Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes

Hongsheng Gui,Rutger W W Brouwer,Paul K H Tam,Rajendra K Chauhan,Alan J Burns,Aravinda Chakravarti,Ivana Matera,Salud Borrego,Wilfred F J Van Ijcken,Berta Lasa ,Mirjam C G N Van Den Hout ,Stanislas Lyonnet,Joaquín Dopazo ,Maria Mercè García-Barceló ,Anna Pelet,Marta Bleda,Alice S Brooks,Stacey S Cherny,Jeanne Amiel,Duco Schriemer,Man Ting So ,Clara S Tang ,Binta Jalloh,Thuy Linh Le ,Macarena Ruíz-Ferrer ,Isabella Ceccherini,Yunia Sribudiani,Courtney Berrios,Bart J L Eggen,Paola Griseri,William Cheng ,Pak C Sham,Herma C Van Der Linde,Vincent Chi-Hang Lui ,Tjakko J Van Ham,Elly Sau-Wai Ngan ,Joanne Verheij ,Guillermo Antiñolo ,Iain T Shepherd,Robert M.w Hofstra

doi:10.1186/s13059-017-1174-6

Abstract

BackgroundHirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human.ResultsWe performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS.ConclusionsOur data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.

Highlights

Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine
The coverage of the targeted sequences per sample ranged from 18× to 74×, and the targeted exome was covered by at least ten sequence reads which ranged from 65 to 98%. All these quality metrics or statistics showed data quality at exonic regions that were comparatively good for trios from different platforms or resources and justified our unbiased searching of de novo mutations in the following stages
Several studies found that patients have a significantly higher fraction of loss of function (LOF) de novo mutation (DNM) than healthy controls [6, 7]

Summary

Introduction

Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. HSCR results from a failure of the neural crest (NC) cells, which give rise to the enteric nervous system (ENS), to migrate, proliferate, differentiate, or survive in the bowel wall, resulting in aganglionosis of the distal part of the gastrointestinal tract. This results in clinically severe and sometimes lifethreatening bowel obstruction. These findings indicate that the majority of the disease risk must be due to as yet unidentified rare or common variants in the known HSCR genes or, more likely, variants in yet unknown genes, acting alone or in combination

Objectives

Methods

Results

Discussion

Conclusion