U.S. patent application number 12/553330 was filed with the patent office on 2010-03-04 for sequence variations in pnpla3 associated with hepatic steatosis.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Jonathan C. Cohen, Helen H. Hobbs.
Application Number | 20100056384 12/553330 |
Document ID | / |
Family ID | 41726333 |
Filed Date | 2010-03-04 |
United States Patent
Application |
20100056384 |
Kind Code |
A1 |
Hobbs; Helen H. ; et
al. |
March 4, 2010 |
Sequence Variations in PNPLA3 Associated with Hepatic Steatosis
Abstract
Disclosed are methods of identifying a genetic variant in a
person determined to have or be predisposed having a fatty liver by
determining whether the person has PNPLA3-I148M. Also disclosed are
methods of identifying a genetic variant in a person by determining
whether the person has PNPLA3-I148M; and prescribing to the person
a treatment to reduce liver fat or associated inflammation.
Inventors: |
Hobbs; Helen H.; (Dallas,
TX) ; Cohen; Jonathan C.; (Dallas, TX) |
Correspondence
Address: |
RICHARD ARON OSMAN
3525 Del Mar Heights Rd. #915
San Diego
CA
92130
US
|
Assignee: |
Board of Regents, The University of
Texas System
|
Family ID: |
41726333 |
Appl. No.: |
12/553330 |
Filed: |
September 3, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61094408 |
Sep 4, 2008 |
|
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Current U.S.
Class: |
506/7 ;
435/6.11 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6883 20130101; C12Q 2600/172 20130101 |
Class at
Publication: |
506/7 ;
435/6 |
International
Class: |
C40B 30/00 20060101
C40B030/00; C12Q 1/68 20060101 C12Q001/68 |
Goverment Interests
[0002] This work was supported by grants from the NIH and NHLBI
Program for Genomic Applications (HL-066681); the Government has
certain rights in this invention.
Claims
1. A method of identifying a genetic variant in a person determined
to have or be predisposed to having a liver disease selected from
fatty liver, an increase in hepatic fat, hepatic steatosis,
nonalcoholic fatty liver disease, and associated inflammation, the
method comprising: (a) determining whether the person has
PNPLA3-I148M.
2. The method of claim 1 further comprising the step of (b)
determining whether the person has PNPLA3-S453I.
3. The method of claim 1 wherein the determining step (a) comprises
detecting using a method selected from the group consisting of:
mass spectroscopy, oligonucleotide microarray analysis,
allele-specific hybridization, allele-specific PCR, and sequencing,
a marker of PNPLA3-I148M that is SNP rs738408, or a surrogate SNP
in linkage disequilibrium with the PNPLA3-I148M and having a
r.sup.2 value greater than 0.5, wherein the surrogate SNP is
selected from: TABLE-US-00002 rs12483959, rs1977081, rs2072905,
rs1010022, rs11090617, rs1883349, rs2896019, rs926633, rs4823173,
rs2281135, rs2073081, rs2294916, and rs2076211, rs2072907,
rs1010023, rs4823179.
4. The method of claim 2 wherein the determining step (a) comprises
detecting using a method selected from the group consisting of:
mass spectroscopy, oligonucleotide microarray analysis,
allele-specific hybridization, allele-specific PCR, and sequencing,
a marker of PNPLA3-I148M that is SNP rs738408, or a surrogate SNP
in linkage disequilibrium with the PNPLA3-I148M and having a
r.sup.2 value greater than 0.5, wherein the surrogate SNP is
selected from: TABLE-US-00003 rs12483959, rs1977081, rs2072905,
rs1010022, rs11090617, rs1883349, rs2896019, rs926633, rs4823173,
rs2281135, rs2073081, rs2294916, and rs2076211, rs2072907,
rs1010023, rs4823179.
5. The method of claim 3 wherein the determining step (a) comprises
detecting a plurality of the SNP and surrogate SNP markers.
6. The method of claim 4 wherein the determining step (a) comprises
detecting a plurality of the SNP and surrogate SNP markers.
7. A method of identifying a genetic variant in a person, the
method comprising: (a) determining whether the person has
PNPLA3-I148M; and (b)(i) providing the person with information
about risk of developing a liver disease selected from fatty liver,
an increase in hepatic fat, hepatic steatosis, nonalcoholic fatty
liver disease, and associated inflammation, or (ii) providing the
person with a recommendation for an additional diagnostic test or
monitoring to detect an indication of the liver disease, or (iii)
prescribing to the person a treatment for the liver disease.
8. The method of claim 7 further comprising the step of (c)
determining whether the person has PNPLA3-S453I.
9. The method of claim 7 wherein the determining step (a) comprises
detecting using a method selected from the group consisting of:
mass spectroscopy, oligonucleotide microarray analysis,
allele-specific hybridization, allele-specific PCR, and sequencing,
a marker of PNPLA3-I148M that is SNP rs738408, or a surrogate SNP
in linkage disequilibrium with the PNPLA3-I148M and having a
r.sup.2 value greater than 0.5, wherein the surrogate SNP is
selected from: TABLE-US-00004 rs12483959, rs1977081, rs2072905,
rs1010022, rs11090617, rs1883349, rs2896019, rs926633, rs4823173,
rs2281135, rs2073081, rs2294916, and rs2076211, rs2072907,
rs1010023, rs4823179.
10. The method of claim 8 wherein the determining step (a)
comprises detecting using a method selected from the group
consisting of: mass spectroscopy, oligonucleotide microarray
analysis, allele-specific hybridization, allele-specific PCR, and
sequencing, a marker of PNPLA3-I148M that is SNP rs738408, or a
surrogate SNP in linkage disequilibrium with the PNPLA3-I148M and
having a r.sup.2 value greater than 0.5, wherein the surrogate SNP
is selected from: TABLE-US-00005 rs12483959, rs1977081, rs2072905,
rs1010022, rs11090617, rs1883349, rs2896019, rs926633, rs4823173,
rs2281135, rs2073081, rs2294916, and rs2076211, rs2072907,
rs1010023, rs4823179.
11. The method of claim 9 wherein the determining step comprises
detecting a plurality of the SNP and surrogate SNP markers.
12. The method of claim 10 wherein the determining step comprises
detecting a plurality of the SNP and surrogate SNP markers.
13. A method of identifying a genetic variant in a person
determined to have or be predisposed to having a subnormal hepatic
fat or triglyceride content, or a subnormal susceptibility to
hepatic steatosis or nonalcoholic fatty liver disease, the method
comprising: determining whether the person has PNPLA3-S4531.
14. The method of claim 13 wherein the determining step comprises
detecting using a method selected from the group consisting of:
mass spectroscopy, oligonucleotide microarray analysis,
allele-specific hybridization, allele-specific PCR, and sequencing,
a marker of PNPLA3-S453I that is SNP rs6006460, or a surrogate SNP
in linkage disequilibrium with the PNPLA3-S453I and having a
r.sup.2 value greater than 0.5.
15. The method of claim 13 wherein the determining step comprises
detecting a plurality of the SNP and surrogate SNP markers.
16. A method of identifying a genetic variant in a person, the
method comprising: determining whether the person has PNPLA3-S453I;
and providing the person with information about predisposition to
have a subnormal hepatic fat or triglyceride content or a subnormal
susceptibility to hepatic steatosis or nonalcoholic fatty liver
disease, or providing the person with a recommendation for an
additional diagnostic test or monitoring to detect an indication of
liver disease, or prescribing to the person an alternative
treatment for liver disease.
17. The method of claim 16 wherein the determining step comprises
detecting using a method selected from the group consisting of:
mass spectroscopy, oligonucleotide microarray analysis,
allele-specific hybridization, allele-specific PCR, and sequencing,
a marker of PNPLA3-S453I that is SNP r s6006460, or a surrogate SNP
in linkage disequilibrium with the PNPLA3-S453I and having a
r.sup.2 value greater than 0.5.
18. The method of claim 16 wherein the determining step comprises
detecting a plurality of the SNP and surrogate SNP markers.
Description
[0001] This application claims priority to U.S. Ser No. 61/094,408,
filed: Sep. 4, 2008.
[0003] The field of the invention is sequence variations in the
human gene PNPLA3 as risk factors for hepatic steatosis and hepatic
injury.
BACKGROUND OF THE INVENTION
[0004] Nonalcoholic fatty liver disease (NAFLD) is a burgeoning
health problem of unknown etiology that varies in prevalence among
ethnic groups. To identify genetic variants/polymorphisms
contributing to differences in hepatic fat content, we performed a
genome-wide association scan of nonsynonymous sequence variations
in a multiethnic population. A variant (I148M) in PNPLA3
(Patatin-like phospholipase domain containing 3) was strongly
associated with increased hepatic fat levels and with hepatic
inflammation. The variant was most common in Hispanics, the group
most susceptible to NAFLD; homozygotes had a >2-fold higher
hepatic fat content. Resequencing revealed another variant
associated with lower hepatic fat content in African-Americans, the
group at lowest risk of NAFLD. Thus, variation in PNPLA3
contributes to ethnic and inter-individual differences in hepatic
fat content and susceptibility to NAFLD.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the invention provides methods and
compositions for identifying a genetic variant in a person
determined to have or be predisposed to having a liver disease, the
method comprising the step of determining whether the person has
PNPLA3-I148M.
[0006] In another embodiment, the invention provides methods and
compositions for identifying a genetic variant in a person, the
method comprising the steps of (a) determining whether the person
has PNPLA3-I148M; and (b)(i) providing the person with information
about risk of developing a liver disease, or (ii) providing the
person with a recommendation for an additional diagnostic test or
monitoring to detect an indication of the liver disease, or (iii)
prescribing to the person a treatment for the liver disease.
[0007] In particular embodiments, the liver disease is fatty liver,
an increase in or supra-normal hepatic fat, hepatic steatosis,
steatohepatitis, nonalcoholic fatty liver disease, or associated
inflammation.
[0008] In particular embodiments, the methods further comprise the
step of determining whether the person has PNPLA3-S453I.
[0009] In particular embodiments, the determining step comprises
detecting the PNPLA3-I148M and/or PNPLA3-S453I using a method
selected from the group consisting of: mass spectroscopy,
oligonucleotide microarray analysis, allele-specific hybridization,
allele-specific PCR, and sequencing.
[0010] In particular embodiments, the determining step comprises
detecting a marker of PNPLA3-I148M that is SNP rs738408, or a
surrogate SNP in linkage disequilibrium with the PNPLA3-I148M and
having a r.sup.2 value greater than 0.5.
[0011] As described further below, the variant allele PNPLA3-I148M
is readily detected by associated SNPs that are in linkage
disequilibrium with PNPLA3-I148M, and provide markers for this
allele: SNP markers for PNPLA3-I148M include rs738408 (r.sup.2=1)
and associated SNPs having a r.sup.2 value greater than 0.5. In
Table 1 below, the rs number is shown, followed by the correlation
with the 1148M allele (r.sup.2 value). These data are from
Caucasians in the HapMap.
TABLE-US-00001 TABLE 1 SNP markers for I148M rs12483959 0.657
rs2072905 0.609 rs11090617 0.624 rs2896019 0.607 rs4823173 0.598
rs2073081 0.568 rs2076211 0.657 rs1010023 0.609 rs1977081 0.609
rs1010022 0.609 rs1883349 0.643 rs926633 0.609 rs2281135 0.609
rs2294916 0.609 rs2072907 0.609 rs4823179 0.579
[0012] In particular embodiments, the determining step comprises
detecting a plurality of the SNP and surrogate SNP markers.
[0013] In particular embodiments, the methods further comprise an
antecedent step of determining that the person has or is
predisposed to having a subject liver disease. The subject liver
diseases are amenable to convention clinical diagnosis; for
example, fatty liver or hepatic steatosis may be determined inter
alia using computer-aided tomography (CAT) scan or NMR, such as
proton magnetic resonance spectroscopy, and is generally clinically
defined as hepatic triglyceride greater than 5.5%. Indicators of
predisposition to fatty liver include obesity, diabetes, insulin
resistance, and alcohol ingestion.
[0014] In particular embodiments, the methods may further comprise
the step of prescribing to the person a treatment for the liver
disease or treating the person with a therapy for the liver
disease, such an anti-obesity drug such as Orlistat, Sibutramine,
Byetta, Symlin, Rimonabant, or an anti-diabetic drugs such as a
thiazolidinedione (e.g. rosiglitazone), or metformin or
glimepiride, or anti-inflammatory drugs.
[0015] In another embodiment, the invention provides methods and
compositions for identifying a genetic variant in a person
determined to have or be predisposed to having a subnormal hepatic
fat or triglyceride content, or a subnormal susceptibility to
hepatic steatosis or nonalcoholic fatty liver disease, the method
comprising the step of determining whether the person has
PNPLA3-S453I.
[0016] In another embodiment, the invention provides methods and
compositions for identifying a genetic variant in a person, the
method comprising the steps of: (a) determining whether the person
has PNPLA3-S453I; and (b) providing the person with information
about predisposition to have a subnormal hepatic fat or
triglyceride content or a subnormal susceptibility to hepatic
steatosis or nonalcoholic fatty liver disease, or providing the
person with a recommendation for an additional diagnostic test or
monitoring to detect an indication of liver disease, or prescribing
to the person an alternative treatment for liver disease.
[0017] In particular embodiments, the determining step comprises
detecting the variant using a method selected from the group
consisting of: mass spectroscopy, oligonucleotide microarray
analysis, allele-specific hybridization, allele-specific PCR, and
sequencing.
[0018] In particular embodiments, the determining step comprises
detecting a marker of PNPLA3-S453I that is SNP rs6006460, or a
surrogate SNP in linkage disequilibrium with the PNPLA3-S453I and
having a r.sup.2 value greater than 0.5.
[0019] In particular embodiments, the determining step comprises
detecting a plurality of the SNP and surrogate SNP markers.
[0020] In other embodiments, the invention provides reagents and
kits for practicing the disclosed methods.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0021] Genetic variation in PNPLA3 confers susceptibility to fatty
liver disease. In humans, adipose tissue serves as a reservoir to
limit the deposition of triglyceride (TG) in the liver and other
metabolically active tissues.sup.1. The effectiveness of this
buffer in protecting against the accumulation of fat in the liver
varies widely among individuals: hepatic fat content ranges from
less than 1% to more than 50% of liver weight in the general
population.sup.2. The accumulation of excess TG in the liver, a
condition known as hepatic steatosis (or fatty liver), is
associated with adverse metabolic consequences, including insulin
resistance and dyslipidemia.sup.3,4. In a subset of individuals
hepatic steatosis promotes an inflammatory response in the liver,
referred to as steatohepatitis, which can progress to cirrhosis and
liver cancer.sup.3,5. Nonalcoholic fatty liver disease (NAFLD) is
the most common form of liver disease in Western countries.sup.6.
Approximately 10% of liver transplants performed in the United
States are for cirrhosis related to NAFLD.sup.4.
[0022] Factors promoting deposition of fat in the liver include
obesity, diabetes, insulin resistance, and alcohol
ingestion.sup.3,6. Hispanics are particularly susceptible to
develop fatty liver and also have a higher prevalence of
steatohepatitis and cirrhosis, whereas African-Americans tend to be
resistant to the accumulation of liver fat and are less prone to
develop liver failure.sup.2,7-9.
[0023] To identify DNA sequence variations that contribute to
inter-individual differences in NAFLD, we performed a genome-wide
survey of nonsynonymous (NS) sequence variations in a multiethnic
population-based study, the Dallas Heart Study.sup.10, in which
hepatic TG content was measured using proton magnetic resonance
spectroscopy (.sup.1H-MRS), the most accurate, quantitative
noninvasive method available.sup.2,11,12. We assayed directly the
sequence variations with a higher likelihood of affecting gene
function. Of the 12,138 NS variants assayed using chip-based
oligonucleotide hybridization.sup.13, 9,229 exceeded the quality
control threshold for the study (see METHODS) and were included in
the analysis.
[0024] Each variant was tested for association with hepatic fat
content in the 1,032 African-American, 696 European-American and
383 Hispanic study participants in the Dallas Heart Study who
obtained .sup.1H-MRS of the liver.sup.2. To maximize statistical
power, the three ethnic groups were pooled and global ancestry was
included as a covariate to control for population stratification
(see METHODS). The quantile-quantile plot of P-values showed no
systematic deviation from the null distribution.
[0025] A single variant in PNPLA3 (rs738409) was strongly
associated with liver fat content (P=5.9.times.10.sup.-10). The
variant is a cytosine to guanine substitution that changes codon
148 from isoleucine to methionine; this residue is highly conserved
in vertebrates. PNPLA3 encodes a 481 amino acid protein of unknown
function that belongs to the patatin-like phospholipase
family.sup.14. The progenitor of this family, patatin, is a major
protein of potato tubers and has nonspecific lipid acyl hydrolase
activity.sup.15,16. None of the other NS sequence variants tested
in the genome-wide scan exceeded the Bonferroni-corrected threshold
for significance (P=5.4.times.10.sup.-6).
[0026] The association between PNPLA3-I148M and hepatic fat content
remained highly significant (P=7.0.times.10.sup.-14) after
adjusting for BMI, diabetes status, ethanol use, as well as global
and local ancestry, and was associated with a significant increase
in liver TG content in all three ethnic groups. Thus, the
association between rs738409 and liver fat content was not
attributable either to the effect of known risk factors for liver
fat accumulation or to population stratification.
[0027] The frequencies of the PNPLA3-I148M allele mirrored the
relative prevalence of NAFLD in the three ethnic groups.sup.2; the
highest frequency was in Hispanics (0.49), with lower frequencies
observed in European Americans (0.23) and African-Americans (0.17).
Accordingly, we examined the relationship between PNPLA3-I148M and
evidence of hepatic inflammation, as indicated by release of liver
enzymes into the circulation. A significant elevation in serum
levels of alanine aminotransferase (ALT) was found in association
with the PNPLA3-I148M allele in the entire sample
(P=3.7.times.10.sup.-4). Analysis of the three ethnic groups
revealed that the association with ALT was limited to the Hispanics
(P=1.3.times.10.sup.-5), the group with the greatest prevalence of
hepatic steatosis and susceptibility to cirrhosis.sup.2,7. The
PNPLA3-I148M allele was also associated with serum aspartate
aminotransferase levels in Hispanics (P=0.002). These findings are
consistent with our prior observation that a higher proportion of
Hispanics with hepatic steatosis have evidence of associated
inflammation.sup.7, and indicates that PNPLA3-I148M allele
adversely affects liver function.
[0028] Increased hepatic fat content is associated with insulin
resistance and dyslipidemia (increased plasma levels of TG and
lower levels of high density lipoprotein-cholesterol), but the
causal nature of these relationships remains poorly defined.sup.3.
No association was found between the PNPLA3-I148M allele and body
mass index (BMI) or indices of insulin sensitivity, including
fasting glucose and insulin or homeostatic model assessment of
insulin resistance (HOMA-IR) in the Dallas Heart Study. No
associations were observed between PNPLA3 genotype and plasma
levels of TG, total cholesterol, HDL-cholesterol or
LDL-cholesterol. A corresponding analysis in a larger, biracial
sample (n=14,821), the Atherosclerosis Risk in Communities
Study.sup.17, also revealed no association of PNPLA3-I148M with
BMI, indices of insulin sensitivity, or plasma levels of TG or
HDL-C. The data from these studies indicate that the PNPLA3-I148M
allele is associated with a systematic increase in liver fat
content but not with major alterations in glucose homeostasis or
lipoprotein metabolism. Thus, increased liver fat content does not
inevitably lead to insulin resistance, which is consistent with
recent observations in various animal models.sup.18,19.
[0029] To determine if other sequence variations in PNPLA3
contribute to differences in hepatic fat content, we resequenced
the coding region of PNPLA3 in the 80 men (32 African-Americans, 32
European-Americans, and 16 Hispanics) and 80 women who had the
highest levels of liver fat in the Dallas Heart Study, and in a
sex- and ethnicity-matched group with the lowest levels.sup.2. The
number of individuals with NS variants found only in the high group
(n=10) was similar to the number found only in the low group (n=8),
but the three subjects with likely null mutations (Fs-Y21 and
IVS7+1) were all in the high group, which is consistent with
loss-of-function of PNPLA3 causing an increase in hepatic TG
content.
[0030] Eight variants were present in both the low and the high
hepatic fat groups and the six most common were genotyped in the
sample. One variant, PNPLA3-S453I was common in African-Americans
(MAF=0.104) but rare in European-Americans (0.003) and Hispanics
(0.008). Median liver fat content was 18% lower in
African-Americans with the PNPLA3-S453I allele when compared to
African-Americans homozygous for the wild-type allele (3.3% versus
2.7%, P=6.times.10.sup.-4). Further evidence that the variant was
associated with lower hepatic fat content was the finding that a
significantly greater number of individuals with the PNPLA3-S453I
allele were present in the individuals with a hepatic fat content
in the lowest decile when compared to the highest decile of the
population. No significant differences in the number of individuals
identified in the extremes were found for any of the other five
SNPs.
[0031] The identification of a second allele of PNPLA3 that was
independently associated with liver fat content further supports a
role for PNPLA3 in determining liver fat levels, and indicates the
presence of both loss-of-function and gain-of-function alleles at
this locus.
[0032] The frequencies of PNPLA3-I148M and of PNPLA3-S453I in the
three ethnic groups represented in the Dallas Heart Study correlate
with ethnic differences in the relative propensity to develop NAFLD
(Hispanics>Caucasians>African-Americans).sup.2. Exclusion of
the individuals carrying either of these two alleles (PNPLA3-I148M
and PNPLA3-S453I) attenuated the differences in liver fat content
between the ethnic groups; regression analysis indicated that these
two sequence variations accounted for 72% of the observed ethnic
differences in hepatic fat content. Thus, genetic variation in
PNPLA3 accounts for a large fraction of the ethnic differences in
the propensity to accumulate excess fat in the liver.
[0033] Expression of PNPLA3 is under metabolic control in adipose
tissue and the liver, with levels being low in the fasted state and
increases dramatically with carbohydrate feeding.sup.20,21. PNPLA3
structurally resembles calcium-independent phospholipase A.sub.2
but the recombinant protein has low phospholipase activity when
expressed in insect (Sf9) cells.sup.22. PNPLA3 has more robust
activity against TG in vitro and can also transfer fatty acids to
and from mono- and diacylglycerol.sup.22.
[0034] Our finding that markers of liver inflammation (serum levels
of liver-derived enzymes) were elevated in 148M carriers, indicates
that genetic variation PNPLA3-I148M allele can confer
susceptibility to disease progression. Patatin-like phospholipase
family members in other organisms are up-regulated in response to
environmental insults.sup.23. The sequence variations we have
identified in PNPLA3 provides predictive information regarding the
risk of developing hepatic steatosis and liver injury in response
to environmental stresses such as caloric excess, infections, or
drugs.
[0035] METHODS; Study populations. The Dallas Heart Study is a
population-based probability sample of Dallas County. The sampling
frame and the study design are described in detail in Victor et al.
10 African-Americans were over-sampled (52% African American,
self-identified as `black`, 29% European American, self-identified
as `white`, 17% Hispanic self identified as "Hispanic" and 2% other
ethnicities). The institutional review board of University of Texas
Southwestern Medical Center approved the study. Alcohol consumption
was determined according to answers to previously validated
questions.sup.2. Blood pressure, height, weight and BMI and
calculated variables were measured as described 10 Fasting blood
samples were obtained from 3,551 subjects (ages 30-65) and 2,971 of
these individuals completed a clinic visit; hepatic TG content was
measured using .sup.1H-MRS in 2,240 African-Americans,
European-Americans and Hispanics.sup.7,12.
[0036] The association between PNPLA3-I148M and metabolic
phenotypes were also examined in the Atherosclerosis Risk in
Communities Study (ARIC), a large prospective study that focuses on
cardiovascular disease in European-Americans and African-Americans.
Details of the ARIC study design and the methods used to measure
plasma lipid levels have been published previously.sup.17,24. The
data used in this analysis was collected from the baseline
examination.
[0037] Whole-genome association and other statistical methods. A
genome-wide association analysis was performed using 12,138 NS
sequence variations from db SNP and the Perlegen SNP database. SNPs
were assayed in 3,383 Africans-American, Caucasian and Hispanic
participants of the Dallas Heart Study using high-density
oligonucleotide arrays (Perlegen Sciences, Mountain View, Calif.).
SNPs that met any of the following criteria were excluded
(n=2,623): error probability >20%, genotype call rate <80%,
or a significant deviation from Hardy-Weinberg Equilibrium (p-value
<0.0001). Of the 9,515 SNPs that were successfully assayed, 286
were monomorphic in the Dallas Heart Study sample. The remaining
9,229 variants were tested for association with hepatic fat content
in the 2,111 African-Americans, Caucasians and Hispanic subjects in
the Dallas Heart Study who underwent .sup.1H-MRS of the liver.sup.2
and in whom ancestry-informative SNPs had been assayed previously;
global and local ancestries were inferred for each individual using
STRUCTURE.sup.25 with 2,270 ancestry-informative SNPs.sup.26. The
results were almost identical when ancestry adjustment was
performed with the same SNPs using principal components analysis.
Though the ancestry-informative SNP panel was primarily designed
for African-Americans [the mean multipoint information content (
IC)=0.82], it was also adequately informative in European-Americans
( IC=0.63) and Hispanics ( I/C=0.66). We pooled all participants
together and inferred global ancestry setting the number of
clusters K equal to 3.
[0038] The statistical significance of 9,229 SNPs in the whole
genome association study was assessed using analysis of variance
(ANOVA). To accommodate confounding factors, we included age, sex,
and global ancestry as covariates in the model. The additive effect
of each variant was tested by encoding the genotype variable as 0,
1, and 2. Since the distribution of hepatic TG levels is highly
skewed, a power transformation (.lamda.=1/4) was applied to the
trait before the analysis. To account for multiple testing, we
adjusted the significance threshold for the number of tests
performed using the Bonferroni method. SNPs with a nominal P-value
<5.4.times.10.sup.-06 were considered significant on a
genome-wide scale.
[0039] The association between PNPLA3 variants and hepatic fat
content within each ethnic group was tested using ANOVA, including
age, gender, BMI, diabetes status, ethanol use and local ancestry
as covariates. Individuals whose genetic ancestry was not
consistent with their self-reported ancestry (n=11, 5, and 16 for
African-Americans, European-Americans and Hispanics, respectively)
and had a fractional ancestry was more than 3 times the
inter-quartile range below the 25h percentile for their reference
group were excluded from the analysis. Because the distribution of
hepatic TG content is skewed, we reported medians and
inter-quartile ranges.
[0040] The association of PNPLA3-I148M with BMI, HOMA-IR and plasma
TG levels was analyzed in the African-Americans, Caucasians and
Hispanics together using the ANOVA including age, gender, and local
ancestry as covariates. HOMA-IR was adjusted for BMI and plasma TG
levels were adjusted for BMI and diabetes.
[0041] To determined the contribution of PNPLA3 to the ethnic
differences in liver fat content, we examined the proportion of
variance explained by ancestry (R1) using a linear model. We then
determined the proportion of variance explained by ancestry after
adjusting for the PNPLA3 genotypes (148M and S453I) (R2). The
proportion of variance due to ancestry and explained by 148M and
S453I was determined from (R1-R2)/R1.
[0042] Resequencing PNPLA3. The exons and flanking introns of
PNPLA3 were sequenced as described previously.sup.28 in the
African-American, European-American and Hispanic men and women in
the Dallas Heart Study with the highest and lowest hepatic TG
content. All sequence variants identified were verified by manual
inspection of the chromatograms and missense changes were confirmed
by an independent resequencing reaction.
[0043] Genotyping assays. Fluorogenic 5'-nucleotidase assays were
developed for PNPLA3-I148M and for the sequence variants identified
in both the high and low hepatic TG groups in the resequencing
experiments. Sequence variations in PNPLA3 were assayed using the
TaqMan assay system (Applied Biosystems) on a 7900HT Fast Real-Time
PCR instrument. Probes and reagents were purchased from Applied
Biosystems.
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[0072] The examples and detailed description herein are offered by
way of illustration and not by way of limitation. All publications
and patent applications cited in this specification are herein
incorporated by reference as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference. Although the foregoing invention has
been described in some detail by way of illustration and example
for purposes of clarity of understanding, it will be readily
apparent to those of ordinary skill in the art in light of the
teachings of this invention that certain changes and modifications
may be made thereto without departing from the spirit or scope of
the appended claims.
* * * * *