Human Mutation RESEARCH ARTICLE Mutations in GDF5 Presenting as Semidominant Brachydactyly A1 Ashley M. Byrnes, 1,2 Lemuel Racacho, 1,2 Sarah M. Nikkel, 4,5 Fengxia Xiao, 1 Heather MacDonald, 1 T. Michael Underhill, 6 and Dennis E. Bulman 1–3Ã 1 Department of Regenerative Medicine, Ottawa Hospital Research Institute, and the University of Ottawa Centre for Neuromuscular Disease, Ottawa, ON, Canada; 2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada; 3 Department of Medicine, Division of Neurology, University of Ottawa, Ottawa, ON, Canada; 4 Department of Genetics, Children’s Hospital of Eastern Ontario, ON, Canada; 5 Department of Pediatrics, University of Ottawa, Ottawa, ON, Canada; 6 Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, BC, Canada Communicated by Iain McIntosh Received 19 May 2010; accepted revised manuscript 20 July 2010. Published online 3 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/humu.21338 ABSTRACT: Brachydactyly A1 (BDA1) is an autosomal dominant disorder characterized by shortness of all middle phalanges of the hands and toes, shortness of the proximal phalanges of the first digit, and short stature. Missense mutations in the Indian Hedgehog gene (IHH) are known to cause BDA1, and a second locus has been mapped to chromosome 5p. In a consanguineous French Canadian kindred with BDA1, both IHH and the 5p locus were excluded. Microsatellites flanking GDF5 on chromosome 20q were found to cosegregate with the disease. Sequencing of the GDF5 coding region revealed that a mildly affected individual in the family was heterozygous, and that all of the severely affected individuals were homozygous for a novel missense c.1195C4T mutation that predicts a p.Arg399Cys substitution at a highly conserved amino acid. Functional analysis demonstrated that although the p.Arg399Cys mutant is able to stimulate chondrogenesis, it is much less effective than wild-type GDF5. This data confirms genetic heterogeneity in BDA1, demonstrates that mutations up- stream of IHH can result in BDA1, and shows that BDA1 can result from semidominant mutations in GDF5. Hum Mutat 31:1155–1162, 2010. & 2010 Wiley-Liss, Inc. KEY WORDS: Brachydactyly A1; GDF5; IHH; semido- minant Introduction Brachydactyly type A1 (BDA1; MIM] 112500) was the first disorder described in terms of autosomal dominant inheritance in humans [Farabee, 1903] and is characterized by shortness of all middle phalanges of the hands and feet. Although all of the small tubular bones tend to be reduced in size, there is a dispropor- tionate and significant shortness of the middle phalanges and the proximal first phalanges. The second and fifth middle phalanges tend to be the most severely affected, while the fourth and fifth are the most likely of the metacarpals and metatarsals to be involved [Armour et al., 2000]. To date, BDA1 has been described as a fully penetrant, autosomal dominant condition. Mutations have been identified in the gene Indian hedgehog (IHH; MIM] 600726) [Byrnes et al., 2009; Gao et al., 2001; Giordano et al., 2003; Kirkpatrick et al., 2003; Liu et al., 2006; McCready et al., 2002, 2005; Stattin et al., 2009; Zhu et al., 2007], whose protein product is critical for growth and differentiation of long bone cartilage templates [Vortkamp et al., 1996]. However, mutations in IHH account for approximately 40% of the cases of BDA1. A second locus for BDA1 was reported at chromosome 5p13.3–13.2 (designated BDA1B; MIM] 607004) in a large Canadian family [Armour et al., 2002]. Furthermore, an inversion of the Sonic hedgehog (Shh) locus has yielded a murine BDA1 phenotype in heterozygous Short digits (Dsh/1) mice [Niedermaier et al., 2005]. Brachydactyly type A2 (BDA2; MIM] 112600) is characterized by the shortening of the middle phalange in digit 2 and to a variable degree clinodactyly of the fifth finger. Recently, it has been shown that mutations in growth/differentiation factor 5 (GDF5; MIM] 601146) and its receptor, bone morphogenetic receptor 1B (BMPR1B; MIM] 603248), both cause autosomal dominant BDA2 [Kjaer et al., 2006; Lehmann et al., 2003]. Brachydactyly type C (BDC; MIM] 113100) is similar to brachydactyly types A1 and A2 in that they each involve malformation of various middle phalanges; however in BDC, the fourth finger is spared; the primary characteristics observed are shortening of the middle phalanges in digits 2, 3, and 5, and of the first metacarpal. Mutations in GDF5 have been shown to cause both autosomal dominant and semi- dominant BDC [Polinkovsky et al., 1997; Schwabe et al., 2004]. Materials and Methods DNA Collection and Extraction Genomic DNA was extracted from peripheral venous blood or saliva samples using the QIAamp DNA blood minikit and the Oragene DNA self-collection kit (Qiagen, Mississauga, Ontario; DNA Genotek, Ottawa, Ontario). Sequencing The primer sequences and conditions for the screening of IHH can be found elsewhere [McCready et al., 2002]. Exons of the GDF5 gene were amplified by PCR using oligonucleotide primers described in Table 1. The PCR products were treated with ExoSAP-IT TM (USB, OFFICIAL JOURNAL www.hgvs.org & 2010 WILEY-LISS, INC. Ã Correspondence to: Dennis E. Bulman, Ottawa Hospital Research Institute, 501 Smyth Road, Ottawa, ON, Canada K1G 5P1. E-mail: dbulman@ohri.ca