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Author Notes:

Maria-Mercè Garcia-Barceló, Phone: +852-28315073, Email: mmgarcia@hku.hk

HG and DS performed the exome sequencing analyses and wrote the manuscript.

WC together with AJB, RC, VL, BJ, TJvH, HCvdL, and ITS performed the zebrafish experiments and prepared the figures for the manuscript.

YS and CST conducted the CNV analyses.

Sanger sequencing validation was performed by PG, IM, AP, MTS, MRF, BL-T and DS.

Statistical support was provided by HG, MB, RWWB, TL, SC, PS, and AC.

Expression data were obtained and analyzed by YS and ESWN.

Bioinformatics support was provided by SC, JD, PS, MvdH, WvIJ, and JBGMV. ASB, CB, PT, JA, SL, RH, BE, MMGB, GA, SB, and IC were involved in patient recruitment and clinical aspects of the study.

PT, JA, SL, RH, BE, MMGB, SB, IC, and AC conceived and designed the project.

All authors contributed to writing and editing.

The authors would like to thank the patients and families involved in this study. We thank W. W. Pijnpappel and M. Broeders for providing the Cas9 protein.

The authors declare that they have no competing interests.


Research Funding:

DS, AB, RC, BE,YS, and RH are supported by research grants from ZonMW (TOP-subsidie 40-00812-98-10042 to BJLE/RMWH) and the Maag Lever Darm stichting (WO09-62 to RMWH); AC is supported by NIH grant R37 HD28088; HG, WC, VL, EN, PS, MS, CT, PT, and MMG-B are supported by Health and Medical Research Fund (HMRF 01121326 to VCHL; HMRF 02131866 to MMGB; and 01121476 to ESWN); General Research Fund (HKU 777612 M to PKT); HKU seed funding for basic research (201110159001 to PKT) and small project funding (201309176158 to CSMT). The work described was also partially supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region, China, project number T12C-714/14R to PKT; CH and PG are supported by grants from the Italian Ministry of Health through “Cinque per mille” and Ricerca Corrente to the Gaslini Institute; GA, MB, BL-T, MR-F, and SB are supported by the Spanish Ministry of Economy and Competitiveness (Institute of Health Carlos III (ISCIII), PI13/01560 and CDTI, FEDER-Innterconecta EXP00052887/ITC-20111037), and the Regional Ministry of Innovation, Science and Enterprise of the Autonomous Government of Andalusia (CTS-7447); NINDS (5R21NS082546) awarded to ITS.


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

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

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Journal Title:

Genome Biology


Volume 18, Number 1


, Pages 48-48

Type of Work:

Article | Final Publisher PDF


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.

Copyright information:

© The Author(s). 2017

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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