In humans, adipose tissue serves as a reservoir to limit the deposition of triglyceride (TG) in the liver and other metabolically active tissues1
. 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 population2
. 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 dyslipidemia3,4
. 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 cancer3,5
. Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease in Western countries6
. Approximately 10% of liver transplants performed in the United States are for cirrhosis related to NAFLD4
Factors promoting deposition of fat in the liver include obesity, diabetes, insulin resistance, and alcohol ingestion3,6
. The propensity to develop hepatic steatosis differs among ethnic groups, with African-Americans having the lower (24%) and Hispanics a higher (45%) frequency of the disorder than European-Americans (33%) in a large US urban population3
. Hispanics also have a higher prevalence of steatohepatitis and cirrhosis, whereas African-Americans are less prone to develop liver failure2,7–9
. The factors responsible for these ethnic differences in prevalence of hepatic steatosis and liver injury are not known.
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 Study10
. We limited our analysis to NS sequence variations to screen directly the variants with a higher likelihood of affecting gene function. Hepatic fat content was measured in the Dallas Heart Study using proton magnetic resonance spectroscopy (1
H-MRS), the most accurate, quantitative noninvasive method available2,11,12
. Of the 12,138 NS variants assayed using chip-based oligonucleotide hybridization13
, 9,229 exceeded the quality control threshold for the study (see METHODS) and were included in the analysis.
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 1
H-MRS of the liver2
. To maximize statistical power, the three ethnic groups were pooled and a global ancestry score (calculated using a panel of 2,270 ancestry informative SNPs) was included in the model to control for population stratification (see METHODS). The quantile-quantile plot of P-values showed no systematic deviation from the null distribution ().
Figure 1 Whole-genome scan of liver triglyceride content measured by proton magnetic resonance imaging in the Dallas Heart Study (n=2,111). (a) Quantile-quantile plot of P-values. (b) Scatter plot of P-values. The dashed line denotes the Bonferroni corrected significance (more ...)
A single variant in PNPLA3
(rs738409) was strongly associated with hepatic fat content (P=5.9×10−10
) (). 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 family14
. The progenitor of this family, patatin, is a major protein of potato tubers and has nonspecific lipid acyl hydrolase activity15,16
. None of the other NS sequence variants tested in the genome-wide scan exceeded the Bonferroni-corrected threshold for significance (P=5.4×10−6
Figure 2 Association between a sequence variant in PNPLA3 (I148M) and hepatic triglyceride (TG) content. (a) PNPLA3 is a 481 residue protein that contains a patatin-like domain at the N terminus (residues 10-179, UniProt http://pir.uniprot.org/uniprot/) with consensus (more ...)
The association between PNPLA3
-I148M and hepatic fat content remained highly significant (P=7.0 × 10−14
) after adjusting for BMI, diabetes status, ethanol use, as well as global and local ancestry (), and was apparent in all three ethnic groups ( and Supplementary Table 1
online). Thus, the association between rs738409 and hepatic fat content was not attributable either to the effect of known risk factors for liver fat accumulation or to population stratification.
The frequencies of the PNPLA3
-148M allele were concordant with the relative prevalence of NAFLD in the three ethnic groups2
; the highest frequency of the allele 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
-148M allele (P=3.7 × 10−4
). Analysis of the three ethnic groups revealed that the association with ALT was limited to the Hispanics (P=1.3 × 10−5
), the group with the greatest prevalence of hepatic steatosis and susceptibility to cirrhosis (Supplementary Table 1
. The PNPLA3
-148M 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 hepatic inflammation7
, and suggests that PNPLA3
-148M allele adversely affects liver function.
Increased hepatic fat content is associated with insulin resistance and dyslipidemia [increased plasma levels of TG and lower levels of high density lipoprotein-cholesterol (HDL-C)], but the causal nature of these relationships remains poorly defined3
. No association was found between the PNPLA3
-148M allele and body mass index (BMI) or indices of insulin sensitivity, including fasting glucose and insulin (Supplementary Table 1
online) 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-C or low density lipoprotein (LDL)-C (Supplementary Table 1
online). A corresponding analysis in a larger, biracial sample (n=14,821), the Atherosclerosis Risk in Communities Study (Supplementary Table 2
, also revealed no association of PNPLA3
-I148M with BMI, indices of insulin sensitivity, or plasma levels of TG or HDL-C (Supplementary Table 2
online). Based on the observed associations between both the SNP and hepatic fat and between hepatic fat and HOMA-IR in the Dallas Heart Study, the power to detect an association with HOMA-IR was >96% in the African-Americans and 91% in the European-Americans in ARIC.
The data from these studies indicate that the PNPLA3-148M
allele is associated with a systematic increase in hepatic fat content but not with major alterations in glucose homeostasis or lipoprotein metabolism. Thus, increased hepatic fat content does not inevitably lead to insulin resistance, which is consistent with recent observations in some animal models18,19
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 hepatic fat in the Dallas Heart Study, and in a sex- and ethnicity-matched group with the lowest levels2
. The number of individuals with NS variants found only in the high group (n=8) was similar to the number found only in the low group (n=9), 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.
Figure 3 Identification of a PNPLA3 allele (S453I) associated with lower hepatic fat content in African-Americans in the Dallas Heart Study. (a) Exons and flanking introns of PNPLA3 were sequenced in the 32 European-American and African-American men and women (more ...)
Eight variants were present in both the low and the high hepatic fat groups ( and Supplementary Table 3
online) and the six most common of these sequence variations were genotyped in the entire sample. One variant, PNPLA3
-S453I, that was common in African-Americans (MAF=0.104) but rare in European-Americans (0.003) and Hispanics (0.008) (Supplementary Table 3
online) was associated with a significantly lower liver fat content. Median hepatic TG content was 18% lower in African-Americans with the PNPLA3
-453I allele when compared to African-Americans homozygous for the wild-type allele (3.3% versus 2.7%, P=6.0 × 10−4
) (). 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
-453I allele had a hepatic fat content in the lowest decile when compared to the highest decile of the population (). No significant difference in the number of individuals identified in the extremes was found for any of the other SNPs (data not shown).
The effect of PNPLA3-S453I on hepatic fat content was independent of the PNPLA3-I148M polymorphism. Both variants were statistically significant when included in a multiple regression model and the S453I was significantly associated with hepatic fat in African-Americans homozygous for the PNPLA3-148I allele (data not shown). The identification of a second allele of PNPLA3 (i.e. S453I) that was independently associated with hepatic fat content further supports a role for PNPLA3 in determining hepatic TG levels, and indicates the presence of both loss-of-function and gain-of-function alleles at this locus. The mechanisms by which these alleles affect hepatic fat content are not known.
The frequencies of both PNPLA3
-148M and of PNPLA3
-453I in the three ethnic groups represented in the Dallas Heart Study are concordant with ethnic differences in hepatic fat content2
. Exclusion of the individuals carrying either of these two alleles (148M and 453I) substantially attenuated the differences in hepatic 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 in the Dallas Heart Study. Thus, genetic variation in PNPLA3
accounts for a large fraction of the ethnic differences in the propensity to accumulate excess fat in the liver.
The physiological substrate(s) of PNPLA3 has not been defined. Expression of PNPLA3
is under metabolic control in adipose tissue and the liver; mRNA levels are low in the fasted state and increase dramatically with carbohydrate feeding20,21
. PNPLA3 structurally resembles calcium-independent phospholipase A2
but the recombinant protein has low phospholipase activity when expressed in insect (Sf9) cells22
. PNPLA3 has more robust activity against TG in vitro
and can also transfer fatty acids to and from mono- and diacylglycerol22
. It is not known if the major effect of PNPLA3 in the liver is to hydrolyze TG or to transfer fatty acids between lipids (transacetylation). Studies are in progress to determine the specific effects on lipid metabolism of the NS variants in PNPLA3 identified in this study.
Currently, we cannot accurately predict which individuals with fatty liver will develop steatohepatitis and progress to cirrhosis and liver failure. The finding that markers of liver inflammation (serum levels of liver-derived enzymes) were elevated in PNPLA3
-148M carriers, which was also observed in an independent genome-wide association study23
, suggests that this genetic variant may confer increased susceptibility to hepatic injury. Patatin-like phospholipase family members in other organisms are up-regulated in response to environmental insults24
. The sequence variations we have identified in PNPLA3
may provide predictive information regarding the risk of developing hepatic steatosis and liver injury in response to environmental stresses such as caloric excess, infections, or drugs.