Genome-wide interaction study with body mass index identifies CYP7A1 and GIPR as genetic modulators of metabolic dysfunction-associated steatotic liver disease

Abstract

Background/aims: Metabolic dysfunction-associated steatotic liver disease (MASLD) may progress to liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma. So far, genome-wide association studies explain a small fraction of MASLD heritability.

Methods: We sought to identify novel genetic determinants of MASLD by exploring interactions between genetic variants and body mass index (BMI). First, we examined genome-wide interactions with BMI for circulating alanine aminotransferase (ALT) levels using UK Biobank data. For identified loci, we next examined associations with hepatic proton density fat fraction (PDFF) in 35,146 independent UK Biobank participants. Associations with PDFF were replicated in four independent European cohorts, followed by a phenome-wide association study. Finally, we used human liver epigenomic maps and CRISPR/Cas9 experiments in vitro and in vivo to functionally characterize the CYP7A1 locus.

Results: Thirteen loci interact with BMI for ALT (P<5E-8), including eight well-known genetic modulators of MASLD. Two loci-UBXN2B/CYP7A1 and GIPR-are additionally associated with PDFF. For the intronic rs34783010 in GIPR, the minor T allele is associated with lower BMI and higher HbA1c and liver triglyceride content in humans. The UBXN2B/CYP7A1 locus is associated with PDFF in four additional European cohorts. Epigenomic data and in vitro experiments in human liver cells prioritise rs10504255 and CYP7A1 as the functional effectors in this locus. Perturbation of CYP7A1 orthologues using CRISPR/Cas9 results in less liver fat in 10-day-old, metabolically challenged zebrafish larvae.

Conclusion: A genome-wide single nucleotide polymorphism×BMI design fuelled identification of two MASLD genes: CYP7A1 and GIPR.

Keywords: CYP7A1; Gastric inhibitory polypeptide receptor; Gene-environment interaction; Genome-wide association study; MASLD.

Figure 1.

13 loci interact with BMI for ALT. Top: Manhattan plot of genome-wide interaction analysis with BMI for ALT in European ancestry participants (UK Biobank). P-values were calculated by using a whole-genome regression model in REGENIE. Red dashed line represents the genome-wide significance level, 5E-8. Bottom: association of ALT (left) and PDFF (right) with 13 loci that interact with BMI for ALT stratified by BMI. Associations were examined using linear regression analysis adjusted for BMI, age, sex, age×sex, age² and age²×sex, first 10 genomic principal components and array batch. For ALT, the association analyses have been performed after excluding individuals with PDFF data. The x-axis represents beta coefficients, with error bars showing 95% confidence intervals. Greyed-out circles represent associations with a nominal P-value>0.05. The SNP-BMI interaction P-values are displayed as a secondary y-axis on the right-hand side of each panel. ALT, alanine aminotransferase; BMI, body mass index; PDFF, proton density fat fraction; SNP, single nucleotide polymorphism.

Figure 2.

Regional plots for association of 3 new loci with PDFF. For each locus, a window of ±200 kb around lead variants from GEWIS on ALT was considered. The lead variants from the GEWIS for ALT and GWAS on PDFF are marked with a square and black diamond, respectively. Credible set indicates putative causal variants from the GEWIS for ALT. Red dashed line represents the FDR threshold using the Benjamini-Hochberg method. Data from a total of 337,000 unrelated White-British participants from UK Biobank were used for insample LD structure. ALT, alanine aminotransferase; GEWIS, genome-wide-environment interaction study; GWAS, genome-wide association study; PDFF, proton density fat fraction; FDR, false discovery rate; LD, linkage disequilibrium.

Figure 3.

Forest plot for association of controlled attenuation parameter and magnetic resonance spectroscopy hepatic fat content with the UBXN2B/CYP7A1 rs7826120 T allele in four European replication cohorts. The association was examined by a linear regression analysis under an additive genetic model adjusted for age, sex, and BMI. Pooled effect estimates were calculated using inverse-variance–weighted fixed- and random-effects meta-analysis. I², τ² (between-study variance) and P-value for Cochran’s Q heterogeneity test have been reported to assess the betweenstudy heterogeneity. BMI, body mass index; CI, confidence interval; MAFALDA, Molecular Architecture of FAtty Liver Disease in patients with obesity undergoing bariatric surgery; NEO, the Netherlands Epidemiology of Obesity Study.

Figure 4.

The association of a set of metabolic biomarkers and relevant diseases with the GIPR rs34783010 T allele in the UK Biobank. The association was examined by a linear or logistic regression analysis under an additive genetic model adjusted for BMI, age, sex, age×sex, age² and age²×sex, the first 10 genomic principal components, and array batch. For binary traits, log odds of effects are shown. The colour represents the –log₁₀-transformed P-values. BMI, body mass index; LDL, low-density lipoprotein.

Figure 5.

The association of relevant traits with the rs7826120 T allele (UK Biobank). (A) Associations were examined by additive linear or logistic regression analyses adjusted for BMI, age, sex, age×sex, age² and age²×sex, first 10 genomic principal components and array batch. For binary traits, log odds of effects are shown. The colour represents the –log₁₀-transformed P-values. (B) Overview of the metabolic- dysfunction associated steatotic liver disease (MASLD) locus UBXN2B/CYP7A1. Of all MASLD variants in the fine-mapped credible set at this locus, only rs10504255 (red circle) resides within an active liver cis-regulatory element (CRE). The lead variant (purple diamond) resides adjacent to an open chromatin region not enriched for the active CRE histone mark H3K27ac. Tracks show pooled normalised signal for H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) and chromatin accessibility detected by ATAC-seq across 4–6 independent samples. (C) MotifbreakR results showing position weight matrices (PWMs) of transcription factor (TF) motifs predicted to be affected by the variant rs10504255. PWMs matching the G allele, associated with higher proton density fat fraction, are shown at the top, and those matching the A allele are shown at the bottom. TFs with P<1E-3 were prioritised as likely hits if they were expressed in both liver tissue and hepatocytes and are shown ranked top to bottom for each allele by significance. *Indicates TFs for which there is ChIP-seq evidence of binding at the enhancer element. (D) Schematic (left) of CRISPRa complex targeting the enhancer at the UBXN2B/CYP7A1 locus. Bar plot (right) of relative expression detected by RT-qPCR of CYP7A1 and UBXN2B upon CRISPRa targeting of the enhancer vs. negative control guides. Data are presented as the mean±standard deviation of biological replicates (n=3 independent transductions) and statistically analysed by Student’s t-test.

Figure 6.

The effect of CRISPR/Cas9-induced mutations in both orthologues of CYP7A1 on liver area and liver fat area in 10-dayold zebrafish larvae stratified by dietary condition. 4%EC, diet enriched with 4% extra cholesterol; CD, control diet; OF, overfeeding (3× more); SD, standard deviation. Orange, larvae carrying CRISPR/Cas9-induced mutations in both CYP7A1 orthologues; grey, sibling controls free from such mutations.