The ubiquitin E3 ligase BFAR promotes degradation of PNPLA3.

A missense variant in patatin-like phospholipase domain-containing protein 3 [PNPLA3(I148M)] is the most impactful genetic risk factor for fatty liver disease (FLD). We previously showed that PNPLA3 is ubiquitylated and subsequently degraded by proteasomes and autophagosomes and that the PNPLA3(148M) variant interferes with this process. To define the machinery responsible for PNPLA3 turnover, we used small interfering (si)RNAs to inactivate components of the ubiquitin proteasome system. Inactivation of bifunctional apoptosis regulator (BFAR), a membrane-bound E3 ubiquitin ligase, reproducibly increased PNPLA3 levels in two lines of cultured hepatocytes. Conversely, overexpression of BFAR decreased levels of endogenous PNPLA3 in HuH7 cells. BFAR and PNPLA3 co-immunoprecipitated when co-expressed in cells. BFAR promoted ubiquitylation of PNPLA3 in vitro in a reconstitution assay using purified, epitope-tagged recombinant proteins. To confirm that BFAR targets PNPLA3, we inactivated Bfar in mice. Levels of PNPLA3 protein were increased twofold in hepatic lipid droplets of Bfar-/- mice with no associated increase in PNPLA3 mRNA levels. Taken together these data are consistent with a model in which BFAR plays a role in the post-translational degradation of PNPLA3. The identification of BFAR provides a potential target to enhance PNPLA3 turnover and prevent FLD.

Interaction between estrogen receptor-α and PNPLA3 p.I148M variant drives fatty liver disease susceptibility in women.

Fatty liver disease (FLD) caused by metabolic dysfunction is the leading cause of liver disease and the prevalence is rising, especially in women. Although during reproductive age women are protected against FLD, for still unknown and understudied reasons some develop rapidly progressive disease at the menopause. The patatin-like phospholipase domain-containing 3 (PNPLA3) p.I148M variant accounts for the largest fraction of inherited FLD variability. In the present study, we show that there is a specific multiplicative interaction between female sex and PNPLA3 p.I148M in determining FLD in at-risk individuals (steatosis and fibrosis, P < 10-10; advanced fibrosis/hepatocellular carcinoma, P = 0.034) and in the general population (P < 10-7 for alanine transaminase levels). In individuals with obesity, hepatic PNPLA3 expression was higher in women than in men (P = 0.007) and in mice correlated with estrogen levels. In human hepatocytes and liver organoids, PNPLA3 was induced by estrogen receptor-α (ER-α) agonists. By chromatin immunoprecipitation and luciferase assays, we identified and characterized an ER-α-binding site within a PNPLA3 enhancer and demonstrated via CRISPR-Cas9 genome editing that this sequence drives PNPLA3 p.I148M upregulation, leading to lipid droplet accumulation and fibrogenesis in three-dimensional multilineage spheroids with stellate cells. These data suggest that a functional interaction between ER-α and PNPLA3 p.I148M variant contributes to FLD in women.

Integrated analysis of the transcriptome and its interaction with the metabolome in metabolic associated fatty liver disease: Gut microbiome signatures, correlation networks, and effect of PNPLA3 genotype.

Interactions between communities of the gut microbiome and with the host could affect the onset and progression of metabolic associated fatty liver disease (MAFLD), and can be useful as new diagnostic and prognostic biomarkers. In this study, we performed a multi-omics approach to unravel gut microbiome signatures from 32 biopsy-proven patients (10 simple steatosis -SS- and 22 steatohepatitis -SH-) and 19 healthy volunteers (HV). Human and microbial transcripts were differentially identified between groups (MAFLD vs. HV/SH vs. SS), and analyzed for weighted correlation networks together with previously detected metabolites from the same set of samples. We observed that expression of Desulfobacteraceae bacterium, methanogenic archaea, Mushu phage, opportunistic pathogenic fungi Fusarium proliferatum and Candida sorbophila, protozoa Blastocystis spp. and Fonticula alba were upregulated in MAFLD and SH. Desulfobacteraceae bacterium and Mushu phage were hub species in the onset of MAFLD, whereas the activity of Fonticula alba, Faecalibacterium prausnitzii, and Mushu phage act as key regulators of the progression to SH. A combination of clinical, metabolomic, and transcriptomic parameters showed the highest predictive capacity for MAFLD and SH (AUC = 0.96). In conclusion, faecal microbiome markers from several community members contribute to the switch in signatures characteristic of MAFLD and its progression towards SH.

PNPLA3 GENE POLYMORPHISM AND RED MEAT CONSUMPTION INCREASED FIBROSIS RISK IN NASH BIOPSY-PROVEN PATIENTS UNDER MEDICAL FOLLOW-UP IN A TERTIARY CENTER IN SOUTHWEST BRAZIL.

BACKGROUND: Recent studies show an increase in nonalcoholic fatty liver disease (NAFLD) in populations with higher consumption of red meat, processed and cooked at high temperatures. On the other hand, the single nucleotide polymorphism rs738409 in the Patatin-like phospholipase domain containing 3 (PNPLA3) gene has been implicated in susceptibility to NAFLD and liver fibrosis. However, the synergistic effect between red meat consumption and the PNPLA3 gene polymorphism in NAFLD has not yet been evaluated. OBJECTIVE: To evaluate the association between the presence of the polymorphism in the PNPLA3 gene and the consumption of macronutrients, including meat consumption and its cooking method among NAFLD patients. METHODS: This was a cross-sectional study with 91 patients diagnosed with NAFLD by liver biopsy with genotyping for the polymorphism in the PNPLA3 gene were included. The consumption of calories and macronutrients was verified using the semi-quantitative food frequency questionnaire and the specific questionnaire on meat consumption. PNPLA3 gene polymorphism was analyzed by real-time polymerase chain reaction (RT-PCR) and anthropometric evaluation was realized. RESULTS: The mean BMI was 32.38±4.58 kg/m² and the waist circumference was 107±10 cm. On liver biopsy, 42% of patients had significant fibrosis (F≥2). The odds ratio of F≥2 was 2.12 for the GG group and 1.54 for the CG group, compared to the CC group. The mean caloric intake was 1170±463.20 kcal/d. The odds ratio in the CC group concerning high red meat consumption in comparison to low consumption was 1.33. For white meat, the odds ratio was 0.8 when comparing high and low intake, also in the CC group. CONCLUSION: High red meat intake and PNPLA3 gene polymorphism seem to synergistically affect NAFLD and liver fibrosis, requiring confirmation in a larger number of patients and in different populations.

Long-term hypercaloric diet exacerbates metabolic liver disease in PNPLA3 I148M animals.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is a major health burden associated with the metabolic syndrome leading to liver fibrosis, cirrhosis and ultimately liver cancer. In humans, the PNPLA3 I148M polymorphism of the phospholipase patatin-like phospholipid domain containing protein 3 (PNPLA3) has a well-documented impact on metabolic liver disease. In this study, we used a mouse model mimicking the human PNPLA3 I148M polymorphism in a long-term high fat diet (HFD) experiment to better define its role for NAFLD progression. METHODS: Male mice bearing wild-type Pnpla3 (Pnpla3WT ), or the human polymorphism PNPLA3 I148M (Pnpla3148M/M ) were subjected to HFD feeding for 24 and 52 weeks. Further analysis concerning basic phenotype, inflammation, proliferation and cell death, fibrosis and microbiota were performed in each time point. RESULTS: After 52 weeks HFD Pnpla3148M/M animals had more liver fibrosis, enhanced numbers of inflammatory cells as well as increased Kupffer cell activity. Increased hepatocyte cell turnover and ductular proliferation were evident in HFD Pnpla3148M/M livers. Microbiome diversity was decreased after HFD feeding, changes were influenced by HFD feeding (36%) and the PNPLA3 I148M genotype (12%). Pnpla3148M/M mice had more faecal bile acids. RNA-sequencing of liver tissue defined an HFD-associated signature, and a Pnpla3148M/M specific pattern, which suggests Kupffer cell and monocytes-derived macrophages as significant drivers of liver disease progression in Pnpla3148M/M animals. CONCLUSION: With long-term HFD feeding, mice with the PNPLA3 I148M genotype show exacerbated NAFLD. This finding is linked to PNPLA3 I148M-specific changes in microbiota composition and liver gene expression showing a stronger inflammatory response leading to enhanced liver fibrosis progression.

Association of PNPLA3 SNP With the Development of HBV-related Hepatocellular Carcinoma.

BACKGROUND/AIM: Concomitant nonalcoholic fatty liver disease (NAFLD)/hepatic steatosis (HS) is suggested to increase the risk of hepatocellular carcinoma (HCC) in hepatitis virus B (HBV)-infected patients. Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 gene single-nucleotide polymorphism (SNP) is well-known to be associated with the development of NAFLD/HS; however, it is still unclear whether this SNP is related to the development of HCC in HBV-infected patients. PATIENTS AND METHODS: We investigated a total of 202 HBV-infected patients who received percutaneous liver biopsy, and simultaneously assessed biopsy-proven HS, insulin resistance, and the PNPLA3 SNP status. We further investigated the relationships of these factors with the development of HCC in HBV-infected patients. RESULTS: Most of the enrolled cases (196/202: 97.0%) were non-cirrhotic patients. One hundred seventy-three patients (85.6%) received antiviral therapy. A Kaplan-Meier analysis showed that the incidence of HCC development in patients with HS was higher than that in patients without HS (p<0.01). An increased homeostasis model assessment as an index of insulin resistance (HOMA-IR) value (≥1.6) was associated not only with the presence of HS (p<0.0001) but also with the development of HCC (p<0.01). The PNPLA3 rs738409 SNP was also associated with the presence of HS (p<0.01) and the development of HCC (p<0.05) in HBV-infected patients. CONCLUSION: In addition to HS and IR, PNPLA3 rs738409 SNP was suggested to be associated with the development of HCC in Japanese patients with HBV infection.

IL-6/STAT3 axis dictates the PNPLA3-mediated susceptibility to non-alcoholic fatty liver disease.

BACKGROUND & AIMS: A number of genetic polymorphisms have been associated with susceptibility to or protection against non-alcoholic fatty liver disease (NAFLD), but the underlying mechanisms remain unknown. Here, we focused on the rs738409 C>G single nucleotide polymorphism (SNP), which produces the I148M variant of patatin-like phospholipase domain-containing protein 3 (PNPLA3) and is strongly associated with NAFLD. METHODS: To enable mechanistic dissection, we developed a human pluripotent stem cell (hPSC)-derived multicellular liver culture by incorporating hPSC-derived hepatocytes, hepatic stellate cells, and macrophages. We first applied this liver culture to model NAFLD by utilising a lipotoxic milieu reflecting the circulating levels of disease risk factors in affected individuals. We then created an isogenic pair of liver cultures differing only at rs738049 and compared NAFLD phenotype development. RESULTS: Our hPSC-derived liver culture recapitulated many key characteristics of NAFLD development and progression including lipid accumulation and oxidative stress, inflammatory response, and stellate cell activation. Under the lipotoxic conditions, the I148M variant caused the enhanced development of NAFLD phenotypes. These differences were associated with elevated IL-6/signal transducer and activator of transcription 3 (STAT3) activity in liver cultures, consistent with transcriptomic data of liver biopsies from individuals carrying the rs738409 SNP. Dampening IL-6/STAT3 activity alleviated the I148M-mediated susceptibility to NAFLD, whereas boosting it in wild-type liver cultures enhanced NAFLD development. Finally, we attributed this elevated IL-6/STAT3 activity in liver cultures carrying the rs738409 SNP to increased NF-κB activity. CONCLUSIONS: Our study thus reveals a potential causal link between elevated IL-6/STAT3 activity and 148M-mediated susceptibility to NAFLD. IMPACT AND IMPLICATIONS: An increasing number of genetic variants manifest in non-alcoholic fatty liver disease (NAFLD) development and progression; however, the underlying mechanisms remain elusive. To study these variants in human-relevant systems, we developed an induced pluripotent stem cell-derived multicellular liver culture and focused on a common genetic variant (i.e. rs738409 in PNPLA3). Our findings not only provide mechanistic insight, but also a potential therapeutic strategy for NAFLD driven by this genetic variant in PNPLA3. Our liver culture is therefore a useful platform for exploring genetic variants in NAFLD development.

PNPLA3 allele frequency has no impact on biliary bile acid composition or disease course in patients with primary sclerosing cholangitis.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic inflammatory disease that leads to bile duct strictures, cholestasis, and biliary cirrhosis. PNPLA3 (patatin-like phospholipase domain containing 3), regulates cellular lipid synthesis by converting lysophosphatidic acid into phosphatidic acid. Isoleucine mutation to methionine at position 148 (I148M) causes a loss of this function. Only two studies, with contradictory results, have evaluated the role of PNPLA3 in PSC. The rs738409(G) variant of PNPLA3 has been associated with an increased risk for transplantation in male patients with dominant strictures (DS). The study aimed to evaluate the PNPLA3 allele frequency effect on the clinical outcomes, progression, and prognosis of PSC. Furthermore, we analyzed the impact of PNPLA3 on phospholipid and bile acid composition to evaluate the effect of the PNPLA3 status on UDCA response. PATIENTS AND METHODS: We recruited 560 patients prospectively and collected clinical and laboratory data as well as liver histology and imaging findings. PNPLA3 (CC, CG, GG) alleles were analyzed with TaqManTM. We also analyzed bile acids (BA), cholesterol and phospholipids and individual BA from a sample aspirated during endoscopic retrograde cholangiography (ERC). RESULTS: Among the recruited patients, 58.4%, 35.7% and 5.9% had the wild (CC), heterozygous (CG) and homozygous (GG) alleles, respectively. The PNPLA3 haplotype did not impact bile composition or individual BA. In addition, we found no differences in age at diagnosis, disease progression, liver fibrosis or survival between the cohorts. CONCLUSIONS: The PNPLA3 I148M variant had no significant impact on on bile composition, including UDCA content, clinical outcomes, progression of liver fibrosis, hepatobiliary cancer risk, liver transplantation, or overall survival.

miR-516a-3P, a potential circulating biomarker in hepatocellular carcinoma, correlated with rs738409 polymorphism in PNPLA3.

Aim: The aim was to investigate the expression profile of miR-516a-3P and its correlation with the PNPLA3 rs738409 polymorphism in Egyptian hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) patients. Materials & methods: miR-516a-3P was quantified and rs738409 was genotyped by quantitative reverse transcription PCR. Results: miR-516a-3P was significantly upregulated in HCC patients compared with HCV patients (p = 0.001). Receiver operating characteristic curve analysis confirmed that miR-516a-3P discriminates HCC from HCV (p = 0.001). A significant (p = 0.015) correlation between miR-516a-3p level and PNPLA3 rs738409 genotypes was recorded in HCV patients, yet it was not recorded in either healthy individuals or HCC patients. miR-516a-3p level was significantly (p = 0.001) higher in HCV patients carrying the rs738409 GG genotype than in those carrying the CC genotype. Conclusion: miR-516a-3P is a potential biomarker in HCC. PNPLA3 rs738409 GG carriers affect miR-516a-3P expression in HCV, and this may highlight a new mechanism in liver disease.

In the past decade, miRNAs were established as biomarkers in various cancers, including liver cancer. Information about the deregulation of miR-516a-3p and its efficiency as a biomarker in hepatitis C virus (HCV) and liver cancer patients is lacking globally and in Egypt. This study proved for the first time that miR-516a-3p is differentially expressed in HCV and liver cancer patients and is statistically efficient in discriminating between them, so it might be used for early detection of HCC. Genetic variants that affect expression levels of genes are called expression quantitative trait loci (eQTL), and those acting on genes on other chromosomes are called trans-eQTL. The authors speculate that rs738409 is a genetic variant that might have a trans-eQTL effect on the miR-516a-3p gene in HCV patients.

Synergistic Associations of PNPLA3 I148M Variant, Alcohol Intake, and Obesity With Risk of Cirrhosis, Hepatocellular Carcinoma, and Mortality.

Importance: Alcohol drinking and obesity are associated with an increased risk of cirrhosis and hepatocellular carcinoma (HCC), but the risk is not uniform among people with these risk factors. Genetic variants, such as I148M in the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene, may play an important role in modulating cirrhosis and HCC risk. Objective: To investigate the joint associations of the PNPLA3 I148M variant, alcohol intake, and obesity with the risk of cirrhosis, HCC, and liver disease-related mortality. Design, Setting, and Participants: This prospective cohort study analyzed 414 209 participants enrolled in the UK Biobank study from March 2006 to December 2010. Participants had no previous diagnosis of cirrhosis and HCC and were followed up through March 2021. Exposures: Self-reported alcohol intake (nonexcessive vs excessive), obesity (body mass index ≥30 [calculated as weight in kilograms divided by height in meters squared]), and PNPLA3 I148M variant status (noncarrier, heterozygous carrier, or homozygous carrier) from initial assessment. Main Outcomes and Measures: The primary outcomes were incident cirrhosis and HCC cases and liver disease-related death ascertained from inpatient hospitalization records and death registry. The risks were calculated by Cox proportional hazards regression models. Results: A total of 414 209 participants (mean [SD] age, 56.3 [8.09] years; 218 567 women [52.8%]; 389 452 White race and ethnicity [94.0%]) were included. Of these participants, 2398 participants (0.6%) developed cirrhosis (5.07 [95% CI, 4.87-5.28] cases per 100 person-years), 323 (0.1%) developed HCC (0.68 [95% CI, 0.61-0.76] cases per 100 person-years), and 878 (0.2%) died from a liver disease-related cause (1.76 [95% CI, 1.64-1.88] cases per 100 person-years) during a median follow-up of 10.9 years. Synergistic interactions between the PNPLA3 I148M variant, obesity, and alcohol intake were associated with the risk of cirrhosis, HCC, and liver disease-related mortality. The risk of cirrhosis increased supramultiplicatively (adjusted hazard ratio [aHR], 17.52; 95% CI, 12.84-23.90) in individuals with obesity, with excessive drinking, and who were homozygous carriers compared with those with no obesity, with nonexcessive drinking, and who were noncarriers. Supramultiplicative associations between the 3 factors and risks of HCC were found in individuals with 3 risk factors (aHR, 30.13; 95% CI, 16.51-54.98) and liver disease-related mortality (aHR, 21.82; 95% CI, 13.78-34.56). The PNPLA3 I148M variant status significantly differentiated the risk of cirrhosis, HCC, and liver disease-related mortality in persons with excessive drinking and obesity. Conclusions and Relevance: This study found synergistic associations of the PNPLA3 I148M variant, excessive alcohol intake, and obesity with increased risk of cirrhosis, HCC, and liver disease-related death in the general population. The PNPLA3 I148M variant status may help refine the risk stratification for liver disease in persons with excessive drinking and obesity who may need early preventive measures.

The PNPLA3 variant I148M reveals protective effects toward hepatocellular carcinoma in mice via restoration of omega-3 polyunsaturated fats.

Alcohol consumption and high caloric diet are leading causes of progressive fatty liver disease. Genetic variant rs738409 in patatin-like phospholipase domain-containing protein 3 (PNPLA3 rs738409 C>G) has been repeatedly described as one of the major risk loci for alcoholic liver cirrhosis (ALC) and hepatocellular carcinoma (HCC) in humans, however, the mechanism behind this association is incompletely understood. We generated mice carrying the rs738409 variant (PNPLA3 I148M) in order to detect genotype-phenotype relationships in mice upon chow and alcohol-high fat/high sugar diet (EtOH/WD). We could clearly demonstrate that the presence of rs738409 per se is sufficient to induce spontaneous development of steatosis after 1 year in mice on a chow diet, whereas in the setting of unhealthy diet feeding, PNPLA3 I148M did not affect hepatic inflammation or fibrosis, but induced a striking lipid remodeling, microvesicular steatosis and protected from HCC formation. Using shot gun lipidomics, we detected a striking restoration of reduced long chain-polyunsaturated fatty acids (LC-PUFA)-containing TGs, docosapentaenoic acid (C22:5 n3) and omega-3-derived eicosanoids (5-HEPE, 20-HEPE, 19,20-EDP, 21-HDHA) in PNPLA3 I148M mice upon EtOH/WD. At the molecular level, PNPLA3 I148M modulated enzymes for fatty acid and TG transport and metabolism. These findings suggest (dietary) lipids as an important and independent driver of hepatic tumorigenesis. Genetic variant in PNPLA3 exerted protective effects in mice, conflicting with findings in humans. Species-related differences in physiology and metabolism should be taken into account when modeling unhealthy human lifestyle, as genetic mouse models may not always allow for translation of insight gained in humans.

Impact of the PNPLA3 genotype on the risk of hepatocellular carcinoma after hepatitis C virus eradication.

Almost all chronic hepatitis C (CHC) patients can achieve sustained virological response (SVR) with direct-acting antivirals. However, the development of hepatocellular carcinoma (HCC), even after the achievement of SVR, continues to be a serious problem. The aim of this study was to assess the association between host genetic factors and de novo HCC after SVR. This single-center, retrospective study consisted of 442 consecutive CHC patients without a history of HCC who achieved SVR through interferon (IFN)-based and IFN-free therapy. Predictive factors associated with the development of HCC were determined by the Cox proportional hazards model. The single-nucleotide polymorphism (SNP) genotyping data of 223 patients were available for analysis. Of the seven SNPs analyzed in this study, only the patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 GG genotype was significantly, positively associated with the development of de novo HCC after adjusting for age, sex, and fibrosis status (adjusted hazard ratio [aHR] 5.66, p = 0.003). In multivariable analysis, age (aHR 1.05, p = 0.007), advanced fibrosis (aHR 2.69, p = 0.019), α-fetoprotein at post-12 weeks of treatment ≥7.0 ng/ml (aHR 3.85, p = 0.001), and PNPLA3 GG genotype (aHR 3.02, p = 0.004) were extracted as independent predictors of the development of de novo HCC. In conclusion, the PNPLA3 genotype was independently associated with the de novo HCC of CHC patients who achieved SVR. Such detailed knowledge of host genetic factors will be useful for HCC surveillance after HCV elimination.

African genetic ancestry is associated with lower frequency of PNPLA3 G allele in non-alcoholic fatty liver in an admixed population.

INTRODUCTION AND OBJECTIVES: PNPLA3 (rs738409) and TM6SF2 (rs58542926) variants, interindividual and ethnic differences may be risk factors for non-alcoholic fatty liver disease (NAFLD). The PNPLA3 G allele is associated with worse NAFLD evolution in Hispanics and Caucasians. TM6SF2 is associated with hypertriglyceridemia, NAFLD, and cardiovascular disease. We aimed to evaluate the association between genetic ancestry by Ancestry Informative Markers (AIM), PNPLA3 and TM6SF2 polymorphisms in patients with biopsy-proven NAFLD in an admixed population. METHODS: We included adults with biopsy-proven NAFLD and excluded patients with the presence of other chronic liver disease, alcohol intake >100g/week, HIV, drug-induced fatty liver disease, or liver transplantation. We classified NAFLD using the Non-Alcoholic Steatohepatitis Clinical Research Network (NASH-CRN) histological scoring system. The PNPLA3 (rs738409 c.444C>G) and TM6SF2 (rs58542926 c.449C>T) genotyping were performed by RT-PCR. Genetic ancestry was determined using 46 insertion-deletion AIM; α<0.05 was considered significant. RESULTS: A total of 248 patients with NAFLD were enrolled [34 with simple steatosis (NAFL); 214 with NASH]. Overall, we detected a greater European ancestry contribution (0.645), followed by African (0.173), Amerindian (0.095), and East Asian (0.087) ancestry contribution, without differences between NAFL and NASH patients. However, we found a higher African genetic ancestry contribution among patients with NAFL who had the PNPLA3 C/C genotype than those with the G allele (0.216 ± 0.205 versus 0.105 ± 0.101, respectively; p=0.047). Ancestry contributions did not differ among TM6SF2 genotypes. CONCLUSION: Among NAFL patients, greater African genetic ancestry was associated to a lower frequency of the PNPLA3 G allele, demonstrating a possible NASH ancestry-related protective factor.

A PNPLA3 Polymorphism Confers Lower Susceptibility to Incident Diabetes Mellitus in Subjects With Nonalcoholic Fatty Liver Disease.

BACKGROUND AND AIMS: We investigated the association between the patatin-like phospholipase domain-containing-3 (PNPLA3) rs738409 genotype and the risk of diabetes mellitus (DM) using a biopsy-confirmed nonalcoholic fatty liver disease (NAFLD) cohort and a longitudinal observational cohort. METHODS: Associations between genotypes and the prevalence of DM were evaluated with stratification according to the histological severity of NAFLD in the Boramae cohort (n = 706). A longitudinal cohort consisting of nondiabetic individuals with ≥2 health checkups was then selected to investigate the risk of incident DM according to the genotype (the GENIE cohort; n = 4998). RESULTS: Among subjects with NAFLD in the Boramae cohort, the G allele was independently associated with a lower prevalence of DM in both NAFL (odds ratio [OR] per 1 allele, 0.66; 95% confidence interval [CI], 0.46-0.97) and nonalcoholic steatohepatitis (OR per 1 allele, 0.59; 95% CI, 0.38-0.92). This result was replicated in the longitudinal GENIE cohort. The G allele was associated with a lower risk of incident DM during the median follow-up of 60 months in subjects with NAFLD (hazard ratio, 0.65; 95% CI, 0.45-0.93). In contrast, G allele carriers without NAFLD showed higher odds for DM in the context of the Boramae cohort (OR, 2.44; 95% CI, 1.00-5.95). CONCLUSIONS: These findings clarify conflicting results regarding the association between the PNPLA3 rs738409 genotype and the risk of DM, demonstrating a clear difference between subjects with and without NAFLD; this difference might be explained by the low metabolic risk in genetic NAFLD.

Common genetic variation in alcohol-related hepatocellular carcinoma: a case-control genome-wide association study.

BACKGROUND: Hepatocellular carcinoma is a frequent consequence of alcohol-related liver disease, with variable incidence among heavy drinkers. We did a genome-wide association study (GWAS) to identify common genetic variants for alcohol-related hepatocellular carcinoma. METHODS: We conducted a two-stage case-control GWAS in a discovery cohort of 2107 unrelated European patients with alcohol-related liver disease aged 20-92 years recruited between Oct 22, 1993, and March 12, 2017. Cases were patients with alcohol-related hepatocellular carcinoma diagnosed by imaging or histology. Controls were patients with alcohol-related liver disease without hepatocellular carcinoma. We used an additive logistic regression model adjusted for the first ten principal components to assess genetic variants associated with alcohol-related hepatocellular carcinoma. We did another analysis with adjustment for age, sex, and liver fibrosis. New candidate associations (p<1 × 10-6) and variants previously associated with alcohol-related hepatocellular carcinoma were evaluated in a validation cohort of 1933 patients with alcohol-related liver disease aged 29-92 years recruited between July 21, 1995, and May 2, 2019. We did a meta-analysis of the two case-control cohorts. FINDINGS: The discovery cohort included 775 cases and 1332 controls. Of 7 962 325 variants assessed, we identified WNT3A-WNT9A (rs708113; p=1·11 × 10-8) and found support for previously reported regions associated with alcohol-related hepatocellular carcinoma risk at TM6SF2 (rs58542926; p=6·02 × 10-10), PNPLA3 (rs738409; p=9·29 × 10-7), and HSD17B13 (rs72613567; p=2·49 × 10-4). The validation cohort included 874 cases and 1059 controls and three variants were replicated: WNT3A-WNT9A (rs708113; p=1·17 × 10-3), TM6SF2 (rs58542926; p=4·06 × 10-5), and PNPLA3 (rs738409; p=1·17 × 10-4). All three variants reached GWAS significance in the meta-analysis: WNT3A-WNT9A (odds ratio 0·73, 95% CI 0·66-0·81; p=3·93 × 10-10), TM6SF2 (1·77, 1·52-2·07; p=3·84×10-13), PNPLA3 (1·34, 1·22-1·47; p=7·30 × 10-10). Adjustment for clinical covariates yielded similar results. We observed an additive effect of at-risk alleles on alcohol-related hepatocellular carcinoma. WNT3A-WNT9A rs708113 was not associated with liver fibrosis. INTERPRETATION: WNT3A-WNT9A is a susceptibility locus for alcohol-related hepatocellular carcinoma, suggesting an early role of the Wnt-ß-catenin pathway in alcohol-related hepatocellular carcinoma carcinogenesis. FUNDING: Ligue Nationale contre le Cancer, Bpifrance, INSERM, AFEF, CARPEM, Labex OncoImmunology, and Agence Nationale de la Recherche.

GWAS loci associated with Chagas cardiomyopathy influences DNA methylation levels.

A recent genome-wide association study (GWAS) identified a locus in chromosome 11 associated with the chronic cardiac form of Chagas disease. Here we aimed to elucidate the potential functional mechanism underlying this genetic association by analyzing the correlation among single nucleotide polymorphisms (SNPs) and DNA methylation (DNAm) levels as cis methylation quantitative trait loci (cis-mQTL) within this region. A total of 2,611 SNPs were tested against 2,647 DNAm sites, in a subset of 37 chronic Chagas cardiomyopathy patients and 20 asymptomatic individuals from the GWAS. We identified 6,958 significant cis-mQTLs (False Discovery Rate [FDR]<0.05) at 1 Mb each side of the GWAS leading variant, where six of them potentially modulate the expression of the SAC3D1 gene, the reported gene in the previous GWAS. In addition, a total of 268 cis-mQTLs showed differential methylation between chronic Chagas cardiomyopathy patients and asymptomatic individuals. The most significant cis-mQTLs mapped in the gene bodies of POLA2 (FDR = 1.04x10-11), PLAAT3 (FDR = 7.22x10-03), and CCDC88B (FDR = 1.89x10-02) that have been associated with cardiovascular and hematological traits in previous studies. One of the most relevant interactions correlated with hypermethylation of CCDC88B. This gene is involved in the inflammatory response, and its methylation and expression levels have been previously reported in Chagas cardiomyopathy. Our findings support the functional relevance of the previously associated genomic region, highlighting the regulation of novel genes that could play a role in the chronic cardiac form of the disease.

TGF-Β1 & PNPLA3 Genetic Variants and the Risk of Hepatic Fibrosis and HCC in Egyptian Patients with HCV-Related Liver Cirrhosis.

OBJECTIVE: The clinical outcomes of hepatitis C virus (HCV) infection and its sequelae including liver cirrhosis and hepatocellular carcinoma (HCC) are greatly affected by host genetic factors; however, the possible mechanisms are still largely unclear. This work aimed to assess transforming growth factor-ß1 (TGF-ß1), and patatin-like phospholipase domain containing-protein 3 (PNPLA3) genetic variants as risk factors for hepatic fibrosis and hepatocellular carcinoma (HCC) in Egyptian patients with HCV-related liver cirrhosis. METHODS: Seventy HCV-related liver cirrhosis patients (Total cirrhosis) who were divided into two groups; 34 patients with HCC (HCC group), and 36 patients without HCC (LC group) and 20 healthy volunteers (control group) were included. Routine laboratory investigations and imaging studies were determined. TGF-ß1 (Arg25Pro; 915G>C) and PNPLA3 (I148M; C>G) variants were evaluated using real-time polymerase chain reaction (real-time PCR). RESULTS: HCC group showed a significantly higher GG genotype distribution of TGF-ß1 (Arg25Pro) than the LC group (P= 0.008, OR: 7.083, CI 95%: 1.422 - 35.282). The distributions of GG genotype (P= 0.047) and G allele (P= 0.002, OR: 4.395, CI 95%: 1.622 - 11.911) of PNPLA3 (I148M) were significantly higher in total cirrhosis patients than controls. CONCLUSION: TGF-ß1 (Arg25Pro) GG variant may be associated with HCC risk in HCV-related liver cirrhosis patients, while PNPLA3 (I148M) GG variant may be associated with cirrhosis development but not HCC risk in HCV-related liver cirrhosis patients.

The Protection Conferred by HSD17B13 rs72613567 Polymorphism on Risk of Steatohepatitis and Fibrosis May Be Limited to Selected Subgroups of Patients With NAFLD.

INTRODUCTION: Our study aimed to explore how PNPLA3 rs738409 or phenotypic risk factors may moderate the relationship between HSD17B13 rs72613567 and risk of steatohepatitis and fibrosis. METHODS: This analysis consisted of 1,153 non-Hispanic whites with biopsy-proven nonalcoholic fatty liver disease enrolled in the nonalcoholic steatohepatitis Clinical Research Network studies. Nonalcoholic fatty liver disease severity was determined by liver histology scored centrally according to the nonalcoholic steatohepatitis Clinical Research Network criteria. Moderation and logistic regression analyses were performed to identify the influence of moderators (PNPLA3 rs738409, age, sex, body mass index, and diabetes) on the relationship between HSD17B13 rs72613567 and risk of steatohepatitis and fibrosis. RESULTS: HSD17B13 rs72613567 genotype frequency was as follows: (-/-), 64%; (-/A), 30%; (A/A), 6%. Moderation analysis showed that the protective effect of HSD17B13 rs72613567 A-allele on risk of steatohepatitis remained only significant among patients with PNPLA3 rs738409 genotype CC (ß coeff: -0.19, P = 0.019), women (ß coeff: -0.18, P < 0.001), patients of age ≥ 45 years (ß coeff: -0.18, P < 0.001), patients with body mass index ≥ 35 kg/m2 (ß coeff: -0.17, P < 0.001), and patients with diabetes (ß coeff: -0.18, P = 0.020). Among women, the protective effect of HSD17B131 rs72613567 A-allele on risk of steatohepatitis was stronger in those aged ≥ 51 years. Logistic regression-based sensitivity analysis including various important subgroups confirmed our observations. DISCUSSION: The protection conferred by HSD17B13 rs72613567 A-allele on risk of steatohepatitis and fibrosis may be limited to selected subgroups of individuals who are aged ≥ 45 years, women and have class ≥ 2 obesity or diabetes, and those with PNPLA3 rs738409 CC genotype.

Single nucleotide polymorphisms in PNPLA3, ADAR-1 and IFIH1 are associated with advanced liver fibrosis in patients co-infected with HIV-1//hepatitis C virus.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD), insulin resistance and liver fibrosis are prevalent in individuals co-infected with HIV type 1 (HIV-1)/hepatitis C virus (HCV), even after HCV eradication. Our aim was to evaluate single nucleotide polymorphisms (SNPs) associated with advanced liver fibrosis in HIV-1/HCV co-infected patients. DESIGN/METHODS: In a cohort of 102 participants, we genotyped 16 SNPs in 10 genes previously associated with NAFLD and the innate immune response and correlated the genotypes with liver fibrosis and fat accumulation. RESULTS: Multinomial logistic regression analysis identified three metabolic parameters that were significantly associated with advanced liver fibrosis (stage F3-F4): albumin [odds ratio (OR) 0.80, 95% confidence interval (CI) 0.69-0.91, P = 0.001], percentage of visceral fat area (PVFA) (OR 1.06, 95% CI 1.01-1.12, P = 0.03) and BMI (OR 1.47, 95% CI 1.22-1.77, P < 0.0001). After adjustment for sex, albumin, PVFA and BMI, we found that three SNPs were significantly associated with advanced fibrosis, one each in PNPLA3/rs738409 (P = 0.016), ADAR-1/rs1127313 (P = 0.029) and IFIH1/rs1990760 (P = 0.033). CONCLUSION: Our results indicate that genotyping for these SNPs can be a useful predictive tool for liver fibrosis progression and liver fat accumulation in patients co-infected with HIV-1/HCV.

Evidence of multiple hepatic mechanisms to mobilize docosahexaenoic acid into dam plasma during pregnancy in chow-fed sprague dawley rats.

Fetal brain growth requires considerable amounts of docosahexaenoic acid (DHA) during late pregnancy that is associated with increased maternal/dam plasma levels of PC 16:0_22:6 (palmitoyl docosahexaenoyl phosphatidylcholine). While biosynthesis of DHA during pregnancy is upregulated, the mechanisms responsible for the incorporation of dam DHA into PC 16:0_22:6 are not understood. The present study used a discovery approach combining untargeted lipidomics of plasma and liver (n = 3/group) with semi-targeted qPCR of hepatic gene products (n = 6/group) to identify metabolic pathways related to DHA metabolism, with a hypothesis that an upregulated acyltransferase involved in PC remodeling would be identified. Sprague Dawley rats were fed a commercial rodent chow throughout the study and samples were collected before pregnancy (baseline), at 15 and 20 days of pregnancy, and 7 days postpartum. Plasma and hepatic PC 16:0_22:6 was significantly increased (by 79% and 194%, respectively) at day 20 of pregnancy. An increase in hepatic DG (diacylglycerol) 16:0_22:6 (by 243%) and significant decreases in Pla2G15 (0.4-fold) and Pla2G16 (0.6-fold) at day 20 of pregnancy, no changes in Lpcat1-4, and an abundant pool of hepatic pool PE (phosphatidylethanolamine) 16:0_22:6 suggest that plasma PC 16:0_22:6 is not being produced by fatty acyl remodeling during pregnancy. The increase in plasma PC 16:0_22:6 during pregnancy appears to be due to an increase in de novo synthesis of PC and both the CDP-choline and phosphatidylcholine methyltransferase pathways are implicated. There was also evidence suggesting channeling of DHA into PC and lipoprotein assembly may be occurring. Targeted research is necessary to confirm these findings, but the results of this study indicate metabolic adaptions to enable maternal/dam resiliency towards meeting the fetal/pup demand for DHA during pregnancy.