Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function

The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3 L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.     

A Sequence Variation (I148M) in PNPLA3 Associated with Nonalcoholic Fatty Liver Disease Disrupts Triglyceride Hydrolysis

Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that ∼90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.     

PNPLA3 mediates hepatocyte triacylglycerol remodeling

The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3^I148M) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3^WT) and PNPLA3^I148M on HuH7 cell lipidome after [¹³C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3^I148M induced a net accumulation of unlabeled TAGs, but not newly synthesized total [¹³C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3^WT and PNPLA3^I148M induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3^WT associated in PCA with newly synthesized [¹³C]TAGs, particularly 52:1 and 50:1, while PNPLA3^I148M associated with similar preexisting TAGs. PNPLA3^WT overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3^I148M. PNPLA3^I148M localized more extensively to lipid droplets (LDs) than PNPLA3^WT, suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3^I148M.     

Expression and characterization of a PNPLA3 protein isoform (I148M) associated with nonalcoholic fatty liver disease

A genetic variant of PNPLA3 (patatin-like phospholipase domain-containing 3; PNPLA3-I148M), a serine protease of unknown function, is associated with accumulation of triacylglycerol (TAG) in the liver. To determine the biological substrates of PNPLA3 and the effect of the I148M substitution on enzymatic activity and substrate specificity, we purified and characterized recombinant human PNPLA3 and PNPLA3-I148M. Maximal hydrolytic activity of PNPLA3 was observed against the three major glycerolipids, TAG, diacylglycerol, and monoacylglycerol, with a strong preference for oleic acid as the acyl moiety. Substitution of methionine for isoleucine at position 148 markedly decreased the V(max) of the enzyme for glycerolipids but had only a modest effect on the K(m). Purified PNPLA3 also catalyzed the hydrolysis of oleoyl-CoA, but the V(max) was 100-fold lower for oleoyl-CoA than for triolein. The thioesterase activity required the catalytic serine but was only modestly decreased by the I148M substitution. The enzyme had little or no hydrolytic activity against the other lipid substrates tested, including phospholipids, cholesteryl ester, and retinyl esters. Neither the wild-type nor mutant enzyme catalyzed transfer of oleic acid from oleoyl-CoA to glycerophosphate, lysophosphatidic acid, or diacylglycerol, suggesting that the enzyme does not promote de novo TAG synthesis. Taken together, our results are consistent with the notion that PNPLA3 plays a role in the hydrolysis of glycerolipids and that the I148M substitution causes a loss of function, although we cannot exclude the possibility that the enzyme has additional substrates or activities.     

PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes

The mechanism of how patatin-like phospholipase domain-containing protein 3 (PNPLA3) variant M148 is associated with increased risk of development of hepatic steatosis is still debated. Here, we propose a novel role of PNPLA3 as a key player during autophagosome formation in the process of lipophagy. A human hepatocyte cell line, HepG2 cells, expressing recombinant I148 or 148M, was used to study lipophagy under energy deprived conditions, and lipid droplet morphology was investigated using florescence microscopy, image analysis and biochemical assays. Autophagic flux was studied using the golden-standard of LC3-II turnover in combination with the well characterized GFP-RFP-LC3 vector. To discriminate between, perturbed autophagic initiation and lysosome functionality, lysosomes were characterized by Lysotracker staining and LAMP1 protein levels as well as activity and activation of cathepsin B. For validation, human liver biopsies genotyped for I148 and 148M were analyzed for the presence of LC3-II and PNPLA3 on lipid droplets. We show that the M148-PNPLA3 variant is associated with lipid droplets that are resistant to starvation-mediated degradation. M148 expressing hepatocytes reveal decreased autophagic flux and reduced lipophagy. Both I148-PNPLA3 and M148-PNPLA3 colocalize and interact with LC3-II, but the M148-PNPLA3 variant has lower ability to bind LC3-II. Together, our data indicate that PNPLA3 might play an essential role in lipophagy in hepatocytes and furthermore that the M148-PNPLA3 variant appears to display a loss in this activity, leading to decreased lipophagy.     

The PNPLA3 rs738409 148M/M genotype is a risk factor for liver cancer in alcoholic cirrhosis but shows no or weak association in hepatitis C cirrhosis

Background

An isoleucine>methionine mutation at position 148 in the PNPLA3 gene (p.I148M, rs738409) has recently been identified as a susceptibility factor for liver damage in steatohepatitis. Here, we studied whether the PNPLA3 rs738409 polymorphism also affects predisposition to hepatocellular carcinoma (HCC).

Methods

We compared distributions of PNPLA3 genotypes in 80 and 81 Caucasian patients with alcoholic and hepatitis C virus (HCV)-associated HCC to 80 and 81 age- and sex-matched patients with alcohol-related and HCV-related cirrhosis without HCC, respectively. PNPLA3 genotypes in 190 healthy individuals from the same population served as reference. Potential confounders obesity, diabetes, HCV genotype and HBV co-infection were controlled by univariate and multivariate logistic regression with forward variable selection.

Results

PNPLA3 genotypes were in Hardy-Weinberg equilibrium for all study groups. The frequency of the 148M allele was significantly (p<0.001) increased in alcoholic cirrhosis with (53.7%) and without HCC (36.2%) but was not different between healthy controls (22.9%) and patients with cirrhosis (25.3%; p = 0.545) and HCC (30.2%; p = 0.071) due to hepatitis C. HCC risk was highest in 148M/M homozygous patients with alcoholic liver disease (odds ratio (OR) 16.8 versus healthy controls; 95% confidence interval (CI) 6.68–42.43, p<0.001). Finally, multivariate regression confirmed 148M/M homozygosity (OR 2.8; 95%-CI: 1.24–6.42; p = 0.013) as HCC risk factor in alcoholic cirrhosis. In HCV-related cirrhosis only HCV genotype 1 was confirmed as a HCC risk factor (OR 4.2; 95%-CI: 1.50–11.52; p = 0.006).

Conclusion

The PNPLA3 148M variant is a prominent risk factor for HCC in patients with alcoholic cirrhosis, while its effects are negligible in patients with cirrhosis due to HCV. This polymorphism provides an useful tool to identify individuals with particularly high HCC risk in patients with alcoholic liver disease that should be taken into account in future HCC prevention studies.     

A Frequent PNPLA3 Variant Is a Sex Specific Disease Modifier in PSC Patients with Bile Duct Stenosis

Background & Aims

Primary sclerosing cholangitis predominantly affects males and is an important indication for liver transplantation. The rs738409 variant (I148M) of the PNPLA3 gene is associated with alcoholic and non-alcoholic liver disease and we evaluated its impact on the disease course of PSC.

Methods

The I148M polymorphism was genotyped in 121 German PSC patients of a long-term prospective cohort and 347 Norwegian PSC patients.

Results

In the prospective German cohort, actuarial survival free of liver transplantation was significantly reduced for I148M carriers (p = 0.011) compared to wildtype patients. This effect was restricted to patients with severe disease, as defined by development of dominant stenosis (DS) requiring endoscopic intervention. DS patients showed markedly decreased survival (p = 0.004) when carrying the I148M variant (I148M: mean 13.8 years; 95% confidence interval: 11.6–16.0 vs. wildtype: mean 18.6 years; 95% confidence interval: 16.3–20.9) while there was no impact on survival in patients without a DS (p = 0.87). In line with previous observations of sex specific effects of the I148M polymorphism, the effect on survival was further restricted to male patients (mean survival 11.9 years; 95% confidence interval: 10.0–14.0 in I148M carriers vs. 18.8 years; 95% confidence interval: 16.2–21.5 in wildtype; p<0.001) while female patients were unaffected by the polymorphism (p = 0.65). These sex specific findings were validated in the Norwegian cohort (p = 0.013).

Conclusions

In male PSC patients with severe disease with bile duct stenosis requiring intervention, the common I148M variant of the PNPLA3 gene is a risk factor for reduced survival.     

Association of the I148M/PNPLA3 variant with elevated alanine transaminase levels in normal-weight and overweight/obese Mexican children

Background and aims

Non-alcoholic fatty liver disease (NAFLD) and elevated alanine transaminase (ALT) levels are common in obese Hispanic adults and children. Recently, a PNPLA3 gene variant (I148M) was strongly associated with NAFLD and higher ALT levels in obese adults, including Hispanics. The aims of this study were to estimate the frequency of elevated ALT levels, and to address the influence of obesity and PNPLA3/I148M on ALT levels in a general population sample of Mexican school-aged children.

Methods

A total of 1037 non-related Mexican children aged 6 to 12 years were genotyped for the I148M variant. Anthropometric, clinical and metabolic parameters were collected from all participants.

Results

Elevated ALT levels (> 35 U/L) were more frequent in obese (26.9%) and overweight (9.3%) than in normal weight children (2.2%). The M148M genotype was significantly associated with elevated ALT levels in this population (OR = 3.7, 95% CI 2.3–5.9; P = 3.7 × 10^− 8), and children carrying the M148M genotype showed significantly lower HDL cholesterol levels and BMI z-core (P = 0.036 and 0.015, respectively). On stratifying by BMI percentile, this genotype conferred a much greater risk of elevated ALT levels in normal weight (OR = 19.9, 95% CI 2.5–157.7; P = 0.005) than overweight and obese children (OR = 3.4, 95% CI 1.3–8.9; P = 0.014 and OR = 3.1, 95% CI 1.7–5.5; P = 1.4 x10^− 4, respectively).

Conclusions

The I148M PNPLA3 variant is strongly associated with elevated ALT levels in normal weight and overweight/obese Mexican children. Thus, the M148M genotype may be considered as an important risk factor for liver damage in this population.     

Interaction between PNPLA3 I148M Variant and Age at Infection in Determining Fibrosis Progression in Chronic Hepatitis C

Background and Aims

The PNPLA3 I148M sequence variant favors hepatic lipid accumulation and confers susceptibility to hepatic fibrosis and hepatocellular carcinoma. The aim of this study was to estimate the effect size of homozygosity for the PNPLA3 I148M variant (148M/M) on the fibrosis progression rate (FPR) and the interaction with age at infection in chronic hepatitis C (CHC).

Methods

FPR was estimated in a prospective cohort of 247 CHC patients without alcohol intake and diabetes, with careful estimation of age at infection and determination of fibrosis stage by Ishak score.

Results

Older age at infection was the strongest determinant of FPR (p<0.0001). PNPLA3 148M/M was associated with faster FPR in individuals infected at older age (above the median, 21 years; −0.64±0.2, n = 8 vs. −0.95±0.3, n = 166 log₁₀ FPR respectively; p = 0.001; confirmed for lower age thresholds, p<0.05), but not in those infected at younger age (p = ns). The negative impact of PNPLA3 148M/M on fibrosis progression was more marked in subjects at risk of altered hepatic lipid metabolism (those with grade 2–3 steatosis, genotype 3, and overweight; p<0.05). At multivariate analysis, PNPLA3 148M/M was associated with FPR (incremental effect 0.08±0.03 log₁₀ fibrosis unit per year; p = 0.022), independently of several confounders, and there was a significant interaction between 148M/M and older age at infection (p = 0.025). The association between 148M/M and FPR remained significant even after adjustment for steatosis severity (p = 0.032).

Conclusions

We observed an interaction between homozygosity for the PNPLA3 I148M variant and age at infection in determining fibrosis progression in CHC patients.     

The association of PNPLA3 variants with liver enzymes in childhood obesity is driven by the interaction with abdominal fat

Background and Aims

A polymorphism in adiponutrin/patatin-like phospholipase-3 gene (PNPLA3), rs738409 C->G, encoding for the I148M variant, is the strongest genetic determinant of liver fat and ALT levels in adulthood and childhood obesity. Aims of this study were i) to analyse in a large group of obese children the role of the interaction of not-genetic factors such as BMI, waist circumference (W/Hr) and insulin resistance (HOMA-IR) in exposing the association between the I148M polymorphism and ALT levels and ii) to stratify the individual risk of these children to have liver injury on the basis of this gene-environment interaction.

Methods

1048 Italian obese children were investigated. Anthropometric, clinical and metabolic data were collected and the PNPLA3 I148M variant genotyped.

Results

Children carrying the 148M allele showed higher ALT and AST levels (p = 0.000006 and p = 0.0002, respectively). Relationships between BMI-SDS, HOMA-IR and W/Hr with ALT were analysed in function of the different PNPLA3 genotypes. Children 148M homozygous showed a stronger correlation between ALT and W/Hr than those carrying the other genotypes (p: 0.0045) and, therefore, 148M homozygotes with high extent of abdominal fat (W/Hr above 0.62) had the highest OR (4.9, 95% C. I. 3.2–7.8, p = 0.00001) to develop pathologic ALT.

Conclusions

We have i) showed for the first time that the magnitude of the association of PNPLA3 with liver enzymes is driven by the size of abdominal fat and ii) stratified the individual risk to develop liver damage on the basis of the interaction between the PNPLA3 genotype and abdominal fat.     

Influence of dietary pattern,physical activity,and I148M PNPLA3 on steatosis severity in at-risk adolescents

Evidence relating dietary patterns to obesity and related disorders such as non-alcoholic fatty liver disease (NAFLD) is limited in pediatric age. Aim of this study was to analyze the association between dietary patterns, obesity and development of severe steatosis and the metabolic syndrome in a series of children and adolescents referred for suspected NAFLD, and the interaction with the rs738409 I148M PNPLA3 polymorphism. Two hundred patients (112 females) had completed a food frequency and demographic questionnaire. Nearly 58 % were obese, and 32 % were overweight. Mild, moderate, and severe fatty liver was present in 60 (30 %), 87 (44 %), and 51 (26 %) participants, respectively. A great proportion of overweight/obese children and adolescents reported a correct dietary pattern. At multivariate ordinal regression analysis considering demographic, anthropometric, genetic, and behavioral determinants, the major determinant of steatosis severity was PNPLA3 I148M genotype (p < 0.0001), followed by older age (p = 0.017), higher waist circumference (p = 0.016), and less time spent practising physical exercise (p = 0.034). Furthermore, there was a significant interaction between PNPLA3 I148M and intake of sweetened beverages (p = 0.033) and of vegetables (p = 0.038). In conclusion, although dietary pattern was reportedly correct in at-risk overweight adolescents with NAFLD, we report a novel interaction between PNPLA3 I148M and dietary components with the severity of steatosis.     

PNPLA3 I148M polymorphism is associated with elevated alanine transaminase levels in Mexican Indigenous and Mestizo populations

The patatin like phospholipase domain-containing (PNPLA3) I148M variant is the strongest genetic factor associated with elevated alanine transaminase (ALT) levels in different populations, particularly in Hispanics who have the highest 148M risk allele frequency reported to date. It has been suggested that Indigenous ancestry is associated with higher ALT levels in Mexicans. The aim of the present study was to assess the frequency of the PNPLA3 148M risk allele in Mexican indigenous and Mestizo individuals, and to examine its association with serum ALT levels. The study included a total of 1624 Mexican individuals: 919 Indigenous subjects from five different native groups and 705 Mexican Mestizo individuals (141 cases with ALT levels ≥40 U/L and 564 controls with ALT <40 U/L). The I148M polymorphism was genotyped by TaqMan assays. The frequency of elevated ALT levels in Indigenous populations was 18.7 %, and varied according to obesity status: 14.4 % in normal weight, 19.9 % in overweight and 24.5 % in obese individuals. The Mexican indigenous populations showed the highest reported frequency of the PNPLA3 148M risk allele (mean 0.73). The M148M genotype was significantly associated with elevated ALT levels in indigenous individuals (OR = 3.15, 95 % CI 1.91–5.20; P = 7.1 × 10^?6) and this association was confirmed in Mexican Mestizos (OR = 2.24, 95 % CI 1.50–3.33; P = 8.1 × 10^?5). This is the first study reporting the association between M148M genotype and elevated ALT levels in Indigenous Mexican populations. The 148M allele risk may be considered an important risk factor for liver damage in Mexican indigenous and Mestizo populations.     

中国汉族人群PNPLA3 I148M 多态性影响脂联素水平及非酒精性脂肪性 肝病遗传易感性的相关性研究*

目的:探讨在中国人群中PNPLA3 I148M基因型、脂联素与非酒精性脂肪性肝病的遗传易感性的相关性,及PNPLA3基因型与空腹血清脂联素水平的关系。方法:对96例NAFLD患者和76名正常对照,采用多聚酶链反应(PCR)及直接测序法检测PNPLA3基因型。计量资料结果均用均数±标准差(X±S)表示,经方差齐性检验后,行t检验;性别、基因型及等位基因频率的比较行X2检验。结果:中国汉族人群中,存在PNPLA3基因I148M多态性,I148M G等位基因频率分布在NAFLD(64.89%)与正常对照组(34.87%)、NASH组(71.70%)与SS组(56.09%)中比较差异均有统计学意义(P<0.05)。病例对照分析显示:148GG基因携带者与148CC基因携带者相比较,前者发生NAFLD的比值比(OR)为3.45(95%CI:2.21~5.41,P<0.05),发生NASH的OR为1.98(95%CI=1.08~3.64,P<0.05)。PNPLA3基因rs738409多态性与血清ALT水平有关(P<0.05),对NASH组分层分析,148GG基因型BMI、ALT、FINS均高于148CC基因型(P<0.05),血清HDL水平低于148CC基因型和148GC基因型(P<0.05),这些结果提示等位基因G与肝脏炎症和肝脏脂肪增加有相关性.Ordinal Logistic回归分析显示PNPLA3 I148M多态性与低浓度血清脂联素水平相关(<6μg/ml)(OR=2.78,95%CI=1.765~4.384,P<0.05)。结论:中国汉族人群中,PNPLA3基因I148M多态性与NAFLD的遗传易感性及脂联素的分泌调节相关,是决定NAFLD个体遗传易感性的重要因素。     

The common adiponutrin variant p.I148M does not confer gallstone risk but affects fasting glucose and triglyceride levels

Recently the common adiponutrin (PNPLA3) polymorphism p.I148M has been identified as a genetic determinant of severe forms of non-alcoholic fatty liver disease and alcoholic liver disease. Additionally, insulin resistance - linked to the development of non-alcoholic steatohepatitis - increases the risk of developing gallstones. Here we assessed whether the PNPLA3 p.I148M (c.444 C-G) polymorphism affects glucose and lipid levels and increases gallstone risk. We analysed 229 individuals with gallstones from 108 families (age 24-80 years, BMI 17-55 kg/m(2)) and 258 gallstone-free controls (age 20-70 years, BMI 14-43 kg/m(2)). Fasting glucose, triglyceride and cholesterol serum levels were determined. The p.I148M polymorphism was genotyped using a PCR-based assay with 5'-nuclease and fluorescence detection. Case-control association tests and nonparametric linkage (NPL) analysis in sib-pairs were performed. Individuals carrying the [GG] genotype had significantly (P<0.0001) higher median fasting glucose levels as compared to [GC] and [CC] carriers. After adjustment for multiple testing, we detected a trend for an association between triglyceride levels and variant adiponutrin in gallstone patients (P=0.032), and gallstone cases carrying the genotype [CC] presented with significantly higher triglyceride levels than the corresponding controls (P<0.003). No significant effects on cholesterol metabolism were detected. Neither genotype distributions nor NPL scores provided evidence for association or linkage between the PNPLA3 variant and gallstones. In conclusion, homozygous carriers of the PNPLA3 risk allele display higher fasting glucose. Although this adiponutrin variant may affect triglyceride homeostasis, it does not increase the risk of cholelithiasis.     

PNPLA3 I148M Polymorphism,Clinical Presentation,and Survival in Patients with Hepatocellular Carcinoma

Background & Aims

Aim of this study was to evaluate whether the PNPLA3 I148M polymorphism, previously associated with hepatocellular carcinoma (HCC) risk, influences the clinical presentation of HCC and survival.

Methods

we considered 460 consecutive HCC patients referred to tertiary care centers in Northern Italy, 353 with follow-up data.

Results

Homozygosity for PNPLA3 148M at risk allele was enriched in HCC patients with alcoholic liver disease or nonalcoholic fatty liver disease (ALD&NAFLD: relative risk 5.9, 95% c.i. 3.5–9.9; other liver diseases: relative risk 1.9, 95% c.i. 1.1–3.4). In ALD&NAFLD patients, the PNPLA3 148M allele was associated with younger age, shorter history of cirrhosis, less advanced (Child A) cirrhosis at HCC diagnosis, and lower HCC differentiation grade (p<0.05). Homozygosity for PNPLA3 148M was associated with reduced survival in the overall series (p = 0.009), and with a higher number of HCC lesions at presentation (p = 0.007) and reduced survival in ALD&NAFLD patients (p = 0.003; median survival 30, 95% c.i. 20–39 vs. 45, 95% c.i. 38–52 months), but not in those with HCC related to other etiologies (p = 0.86; 48, 95% c.i. 32–64 vs. 55, 95% c.i. 43–67 months). At multivariate Cox regression analysis, homozygosity for PNPLA3 148M was the only negative predictor of survival in ALD&NAFLD patients (HR of death 1.57, 95% c.i. 1.12–2.78).

Conclusions

PNPLA3 148M is over-represented in ALD&NAFLD HCC patients, and is associated with occurrence at a less advanced stage of liver disease in ALD&NAFLD. In ALD&NAFLD, PNPLA3 148M is associated with more diffuse HCC at presentation, and with reduced survival.     

Interactions of allelic variance of PNPLA3 with nongenetic factors in predicting nonalcoholic steatohepatitis and nonhepatic complications of severe obesity

Objective : Allelic variation (rs738409C→G) in adiponutrin (patatin‐like phospholipase domain‐containing protein 3, PNPLA3) has been associated with hepatic steatosis and liver fibrosis. The physiologic impact of the PNPLA3 G allele may be exacerbated in patients with severe obesity. In this study, we investigated the interactions of PNPLA3 rs738409 with a broad panel of metabolic and histologic characteristics of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH) in patients with medically complicated obesity. Design and Methods : Consecutive patients undergoing bariatric surgery were selected for a prospective study. They underwent extensive laboratory and histologic (liver biopsy) assessment, as well as evaluation of rs738409 polymorphism by TaqMan assay. Results : Only 12 (8.3%) of the 144 patients had normal liver histology, with 72 (50%) NASH, of whom 15 (10.4% of total patients) had fibrosis stage 2‐3. PNPLA3 GG genotype correlated positively (P < 0.05) with serum levels of alanine aminotransferase (ALT), asparate aminotransferase (AST), glucose, fibrinogen, and insulin‐dependent diabetes mellitus, homeostasis model assessment—insulin resistance, and presence of NASH. Multivariate analysis indicated that PNPLA3 rs738409 G versus C allele remained an (independent) risk factor for NASH, in addition to CK‐18 >145 IU/l, glucose >100 mg/dl, and C‐reactive protein (CRP) >0.8 mg/dl. The probability of NASH increased from 9% (no risk factor) to 82% if all four risk factors were present. Conclusions : In this cohort of patients with medically complicated obesity, PNPLA3 rs738409 G allelic expression is associated with hepatic (NASH) and nonhepatic complications of obesity, such as insulin resistance. These novel findings may be related to a greater impact of PNPLA3 variant in magnitude and scope in patients with severe obesity than in less obese populations. Further studies are needed to characterize the nature of these associations.