chi-squared statistic can be calculated.
With these large contingency tables, it is likely that the validity
of the chi-square test is violated (<80% of expected values
>5, and expected values <1). With smaller contingency tables,
the usual remedy to violations of validity is to use Fishers Exact
test, but in this larger case, it is not viable. Instead a null
distribution for the evaluated chi-square statistic is simulated,
and significance assessed from this. This test has been named the
Monte Carlo Composite Genotype (MCCG) test.
5.12 Example 12
[0232] In this example, haplotype relative risk (HRR) analysis is
discussed. This analysis is only suitable for qualitative traits
(quantitative traits may be used if, dichotomised), and as with all
association tests, the candidate gene approach. Haplotype analysis
investigates the association between specific genetic markers for
diseases and the way a set of markers may influence the outcome of
the disease. Analyzing the relationship between specific genetic
markers and disease is an extremely complex process. The analysis
needs to take into account (i) the relation between genetic markers
in neighboring genes, (ii) the way the polymorphic markers affect
expression of the gene in question, (iii) the distribution of the
genetic markers for a specific polymorphism over both chromosomes,
and (iv) the way the expressed gene product(s) affect the disease
process.
[0233] The relationship between these factors can be identified by
statistical equations that look at multipoint linkage analysis,
transmission/disequilibrium test (TDT), multipoint quantitative
trait loci (QTL) analysis, identity-by-state (IBS),
identity-by-descent (IBD), and grouping of multiallelic markers for
biological functions related to disease. This approach has been
described by Camp ((1997) American Journal of Human Genetics 61:
1424-30); Cox et al ((1998) American Journal of Human Genetics 62:
1180-88); and Almasy and Blangero ((1998) American Journal of Human
Genetics 62: 1198-1211).
[0234] Genomic DNA was extracted from whole blood using standard
methods and PCR for variants within the IL-1 locus performed as
previously described (Francis SE, et al. Circulation
1999;99:861-866) or using an automated Taqman.TM. FRET-based
system. The less common IL-RN gene variant is referred to as
IL-1RN.*2.
[0235] Differences in genotype distribution were assessed by
chi-square analysis of the relevant 2*2 contingency table (table
2). Odds ratios with 95% confidence intervals were also calculated.
To summarize results over the Leicester and Sheffield cohorts
Mantel Haenszel analyses were performed. A p-value of less than
0.05 was used to indicate nominal significance. For an overall type
1 error of 0.05, a corrected critical p-value of 0.013 should be
used to account for muliple testing. Here we have corrected
accounting for the 4 loci tested. However, due to linkage
disequilibrium between these loci, this correction is likely to be
conservative. IL-1RN (VNTR) was collapsed and analysed as a
biallelic marker since very few genotypes were recorded with the
rarer alleles. Neither of the cohorts studied were significantly
different from the Hardy Weinberg Equilibrium for any of the
polymorphisms.
[0236] Demographic data were expressed as percent with actual
counts in parentheses. These variables were compared by .chi..sup.2
test.
Results
[0237] Demographics
[0238] The Sheffield and Leicester combined cohorts were well
matched for baseline clinical features (Table 1).
[0239] Genetic analysis
[0240] The Mantel-Haenzel results summarized over the Leicester and
Sheffield cohorts showed no significant differences in genotypic
distributions at the IL-1A (-889), IL-1B (+3954) and IL-1B (-511)
loci between restenosers and non-restenosers (Table II).
[0241] The frequency of allele 2 (IL-1RN *2) was however increased
in the non-restenosers: 34% versus 23% in restenosers (FIG. 1,
Table II). Genotype distribution analysis indicated a significant
association between homozygosity for allele *2 and non-restenosis
(MH, p=0.0196 (L+S); p=0.0131 (L+S, SVD only, Table III)). When the
populations are analysed separately, the data trends concur, but
are only significant in the Sheffield SVD cohort (p=0.0384 (S);
p=0.1573 (L)). This is most likely because of the low statistical
power of these tests, since sample sizes are small due to data
subdivision.
[0242] Interestingly, and a further implication that the results
are more specifically applicable to SVD only, when carriage of
IL-1RN*2 is compared between SVD and MVD groups in the Leicester
cohort, there is a significant increase of carriage of IL-RN*2 in
the SVD group (p=0.0342). This result is strengthened when the
Sheffield SVD patients are added (p=0.0314).
Discussion
[0243] These data suggest a genetic susceptibility to restenosis
mediated by polymorphism at the IL-1 locus.
[0244] Specifically, the data presented here indicate that IL-1RN*2
is associated with a lower restenosis rate in patients with SVD.
This supports previous data indicating that distinct populations
with different prepensitites to restenosis exist, and that the
precess is at least to some extent patient-related rather than
lesion dependent or both (Lehmann K G, et al. Circulation,
1996;93:1123-1132; Weintraub W S, et al. Am J Cardiol.
1993;72:1107-1113). Our previous data (Francis S E, et al.
Circulation 1999;99:861-866)., that IL-1RN*2 is associated with SVD
on the basis of angiography, led us to speculate that there may be
a true genetic distinction between SVD and MVD. If so, this might
indicate that IL-1RN*2 genotype could either lead more rapidly to
SVD or protest against progression to MVD. The data presented here
add to this.
[0245] Since restenosis is a biological phenomenon characterized by
an early inflammatory response, these new data suggest that
IL-1RN*2 may modulate the arterial wall response to injury in such
a way as to reduce the likelihood of restenosis. Whilst there are
many potential mechanisms by which this could occur, a protection
or beneficial effect of IL-1RN*2 upon vessel wall healing in
response to injury is suggested. This might also support the
hypothesis that IL-1RN*2 slows progression toward MVD made in our
earlier study (Francis S E, et al. Circulation
1999;99:861-866).
[0246] The mechanism by which IL-1RN*2 modulates the vessel wall
response to injury is unclear. This polymorphism has functional
correlates but these appear highly cell-type specific. In
monocytes, IL-1RN*2 is associated with increased IL-1ra production
under basal and stimulated conditions (Wilkinson R J, et al. J Exp
Med. 1999;189:1863-1873). In contrast, within cells of the columnar
epithelium in inflammatory bowel disease (Carter M J,
Gastroenterology. 1998;114(4):3882), and in endothelial cells
(Dewberry R M, et al. Heart. 1999;81(Suppl 1); 78 [abstract]),
IL-1RN*2 os associated with reduced production of IL-1ra. Since the
inflammatory influx seen following experimental PTCA in pigs is
highly neutrophilic and IL-1B staining abundant, predominantly in
the luminal endothelium even into the late phase of healing
(Chamberlin J, et al. Cardiovasc Res. 1999;44(1):156-165), we
speculate that the relatively pro-inflammatory endothelial cell
phenotype created by the IL-1RN*2 genotype may be important to
PTCA. This suggests that modifying the inflammatory response at the
time of injury may indeed be beneficial acting to limit the healing
response that leads to luminal re-narrowing.
[0247] The IL-1RN VNTR polymorphism is known to be in linkage
disequilibrium with other genes in the IL-1 locus (Cox A, et al. Am
J Hum Genet. 1998;62(5):1180-1188), and although there are some
weakly consistent trends which exist for IL-1A (=4845) and IL-1B
(+3954), there are no other significant associations with
restenosis or non-restenosis for the other IL-1 polymorphisms
within the cluster. Hence, a specific complex haplotype is not
supported by these data. However, linkage disequilibrium between
this polymorphism and other unidentified gene polymorphisms cannot
be excluded.
[0248] Due to sub-division of the data, this study has small sample
sizes for many of the analyses performed. This reduces power and to
some extent the reliability and confidence in these findings.
However, the results here are strengthened by the fact that two
separate cohorts were collected, and that very similar directional
trends were found in both populations. It was consistently found
that evidence for association was strengthened by summarizing over
the two cohorts, which further illustrates the concordance. It is,
of course, possible that spurious results could have arisen due to
genetic admixture within the cohorts, but again the consistence
between the two populations argues away from this.
[0249] We favor the interpretation that polymorphic variation
within the IL-1 locus has an important inpact on arterial disease.
Our original published work (Francis S E, et al. Circulation
1999;99:861-866), showed an association with single vessel coronary
disease in two independent populations (Sheffield and London). The
study reported here shows association with a different clinical
phenotype in a population predominantly from Leicester. Other
investigators have demonstrated association between IL-1RN+2016, a
single nucleotide polymorphism (SNP) in linkage disequilibrium with
IL-1RN*2, and carotid intimal/medial changes in African Americans
(Pankow J S, et al. Association of Interleukin-1 gene variants and
carotid arterial wall thickness: the ARID Study. 71st EAS Congress
and Satellite Symposia) These all argue strongly that polymorphism
within the IL-1 locus does have an impact on the pathogenesis of
atherosclerotic lesions, although the mechanism remains to be
elucidated.
[0250] The biological control of IL-1 is complex (Dinarello C A.
Blood. 1991;77:1627-1632). IL-1 actions are inhibited by a
non-signaling receptor IL-1 RII in membrane bound or soluble form
and also by IL-1ra (Symons J A, et al. J Exp Med. 1991;177:557-560)
which binds without agonist activity to be signaling receptor
IL-1RI (Symons J A, et al. Proc Natl Acad Sci. 1995;92:1714-1718).
IL-1ra is an acute phase protein and induced by cytokines and
bacterial products (Arend W P. Adv Immunol. 1993;54:167-227).
Levels of IL-1 and IL-1ra in vivo vary in parallel suggesting a
coordinated pattern of regulation (Arend W P. Adv Immunol.
1993;54:167-227). IL-1ra is detected in the endothelium of diseased
coronary arteries (Dewberry R M, et al. Heart. 1999;81(Suppl 1); 78
[abstract]) and inhibits fatty streak formation in the
apolipoprotein E deficient mouse (Hirsch E, et al. Proc Natl Acad
Sci. 1996;93:11008-11013). These data taken together strongly
implicate IL-1ra in the control of inflammation in the arterial
wall.
[0251] In conclusion, the results reported here suggest an
important association between IL-1RN*2 and protection from
restenosis in individuals with SVD. They also might suggest that
inflammation may be a positive influence rather than wholly
negative after arterial injury. Validation studies in larger study
groups including a post-stenting and a reappraisal of the complex
injury-repair mechanisms employed by the arterial wall are
indicated.
7TABLE I Clinical Characteristics of Patients with and without
Restenosis Restenosis Non-restenosis P Leicester no. of patients 49
69 age (yrs) mean .+-. SEM 59.08 .+-. 1.19 57.08 .+-. 0.91 ns Women
(%) 12.2 [6] 17.4 [12] nd Hypertnesion (%) 24 [12] 17.3 [12] nd
Smoking (%) 29 [14] 34.7 [24] nd Diabetes (%) 2.04 [1] 4.34 [3] nd
MI (%) 48.9 [24] 43.4 [30] nd Multivessel 48.9 [24] 39.1 [27] nd
disease (%) Sheffield no. of patients 18 35 age (yrs) mean .+-. SEM
53.61 .+-. 1.77 53.88 .+-. 1.45 ns Women (%) 17 [3] 11.4 [4] nd
Hypertension (%) 61.1 [11] 37.1 [13] nd Smoking (%) 77.7 [14] 74.2
[26] nd Diabetes (%) 5.5 [1] 11.4 [4] nd MI (%) 57.1 [8] 42.8 [15]
nd Multivessel 0 0 nd disease (%) Sheffield and Leicester no. of
patients 67 104 age (yrs) mean .+-. SEM 57.97 .+-. 1.44 55.98 .+-.
0.98 ns Women (%) 13.4 [9] 15.3 [16] ns Hypertension (%) 34.3 [23]
24.0 [25] ns Smoking (%) 41.7 [28] 48.0 [50] ns Diabetes (%) 2.98
[2] 6.7 [7] ns MI (%) 47.7 [32] 43.2 [45] ns Multivessel 35.8 [24]
25.9 [27] ns disease (%)
[0252] Values in parentheses are the number of patients affected in
that cohort.
[0253] Hypertension defined as diastolic bp>95 mmHg (Leicester);
Sytolic bp>160 mmHg.
[0254] Smoking: current or former (Sheffield), current
(Leicester).
[0255] ns--not significant, where normal statistical significance,
P<0.05.nd--not done.
8TABLE II Carriage of alleles within the IL-1 locus in Sheffield
and Leicester restenosis and non-restenosis cohorts. 11 12/22 11
12/22 11/12 22 11/12 22 Leicester SVD & MVD restenosis 20 24 26
19 36 7 46 3 non 34 27 42 19 47 12 58 10 p-value 0.2399 0.2982
0.6194 0.6191 OR 1.6 1.5 1.3 2.6 95% CI 0.7, 3.6 0.7, 3.3 0.5, 3.7
0.7, 10.2 Sheffield SVD only restenosis 8 10 10 8 15 3 16 0 non 20
14 22 11 29 4 25 7 p-value 0.4329 0.3244 0.6691 0.0384 OR 1.6 1.8
0.7 N/A 95% CI 0.5, 5.2 0.6, 5.7 01., 3.5 N/A MH p-value 0.1604
0.1594 0.8333 0.0196
[0256] MH Mantel-Haenzsel summary statistic
[0257] N/A OR and p-value not applicable since one of the values in
the contingency table is 0.
[0258] Note: Alleles are grouped according to previously described
commonest haplotype (Cox A, et al. Am J Hum Genet.
1998;62(5):1180-1188), carriage of *2 for IL-1A [+4845]; IL1 B
[3954] and carriage of *1 for IL-1B [-511] and IL-1Rn [VNTR].
9TABLE III Homozygosity at IL-1RN*2 illustrates the difference
between SVD and MVD in the Sheffield and Leicester cohorts. SVD MVD
11/12 22 11/12 22 Leicester restenosis 24 1 22 2 non 35 7 23 3
p-value 0.1573 0.7699 OR 4.8 1.4 95% CI 0.6, 41.6 0.1, 4.6
Sheffield restenosis 16 0 N/A non 25 7 p-value 0.0384 OR N/A 95% CI
MH p-value 0.0131
[0259] MH Mantel Naenszel summary statistic
[0260] N/A OR and p-value not applicable since one of the values in
the contingency table is 0.
5.2. Protective Role Against Restenosis from an Interleukin-1
Receptor Antagonist Gene Polymorphism in Patients Treated with
Coronary Stenting (The Munich Study)
[0261] Patients
[0262] The study included 1850 consecutive Caucasian patients with
symptomatic coronary artery disease who underwent coronary stent
implantation at Deutsches Herzzentrum Munchen and 1. Medizinische
Klinik rechts der Isar der Technischen Universitt Munchen. All
patients were scheduled for angiographic follow-up at 6 months. All
patients participating in this study gave written informed consent
for the intervention, follow-up angiography, and genotype
determination. The study protocol conformed to the Declaration of
Helsinki and was approved by the institutional ethics
committee.
10TABLE 3 Baseline clinical characteristics. IL-1RN 1/2 or 2/2
IL-1RN 1/1 (n = 896) (n = 954) P Age - yr 63.4 .+-. 10.0 62.6 .+-.
10.0 0.11 Women - % 22.4 19.9 0.19 Arterial hypertension - % 67.2
68.9 0.44 Diabetes - % 22.7 19.4 0.08 Current or former smoker - %
38.7 41.2 0.28 Elevated total cholesterol - % 42.5 43.1 0.81 Acute
myocardial infarction - % 20.3 20.2 0.97 Unstable angina - % 27.9
27.8 0.95 Prior bypass surgery - % 10.6 11.5 0.53 Reduced left
ventricular function - % 31.3 27.7 0.09 Number of diseased coronary
vessels 0.39 1 vessel - % 29.2 27.3 2 vessels - % 32.9 31.9 3
vessels - % 37.8 40.9 Periprocedural abciximab therapy - % 19.8
19.6 0.93 Data are proportions or mean SD
[0263] The protocol of stent placement and poststenting therapy is
familiar to practitioners in the arts. Most of the stents were
implanted hand-mounted on conventional angioplasty balloons.
Postprocedural therapy consisted of aspirin (100 mg twice daily,
indefinitely) and ticlopidine (250 mg twice daily for 4 weeks).
Patients with suboptimal results due to residual thrombus or
dissection with flow impairment after stent implantation received
additional therapy with abciximab given as bolus injection during
stent insertion procedure and as a 12-hours continuous infusion
thereafter. The decision to give abciximab was taken at the
operator's discretion.
[0264] Determination of the IL-1RN Genotype
[0265] Genomic DNA was extracted from 200 ml of peripheral blood
leukocytes with the QIAamp Blood Kit (Qiagen, Hilden, Germany) and
the High Pure PCR Template Preparation Kit (Boehringer Mannheim,
Mannheim, Germany).
[0266] IL-1RN genotyping was performed with the ABI Prism Sequence
Detection System (PE Applied Biosystems, Weiterstadt, Germany). The
use of allele-specific fluorogenic probes in the 5' nuclease
reaction combines DNA amplification and genotype determination into
a single assay 33. IL-1RN (+2018), a single base pair polymorphism
in exon 2, was the polymorphism typed for this study 26. The
nucleotide sequences of primers and probes were as follows: forward
primer 5' GGG ATG TTA ACC AGA AGA CCT TCT ATC T 3'(SEQ ID NO. 22),
reverse primer 5' CAA CCA CTC ACC TTC TAA ATT GAC ATT 3' (SEQ ID
NO. 23), allele 1 probe 5' AAC AAC CAA CTA GTT GCT GGA TAC TTG CAA
3'(SEQ ID NO. 24), allele 2 probe 5' ACA ACC AAC TAG TTG CCG GAT
ACT TGC 3'(SEQ ID NO. 25). The probes for allele 1 were labeled
with the fluorescent dye 6-carboxy-fluorescein (FAM) and for allele
2 with the fluorescent dye tetrachloro-6-carboxy-fluorescein (TET)
at the 5' end. Both probes were labeled with the quencher
6-carboxy-tetramethyl-rhodamine (TAMRA) at their 3' ends. The
thermocycling protocol consisted of 40 cycles of denaturation at 95
C for 15 seconds and annealing/extension at 64 C for 1 minute.
Genotype validation was performed by repeating the determination in
20% of the patients using a duplicate DNA sample with a novel
subject code unrelated to the original subject code. There was a
100% matching between the 2 results.
[0267] Angiographic Assessment
[0268] Coronary lesions were classified according to the modified
American College of Cardiology/American Heart Association grading
system. Left ventricular function was assessed qualitatively on the
basis of biplane angiograms using a 7 segment division; the
diagnosis of reduced left ventricular function was established in
the presence of at least two hypokinetic segments in the contrast
angiogram. Quantitative computer-assisted angiographic analysis was
performed off-line on angiograms obtained just before stenting,
immediately after stenting, and at follow up using the automated
edge-detection system CMS (Medis Medical Imaging Systems, Nuenen,
The Netherlands). Operators were unaware of the patient's IL-1RN
genotype. Identical projections of the target lesion were used for
all assessed angiograms. Minimal lumen diameter, interpolated
reference diameter, diameter stenosis, lesion length and diameter
of the maximally inflated balloon were the angiographic parameters
obtained with this analysis system. Acute lumen gain was calculated
as the difference between minimal lumen diameter at the end of
intervention and minimal lumen diameter before the intervention.
Late lumen loss was calculated as the difference between minimal
lumen diameter at the end of intervention and minimal lumen
diameter at the time of follow-up angiography. Loss index was
calculated as the ratio between late lumen loss and acute lumen
gain.
Definitions and Study Endpoints
[0269] Primary endpoint of the study was restenosis. Two measures
of restenosis were assessed: the incidence of angiographic
restenosis defined as a diameter stenosis of 50% at 6-month
follow-up angiography, and the need for target vessel
revascularization (PTCA or aortocoronary bypass surgery [CABG]) due
to symptoms or signs of ischemia in the presence of angiographic
restenosis at the stented site over 1 year after the intervention.
Other major adverse events evaluated were: death from any cause and
myocardial infarction. All deaths were considered due to cardiac
causes unless an autopsy established a noncardiac cause. The
diagnosis of acute myocardial infarction was based on the criteria
applied in the EPISTENT trial (new pathological Q waves or a value
of creatine kinase [CK] or its MB isoenzyme at least 3 times the
upper limit) 35. CK was determined systematically over the 48 hours
following stenting procedure. Clinical events were monitored
throughout the 1-year follow-up period. The assessment was made on
the basis of the information provided by hospital readmission
records, referring physician or phone interview with the patient.
For all those patients who revealed cardiac symptoms during the
interview, at least one clinical and electrocardiographic check-up
was performed at the outpatient clinic or by the referring
physician.
Statistical Analysis
[0270] Discrete variables are expressed as counts or percentages
and compared with Chi-square or Fisher's exact test, as
appropriate. Continuous variables are expressed as mean SD and
compared by means of the unpaired, two-sided t-test or analysis of
variance for more than 2 groups. Risk analysis was performed
calculating the odds ratio and the 95% confidence interval. The
main analysis consisted in comparing combined heterozygous and
homozygous carriers of the IL-11RN*2 allele with homozygous
carriers of the IL-11RN*1 allele. Moreover, the association between
IL-1RN genotype and restenosis was assessed in a multivariate
logistic regression model including also those clinical and
lesion-related characteristics for which the comparison between
carriers and noncarriers of the IL-1RN*2 allele showed a P-value
0.30. In this multivariate model, we tested for the possible
interaction between IL-1RN genotype and age. Since the relative
contribution of genetic factors to multifactorial processes such as
restenosis may decrease with the age, we carried out an additional
analysis for a prespecified subgroup of patients<60 years.
Successively, we used test for trend for assessing gene dose
effect, i.e. a stepwise increasing phenotypic response with the
presence of 0, 1 or 2 putative alleles. Statistical significance
was accepted for P-values 0.05.
Results
[0271] Patients Characteristics
[0272] The observed IL-1RN genotypes in the study population were
1/1 in 954 (51.6%), 1/2 in 742 (40.1%) and 2/2 in 154 (8.3%). Thus,
allele 2 frequency was 0.28. The observed distribution complied
with Hardy-Weinberg equilibrium. Main baseline characteristics of
the patients are listed in Table 6 and compared between carriers
and noncarriers of the IL-1RN*2 allele. There was a trend to a
higher frequency of diabetes and reduced left ventricular function
among carriers of the IL-1RN*2 allele. The other characteristics
were evenly distributed between the 2 groups. The angiographic and
procedural characteristics at the time of intervention are listed
in Table 7 and show no significant differences between carriers and
noncarriers of the IL-1RN*2 allele.
11TABLE 5 Lesion and procedural characteristics at the time of
intervention. IL-1RN 1/2 or 2/2 IL-1RN 1/1 (n = 896) (n = 954) P
Target coronary vessels 0.89 Left main - % 1.3 1.6 LAD - % 40.1
39.3 LCx - % 19.9 20.0 RCA - % 32.6 31.9 Venous bypass graft - %
6.1 7.2 Complex lesions - % 75.2 74.1 0.58 Restenotic lesions - %
25.3 23.3 0.30 Before stenting Reference diameter, mm 3.02 .+-.
0.53 3.05 .+-. 0.54 0.29 Diameter stenosis - % 79.1 .+-. 14.9 78.7
.+-. 15.7 0.57 Lesion length - mm 12.1 .+-. 6.9 12.1 .+-. 6.6 0.98
Procedural data Measured balloon diameter - mm 3.2 .+-. 0.5 3.2
.+-. 5 0.45 Maximal balloon pressure - atm 13.9 .+-. 3.3 13.8 .+-.
3.2 0.20 Stented segment length - mm 20.0 .+-. 14.3 20.3 .+-. 13.6
0.70 Immediately after stenting Diameter stenosis - % 5.2 .+-. 9.1
5.4 .+-. 7.6 0.47 Data are proportions or mean .+-. SD LAD
indicates left anterior descending coronary artery; LCx, left
circumflex coronary artery; RCA, right coronary artery; complex
lesions were defined as ACC/AHA lesion types B2 and C, according to
the American College of Cardiology/American Heart Association
grading system.
[0273] IL-1RN Polymorphism, Mortality and Myocardial Infarction
After Stenting
[0274] Table 6 shows the adverse clinical events observed within
the first 30 days after coronary stenting in carriers and
noncarriers of the IL-1RN*2 allele. There was no association
between the presence of the IL-1RN*2 allele and death, myocardial
infarction or target vessel revascularization, showing no
significant influence of the polymorphism in the IL-1ra gene in the
risk for early thrombotic events after coronary stenting.
12TABLE 6 Incidence of adverse events recorded during the early 30
days IL-1RN 1/2 or 2/2 IL-1RN 1/1 (n = 896) (n = 954) P Death - %
0.9 0.9 0.91 Nonfatal myocardial infarction - % 3.3 2.6 0.52 Q-wave
- % 1.1 0.7 0.39 non-Q-wave - % 2.2 1.9 0.60 Target vessel
revascularization - % 3.0 2.3 0.34
[0275] One-year follow-up indicated also that there is no
correlation between the presence of the IL-1RN*2 allele and
mortality or incidence of myocardial infarction after the
intervention. During the 1-year period, mortality rate was 2.8% in
the combined group of IL-1RN 1/2 and IL-1RN 2/2 patients and 2.2%
in IL-1 1/1 patients (P=0.42), yielding an odds ratio of 1.28 (95%
confidence interval, 0.71-2.29). The incidence of nonfatal
myocardial infarction was 3.5% in IL-1RN*2 allele carriers and 3.9%
in homozygous carriers of the IL-1RN*1 allele (P=0.54), and the
respective odds ratio was 0.86 (0.53-1.4).
[0276] IL-1RN Polymorphism and Restenosis After Stenting
[0277] Control angiography was performed in 84% of the patients
after a median of 188 days (interquartile range, 171-205 days). The
proportion of patients with control angiography was similar in the
2 groups defined by the presence or absence of the IL-1RN*2 allele.
Table 7 lists the results of the quantitative assessment of 6-month
angiograms.
13TABLE 7 Results at follow-up angiography. IL-1RN 1/2 or 2/2
IL-1RN 1/1 (n = 758) (n = 798) P Late lumen loss - mm 1.16 .+-.
0.82 1.24 .+-. 0.86 0.07 Loss index 0.53 .+-. 0.38 0.59 .+-. 0.45
0.009 Diameter stenosis - % 41.8 .+-. 26.2 45.2 .+-. 28.7 0.015
Restenosis rate - % 30.2 35.6 0.024 Data are proportions or mean
.+-. SD
[0278] Of note, loss index which reflects the hyperplastic response
after stenting was significantly lower in patients who carried the
IL-1RN*2 allele. The incidence of angiographic restenosis was also
significantly lower in carriers of the IL-1RN*2 allele, with 30.2%
vs. 35.6% in patients of the IL-1RN 1/1 genotype. Thus, the
presence of the IL-1RN*2 allele was associated with a 22% decrease
in restenosis rate (odds ratio, 0.78 [0.63-0.97]; FIG. 9, left
panel). Clinical restenosis expressed as the need for target vessel
revascularization was also significantly lower, with 17.7% in
IL-1RN*2 allele carriers vs. 22.7% in homozygous patients for the
IL-1RN*1 allele (P=0.026), yielding an odds ratio of 0.73
(0.58-0.92) as shown in FIG. 9, left panel.
[0279] Age, gender, the presence or absence of diabetes, smoking
habit, reduced left ventricular function and restenotic lesions,
vessel size (all variables differing in univariate analysis by a
P-value 0.30) were entered into the multivariate model for
angiographic restenosis along with the presence or absence of the
IL-1RN*2 allele. Older age (P=0.005), the presence of diabetes
(P<0.001), restenotic lesion (P<0.001) and small vessel size
(P<0.001) were independently correlated with an increased risk
of restenosis. On the opposite, the presence of the IL-1RN*2 allele
was independently (P<0.001) correlated with a decreased risk for
restenosis with an adjusted odds ratio of 0.81 (0.71-0.92). In
addition, there was a significant interaction between the presence
of the IL-1RN*2 allele and age (P=0.009) as reflected by a
progressively stronger protective effect of this allele in younger
patients.
[0280] The results of the analysis in the prespecified subgroup of
patients<60 years (n=696) are presented in Table 8, FIG. 9,
right panel and FIG. 10. During the 1-year follow-up period, 17.1%
of the IL-1RN*2 allele carriers and 24.9% of the homozygous
IL-1RN*1 allele carriers needed target vessel revascularization
(P=0.013). Thus, the presence of the IL-1RN*2 allele was associated
with a 37% reduction (odds ratio: 0.63 [0.43-0.91]; FIG. 1, right
panel) of the need of ischemia-driven reinterventions. Quantitative
angiographic data obtained for the control study at 6 months
(performed in 590 or 85% of patients<60 years) are displayed in
Table 8.
14TABLE 8 Results at follow-up angiography in patients <60
years. IL-1RN 1/2 or 2/2 IL-1RN 1/1 (n = 273) (n = 317) P Late
lumen loss - mm 1.08 .+-. 0.77 1.27 .+-. 0.93 0.008 Loss index 0.49
.+-. 0.35 0.59 .+-. 0.48 0.003 Diameter stenosis - % 39.3 .+-. 24.1
46.7 .+-. 30.5 0.001 Restenosis rate - % 25.6 38.5 <0.001 Data
are proportions or mean .+-. SD
[0281] The incidence of angiographic restenosis was 25.6% in the
combined group of IL-1RN 1/2 and IL-1RN 2/2 patients and 38.5%
among IL-1RN 1/1 patients (P<0.001), which corresponds to a 45%
reduction (odds ratio: 0.55 [0.39-0.78]; FIG. 1, right panel). FIG.
2 illustrates the gene dose effect verified in the subgroup of
younger patients. The incidence of restenosis decreased
progressively with heterozygosity and homozygosity for the IL-1RN*2
allele. The rate of angiographic restenosis was 38.5% in IL-1RN 1/1
patients, 26.3% in IL-1RN 1/2 patients and 22.4% in IL-1RN 2/2
patients (P=0.001, test for trend). The target vessel
revascularization rate was 24.9% in IL-1RN 1/1 patients, 17.9% in
IL-1RN 1/2 patients and 13.2% in IL-1RN 2/2 patients (P=0.01, test
for trend; FIG. 2).
5.3. Example 3: The IL-1 Haplotype Patterns Associated with
Occlusive Cardiovascular Disorders and Periodontitis
[0282] The association between periodontitis, cardiovascular
disease and four basic biallelic markers (IL-1A (+4845), IL-1B
(+3954), IL-1B (-511), and IL-1RN (+2018)) in the interleukin-1
(IL-1) gene cluster on chromosome 2 was investigated.
[0283] Two haplotype patterns may be defined by four polymorphic
loci in the IL-1 gene cluster as shown in Table 9 (IL-1A(+4845),
IL-1B(+3954), IL-1B (-511), IL-1RN(+2018)). One pattern includes
allele 2 at both the IL-1A (+4845) and at the IL-1B (+3954) loci.
The other pattern includes allele 2 at both the IL-1B(-511), and at
the IL-1RN(+2018) loci.
15TABLE 9 IL-1RN Haplotypes IL-1A (+4845) IL-1B (+3954) IL-1B
(-511) (+2018) Pattern 1 Allele 2 Allele 2 Allele 1 Allele 1
Pattern 2 Allele 1 Allele 1 Allele 2 Allele 2
[0284] The haplotype pattern indicates that when allele 2 is found
at one locus, it is highly likely that it will be found at other
loci. Previous data (Cox et al. (1998) Am. J. Hum. Genet.
62:1180-1188) indicate that when allele 2 is found at the IL-1A
(+4845) locus allele 2 will also be present at the IL-1B (+3954)
locus approximately 80% of the time. Haplotype patterns are
relevant only for a single copy of a chromosome. Since there are
two copies of chromosome 2 and standard genotyping procedures are
unable to identify on which chromosome copy a specific allele is
found, special statistical programs are used to infer haplotype
patterns from the genotype pattern that is determined.
[0285] The distribution of these genetic patterns was evaluated in
a new population that was part of a study of atherosclerosis
(Pankow et al. (1999) The ARIC study. European Atherosclerosis
Society Annual Meeting, Abstract, #646). In this population
(N=1,368), IL-1A(+4845) genotype 2.2 was found in 10.2% of the
subjects. However, in the subjects with genotype IL-1B (+3954)=2.2
(N=95), the IL-1A (+4845) genotype 2.2 was found in 71.6% of the
subjects. This indicates that allele 2 at IL-1A (+4845) is
inherited together with allele 2 at IL-1B (3954) at a much higher
rate than one would expect given the distribution of each of these
markers in the population. Similar data exists for allele 2 at the
2 loci that are characteristic of Pattern 2. In addition, when
genotype Pattern 1 is found it is highly unlikely that allele 2
will be present at either of the loci that are characteristic of
the other pattern.
[0286] The two genotype patterns are also associated with specific
differences in the functional biology of interleukin-1. For
example, peripheral monocytes from individuals with one or two
copies of allele 2 at IL-1B (+3954) produced 2 to 4 times as much
IL-1.beta. when stimulated with LPS as monocytes from individuals
who have the genotype pattern IL-1B (+3954)=1.1 (DiGiovini, F S et
al. (1995) Cytokine, 7:606). Similar data have recently been
reported for peripheral blood polymorphonuclear leukocytes isolated
from individuals with severe periodontitis (Gore, E A et al. (1998)
J. Clin. Periodontol., 25:781). In addition gingival crevice fluid
(GCF) from subjects with the composite genotypes indicative of
Pattern 1 have 2 to 3 times higher levels of IL-1.beta. than GCF
from individuals who are negative for those genotypes
(Engelbretson, S P et al. (1999) J. Periodontol., in press). There
are also data indicating that for Pattern 2, allele 2 at
IL-1RN+2018 is associated with decreased levels of IL-1 receptor
antagonist protein. Thus, Pattern 1 genotypes appear to be
associated with increased IL-1 agonists, and Pattern 2 appears to
be associated with decreased levels of IL-1 receptor
antagonist.
[0287] The composite IL-1 genotypes that are consistent with
Pattern 1 are associated with increased susceptibility to severe
adult periodontitis (Kornman, K S et al. (1997), supra; Gore, E A
et al. (1998), supra; McGuire, M K et al. (1999) J. Periodontol.,
in press; McDevitt, M J et al. (1999) J. Periodontol., in press).
One aspect of the IL-1 genotype influence on periodontitis appears
to be an enhancement of the subgingival levels of specific
bacterial complexes that include accepted periodontal pathogens
(Socransky, S S et al. (1999) IADR Annual Meeting, Abstract#3600).
Pattern 1 genotypes were not, however, associated with increased
risk for occlusive cardiovascular disease. In data from the
Atherosclerosis Risk in Communities (ARIC) study that was presented
by Pankow and co-workers (see Pankow et al., supra), individuals
with ultrasound measurements of carotid wall intima-medial
thickness (IMT) that were indicative of occlusive cardiovascular
disorders were compared to a stratified random control population
for IL-1 gene polymorphisms. Neither IL-1A (+4845) or IL-1B (+3954)
showed any association with risk for high IMT.
[0288] Genotypes that are characteristic of pattern 2 have recently
been associated with increased susceptibility to occlusive coronary
artery disease, but not increased risk for periodontitis. In a
report on coronary artery disease, patients with angiographic
evidence of coronary stenoses were significantly more likely to be
carriers of allele 2 at either the IL-1RN (+2018) locus or the
IL-1B (-511) locus (see Francis et al., supra). Both loci are
characteristic of the haplotype Pattern 2. In the ARIC study, as
discussed above, carriage of IL-1RN (+2018) allele 2 in
African-Americans with high IMT measurements was significantly
higher than ethnically matched controls. In Caucasians with high
IMT measurements the carriage of one copy of allele 2 at IL-1RN
(+2018) was significantly greater than in controls, however
individuals homozygous at this locus were not different from
controls. It should be noted that the prevalence of individuals
homozygous for allele 2 at IL-1RN (+2018) in Caucasians in the
study was substantially lower than that observed in other
populations.
[0289] When individuals with periodontitis and gingival health were
evaluated for genotype patterns consistent with Pattern 1 and
Pattern 2, individuals with severe adult periodontitis were found
to have a predominance of genotypes consistent with Pattern 1,
whereas individuals with a healthy periodontal condition had
genotype patterns that were dominated by neither Pattern 1 nor
Pattern 2. It appears therefore that IL-1 genotypes consistent with
the haplotype Pattern 1 are associated with severe periodontitis
and plaque fragility disorders and not occlusive cardiovascluar
diseases whereas IL-1 genotypes consistent with the haplotype
Pattern 2 are associated with occlusive cardiovascular diseases but
not periodontitis or plaque fragility. One mechanism may be that
IL-1 genotype Pattern 1 directly influences plaque fragility;
another mechanism may be that Pattern 1 influences periodontitis
directly, which may lead to indirect influences on cardiovascular
disease through the periodontal micororganisms found as part of the
oral chronic inflammatory process. Another mechanism may be that
IL-1 genotype Pattern 2 directly influences cardiovascular
occlusive disorders but has no influence on periodontitis. It is
thus likely that IL-1 genetic polymorphisms can influence both
cardiovascular disease and severe periodontitis, by a common
underlying mechanism that directly alters the immunoinflammatory
responses in both diseases in an identical fashion and by an
indirect mechanism that enhances the oral bacterial load and then
influences cardiovascular disease. The IL-1 genotypes that are
consistent with haplotype Pattern 1 may influence the association
between periodontidis and cardiovascular disease in one segment of
the population by amplifying both the immuno-inflammatory response
and the subgingival bacterial load.
5.4 Example 4 Genotyping Methods
[0290] Preparation of DNA
[0291] Blood is taken by venipuncture and stored uncoagulated at
-20.degree. C. prior to DNA extraction. Ten milliliters of blood
are added to 40 ml of hypotonic red blood cell (RBC) lysis solution
(10 mM Tris, 0.32 Sucrose, 4 mM MgCl.sub.2, 1% Triton X-100) and
mixed by inversion for 4 minutes at room temperature (RT). Samples
are then centrifuged at 1300 g for 15 minutes, the supernatant
aspirated and discarded, and another 30 ml of RBC lysis solution
added to the cell pellet. Following centrifugation, the pellet is
resuspended in 2 ml white blood cell (WBC) lysis solution (0.4 M
Tris, 60 mM EDTA, 0.15 M NaCl, 10% SDS) and transferred into a
fresh 15 ml polypropylene tube. Sodium perchlorate is added at a
final concentration of 1M and the tubes are first inverted on a
rotary mixer for 15 minutes at RT, then incubated at 65.degree. C.
for 25 minutes, being inverted periodically. After addition of 2 ml
of chloroform (stored at -20.degree. C.), samples are mixed for 10
minutes at room temperature and then centrifuged at 800 G for 3
minutes. At this stage, a very clear distinction of phases can be
obtained using 300 l Nucleon Silica suspension (Scotlab, UK) and
centrifugation at 1400 G for 5 minutes. The resulting aqueous upper
layer is transferred to a fresh 15 ml polypropylene tube and cold
ethanol (stored at -20.degree. C.) is added to precipitate the DNA.
This is spooled out on a glass hook and transferred to a 1.5 ml
eppendorf tube containing 500 l TE or sterile water. Following
overnight resuspension in TE, genomic DNA yield is calculated by
spectrophotometry at 260 nm. Aliquots of samples are diluted at 100
ug/ml, transferred to microtiter containers and stored at 4.degree.
C. Stocks are stored at -20.degree. C. for future reference.
[0292] 5.4.1 Polymerase Chain Reaction
[0293] Oligonucleotide primers designed to amplify the relevant
region of the gene spanning the polymorphic site (as detailed
below) are synthesized, resuspended in Tris-EDTA buffer (TE), and
stored at -20.degree. C. as stock solutions of 200 uM. Aliquots of
working solutions (1:1 mixture of forward and reverse, 20 .mu.M of
each in water) are prepared in advance.
[0294] Typically, PCR reaction mixtures are prepared as detailed
below.
16 Stock Final Concentration Volume Concentration Sterile H.sub.20
29.5 .mu.l 10 .times. PCR buffer 200 mM Tris-HCl 5.00 .mu.l 20 mM
Tris-HCl, (pH 8.4) MgCl.sub.2 50 mM 1.75 .mu.l 1.75 mM dNTP mix 10
mM of 4.00 .mu.l 0.2 mM of each each primer forward 20 uM 2.5 .mu.l
1 uM prime reverse 20 uM 2.5 .mu.l 1 uM Taq polymerase 5 U/.mu.l
0.25 .mu.l 1.25 units/50 .mu.l Detergent (eg W-1, 1% 2.5 .mu.l
0.05% Gibco) Template 200 ng/.mu.l 2.00 .mu.l 2 ng/l Final Volume
50.00 .mu.l
[0295] DNA template is dotted at the bottom of 0.2 ml tubes or
microwells. The same volume of water or negative control DNA is
also randomly tested. A master-mix (including all reagents except
templates) is prepared and added to the wells or tubes, and samples
are transferred to the thermocycler for PCR.
[0296] PCR can be performed in 0.5 ml tubes, 0.2 ml tubes or
microwells, according to the thermocycler available. The reaction
mixture is overlaid with mineral oil if a heated lid (to prevent
evaporation) is not available.
[0297] 5.4.2 Restriction Enzyme Digestion
[0298] A master mix of restriction enzyme buffer and enzyme is
prepared and aliquotted in suitable volumes in fresh microwells.
Digestion is carried out with an oil overlay or capped microtubes
at the appropriate temperature for the enzyme on a dry block.
[0299] Restriction buffer dilutions are calculated on the whole
reaction volume (i.e. ignoring salt concentrations of PCR buffer).
Restriction enzymes are used 3-5 times in excess of the recommended
concentration to compensate for the unfavorable buffer conditions
and to ensure complete digestion.
[0300] 5.4.3 Electrophoresis
[0301] Polyacrylamide-gel electrophoresis (PAGE) of the PCR sample
is carried out in Tris-HCl-EDTA buffer and at constant voltage.
Depending on the size discrimination need, different PAGE
conditions are used (9 to 12% acrylamide, 1.5 mm.times.200) and
different DNA size marker (X174-Hae III or X 174-Hinf 1). A 2%
agarose horizontal gel can be used for genotyping the IL-1RN (VNTR)
marker.
[0302] 5.4.4 Allele Detection Methods
[0303] The following Table 10 provides methods for detecting
particular alleles that are associated with the existence of or
susceptibility to developing restenosis.
17TABLE 10 IL-1A (+4845) 5' Primer
ATG.GTT.TTA.GAA.ATC.ATC.AAG.CCT.AGG.GCA (+4814/+4843) (SEQ ID No.
1) 3' Primer AAT.GAA.AGG.AGG.GGA.GGA.TGA.CAG.- AAA.TGT
(+5015/+5044) (SEQ ID No. 2) PCR MgCl.sub.2 is used at 1 mM final,
and PCR Conditions primers at 0.8 mM. DMSO is added at 5%, DNA
template at 150 ng/50 ml, and TaqMan 1.25 u/50 .mu.l. Cycling 1X
[95.degree. C. 1 min.]; 35X [94.degree. C. 1 min., conditions
56.degree. C. 1 min., 72.degree. C. 2 min.]; 1X [72.degree. C. 5
min.]; 4.degree. C. Analysis Cleavage with 2.5 units of Fnu4H1 in
addition to 2 ml of the specific 10 restriction buffer at
37.degree. C. overnight, followed by 9% PAGE analysis yields a
constant band of 76 bp (absence indicates incomplete digestion) and
two further bands of 29 and 124 bp (allele 1), or a single band of
153 bp (allele 2). Allele frequencies in North British Caucasian
population are 0.71 and 0.29. Reference Gubler, et al.(1989)
Interleukin, inflammation and disease (Bomford and Henderson, eds.)
p. 31-45, Elsevier publishers; and Van den velden and Reitsma
(1993) Hum Mol Genetics 2: 1753-50). GenBank Accession No. X03833.
IL-1B (-511) 5' Primer TGG.CAT.TGA.TCT.GGT.TCA.TC (-702/-682) (SEQ
ID No: 3) 3' Primer GTT.TAG.GAA.TCT.TCC.CAC.TT (-417/-397) (SEQ ID
No: 4) PCR 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 1.5 Conditions mM
MgCl.sub.2, 200 mM dNTPs, 25 ng primers, 50 ng template, 0.004% W-1
(Gibco-BRL), 0.2 U Taq polymerase, 50 .mu.l total volume Cycling 1X
[95.degree. C. 2 min.]; 35X [95.degree. C. 1 min., conditions
53.degree. C. 1 min., 72.degree. C. 1 min.]; 1X [72.degree. C., 5
min.]; 4.degree. C. Analysis Each PCR reaction is divided into two
25 .mu.l aliquots: one is added of 3 units of Ava I restriction
endonuclease, the other 3.7 units of Bsu 36 I, in addition to 3
.mu.l of the specific 10x restriction buffer. Incubation is at
37.degree. C. overnight. Electrophoresis is by PAGE 9%. Cleavage
with Ava I and Bsu 36I. Allele 1 (C) produces 190 and 114 bp
fragments when digested with Ava I and a 304 bp fragment when
digested with Bsu 36I. Allele 2 (T) produces a 304 bp fragment when
digested with Ava I and 190 and 114 bp fragments when digested with
Bsu 36I. The restriction pattern obtained should be the inverse in
the two aliquots (identifying homozygotyes) or identical
(heterozygotes). Frequencies in North British Caucasian population
are 0.61 and 0.39 for allele 1 and 2 respectively. Reference
diGiovine, Hum. Molec. Genet., 1(6): 450 (1992); Clark, et al.,
Nucl. Acids. Res., 14: 7897-7914 (1986) [published erratum appears
in Nucleic Acids Res., 15(2): 868 (1987)]; GenBank Accession No.
X04500. IL-1B (+3954) 5' Primer CTC.AGG.TGT.CCT.CGA.AGA.AAT.CAA.A
(+3844/+3868) (SEQ ID No: 5) 3' Primer GCT.TTT.TTG.CTG.TGA.GTC.CCG
(+4017/+4037) (SEQ ID No: 6) PCR 50 mM KCl, 10 mM Tris-HCl, pH 9.0,
1.5 Conditions mM MgCl.sub.2, 200 mM dNTPs, 25 ng primers, 50 ng
template, 0.004% W-1 (Gibco-BRL), 0.2 U Taq polymerase, 50 .mu.l
total volume Cycling 1X [95.degree. C. 2 min.]; 35X [95.degree. C.
1 min., conditions 67.5.degree. C. 1 min., 72.degree. C. 1 min]; 1X
[72.degree. C., 5 min.]; 4.degree. C. Analysis Each PCR reaction is
added of 10 u of Taq 1 restriction endonuclease in addition to 3
.mu.l of the specific 10x restriction buffer. Incubation is at
65.degree. C. overnight. Electrophoresis is by PAGE 9%. Following
digestion with Taq I, Allele 1 produces 97, 85 and 12 bp fragments;
Allele 2 produces 182 and 12 bp fragments. The absence of the 12 bp
band indicates incomplete digestion. Frequencies in a North British
Caucasian population are 0.82 (allele 1) and 0.18 (allele 2). For
90% power at 0.05 level of significance in a similar genetic pool,
408 cases should be studied to detect 1.5 fold increase in the
frequency, or 333 for 0.1 absolute increase in frequency. Reference
di Giovine, et al. Cytokine 7(6): 606 (1995) IL-1RN (VNTR) 5'
Primer CTC.AGC.AAC.ACT.CCT.AT (+2879/+2895) (SEQ ID NO. 7) 3'
Primer TCC.TGG.TCT.GCA.GCT.AA (+3274/+3290) (SEQ ID NO. 8) PCR 50
mM KCl, 10 mM Tris-HCl pH 9.0, 1.7 Conditions mM MgCl.sub.2, 200 mM
dNTPs, 25 ng primers, 50 ng template, 0.004% W-1 (Gibco-BRL) 0.2 u
Taq polymerase Cycling 1X [96.degree. C. for 1 min.]; 30X
[94.degree. C. for conditions 1 min., 60.degree. C. for 1 min.,
70.degree. C. for 1 min.]; 1 [70.degree. C. for 2 min.]. Analysis
The variable number of tandem repeats (VNTR) in intron 2 of IL1-RN
corresponds to a variable number (2 to 6) of an 86 bp repeat and so
the PCR product sizes are a direct indication of the number of
repeats. Electro- phoresis is by 2% agarose, 90V, 30 min. Allele 1
4 repeats 412 bp PCR product Allele 2 2 repeats 240 bp PCR product
Allele 3 3 repeats 326 bp PCR product Allele 4 5 repeats 498 bp PCR
product Allele 5 6 repeats 584 bp PCR product Frequencis in a North
British Caucasian population for the four most frequent alleles are
0.734, 0.241, 0.021 and 0.004. Reference Steinkasserer et al.
(1991) Nucleic Acids Research 19: 5090-95; Tarlow, et al.. Hum.
Genet. 91: 403-4 (1993) IL-1RN (+2018) 5' Primer
CTA.TCT.GAG.GAA.CAA.CCA.ACT.AGT.AGC-3' (+1992/+2017) (SEQ ID No. 9)
3' Primer TAG.GAC.ATT.GCA.CCT.AGG.GTT.TGT-- 3' (+2135/+2158) (SEQ
ID No. 10) PCR Each PCR reaction is divided in two 25 Conditions
.mu.l aliquots; to one is added 5 Units of Alu I, the other 5 Units
of Msp I, in addition to 3 .mu.l of the specific 10X restriction
buffer. Incubation is a 37.degree. C. overnight. Electrophoresis is
by PAGE 9%. Cycling 1X [96.degree. C. for 1 min]; 35X [94.degree.
C. for conditions 1, min., 57.degree. C. for 1 min 70.degree. C.
for 2 min.]; 1X [70.degree. for 5 min.]; 4.degree. C. Allele The
above described PCR primers Detection incorporate mismatches to the
genomic sequence so as to engineer two different restriction sites
on the alleles. The two alleles are 100% in linkage disequilibrium
with the two most frequent alleles of IL-1RN (VNTR). Alu I will
produce 126 + 28 bp fragments for Allele 1, while it does not
digest Allele 2 (154 bp). Msp I will produce 125 + 29 bp with
Allele 2, while Allele 1 is uncut (154 bp). Hence the two reactions
(separated side by side in PAGE) will give inverted patterns of
digestion for homozygote individuals, and identical patterns in
heterozygotes. Allelic frequencies in a North British Caucasion
population are 0.74 and 0.26. For 90% power at 0.05 level of
significance in a similar genetic pool, 251 cases should be studied
to detect 1.5 fold increase in frequency, or 420 for 0.1 absolute
increase in frequency. Reference Clay, et al.(1996) Hum. Genet. 97:
723-26.
[0304] Results: Typing of additional numbers of individuals is
required to bring the results to significance, but preliminary
results indicate that allele 2 of the 4845, -511, +3954 and VNTR
markers in the IL-1RN gene will be over-represented in restenosis.
It is predicted that individuals with at least one copy of allele 2
from one of the above markers are more likely to have restenosis
than those who are negative for allele 2. Individuals who are
homozygous for any of these alleles, or have allele 2 from more
than one marker are estimated to have even higher risk for
restenosis.
5.5 Example 5
[0305] In this example, the preparation of template DNA is
described. PCR-based genotyping does not require particularly
high-MW DNA (<20 Kb DNA is often an excellent template). As 100
ng genomic DNA is more than sufficient for single-copy gene
amplification, direct amplification from dried blood spots or cell
lysates can be used for genotyping, and two of the protocols that
we have used are here described below.
[0306] However, if DNA banks need to be established for population
studies where DNA needs to be stored for future reference or
genotyping at different loci, or where genomic Southern blotting
might be needed, good quality high-MW genomic DNA needs to be
extracted. Basic buffers and the composition of chemical solutions
can be found in major protocol textbooks (Sambrook et al. (1989)
Molecular cloning: a laboratory manual, Cold Spring Harbor Press;
Ausubel and Frederick (1994) Current protocols in molecular
biology, John Wiley and Sons).
[0307] Sample DNA can also be obtained from dried blood spots. Such
a means of sample collection (Guthrie spots) has been used for many
years in neonatal diagnosis of phenylketonuria. In the last few
years dried blood spots have proved useful in PCR-based diagnostics
(Raskin et al. (1991) Am J Hum Genet 49: 320-29). Uncoagulated
blood is spotted evenly using a sterile Pasteur pipette onto a
clean sheet of filter paper. This is left to dry overnight in a
clean area (physically isolated from post-PCR events) and stored
subsequently at room temperature.
[0308] For PCR, a mastermix is prepared as described later in this
chapter, where Taq polymerase is omitted. This is aliquotted in
reaction tubes, and approximately 1 mm.sup.2 of the blood spot is
cut out and placed into the reaction mix. This is overlaid with 40
.mu.l mineral oil. The lid of each tube is pierced with a sterile
needle, and samples are then heated at 98.degree. C. for 15
minutes. Following cooling for a few minutes, Taq polymerase is
added and standard PCR cycling follows.
[0309] Sample DNA can also be obtained from cell lysates. White
blood cells, buccal cells or homogenised tissue is suspended in PK
buffer (0.1 M NaCl, 10 mM Tris-HCl, 25 mM EDTA, 0.5% SDS pH 8.0,
0.1 mg/ml fresh Proteinase K) and incubated on a tumbler at
37.degree. C. for 1 hour. Samples are heated at 95.degree. C. for
10 mins, spun at 13,000 rpm in a microfuge and supernatant stored
at -20.degree. C. prior to PCR. For higher quality DNA, a
phenol/chloroform extraction followed by ethanol precipitation can
be added.
[0310] Sample genomic DNA can also be obtained from whole blood.
Blood is taken by venepuncture and stored uncoagulated at
-20.degree. C. prior to DNA extraction. When possible, we prefer to
collect two 10 ml samples, extract DNA form the first and keep the
second for future reference. Ten milliliters of blood are added to
40 ml of hypotonic red blood cell (RBC) lysis solution (10 mM
Tris-HCl, 0.32 Sucrose, 4 mM MgC.sub.2, 1% Triton X-100) and mixed
by inversion for 4 minutes at room temperature. Samples are then
centrifuged at 1300 g for 15 minutes, the supernatant aspirated and
discarded, and another 30 ml of RBC lysis solution added to the
cell pellet. Following centrifugation, the pellet is resuspended in
2 ml white blood cell (WBC) lysis solution (0.4M Tris-HCl, 60 mM
EDTA, 0.15M NaCl, 10% SDS) and transferred into a fresh 15 ml
polypropylene tube. Sodium perchlorate is added at a final
concentration of 1M and the tubes are first inverted on a rotary
mixer for 15 minutes at room temperature (RT), then incubated at
65.degree. C. for 25 minutes, being inverted periodically. After
addition of 2 ml of chloroform (stored at -20.degree. C.), samples
are mixed for 10 minutes at room temperature and then centrifuiged
at 800 g for 3 minutes. At this stage a very clear distinction of
phases can be obtained using 300 .mu.l Nucleon Silica suspension
(Scotlab, UK) and centrifugation at 1400 G for 5 minutes. The
resulting aqueous upper layer is transferred to a fresh 15 ml
polypropylene tube and cold ethanol (stored at -20.degree. C.) is
added to precipitate the DNA. This is spooled out on a glass hook
and transferred to a 1.5 ml eppendorf tube or containing 500 .mu.l
TE or sterile water. Following overnight resuspension in TE,
genomic DNA yield is calculated by spectrophotometry at 260 nm.
Aliquots of samples are diluted at 100 .mu.g/ml, transferred to
microtiter containers and stored at 4.degree. C. Stocks are stored
at -20.degree. C. for future reference.
5.6 Example 6
[0311] In this example, the conditions for conducting appropriate
polymerase chain reactions on the collected samples are described.
Oligonucleotide primers designed to amplify the relevant region of
the gene spanning the polymorphic site (as detailed below) are
synthesised, resuspended in Tris-HCl-EDTA buffer (TE) and stored at
-20.degree. C. as stock solutions of 200 .mu.M. Aliquots of working
solutions (1:1 mixture of forward and reverse, 20 .mu.M of each in
water) are prepared in advance of the experiment. Typically PCR
reaction mixtures are prepared as detailed below. Divergence from
the scheme below can be made for each specific protocol.
18 Stock Final Concentration Volume Concentration Sterile H.sub.20
29.5 .mu.l 10 .times. PCR buffer 200 mM Tris-HCl 5.00 .mu.l 20 mM
Tris-HCl, (pH 8.4), 50 mM KCl 500 mM KCl MgCl.sub.2 50 mM 1.75
.mu.l 1.75 mM dNTP mix 10 mM of 4.00 .mu.l 0.2 mM of each each
primer forward 20 .mu.M 2.5 .mu.l 1 .mu.M primer reverse 20 .mu.M
2.5 .mu.l 1 .mu.M Taq polymerase 5 U/.mu.l 0.25 .mu.l 1.25 units/50
.mu.l Detergent (eg W-1 1% 2.5 .mu.l 0.05% Gibco) Template 200
ng/.mu.l 2.00 .mu.l 2 ng/.mu.l Final volume 50.00 .mu.l
[0312] DNA template is dotted at the bottom of 0.2 ml tubes or
microwells. The same volume of water or negative control DNA is
also randomly tested. A master-mix (including all reagents except
templates) is prepared and added to the wells or tubes, and samples
are transferred to the thermocycler for PCR.
[0313] PCR can be performed in 0.5 ml tubes, 0.2 ml tubes or
microwells, according to the thermocycler available and to the
needs of the project. The reaction mixture is overlaid with mineral
oil if a heated lid (to prevent evaporation) is not available. We
use 96-well format microplates, because they allow use of
multichannel pipettes both for transfer of template DNA (stored in
1 ml/microwell plates) and for dispensing of the reaction
mastermix.
5.7 Example 7
[0314] In this example, the conditions for conducting appropriate
polymerase chain reactions on the collected samples are described.
A master mix of restriction enzyme buffer and enzyme is prepared
and aliquotted in suitable volumes in fresh microwells. We use a
multichannel pipette to transfer and mix 25-30 .mu.l of PCR product
in the microwells. Digestion is carried out with an oil overlay or
capped microtubes at the appropriate temperature for the enzyme on
a dry block. Restriction buffer dilutions are calculated on the
whole reaction volume (i.e. ignoring salt concentrations of PCR
buffer). Restriction enzymes are used 3-5 times in excess of the
recommended concentration, to compensate for the unfavorable buffer
conditions and to ensure complete digestion.
5.8 Example 8
[0315] In this example, the conditions for conducting gel
electorphoresis analysis of the products of pcr amplification and
restriction endonuclease digestion are considered.
Polyacrylamide-gel electrophoresis (PAGE) of 20-40 .mu.l PCR sample
is carried out in Tris-HCl-EDTA buffer and at constant voltage.
Depending on the size discrimination needed, different PAGE
conditions are used (9 to 12% acrylamide, 1.5 mm.times.200) and
different DNA size markers (.phi.X174-Hae III or .phi.X 174-Hinf
I). A 2% agarose horizontal gel can be used for IL-1RN (VNTR).
5.9 Example 9
[0316] In this example, quality controls for these genotyping
protocols are considered. Incomplete digestion is the most common
cause of mis-typing in PCR-RFLP genotyping methods. Most of the
protocols described herein are based on a double-cut strategy, for
which either a second restriction cutting site is used for
digestion control on the diagnostic cleavage, or one enzyme cuts
one allelic DNA form, and a different enzyme cuts the other allele.
In this case each reaction is the control for the other. PCR
conditions are tested (and, if necessary, re-optimised) for each
DNA preparation not performed in our laboratory. Template DNA
quality is assessed by spectrophotometry and by gel
electrophoresis.
[0317] The possibility of cross-contamination is very high in
PCR-based techniques. Although the genotyping is physically
separated from any lab where relevant cloned fragments are being
handled, it is still possible to have PCR-product carryover from
previous experiments (from labcoat, hair, skin, etc.). A
"PCR-carryover prevention kit" is available from Perkin-Elmer. This
is based on UNG treatment of samples prior to PCR, which will
cleave all dUTP-containing DNA. As all PCRs are performed using
dUTP instead of dTTP, all previous PCR products, but not native
templates, will be cleaved in this digestion step. This enzyme is
inactivated by the first temperature ramping (94.degree. C.) and
therefore normal PCR can take place without UNG activity. If
laboratories do not use this system (which is expensive), there are
stringent rules that can be used to reduce the risk of artefacts
due to contamination.
5.10 Example 10
[0318] In this example, the prevention of contamination in these
genotyping protocols is considered. Incomplete digestion is the
most common cause of mis-typing in PCR-RFLP genotyping methods.
[0319] Laboratories are divided into GREEN (Pre-PCR) and RED
(Post-PCR) areas. All laboratories have dedicated white coats, and
workers are encouraged to change lab gloves as frequently as
possible. GREEN laboratories have the most stringent requirements.
Only goods coming from other green areas can enter, anything
(equipment included) that leaves them cannot re-enter. These
usually include a store-room, a "sample reception" area, a "clean
DNA room" (where DNA extraction and PCR preparation are performed)
and offices. RED laboratories have open access, but material and
equipment can only move to other red areas or disposed of in bags
for autoclaving or incineration. Red areas are where PCR and
electrophoresis take place. Results and images are stored in
computer files and transferred to the offices by local network.
[0320] All PCR's carry 10% negative controls which are randomly
placed within the experiment. These are routinely represented by
water controls. In the case of amplicards, negative controls are
represented also by fragments (2-3-mm.sup.2) of paper from the edge
of the card. For human blood DNA preparations, murine T cell
lysates are extracted at the same time as each new batch of frozen
blood, and resulting DNA used as negative control.
5.11 Example 11
[0321] In this example, the design of human polymorphic marker
association studies are examined and the resulting data is
analyzed. Traditional parametric analyses (requiring the
specification of a distribution and/or the mode of inheritance)
have been used successfully to locate genes for monogenic diseases
following simple Mendelian modes of inheritances. More commonly
used in the genetic analysis of complex diseases are non-parametric
methods since these work independently of inheritance
specifications, and are generally more powerful than parametric
methods when parameters are mis-specified. The choice of method of
analysis depends on whether the investigator wishes to perform a
whole genome screen or use a candidate gene approach, since certain
methods are best suited to just one of these two approaches or to
specific pedigree structures. The following sections contain an
outline of most commonly used non-parametric methods of analysis
and their suitability to the candidate gene approach.
[0322] An allele at a certain locus is said to be associated with a
disease if the frequency for that allele is significantly increased
in the disease population over that of the normal healthy control
population. True associations are due to linkage disequilibrium,
where the disease causing allele at the `disease` locus remains on
the same haplotype as those alleles which were present at closely
flanking loci when the ancestral mutation occurred. Thus, the
frequency of any allele on the `disease haplotype` (including, of
course, the disease allele itself) will be increased in the disease
population. Recombination over extremely small distances is very
low, but as the time from the ancestral mutation increases, the
distance over which linkage disequilibrium acts decreases reducing
the length of the `disease haplotype`. It is therefore easier to
detect association in young, isolated populations with a single
founder mutation effect where linkage extends over larger
distances, than in large mixed populations.
[0323] Association studies are at present only suited to the
candidate gene approach due to the small distances over which
associations are detectable. In the future it is proposed that
genome-wide association studies will be performed using several
biallelic markers in every gene. Care must be taken when selecting
the disease population in an association study, since spurious
positive results may occur as an artefact of population admixture.
It is usually advisable to investigate within a single ethnic
group, since allele frequencies may vary between different groups.
Similarly, if a control population is needed, it must be matched to
the disease group for ethnicity, and ideally sex and age.
[0324] Case control studies can be performed for both qualitative
and quantitative phenotypes. Obvious advantages of this approach
include the ease of collection of large populations, the
possibility of recruitment of patients with "early disease"
phenotypes, and the possibility of analyzing late-onset diseases,
where parental DNA may not be available.
[0325] For qualitative phenotypic studies, the candidate gene
locus, allele frequencies or alternatively genotype frequencies,
within the disease and control populations are calculated.
[0326] The analysis is simple, comprising of a 2.times.n
contingency table (n denoting the number of categories, 2 for
allele frequencies or 3 for genotypes at a biallelic locus), which
a chi-square test may be used to determine whether the proportions
differ significantly between the disease and control
populations.
[0327] For quantitative phenotypic studies looking for a disease
susceptibility allele, the individuals in both populations are
first phenotyped quantitatively (usually the disease is classified
as attaining a certain threshold value, therefore the unaffected
controls are individuals failing below this). All individuals are
then subdivided into the three (or more) genotypes. If an allele
responsible for the inflated phenotype value of the diseased
individuals exists, it would be expected that these individuals
carry at least, one copy of it. Thus the median of these genotype
groups would be higher than those of the non-carrier groups. The
non-parametric test involves testing for significant difference
between the medians of the different genotype (or carriage) groups.
This may be done via a Mann-Whitney test (for 2 groups), or a
Kruskall-Wallis (for >2 groups), although several other tests
also exist. In exactly the same way, this type of analysis may also
be performed solely within the disease group.
[0328] For use of qualitative traits in studies employing more than
one IL-1 polymorphic locus, the simple one locus case-control
analysis can be extended to one involving several loci (given a
sufficient sample size). In a similar way, a larger contingency
table can be calculated, with groups corresponding now to composite
genotypes. As before, a chi-squared statistic can be calculated.
With these large contingency tables, it is likely that the validity
of the chi-square test is violated (<80% of expected
values>5, and expected values<1). With smaller contingency
tables, the usual remedy to violations of validity is to use
Fishers Exact test, but in this larger case, it is not viable.
Instead a null distribution for the evaluated chi-square statistic
is simulated, and significance assessed from this. This test has
been named the Monte Carlo Composite Genotype (MCCG) test.
5.12 Example 12
[0329] In this example, haplotype relative risk (HRR) analysis is
discussed. This analysis is only suitable for qualitative traits
(quantitative traits may be used if, dichotomised), and as with all
association tests, the candidate gene approach. Haplotype analysis
investigates the association between specific genetic markers for
diseases and the way a set of markers may influence the outcome of
the disease. Analyzing the relationship between specific genetic
markers and disease is an extremely complex process. The analysis
needs to take into account (i) the relation between genetic markers
in neighboring genes, (ii) the way the polymorphic markers affect
expression of the gene in question, (iii) the distribution of the
genetic markers for a specific polymorphism over both chromosomes,
and (iv) the way the expressed gene product(s) affect the disease
process.
[0330] The relationship between these factors can be identified by
statistical equations that look at multipoint linkage analysis,
transmission/disequilibrium test (TDT), multipoint quantitative
trait loci (QTL) analysis, identity-by-state (IBS),
identity-by-descent (IBD), and grouping of multiallelic markers for
biological functions related to disease. This approach has been
described by Camp ((1997) American Journal of Human Genetics 61:
1424-30); Cox et al ((1998) American Journal of Human Genetics 62:
1180-88); and Almasy and Blangero ((1998) American Journal of Human
Genetics 62: 1198-1211).
[0331] To perform a HRR analysis (Falk et al. (1987) Ann Hum Genet
51: 27-233) nuclear families with affected offspring are needed.
This type of analysis uses an artificial internal control, and
therefore the problem of collecting an independent matched control
population is removed. The parents and affected offspring are
genotyped. It is then established which parental alleles were
passed on to the affected offspring and which were not. From this
the transmitted genotype and the non-transmitted genotype (internal
control) are determined and recorded in the transmitted and
non-transmitted groups, respectively. The two groups are then
tested for significant differences in the proportions of their
genotypes.
5.13 Example 13
[0332] In this example, the transmission/ disequilibrium test (TDT)
is discussed. This analysis is suitable for qualitative traits
investigated using a candidate gene approach. Nuclear families are
needed, including at least one parent, all affected offspring, and
if possible an unaffected sibling.
[0333] The TDT (Spielman et al. (1993) Am J Hum Genet 52: 506-16)
is a test for both association and for linkage, more specifically,
it tests for linkage in the presence of association. Thus, if
association does not exist at the locus of interest, linkage will
not be detected even if it exists. It is for this reason that the
test has been included in this section. It may be used as an
initial test, but is more commonly used when tentative evidence for
association has already been identified. In this case, a positive
result will not only confirm the initial association, but also
provide evidence for linkage.
[0334] All parents and affected offspring are genotyped. Only
parents heterozygous for the allele of interest may be used in the
analysis. If the allele of interest is, or is linked to, the
disease allele, the transmission rate for that allele from
heterozygous parents to their affected offspring should be
elevated. To test if the transmission rate of the allele of
interest is significantly elevated, the number of times it is
transmitted, b, and the number of times other alleles are
transmitted, c, are counted. The squared difference of b and c
divided by their sum provides a statistic that follows a chi-square
distribution with one degree of freedom, and can thus be assessed
for significant deviation from the expected under no association or
linkage. It is often advised to repeat this procedure using the
unaffected offspring from the same parents to rule out the
possibility of a spurious result due to biased meioses.
[0335] The TDT may also be used once linkage on a coarse scale has
been shown to provide the fine scale mapping that is necessary to
pin-point more accurately the disease locus. Of course, these tests
are only valid when associations within the area also exist.
5.14 Example 14
[0336] In this example, the non-parametric linkage analysis is
discussed. Non-parametric linkage analysis methods (such as
Affected Sib-Pair analysis, the Haseman-Elston method and Variance
Component Method) are based on the allele sharing status of
affected relative pairs, usually sibs. These methods are suitable
for whole genome screens (commonly done at 10 cM intervals) and
also a candidate gene approach (although for fine localisation
alternative methods such as the TDT (section 4.2.1.3) should be
used).
5.15 Example 15
[0337] In this example the analysis of significance and power of
the data is examined. Throughout this section, evidence strong
enough to suggest association or linkage has been termed
significant. The significance level of a test is left to the
discretion of the investigator, but conventionally a 5%
significance level is used. This means that it is accepted that
there is enough evidence to suggest an association (or linkage) if
the result would have occurred only 1 in 20 (0.05) times by chance
in data where no association (linkage) existed, that is, there is
only a 0.05 chance that the result is a false-positive. For each
test a p-value may be calculated which indicates the probability of
the result occurring by chance. In a single test, if this value is
less than 0.05 then significant evidence may be claimed. This
concept becomes more complicated when multiple, independent tests
are performed. For example, if two tests were performed, and each
was tested at the 5% level of significance, overall there is a 2 in
20 (0.1) chance of at least one result being a false-positive.
Thus, for two independent tests, to maintain an overall
significance level of 0.05 (0.05 chance of at least one test being
a false positive) either the individual significance level for each
test must be lowered to 0.05/2=0.025, or the p-values doubled
before assessing the result. This method of correction is called
the Bonferroni correction. More generally, if n independent tests
were carried out, each individual test should be tested at the
0.05/n level, or alternatively, every p-value multiplied by n
before assessing the results. With non-independent tests, however,
the Bonferroni correction may be too conservative.
[0338] Many investigators may find that they lose their potential
significances through the dilution of p-values due to the
correction criteria for multiple tests. Unfortunately these
corrections are necessary for statistical correctness and cannot be
discarded. However, if the results from the first set of
observations are real, a second replication sample need only test
those interesting results found from the first. This reduces the
number of tests necessary on the second set of observations and
thus reduces the dilution, increasing the chance of maintaining the
statistical significance that may have been lost the first time.
For complex diseases where there are so many questions to be
answered it is perhaps unreasonable to expect that a single sample
would be sufficient, and instead anticipate the necessity for a
two-stage analysis and prepare accordingly. This is especially true
for whole genome screens where the corrections necessary are
massive. Lander et al. ((1995) Nature Genet 11: 241-7) list
sensible guidelines for claiming significance in linkage analyses,
specifically in the case of genome screens.
[0339] Along with significance, a second, and equally important
issue is that of power, the ability to pick up significant evidence
where it actually exists. Given the phenotype, data structure and
number of observations, it is important to choose the method of
analysis which is most likely to determine associations or linkages
if they exist. In fact, it is advisable that in the planning stages
of these studies the number of observations that are necessary to
reach a predetermined power level are calculated. Unfortunately,
this task is not as simple as it sounds, since power depends on
several factors, of which some may be unknown, for example, allele
frequencies, marker informativeness, familial clustering of the
disease, recombination between marker and disease locus. Even if
these factors are known, the power cannot be explicitly calculated
for some methods, and instead empirical powers must be worked out
via simulations.
[0340] There is no clear answer to which analyses should be done in
different situations because of the many variables that are
involved. However, it is strongly advisable to make the most
informed choice possible, using previous work that has been done,
to increase the chances of detection and location of genes
responsible, or involved in complex diseases.
Sequence CWU 1
1
29 1 30 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 atggttttag aaatcatcaa gcctagggca 30 2 30 DNA Artificial
Sequence Description of Artificial Sequence Primer 2 aatgaaagga
ggggaggatg acagaaatgt 30 3 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 3 tggcattgat ctggttcatc 20 4 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 4
gtttaggaat cttcccactt 20 5 25 DNA Artificial Sequence Description
of Artificial Sequence Primer 5 ctcaggtgtc ctcgaagaaa tcaaa 25 6 21
DNA Artificial Sequence Description of Artificial Sequence Primer 6
gcttttttgc tgtgagtccc g 21 7 17 DNA Artificial Sequence Description
of Artificial Sequence Primer 7 ctcagcaaca ctcctat 17 8 17 DNA
Artificial Sequence Description of Artificial Sequence Primer 8
tcctggtctg cagctaa 17 9 27 DNA Artificial Sequence Description of
Artificial Sequence Primer 9 ctatctgagg aacaaccaac tagtagc 27 10 24
DNA Artificial Sequence Description of Artificial Sequence Primer
10 taggacattg cacctagggt ttgt 24 11 30 DNA Artificial Sequence
Description of Artificial Sequence Primer 11 atttttttat aaatcatcaa
gcctagggca 30 12 30 DNA Artificial Sequence Description of
Artificial Sequence Primer 12 aattaaagga gggaagaatg acagaaatgt 30
13 27 DNA Artificial Sequence Description of Artificial Sequence
Primer 13 aagcttgttc taccacctga actaggc 27 14 20 DNA Artificial
Sequence Description of Artificial Sequence Primer 14 ttacatatga
gccttccatg 20 15 11970 DNA Homo sapiens 15 aagcttctac cctagtctgg
tgctacactt acattgctta catccaagtg tggttatttc 60 tgtggctcct
gttataacta ttatagcacc aggtctatga ccaggagaat tagactggca 120
ttaaatcaga ataagagatt ttgcacctgc aatagacctt atgacaccta accaacccca
180 ttatttacaa ttaaacagga acagagggaa tactttatcc aactcacaca
agctgttttc 240 ctcccagatc catgcttttt tgcgtttatt attttttaga
gatgggggct tcactatgtt 300 gcccacactg gactaaaact ctgggcctca
agtgattgtc ctgcctcagc ctcctgaata 360 gctgggacta caggggcatg
ccatcacacc tagttcattt cctctattta aaatatacat 420 ggcttaaact
ccaactggga acccaaaaca ttcatttgct aagagtctgg tgttctacca 480
cctgaactag gctggccaca ggaattataa aagctgagaa attctttaat aatagtaacc
540 aggcaacatc attgaaggct catatgtaaa aatccatgcc ttcctttctc
ccaatctcca 600 ttcccaaact tagccactgg ttctggctga ggccttacgc
atacctcccg gggcttgcac 660 acaccttctt ctacagaaga cacaccttgg
gcatatccta cagaagacca ggcttctctc 720 tggtccttgg tagagggcta
ctttactgta acagggccag ggtggagagt tctctcctga 780 agctccatcc
cctctatagg aaatgtgttg acaatattca gaagagtaag aggatcaaga 840
cttctttgtg ctcaaatacc actgttctct tctctaccct gccctaacca ggagcttgtc
900 accccaaact ctgaggtgat ttatgcctta atcaagcaaa cttccctctt
cagaaaagat 960 ggctcatttt ccctcaaaag ttgccaggag ctgccaagta
ttctgccaat tcaccctgga 1020 gcacaatcaa caaattcagc cagaacacaa
ctacagctac tattagaact attattatta 1080 ataaattcct ctccaaatct
agccccttga cttcggattt cacgatttct cccttcctcc 1140 tagaaacttg
ataagtttcc cgcgcttccc tttttctaag actacatgtt tgtcatctta 1200
taaagcaaag gggtgaataa atgaaccaaa tcaataactt ctggaatatc tgcaaacaac
1260 aataatatca gctatgccat ctttcactat tttagccagt atcgagttga
atgaacatag 1320 aaaaatacaa aactgaattc ttccctgtaa attccccgtt
ttgacgacgc acttgtagcc 1380 acgtagccac gcctacttaa gacaattaca
aaaggcgaag aagactgact caggcttaag 1440 ctgccagcca gagagggagt
catttcattg gcgtttgagt cagcaaaggt attgtcctca 1500 catctctggc
tattaaagta ttttctgttg ttgtttttct ctttggctgt tttctctcac 1560
attgccttct ctaaagctac agtctctcct ttcttttctt gtccctccct ggtttggtat
1620 gtgacctaga attacagtca gatttcagaa aatgattctc tcattttgct
gataaggact 1680 gattcgtttt actgagggac ggcagaacta gtttcctatg
agggcatggg tgaatacaac 1740 tgaggcttct catgggaggg aatctctact
atccaaaatt attaggagaa aattgaaaat 1800 ttccaactct gtctctctct
tacctctgtg taaggcaaat accttattct tgtggtgttt 1860 ttgtaacctc
ttcaaacttt cattgattga atgcctgttc tggcaataca ttaggttggg 1920
cacataagga ataccaacat aaataaaaca ttctaaaaga agtttacgat ctaataaagg
1980 agacaggtac atagcaaact aattcaaagg agctagaaga tggagaaaat
gctgaatgtg 2040 gactaagtca ttcaacaaag ttttcaggaa gcacaaagag
gaggggctcc cctcacagat 2100 atctggatta gaggctggct gagctgatgg
tggctggtgt tctctgttgc agaagtcaag 2160 atggccaaag ttccagacat
gtttgaagac ctgaagaact gttacaggta aggaataaga 2220 tttatctctt
gtgatttaat gagggtttca aggctcacca gaatccagct aggcataaca 2280
gtggccagca tgggggcagg ccggcagagg ttgtagagat gtgtactagt cctgaagtca
2340 gagcaggttc agagaagacc cagaaaaact aagcattcag catgttaaac
tgagattaca 2400 ttggcaggga gaccgccatt ttagaaaaat tatttttgag
gtctgctgag ccctacatga 2460 atatcagcat caacttagac acagcctctg
ttgagatcac atgccctgat ataagaatgg 2520 gttttactgg tccattctca
ggaaaacttg atctcattca ggaacaggaa atggctccac 2580 agcaagctgg
gcatgtgaac tcacatatgc aggcaaatct cactcagatg tagaagaaag 2640
gtaaatgaac acaaagataa aattacggaa catattaaac taacatgatg tttccattat
2700 ctgtagtaaa tactaacaca aactaggctg tcaaaatttt gcctggatat
tttactaagt 2760 ataaattatg aaatctgttt tagtgaatac atgaaagtaa
tgtgtaacat ataatctatt 2820 tggttaaaat aaaaaggaag tgcttcaaaa
cctttctttt ctctaaagga gcttaacatt 2880 cttccctgaa cttcaattaa
agctcttcaa tttgttagcc aagtccaatt tttacagata 2940 aagcacaggt
aaagctcaaa gcctgtcttg atgactacta attccagatt agtaagatat 3000
gaattactct acctatgtgt atgtgtagaa gtccttaaat ttcaaagatg acagtaatgg
3060 ccatgtgtat gtgtgtgacc cacaactatc atggtcatta aagtacattg
gccagagacc 3120 acatgaaata acaacaatta cattctcatc atcttatttt
gacagtgaaa atgaagaaga 3180 cagttcctcc attgatcatc tgtctctgaa
tcaggtaagc aaatgactgt aattctcatg 3240 ggactgctat tcttacacag
tggtttcttc atccaaagag aacagcaatg acttgaatct 3300 taaatacttt
tgttttaccc tcactagaga tccagagacc tgtctttcat tataagtgag 3360
accagctgcc tctctaaact aatagttgat gtgcattggc ttctcccaga acagagcaga
3420 actatcccaa atccctgaga actggagtct cctggggcag gcttcatcag
gatgttagtt 3480 atgccatcct gagaaagccc cgcaggccgc ttcaccaggt
gtctgtctcc taacgtgatg 3540 tgttgtggtt gtcttctctg acaccagcat
cagaggttag agaaagtctc caaacatgaa 3600 gctgagagag aggaagcaag
ccagctgaaa gtgagaagtc tacagccact catcaatctg 3660 tgttattgtg
tttggagacc acaaatagac actataagta ctgcctagta tgtcttcagt 3720
actggcttta aaagctgtcc ccaaaggagt atttctaaaa tattttgagc attgttaagc
3780 agatttttaa cctcctgaga gggaactaat tggaaagcta ccactcacta
caatcattgt 3840 taacctattt agttacaaca tctcattttt gagcatgcaa
ataaatgaaa aagtcttcct 3900 aaaaaaatca tctttttatc ctggaaggag
gaaggaaggt gagacaaaag ggagagaggg 3960 agggaagcct aatgaaacac
cagttaccta agaccagaat ggagatcctc ctcactacct 4020 ctgttgaata
cagcacctac tgaaagaact ttcattccct gaccatgaac agcctctcag 4080
cttctgtttt ccttcctcac agaaatcctt ctatcatgta agctatggcc cactccatga
4140 aggctgcatg gatcaatctg tgtctctgag tatctctgaa acctctaaaa
catccaagct 4200 taccttcaag gagagcatgg tggtagtagc aaccaacggg
aaggttctga agaagagacg 4260 gttgagttta agccaatcca tcactgatga
tgacctggag gccatcgcca atgactcaga 4320 ggaaggtaag gggtcaagca
caataatatc tttcttttac agttttaagc aagtagggac 4380 agtagaattt
aggggaaaat taaacgtgga gtcagaataa caagaagaca accaagcatt 4440
agtctggtaa ctatacagag gaaaattaat ttttatcctt ctccaggagg gagaaatgag
4500 cagtggcctg aatcgagaat acttgctcac agccattatt tcttagccat
attgtaaagg 4560 tcgtgtgact tttagccttt caggagaaag cagtaataag
accacttacg agctatgttc 4620 ctctcatact aactatgcct ccttggtcat
gttacataat cttttcgtga ttcagtttcc 4680 tctactgtaa aatggagata
atcagaatcc cccactcatt ggattgttgt aaagattaag 4740 agtctcaggc
tttacagact gagctagctg ggccctcctg actgttataa agattaaatg 4800
agtcaacatc ccctaacttc tggactagaa taatgtctgg tacaaagtaa gcacccaata
4860 aatgttagct attactatca ttattattat tattttattt tttttttttg
agatggagtc 4920 tggctctgtc acccaggctg gagtgcagtg gcacaatctc
ggctcactgc aagctctgcc 4980 tcctgggttc atgccattct cctgcctcag
cctcccgagt aagctgggaa tacaggcacc 5040 cgccactgtt cccggctaat
tttttgtatt tttagtagag acggagtttc accgtggtct 5100 ccatctcctc
gtgatccacc caccttggcc tcccaaagtg ccgggattac aggcgtgagc 5160
caccgcgccc ggcctattat tattattatt actactacta ctacctatat gaatactacc
5220 agcaatacta atttattaat gactggatta tgtctaaacc tcacaagaat
cctaccttct 5280 cattttacat aaaaggaaac taagctcatt gagataggta
aactgcccaa tggcatacat 5340 ctgtaagtgg gagagcctca aatctaattc
agttctacct gagtaaaaaa atcatggttt 5400 ctcctccatc cctttactgt
acaagcctcc acatgaacta taaacccaat attcctgttt 5460 ttaagataat
acctaagcaa taacgcatgt tcacctagaa ggttttaaaa tgtaacaaaa 5520
tataagaaaa taaaaatcac tcatatcgtc agtgagagtt tactactgcc agcactatgg
5580 tatgtttcct taaaatcttt gctatacaca tacctacatg tgaacaaata
tgtctaacat 5640 caagaccaca ctatttacaa ctttatatcc agcttttctt
acttagcaat gtattgagga 5700 cattttagag tgcccgtttt tcaccattat
aagcaatgca acaatgaaca tctgtataaa 5760 taaatattca tttctctcac
cctttatttc cttagaatat attcctagaa gtagaatttc 5820 ccagagccat
gaggatttgt gacgctattg atatgtgcca ctttgcactc tctgtgacat 5880
atataattat ttttaatgca ttcatttttt tctcagagtg cattcgtttg aaaacataga
5940 cgggaaatac tggtagtctt ccttgtcagt tagaaacacc caaacaatga
aaaatgaaaa 6000 agttgcacaa atagtctcta aaaacaatga aactattgcc
tgaggaattg aagtttaaaa 6060 agaagcacat aagcaacaac aaggataatc
ctagaaaacc agttctgctg actgggtgat 6120 ttcacttctc tttgcttcct
catctggatt ggaatattcc taataccccc tccagaacta 6180 ttttccctgt
ttgtactaga ctgtgtatat catctgtgtt tgtacataga cattaatctg 6240
cacttgtgat catggtttta gaaatcatca agcctaggtc atcacctttt agcttcctga
6300 gcaatgtgaa atacaacttt atgaggatca tcaaatacga attcatcctg
aatgacgccc 6360 tcaatcaaag tataattcga gccaatgatc agtacctcac
ggctgctgca ttacataatc 6420 tggatgaagc aggtacatta aaatggcacc
agacatttct gtcatcctcc cctcctttca 6480 tttacttatt tatttatttc
aatctttctg cttgcaaaaa acatacctct tcagagttct 6540 gggttgcaca
attcttccag aatagcttga agcacagcac ccccataaaa atcccaagcc 6600
agggcagaag gttcaactaa atctggaagt tccacaagag agaagtttcc tatctttgag
6660 agtaaagggt tgtgcacaaa gctagctgat gtactacctc tttggttctt
tcagacattc 6720 ttaccctcaa ttttaaaact gaggaaactg tcagacatat
taaatgattt actcagattt 6780 acccagaagc caatgaagaa caatcactct
cctttaaaaa gtctgttgat caaactcaca 6840 agtaacacca aaccaggaag
atctttatta tctctgataa catatttgtg aggcaaaacc 6900 tccaataagc
tacaaatatg gcttaaagga tgaagtttag tgtccaaaaa cttttatcac 6960
acacatccaa ttttcatggc ggacatgttt tagtttcaac agtatacata ttttcaaagg
7020 tccagagagg caattttgca ataaacaagc aagacttttt ctgattggat
gcacttcagc 7080 taacatgctt tcaactctac atttacaaat tattttgtgt
tctatttttc tacttaatat 7140 tatttctgca attttcccaa tattgacatc
gtgtatgtat ttgccatttt taatatcact 7200 agacaattca atcaggttgc
tacgttggtc ccttgggttt actctaaata gcttgattgc 7260 aaatatcttt
gtatatatta ttgttttttc tcctatcttg taatttcttt gagcacatcc 7320
caaagaggaa tgcctagatc aatgggcaca aataatttga cagctcttat taaacattat
7380 tctgtaagta aaaactgaac tacttttcag tatcactagc aacatatgag
tgtatcagct 7440 tcctaaaccc ctccatgtta ggtcattatg aacttatgat
ctaacaaatt acagggtctt 7500 atcccactaa tgaaattata agagattcaa
cacttattca gccccgaagg attcattcaa 7560 cgtagaaaat tctaagaaca
ttaaccaagt atttacctgc ctagtgagtg tggaagacat 7620 tgtgaaggac
acaaagatgt atagaattcc attcctgact tccaggtatt tacaccatag 7680
gtggggacct aactacacac acacacacac acacacacac acacacacac accatgcaca
7740 cacaatctac atcaacactt gattttatac aaatacaatg aatttacttt
ctttttggtt 7800 cttctcttca ccagtgaaat ttgacatggg tgcttataag
tcatcaaagg atgatgctaa 7860 aattaccgtg attctaagaa tctcaaaaac
tcaattgtat gtgactgccc aagatgaaga 7920 ccaaccagtg ctgctgaagg
tcagttgtcc tttgtctcca acttaccttc atttacatct 7980 catatgtttg
taaataagcc caataggcag acacctctaa caaggtgaca ctgtcctctt 8040
tccttcctac cacagccccc acctacccac cccactccca ttgattccag aggcgtgcct
8100 aggcaggatc tatgagaaaa tataacagag agtaagagga aaattacctt
ctttcttttt 8160 cctttccctg cctgacctta ttcacctccc atcccagagc
atccatttat tccattgatc 8220 tttactgaca tctattatct gacctacaca
atactagaca ttaggacaat gtggcctgcc 8280 tccaagaaac tcaaataagc
caactgagat cagagaggat taatcacctg ccaatgggca 8340 caaagcaaca
agctgggagc caagtcccaa aatggggcct gctgcttcca gttcccctct 8400
ctctgcattg atgtcagcat tatccttcgt cccagtcctg tctccactac cactttcccc
8460 ctcaaacaca cacacacaca acagccttag atgttttctc cactgataag
taggtgactc 8520 aatttgtaag tatataatcc aagaccttct attcccaagt
agaatttatg tgcctgcctg 8580 tgcttttcta cctggatcaa gtgatgtcta
cagagtaggg cagtagcttc attcatgaac 8640 tcattcaaca agcattattc
actgagagcc ttgtattttt caggcatagt gccaacagca 8700 gtgtggacag
tggtgcatca aagcctctag tctcatagaa cttagtcttc tggaggatat 8760
ggaaaacaga caacccaaac aaccaacaaa agagcaagat gctgcaaaaa aaaaaaaaat
8820 gaatagggtg ctaagataga gaaaagtggg agagtgctat ttagacaaag
tggtaaaaac 8880 aaagcccctt gtgagatgag agctgccgac agagggggcg
ggtcatggtt gtgggttttt 8940 gggtaggaca ttcagaggag ggggcgggtc
gtggttgtgg gtttttgggt aggacattca 9000 gaggaggggg cgggtcgtgg
ttgtgggttt ttgggtagga cattcagagg agggggcggg 9060 tcgtggttgt
gggtttttgg gtaggacatt cagaggaggg ggcgggtcgt ggttgtgggt 9120
ttttgggaca ttcagaggag tctgaatgca cccaggccta caacttcaag atggtaaagg
9180 acagctccaa ggatcagaag aagcattctt ggaactgggg cattttgaga
aggaggaaaa 9240 atatgcagag actagtgctt gcagagcttg catttggatt
tcatttgagg tacaatgaaa 9300 acccattaat gggtttcaca cagtgcaatg
gcctgacctc acttatattt cctaaaatag 9360 aaaacagatc agaaggaagg
caatagagaa gcagaaagtc caatgaggag gtttcacagc 9420 agtcatgggg
gtggggtaag gaaaagaagt ggaaagaaac agacagaatt gggttatatt 9480
ttggagatag aaccaacaga aggaagagga gaaacaacat ttactgagaa gggaaaaagt
9540 aggagaggaa taggtttggg aaataaatcc tgctgacatt ggaaacccca
aggaagcctc 9600 aaaagtatat ttacttgctt tagatttaaa agaataggaa
agaagcatct caacttggaa 9660 tttgaaatct atttttccat aaaagtattg
ttaaattcta ctcatactca caagaaaagt 9720 acattctaaa gagtatattg
aaagagttta ctgatatact taggaatttt gtgtgtatgt 9780 gtgtgtgtgt
atgtgtgtgt gtgtgtttaa ccttcaattg ttgacttaaa tactgagata 9840
aatgtcatct aaatgctaaa ttgatttccc aaaggtatga tttgttcact tggagatcaa
9900 aatgtttagg gggcttagaa tcactgtagt gctcagattt gatgcaaaat
gtcttaggcc 9960 tatgttgaag gcaggacaga aacaatgttt ccctcctacc
tgcctggata cagtaagata 10020 ctagtgtcac tgacaatctt cataactaat
ttagatctct ctccaatcaa ctaaggaaat 10080 caactcttat taatagactg
ggccacacat ctactaggca tgtaataaat gcttgctgaa 10140 tgaacaaatg
aatgaagagc ctatagcatc atgttacagc catagtccta aagtggtgtt 10200
tctcatgaag gccaaatgct aagggattga gcttcagtcc tttttctaac atcttgttct
10260 ctaacagaat tctcttcttt tcttcatagg agatgcctga gatacccaaa
accatcacag 10320 gtagtgagac caacctcctc ttcttctggg aaactcacgg
cactaagaac tatttcacat 10380 cagttgccca tccaaacttg tttattgcca
caaagcaaga ctactgggtg tgcttggcag 10440 gggggccacc ctctatcact
gactttcaga tactggaaaa ccaggcgtag gtctggagtc 10500 tcacttgtct
cacttgtgca gtgttgacag ttcatatgta ccatgtacat gaagaagcta 10560
aatcctttac tgttagtcat ttgctgagca tgtactgagc cttgtaattc taaatgaatg
10620 tttacactct ttgtaagagt ggaaccaaca ctaacatata atgttgttat
ttaaagaaca 10680 ccctatattt tgcatagtac caatcatttt aattattatt
cttcataaca attttaggag 10740 gaccagagct actgactatg gctaccaaaa
agactctacc catattacag atgggcaaat 10800 taaggcataa gaaaactaag
aaatatgcac aatagcagtt gaaacaagaa gccacagacc 10860 taggatttca
tgatttcatt tcaactgttt gccttctgct tttaagttgc tgatgaactc 10920
ttaatcaaat agcataagtt tctgggacct cagttttatc attttcaaaa tggagggaat
10980 aatacctaag ccttcctgcc gcaacagttt tttatgctaa tcagggaggt
cattttggta 11040 aaatacttct cgaagccgag cctcaagatg aaggcaaagc
acgaaatgtt attttttaat 11100 tattatttat atatgtattt ataaatatat
ttaagataat tataatatac tatatttatg 11160 ggaacccctt catcctctga
gtgtgaccag gcatcctcca caatagcaga cagtgttttc 11220 tgggataagt
aagtttgatt tcattaatac agggcatttt ggtccaagtt gtgcttatcc 11280
catagccagg aaactctgca ttctagtact tgggagacct gtaatcatat aataaatgta
11340 cattaattac cttgagccag taattggtcc gatctttgac tcttttgcca
ttaaacttac 11400 ctgggcattc ttgtttcatt caattccacc tgcaatcaag
tcctacaagc taaaattaga 11460 tgaactcaac tttgacaacc atgagaccac
tgttatcaaa actttctttt ctggaatgta 11520 atcaatgttt cttctaggtt
ctaaaaattg tgatcagacc ataatgttac attattatca 11580 acaatagtga
ttgatagagt gttatcagtc ataactaaat aaagcttgca acaaaattct 11640
ctgacacata gttattcatt gccttaatca ttattttact gcatggtaat tagggacaaa
11700 tggtaaatgt ttacataaat aattgtattt agtgttactt tataaaatca
aaccaagatt 11760 ttatattttt ttctcctctt tgttagctgc cagtatgcat
aaatggcatt aagaatgata 11820 atatttccgg gttcacttaa agctcatatt
acacatacac aaaacatgtg ttcccatctt 11880 tatacaaact cacacataca
gagctacatt aaaaacaact aataggccag gcacggtggc 11940 tcagacctgt
aatcccagca ctttgggagg 11970 16 9721 DNA Homo sapiens modified_base
(135)..(136) a, t, c, g, other or unknown 16 agaaagaaag agagagagaa
agaaaagaaa gaggaaggaa ggaaggaagg aagaaagaca 60 ggctctgagg
aaggtggcag ttcctacaac gggagaacca gtggttaatt tgcaaagtgg 120
atcctgtgga ggcanncaga ggagtcccct aggccaccca gacagggctt ttagctatct
180 gcaggccaga caccaaattt caggagggct cagtgttagg aatggattat
ggcttatcaa 240 attcacagga aactaacatg ttgaacagct tttagatttc
ctgtggaaaa tataacttac 300 taaagatgga gttcttgtga ctgactcctg
atatcaagat actgggagcc aaattaaaaa 360 tcagaaggct gcttggagag
caagtccatg aaatgctctt tttcccacag tagaacctat 420 ttccctcgtg
tctcaaatac ttgcacagag gctcactccc ttggataatg cagagcgagc 480
acgatacctg gcacatacta atttgaataa aatgctgtca aattcccatt cacccattca
540 agcagcaaac tctatctcac ctgaatgtac atgccaggca ctgtgctaga
cttggctcaa 600 aaagatttca gtttcctgga ggaaccagga gggcaaggtt
tcaactcagt gctataagaa 660 gtgttacagg ctggacacgg tggctcacgc
ctgtaatccc aacatttggg aggccgaggc 720 gggcagatca caaggtcagg
agatcgagac catcctggct aacatggtga aaccctgtct 780 ctactaaaaa
tacaaaaaat tagccgggcg ttggcggcag gtgcctgtag tcccagctgc 840
tggggaggct gaggcaggag aatggtgtga acccgggagg cggaacttgc agggggccga
900 gatcgtgcca ctgcactcca gcctgggcga cagagtgaga ctctgtctca
aaaaaaaaaa 960 aaaagtgtta tgatgcagac ctgtcaaaga ggcaaaggag
ggtgttccta cactccaggc 1020 actgttcata acctggactc tcattcattc
tacaaatgga gggctcccct gggcagatcc 1080 ctggagcagg cactttgctg
gtgtctcggt taaagagaaa ctgataactc ttggtattac 1140 caagagatag
agtctcagat ggatattctt acagaaacaa tattcccact tttcagagtt 1200
caccaaaaaa tcattttagg cagagctcat ctggcattga
tctggttcat ccatgagatt 1260 ggctagggta acagcacctg gtcttgcagg
gttgtgtgag cttatctcca gggttgcccc 1320 aactccgtca ggagcctgaa
ccctgcatac cgtatgttct ctgccccagc caagaaaggt 1380 caattttctc
ctcagaggct cctgcaattg acagagagct cccgaggcag agaacagcac 1440
ccaaggtaga gacccacacc ctcaatacag acagggaggg ctattggccc ttcattgtac
1500 ccatttatcc atctgtaagt gggaagattc ctaaacttaa gtacaaagaa
gtgaatgaag 1560 aaaagtatgt gcatgtataa atctgtgtgt cttccacttt
gtcccacata tactaaattt 1620 aaacattctt ctaacgtggg aaaatccagt
attttaatgt ggacatcaac tgcacaacga 1680 ttgtcaggaa aacaatgcat
atttgcatgg tgatacattt gcaaaatgtg tcatagtttg 1740 ctactccttg
cccttccatg aaccagagaa ttatctcagt ttattagtcc cctcccctaa 1800
gaagcttcca ccaatactct tttccccttt cctttaactt gattgtgaaa tcaggtattc
1860 aacagagaaa tttctcagcc tcctacttct gcttttgaaa gctataaaaa
cagcgaggga 1920 gaaactggca gataccaaac ctcttcgagg cacaaggcac
aacaggctgc tctgggattc 1980 tcttcagcca atcttcattg ctcaagtatg
actttaatct tccttacaac taggtgctaa 2040 gggagtctct ctgtctctct
gcctctttgt gtgtatgcat attctctctc tctctctctt 2100 tctttctctg
tctctcctct ccttcctctc tgcctcctct ctcagctttt tgcaaaaatg 2160
ccaggtgtaa tataatgctt atgactcggg aaatattctg ggaatggata ctgcttatct
2220 aacagctgac accctaaagg ttagtgtcaa agcctctgct ccagctctcc
tagccaatac 2280 attgctagtt ggggtttggt ttagcaaatg cttttctcta
gacccaaagg acttctcttt 2340 cacacattca ttcatttact cagagatcat
ttctttgcat gactgccatg cactggatgc 2400 tgagagaaat cacacatgaa
cgtagccgtc atggggaagt cactcatttt ctccttttta 2460 cacaggtgtc
tgaagcagcc atggcagaag tacctgagct cgccagtgaa atgatggctt 2520
attacaggtc agtggagacg ctgagaccag taacatgagc aggtctcctc tttcaagagt
2580 agagtgttat ctgtgcttgg agaccagatt tttcccctaa attgcctctt
tcagtggcaa 2640 acagggtgcc aagtaaatct gatttaaaga ctactttccc
attacaagtc cctccagcct 2700 tgggacctgg aggctatcca gatgtgttgt
tgcaagggct tcctgcagag gcaaatgggg 2760 agaaaagatt ccaagcccac
aatacaagga atccctttgc aaagtgtggc ttggagggag 2820 agggagagct
cagattttag ctgactctgc tgggctagag gttaggcctc aagatccaac 2880
agggagcacc agggtgccca cctgccaggc ctagaatctg ccttctggac tgttctgcgc
2940 atatcactgt gaaacttgcc aggtgtttca ggcagctttg agaggcaggc
tgtttgcagt 3000 ttcttatgaa cagtcaagtc ttgtacacag ggaaggaaaa
ataaacctgt ttagaagaca 3060 taattgagac atgtccctgt ttttattaca
gtggcaatga ggatgacttg ttctttgaag 3120 ctgatggccc taaacagatg
aaggtaagac tatgggttta actcccaacc caaggaaggg 3180 ctctaacaca
gggaaagctc aaagaaggga gttctgggcc actttgatgc catggtattt 3240
tgttttagaa agactttaac ctcttccagt gagacacagg ctgcaccact tgctgacctg
3300 gccacttggt catcatatca ccacagtcac tcactaacgt tggtggtggt
ggccacactt 3360 ggtggtgaca ggggaggagt agtgataatg ttcccatttc
atagtaggaa gacaaccaag 3420 tcttcaacat aaatttgatt atccttttaa
gagatggatt cagcctatgc caatcacttg 3480 agttaaactc tgaaaccaag
agatgatctt gagaactaac atatgtctac cccttttgag 3540 tagaatagtt
ttttgctacc tggggtgaag cttataacaa caagacatag atgatataaa 3600
caaaaagatg aattgagact tgaaagaaaa ccattcactt gctgtttgac cttgacaagt
3660 cattttaccc gctttggacc tcatctgaaa aataaagggc tgagctggat
gatctctgag 3720 attccagcat cctgcaacct ccagttctga aatattttca
gttgtagcta agggcatttg 3780 ggcagcaaat ggtcattttt cagactcatc
cttacaaaga gccatgttat attcctgctg 3840 tcccttctgt tttatatgat
gctcagtagc cttcctaggt gcccagccat cagcctagct 3900 aggtcagttg
tgcaggttgg aggcagccac ttttctctgg ctttatttta ttccagtttg 3960
tgatagcctc ccctagcctc ataatccagt cctcaatctt gttaaaaaca tatttcttta
4020 gaagttttaa gactggcata acttcttggc tgcagctgtg ggaggagccc
attggcttgt 4080 ctgcctggcc tttgcccccc attgcctctt ccagcagctt
ggctctgctc caggcaggaa 4140 attctctcct gctcaacttt cttttgtgca
cttacaggtc tctttaactg tctttcaagc 4200 ctttgaacca ttatcagcct
taaggcaacc tcagtgaagc cttaatacgg agcttctctg 4260 aataagagga
aagtggtaac atttcacaaa aagtactctc acaggatttg cagaatgcct 4320
atgagacagt gttatgaaaa aggaaaaaaa agaacagtgt agaaaaattg aatacttgct
4380 gagtgagcat aggtgaatgg aaaatgttat ggtcatctgc atgaaaaagc
aaatcatagt 4440 gtgacagcat tagggataca aaaagatata gagaaggtat
acatgtatgg tgtaggtggg 4500 gcatgtacaa aaagatgaca agtagaatcg
ggatttattc taaagaatag cctgtaaggt 4560 gtccagaagc cacattctag
tcttgagtct gcctctacct gctgtgtgcc cttgagtaca 4620 cccttaacct
ccttgagctt cagagaggga taatcttttt attttatttt attttatttt 4680
gttttgtttt gttttgtttt gttttatgag acagagtctc actctgttgc ccaggctgga
4740 gtgcagtggt acaatcttgg cttactgcat cctccacctc ctgagttcaa
gcgattctcc 4800 ttcctcagtc tcctgaatag ctaggattac aggtgcaccc
caccacaccc agctaatttt 4860 tgtattttta gtagagaagg ggtttcgcca
tgttggccag gctggttttg aagtcctgac 4920 ctaaatgatt catccacctc
ggcttcccaa agtgctggga ttacaggcat gagccaccac 4980 gcctggccca
gagagggatg atctttagaa gctcgggatt ctttcaagcc ctttcctcct 5040
ctctgagctt tctactctct gatgtcaaag catggttcct ggcaggacca cctcaccagg
5100 ctccctccct cgctctctcc gcagtgctcc ttccaggacc tggacctctg
ccctctggat 5160 ggcggcatcc agctacgaat ctccgaccac cactacagca
agggcttcag gcaggccgcg 5220 tcagttgttg tggccatgga caagctgagg
aagatgctgg ttccctgccc acagaccttc 5280 caggagaatg acctgagcac
cttctttccc ttcatctttg aagaaggtag ttagccaaga 5340 gcaggcagta
gatctccact tgtgtcctct tggaagtcat caagccccag ccaactcaat 5400
tcccccagag ccaaagccct ttaaaggtag aaggcccagc ggggagacaa aacaaagaag
5460 gctggaaacc aaagcaatca tctctttagt ggaaactatt cttaaagaag
atcttgatgg 5520 ctactgacat ttgcaactcc ctcactcttt ctcaggggcc
tttcacttac attgtcacca 5580 gaggttcgta acctccctgt gggctagtgt
tatgaccatc accattttac ctaagtagct 5640 ctgttgctcg gccacagtga
gcagtaatag acctgaagct ggaacccatg tctaatagtg 5700 tcaggtccag
tgttcttagc caccccactc ccagcttcat ccctactggt gttgtcatca 5760
gactttgacc gtatatgctc aggtgtcctc caagaaatca aattttgcca cctcgcctca
5820 cgaggcctgc ccttctgatt ttatacctaa acaacatgtg ctccacattt
cagaacctat 5880 cttcttcgac acatgggata acgaggctta tgtgcacgat
gcacctgtac gatcactgaa 5940 ctgcacgctc cgggactcac agcaaaaaag
cttggtgatg tctggtccat atgaactgaa 6000 agctctccac ctccagggac
aggatatgga gcaacaaggt aaatggaaac atcctggttt 6060 ccctgcctgg
cctcctggca gcttgctaat tctccatgtt ttaaacaaag tagaaagtta 6120
atttaaggca aatgatcaac acaagtgaaa aaaaatatta aaaaggaata tacaaacttt
6180 ggtcctagaa atggcacatt tgattgcact ggccagtgca tttgttaaca
ggagtgtgac 6240 cctgagaaat tagacggctc aagcactccc aggaccatgt
ccacccaagt ctcttgggca 6300 tagtgcagtg tcaattcttc cacaatatgg
ggtcatttga tggacatggc ctaactgcct 6360 gtgggttctc tcttcctgtt
gttgaggctg aaacaagagt gctggagcga taatgtgtcc 6420 atccccctcc
ccagtcttcc ccccttgccc caacatccgt cccacccaat gccaggtggt 6480
tccttgtagg gaaattttac cgcccagcag gaacttatat ctctccgctg taacgggcaa
6540 aagtttcaag tgcggtgaac ccatcattag ctgtggtgat ctgcctggca
tcgtgccaca 6600 gtagccaaag cctctgcaca ggagtgtggg caactaaggc
tgctgacttt gaaggacagc 6660 ctcactcagg gggaagctat ttgctctcag
ccaggccaag aaaatcctgt ttctttggaa 6720 tcgggtagta agagtgatcc
cagggcctcc aattgacact gctgtgactg aggaagatca 6780 aaatgagtgt
ctctctttgg agccactttc ccagctcagc ctctcctctc ccagtttctt 6840
cccatgggct actctctgtt cctgaaacag ttctggtgcc tgatttctgg cagaagtaca
6900 gcttcacctc tttcctttcc ttccacattg atcaagttgt tccgctcctg
tggatgggca 6960 cattgccagc cagtgacaca atggcttcct tccttccttc
cttcagcatt taaaatgtag 7020 accctctttc attctccgtt cctactgcta
tgaggctctg agaaaccctc aggcctttga 7080 ggggaaaccc taaatcaaca
aaatgaccct gctattgtct gtgagaagtc aagttatcct 7140 gtgtcttagg
ccaaggaacc tcactgtggg ttcccacaga ggctaccaat tacatgtatc 7200
ctactctcgg ggctaggggt tggggtgacc ctgcatgctg tgtccctaac cacaagaccc
7260 ccttctttct tcagtggtgt tctccatgtc ctttgtacaa ggagaagaaa
gtaatgacaa 7320 aatacctgtg gccttgggcc tcaaggaaaa gaatctgtac
ctgtcctgcg tgttgaaaga 7380 tgataagccc actctacagc tggaggtaag
tgaatgctat ggaatgaagc ccttctcagc 7440 ctcctgctac cacttattcc
cagacaattc accttctccc cgcccccatc cctaggaaaa 7500 gctgggaaca
ggtctatttg acaagttttg cattaatgta aataaattta acataatttt 7560
taactgcgtg caaccttcaa tcctgctgca gaaaattaaa tcattttgcc gatgttatta
7620 tgtcctacca tagttacaac cccaacagat tatatattgt tagggctgct
ctcatttgat 7680 agacaccttg ggaaatagat gacttaaagg gtcccattat
cacgtccact ccactcccaa 7740 aatcaccacc actatcacct ccagctttct
cagcaaaagc ttcatttcca agttgatgtc 7800 attctaggac cataaggaaa
aatacaataa aaagcccctg gaaactaggt acttcaagaa 7860 gctctagctt
aattttcacc cccccaaaaa aaaaaaattc tcacctacat tatgctcctc 7920
agcatttggc actaagtttt agaaaagaag aagggctctt ttaataatca cacagaaagt
7980 tgggggccca gttacaactc aggagtctgg ctcctgatca tgtgacctgc
tcgtcagttt 8040 cctttctggc caacccaaag aacatctttc ccataggcat
ctttgtccct tgccccacaa 8100 aaattcttct ttctctttcg ctgcagagtg
tagatcccaa aaattaccca aagaagaaga 8160 tggaaaagcg atttgtcttc
aacaagatag aaatcaataa caagctggaa tttgagtctg 8220 cccagttccc
caactggtac atcagcacct ctcaagcaga aaacatgccc gtcttcctgg 8280
gagggaccaa aggcggccag gatataactg acttcaccat gcaatttgtg tcttcctaaa
8340 gagagctgta cccagagagt cctgtgctga atgtggactc aatccctagg
gctggcagaa 8400 agggaacaga aaggtttttg agtacggcta tagcctggac
tttcctgttg tctacaccaa 8460 tgcccaactg cctgccttag ggtagtgcta
agaggatctc ctgtccatca gccaggacag 8520 tcagctctct cctttcaggg
ccaatcccca gcccttttgt tgagccaggc ctctctcacc 8580 tctcctactc
acttaaagcc cgcctgacag aaaccacggc cacatttggt tctaagaaac 8640
cctctgtcat tcgctcccac attctgatga gcaaccgctt ccctatttat ttatttattt
8700 gtttgtttgt tttgattcat tggtctaatt tattcaaagg gggcaagaag
tagcagtgtc 8760 tgtaaaagag cctagttttt aatagctatg gaatcaattc
aatttggact ggtgtgctct 8820 ctttaaatca agtcctttaa ttaagactga
aaatatataa gctcagatta tttaaatggg 8880 aatatttata aatgagcaaa
tatcatactg ttcaatggtt ctgaaataaa cttcactgaa 8940 gaaaaaaaaa
aaagggtctc tcctgatcat tgactgtctg gattgacact gacagtaagc 9000
aaacaggctg tgagagttct tgggactaag cccactcctc attgctgagt gctgcaagta
9060 cctagaaata tccttggcca ccgaagacta tcctcctcac ccatcccctt
tatttcgttg 9120 ttcaacagaa ggatattcag tgcacatctg gaacaggatc
agctgaagca ctgcagggag 9180 tcaggactgg tagtaacagc taccatgatt
tatctatcaa tgcaccaaac atctgttgag 9240 caagcgctat gtactaggag
ctgggagtac agagatgaga acagtcacaa gtccctcctc 9300 agataggaga
ggcagctagt tataagcaga acaaggtaac atgacaagta gagtaagata 9360
gaagaacgaa gaggagtagc caggaaggag ggaggagaac gacataagaa tcaagcctaa
9420 agggataaac agaagatttc cacacatggg ctgggccaat tgggtgtcgg
ttacgcctgt 9480 aatcccagca ctttgggtgg caggggcaga aagatcgctt
gagcccagga gttcaagacc 9540 agcctgggca acatagtgag actcccatct
ctacaaaaaa taaataaata aataaaacaa 9600 tcagccaggc atgctggcat
gcacctgtag tcctagctac ttgggaagct gacactggag 9660 gattgcttga
gcccagaagt tcaagactgc agtgagctta tccgttgacc tgcaggtcga 9720 c 9721
17 12565 DNA Homo sapiens 17 gtcgacctgc aggtcaacgg atctgagagg
agagtagctt cttgtagata acagttggat 60 tatataccat gtcctgatcc
ccttcatcat ccaggagagc agaggtggtc accctgatag 120 cagcaagcct
gggggctgca gcttggtggg tagaggtact caggggtaca gatgtctcca 180
aacctgtcct gctgccttag ggagcttcta ataagttgat ggatttggtt aaaattaact
240 tggctacttg gcaggactgg gtcagtgagg accaacaaaa agaagacatc
agattatacc 300 ctgggggttt gtatttcttg tgtttctttc tcttctttgt
actaaaatat ttacccatga 360 ctgggaaaga gcaactggag tctttgtagc
attatcttag caaaaattta caaagtttgg 420 aaaacaatat tgcccatatt
gtgtggtgtg tcctgtgaca ctcaggattc aagtgttggc 480 cgaagccact
aaatgtgaga tgaagccatt acaaggcagt gtgcacatct gtccacccaa 540
gctggatgcc aacatttcac aaatagtgct tgcgtgacac aaatgcagtt ccaggaggcc
600 caaatgaaaa tgtttgtact gaaatttgtt aaagcttccc gacaaactag
atttatcagt 660 aaggattgtt ttctgcaagg gggatgaaac ttgtggggtg
agccatttgg gctgaggagg 720 agggaggttg gagctgagaa atgtggagac
aatttccctt tagaaggact gaatctccct 780 gcctctctgg ggtgcggcag
ccagcaggat ccaatggtgt atatgtctcc ccagctcccc 840 attcagtgat
atcatgtcag tagcttgaaa ttatccgtgg tgggagtatt atgtcatgga 900
aattggcaaa tggaaacttt tattggagat tcaattgtta aacttttacc agcacaacac
960 tgccctgcct tcagagtcaa tgaccctatc caagtttaat ccatctgtcc
actgtctcca 1020 acacgatctt tataaaacac acctgacaac attacccttt
tattcagttt tttaaaagat 1080 aagtttccag ctcatcgggg tggctttaaa
ggccatttct cctctggacc tcacccaact 1140 tttcaaatca cttttcctac
ccctacctct aaatgctact caaactccag ccatcctgaa 1200 taataagact
tttgaaaagt agattatggg ctgggcacag tggctcacac ctgtaatccc 1260
agcactttgg gaggccaaga tgggtggatc acctgaggtc gggagttcga gaccagcctg
1320 actaacatag tgaaaccctg tctctactaa aaatacaaaa ttagttgggg
gtggtggcac 1380 aagcctgtaa tcccagctac tcaggaggtt gaggcagggg
aattgcttga acctgggagg 1440 cggaggttgc ggtgagccta gattgctcca
ctgcactcca gcctgggcaa caagagcgaa 1500 actccatctc aaaaaaataa
ataaataaat aaagtagatt acatcagata cctctggcct 1560 aggttgttta
tgaccaactc tcctgctgag aataactaga aaagctagac aaaacatatt 1620
tccaaaagat ctctttggag gcatcagaga atggccaagg ctgtaaggaa ctgcctgagc
1680 ccagagaggt ggagcccagc actggtgccc tttactcctg gggacatgtg
ctggtttcaa 1740 aaacttcagc tgagcttttg agcattcatg gaacttggtg
ggggagatga aatttgtacc 1800 ttaaatcctg cctacaggga gggtccctga
taatccccac ccaatttgga aatctgggtc 1860 agccttcaca ggtactgaag
ccctcctctg aatgatctca agtcctgcta gggtagaggt 1920 tacctgcttt
tgaaaggctc ctggcctacc tgtgcagcag gagcaaaagt gaaccatctc 1980
agggtacaga taacaatcat ccagagcctt gaatgacctc tactgtgctt aatatatagt
2040 attcagcagt cagtaaaaag gatttaggca catgcaagat gacctgtgta
tcagggagaa 2100 ataggcaata aattgagatc cagcagggat ttgaatcatg
gatttgaatc aggggcagcc 2160 ttcgaaagaa ctatggagaa tatactcaga
tttaaaacat aagattggaa tttttggcag 2220 agaactaaca actgtacaaa
aaaggaacca aatggaaatc ctagaactga aagatgcaat 2280 taaccgatgt
tgagaaatag ccaacatcta ttgaacactt cccatgtgga cagctgtgct 2340
aaacacttta caggcatcaa cataagatgt gtccccttac agcagtgcag tgtccctcct
2400 aagacatgga cagcctggtt tccctatctc tctgcttcat caaaacccct
ttacgtgggg 2460 cttagacact cctgttgtct ctagtgtcta gtagcacagg
gctcagcaca tggaagccac 2520 tagatacaat ttgatgacca ggacctccga
tgaaagccat gggtgctgat tgggaaggca 2580 ttgtctttta tgtgctatgg
tcttaaagct tcatccagga agcagaactc ggggggtgct 2640 gaggacccag
aaccgagaat aagattagtc agagatttcc tgtgggcaga aatcataagg 2700
acgccaactg tttgggtgag ataagacgaa accaagagtg gacttgtggc cagaagcgtg
2760 aggaagaggg agagagcttc ccttgtcccc tttcttcctc tccctaagcc
acagtgattg 2820 acagcccccc cgctttggag tcagagcagg cttgagactg
gactgggaaa ggagggtggg 2880 tcaggataca gagcaggaag gctgggagtg
cagggcagga gcaaggggct ggggcattca 2940 ttgtgcctga tctctcccac
tttacctggg gtaaagaagc atatgcaaaa gccacggtgt 3000 gagtatttcc
caagtgccag ggtcagggca tgattcatca cgtgcagcat ttcattcaat 3060
ccttatagta accgatgatg tggcttctat tattagctct atcagataat gaaactgaga
3120 ccaagacagg ctctgcacat tgtgtggggt aatgacacag ggggattcag
acctagactc 3180 cataactcct gccccaggga ccacccccac cctcaccctg
tgcatgtcga caaaggacag 3240 actgggccac ttctcaggac acagcgggga
aatgacacag agcagggagg ttccaggagc 3300 cccgagcgtc ttttctccag
gagaatactc tctgaattca gactggggtc agagaaacat 3360 ttacccagga
gccgcagtgt gggtggggct ttttacttga aacgctgtct gaaggcagtg 3420
gcaggatgaa ctctccaccc taccttggca agccacttct cttctgcaat ctgtaaggac
3480 attgttgaga gaattatggt cttccaattc cggagggttg aagaaagaca
aataggagag 3540 aacctatcat agtcaggtgc tagctgcctt ctctttcaga
gagtgtgaga ataaagtgat 3600 acacttgatt attagcaaat actttggaaa
ttttaaacgc taatattcaa cacactctgg 3660 aagaggcaaa taagtagaca
ggttcatata catcatctcc ttcagctagt cctcacaaaa 3720 acaaacaaat
gaataaacaa aattcttctt tggccctcat aggaagacac tgtttcttga 3780
acgtgtttca aaaaggatgg gtgactcact caaggtcaca ctgtttatga ggacagtaca
3840 ggaatacaga catgccattt tgcctgaaaa aatccatcac ccagggaggt
gacacaattt 3900 tgcagaaatg ttctatttcc tctgaaggat acattcttta
aacctttggg aaattcattc 3960 atagtcttcc tcctttgaag gattactctc
tggacacaaa gtgtttgatt ctgatttgtt 4020 ggttggaaga tgtgttggtt
gagagaaaga ttctgatttg ttggttgaaa atagactcat 4080 caagatcaac
tgctgtagta gtaaatattt tgacattttg tctgtattcc tgtgctgccc 4140
tcacaagctg catcaccttg agtgagtcat tcatactttt ttgtttgttt ttgttttgga
4200 gatggagtct tactctgttg cctaggctgg agtgcggtgg cgtgatcttg
gctcactgcg 4260 acctccatct cctgggttca agtgatcctc ctgcctcagc
ctcccgagta gctgggatta 4320 caggcacatg ccaccatccc tgctaatttt
tgcattttca gtagagacgg agtttcacca 4380 tgttggtcag gttggtcttg
aactcctgac ctcaggtgat ccgcccacct cagcctcccc 4440 aagtgctggg
attacaggtg tgagccaccg tgcccagccc agccatcatt tttgaaacac 4500
gtttgagaaa tagtgtcttc ctttgagggc caaggagaca ttttttttgt ttatttgttt
4560 gtttttgtga ggactagctg aagggggtga tgtatattaa cctgcctact
tatttgcctc 4620 ttcccagagt gtgatgaata ttagggttta aagtttctga
agcatttgtt aataaagccc 4680 ggggctggag gtcagaagac ctggatttct
ctgcatactt ttgccatcag caagctgtgt 4740 gaccttggac agatcccttt
tttgtctaaa tctttctgag tcttcttgaa aacaatgcca 4800 ggttgggaca
ggatgattgc caagctcccg tccagctcta aaacactgca acgtatgctt 4860
ctgcaccagc actgtccatc ctgtagatca tgcagaaatt ctcttcaact ttttcctacc
4920 cataaaatag gagcatgctt acctttttcc taatgttcca ggccccgggt
ctagatattg 4980 taagtaagga agttaatgtg tatcagagcc cattatgggc
cagaagttct cctcttcctt 5040 cctacacctg cttcctccct ccctccctcc
ctctttccct tccttccttc catccatttg 5100 tgaagaagac atgatcaccc
tcattctgag agtgaagaga cagaggctca actaatgaaa 5160 tgatttgttc
aaggtcacac gggtggcaca aggcaagtgg cagaggttga atttagaccc 5220
attcctgtcc aaatgctgag tttatgtcat cgtcccgaga ccataacttt aaagatgtaa
5280 gatagtggga aaagagttga tttcaaagca cctctcagaa ggactcactt
tacatcaggg 5340 gtcagcagac tcaggccaaa tccggtccat tccccgcttt
tgcaaagaaa gttgtagtgg 5400 aacacagcta ggcttattga tttatggatt
gccaacgtcc ttttgtgaaa cagacagctg 5460 agctgagtaa tcgtggcgca
caaaacctaa aatatttact atctcgtcct ttacagaatg 5520 tttgccaatc
tatggtccgg agtccaaggc tgtccatttt tcaaagaaca caaagtgaca 5580
tgagactgtc ccatgtgcag ggagccctat cattttatta tgaaaaaacg gcctttctgc
5640 tcaaatctgt tttttaaaaa gtcaacaaac agactctggg tacctgtcag
gaacagtagg 5700 gagtttggtt tccattgtgc tcttcttccc aggaactcaa
tgaaggggaa atagaaatct 5760 taattttggg gaaattgcac aggggaaaaa
ggggagggaa tcagttacaa cactccattg 5820 cgacacttag tggggttgaa
agtgacaaca gcaagggttt ctctttttgg aaatgcgagg 5880 agggtatttc
cgcttctcgc agtggggcag ggtggcagac gcctagcttg ggtgagtgac 5940
tatttcttta taaaccacaa ctctgggccc gcaatggcag tccactgctt gctgcagtca
6000 cagaatggaa atctgcagag gcctccgcag tcacctaatc actctcctcc
tcttcctgtt 6060 ccattcagag acgatctgcc gaccctctgg gagaaaatcc
agcaagatgc aagccttcag 6120 gtaaggctac cccaaggagg agaaggtgag
ggtggatcag ctggagactg gaaacatatc 6180 acagctgcca gggctgccag
gccagagggc ctgagaactg ggtttgggct ggagaggatg 6240 tccattattc
aagaaagagg ctgttacatg catgggcttc aggacttgtg tttcaaaata 6300
tcccagatgt ggatagtgcg accggagggc tgtcttactt tcccagagac tcaggaaccc
6360 agtgagtaat agatgcatgc caaggagtgg gactgcgatt caggcctagt
tgaatgtgct 6420 gacagagaag cagagagggg caccaggggc acagcccgaa
ggcccagact gatatgggca 6480 aggcctgtct
gtgctgacat gtcggagggt cccactctcc agggaccttg gtttccccgt 6540
ctgtgacatc tgtgacatga gagtcacgat aactccttgt gtgccttaca gggttgttgt
6600 gaaaattaaa tgcacagata atagcgtaac agtattccgt gcattgtaaa
gagcctgaaa 6660 accattatga tttgaaaatg gaatcggctt tgtgagacca
tcactattgt aaagatgtga 6720 tgctgataga aatgacagga ctgcttgtgc
atgccctctg cagtgtgaca ttccagcagt 6780 gaaatcatgt tggggtgact
tctcccccac tctgaccttt atgtttgtct gggccgaggc 6840 tgcaagtcgg
gctctgtggg tgtatgagtg acaagtctct cccttccaga tatggggact 6900
gtctgcttcc ctaggttgcc tctccctgct ctgatcagct agaagctcca ggagatcctc
6960 ctggaggccc cagcaggtga tgtttatccc tccagactga ggctaaatct
agaaactagg 7020 ataatcacaa acaggccaat gctgccatat gcaaagcact
ttggtttgcc tggccacccc 7080 tcgtcgagca tgtgggctct tcagagcacc
tgatgaggtg ggtacagtta gccacacttc 7140 acaggtgaag aggtgaggca
caggtcccag gtcaggctgg ccggagctct gtttattacg 7200 tctcacagct
ttgagtcctg ctctcaacca gagaggccct ttaccaagaa gaaaggattg 7260
ggacccagaa tcaggtcact ggctgaggta gagaggaagc cgggttgttc ccaagggtag
7320 ctgctcctgc aggactctga gcaggtcacc agctaatgga ggaaaggctc
tagggaaaga 7380 cccttctggt ctcagactca gagcgagtta gctgcaaggt
gttccgtctc ttgaaacttc 7440 tacctaggtg ctatggtagc cactagtctc
aggtggctat ttaaatttat acttaaatga 7500 atgaaaatag aagaaaattt
aaaatccaga cccttggtca cactatccac atttaaagag 7560 gtcaatagcc
acatgtggtt agtggccacc ctattgggca gtgcagctac agaacatttt 7620
tgcatcccag aaagttcttt tggatgttgc tgctctacag catgctttgc tgaaacagaa
7680 gtgccttccc tgggaatctc agatgggaag caagtaagga ggggagtcaa
atgtgggctc 7740 actgctcacc agctgtgagg gttgggcctg cctcttaacc
attgtcagcc tcagtcttct 7800 catccatgca tgccgtgggt atactaaaat
actatacccc tggaagagct ggatgcaaat 7860 ttgacaagtt ctgggggaca
caggaaggtg ccaagcacaa ggctgggcac atggtggctg 7920 tgcactacag
ctgagtcctt ttccttttca gaatctggga tgttaaccag aagaccttct 7980
atctgaggaa caaccaacta gttgctggat acttgcaagg accaaatgtc aatttagaag
8040 gtgagtggtt gccaggaaag ccaatgtatc tgggcatcac gtcactttgc
ccgtctgtct 8100 gcagcagcat ggcctgcctg cacaaaccct aggtgcaatg
tcctaatcct tgttgggtct 8160 ttgtattcaa gtttgaagct gggagggcct
ggctactgaa gggcacatat gagggtagcc 8220 tgaagagggt gtggagaggt
agagtctagg tcagaggtca gtgcctatag gcaagtggtc 8280 ccagggccac
agctgggaag ggcaaatacc agaaggcaag gttgaccatt cccttcctca 8340
agtgcctatt aaggctccat gttcctatgt tgttcaaacc ctaactcaat cccaaattaa
8400 tccaccatgt ataaggttga gctatgtctc ttattcctgg acaccatact
cagccatatc 8460 tggtccacac attaacagct ggatgacctt gaagaagctt
cacccactct gttcctcagc 8520 tttcccttca gtgggatgat atcaactgga
caacaggatg tgcgattctt ttagttccag 8580 ccttccagga tgttttcact
cccctgtttg ttgttgtagg atggtattac ctccaccttc 8640 ccaccttccc
tatgccctgg ttctgtctcc tgtgcctcgc tctgaaagtg gatgagacct 8700
acaattcctg tcctggtagt tctcctaatg aacacactga agcacgagga agctgagatt
8760 tttgttgcta catgagagca tggaggcctc ttagggagag aggaggttca
gagactccta 8820 ggctcctggt ggagccccac tcatggcctt gttcattttc
cctgcccctc agcaacactc 8880 ctattgacct ggagcacagg tatcctgggg
aaagtgaggg aaatatggac atcacatgga 8940 acaacatcca ggagactcag
gcctctagga gtaactgggt agtgtgcatc ctggggaaag 9000 tgagggaaat
atggacatca catggaacaa catccaggag actcaggcct ctaggagtaa 9060
ctgggtagtg tgcatcctgg ggaaagtgag ggaaatatgg acatcacatg gaacaacatc
9120 caggagactc aggcctctag gagtaactgg gtagtgtgca tcctggggaa
agtgagggaa 9180 atatggacat cacatggaac aacatccagg agactcaggc
ctctaggagt aactgggtag 9240 tgtgcttggt ttaatcttct atttacctgc
agaccaggaa gatgagacct ctctgccctt 9300 ctgacctcgg gattttagtt
ttgtggggac caggggagat agaaaaatac ccggggtctc 9360 ttcattattg
ctgcttcctc ttctattaac ctgaccctcc cctctgttct tccccagaaa 9420
agatagatgt ggtacccatt gagcctcatg ctctgttctt gggaatccat ggagggaaga
9480 tgtgcctgtc ctgtgtcaag tctggtgatg agaccagact ccagctggag
gtaaaaacat 9540 gctttggatc tcaaatcacc ccaaaaccca gtggcttgaa
acaaccaaaa ttttttctta 9600 tgattctgtg ggttgaccag gattagctgg
gtagttctgt tccatgtggt ggaacatgct 9660 ggggtcactt tggaagctgc
attcagcaga gtgccaggct tgcgctgggc atccaaggtg 9720 gtccctcatc
ctccaggctc tctttccatg tgatctctca gtgtttaaga gttagttgga 9780
gcttccttac agcatggcgg ctgacttcca aaagggatta ttccaaaaag agcctcaaca
9840 tgcaggcgct tattatgact tctgcttgca tcatcctatt ggccaaagcc
agtcacgtgg 9900 ctaagtctag ccccctgtga gaggagactg cataagagtg
tgaacaccag gagacacggt 9960 cactgggggc caccactgta accatctacc
acaggacctg aatctctgtg tgctactccc 10020 ttgctcaagg gcccccctac
ccacgcagac ctgctgtctt ctagcaaagc ccatcctcag 10080 gacctttctc
ttccaatcct tattgactca aattgattag ttggtgctcc acccagagcc 10140
ctgtgctcct ttatctcatg taatgttaat gggtttccca gccctgggaa aacatggctt
10200 tgtctcaggg gcttgctgga tgcaacctta acctcaatgt gagtggccat
actgtggcac 10260 tgtcccatcc ctcaccaggg acactgttct ggagggtgac
tgcctgttct gtgaggagtg 10320 gggatggcta ggacattgca tggaacacac
caccacccca tcttctcaga gctcaaaccc 10380 tgacagaaca ccagctccac
aggccttggc ttctgctgat ggtgccgtgt atttaccaga 10440 cttagtggtc
caaggccaga gtggcagatt tcccaaagtc aaggtgtgac agtgggacag 10500
cctctttgtg tctttgctgt cctaagaaac ctgggccagg ccaggcgcag tggctcacgc
10560 cttgtaatcc cagcactttg agaggccaag gtgggcagat cacgaggtca
ggagtttgag 10620 accagcctgg ccaacattgg tgaaaccctg tctctattaa
aaatagaaaa cattagacag 10680 gtgtggtggt gcatgcctgt aatcccagct
actcaggagg ctgaggcagg agaatcgctt 10740 gaacccagga ggtggaggtt
gcagtgagcc gagattgtgc cactgcactc cagcctaggc 10800 gacagagcaa
gactccgtct cgggaaaatt aattaataaa taaataaacc taggtcccag 10860
agtcccacag aatggcagac aggagcacct gggggctttt agggtatggc atttcccctg
10920 tactaactct gggctgtcca gaggcgattt catggcgtgg agtggagagg
gaggcagcac 10980 aggacttcct aggcctcagc tctcacctgc ccatcttttg
atttccaggc agttaacatc 11040 actgacctga gcgagaacag aaagcaggac
aagcgcttcg ccttcatccg ctcagacagt 11100 ggccccacca ccagttttga
gtctgccgcc tgccccggtt ggttcctctg cacagcgatg 11160 gaagctgacc
agcccgtcag cctcaccaat atgcctgacg aaggcgtcat ggtcaccaaa 11220
ttctacttcc aggaggacga gtagtactgc ccaggcctgc ctgttcccat tcttgcatgg
11280 caaggactgc agggactgcc agtccccctg ccccagggct cccggctatg
ggggcactga 11340 ggaccagcca ttgaggggtg gaccctcaga aggcgtcaca
acaacctggt cacaggactc 11400 tgcctcctct tcaactgacc agcctccatg
ctgcctccag aatggtcttt ctaatgtgtg 11460 aatcagagca cagcagcccc
tgcacaaagc ccttccatgt cgcctctgca ttcaggatca 11520 aaccccgacc
acctgcccaa cctgctctcc tcttgccact gcctcttcct ccctcattcc 11580
accttcccat gccctggatc catcaggcca cttgatgacc cccaaccaag tggctcccac
11640 accctgtttt acaaaaaaga aaagaccagt ccatgaggga ggtttttaag
ggtttgtgga 11700 aaatgaaaat taggatttca tgattttttt ttttcagtcc
ccgtgaagga gagcccttca 11760 tttggagatt atgttctttc ggggagaggc
tgaggactta aaatattcct gcatttgtga 11820 aatgatggtg aaagtaagtg
gtagcttttc ccttcttttt cttctttttt tgtgatgtcc 11880 caacttgtaa
aaattaaaag ttatggtact atgttagccc cataattttt tttttccttt 11940
taaaacactt ccataatctg gactcctctg tccaggcact gctgcccagc ctccaagctc
12000 catctccact ccagattttt tacagctgcc tgcagtactt tacctcctat
cagaagtttc 12060 tcagctccca aggctctgag caaatgtggc tcctgggggt
tctttcttcc tctgctgaag 12120 gaataaattg ctccttgaca ttgtagagct
tctggcactt ggagacttgt atgaaagatg 12180 gctgtgcctc tgcctgtctc
cccaccaggc tgggagctct gcagagcagg aaacatgact 12240 cgtatatgtc
tcaggtccct gcagggccaa gcacctagcc tcgctcttgg caggtactca 12300
gcgaatgaat gctgtatatg ttgggtgcaa agttccctac ttcctgtgac ttcagctctg
12360 ttttacaata aaatcttgaa aatgcctata ttgttgacta tgtccttggc
cttgacaggc 12420 tttgggtata gagtgctgag gaaactgaaa gaccaatgtg
tyttycttac cccagaggct 12480 ggcgcctggc ctcttctctg agagttcttt
tcttccttca gcctcactct ccctggataa 12540 catgagagca aatctctctg cgggg
12565 18 25 DNA Homo sapiens 18 tgtacctaag cccacccttt agagc 25 19
20 DNA Artificial Sequence Description of Artificial Sequence
Primer 19 tggcctccag aaacctccaa 20 20 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 20 gctgatattc tggtgggaaa
20 21 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 21 ggcaagagca aaactctgtc 20 22 28 DNA Artificial Sequence
Description of Artificial Sequence Primer 22 gggatgttaa ccagaagacc
ttctatct 28 23 27 DNA Artificial Sequence Description of Artificial
Sequence Primer 23 caaccactca ccttctaaat tgacatt 27 24 30 DNA
Artificial Sequence Description of Artificial Sequence Probe 24
aacaaccaac tagttgctgg atacttgcaa 30 25 27 DNA Artificial Sequence
Description of Artificial Sequence Probe 25 acaaccaact agttgccgga
tacttgc 27 26 4 PRT Artificial Sequence Description of Artificial
Sequence Illustrative zinc finger peptide 26 Thr Lys Pro Arg 1 27 5
PRT Artificial Sequence Description of Artificial Sequence
Illustrative zinc finger peptide 27 Ile Thr Gly Ser Glu 1 5 28 6
PRT Artificial Sequence Description of Artificial Sequence
Illustrative zinc finger peptide 28 Val Thr Lys Phe Tyr Phe 1 5 29
6 PRT Artificial Sequence Description of Artificial Sequence
Illustrative zinc finger peptide 29 Val Thr Asp Phe Tyr Phe 1 5
* * * * *
References