title: Whole exome sequencing coupled with unbiased functional analysis reveals new Hirschsprung disease genes.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.|Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Gui
authors.affiliation: Department of Neuroscience, section Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Duco
authors.initials: D
authors.lastname: Schriemer
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.|Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands.
authors.forename: William W
authors.initials: WW
authors.lastname: Cheng
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands.
authors.forename: Rajendra K
authors.initials: RK
authors.lastname: Chauhan
authors.affiliation: Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.|Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Antiňolo
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, USA.
authors.forename: Courtney
authors.initials: C
authors.lastname: Berrios
authors.affiliation: Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain.|Department of Medicine, School of Clinical Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.
authors.forename: Marta
authors.initials: M
authors.lastname: Bleda
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands.
authors.forename: Alice S
authors.initials: AS
authors.lastname: Brooks
authors.affiliation: Erasmus Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Rutger W W
authors.initials: RW
authors.lastname: Brouwer
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands.|Stem Cells and Regenerative Medicine, Birth Defects Research Centre, UCL Institute of Child Health, London, UK.
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Burns
authors.affiliation: Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Stacey S
authors.initials: SS
authors.lastname: Cherny
authors.affiliation: Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.|Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain.
authors.forename: Joaquin
authors.initials: J
authors.lastname: Dopazo
authors.affiliation: Department of Neuroscience, section Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Bart J L
authors.initials: BJ
authors.lastname: Eggen
authors.affiliation: UOC Genetica Medica, Istituto Gaslini, Genoa, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Griseri
authors.affiliation: Department of Biology, Emory University, Atlanta, USA.
authors.forename: Binta
authors.initials: B
authors.lastname: Jalloh
authors.affiliation: Laboratory of embryology and genetics of human malformations, INSERM UMR 1163, Institut Imagine, Paris, France.|Department of Genetics, Paris Descartes-Sorbonne Paris Cité University, Hôpital Necker-Enfants Malades (APHP), Paris, France.
authors.forename: Thuy-Linh
authors.initials: TL
authors.lastname: Le
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Vincent C H
authors.initials: VC
authors.lastname: Lui
authors.affiliation: Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.|Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain.
authors.forename: Berta
authors.initials: B
authors.lastname: Luzón-Toro
authors.affiliation: UOC Genetica Medica, Istituto Gaslini, Genoa, Italy.
authors.forename: Ivana
authors.initials: I
authors.lastname: Matera
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Elly S W
authors.initials: ES
authors.lastname: Ngan
authors.affiliation: Laboratory of embryology and genetics of human malformations, INSERM UMR 1163, Institut Imagine, Paris, France.|Department of Genetics, Paris Descartes-Sorbonne Paris Cité University, Hôpital Necker-Enfants Malades (APHP), Paris, France.
authors.forename: Anna
authors.initials: A
authors.lastname: Pelet
authors.affiliation: Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.|Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain.
authors.forename: Macarena
authors.initials: M
authors.lastname: Ruiz-Ferrer
authors.affiliation: Centre for Genomic Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Pak C
authors.initials: PC
authors.lastname: Sham
authors.affiliation: Department of Biology, Emory University, Atlanta, USA.
authors.forename: Iain T
authors.initials: IT
authors.lastname: Shepherd
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Man-Ting
authors.initials: MT
authors.lastname: So
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands.|Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia.
authors.forename: Yunia
authors.initials: Y
authors.lastname: Sribudiani
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Clara S M
authors.initials: CS
authors.lastname: Tang
authors.affiliation: Erasmus Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Mirjam C G N
authors.initials: MC
authors.lastname: van den Hout
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands.
authors.forename: Herma C
authors.initials: HC
authors.lastname: van der Linde
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands.
authors.forename: Tjakko J
authors.initials: TJ
authors.lastname: van Ham
authors.affiliation: Erasmus Center for Biomics, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Wilfred F J
authors.initials: WF
authors.lastname: van IJcken
authors.affiliation: Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Joke B G M
authors.initials: JB
authors.lastname: Verheij
authors.affiliation: Laboratory of embryology and genetics of human malformations, INSERM UMR 1163, Institut Imagine, Paris, France.|Department of Genetics, Paris Descartes-Sorbonne Paris Cité University, Hôpital Necker-Enfants Malades (APHP), Paris, France.
authors.forename: Jeanne
authors.initials: J
authors.lastname: Amiel
authors.affiliation: Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain.|Centre for Biomedical Network Research on Rare Diseases (CIBERER), Seville, Spain.
authors.forename: Salud
authors.initials: S
authors.lastname: Borrego
authors.affiliation: UOC Genetica Medica, Istituto Gaslini, Genoa, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Ceccherini
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, USA.
authors.forename: Aravinda
authors.initials: A
authors.lastname: Chakravarti
authors.affiliation: Laboratory of embryology and genetics of human malformations, INSERM UMR 1163, Institut Imagine, Paris, France.|Department of Genetics, Paris Descartes-Sorbonne Paris Cité University, Hôpital Necker-Enfants Malades (APHP), Paris, France.
authors.forename: Stanislas
authors.initials: S
authors.lastname: Lyonnet
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China.
authors.forename: Paul K H
authors.initials: PK
authors.lastname: Tam
authors.affiliation: Department of Surgery, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, SAR, China. mmgarcia@hku.hk.
authors.forename: Maria-Mercè
authors.initials: MM
authors.lastname: Garcia-Barceló
authors.affiliation: Department of Clinical Genetics, Erasmus University Medical Center, PO Box 2040, 3000CA, Rotterdam, The Netherlands. r.hofstra@erasmusmc.nl.|Stem Cells and Regenerative Medicine, Birth Defects Research Centre, UCL Institute of Child Health, London, UK. r.hofstra@erasmusmc.nl.
authors.forename: Robert M W
authors.initials: RM
authors.lastname: Hofstra
mesh_terms: D000483:Alleles; D000818:Animals; D016022:Case-Control Studies; D019295:Computational Biology / Q000379:methods; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D059472:Exome; D055786:Gene Knockout Techniques; D020022:Genetic Predisposition to Disease; D055106:Genome-Wide Association Study; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006627:Hirschsprung Disease / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010641:Phenotype; D015027:Zebrafish
keywords: De novo mutations; ENS; Hirschsprung disease; Neural crest