title: RET and EDNRB mutation screening in patients with Hirschsprung disease: Functional studies and its implications for genetic counseling.
abstract: Hirschsprung disease (HSCR) is a major cause of chronic constipation in children. HSCR can be caused by germline mutations in RET and EDNRB. Defining causality of the mutations identified is difficult and almost exclusively based on in silico predictions. Therefore, the reported frequency of pathogenic mutations might be overestimated. We combined mutation analysis with functional assays to determine the frequencies of proven pathogenic RET and EDNRB mutations in HSCR. We sequenced RET and EDNRB in 57 HSCR patients. The identified RET-coding variants were introduced into RET constructs and these were transfected into HEK293 cells to determine RET phosphorylation and activation via ERK. An exon trap experiment was performed to check a possible splice-site mutation. We identified eight rare RET-coding variants, one possible splice-site variant, but no rare EDNRB variants. Western blotting showed that three coding variants p.(Pr270Leu), p.(Ala756Val) and p.(Tyr1062Cys) resulted in lower activation of RET. Moreover, only two RET variants (p.(Ala756Val) and p.(Tyr1062Cys)) resulted in reduced ERK activation. Splice-site assays on c.1880-11A>G could not confirm its pathogenicity. Our data suggest that indeed almost half of the identified rare variants are proven pathogenic and that, hence, functional studies are essential for proper genetic counseling.
authors.affiliation: Department of Pediatric, Faculty of Medicine, Universitas Gadjah Mada, Prof.Dr Sardjito Hospital, Yogyakarta, Indonesia.
authors.forename: Titis
authors.initials: T
authors.lastname: Widowati
authors.affiliation: Department of Clinical Genetic, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Shamiram
authors.initials: S
authors.lastname: Melhem
authors.affiliation: Department of Pediatric, Faculty of Medicine, Universitas Gadjah Mada, Prof.Dr Sardjito Hospital, Yogyakarta, Indonesia.
authors.forename: Suryono Y
authors.initials: SY
authors.lastname: Patria
authors.affiliation: Department of Clinical Genetic, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Bianca M
authors.initials: BM
authors.lastname: de Graaf
authors.affiliation: Department of Genetic, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Sinke
authors.affiliation: Department of Genetic, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Martijn
authors.initials: M
authors.lastname: Viel
authors.affiliation: Department of Genetic, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
authors.forename: Jos
authors.initials: J
authors.lastname: Dijkhuis
authors.affiliation: Department of Biochemistry, Faculty of Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia.
authors.forename: Ahmad H
authors.initials: AH
authors.lastname: Sadewa
authors.affiliation: Department of Pediatric Surgery, Faculty of Medicine, Universitas Gadjah Mada, Prof.Dr Sardjito Hospital, Yogyakarta, Indonesia.
authors.forename: Rochadi
authors.initials: R
authors.lastname: Purwohardjono
authors.affiliation: Department of Pediatric, Faculty of Medicine, Universitas Gadjah Mada, Prof.Dr Sardjito Hospital, Yogyakarta, Indonesia.
authors.forename: Yati
authors.initials: Y
authors.lastname: Soenarto
authors.affiliation: Department of Clinical Genetic, Erasmus Medical Center, Rotterdam, The Netherlands.
authors.forename: Robert Mw
authors.initials: RM
authors.lastname: Hofstra
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia.
authors.forename: Yunia
authors.initials: Y
authors.lastname: Sribudiani
mesh_terms: D000293:Adolescent; D002648:Child; D002675:Child, Preschool; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D005817:Genetic Counseling; D057809:HEK293 Cells; D006627:Hirschsprung Disease / Q000175:diagnosis / Q000235:genetics*; D006801:Humans; D007223:Infant; D008297:Male; D020125:Mutation, Missense; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051096:Proto-Oncogene Proteins c-ret / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D044023:Receptor, Endothelin B / Q000235:genetics*
chemical_list: C000588596:EDNRB protein, human; D044023:Receptor, Endothelin B; D051096:Proto-Oncogene Proteins c-ret; C099282:RET protein, human; D048049:Extracellular Signal-Regulated MAP Kinases