title: Functional loss of semaphorin 3C and/or semaphorin 3D and their epistatic interaction with ret are critical to Hirschsprung disease liability.
abstract: Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA; Department of Medical Genetics, Capital Institute of Pediatrics, Beijing 100020, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Jiang
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Arnold
authors.affiliation: Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Tiffany
authors.initials: T
authors.lastname: Heanue
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Krishna Praneeth
authors.initials: KP
authors.lastname: Kilambi
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Betty
authors.initials: B
authors.lastname: Doan
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Ashish
authors.initials: A
authors.lastname: Kapoor
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Albee Yun
authors.initials: AY
authors.lastname: Ling
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Maria X
authors.initials: MX
authors.lastname: Sosa
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Moltu
authors.initials: M
authors.lastname: Guy
authors.affiliation: Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Qingguang
authors.initials: Q
authors.lastname: Jiang
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Burzynski
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Kristen
authors.initials: K
authors.lastname: West
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Seneca
authors.initials: S
authors.lastname: Bessling
authors.affiliation: U.O.C. Genetica Medica, Istituto Gaslini, 16147 Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Griseri
authors.affiliation: INSERM UMR 1163, Laboratory of Embryology and Genetics of Congenital Malformations, 75015 Paris, France; Paris Descartes University-Sorbonne Paris Cité, Imagine Institute, 75015 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, 41013 Seville, Spain; Centre for Biomedical Network Research on Rare Diseases (CIBERER), 41013 Seville, Spain.
authors.forename: Raquel M
authors.initials: RM
authors.lastname: Fernandez
authors.affiliation: Department of Medical Genetics, University of Groningen, Groningen 9700, Germany.
authors.forename: Joke B G M
authors.initials: JB
authors.lastname: Verheij
authors.affiliation: Department of Clinical Genetics, University of Rotterdam, Erasmus Medical Center, 3015 CE Rotterdam, the Netherlands; Neural Development and Gastroenterology Units, UCL Institute of Child Health, London WC1 NEH, UK.
authors.forename: Robert M W
authors.initials: RM
authors.lastname: Hofstra
authors.affiliation: Department of Genetics, Reproduction and Fetal Medicine, Institute of Biomedicine of Seville (IBIS), University Hospital Virgen del Rocío/CSIC/University of Seville, 41013 Seville, Spain; Centre for Biomedical Network Research on Rare Diseases (CIBERER), 41013 Seville, Spain.
authors.forename: Salud
authors.initials: S
authors.lastname: Borrego
authors.affiliation: INSERM UMR 1163, Laboratory of Embryology and Genetics of Congenital Malformations, 75015 Paris, France; Paris Descartes University-Sorbonne Paris Cité, Imagine Institute, 75015 Paris, France.
authors.forename: Stanislas
authors.initials: S
authors.lastname: Lyonnet
authors.affiliation: U.O.C. Genetica Medica, Istituto Gaslini, 16147 Genova, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Ceccherini
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Gray
authors.affiliation: Division of Molecular Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Vassilis
authors.initials: V
authors.lastname: Pachnis
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: McCallion
authors.affiliation: McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Electronic address: aravinda@jhmi.edu.
authors.forename: Aravinda
authors.initials: A
authors.lastname: Chakravarti
mesh_terms: D000818:Animals; D001483:Base Sequence; D004843:Epistasis, Genetic / Q000235:genetics*; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D014644:Genetic Variation; D055106:Genome-Wide Association Study; D006627:Hirschsprung Disease / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics*; D039961:Semaphorins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: C530499:Sema 3D protein, mouse; D039961:Semaphorins; C510203:semaphorin 3C protein, mouse; D051096:Proto-Oncogene Proteins c-ret; C494999:Ret protein, mouse
references.citation: Development. 2006 Mar;133(6):1035-44
references.pmid: 16467361
references.citation: Proc Natl Acad Sci U S A. 2006 May 2;103(18):6919-24
references.pmid: 16632597
references.citation: Cell. 1993 Dec 31;75(7):1401-16
references.pmid: 8269518
references.citation: Nature. 1994 Jan 27;367(6461):380-3
references.pmid: 8114940
references.citation: Methods Enzymol. 2000;327:198-210
references.pmid: 11044984
references.citation: Development. 2001 Aug;128(16):3061-70
references.pmid: 11688556
references.citation: Development. 2001 Aug;128(16):3071-80
references.pmid: 11688557
references.citation: Science. 2002 Apr 12;296(5566):261-2
references.pmid: 11954565
references.citation: Nat Genet. 2002 Oct;32(2):237-44
references.pmid: 12355085
references.citation: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1826-31
references.pmid: 12574515
references.citation: Hum Mol Genet. 2003 Jul 15;12(14):1725-35
references.pmid: 12837695
references.citation: Nucleic Acids Res. 2004 Jan 1;32(Database issue):D493-6
references.pmid: 14681465
references.citation: J Neurosci. 2004 Sep 29;24(39):8428-35
references.pmid: 15456815
references.citation: Cell. 1990 Oct 5;63(1):185-94
references.pmid: 1698555
references.citation: Cell. 1993 Dec 31;75(7):1389-99
references.pmid: 8269517
references.citation: Genome Biol. 2008;9(4):R63
references.pmid: 18387188
references.citation: Am J Hum Genet. 2010 Apr 9;86(4):526-39
references.pmid: 20303063
references.citation: Am J Hum Genet. 2010 Jul 9;87(1):60-74
references.pmid: 20598273
references.citation: Wiley Interdiscip Rev Syst Biol Med. 2009 Sep-Oct;1(2):220-7
references.pmid: 20490374
references.citation: Neuron. 2010 Oct 6;68(1):32-44
references.pmid: 20920789
references.citation: Nature. 2010 Oct 28;467(7319):1061-73
references.pmid: 20981092
references.citation: Dev Dyn. 2011 Jan;240(1):9-22
references.pmid: 21108318
references.citation: Dev Dyn. 2005 Apr;232(4):939-49
references.pmid: 15729704
references.citation: Nature. 2005 Apr 14;434(7035):857-63
references.pmid: 15829955
references.citation: Dev Biol. 2005 Apr 15;280(2):373-85
references.pmid: 15882579
references.citation: Nat Rev Mol Cell Biol. 2005 Oct;6(10):789-800
references.pmid: 16314868
references.citation: Neuron. 2005 Dec 22;48(6):949-64
references.pmid: 16364899
references.citation: Dev Biol. 1999 Aug 1;212(1):42-53
references.pmid: 10419684
references.citation: Bioinformatics. 2005 Jan 15;21(2):263-5
references.pmid: 15297300
references.citation: Cold Spring Harb Perspect Biol. 2011 Jun;3(6). pii: a001727. doi: 10.1101/cshperspect.a001727
references.pmid: 21123392
references.citation: PLoS One. 2011;6(8):e22477
references.pmid: 21829626
references.citation: Hum Mutat. 2012 Jan;33(1):281-9
references.pmid: 21898659
references.citation: Nat Struct Mol Biol. 2012 Dec;19(12):1293-9
references.pmid: 23104057
references.citation: Nat Med. 2013 Jun;19(6):760-5
references.pmid: 23685842
references.citation: Dev Biol. 2013 Oct 1;382(1):320-9
references.pmid: 23707863
references.citation: Nature. 2007 Oct 18;449(7164):851-61
references.pmid: 17943122
references.citation: Annu Rev Cell Dev Biol. 2007;23:263-92
references.pmid: 17539753
references.citation: Curr Protoc Protein Sci. 2007 Nov;Chapter 2:Unit 2.9
references.pmid: 18429317
references.citation: Mech Dev. 2008 Aug;125(8):687-99
references.pmid: 18565740
references.citation: Proc Natl Acad Sci U S A. 2009 Feb 24;106(8):2694-9
references.pmid: 19196962
references.citation: Hum Mutat. 2009 May;30(5):771-5
references.pmid: 19306335
references.citation: Dev Biol. 2009 Dec 15;336(2):257-65
references.pmid: 19833121
references.citation: Ann Hum Genet. 1995 Jul;59(Pt 3):323-36
references.pmid: 7486838
references.citation: Development. 1996 Jan;122(1):349-58
references.pmid: 8565847
references.citation: Protein Sci. 1997 Aug;6(8):1661-81
references.pmid: 9260279
references.citation: Neuron. 1998 Nov;21(5):1079-92
references.pmid: 9856463
references.citation: Development. 1999 Jun;126(12):2785-97
references.pmid: 10331988
references.citation: FASEB J. 2006 Oct;20(12):2150-2
references.pmid: 16940438
references.citation: Protein Sci. 2006 Nov;15(11):2507-24
references.pmid: 17075131
references.citation: Dev Biol. 2007 May 1;305(1):287-99
references.pmid: 17362911
references.citation: Nat Rev Neurosci. 2007 Jun;8(6):466-79
references.pmid: 17514199
references.citation: PLoS Genet. 2007 May 25;3(5):e78
references.pmid: 17530925
references.citation: Hum Mutat. 2007 Aug;28(8):790-6
references.pmid: 17397038