Whole-Exome Sequencing Identifies a Variant in Phosphatidylethanolamine N-Methyltransferase Gene to be Associated With Lean-Nonalcoholic Fatty Liver Disease

Govardhan Bale; Ravikanth V Vishnubhotla; Sasikala Mitnala; Mithun Sharma; Rao N Padaki; Smita C Pawar; Reddy N Duvvur

doi:10.1016/j.jceh.2019.02.001

Whole-Exome Sequencing Identifies a Variant in Phosphatidylethanolamine N-Methyltransferase Gene to be Associated With Lean-Nonalcoholic Fatty Liver Disease

J Clin Exp Hepatol. 2019 Sep-Oct;9(5):561-568. doi: 10.1016/j.jceh.2019.02.001. Epub 2019 Feb 11.

Authors

Govardhan Bale¹, Ravikanth V Vishnubhotla¹, Sasikala Mitnala¹, Mithun Sharma², Rao N Padaki², Smita C Pawar³, Reddy N Duvvur²

Affiliations

¹ Institute of Basic Sciences and Translational Research, Asian Healthcare Foundation, Hyderabad 500082, Telangana, India.
² Department of Medical Gastroenterology, Asian Institute of Gastroenterology, Hyderabad 500082, Telangana, India.
³ Department of Genetics, Osmania University, Hyderabad, Telangana, India.

Abstract

Background and aim: Nonalcoholic fatty liver disease (NAFLD) is a spectrum of liver diseases with simple steatosis on one end and hepatocellular carcinoma on the other. Although obesity is a known risk factor for NAFLD, individuals with normal body mass index (BMI) also have hepatic fatty infiltration, now termed "lean-NAFLD". It represents a distinct entity with a strong underlying genetic component. The present study aimed to sequence the complete exonic regions of individuals with lean-NAFLD to identify germline causative variants associated with disrupted hepatic fatty acid metabolism, thereby conferring susceptibility to NAFLD.

Methods: Whole blood was collected from patients with lean-NAFLD (n = 6; BMI < 23.0 kg/m²) and matched lean controls (n = 2; discovery set). Liver fat was assessed using acoustic radiation force impulse (ARFI) imaging. Patients with ultrasound-detected NAFLD (n = 191) and controls (n = 105) were part of validation set. DNA was isolated, and whole-exome sequencing (WES) was performed in the discovery cohort (Ion Proton™; Ion AmpliSeq™ Exome RDY Kit). Data were analyzed (Ion Reporter software; Life Technologies), and variants identified. Validation of variants was carried out (Taqman probes; Real time-PCR). Student's t test and Fisher's exact test were used to analyze the statistical significance.

Results: Although WES identified ∼74,000 variants in individual samples, using various pipelines. variants in genes namely phosphatidylethanolamine N-methyltransferase (PEMT) and oxysterol-binding protein-related protein10 (OSBPL10) that have roles in dietary choline intake and regulation of cholesterol homeostasis, respectively, were identified (discovery set). Furthermore, significant differences were noted in BMI (p = 0.006), waist/hip circumference (p > 0.001), waist/hip ratio (p > 0.001), aspartate aminotransferase (p > 0.001), alanine aminotransferase (p > 0.001), and triglycerides (p = 0.002) between patients and controls. Validation of variants (rs7946-PEMT and rs2290532-OSBPL10) revealed that variant in PEMT but not OSBPL10 gene was associated (p = 0.04) with threefold increased risk of NAFLD in lean individuals.

Conclusion: Our results demonstrate the association of rs7946 with lean-NAFLD. WES may be an effective strategy to identify causative variants underlying lean-NAFLD.

Keywords: ALT, alanine aminotransferase; ARFI, Acoustic Radiation Force Impulse; AST, aspartate aminotransferase; BMI, Body mass index; CI, confidence interval; DNA, Deoxyribonucleic acid; FFAs, free fatty acids; GWAS, Genome-wide association studies; HCC, Hepatocellular carcinoma; HDL, high-density lipoproteins; NAFLD, Nonalcoholic fatty Liver disease; NASH, nonalcoholic steatohepatitis; OSBPL10; OSBPL10, Oxysterol-binding protein-related protein10; PC, phosphatidylcholine; PCR, Polymerase chain reaction; PE, phosphatidylethanolamine; PEMT; PEMT, Phosphatidylethanolamine N-methyltransferase; PHRED, Phil's Read Editor; SIFT, Sorting Intoleratnt from Tolerant; SNPs, Single-nucleotide polymorphisms; SNVs, Single-nucleotide variants; WC, Waist circumference; WES, Whole-Exome Sequencing; gDNA, genomic Deoxyribonucleic acid; indel, insertion deletion; lean-NAFLD; ng, nano gram; nonalcoholic fatty liver disease; pM, pico mole; whole-exome sequencing.