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

Hongsheng Gui, Duco Schriemer, William W. Cheng, Rajendra K. Chauhan, Guillermo Antiňolo, Courtney Berrios, Marta Bleda, Alice S. Brooks, Rutger W.W. Brouwer, Alan J. Burns, Stacey S. Cherny, Joaquin Dopazo, Bart J.L. Eggen, Paola Griseri, Binta Jalloh, Thuy Linh Le, Vincent C.H. Lui, Berta Luzón-Toro, Ivana Matera, Elly S.W. NganAnna Pelet, Macarena Ruiz-Ferrer, Pak C. Sham, Iain T. Shepherd, Man Ting So, Yunia Sribudiani, Clara S.M. Tang, Mirjam C.G.N. van den Hout, Herma C. van der Linde, Tjakko J. van Ham, Wilfred F.J. van IJcken, Joke B.G.M. Verheij, Jeanne Amiel, Salud Borrego, Isabella Ceccherini, Aravinda Chakravarti, Stanislas Lyonnet, Paul K.H. Tam, Maria Mercè Garcia-Barceló*, Robert M.W. Hofstra

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

67 Scopus citations

Abstract

Background: 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. 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. Results: We 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. Conclusions: Our 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.

Original languageEnglish
Article number48
JournalGenome Biology
Volume18
Issue number1
DOIs
StatePublished - 8 Mar 2017
Externally publishedYes

Funding

FundersFunder number
FEDER-InnterconectaEXP00052887/ITC-20111037
Gaslini Institute
JBGMV
Regional Ministry of Innovation, Science and Enterprise of the Autonomous Government of AndalusiaCTS-7447
National Institutes of Health
National Institute of Neurological Disorders and Stroke5R21NS082546
National Institute of Child Health and Human DevelopmentR37HD028088
ZonMwTOP-subsidie 40-00812-98-10042
Centro para el Desarrollo Tecnológico Industrial
Research Grants Council, University Grants CommitteeT12C-714/14R
Ministero della Salute
Ministerio de Economía y Competitividad
Instituto de Salud Carlos IIIPI13/01560
Health and Medical Research FundHMRF 01121326, 01121476, HMRF 02131866
Maag Lever Darm StichtingWO09-62
General Research Fund of Shanghai Normal University201309176158, HKU 777612 M, 201110159001

    Keywords

    • De novo mutations
    • ENS
    • Hirschsprung disease
    • Neural crest

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