U.S. patent application number 10/493881 was filed with the patent office on 2005-03-10 for acid phosphatase (acp1)gene as a susceptibility locus for hyperlipidemia.
This patent application is currently assigned to City of Hope. Invention is credited to Bottini, Nunzio, Comings, David E, MacMurray, James P..
Application Number | 20050055732 10/493881 |
Document ID | / |
Family ID | 23290894 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050055732 |
Kind Code |
A1 |
Comings, David E ; et
al. |
March 10, 2005 |
Acid phosphatase (acp1)gene as a susceptibility locus for
hyperlipidemia
Abstract
The ACP1 *A allele provides a means for diagnosing
susceptability of a human subject to hyperlipidemia, especially
hyperlipidemia associated with metabolic syndrome, a means for
treating, or preventing the onset of, hyperlipidemia and metabolic
syndrome, and a means for screening and identifying drugs suitable
for use in treating or preventing hyperlipidemia, especially
hyperlipidemia associated with metabolic syndrome. Diagnostic kits
are also provided.
Inventors: |
Comings, David E; (Duarte,
CA) ; MacMurray, James P.; (Loma Linda, CA) ;
Bottini, Nunzio; (La Jolla, CA) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
City of Hope
|
Family ID: |
23290894 |
Appl. No.: |
10/493881 |
Filed: |
September 17, 2004 |
PCT Filed: |
October 29, 2002 |
PCT NO: |
PCT/US02/34500 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60330693 |
Oct 29, 2001 |
|
|
|
Current U.S.
Class: |
800/3 ;
435/6.11 |
Current CPC
Class: |
C12Q 1/6883 20130101;
A61P 3/06 20180101; C12Q 2600/156 20130101 |
Class at
Publication: |
800/003 ;
435/006 |
International
Class: |
C12Q 001/68; A01K
067/027 |
Claims
What is claimed is:
1. A method for identifying a human subject that is at risk of
developing hyperlipidemia, comprising screening for the presence or
absence of a non-*A allele of the ACP1 gene, and identifying the
presence of said non-*A allele, such presence being an indication
that said human subject is at risk of developing
hyperlipidemia.
2. The method of claim 1, wherein the presence of said non-*A
allele is determined by identifying the presence in the genome of
said human subject, a DNA sequence encoding a non-*A ACP1
polypeptide.
3. The method of claim 1, wherein the presence of said non-*A
allele is determined by measuring the activity of an ACP1enzyme
obtained from said human subject relative to a known ACP1 *A enzyme
standard.
4. The method of any of claims 1-3, wherein the hyperlipidemia is
associated with metabolic disorder.
5. A method for identifying a drug product having preventative or
curative activity against hyperlipidemia, comprising measuring the
activity of an ACP1 enzyme in cells expressing a non-*A allele of
the ACP1 gene, to obtain a first enzyme activity value, exposing
cells expressing said non-*A allele of the ACP1 enzyme to a drug
candidate, measuring ACP1 enzyme activity to obtain a second enzyme
activity value, comparing the first enzyme activity value to the
second enzyme activity value to obtain an enzyme activity ratio, a
ratio of greater than 1 being an indicator of preventative or
curative activity against hyperlipidemia.
6. The method of claim 5, wherein said cells are in an animal.
7. The method of claim 5, wherein said cells comprise a cell
culture.
8. The method of any of claims 5-7, wherein the hyperlipidemia is
associated with metabolic disorder.
9. A method for preventing or treating hyperlipidemia comprising
administering to a person at risk of developing hyperlipidemia or
suffering from hyperlipidemia, a PTP inhibitor in an amount
sufficient to reduce ACP1 enzyme activity relative to the activity
of that enzyme in the absence of said inhibitor.
10. The method of claim 9, wherein the hyperlipidemia is associated
with metabolic disorder.
11. A diagnostic kit for use in identifying persons who are at risk
of developing hyperlipidemia, comprising a means for screening for
the presence or absence of a non-*A allele of the ACP1 gene, and a
means for identifying the presence of said non-*A allele, such
presence indicating that the person is at risk of developing
hyperlipidemia.
12. The method of claim 11, wherein the hyperlipidemia is
associated with metabolic disorder.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to the field of human
genetics. More specifically, the invention relates to diagnosis of
susceptibility to hyperlipidemia, especially hyperlipidemia
associated with metabolic syndrome. The invention further relates
to methods for screening drug candidates for suitability in the
treatment of hyperlipidemia and metabolic syndrome, and to methods
for treating or preventing hyperlipidemia, especially
hyperlipidemia associated with metabolic syndrome.
[0002] The publications and other materials used herein to
illuminate the background of the invention or provide additional
details respecting the practice are incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] Increased incidence of obesity, hyperlipidemia,
hypertension, non-insulin dependent diabetes and coronary artery
disease often cluster in the same individuals, and it has been
frequently asserted that a common mechanism may be responsible for
the comorbidity of these conditions in a subset of the population.
The risk factor constellation for this group is often referred to
as the "metabolic syndrome", "insulin resistance syndrome" or
"syndrome X". The prevalence of the metabolic syndrome is roughly
2.5% in individuals under 40 years of age, rising to 5 to 10% in
middle aged and older persons (1). The reasons for this increase in
risk is largely due to the corollary age-related increase in
obesity, and in particular abdominal obesity in the U.S.
population. Obesity is now estimated to be the second leading
preventable cause of death after cigarette smoking in the U.S.(2).
Thirty-nine million Americans are estimated to be obese (having a
body-mass-index (BMI) of .gtoreq.30) and an additional 57 million
are estimated to be overweight (BMI between 25 and 29). Abdominal
obesity, which increases with age among men and postmenopausal
women, is responsible for most of the association of obesity with
the metabolic syndrome and associated diseases (3). In addition to
insulin resistance and hypertension, the principal abnormalities
associated with the metabolic syndrome in obese individuals include
elevated triglyceride levels and elevated cholesterol/HDL ratio.
Increases both in triglyceride and cholesterol/HDL ratio are now
recognized as independent risk factors for coronary artery disease
(CAD) as well as overall 5-year mortality (4). However, it has
remained unclear what distinguishes those individuals whose weight
gain leads to the development of the metabolic syndrome from those
more fortunate persons who appear capable of considerable weight
gain without experiencing the dyslipidemia and insulin resistance
that trigger increased risk of developing CAD and
non-insulin-dependent diabetes mellitis (NIDDM).
[0004] The acid phosphatase locus 1 (ACP1) encodes a low molecular
weight protein tyrosine phosphatase (LMPTP ) involved in the
negative modulation of insulin signal transduction (5). The ACP1
gene product is present ubiquitously in human tissues in two
isoforms, called LMPTP-A and -B (6). The same locus also encodes
the adipocyte LMPTP, which also is indicated as adipocyte acid
phosphatase (HAAP), and is able to dephosphorylate in vitro the
tyrosine phosphorylated adipocyte lipid binding protein (ALBP)
(7).
[0005] ACP1 shows genetic polymorphism corresponding to strong
variations in total enzymatic activity and in the ratio between the
activity of the two isoforms associated with the different
genotypes (8). A positive association between those ACP1 genotypes
associated with a low total enzymatic activity and extreme values
of BMI in obese children and adult subjects (9,10,11) and in
non-dyslipidemic NIDDM subjects (12) has been reported in the
Italian population. In 11 Italian studies the ACP1 polymorphism has
been found to be associated with clinical variability of obesity,
but not with the disease itself. The ACP1 *A allele is a variation
of the A allele, distinguished by a Gln to Arg substitution at
position 105 of the encoded protein, and lower enzymatic activity.
In a recent study by Lucarini et al., a highly significant positive
association between the ACP1 *A allele (associated with a reduced
total enzymatic activity) and BMI has been described, but only in
those cases with blood lipid levels (BLL) in the normal range
(12).
SUMMARY OF THE NVENTION
[0006] The present invention provides diagnostic and prognostic
methods for detecting a predisposition to hyperlipidemia,
especially hyperlipidemia associated with metabolic disease, by
detecting a non-*A allele at the APC1 locus, or confirming the lack
of a predisposition by detecting the presence of the *A allele at
the APC1 locus. Methods of treating, or reducing the probability of
developing hyperlipidemia and metabolic disease are also
provided.
[0007] In one embodiment, the invention provides a non-human animal
which carries a human ACP1 allele in its genome. In another
embodiment, the invention provides a cell line derived from one or
more cells from a non-human animal.
[0008] The invention further provides a method for diagnosing in a
human subject a susceptability to hyperlipidemia, especially
hyperlipidemia associated with metabolic syndrome, comprising
testing the subject for the presence of a non-*A ACP1 allele, such
presence being an indicator of susceptability to
hyperlipidemia.
[0009] The invention further provides methods for treating, or
preventing the onset of, hyperlipidemia and metabolic disorder in
susceptible individuals, comprising inhibiting the activity of the
ACP1 enzyme. An alternative method comprises inhibiting the
transcription or translation of a non-*A ACP1 gene allele.
[0010] The invention further provides a method for identifying a
drug product having preventative or curative activity against
hyperlipidemia and metabolic syndrome, comprising measuring the
activity of an ACP1 enzyme in cells expressing a non-*A allele of
the ACP1 gene, to obtain a first enzyme activity value, exposing
cells expressing the non-*A allele of the ACP1 enzyme to a drug
candidate, measuring ACP1 enzyme activity to obtain a second enzyme
activity value, comparing the first enzyme activity value to the
second enzyme activity value to obtain an enzyme activity ratio, a
ratio of greater than 1 being an indicator of preventative or
curative activity against hyperlipidemia, especially
hyperlipidernia associated with metabolic syndrome.
[0011] The invention further provides diagnostic kits for use in
identifying persons who are at risk of developing hyperlipidemia,
especially hyperlipidemia associated with metabolic syndrome,
comprising a means for screening for the presence or absence of a
non-*A allele of the ACP1 gene, and a means for identifying the
presence of said non-*A allele, such presence indicating that the
person is at risk of developing hyperlipidemia. Screening can be
accomplished by providing a means for identifying in a sample the
presence of DNA encoding a non-*A ACP1 enzyme. Alternatively,
screening can be accomplished by providing a means suitable for
measuring the activity of an ACP1 enzyme in a sample, and comparing
the activity measurement to the activity of an ACP1 *A
standard.
[0012] The invention further provides a method of screening for
drug candidates useful in treating a disease or condition
associated with a non-*A ACP1 allele, wherein the method comprises
administering a drug to an animal which is heterozygous or
homozygous for the allele, wherein if the animal shows a decrease
in signs or symptoms associated with the disease when compared to
an animal that is heterozygous or homozygous for that allele and
that does not receive the drug, the drug is a drug candidate for
treating that disease. In a preferred embodiment, the condition is
metabolic syndrome. An alternative method comprises exposing a
cell, or culture of cells, comprising a non-*A ACP1 allele to a
drug candidate, and subsequently measuring ACP1 activity, a
reduction in activity relative to an untreated control indicating
suitability of the drug candidate for treatment of hyperlipidemia,
especially hyperlipidemia associated with metabolic disorder, or
prevention of its onset.
BRIEF DESCRIPTION OF THE FIGURE
[0013] FIG. 1 shows the relationship between ACP1 polymorphism and
triglyceride levels for subjects grouped according to their
classification as non-obese, obese or morbidly obese.
DETAILED DESCRIPTION OF THE INVENTION
[0014] In an investigation of the relationship between ACP1
polymorphism and metabolic variables in 173 Caucasian American
post-menopausal obese subjects and 76 age matched non-obese control
subjects, ACP1 genotypes were found to be significantly associated
with elevated levels of total cholesterol (p=0.005) and
triglyceride (p=0.006) in obese women only. The association between
ACP1 polymorphism and blood lipid levels in obese women was mainly
due to an age-independent protective effect of the *A allele
against hyperlipidemia. The protective effect on tryglicerides was
positively correlated with BMI in obese subjects and was marked in
morbidly obese subjects. It has been unclear why some individuals
who gain weight develop dyslipidemia. and other aspects of the
metabolic syndrome, while others do not. The strong protective
effect that the ACP1 *A allele exerts against the development of
hypertrigliceridemia in obese subjects, indicates that those who
gain weight and carry the ACP1 *A allele are the ones who do not
develop the metabolic syndrome.
[0015] Obese women carrying the *A allele, associated with a
reduced total enzymatic activity, show a tendency toward higher
degrees of obesity. The weaker association between ACP1
polymorphism and degree of obesity in the present data compared to
the Italian samples of an earlier study could be due to differences
in the BMI distribution that is very different between the two
samples (91% of the present study subjects show a BMI value over
32).
[0016] A highly significant association of ACP1 polymorphism with
BLL is present in the obese sample and the data indicate that most
of the association of ACP1 polymorphism with BLL was at the TaqI
SNP. This SNP distinguishes the presence or absence of the *A
allele, which is associated with low levels of total enzymatic
activity and a high ratio between A and B isoform production. The
TaqI SNP leads also to the incorporation of an Arg instead of a Gln
in position 105 of both the LMPTP isoforms in the protein product
of the ACP1 *A allele (16).
[0017] Interestingly, the effect of the ACP1 gene on triglyceride
levels depends on BMI: individuals carrying the ACP1 *A allele are
protected from the hypertriglyceridemia that follows the increase
of BMI between overweight and obese subjects, suggesting that ACP1
is a gene influencing the predisposition at least to some features
of the metabolic syndrome that is associated with central obesity
(3). It seems possible that an ACP1 isoform is acting with high
specificity on some pathway responsible for the regulation of fatty
acid absorption and/or metabolism in obese subjects. The results of
correlation analysis with the enzymatic parameters associated with
ACP1 genotypes suggest that the LMPTP *A isoform is involved in
mediating the association between ACP1 and the clinical variability
of obesity. Another possibility that doesn't exclude the latter is
that such association is due to the different affinity of the two
enzyme variants (Gln105 and Argl105) for specific substrates in the
adipocytes and/or in other tissues.
[0018] LMPTP is involved in the in vitro negative modulation of
insulin signal transduction (17). LMPTP is also able to in vitro
dephosphorylate the adipocyte lipid binding protein (ALBP or pp15)
(7). ALBP belongs to a family of lipid binding proteins present in
various isoforms in many human tissues. In adipose tissue ALBP is
phosphorylated on Tyr19 after insulin stimulation and this
phenomenon seems to impair its fatty acid binding ability (18). In
the adipose tissue the double activity of LMPTP (on insulin
receptor signal transduction and ALBP phosphorylation) could partly
compensate each other, thus explaining the weaker association
between ACP1 genotypes and overall BMI. In fact, LMPTP could at the
same time counteract the adipogenic stimulus mediated by the
insulin receptor and contribute to ALBP dephosphorylation which
causes an increase of its lipid binding activity.
[0019] As a result of previous studies, the ACP1 locus is currently
included among the candidate "modifier" loci in obesity (19). The
association of ACP1 with BLL and/or BMI is present only in obese
subjects in all the samples studied to date, and as shown in FIG.
1, the effect of ACP1 on tryglicerides is more evident in higher
classes of BMI. No linkage study has shown the 2p25 region (the
locus of ACP1) to be associated with obesity. Indeed, linkage
analysis has less chance of revealing the role of "modifier" genes
that are acting only when other genes involved in the disease
predisposition are present, i. e. epistasis.
[0020] Insulin signal transduction is well known to be modulated by
other cytosolic tyrosine phosphatases that act with higher affinity
than LMPTP (20). Two transmembrane phosphatases in adipocytes have
been isolated that are responsible for dephosphorylating ALBP with
high affinity (21). We propose that the effect of LMPTP becomes
evident in the regulation of metabolic signaling only in
pathological situations, when other control systems usually acting
with higher affinity are failing. Recently the tyrosine
phosphorylated caveolin has been proposed as a possible
physiological substrate of LMPTP (22). Caveolin is expressed at
high levels only in very differentiated tissues such as endothelial
tissue and adipocytes. In these tissues the reversible
phosphorylation of caveolin on tyrosine residues is involved in the
modulation of signal transduction through many receptors (23).
Following the expansion of adipose tissue in obese subjects the
interaction between caveolin and LMPTP could become the limiting
factor for one or more signal transduction pathways affecting lipid
absorption and/or metabolism that are normally regulated mainly
through other mechanisms.
[0021] In summary, the presence of the ACP1 *A allele exerts an
independent protective effect against the hypertrigliceridemia
associated with increases in BMI into the obese range. Because most
of the lethality and morbidity connected with obesity comes from
the associated diseases and not from the weight gain itself, the
confirmation of ACP1 as a modifier gene of metabolic complications
of obesity opens the door to possible modulation of this gene
product in the treatment of obesity as a safeguard against
hyperlipidemia and metabolic syndrome. Today there is intense
research going on in the role of tyrosine phosphatases in the
pathogenesis of metabolic diseases and/or their clinical
variability (24). Until now LMPTP is the only identified PTPase
whose polymorphism has been demonstrated to be associated with the
clinical variability of obesity in different populations.
[0022] Predisposition to hyperlipidemia, especially hyperlipidemia
associated with metabolic syndrome, can be ascertained by testing
any tissue of a human for the presence of a non-*A allele at the
ACP1 locus. For example, a person who has inherited a germuline
non-*A ACP1 allele would be prone to develop hyperlipidemia, and
perhaps metabolic syndrome, if they became obese. The presence of a
non-*A ACP1 allele can be determined by testing DNA from any tissue
of the person's body. Most simply, blood can be drawn and DNA
extracted from the cells of the blood. In addition, prenatal
diagnosis can be accomplished by testing fetal cells, placental
cells or amniotic cells for a non-*A ACP1 allele. The presence of
an *A or a non-*A allele at the ACP1 locus can be detected by any
of the means discussed herein.
[0023] Useful diagnostic techniques include, but are not limited to
fluorescent in situ hybridization (FISH), direct DNA sequencing,
PFGE analysis, Southern blot analysis, single stranded conformation
analysis (SSCA), RNase protection assay, allele-specific
oligonucleotide (ASO), dot blot analysis and PCR-SSCP, as discussed
in detail further below. Also useful are techniques employing DNA
microchip technology. (37)
[0024] There are several methods well known to persons of ordinary
skill in the art that can be used to detect DNA sequence variation,
including direct DNA sequencing, clamped denaturing gel
electrophoresis, heteroduplex analysis and chemical mismatch
cleavage. None of these methods will detect large deletions,
duplications or insertions, nor will they detect a regulatory
mutation which affects transcription or translation of the protein.
Other methods which might detect these classes of mutations, such
as a protein truncation assay or the asymmetric assay, detect only
specific types of mutations and would not detect missense
mutations. Once a mutation is known, an allele-specific detection
approach such as allele-specific oligonucleotide (ASO)
hybridization can be utilized to rapidly screen large numbers of
other samples for that same mutation
[0025] Detection of point mutations can be accomplished by
molecular cloning of the allele(s) and sequencing the allele(s)
using techniques well known to persons of ordinary skill in the
art. Alternatively, the gene sequences can be amplified directly
from a genomic DNA preparation using known techniques. The DNA
sequence of the amplified sequences then can be determined.
[0026] DNA sequences of the gene which have been amplified by use
of PCR may also be screened using allele-specific oligomer probes,
each of which contains a region of the gene sequence harboring a
known mutation. For example, one oligomer may be about 30
nucleotides in length (although shorter and longer oligomers can be
used, as recognized by those of ordinary skill in the art),
corresponding to a portion of the gene sequence. By use of a
battery of such allele-specific probes, PCR amplification products
can be screened to identify the presence in an individual of a
previously identified gene mutation. Hybridization of
allele-specific probes with nucleic acids amplified from cells can
be performed, for example, on a nylon filter. Hybridization to a
particular probe under high stringency hybridization conditions
indicates the presence of the same mutation in the cells as in the
allele-specific probe.
[0027] Nucleic acid analysis via microchip technology is also
applicable to the present invention In this technique, literally
thousands of distinct oligonucleotide probes can be applied in an
array on a silicon chip. A nucleic acid to be analyzed is
fluorescently labeled and hybridized to the probes on the chip. It
is also possible to study nucleic acid-protein interactions using
these nucleic acid microchips. Using this technique one can
determine the presence of mutations, sequence the nucleic acid
being analyzed, or measure expression levels of a gene of interest.
The method is one of parallel processing of many, even thousands,
of probes at once and can tremendously increase the rate of
analysis.
[0028] An ACP1 *A allele, or a non-*A allele, can be detected by
detection of the corresponding mRNA transcript by any technique
known to persons of ordinary skill in the art. These include
Northern blot analysis, PCR amplification and RNase protection.
[0029] An ACP1 *A allele, or a non-*A allele, also can be detected
by screening for the encoded protein. For example, monoclonal
antibodies irnmunoreactive with the protein encoded by the ACP1 *A
allele can be used to screen a tissue. Lack of cognate antigen
would indicate the presense of a non-*A allele. Antibodies specific
for products of non-*A alleles also could be used to detect a
non-*A gene product. Such immunological assays can be done in any
convenient format known in the art These include Western blots,
immunohistochemical assays and ELISA assays. Functional assays,
such as protein binding determiinations, also can be used. In
addition, assays which detect biochemical function can be used.
[0030] The nucleic acid probes provided by the present invention
are useful for a number of purposes. They can be used in Southern
hybridization to genomic DNA and in the RNase protection method for
detecting point mutations. The probes can be used to detect PCR
amplification products. They may also be used to detect mismatches
with a particular ACP1 allele or mRNA using other techniques.
[0031] In order to detect an ACP1 gene allele, a biological sample
is prepared and analyzed for a difference between the sequence of
the allele being analyzed and the sequence of a reference allele.
Alternatively, the presence or absence of a particular allele can
be determined by an immunological assay using antibodies specific
for the protein produced by a reference ACP1 allele (e.g., the *A
allele, the A allele, the B allele, etc.).
[0032] "Amplification of Polynucleotides" utilizes methods such as
the polymerase chain reaction (PCR), ligation amplification (or
ligase chain reaction, LCR) and amplification methods based on the
use of Q-beta replicase. Also useful are strand displacement
amplification (SDA) and nucleic acid sequence based amplification
(NASBA). These methods are well known and widely practiced in the
art. Reagents and hardware for conducting PCR are commercially
available. Primers useful to amplify sequences from the region are
preferably complementary to, and hybridize specifically to,
sequences in the region or in regions that flank a target region
therein.
[0033] "Antibodies." The present invention also provides polyclonal
and/or monoclonal antibodies and fragments thereof, and immunologic
binding equivalents thereof, which are capable of specifically
binding to the polypeptides and fragments thereof of an ACP1 enzyme
encoded by a particular ACP1 allele, or to polynucleotide sequences
from the region, particularly from the ACP1 locus or a portion
thereof. The term "antibody" is used both to refer to a homogeneous
molecular entity, or a mixture such as a serum product made up of a
plurality of different molecular entities. Antibodies will be
useful in assays as well as pharmaceuticals.
[0034] Once a sufficient quantity of desired polypeptide has been
obtained, it may be used for various purposes. A typical use is the
production of antibodies specific for binding. These antibodies may
be either polyclonal or monoclonal, and may be produced by in vitro
or in vivo techniques well known by persons of ordinary skill in
the art.
[0035] An immunological response is usually assayed with an
immunoassay. Normally, such immunoassays involve some purification
of a source of antigen, for example, that produced by the same
cells and in the same fashion as the antigen. A variety of
immunoassay methods are well known by persons of ordinary sill in
the art.
[0036] Frequently, polypeptides and antibodies will be labeled by
joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionuclides, enzymes, substrates, cofactors, inhibitors
fluorescent agents, chemiluminescent agents, magnetic particles and
the like.
[0037] A polynucleotide is said to "encode" a polypeptide if, in
its native state or when manipulated by methods well known to those
skilled in the art, it can be transcribed and/or translated to
produce the MRNA for and/or the polypeptide or a fragment thereof.
The anti-sense strand is the complement of such a nucleic acid, and
the encoding sequence can be deduced therefrom.
[0038] An "isolated" or "substantially pure" nucleic acid (e.g., an
RNA, DNA or a mixed polymer) is one which is substantially
separated from other cellular components which naturally accompany
a native human sequence or protein, e.g., ribosomes, polymerases,
many other human genome sequences and proteins. The term embraces a
nucleic acid sequence or protein which has been removed from its
naturally occurring environrment, and includes recombinant or
cloned DNA isolates and chemically synthesized analogs or analogs
biologically synthesized by heterologous systems.
[0039] The polynucleotide compositions useful in this invention
include RNA, cDNA, genomic DNA, synthetic forms, and mixed
polymers, both sense and antisense strands, and may be chemically
or biochemically modified or may contain non-natural or derivatized
nucleotide bases, as will be readily appreciated by those skilled
in the art. Such modifications include, for example, labels,
methylation, substitution of one or more of the naturally occurring
nucleotides with an analog, intemucleotide modifications such as
uncharged linkages (e.g., methyl phosphonates, phosphotriesters,
phosphoramidates, carbamates, etc.), charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.), pendent moieties
(e.g., polypeptides), intercalators (e.g., acridine, psoralen,
etc.), chelators, alkylators, and modified linkages (e.g., alpha
anomeric nucleic acids, etc.). Also included are synthetic
molecules that mimic polynucleotides in their ability to bind to a
designated sequence via hydrogen bonding and other chemical
interactions. Such molecules are known in the art and include, for
example, those in which peptide linkages substitute for phosphate
linkages in the backbone of the molecule. The polynucleotides
useful in the invention may be isolated or substantially pure.
[0040] The present invention provides for the use of recombinant
nucleic acids comprising the ACP1 *A allele. The recombinant
construct may be capable of replicating autonomously in a host
cell. Alternatively, the recombinant construct may become
integrated into the chromosomal DNA of the host cell. Such a
recombinant polynucleotide comprises a polynucleotide of genomic,
cDNA, semi-synthetic, or synthetic origin which, by virtue of its
origin or manipulation, 1) is not associated with all or a portion
of a polynucleotide with which it is associated in nature; 2) is
linked to a polynucleotide other than that to which it is linked in
nature; or 3) does not occur in nature.
[0041] Therefore, recombinant nucleic acids comprising sequences
otherwise not naturally occurring are useful in this invention.
Although the described sequences may be employed, it will often be
altered, e.g., by deletion, substitution or insertion.
[0042] cDNA or genomic libraries of various types may be screened
as natural sources of the nucleic acids of the present invention,
or such nucleic acids may be provided by amplification of sequences
resident in genomic DNA or other natural sources, e.g., by PCR. The
choice of cDNA libraries normally corresponds to a tissue source
which is abundant in mRNA for the desired proteins. Phage libraries
are normally preferred, but other types of libraries may be used.
Clones of a library are spread onto plates, transferred to a
substrate for screening, denatured and probed for the presence of
desired sequences.
[0043] The recombinant nucleic acid sequences used to produce
fusion proteins of the present invention may be derived from
natural or synthetic sequences. Many natural gene sequences are
obtainable from various cDNA or from genomic libraries using
appropriate probes.
[0044] "Operably linked" refers to ajuxtaposition wherein the
components so described are in a relationship permitting them to
function in their intended manner. For instance, a promoter is
operably linked to a coding sequence if the promoter affects its
transcription or expression.
[0045] "Probes". Polynucleotide polymorphisms associated with
alleles which predispose to metabolic syndrome are detected by
hybridization with a polynucleotide probe which forms a stable
hybrid with that of the target sequence, under highly stringent to
moderately stringent hybridization and wash conditions. If it is
expected that the probes will be perfectly complementary to the
target sequence, high stringency conditions will be used.
Hybridization stringency may be lessened if some mismatching is
expected, for example, if variants are expected with the result
that the probe will not be completely complementary. Conditions are
chosen which rule out nonspecific/adventitious bindings, that is,
which minimize noise.
[0046] Nucleic acid hybridization will be affected by such
conditions as salt concentration, temperature, or organic solvents,
in addition to the base composition, length of the complementary
strands, and the number of nucleotide base mismatches between the
hybridizing nucleic acids, as will be readily appreciated by those
skilled in the art. Stringent temperature conditions will generally
include temperatures in excess of 30.degree. C., typically in
excess of 37.degree. C., and preferably in excess of 45.degree. C.
Stringent salt conditions will ordinarily be less than 1000 mM,
typically less than 500 mM, and preferably less than 200 mM.
However, the combination of parameters is much more important than
the measure of any single parameter. The stringency conditions are
dependent on the length of the nucleic acid and the base
composition of the nucleic acid, and can be determined by
techniques well known by persons of ordinary skill in the art.
[0047] Probes for alleles may be derived from the sequences of the
region or its cDNAs. The probes may be of any suitable length,
which span all or a portion of the region, and which allow specific
hybridization to the region. If the target sequence contains a
sequence identical to that of the probe, the probes may be short,
e.g., in the range of about 8-30 base pairs, since the hybrid will
be relatively stable under even highly stringent conditions. If
some degree of mismatch is expected with the probe, i.e., if it is
suspected that the probe will hybridize to a variant region, a
longer probe may be employed which hybridizes to the target
sequence with the requisite specificity.
[0048] The probes will include an isolated polynucleotide attached
to a label or reporter molecule and may be used to isolate other
polynucleotide sequences having sequence similarity, by standard
methods. Other similar polynucleotides may be selected by using
homologous polynucleotides. Alternatively, polynucleotides encoding
these or similar polypeptides may be synthesized or selected by use
of the redundancy in the genetic code. Various codon substitutions
may be introduced, e.g., by silent changes (thereby producing
various restriction sites) or to optimize expression for a
particular system. Mutations may be introduced to modify the
properties of the polypeptide, perhaps to change ligand-binding
affinities, interchain affinities, or the polypeptide degradation
or turnover rate.
[0049] "Protein modifications or fragments" are provided by the
present invention for ACP1 polypeptides or fragments thereof which
are substantially homologous to primary structural sequence but
which include, e.g., in vivo or in vitro chemical and biochemical
modifications or which incorporate unusual amino acids. Such
modifications include, for example, acetylation, carboxylation,
phosphorylation, glycosylation, ubiquitination, labeling, e.g.,
with radionuclides, and various enzymatic modifications, as will be
readily appreciated by persons of ordinary skill in the art. A
variety of methods for labeling polypeptides and of substituents or
labels usefull for such purposes are well known by persons of
ordinary skill in the art, and include radioactive isotopes such as
.sup.32P, ligands which bind to labeled antiligands (e.g.,
antibodies), fluorophores, chemiluminescent agents, enzymes, and
antiligands which can serve as specific binding pair members for a
labeled ligand. The choice of label depends on the sensitivity
required, ease of conjugation with the primer, stability
requirements, and available instrumentation.
[0050] Besides substantially full-length polypeptides, the present
invention provides for the use of biologically active fragments of
the polypeptides. Significant biological activities include
ligand-binding, immunological activity and other biological
activities characteristic of polypeptides.
[0051] A polypeptide "fragment," "portion" or "segment[ is a
stretch of amino acid residues of at least about five to seven
contiguous amino acids, often at least about seven to nine
contiguous amino acids, typically at least about nine to 13
contiguous amino acids and, most preferably, at least about 20 to
30 or more contiguous amino acids.
[0052] The present invention also provides for the use of fusion
polypeptides, comprising polypeptides and fragments. Homologous
polypeptides may be fusions between two or more polypeptide
sequences or between the sequences of ACP1 and a related protein.
Likewise, heterologous fusions may be constructed which would
exhibit a combination of properties or activities of the derivative
proteins. For example, ligand-binding or other domains may be
"swapped" between different new fusion polypeptides or fragments.
Such homologous or heterologous fusion polypeptides may display,
for example, altered strength or specificity of binding and may
include for example partners such as immunoglobulins, bacterial
b-galactosidase, trpE, protein A, b-lactamase, alpha amylase,
alcohol dehydrogenase and yeast alpha mating factor.
[0053] Fusion proteins will typically be made by either recombinant
nucleic acid methods, as described below, or may be chemically
synthesized. Techniques for the synthesis of polypeptides are well
known by persons of ordinary skill in the art. variants typically
contain the exchange of one amino acid for another at one or more
sites within the protein, and may be designed to modulate one or
more properties of the polypeptide, such as stability against
proteolytic cleavage, without the loss of other functions or
properties. Amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. Preferred substitutions are ones which are conservative,
that is, one amino acid is replaced with one of similar shape and
charge. Conservative substitutions are well known to persons of
ordinary skill in the art and typically include, though not
exclusively, substitutions within the following groups: glycine,
alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and
tyrosine, phenylalanine.
[0054] Certain amino acids may be substituted for other amino acids
in a protein structure without appreciable loss of interactive
binding capacity with structures such as, for example,
antigen-binding regions of antibodies or binding sites on substrate
molecules or binding sites on proteins interacting with an
polypeptide. Since it is the interactive capacity and nature of a
protein which defines that protein's biological functional
activity, certain amino acid substitutions can be made in a protein
sequence, and its underlying DNA coding sequence, and nevertheless
obtain a protein with like properties. In making such changes, the
hydropathic index of amino acids may be considered. The importance
of the hydrophobic amino acid index in conferring interactive
biological function on a protein is generally understood in the art
(Kyte and Doolittle, 1982). Alternatively, the substitution of like
amino acids can be made effectively on the basis of hydrophilicity.
The importance of hydrophilicity in conferring interactive
biological function of a protein is generally understood in the art
(U.S. Pat. No. 4,554,101). The use of the hydrophobic index or
hydrophilicity in designing polypeptides is further discussed in
U.S. Pat. No. 5,691,198.
[0055] "Protein purification" refers to various methods for the
isolation of polypeptides from other biological material, such as
from cells transformed with recombinant nucleic acids encoding
ACP1, and are well known by persons of ordinary skill in the art.
For example, such polypeptides may be purified by immunoaffinity
chromatography employing, e.g., the antibodies provided by the
present invention. Various methods of protein purification are well
known by persons of ordinary skill in the art.
[0056] The terms "isolated", "substantially pure", and
"substantially homogeneous" are used interchangeably to describe a
protein or polypeptide which has been separated from components
which accompany it in its natural state. A monomeric protein is
substantially pure when at least about 60 to 75% of a sample
exhibits a single polypeptide sequence. A substantially pure
protein will typically comprise about 60 to 90% W/W of a protein
sample, more usually about 95%, and preferably will be over about
99% pure. Protein purity or homogeneity may be indicated by a
number of means well known by persons of ordinary skill in the art,
such as polyacrylamide gel electrophoresis of a protein sample,
followed by visualizing a single polypeptide band upon staining the
gel. For certain purposes, higher resolution may be provided by
using HPLC or other means well known by persons of ordinary skill
in the art which are utilized for purification.
[0057] A protein is substantially free of naturally associated
components when it is separated from the native contaminants which
accompany it in its natural state. Thus, a polypeptide which is
chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
substantially free from its naturally associated components. A
protein may also be rendered substantially free of naturally
associated components by isolation, using protein purification
techniques well known by persons of ordinary skill in the art.
[0058] The polypeptides of the present invention, if soluble, may
be coupled to a solid-phase support, e.g., nitrocellulose, nylon,
column packing materials (e.g., Sepharose beads), magnetic beads,
glass wool, plastic, metal, polymer gels, cells, or other
substrates. Such supports may tale the form, for example, of beads,
wells, dipsticlcs, or membranes.
[0059] "Recombinant nucleic acid" is a nucleic acid which is not
naturally occurring, or which is made by the artificial combination
of two otherwise separated segments of sequence. This artificial
combination is often accomplished by either chemical synthesis
means, or by the artificial manipulation of isolated segments of
nucleic acids, e.g., by genetic engineering techniques.
[0060] "Regulatory sequences" refers to those sequences normally
within 100 kb of the coding region of a locus, but they may also be
more distant from the coding region, which affect the expression of
the gene (including transcription of the gene, and translation,
splicing, stability or the like of the messenger RNA).
[0061] Large amounts of the polynucleotides of the present
invention may be produced by a suitable host cell transformed with
a nucleotide sequence encoding the ACP1 protein. Natural or
synthetic polynucleotide fiagments coding for the peptide or a
desired fragment can be incorporated into recombinant
polynucleotide constructs (vectors), usually DNA constructs,
capable of introduction into and replication in a prokaryotic or
eukaryotic cell. Usually the vectors will be suitable for
replication in a unicellular host, such as yeast or bacteria, but
may also be intended for introduction to (with and without
integration within the genome) cultured mammalian or plant or other
eukaryotic cell lines. The most commonly used prokaryotic hosts are
strains of Escherichia coli, although other prokaryotes, such as
Bacillus subtilis or Pseudomnonas may also be used. Mammalian or
other eukaryotic host cells, such as those of yeast, filamentous
fungi, plant, insect, or amphibian or avian species, may also be
useful for production of the proteins of the present invention.
[0062] Vectors will include an appropriate promoter and other
necessary vector sequences that are functional in the selected
host. There may include, when appropriate, those naturally
associated with genes. Many useful vectors are known in the art and
may be obtained from such vendors as Stratagene, New England
BioLabs, Promega Biotech, and others. Promoters such as the trp,
lac and phage promoters, tRNA promoters and glycolytic enzyme
promoters may be used in prokaryotic hosts. Usefll yeast promoters
include promoter regions for metallothionein, 3-phosphoglycerate
idnase or other glycolytic enzymes such as enolase or
glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for
maltose and galactose utilization, and others.
[0063] Expression and cloning vectors preferably contain a
selectable marker gene. Typical marker genes encode proteins that
a) confer resistance to antibiotics or other toxic substances, e.g.
ampicillin, neomycin, methotrexate, etc.; b) complement auxotrophic
deficiencies, or c) supply critical nutrients not available from
complex media, e.g., the gene encoding D-alanine racemase for
Bacilli. The choice of an appropriate proper selectable marker will
depend on the host cell, and appropriate markers for different
hosts are well known to persons of ordinary skill in the art.
[0064] The vectors containing the nucleic acids of interest can be
transcribed in vitro, and the resulting RNA introduced into the
host cell by well-known methods, e.g., by injection, or the vectors
can be introduced directly into host cells by methods well known to
persons of ordinary skill in the art, which vary depending on the
type of cellular host, including electroporation; transfection
employing calcium chloride, rubidium chloride, calcium phosphate,
DEAE-dextran, or other substances; microprojectile bombardment;
lipofection; infection (where the vector is an infectious agent,
such as a retroviral genome); and other methods. The introduction
of the polynucleotides into the host cell by any method known in
the art, including, inter alia, those described above, will be
referred to herein as "transformation." The cells into which have
been introduced nucleic acids described above are meant to also
include the progeny of such cells.
[0065] Clones are selected by using markers, depending on the mode
of the vector construction. The marker may be on the same or a
different DNA molecule, preferably the same DNA molecule. In
prokaryotic hosts, the transformant may be selected, e.g., by
resistance to ampicillin, tetracycline or other antibiotics.
Production of a particular product based on temperature sensitivity
may also serve as an appropriate marker.
[0066] Prokaryotic or eularyotic cells transformed with the
polynucleotides of the present invention are useful not only for
the production of the nucleic acids and polypeptides of the present
invention, but also, for example, in studying the characteristics
of ACP1 polypeptides.
[0067] Antisense polynucleotide sequences are useful in preventing
or diminishing the expression of the locus, as will be appreciated
by those skilled in the art. For example, polynucleotide vectors
containing all or a portion of the locus or other sequences from
the region (particularly those flanking the locus) may be placed
under the control of a promoter in an antisense orientation and
introduced into a cell. Expression of such an antisense construct
within a cell will interfere with transcription and/or translation
and/or replication.
[0068] In order to detect the presence of a non-*A ACP1 allele
predisposing an individual to hyperlipidemia, especially
hyperlipidemia associated with metabolic syndrome, a biological
sample such as blood is prepared and analyzed for the presence or
absence of predisposing alleles of ACP1. Such diagnoses may be
performed by diagnostic laboratories, or, alternatively, diagnostic
kits are manufactured and sold to health care providers or to
private individuals for self-diagnosis.
[0069] Initially, the screening method can involve amplification of
the relevant ACP1 sequences. In another preferred embodiment of the
invention, the screening method involves a non-PCR based strategy.
Such screening methods include two-step label amplification
methodologies that are well known to persons of ordinary skill in
the art. Both PCR and non-PCR based screening strategies can detect
target sequences with a high level of sensitivity.
[0070] Preferred embodiments relating to methods for detecting a
non-*A AC1 allele or its mutations include enzyme linked
immunosorbent assays (ELISA), radioimmunoassays (RIA),
immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA),
including sandwich assays using monoclonal and/or polyclonal
antibodies.
[0071] This invention is particularly useful for screening
compounds by using a non-*A ACP1 polypeptide or binding fragment
thereof in any of a variety of drug screening techniques. There are
a number of protein tyrosine phosphatase (PTP) inhibitors known in
the art (see e.g., ref. 38). It is a matter of routine
experimentation in the pharmaceutical arts to screen such compounds
for activity specifically against ACP1, and subsequently to
evaluate potential drugs for toxicity, side-effects, etc. to
determine their ultimate suitability for in vivo use in humans.
[0072] For example, the polypeptide or fragment employed in such a
test may either be free in solution, affixed to a solid support, or
borne on a cell surface. One method of drug screening utilizes
eukaryotic or prokaryotic host cells which are stably transformed
with recombinant polynucleotides expressing the polypeptide or
fragment, preferably in competitive binding assays. Such cells,
either in viable or fixed form, can be used for standard binding
assays. One may measure, for example, for the formation of
complexes between a non-*A ACP1 polypeptide or fragment and the
agent being tested, or examine the degree to which the formation of
a complex between a non-*ACP1 polypeptide of the invention or
fragment and a known ligand. Alternatively, one may measure the
enzymatic activity of the ACP1 protein in the presence of the agent
being tested, either by measuring the rate of formation of a
reaction product (e.g., dephosphorylated ALBP, or free phospate) or
the disappearance of a substrate, such as ALBP.
[0073] Following identification of a substance which modulates or
affects activity of the non-*A ACP1 enzyme, the substance may be
investigated further. Furthermore, it may be manufactured and/or
used in preparation, i.e., manufacture or formulation, or a
composition such as a medicament, pharmaceutical composition or
drug. These may be administered to individuals. Thus, the present
invention extends, in various aspects, not only to a substance
identified using a nucleic acid molecule as a modulator of
polypeptide activity, in accordance with what is disclosed herein,
but also to a pharmaceutical composition, medicament, drug or other
composition comprising such a substance, methods comprising
administration of such a composition comprising such a substance,
methods comprising administration of such a composition to a
patient, e.g., for treatment of metabolic syndrome, use of such a
substance in the manufacture of a composition for administration,
e.g., for treatment of hyperlipidemia and/or metabolic syndrome,
and a method of making a pharmaceutical composition comprising
admixing such a substance with a pharmaceutically acceptable
excipient, vehicle or carrier, and optionally other
ingredients.
[0074] The present invention contemplates an antisense
polynucleotide that hybridizes with mRNA molecules that encode a
non-*A ACP1 polypeptide, and the use of one or more of those
polynucleotides in treating metabolic syndrome. An antisense
polynucleotide can for example be administered by gene therapy. The
polynucleotide may be introduced into the cell in a vector such
that the polynucleotide remains extrachromosomal. In such a
situation, the polynucleotide will be expressed by the cell from
the extrachromosomal location. Vectors for introduction of
polynucleotides for extrachromosomal maintenance are known in the
art, and any suitable vector may be used. The antisense
polynucleotide may be employed in gene therapy methods in order to
decrease the amount of the expression products of a non-*A ACP1 in
persons predisposed to, or suffering from, hyperlipidemia,
especially hyperlipidemia associated with metabolic syndrome.
[0075] Cells and animals which carry a specific ACP1 allele can be
used as model systems to study and test for substances which have
potential as therapeutic agents. The cells are typically cultured
cells and may be isolated from individuals having the allele of
interest. Alternatively, the cell line or animal can be engineered
to carry the ACP1 allele of interest using standard techniques
well-known in the art. After test substances have been administered
to the animals, the animals are assessed for hyperlipidemia, and/or
expression of other symptoms associated with metabolic disorder,
including obesity, hypertension, non-insulin dependent diabetes and
coronary artery disease. These animal models provide an extremely
important testing vehicle for potential therapeutic products.
Alternatively, as described above, ACP1 activity can be measured in
the cells or animal. Reduction in ACP1 activity relative to
controls indicates suitability as a therapeutic agent for treating
or preventing hyperlipidemia, especially hyperlipidemia associated
with metabolic syndrome. Further analysis of potential drugs thus
identified, to assess specificity, toxicity, side effects, etc., is
a matter of routine experimentation in the pharmaceutical arts.
[0076] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA, genetics,
immunology, cell biology, cell culture and transgenic biology,
which are within the skill of the art. See, e.g., Maniatis et al.,
1982 (28); Sambrook et al., 1989(32); Ausubel et al., 1992 (26);
Anand, 1992 (25); Culture Of Animal Cells (R. I. Freshney, Alan R
Liss, Inc., 1987) (27); Immobilized Cells And Enzymes (IRL Press,
1986) (34); B. Perbal, A Practical Guide To Molecular Cloning
(1984) (30); the treatise, Methods In Enzymology (Academic Press,
Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H.
Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory)
(29); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.)
(33), Immunochemical Methods In Cell And Molecular Biology (Mayer
and Walker, eds., Academic Press, London, 1987) (35); Riott,
Essential Immunology, 6th Edition, Blackwell Scientific
Publications, Oxford, 1988 (31); Hogan et al., Manipulating the
Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1986) (36).
[0077] The present invention is described by reference to the
following Examples, which are offered by way of illustration and
are not intended to limit the invention in any manner. Standard
techniques well known by persons of ordinary skill in the art
and/or the techniques specifically described below were
utilized.
EXAMPLE 1
[0078] A study was performed at the Center for Health Promotion at
Loma Linda University Medical Center, Loma Linda, Calif.
Non-Hispanic Caucasian females (average age 54.5.+-.6.5 [SD] years)
with a lifetime history of obesity (average BMI 39.1.+-.7.5 [SD])
were recruited from the community by newspaper ads. To obtain a
broad range of BMI, each currently obese subject was asked to bring
to the clinic an age matched non-obese friend from her own ethnic,
educational and social class. Using the criteria described by
Trakas et al (13) subjects were classified into three weight
groups: BMI.ltoreq.29=non-obese; BMI30-34=obese;
BMI.gtoreq.35=morbidly obese. The obese and morbidly obese subjects
had an average BMI of 39.1.+-.7.5 [SD]. The non-obese subjects had
an average BMI of 24.0.+-.2.7 [SD]. All subjects were unrelated,
were drug-free, and were specifically not taking lipid lowering
agents or blood pressure medication. Blood lipid levels were
performed on individuals fasted overnight. The cholesterol,
triglycerides and HDL were determined by enzymatic assay (DuPont
Dimension analyzer), and the LDL was calculated from these
results.
[0079] The ACP1 polymorphism was determined with an RFLP-PCR method
as follows: an A to G transversion at the nt 24 of the exon 6
(GenBank GI: 178004) creates in the *A allele a restriction site
for TaqI (14), while a C to T transition at the nt 15 of the exon 3
(GenBank GI:306443) removes in the *C allele a restriction site for
HhaI (Sensabaugh G, unpublished). Both the TaqI and HhaI
restriction polymorphisms have been determined by RFLP-PCR after
amplification respectively of fragments of the exon 6 and exon 3
and digestion of the PCR product with an excess of the relative
restriction enzyme. In both determinations the forward primers
contained a fixed restriction site for HhaI and for TaqI, which was
used as an internal control. One hundred fifty-one obese subjects
were genotyped for both the TaqI and HhaI polymorphisms, 26
additional obese subjects and all non-obese subjects were genotyped
for the non-*A (TaqI) polymorphism only. Chi square test, ANOVA,
Students T-test, Levene's test for homogeneity of variance, MANOVA
and correlation analyses were performed using the SPSS program
(15).
[0080] ACP1 genotype frequencies showed no significant deviation
from Hardy-Weinberg expectation in the samples, and no significant
association was found between ACP1 polymorphism and BMI. Table 1
shows the number of subjects in each of the BMI groups by number of
subjects in each genotypes. Homozygocity for the A allele is fairly
rare. Since this was an uncommon genotype and since there was no
consistent tendecny for the A/A nucleotide genotype to show a lower
triglyceride level than the T/A genotype, in all analyses the T/A
and A/A genotypes were combined into a group termed *A carrier. An
ANOVA analysis was performed in the subgroup of obese subjects
genotyped for both the *A/not*A and the *C/not*C SNPs, with the
ACP1 polymorphism as independent variable. and clinical variables
(BMI, total cholesterol, HDL cholesterol, LDL cholesterol,
Cholesterol/HDL ratio, triglycerides and fasting glucose) as
dependent variables. The analysis showed that a highly significant
association of ACP1 polymorphism with cholesterol and with
triglycerides levels present in the obese sample was due to an
effect of the *A rather than the *C allele (data not shown).
1TABLE I ACP1 Genotypes in the 277 non-Hispanic Caucasian females
studied Non A/Non A Non A/A A/A Total BMI 29 or less 39 (48%) 35
(43%) 8 (10%) 82 BMI 30-34 31 (52%) 27 (45%) 2 (3%) 60 BMI 35 or
more 62 (46%) 56 (42%) 17 (13%) 135 p = n.s.
[0081] Table II shows the results of ANOVA of the association of
the *A carrier (*A/*A, *A/*B and *A/*C) genotypes versus the non-*A
carrier (*B/*B, *B/*C and *C/*C) genotypes for different
biochemical variables in the obese subjects (BMI.gtoreq.30). There
was a significant increase in the total cholesterol, LDL
cholesterol, cholesterol/HDL ratio, and triglyceride levels in
non-*A carriers versus *A allele carriers. There was borderline
association with waist-hip ratio. There was no association of the
*A allele with BMI or blood lipids in the non-obese subjects. A
MANOVA test using age as a co-variate showed these results were
independent of age.
2TABLE II Associationss between *A allele and BMI, BLL and fasting
glucose level in 195 obese post-menopausal women *A carrier non-*A
carrier (N = 102) (N = 93) p BMI 38.7 (8.2) 38.0 (6.0) n.s. Total
Cholesterol (mg/dl) 207.4 (39.6) 224.1 (39.1) 0.002 HDL Cholesterol
(mg/dl) 54.7 (12.8) 53.1 (13.4) n.s. LDL Cholesterol (mg/dl) 117.9
(35.5) 132.7 (37.3) 0.015 Cholesterol/HDL ratio 3.9 (1.1) 4.4 (1.3)
0.006 Triglyceride (mg/dl) 165.2 (74.4) 207.3 (105.1) 0.001 Fasting
Glucose (mg/dl) 105.2 (29.0) 113.8 (50.1) n.s. Waist-Hip Ratio .823
(.13) .857 (.123) 0.09
[0082] Grouping the ACP1 genotypes according to their known
enzymatic activity (8), as shown in Table 1, a Pearson correlation
analysis of putative enzymatic parameters (total activity, A and B
isoform concentration and A/B ratio), associated with ACP1
genotypes, and BMI, total cholesterol and triglyceride showed a
significant positive correlation of both the amount of total
enzymatic activity and of A isoform activity associated with ACP1
genotypes and total cholesterol (R=0.204, p=0.01 for total
activity) and triglyceride (R=0.227, p=0.005 for total activity).
No association was present with B isoform and A/B activity ratio
(data not shown).
[0083] FIG. 1 shows the relationship between ACP1 polymorphism and
triglyceride levels for subjects grouped according to their
classification as non-obese, obese or morbidly obese (13). The
relationship of ACP1 genotype with triglyceride concentrations is
positively associated with BMI in obese subjects and this finding
was more pronounced in morbidly obese subjects. An ANOVA analysis
performed in *A allele carriers and non-*A allele carriers
separately showed that the increase of BMI is significantly
associated with the development of hypertriglyceridemia in non-*A
allele carriers Only (p=0.001 for non-*A carriers, p=0.076 for *A
carriers). These data indicates that the ACP1 *A allele exerts a
strong protective effect against the development of
hypertrigliceridemia in obese subjects. A similar relationship was
found between ACP1 and cholesterol levels in different classes of
BMI, but it didn't reach statistical significance (data not
shown).
References
[0084] 1. Abate N. Obesity and cardiovascular disease. 2000
Pathogenetic role of the metabolic syndrome and therapeutic
implications. J Diabetes Complications. 14: 154-74.
[0085] 2. Allison D B, Fontaine K R, Manson J E, Stevens J,
VanItallie T B. 1999 Annual, deaths attributable to obesity in the
United States . . . AMA. 282:1530-8.
[0086] 3. Alexander J K. 2001 Obesity and coronary artery disease.
Am J Med Sci. 321: 215-24.
[0087] 4. Duriez P, Fruchart J C. 1999. Recent developments in the
treatment of hypertriglyceridemia Curr Atheroscler Rep. 1
:31-7.
[0088] 5. Ramponi G, Stefani M. 1997 Structure and function of the
low Mr phosphotyrosine protein phosphatases. Biochim Biophys Acta.
1341:137-56.
[0089] 6. Cirri P, Fiaschi T, Chiarugi P, Camici G, Manao G, Raugei
G, Ramponi G. 1996 The molecular basis of the differing kinetic
behavior of the two low molecular mass phosphotyrosine protein
phosphatase isoforms. J Bioi Chem. 271 :2604-7.
[0090] 7. Shekels L L, Smith A J, Van Etten R L, Bernlohr D A. 1992
Identification of the adipocyte acid phosphatase as a PAO-sensitive
tyrosyl phosphatase. Protein Sci. 1:710-21.
[0091] 8. Dissing J. 1993 Human "red cell" acid phosphatase (ACP1)
genetic, catalytic and molecular properties. PhD Thesis. Kobenhavn
Universitat, Kobenhavn, Denmark.
[0092] 9. Lucarini N, Finocchi G, Gloria-Bottini F, Macioce M,
Borgiani P, Amante A, Bottini E. 1990 A possible genetic component
of obesity in childhood. Observations on acid phosphatase
polymorphism. Experientia. 46:90-1.
[0093] 10. Botiini E, Lucarini N, Gerlini G, Finocchi G, Scire G,
Gloria-Bottini F. 1990 Enzyme polymorphism and clinical variability
of diseases: study of acid phosphatase locus 1 (ACP1 ) in obese
subjects. Hum Biol. 62:403.
[0094] 11. Paggi A, Borgiani P, Gloria-Bottini F, Russo S, Saponara
I, Banci M, Amante A et al. 1991 Further studies on acid
phosphatase in obese subjects. Dis Markers. 9:1-7.
[0095] 12. Lucarini N, Antonacci E, Bottini N, Gloria Bottini F.
1997 Low-molecular-weight acid phosphatase (ACP1), obesity, and
blood lipid levels in subjects with non-insulin-dependent diabetes
mellitus; Hum Biol. 69:509-15.
[0096] 13. Trakas K, Oh P I, Singh S, Risebrough N, Shear N H. 2001
The health status of obese individuals in Canada. Int J Obes Relat
Metab Disord. 25:662-8.
[0097] 14. Sensabaugh O F and Lazaruk K A. 1993 A TaqI site
identifies the * A allele at the ACP1 locus. Hum Mol Genet.
2:1079.
[0098] 15. SPSS /PC+Version 5.0. 1992 SPSS Inc, Chicago, Ill.
[0099] 16. Dissing J, Johnsen A H. 1992 Human red cell acid
phosphatase (ACP1): the primary structure of the two pairs of
isozynies encoded by the ACP1 * A and ACP1 *C alleles. Biochim
BiophysActa. 1121 :261-8.
[0100] 17. Chiarugi P, Cirri P, Marra F, Raugei G, Canici G, Manao
G, Ramponi G. 1997 LMW-PTP is a negative regulator of
insulin-mediated mitotic and metabolic signalling. Biochem Biophys
Res Commun. 238:676-82.
[0101] 18. Buelt M K, Xu Z, Banaszak L J, Bernlohr D A. 1992
Structural and finctional characterization of the phosphorylated
adipocyte lipid-binding protein (pp 15). Biochemistry.
31:3493-9.
[0102] 19. Chagnon Y C, Perusse L, Weisnagel S J, Rankinen T,
Bouchal. d C. 2000 The human obesity gene map: the 1999 update.
Obes Res. 8:89-117.
[0103] 20. Elchebly M, Cheng A, Tremblay M L. 2000 Modulation of
insulin signaling by protein tyrosine phosphatases. J Mol Med.
78:473-82.
[0104] 21. Liao K, Hoffman R D, Lane M D. 1991 Phosphotyrosyl
turnover in insulin signaling. Characterization of two
membrane-bound pp15 protein tyrosine phosphatases from 3T3-L1
adipocytes. J Biol Chem. 266:6544-53. I
[0105] 22. Caselli A, Taddei M L, Manao G, Camici G, Ramponi G.
2001 Tyrosine-phosphorylated caveolin is a physiological substrate
of the low Mr protein-tyrosine phosphatase. J Biol Chem.
276:18849-54.
[0106] 23. Okamoto T, Schlegel A, Scherer P E, Lisanti M P. 1998
Caveolins, a family of scaffolding proteins for organizing
"preassembled signaling complexes" at the plasma membrane. J Biol.
Chem.273:5419-22.
[0107] 24. Zhang B B, Moller D E. 2000 New approaches in the
treatment of type 2 diabetes. Curr Opin Chem Biol. 4:461-7.
[0108] 25. Anand, R. Techniques for the Analysis of Complex
Geniomes, (Academic Press) 1992.
[0109] 26. Ausubel, F. M., et al. Current Protocols in Molecular
Biology, (J. Wiley and Sons, NY) 1992.
[0110] 27. Freshney, Alan R. Liss, Inc. Culture Of Animal Cells
1987.
[0111] 28. Maniatis. T., et al. Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)
1982.
[0112] 29. Miller, J. H. and M. P. Calos eds.: Gene Transfer
Vectors For Mammalian Cells, 1987, Cold Spring Harbor
Laboratory).
[0113] 30. Perbal, A Practical Guide To Molecular Cloning 1984.
Methods In Enzymology (Academic Press, Inc., N.Y.)
[0114] 31. Roitt, Essential Immunology, 6th Edition, Blackwell
Scientific Publications, (Oxford, 1988.
[0115] 32. Sambrook, J., et al. Molecular Cloning: A Laboratory
Manual, 2nd Ed. (Cold Spring 0Harbor Laboratory, Cold Spring
Harbor, N.Y.) 1989.
[0116] 33. Wu et al. eds.: Methods In Enzymology, Vols. 154 and
155.
[0117] 34. Immobilized Cells And Enzymes (IRL Press, 1986).
[0118] 35. Immunochemical Methuds In Cell And Molecular Biology
(Mayer and Walker, eds., Academic Press, London, 1987) (35).
[0119] 36. Hogan et al., Manipulating the Mouse Embryo, (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) 1986.
[0120] 37. McGall, G. H. and F. C. Christians, 2002, High-density
gene chopolinucleotide prob arrays, Abd. Biochem. Eng. Biotechnol.
77:21-42.
[0121] 38. Heo, Y. S., et al., 2002, Structural basis for
inhibition of protein tyrosine phosphotases by Keggin compounds
phosphomolygdate and phosphotunstate, Exp. Mol. Med.
34:211-213.
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