title: Japanese founder duplications/triplications involving BHLHA9 are associated with split-hand/foot malformation with or without long bone deficiency and Gollop-Wolfgang complex.
abstract: BACKGROUND
  Limb malformations are rare disorders with high genetic heterogeneity. Although multiple genes/loci have been identified in limb malformations, underlying genetic factors still remain to be determined in most patients.
  
  
  METHODS
  This study consisted of 51 Japanese families with split-hand/foot malformation (SHFM), SHFM with long bone deficiency (SHFLD) usually affecting the tibia, or Gollop-Wolfgang complex (GWC) characterized by SHFM and femoral bifurcation. Genetic studies included genomewide array comparative genomic hybridization and exome sequencing, together with standard molecular analyses.
  
  
  RESULTS
  We identified duplications/triplications of a 210,050 bp segment containing BHLHA9 in 29 SHFM patients, 11 SHFLD patients, two GWC patients, and 22 clinically normal relatives from 27 of the 51 families examined, as well as in 2 of 1,000 Japanese controls. Families with SHFLD- and/or GWC-positive patients were more frequent in triplications than in duplications. The fusion point was identical in all the duplications/triplications and was associated with a 4 bp microhomology. There was no sequence homology around the two breakpoints, whereas rearrangement-associated motifs were abundant around one breakpoint. The rs3951819-D17S1174 haplotype patterns were variable on the duplicated/triplicated segments. No discernible genetic alteration specific to patients was detected within or around BHLHA9, in the known causative SHFM genes, or in the exome.
  
  
  CONCLUSIONS
  These results indicate that BHLHA9 overdosage constitutes the most frequent susceptibility factor, with a dosage effect, for a range of limb malformations at least in Japan. Notably, this is the first study revealing the underlying genetic factor for the development of GWC, and demonstrating the presence of triplications involving BHLHA9. It is inferred that a Japanese founder duplication was generated through a replication-based mechanism and underwent subsequent triplication and haplotype modification through recombination-based mechanisms, and that the duplications/triplications with various haplotypes were widely spread in Japan primarily via clinically normal carriers and identified via manifesting patients. Furthermore, genotype-phenotype analyses of patients reported in this study and the previous studies imply that clinical variability is ascribed to multiple factors including the size of duplications/triplications as a critical factor.
authors.affiliation: Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan. amn_e@hama-med.ac.jp.
authors.forename: Eiko
authors.initials: E
authors.lastname: Nagata
authors.affiliation: Laboratory of Bone and Joint Diseases, Center for Integrative Medical Sciences, RIKEN, Tokyo, Japan. kano@src.riken.jp.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kano
authors.affiliation: Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan. fumifumi@hama-med.ac.jp.
authors.forename: Fumiko
authors.initials: F
authors.lastname: Kato
authors.affiliation: Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan. lrs0701@hama-med.ac.jp.
authors.forename: Rie
authors.initials: R
authors.lastname: Yamaguchi
authors.affiliation: Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan. nakashin@hama-med.ac.jp.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Nakashima
authors.affiliation: Department of Orthopedic Surgery, Tokyo, Japan. takayama-s@ncchd.go.jp.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Takayama
authors.affiliation: Division of Medical Genetics, National Center for Child Health and Development, Tokyo, Japan. kosaki-r@ncchd.go.jp.
authors.forename: Rika
authors.initials: R
authors.lastname: Kosaki
authors.affiliation: Section of Clinical Genetics, Department of Pediatrics, Tenshi Hospital, Sapporo, Japan. hidefumi.tonoki@tenshi.or.jp.
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Tonoki
authors.affiliation: Department of Pediatrics, Central Hospital, Aichi Human Service Center, Kasugai, Japan. seiji_mizuno@aichi-colony.jp.
authors.forename: Seiji
authors.initials: S
authors.lastname: Mizuno
authors.affiliation: Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. swatanabe425@yahoo.co.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. kyoshi@nagasaki-u.ac.jp.
authors.forename: Koh-Ichiro
authors.initials: K
authors.lastname: Yoshiura
authors.affiliation: Department of Medical Genetics, Shinshu University School of Medicine, Matsumoto, Japan. ktomoki@shinshu-u.ac.jp.
authors.forename: Tomoki
authors.initials: T
authors.lastname: Kosho
authors.affiliation: Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan. thaseg@a6.keio.jp.
authors.forename: Tomonobu
authors.initials: T
authors.lastname: Hasegawa
authors.affiliation: Department of Orthopedics, National Rehabilitation Center for Disabled Children, Tokyo, Japan. kimiduka@ryouiku.or.jp.
authors.forename: Mamori
authors.initials: M
authors.lastname: Kimizuka
authors.affiliation: Division of Medical Genetics, Saitama Children's Medical Center, Saitama, Japan. suzuki.atsushi.am@pref.saitama.lg.jp.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Suzuki
authors.affiliation: Division of Medical Genetics, Saitama Children's Medical Center, Saitama, Japan. shimizu.kenji@pref.saitama.lg.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Shimizu
authors.affiliation: Division of Medical Genetics, Saitama Children's Medical Center, Saitama, Japan. ohashi.hirofumi@pref.saitama.lg.jp.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Ohashi
authors.affiliation: Department of Rehabilitation Medicine, University of Tokyo, Tokyo, Japan. hagan-reh@h.u-tokyo.ac.jp.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Haga
authors.affiliation: Department of Genetic Counseling, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan. numabe.hironao@ocha.ac.jp.
authors.forename: Hironao
authors.initials: H
authors.lastname: Numabe
authors.affiliation: Department of Orthopedic Surgery, Japanese Red Cross Nagoya Daiichi Hospital, Nagoya, Japan. emikoh_523523@yahoo.co.jp.
authors.forename: Emiko
authors.initials: E
authors.lastname: Horii
authors.affiliation: Department of Pediatrics, Dokkyo Medical University Koshigaya Hospital, Koshigaya, Japan. toshironagai@gmail.com.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Nagai
authors.affiliation: Division of Medical Genetics, Tokyo, Japan. hiroshi_yoshihashi@tmhp.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoshihashi
authors.affiliation: Department of Pediatric Imaging, Tokyo Metropolitan Children's Medical Center, Tokyo, Japan. gen-n@pc4.so-net.ne.jp.
authors.forename: Gen
authors.initials: G
authors.lastname: Nishimura
authors.affiliation: Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe, Japan. toda@med.kobe-u.ac.jp.
authors.forename: Tatsushi
authors.initials: T
authors.lastname: Toda
authors.affiliation: Department of Systems Biomedicine, National Research Institute for Child Health and Development, Tokyo, Japan. takada-s@ncchd.go.jp.
authors.forename: Shuji
authors.initials: S
authors.lastname: Takada
authors.affiliation: Department of Systems Biomedicine, National Research Institute for Child Health and Development, Tokyo, Japan. shigetoshi.yokoyama@nih.gov.|Present address: Laboratory of Metabolism Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA. shigetoshi.yokoyama@nih.gov.
authors.forename: Shigetoshi
authors.initials: S
authors.lastname: Yokoyama
authors.affiliation: Department of Systems Biomedicine, National Research Institute for Child Health and Development, Tokyo, Japan. asahara.syst@tmd.ac.jp.|Department of Systems Biomedicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. asahara.syst@tmd.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Asahara
authors.affiliation: Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan. sanosin2u@yahoo.co.jp.|Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan. sanosin2u@yahoo.co.jp.
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Sano
authors.affiliation: Department of Molecular Endocrinology, National Research Institute for Child Health and Development, Tokyo, Japan. fumaki-m@ncchd.go.jp.
authors.forename: Maki
authors.initials: M
authors.lastname: Fukami
authors.affiliation: Laboratory of Bone and Joint Diseases, Center for Integrative Medical Sciences, RIKEN, Tokyo, Japan. sikegawa@ims.u-tokyo.ac.jp.
authors.forename: Shiro
authors.initials: S
authors.lastname: Ikegawa
authors.affiliation: Department of Pediatrics, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan. tomogata@hama-med.ac.jp.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Ogata
mesh_terms: D000015:Abnormalities, Multiple / Q000235:genetics*; D000328:Adult; D044466:Asian People; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics*; D058674:Chromosome Duplication; D055028:Comparative Genomic Hybridization; D059472:Exome; D005260:Female; D005269:Femur / Q000002:abnormalities*; D055106:Genome-Wide Association Study; D006228:Hand Deformities, Congenital / Q000150:complications / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D007564:Japan; D017880:Limb Deformities, Congenital / Q000150:complications / Q000235:genetics*; D008297:Male; D013977:Tibia / Q000002:abnormalities*; D055815:Young Adult
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; C571985:BHLHA9 protein, human