Genome-wide association analysis identifies variants associated with nonalcoholic fatty liver disease that have distinct effects on metabolic traits

Abstract

Nonalcoholic fatty liver disease (NAFLD) clusters in families, but the only known common genetic variants influencing risk are near PNPLA3. We sought to identify additional genetic variants influencing NAFLD using genome-wide association (GWA) analysis of computed tomography (CT) measured hepatic steatosis, a non-invasive measure of NAFLD, in large population based samples. Using variance components methods, we show that CT hepatic steatosis is heritable (∼26%-27%) in family-based Amish, Family Heart, and Framingham Heart Studies (n = 880 to 3,070). By carrying out a fixed-effects meta-analysis of genome-wide association (GWA) results between CT hepatic steatosis and ∼2.4 million imputed or genotyped SNPs in 7,176 individuals from the Old Order Amish, Age, Gene/Environment Susceptibility-Reykjavik study (AGES), Family Heart, and Framingham Heart Studies, we identify variants associated at genome-wide significant levels (p<5×10(-8)) in or near PNPLA3, NCAN, and PPP1R3B. We genotype these and 42 other top CT hepatic steatosis-associated SNPs in 592 subjects with biopsy-proven NAFLD from the NASH Clinical Research Network (NASH CRN). In comparisons with 1,405 healthy controls from the Myocardial Genetics Consortium (MIGen), we observe significant associations with histologic NAFLD at variants in or near NCAN, GCKR, LYPLAL1, and PNPLA3, but not PPP1R3B. Variants at these five loci exhibit distinct patterns of association with serum lipids, as well as glycemic and anthropometric traits. We identify common genetic variants influencing CT-assessed steatosis and risk of NAFLD. Hepatic steatosis associated variants are not uniformly associated with NASH/fibrosis or result in abnormalities in serum lipids or glycemic and anthropometric traits, suggesting genetic heterogeneity in the pathways influencing these traits.

Figure 1. Study design.

Meta-analysis of genome-wide association data was performed in Stage 1 across the cohorts shown. SNPs representing the best associating loci were genotyped in histology based NAFLD samples (Stage 2) from the NASH CRN matched to genome wide genotyped and imputed MIGen controls. The effects of the five NAFLD associated SNPs on NASH CRN/iCONT, metabolic phenotypes and eQTLs in liver and adipose tissue were then performed (Stage 3).

Figure 2. Genome-wide association results for GOLD (Stage 1).

A. Manhattan plot showing the significance of association of all SNPs in the Stage 1 GOLD meta-analysis with CT hepatic steatosis. SNPs are plotted on the x-axis according to their position on each chromosome against association with CT hepatic steatosis on the y-axis (shown as -log10 p-value). SNPs that also associate with histology based NAFLD are in red, those that only associate with CT hepatic steatosis in blue. B. Quantile-quantile plot of SNPs after Stage 1 GOLD meta-analysis (black) and after removing any SNPs within 500 kb of PNPLA3, PPP1R3B, and NCAN (red).

Figure 3. Regional plots of genome-wide significant or replicating loci of association in GOLD.

SNPs are plotted by position on chromosome against association with CT hepatic steatosis (–log10 p-value). The figures highlight the SNP taken into Stage 2 (diamond). The SNPs surrounding the most significant SNP are color-coded to reflect their LD with this SNP as in the inset (taken from pairwise R2 values from the HapMap CEU database, www.hapmap.org). Estimated recombination rates (from HapMap) are plotted in cyan to reflect the local LD structure. Genes and the direction of transcription, are noted below the plots (data from UCSC genome browser, genome.ucsc.edu). Coding SNPs in high LD with the best SNP are noted with rs number and protein change.

Figure 4. Effects on traits.

Direction of effect on CT fatty liver, histology NAFLD, lipid and glycemic traits of the best associating SNPs at the loci shown. Direction is shown only for significant associations. CT: CT hepatic steatosis; LDL: low density lipoprotein cholesterol; HDL: high density lipoprotein cholesterol; TG: triglycerides; HOMA-IR: homeostatic model of insulin resistance; PNPLA3: patatin-like phospholipase domain-containing protein 3 (HGNC: 18590); NCAN: neurocan (HGNC: 2465); LYPLAL1: lysophospholipase-like 1 (HGNC: 20440); GCKR: glucokinase regulatory protein (HGNC: 4196); PPP1R3B: protein phosphatase 1, regulatory subunit 3b (HGNC: 14942).