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

Abstract

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.

Keywords: Arachidonic Acid; NASH; PNPLA3; TM6SF2.

Fig.1

The MBOAT7/TMC4 rs641738 variant is associated with increased hepatic triglyceride content in the Dallas Heart Study. The association was tested by linear regression analysis adjusted for age, gender, ethnicity, BMI, IFG/T2DM, PNPLA3 I148M and TM6SF2 E167K genotype. Data are shown as median value. Numbers in brackets represent interquartile range. Genetic analyses were performed by using an additive model. P-value represents the significance of a linear trend in median trait value among genotypes.

Fig.2

In the Liver Biopsy cross-sectional Cohort, the MBOAT7/TMC4 rs641738 variant is associated with the severity of histological damage. The histological damage was evaluated by the different components of NAFLD activity score (NAS) and with hepatic fibrosis stage. CC: homozygotes for the C allele; CT: heterozygotes; TT: homozygotes for the T allele. The association between the MBOAT7/TMC4 rs641738 variant and the components of the NAS has been tested by multivariate ordinal regression analysis adjusted for age, gender, BMI, presence of IFG/T2DM, number of PNPLA3 I148M alleles, presence of the TM6SF2 E167K variant, and indication of liver biopsy (severe obesity vs. nonalcoholic fatty liver with increased liver enzymes). Genetic analyses were calculated by using an additive model, except for TM6SF2 where a dominant model was used due to few homozygotes for the 167K mutant allele. P-values represent the significance of a trend in the prevalence of more severe degree of histological damage among genotypes.

Fig.3

MBOAT7 but not TMC4 is highly expressed in human liver and the rs641738 variant is associated with lower circulating levels of Phosphatidylinositol 36:4/34:2 ratio. (A) Distribution of MBOAT7 and TMC4 mRNA in 14 human tissues. (B) MBOAT7 and TMC4 mRNA expression in human primary hepatic cell types. Gene expression level was assessed by qPCR. The tissue or the cell line with the highest CT value was assigned the value of 1. (C) Ratio of phosphatidylinositol species containing different degree of saturation stratified by the rs643718 C>T genotype in the combined Dallas Heart Study (DHS) cohort and stratified by the three ethnic groups composing the study. Abbreviations: MBOAT7, Membrane Bound O-Acyltransferase domain containing 7; TMC4, Transmembrane Channel-like 4; HH, Human Hepatocytes; HSC, Hepatic Stellate Cells; HHSEC, Human Hepatic Sinusoidal Endothelial Cell.

Fig.4

Endogenous MBOAT7 is attached to the ER, MAM, LD and MBOAT7/TMC4 rs641738 variant decreases MBOAT7 mRNA expression and synthesis level in liver biopsies of obese patients. (A) Post-nuclear supernatant (PNS), cytosol and endoplasmic reticulum (ER) were obtained by a differential centrifugation. (B) Crude mitochondria were used to isolate purified mitochondria and mitochondria-associated membranes (MAM) by Percoll density gradient. (C) Post-nuclear supernatant was used to isolate lipid droplets (LD) by sucrose gradient. (D) Hepatic mRNA levels of MBOAT7 and TMC4 in 98 severely obese stratified by the rs643718 C>T genotype (CC n=29; CT n=51; TT n=18). Data are expressed as fold increase as compared to the protective CC genotype. p=0.03 for the effect of the rs641738 T allele on MBOAT7 expression (additive model). Demographic, anthropometric and clinical characteristics of these patients are shown in supplementary Table 7. (E) Western blotting analysis and quantification of MBOAT7 protein levels in obese patients carrying the three MBOAT7/TMC4 different genotypes, of whom hepatic gene expression was available (Supplemental Table 7). Seven samples for each rs641738 genotype (CC/CT/TT) were collected from liver biopsies and pooled according to genotype. All reactions (25 μg of lysates) were performed in triplicate in the same gel. Data are expressed as fold increase of the three MBOAT7 isoforms 1-3 (molecular weight: 52 KDa, 44 KDa and 38 KDa), as compared to the protective CC genotype. p<0.005 (additive model; it represents the difference in the trait examined across MBOAT7 genotypes). Abbreviations: CNX: calnexin; COX IV, cytochrome c oxidase, Complex IV; FACL4, long-chain acyl-CoA synthetase; ADFP, Adipose differentiation-related protein; IN, Infranatant, MBOAT7, Membrane Bound O-Acyltransferase domain containing 7; TMC4, Transmembrane Channel-like 4.