title: Identification of functional variants for cleft lip with or without cleft palate in or near PAX7, FGFR2, and NOG by targeted sequencing of GWAS loci.
abstract: Although genome-wide association studies (GWASs) for nonsyndromic orofacial clefts have identified multiple strongly associated regions, the causal variants are unknown. To address this, we selected 13 regions from GWASs and other studies, performed targeted sequencing in 1,409 Asian and European trios, and carried out a series of statistical and functional analyses. Within a cluster of strongly associated common variants near NOG, we found that one, rs227727, disrupts enhancer activity. We furthermore identified significant clusters of non-coding rare variants near NTN1 and NOG and found several rare coding variants likely to affect protein function, including four nonsense variants in ARHGAP29. We confirmed 48 de novo mutations and, based on best biological evidence available, chose two of these for functional assays. One mutation in PAX7 disrupted the DNA binding of the encoded transcription factor in an in vitro assay. The second, a non-coding mutation, disrupted the activity of a neural crest enhancer downstream of FGFR2 both in vitro and in vivo. This targeted sequencing study provides strong functional evidence implicating several specific variants as primary contributory risk alleles for nonsyndromic clefting in humans.
authors.affiliation: Center for Craniofacial and Dental Genetics, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA. Electronic address: ejl40@pitt.edu.
authors.forename: Elizabeth J
authors.initials: EJ
authors.lastname: Leslie
authors.affiliation: Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Margaret A
authors.initials: MA
authors.lastname: Taub
authors.affiliation: Department of Orthodontics, College of Dentistry, University of Iowa, Iowa City, IA 52242, USA; State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory for Oral Biomedicine of Ministry of Education, School and Hospital of Stomatology, Wuhan University, 430072 Wuhan, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Liu
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Karyn Meltz
authors.initials: KM
authors.lastname: Steinberg
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Koboldt
authors.affiliation: Department of Statistical Genetics, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Qunyuan
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Biostatistics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Jenna C
authors.initials: JC
authors.lastname: Carlson
authors.affiliation: Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Jacqueline B
authors.initials: JB
authors.lastname: Hetmanski
authors.affiliation: Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Hang
authors.initials: H
authors.lastname: Wang
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Larson
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Fulton
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Osteopathic Medicine, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Youssef A
authors.initials: YA
authors.lastname: Kousa
authors.affiliation: Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Walid D
authors.initials: WD
authors.lastname: Fakhouri
authors.affiliation: Department of Diagnostic and Biomedical Sciences, School of Dentistry, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Ali
authors.initials: A
authors.lastname: Naji
authors.affiliation: Department of Biostatistics, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Ingo
authors.initials: I
authors.lastname: Ruczinski
authors.affiliation: Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Ferdouse
authors.initials: F
authors.lastname: Begum
authors.affiliation: Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Parker
authors.affiliation: Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Tamara
authors.initials: T
authors.lastname: Busch
authors.affiliation: Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Standley
authors.affiliation: Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Rigdon
authors.affiliation: Department of Pediatrics, University of Texas Health Science Center at Houston, Houston, TX 77030, USA.
authors.forename: Jacqueline T
authors.initials: JT
authors.lastname: Hecht
authors.affiliation: Institute of Genetic Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Alan F
authors.initials: AF
authors.lastname: Scott
authors.affiliation: Department of Health Management and Policy, College of Public Health, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: George L
authors.initials: GL
authors.lastname: Wehby
authors.affiliation: Department of Epidemiology, Institute of Public Health, University of Southern Denmark, 5230 Odense, Denmark.
authors.forename: Kaare
authors.initials: K
authors.lastname: Christensen
authors.affiliation: Foundation for the Community Control of Hereditary Diseases, Budapest 1148, Hungary.
authors.forename: Andrew E
authors.initials: AE
authors.lastname: Czeizel
authors.affiliation: Department of Surgery, Plastic and Reconstructive Surgery, University of Colorado School of Medicine, Denver, CO 80045, USA.
authors.forename: Frederic W-B
authors.initials: FW
authors.lastname: Deleyiannis
authors.affiliation: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: Brian C
authors.initials: BC
authors.lastname: Schutte
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA.
authors.forename: Richard K
authors.initials: RK
authors.lastname: Wilson
authors.affiliation: Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Cornell
authors.affiliation: Department of Orthodontics, College of Dentistry, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Andrew C
authors.initials: AC
authors.lastname: Lidral
authors.affiliation: The Genome Institute, Washington University School of Medicine, St. Louis, MO 63108, USA; The Jackson Laboratory for Genomic Medicine, Farmington, CT 06117, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Weinstock
authors.affiliation: Department of Epidemiology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
authors.forename: Terri H
authors.initials: TH
authors.lastname: Beaty
authors.affiliation: Center for Craniofacial and Dental Genetics, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA; Department of Human Genetics, Graduate School of Public Health, and Clinical and Translational Science Institute, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address: marazita@pitt.edu.
authors.forename: Mary L
authors.initials: ML
authors.lastname: Marazita
authors.affiliation: Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Murray
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D044466:Asian People / Q000235:genetics; D001921:Brain / Q000002:abnormalities*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002971:Cleft Lip / Q000235:genetics*; D002972:Cleft Palate / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D020022:Genetic Predisposition to Disease; D055106:Genome-Wide Association Study; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D051763:PAX7 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D044465:White People / Q000235:genetics; D015027:Zebrafish / Q000235:genetics; D000099176:Noggin Protein
chemical_list: D002352:Carrier Proteins; D051763:PAX7 Transcription Factor; D051497:Receptor, Fibroblast Growth Factor, Type 2; D014157:Transcription Factors; D000099176:Noggin Protein; C497321:PAX7 protein, human; C496353:FGFR2 protein, human