PNPLA3 variation and kidney disease.

Accumulating epidemiological evidence shows that the patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 G allele, which is the most robust genetic variant associated with greater susceptibility to metabolic dysfunction-associated steatotic liver disease (MASLD), is significantly associated with impaired kidney function in both adults and children, regardless of the presence of common renal risk factors, MASLD severity, and other potential confounders. Although some prospective studies have reported a significant association between the PNPLA3 rs738409 G allele and the increased risk of developing chronic kidney disease (CKD), the epidemiological evidence about a possible direct effect of the PNPLA3 rs738409 G allele on the risk of developing CKD is still limited. Experimentally, PNPLA3 is expressed in renal podocytes, pericytes, and proximal tubule cells, thus supporting the notion that the mutant PNPLA3 protein may play a role in developing renal steatosis and fibrosis. However, it cannot be ruled out that a part of the adverse effect of the PNPLA3 rs738409 G allele on kidney function may be driven by a direct impact of this genetic variant on the development and progression of MASLD. It is possible to hypothesize that identifying the PNPLA3 genotype might help identify individuals at higher risk of CKD and those at greater risk of advanced MASLD. In this narrative minireview, we summarize the current epidemiological data about the association between the PNPLA3 rs738409 G allele and the risk of CKD and abnormal albuminuria. We also briefly discuss the putative biological mechanisms underpinning this association and its potential and future clinical implications.

The Risk of Cirrhosis and Its Complications Based on PNPLA3 rs738409 G Allele Frequency.

BACKGROUND: Data regarding the influence of patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphism for patients with liver cirrhosis (LC) are scarce. OBJECTIVE: This study assesses the role of the PNPLA3 polymorphism for the development of LC and its complications by the findings of genetic examinations. METHODS: Patients with LC caused by virus (n = 157), alcohol (n = 104), nonalcoholic fatty liver disease (NAFLD) (n = 106), or autoimmune disease (n = 33) and without LC (n = 128) were enrolled. LC was composed of the presence and absence of complications, such as variceal bleeding, hepatic ascites, and hepatic encephalopathy. To assess the role of the PNPLA3 polymorphism, odds ratio (OR) for the rs738409 variant was calculated for the patients between (i) with LC and without LC in the entire cohort and (ii) the presence and absence of complications in the patients with LC. RESULTS: There was a significant difference among the patients without LC and those with alcohol, NAFLD-related LC in the frequency of G alleles (p < 0.001, both). According to complications of LC, the OR for NAFLD-related cirrhosis significantly increased in the presence of the two mutated alleles (OR = 3.165; p = 0.046) when the wild type was used as the reference. However, there were no significant risks for the complications in the virus and alcohol-related cirrhosis unless there was a presence of G alleles. CONCLUSION: The PNPLA3 polymorphism was associated with the risk of NAFLD-related LC and its complications.

Role of PNPLA3 in the Assessment and Monitoring of Hepatic Steatosis and Fibrosis in Patients with Chronic Hepatitis C Infection Who Achieved a Sustained Virologic Response.

Background and Objectives: Hepatic diseases are an important public health problem. All patients with chronic hepatitis C virus (HCV) infection receive treatment, regardless of hepatic fibrosis severity. However, evaluation of hepatic fibrosis and steatosis is still useful in assessing evolution, prognosis and monitoring of hepatic disease, especially after treatment with direct-acting antivirals (DAAs). The aim of this study was to assess the link between patatin-like phospholipase domain-containing 3 (PNPLA3) polymorphism and the degree of hepatic steatosis and fibrosis in patients with chronic HCV infection, as well as changes in steatosis and fibrosis three monthsafter obtaining a sustained viral response (SVR). Materials and Methods:Ourstudy included 100 patients with chronic hepatitis C (CHC) infection and compensated cirrhosis who received DAA treatment and who were evaluated using Fibromax prior to and 3 months after SVR. The influence of PNPLA3 (CC, CG, GG) genotype among these patients on the degree of post-treatment regression of steatosis and fibrosis was assessed. Results: Regression was noticed in the degree of both hepatic steatosis and hepatic fibrosis post-DAA treatment (three months after SVR). Analysis of the correlation between PNPLA3 genotype and fibrosis indicated that the average level of fibrosis (F) before DAA treatment was higher in patients with the GG genotype than in patients with the CC or CG genotype. Three months after SVR, the average level of fibrosis decreased; however, it remained significantly increased in GG subjects compared to that in CC or CG patients. The degree of hepatic steatosis before treatment was not significantly different among patients with different PNPLA3 genotypes, and no significant correlations were observed three months after SVR. Conclusions: The genetic variants of PNPLA3 influence the evolution of hepatic fibrosis. The GG subtype plays an important role in the degree of hepatic fibrosis both before and after treatment (three months after SVR)and could be a prognostic marker for assessment of post-SVR evolution.

[Correlation between PNPLA3 rs738409 and TM6SF2 rs58542926 gene polymorphism and primary liver cancer in the Han Population of China's Northeast region].

Objective: To investigate the correlation between patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 and transmembrane 6 superfamily member 2 (TM6SF2) rs58542926 gene polymorphisms and the incidence of primary liver cancer in the Han population of China's Northeast region. Methods: A case-control study was used to enroll 521 patients with primary liver cancer as the case group and 164 healthy people as the control group. The case group was divided into groups with and without liver cirrhosis according to etiology. The polymerase chain reaction (PCR) method was used to detect the genetic polymorphisms of PNPLA3 rs738409 and TM6SF2 rs58542926, respectively. Results: Compared with the control group, the frequency distribution of PNPLA3 rs738409 G allele in the case group was significantly different (OR = 1.583, P = 0.001). Further grouping showed that there was no statistically significant difference between the control and hepatitis C-related liver cancer group (P = 0.161), but there were significant differences in other groups (P < 0.05). Compared with the control group, the frequency of TM6SF2 rs58542926 T allele in the case group was significantly higher than that in the control group (OR = 1.759, P = 0.048). After grouping, the frequency of CT/TT genotype in the liver cancer group combined with liver cirrhosis and the T allele frequency in the alcohol-related liver cancer group had statistically significant difference (P = 0.045 and 0.032, respectively) when compared with control group. The patients were divided into CG/GG group and CC group, and CT/TT group and CC group according to whether they carried PNPLA3 rs738409 G allele, and TM6SF2 rs58542926 T allele. Results showed that there were no statistically significant differences in liver enzyme indexes, albumin (ALB), total bilirubin (TBIL), alpha-fetoprotein (AFP) and fasting blood glucose between CG/GG group and CC group, and CT/TT group and CC group. The patients with liver cirrhosis in the case group were divided into≥7 groups and < 7 groups according to the Child-Pugh score. Results showed that there were no statistically significant difference in the Child-Pugh score between PNPLA3 rs738409 CG/GG group and CC group patients and TM6SF2 rs58542926 CT/TT group and CC group patients (P > 0.05). Conclusion: PNPLA3 rs738409 and TM6SF2 rs58542926 gene polymorphisms are correlated with the occurrence of primary liver cancer in the Han population of China's Northeast region. PNPLA3 rs738409 and TM6SF2 rs58542926 gene polymorphisms have no effect on indexes' such as liver enzymes, ALB, TBIL, AFP and FBS in primary liver cancer..

APOC3rs2854116, PNPLA3rs738409, and TM6SF2rs58542926 polymorphisms might influence predisposition of NAFLD: A meta-analysis.

It is plausible that apolipoprotein C3 (APOC3), patatin-like phospholipase domain containing 3 (PNPLA3), and transmembrane 6 superfamily member 2 (TM6SF2) polymorphisms may affect predisposition of nonalcoholic fatty liver disease (NAFLD), but the results of so far published studies remain controversial. The authors conducted this meta-analysis to clarify relationships between APOC3/PNPLA3/TM6SF2 polymorphisms and predisposition of NAFLD by pooling the findings of eligible studies. A comprehensive search of PubMed, Embase, Web of Science, and CNKI was endorsed by the authors to identify already published studies. Forty-nine studies were found to be eligible for meta-analyses. The pooled meta-analyses results showed that genotypic frequencies of APOC3 rs2854116, PNPLA3 rs738409, and TM6SF2 rs58542926 polymorphisms among patients and controls differed significantly. Moreover, genotypic frequencies of PNPLA3 rs738409 and TM6SF2 rs58542926 polymorphisms among patients and controls from both Caucasians and Asians also differed significantly. But However, no such differences in genotypic frequencies were observed for the APOC3 rs2854117 polymorphism. This meta-analysis suggested that APOC3 rs2854116, PNPLA3 rs738409, and TM6SF2 rs58542926 polymorphisms might affect predisposition of NAFLD.

Epidemiology: Pathogenesis, and Diagnostic Strategy of Diabetic Liver Disease in Japan.

Type 2 diabetes (T2D) is closely associated with nonalcoholic fatty liver disease (NAFLD). Nonalcoholic steatohepatitis (NASH), a severe form of NAFLD, can lead to cirrhosis, hepatocellular carcinoma (HCC), and hepatic decompensation. Patients with T2D have twice the risk of HCC incidence compared with those without T2D. Because the hepatic fibrosis grade is the main determinant of mortality in patients with NAFLD, identifying patients with advanced fibrosis using non-invasive tests (NITs) or imaging modalities is crucial. Globally, the fibrosis-4 index (FIB-4 index), NAFLD fibrosis score, and enhanced liver fibrosis test have been established to evaluate hepatic fibrosis. Two-step algorithms using FIB-4 index as first triaging tool are globally accepted. It remains unknown which kinds of NITs or elastography are best as the second step tool. In Japan, type IV collagen 7s or the CA-fibrosis index (comprising type IV collagen 7s and aspartate aminotransferase (AST)) is believed to precisely predict advanced fibrosis in NAFLD. Patients with NAFLD who have high non-invasive test results should be screened for HCC or esophageal varices. Risk factors of rapid fibrosis progression in NAFLD includes age, severe obesity, presence of T2D, menopause in women, and a patatin-like phospholipase domain containing the 3 GG genotype. Patients with NAFLD who have these risk factors should be intensively treated with lifestyle modification or pharmacotherapies for preventing liver-related mortality.

Identification of Phf16 and Pnpla3 as new adipogenic factors regulated by phytochemicals.

Adipocyte differentiation is controlled by multiple signaling pathways. To identify new adipogenic factors, C3H10T1/2 adipocytes were treated with previously known antiadipogenic phytochemicals (resveratrol, butein, sulfuretin, and fisetin) for 24 hours. Commonly regulated genes were then identified by transcriptional profiling analysis. Three genes (chemokine (C-X-C motif) ligand 1 [ Cxcl1], heme oxygenase 1 [ Hmox1], and PHD (plant homeo domain) finger protein 16 [ Phf16]) were upregulated while two genes (G0/G1 switch gene 2 [ G0s2] and patatin-like phospholipase domain containing 3 [ Pnpla3]) were downregulated by these four antiadipogenic compounds. Tissue expression profiles showed that the G0s2 and Pnpla3 expressions were highly specific to adipose depots while the other three induced genes were ubiquitously expressed with significantly higher expression in adipose tissues. While Cxcl1 expression was decreased, expressions of the other four genes were significantly increased during adipogenic differentiation of C3H10T1/2 cells. Small interfering RNA-mediated knockdown including Phf16 and Pnpla3 indicated that these genes might play regulatory roles in lipid accumulation and adipocyte differentiation. Specifically, the silencing of two newly identified adipogenic genes, Phf16 or Pnpla3, suppressed lipid accumulation and expression of adipocyte markers in both 3T3-L1 and C3H10T1/2 cells. Taken together, these data showed previously uncovered roles of Phf16 and Pnpla3 in adipogenesis, highlighting the potential of using phytochemicals for further investigation of adipocyte biology.

Validation of PNPLA3 polymorphisms as risk factor for NAFLD and liver fibrosis in an admixed population.

INTRODUCTION AND AIM: Studies have shown that two polymorphisms were associated with steatosis and progression of non-alcoholic fatty liver disease (NAFLD) in different populations: the Patatin-like Phospholipase Domain Containing 3 (PNPLA3) and Transmembrane 6 Superfamily Member 2 (TM6SF2). However, the frequency and significance of these polymorphisms in an admixed population, i.e., Brazilian, is unknown. Therefore, we aimed to evaluate them in healthy subjects in comparison to patients with NAFLD. MATERIAL AND METHODS: This was a multicenter cross-sectional study in 248 patients with biopsy-proven NAFLD and in 134 healthy controls from two tertiary centers in Brazil. PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) polymorphisms were evaluated. RESULTS: In controls, the frequencies of PNPLA3 CC and CG+GG were 49.25% and 50.74%, respectively; in NAFLD patients, this was 31.05% and 68.88% (p=0.0044, 95% CI 1.037-2.977). PNPLA3 GG subjects had an increased risk (3.29-fold) of having NAFLD when compared to CC subjects (p=0.0044, 95% CI 1.504-7.225). In patients with nonalcoholic steatohepatitis (NASH), PNPLA3 GG compared to CC was associated with higher AST levels [38.4±25.3 versus 36.7±40.1IU/L, p=0.0395)] and with the presence of liver fibrosis (≥F2 fibrosis, p=0.0272). TM6SF2 polymorphisms were not in Hardy-Weinberg equilibrium in our NAFLD group precluding further analysis. CONCLUSION: We demonstrated for the first time that PNPLA3 CG+GG increase the risk of NAFLD among Brazilian subjects. Moreover, PNPLA3 GG was associated with liver enzyme elevation and fibrosis in NASH patients.

Prevalence and severity of nonalcoholic fatty liver disease by transient elastography: Genetic and metabolic risk factors in a general population.

BACKGROUND & AIMS: The worldwide spread of obesity is leading to a dramatic increase in the prevalence of nonalcoholic fatty liver disease (NAFLD) and its complications. We aimed to evaluate both prevalence and factors associated with NAFLD in a general population in a Mediterranean area. METHODS: We considered 890 consecutive individuals included in the community-based ABCD (Alimentazione, Benessere Cardiovascolare e Diabete) study (ISRCTN15840340). Controlled attenuation parameter (CAP) and liver stiffness measurement (LSM) were measured with FibroScan. Participants were genotyped for PNPLA3 rs738409 and TM6SF2 rs58542926 variants. RESULTS: The prevalence of NAFLD in the cohort was 48%. NAFLD participants exhibited elevated LSM values, suggesting advanced fibrosis (6.5% of cases). Both NAFLD and advanced fibrosis were independently associated with traditional risk factors (NAFLD: age >50 years, obesity, hypertension, elevated ALT and low HDL-cholesterol serum concentrations. Advanced fibrosis: IFG/diabetes, elevated ALT serum concentrations). A high prevalence (>60%) of NAFLD was found in obese people, while it varied widely in non-obese people according to the presence of metabolic risk factors. PNPLA3 G variant (OR = 1.33, 95% C.I. = 1.01-1.8; P < .05) was independently associated with NAFLD. Prevalence of advanced fibrosis (high LSM values) ranged from 3.4% (no risk factors) to 60% (presence of all risk factors). TM6SF2 T variant (OR = 3.06, 95% C.I. = 1.08-8.65, P < .05) was independently associated with advanced fibrosis (high LSM values). CONCLUSIONS: In a cohort of a general population, the prevalence of NAFLD was very high, and among NAFLD patients a significant proportion had advanced fibrosis (high LSM values). Apart from traditional risk factors, genetic factors may have a significant role that needs to be further investigated.

Association between PNPLA3 (rs738409 C>G) variant and hepatocellular carcinoma in Asian chronic hepatitis C patients: A longitudinal study.

BACKGROUND/PURPOSE: Prdevious meta-analyses assess whether or not patatin-like phospholipase domain containing 3 (PNPLA3) (rs738409 C > G) was associated with increased risk of hepatocellular carcinoma (HCC) in Caucasians patients with hepatitis C virus (HCV)-related cirrhosis, these meta-analyses did not provide firm conclusions. Only one cross-sectional study involving Asian patients has previously been conducted to explore this issue. We aim to investigate this in a longitudinal cohort of Asian chronic hepatitis C (CHC) patients. METHODS: We consecutively enrolled 1011 CHC patients who underwent liver biopsy before initiating interferon-based therapy. These patients were followed-up and screened for HCC up to a median of 6.9 years. The influence of rs738409 (GG) genotype on the occurrence of HCC was assessed using the Kaplan-Meier method, then according to the multivariate Cox model. RESULTS: During follow-up, 143 (14.1%) patients developed HCC. rs738409 (GG) genotype was not associated with time-to-HCC development on multivariate Cox regression (P = 0.634). When considering the occurrence of these events over time, rs738409 (GG) genotype did not influence the risk of HCC development (log-rank = 0.12). Among 261 patients with liver cirrhosis, rs738409 (GG) genotype was not associated with time-to-HCC development on multivariate Cox regression (P = 0.737). When considering the occurrence of these events over time, rs738409 (GG) genotype did not influence the risk of HCC development (log-rank = 0.72). CONCLUSION: In this longitudinal study with liver biopsy to stage liver fibrosis, we affirm there is no influence of the rs738409 (GG) genotype on the occurrence of HCC in Asian CHC patients, including cirrhotic patients.

Diagnosis of non-alcoholic fatty liver disease (NAFLD).

Non-alcoholic fatty liver disease (NAFLD) increases risk of mortality from liver and cardiovascular disease (CVD) and is the major cause of hepatocellular carcinoma (HCC), which may develop without cirrhosis. NAFLD predicts type 2 diabetes, even independently of obesity. Globally, the prevalence of NAFLD averages 25% and is as common as the metabolic syndrome. The majority of patients with type 2 diabetes have NAFLD. The challenge for the diabetologist is to identify patients at risk of advanced liver disease and HCC. At a minimum, liver function tests (LFTs), despite being neither specific nor sensitive, should be performed in all patients with the metabolic syndrome or type 2 diabetes. Increases in LFTs, for which the updated reference values are lower (serum ALT ≈30 U/l in men and ≈20 U/l in women) than those hitherto used in many laboratories, should prompt assessment of fibrosis biomarkers and referral of individuals at risk to a NAFLD/hepatology clinic. Preferably, evaluation of NAFLD should be based on measurement of steatosis biomarkers or ultrasound if easily available. A large number of individuals carry the patatin-like phospholipase domain containing 3 (PNPLA3) I148M variant (30-50%) or the transmembrane 6 superfamily member 2 (TM6SF2) E167K variant (11-15%). These variants increase the risk of advanced liver disease and HCC but not of diabetes or CVD. Genotyping of selected patients for these variants is recommended. Many patients have 'double trouble', i.e. carry both a genetic risk factor and have the metabolic syndrome. Excess use of alcohol could be a cause of 'triple trouble', but such patients would be classified as having alcoholic fatty liver disease. This review summarises a presentation given at the symposium 'The liver in focus' at the 2015 annual meeting of the EASD. It is accompanied by two other reviews on topics from this symposium (by Kenneth Cusi, DOI: 10.1007/s00125-016-3952-1 , and by John Jones, DOI: 10.1007/s00125-016-3940-5 ) and a commentary by the Session Chair, Michael Roden (DOI: 10.1007/s00125-016-3911-x ).

The PNPLA3 rs738409 C > G polymorphism is associated with the risk of progression to cirrhosis in NAFLD patients.

BACKGROUND AND AIMS: The patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 C > G single nucleotide polymorphism (SNP) has been associated with steatosis and fibrosis in previous NAFLD populations in which cirrhotic patients were very poorly represented. Since not all NAFLD with fibrosis evolve to cirrhosis, we investigated the specific risk of cirrhosis conferred in NAFLD patients by carrying this SNP. METHODS: Three groups were studied: patients with NASH-cirrhosis; patients with biopsy-proven non-cirrhotic NAFLD; healthy subjects undergoing medicine check-ups. Epidemiological, anthropometric, and clinical data were collected, and the SNP was analyzed by pyrosequencing. RESULTS: Sixty-one patients with NASH-cirrhosis, 60 with non-cirrhotic NAFLD, and 125 healthy controls were included. Frequency of the PNPLA3 minor (G) allele was increased in patients with NASH-cirrhosis compared with non-cirrhotic NAFLD and controls (allele frequency: 0.598 versus 0.367 versus 0.2, respectively, p < 0.001), and different between the latter two groups (p < 0.001). Three-quarters (74%) of NASH cirrhotics carried at least one G allele, and almost half of them (46%) were GG homozygous. By multivariate analysis in the NAFLD population, each copy of the G allele was associated with an almost doubling of the risk of cirrhosis [OR 1.8 (1.02-3.2)], while being GG homozygous with a tripled risk compared with being CC homozygous [3.01 (1.03-10.8)]. CONCLUSIONS: In NAFLD patients, carriage of the PNPLA3G allele, and particularly of the GG genotype, is significantly associated with the risk of cirrhotic evolution. If confirmed in larger series, these results would suggest that most of NASH cases require the contribution of an altered PNPLA3 function to progress until cirrhosis.

The rs738409 (I148M) variant of the PNPLA3 gene and cirrhosis: a meta-analysis.

The human patatin-like phospholipase domain-containing-3 (PNPLA3) gene rs738409 C>G polymorphism is associated with several types of liver disease. The aim of this meta-analysis was to assess the risk of cirrhosis on the basis of rs738409 allele frequency and genotype. Medline, the Cochrane Library, EMBASE, and Google Scholar were searched for prospective and retrospective studies assessing the effect of the rs738409 polymorphism on liver cirrhosis. Seven studies, involving 2,023 patients with cirrhosis, were included. The G allele was associated with a significantly increased risk of cirrhosis versus the C allele [pooled odds ratio (OR) = 1.86, 95% confidence interval (CI) = 1.64-2.12, Z = 9.55, P < 0.001]. Both the GC and GG genotypes were associated with a significantly increased risk of cirrhosis versus the CC genotype (GC vs. CC: pooled OR = 1.73, 95% CI = 1.51-1.98, Z = 7.86, P < 0.001; GG vs. CC: pooled OR = 3.41, 95% CI = 2.77-4.18, Z = 11.65, P < 0.001). There was no evidence of publication bias. Our findings suggest that patients at risk for liver cirrhosis may benefit from PNPLA3 genotyping and thus more intensive monitoring if the rs738409 C>G polymorphism is identified.

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 3L 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.

The impact of PNPLA3 (rs738409 C>G) polymorphisms on liver histology and long-term clinical outcome in chronic hepatitis B patients.

BACKGROUND & AIMS: We aimed to assess the association between the patatin-like phospholipase domain-containing-3 (PNPLA3) I148M polymorphism, liver histology and long-term outcome in chronic hepatitis B (CHB) patients. METHODS: We enrolled 531 consecutive treatment naïve CHB patients diagnosed from 1985 to 2012 with an available liver biopsy for reassessment, and sample for genetic testing. Data on all-cause mortality and hepatocellular carcinoma (HCC) at long-term follow-up were obtained from national database registries. RESULTS: The prevalence of steatohepatitis increased with PNPLA3 CC (14%), CG (20%) and GG (43%) (P < 0.001). The association was altered by both gender (P = 0.010) and overweight (P = 0.015): the effect of PNPLA3 on steatohepatitis was most pronounced among non-overweight females (adjusted OR 13.4, 95%CI: 3.7-51.6, P < 0.001), and non-overweight males (adjusted OR 2.4, 95%CI: 1.4-4.3, P = 0.002). Furthermore, PNPLA3 GG genotype was associated with iron depositions (OR 2.8, 95%CI: 1.2-6.4, P = 0.014) and lobular inflammation (OR 2.2, 95%CI: 1.1-4.5, P = 0.032), but not with advanced fibrosis (OR 1.1, 95%CI: 0.7-1.8, P = 0.566). The median follow-up was 10.1 years (interquartile range 5.6 - 15.8), during which 13 patients developed HCC and 28 died. Steatohepatitis was associated with all-cause mortality [Hazard ratio (HR) 3.1, 95%CI: 1.3-7.3, P = 0.006] and HCC (HR 2.8, 95%CI: 0.9-9.2, P = 0.078), but no significant association was observed for PNPLA3. CONCLUSIONS: In this cohort of biopsied CHB patients, PNPLA3 was independently associated with steatosis, steatohepatitis, lobular inflammation and iron depositions, but not with advanced fibrosis, HCC development or all-cause mortality. The effect of PNPLA3 on steatohepatitis was particularly pronounced among female patients without severe overweight.

Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients.

Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724-1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC.

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 [(13)C]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 [(13)C]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 [(13)C]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).

The effect of dulaglutide on glycated hemoglobin is associated with PNPLA3 Ι148Μ gene polymorphism in patients with type 2 diabetes mellitus.

PURPOSE: The evaluation of the effect of dulaglutide on glycated hemoglobin (HbA1c) and non-invasive indices of hepatic steatosis among different genotypes of the PNPLA3 I148M (rs738409) and CETP Taq1B (rs708272) polymorphisms in patients with type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD). METHODS: Relevant data from patients with inadequately controlled T2DM, also displaying NAFLD, administered 1.5 mg dulaglutide weekly for 6 months were retrospectively retrieved. The non-invasive indices, fatty liver index (FLI) and hepatic steatosis index (HSI), were calculated. Genotyping for rs738409 and rs708272 were performed with polymerase chain reaction. RESULTS: Data from 80 patients (39 females), aged 64.4 ± 9.5 years and displaying a baseline BMI of 34.5 ± 5.8 kg/m2, were retrieved at baseline and after 6 months (endpoint) of dulaglutide treatment. Glycated hemoglobin (HbA1c; -0.72 ± 1.10%; p < 0.001), FLI (-5.8 ± 9.8; p < 0.001) and HSI (-1.18 ± 3.51; p = 0.004) significantly decreased after treatment. Lipid profile and liver function tests also improved after treatment. Overall, homozygotes for the reference rs738409 allele (CC) displayed a 2.4-fold decrease (p = 0.002) and heterozygotes (CG) an 1.6-fold decrease (p = 0.013) compared to GG homozygotes after treatment, but the effect was largely limited to female patients. No similar effect was observed in FLI, HSI and other relevant parameters. No association was observed between rs708272 and any of the parameters studied. CONCLUSIONS: rs738409, but not rs708272, was associated with the effect of dulaglutide on HbA1c, but not on presumed hepatic steatosis or other relevant parameters. Sex-specific effects were also noticed.

A study of genetic variants, genetic risk score and DNA methylation of PNPLA3 and TM6SF2 in alcohol liver cirrhosis.

BACKGROUND: Genetic and epigenetic factors are associated with the development of alcohol-associated liver disease (AALD). The single nucleotide polymorphisms (SNPs), rs738409 in Patatin-like phospholipase domain-containing protein (PNPLA3) and rs58542926 in Transmembrane 6 Superfamily Member 2 (TM6SF2) are strongly associated with AALD in different global populations, Hence, we analyzed the genetic risk score for these variants and deoxyribonucleic acid (DNA) methylation levels of the PNPLA3 and TM6SF2 genes among cases (alcohol liver cirrhosis) and controls (heavy drinkers without cirrhosis). METHOD: We studied patients with alcohol use disorder (AUD) with cirrhosis (AUD-C + ve, n = 136) and without cirrhosis (AUD-C-ve, n = 107) drawn from the clinical services of St. John's Medical College Hospital (SJMCH) (Gastroenterology and Psychiatry) and Centre for Addiction Medicine (CAM), National Institute of Mental Health and Neurosciences, (NIMHANS). Genotype data was generated for rs738409 (PNPLA3) and rs58542926 (TM6SF2) and used to calculate unweighted genetic risk score (uGRS) and weighted genetic risk scores (wGRS). DNA methylation levels were estimated by pyrosequencing at PNPLA3 and TM6SF2 loci. RESULTS: Overall we observed a significantly higher genetic risk score (weighted genetic risk score, wGRS) in individuals with alcohol use disorder compared to control population (p = < 0.01). Further, uGRS and wGRS were associated with the diagnosis of cirrhosis, even after correcting for age of onset, quantity and frequency of drinking. We also found hypomethylation at CpG2 of TM6SF2 gene in AUD-C + ve compared to AUD-C-ve (P = 0.02). CONCLUSION: We found that a genetic risk score based on SNPs in the PNPLA3 and TM6SF2 genes was significantly associated with cirrhosis in patients with AUD, suggesting a potential utility in identifying patients at risk and providing pre-emptive interventions. These may include interventions that aim to alter DNA methylation, which may be one of the mechanisms through which elevated genetic risk may influence the development of cirrhosis.