Marica Meroni

The MBOAT7-TMC4 Variant rs641738 Increases Risk of Nonalcoholic Fatty Liver Disease in Individuals of European Descent.

BACKGROUND & AIMS Nonalcoholic fatty liver disease (NAFLD) is a leading cause of liver damage and is characterized by steatosis. Genetic factors increase risk for progressive NAFLD. A genome-wide association study showed that the rs641738 C>T variant in the locus that contains the membrane bound O-acyltransferase domain-containing 7 gene (MBOAT7, also called LPIAT1) and transmembrane channel-like 4 gene (TMC4) increased the risk for cirrhosis in alcohol abusers. We investigated whether the MBOAT7-TMC4 is a susceptibility locus for the development and progression of NAFLD. METHODS We genotyped rs641738 in DNA collected from 3854 participants from the Dallas Heart Study (a multi-ethnic population-based probability sample of Dallas County residents) and 1149 European individuals from the Liver Biopsy Cross-Sectional Cohort. Clinical and anthropometric data were collected, and biochemical and lipidomics were measured in plasma samples from participants. A total of 2736 participants from the Dallas Heart Study also underwent proton magnetic resonance spectroscopy to measure hepatic triglyceride content. In the Liver Biopsy Cross-Sectional Cohort, a total of 1149 individuals underwent liver biopsy to diagnose liver disease and disease severity. RESULTS The genotype rs641738 at the MBOAT7-TMC4 locus associated with increased hepatic fat content in the 2 cohorts, and with more severe liver damage and increased risk of fibrosis compared with subjects without the variant. MBOAT7, but not TMC4, was found to be highly expressed in the liver. The MBOAT7 rs641738 T allele was associated with lower protein expression in the liver and changes in plasma phosphatidylinositol species consistent with decreased MBOAT7 function. CONCLUSIONS We provide evidence for an association between the MBOAT7 rs641738 variant and the development and severity of NAFLD in individuals of European descent. This association seems to be mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling.

The rs2294918 E434K variant modulates patatin-like phospholipase domain-containing 3 expression and liver damage.

The patatin‐like phosholipase domain‐containing 3 (PNPLA3) rs738409 polymorphism (I148M) is a major determinant of hepatic fat and predisposes to the full spectrum of liver damage in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate whether additional PNPLA3 coding variants contribute to NAFLD susceptibility, first in individuals with contrasting phenotypes (with early‐onset NAFLD vs. very low aminotransferases) and then in a large validation cohort. Rare PNPLA3 variants were not detected by sequencing coding regions and intron‐exon boundaries either in 142 patients with early‐onset NAFLD nor in 100 healthy individuals with alanine aminotransferase <22/20 IU/mL. Besides rs738409 I148M, the rs2294918 G>A polymorphism (E434K sequence variant) was over‐represented in NAFLD (adjusted P = 0.01). In 1,447 subjects with and without NAFLD, the 148M‐434E (P < 0.0001), but not the 148M‐434K, haplotype (P > 0.9), was associated with histological NAFLD and steatohepatitis. Both the I148M (P = 0.0002) and E434K variants (P = 0.044) were associated with serum ALT levels, by interacting with each other, in that the 434K hampered the association with liver damage of the 148M allele (P = 0.006). The E434K variant did not affect PNPLA3 enzymatic activity, but carriers of the rs2294918 A allele (434K) displayed lower hepatic PNPLA3 messenger RNA and protein levels (P < 0.05). Conclusions: Rare loss‐of‐function PNPLA3 variants were not detected in early‐onset NAFLD. However, PNPLA3 rs2294918 E434K decreased PNPLA3 expression, lessening the effect of the I148M variant on the predisposition to steatosis and liver damage. This suggests that the PNPLA3 I148M variant has a codominant negative effect on triglycerides mobilization from lipid droplets, mediated by inhibition of other lipases. (Hepatology 2016;63:787–798)

Liver fat accumulation is associated with circulating PCSK9

Abstract Background: Nonalcoholic fatty liver disease (NAFLD) associates with cardiovascular disease independently of classic risk factors. Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) is secreted by hepatocytes and inhibits the uptake of low-density lipoproteins by targeting the receptor for degradation, and possibly lipogenesis. PCSK9 loss-of-function mutations and anti-PCKS9 drugs reduce LDL-cholesterol. Aim: To evaluate whether hepatic fat content is associated with circulating PCSK9. Materials and methods: In 201 consecutive patients biopsied for suspected nonalcoholic steatohepatitis, liver damage was quantified by NAFLD activity score, circulating PCSK9 by ELISA, and hepatic mRNA by qRT-PCR in a subset (n = 76). Results: Circulating PCSK9 was associated with steatosis grade (p = 0.0011), necroinflammation (p < 0.001), ballooning (p = 0.005), and fibrosis stage (p = 0.001). At multivariate analysis, PCSK9 was associated with steatosis grade (p = 0.012), older age and lower BMI, independently of sex, hyperglycemia, and fibrosis/inflammation. Circulating PCSK9 was associated with hepatic expression of SREBP-1c (p = 0.0002) and FAS (p = 0.03). PCSK9 mRNA levels were also correlated with steatosis severity (p = 0.04) and hepatic APOB (p < 0.001), SREBP-1c (p = 0.047) and FAS expression (p = 0.001). Conclusions: Circulating PCSK9 increases with hepatic fat accumulation and correlates with the severity of steatosis, independently of metabolic confounders and liver damage. Modulation of PCSK9 synthesis and release might be involved in NAFLD pathogenesis. Key Messages Circulating PCSK9 levels increase with hepatic fat accumulation. Circulating PCSK9 levels are associated with increased de novo lipogenesis. Hepatic PCSK9 expression is associated with steatosis severity and activation of lipogenesis.

PNPLA3 overexpression results in reduction of proteins predisposing to fibrosis

Abstract Liver fibrosis is a pathological scarring response to chronic hepatocellular injury and hepatic stellate cells (HSCs) are key players in this process. PNPLA3 I148M is a common variant robustly associated with liver fibrosis but the mechanisms underlying this association are unknown. We aimed to examine a) the effect of fibrogenic and proliferative stimuli on PNPLA3 levels in HSCs and b) the role of wild type and mutant PNPLA3 overexpression on markers of HSC activation and fibrosis. Here, we show that PNPLA3 is upregulated by the fibrogenic cytokine transforming growth factor-beta (TGF-β), but not by platelet-derived growth factor (PDGF), and is involved in the TGF-β-induced reduction in lipid droplets in primary human HSCs. Furthermore, we show that retinol release from human HSCs ex vivo is lower in cells with the loss-of-function PNPLA3 148M compared with 148I wild type protein. Stable overexpression of PNPLA3 148I wild type, but not 148M mutant, in human HSCs (LX-2 cells) induces a reduction in the secretion of matrix metallopeptidase 2 (MMP2), tissue inhibitor of metalloproteinase 1 and 2 (TIMP1 and TIMP2), which is mediated by retinoid metabolism. In conclusion, we show a role for PNPLA3 in HSC activation in response to fibrogenic stimuli. Moreover, we provide evidence to indicate that PNPLA3-mediated retinol release may protect against liver fibrosis by inducing a specific signature of proteins involved in extracellular matrix remodelling.

MBOAT7 rs641738 variant and hepatocellular carcinoma in non-cirrhotic individuals

Nonalcoholic fatty liver disease (NAFLD) represents an emerging cause of hepatocellular carcinoma (HCC), especially in non-cirrhotic individuals. The rs641738 C > T MBOAT7/TMC4 variant predisposes to progressive NAFLD, but the impact on hepatic carcinogenesis is unknown. In Italian NAFLD patients, the rs641738 T allele was associated with NAFLD-HCC (OR 1.65, 1.08–2.55; n = 765), particularly in those without advanced fibrosis (p < 0.001). The risk T allele was linked to 3’-UTR variation in MBOAT7 and to reduced MBOAT7 expression in patients without severe fibrosis. The number of PNPLA3, TM6SF2, and MBOAT7 risk variants was associated with NAFLD-HCC independently of clinical factors (p < 0.001), but did not significantly improve their predictive accuracy. When combining data from an independent UK NAFLD cohort, in the overall cohort of non-cirrhotic patients (n = 913, 41 with HCC) the T allele remained associated with HCC (OR 2.10, 1.33–3.31). Finally, in a combined cohort of non-cirrhotic patients with chronic hepatitis C or alcoholic liver disease (n = 1121), the T allele was independently associated with HCC risk (OR 1.93, 1.07–3.58). In conclusion, the MBOAT7 rs641738 T allele is associated with reduced MBOAT7 expression and may predispose to HCC in patients without cirrhosis, suggesting it should be evaluated in future prospective studies aimed at stratifying NAFLD-HCC risk.

The role of insulin resistance in nonalcoholic steatohepatitis and liver disease development – a potential therapeutic target?

ABSTRACT Insulin resistance (IR) is defined by the inability of insulin to exert its metabolic actions, due to impaired activation of intracellular insulin signaling. This condition is caused by genetic defects or by environmental conditions, among which the most common is obesity. Systemic IR determines the development of hepatic fat accumulation, which can progress to nonalcoholic steatohepatitis, cirrhosis and hepatocellular carcinoma, and is a major determinant of liver disease independently of coexisting factors. Therefore, insulin-sensitizing drugs are currently under evaluation to improve steatohepatitis. Indeed, manipulation of nuclear hormone receptors is already under scrutiny for liver disease prevention by amelioration of IR, whereas NOTCH signaling inhibition represents a novel approach. Nevertheless, further research is warranted to better understand the mechanism linking IR to progressive fibrogenesis in the absence of inflammation and to identify novel drug targets.

The rs2294918 E434K variant modulates PNPLA3 expression and liver damage

The patatin‐like phosholipase domain‐containing 3 (PNPLA3) rs738409 polymorphism (I148M) is a major determinant of hepatic fat and predisposes to the full spectrum of liver damage in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate whether additional PNPLA3 coding variants contribute to NAFLD susceptibility, first in individuals with contrasting phenotypes (with early‐onset NAFLD vs. very low aminotransferases) and then in a large validation cohort. Rare PNPLA3 variants were not detected by sequencing coding regions and intron‐exon boundaries either in 142 patients with early‐onset NAFLD nor in 100 healthy individuals with alanine aminotransferase <22/20 IU/mL. Besides rs738409 I148M, the rs2294918 G>A polymorphism (E434K sequence variant) was over‐represented in NAFLD (adjusted P = 0.01). In 1,447 subjects with and without NAFLD, the 148M‐434E (P < 0.0001), but not the 148M‐434K, haplotype (P > 0.9), was associated with histological NAFLD and steatohepatitis. Both the I148M (P = 0.0002) and E434K variants (P = 0.044) were associated with serum ALT levels, by interacting with each other, in that the 434K hampered the association with liver damage of the 148M allele (P = 0.006). The E434K variant did not affect PNPLA3 enzymatic activity, but carriers of the rs2294918 A allele (434K) displayed lower hepatic PNPLA3 messenger RNA and protein levels (P < 0.05). Conclusions: Rare loss‐of‐function PNPLA3 variants were not detected in early‐onset NAFLD. However, PNPLA3 rs2294918 E434K decreased PNPLA3 expression, lessening the effect of the I148M variant on the predisposition to steatosis and liver damage. This suggests that the PNPLA3 I148M variant has a codominant negative effect on triglycerides mobilization from lipid droplets, mediated by inhibition of other lipases. (Hepatology 2016;63:787–798)

Notch signaling and progenitor/ductular reaction in steatohepatitis

Background and objective Persistent hepatic progenitor cells (HPC) activation resulting in ductular reaction (DR) is responsible for pathologic liver repair in cholangiopathies. Also, HPC/DR expansion correlates with fibrosis in several chronic liver diseases, including steatohepatitis. Increasing evidence indicates Notch signaling as a key regulator of HPC/DR response in biliary and more in general liver injuries. Therefore, we aimed to investigate the role of Notch during HPC/DR activation in a mouse model of steatohepatitis. Methods Steatohepatitis was generated using methionine-choline deficient (MCD) diet. For hepatocyte lineage tracing, R26R-YFP mice were infected with AAV8-TBG-Cre. Results MCD diet promoted a strong HPC/DR response that progressively diffused in the lobule, and correlated with increased fibrosis and TGF-β1 expression. Notch signaling was unchanged in laser-capture microdissected HPC/DR, whereas Notch receptors were down regulated in hepatocytes. However, in-vivo lineage tracing experiments identified discrete hepatocytes showing Notch-1 activation and expressing (the Notch-dependent) Sox9. Stimulation of AML-12 hepatocyte-cell line with immobilized Jag1 induced Sox9 and down-regulated albumin and BSEP expression. TGF-β1 treatment in primary hepatic stellate cells (HSC) induced Jag1 expression. In MCD diet-fed mice, αSMA-positive HSC were localized around Sox9 expressing hepatocytes, suggesting that Notch activation in hepatocytes was promoted by TGF-β1 stimulated HSC. In-vivo Notch inhibition reduced HPC response and fibrosis progression. Conclusion Our data suggest that Notch signaling is an important regulator of DR and that in steatohepatitis, hepatocytes exposed to Jag1-positive HSC, contribute to pathologic DR by undergoing Notch-mediated differentiation towards an HPC-like phenotype. Given the roles of Notch in fibrosis and liver cancer, these data suggest mesenchymal expression of Jag1 as an alternative therapeutic target.

Protein phosphatase 1 regulatory subunit 3B gene variation protects against hepatic fat accumulation and fibrosis in individuals at high risk of nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver damage and has a strong genetic component. The rs4841132 G>A variant, modulating the expression of protein phosphatase 1 regulatory subunit 3B (PPP1R3B), which is involved in glycogen synthesis, has been reported to reduce the risk of NAFLD but at the same time may favor liver disease by facilitating glycogen accumulation. The aim of this study was to assess the impact of rs4841132 on development of histologic steatosis and fibrosis in 1,388 European individuals in a liver biopsy cohort, on NAFLD hepatocellular carcinoma in a cross‐sectional Italian cohort (n = 132 cases), and on liver disease at the population level in the United Kingdom Biobank cohort. We investigated the underlying mechanism by examining the impact of the variant on gene expression profiles. In the liver biopsy cohort, the rs4841132 minor A allele was associated with protection against steatosis (odds ratio [OR], 0.63; 95% confidence interval [CI], 0.42‐0.95; P = 0.03) and clinically significant fibrosis (OR, 0.35; 95% CI, 0.14‐0.87; P = 0.02) and with reduced circulating cholesterol (P = 0.02). This translated into protection against hepatocellular carcinoma development (OR, 0.22; 95% CI, 0.07‐0.70; P = 0.01). At the population level, the rs4841132 variation was not associated with nonalcoholic or nonviral diseases of the liver but was associated with lower cholesterol (P = 1.7 × 10–8). In individuals with obesity, the A allele protecting against steatosis was associated with increased PPP1R3B messenger RNA expression and activation of lipid oxidation and with down‐regulation of pathways related to lipid metabolism, inflammation, and cell cycle. Conclusion: The rs4841132 A allele is associated with protection against hepatic steatosis and fibrosis in individuals at high risk of NAFLD but not in the general population and against dyslipidemia. The mechanism may be related to modulation of PPP1R3B expression and hepatic lipid metabolism. (Hepatology Communications 2018;2:666‐675)

The rs2294918 E434K variant modulates patatin-like phospholipase domain-containing 3 expression and liver damage: Donati et al.

The patatin‐like phosholipase domain‐containing 3 (PNPLA3) rs738409 polymorphism (I148M) is a major determinant of hepatic fat and predisposes to the full spectrum of liver damage in nonalcoholic fatty liver disease (NAFLD). The aim of this study was to evaluate whether additional PNPLA3 coding variants contribute to NAFLD susceptibility, first in individuals with contrasting phenotypes (with early‐onset NAFLD vs. very low aminotransferases) and then in a large validation cohort. Rare PNPLA3 variants were not detected by sequencing coding regions and intron‐exon boundaries either in 142 patients with early‐onset NAFLD nor in 100 healthy individuals with alanine aminotransferase <22/20 IU/mL. Besides rs738409 I148M, the rs2294918 G>A polymorphism (E434K sequence variant) was over‐represented in NAFLD (adjusted P = 0.01). In 1,447 subjects with and without NAFLD, the 148M‐434E (P < 0.0001), but not the 148M‐434K, haplotype (P > 0.9), was associated with histological NAFLD and steatohepatitis. Both the I148M (P = 0.0002) and E434K variants (P = 0.044) were associated with serum ALT levels, by interacting with each other, in that the 434K hampered the association with liver damage of the 148M allele (P = 0.006). The E434K variant did not affect PNPLA3 enzymatic activity, but carriers of the rs2294918 A allele (434K) displayed lower hepatic PNPLA3 messenger RNA and protein levels (P < 0.05). Conclusions: Rare loss‐of‐function PNPLA3 variants were not detected in early‐onset NAFLD. However, PNPLA3 rs2294918 E434K decreased PNPLA3 expression, lessening the effect of the I148M variant on the predisposition to steatosis and liver damage. This suggests that the PNPLA3 I148M variant has a codominant negative effect on triglycerides mobilization from lipid droplets, mediated by inhibition of other lipases. (Hepatology 2016;63:787–798)