Abstract Ref Number = APCP240
NRG1 variant effects in patients with Hirschsprung disease
Gunadi,Nova Yuli Prasetyo Budi,Aditya Rifqi Fauzi,Alvin Santoso Kalim,Taufik Indrawan,Kristy Iskandar,Akhmad Makhmudi,Raman Sethi,Lai Poh San,Indra Adrianto
Universitas Gadjah Mada National University of Singapore and The Khoo Teck PuatNational University Childrens Medical Institute Henry Ford Health System, Detroit, MI, USA
Background : Hirschsprung disease (HSCR) is a heterogeneous genetic disorder characterized by absence of ganglion cells along the intestines resulting in functional bowel obstruction. Mutations in neuregulin 1 (NRG1) gene have been implicated in some cases of intestinal aganglionosis. This study aims to investigate the contribution of the NRG1 gene to HSCR development in an Indonesian population.
Material : We analyzed the entire coding region of the NRG1 gene in 54 histopathologically diagnosed HSCR patients by Sanger sequencing. The in silico tools used to predict coding variant effects on protein function were SIFT, PolyPhen-2, LRT, Mutation Taster, Mutation Assessor, FATHMM, and CADD algorithm. The predicted conservation scores of variants were determined using GERP, PhyloP, and SiPhy tests. The clinical significance of variants was analyzed using ClinVar tool. The HaploReg database was utilized to evaluate the impact of the variant by annotation of epigenomic, conservation and regulatory motif information.
Results : All patients were sporadic non-syndromic HSCR with 53/54 (98%) short-segment and 1/54 (2%) long-segment patients. NRG1 gene analysis identified one rare variant, c.397G>C (p.V133L), and three common variants, rs7834206, rs3735774, and rs75155858. Although the SIFT and PolyPhen-2 analysis of p.V133L showed the variant as being tolerated (0.22) and benign (0.029), respectively, p.V133L was predicted to be disease causing by Mutation Taster. According to the conservation scores prediction using GERP, PhyloP, and SiPhy, the p.V133L variant almost reached a deleterious threshold of 4.26, 1.55, and 10.121, respectively. The p.V133L variant was predicted to reside within a region of high mammalian conservation, overlapping with the promoter and enhancer histone marks of relevant tissues such as digestive and smooth muscle tissues and potentially altering the AP-4_2, BDP1_disc3, Egr-1_known1, Egr-1_known4, HEN1_2 transcription factor binding motifs. This p.V133L variant was absent in 92 non-HSCR controls. Furthermore, the rs7834206 polymorphism was associated with HSCR by case–control analysis (p = 0.037).
Conclusions : This study is the first report of a NRG1 rare variant associated with HSCR patients of South-East Asian ancestry and provides further insights into the contribution of NRG1 in the molecular genetic pathogenesis of HSCR.
Keywords: Hirschsprung disease Indonesia; NRG1 variant transcription factor binding motif