LETTERS TO JMG A novel locus for brachydactyly type A1 on chromosome 5p13.3-p13.2

The brachydactylies are a group of inherited disorders characterised by shortened or malformed digits that are thought to be the result of abnormal growth of the phalanges and/or metacarpals. First classified by Bell into types A, B, C, D, and E, they were reclassified by Temtamy and McKusick and Fitch. Brachydactyly type A1 (BDA1, MIM 112500) is characterised by shortened or absent middle phalanges. Often the second and fifth digits, as well as the first proximal phalanx, are the most severely affected. In addition, all of the small tubular bones tend to be reduced in size and the metacarpals may be shortened, particularly the fifth metacarpal. Radial/ulnar clinodactyly, as well as malformed or absent epiphyses, have also been reported. 2 Complex syndromes have been described in which BDA1 is one of a number of manifestations. Recently, genetic linkage for a BDA1 locus has been reported to map to 2q35-q36 in two unrelated Chinese families. Subsequent sequence analysis identified mutations in the Indian Hedgehog gene (Ihh) in affected subjects. There have been no other reports of linkage for BDA1, although identification of a balanced translocation between 5q11.2 and 17q23 in a girl with Klippel-Feil anomaly and BDA1 suggests that there may be a BDA1 locus on either chromosome 5 or 17. Mastrobattista et al examined a number of candidate genes, including HoxD, Msx1, Msx2, FGF1, and FGF2, in two families with BDA1, but did not find evidence of linkage. Genes involved in two of the other types of brachydactyly have been described. Mutations in CDMP1, a member of the TGF-β superfamily, have been found in a variant of autosomal dominant brachydactyly type C in which the middle phalanges of the second, third, and fifth fingers are shortened. Mutations in this gene also cause Hunter-Thompson and Grebe acromesomelic dysplasias, two autosomal recessive conditions. In addition, dominant mutations in ROR2, an orphan receptor tyrosine kinase, have been shown to cause brachydactyly type B. 10 We have recently described a three generation family with mild BDA1, in which 13 affected subjects exhibited shortened middle and distal phalanges, proximal first phalanx, and fifth metacarpal. Consistently, the middle phalanges of affected members were below 2 SD of age matched norms. Most of the proximal first phalanges and fifth metacarpals of affected subjects were similarly 2 SD below the norms. Affected members also tended to be of short stature. The children who were studied had coned and prematurely fused epiphyses. Several members, such as the proband, had clinodactyly of one or more digits. A number of subjects also had a broad distal hallux and/or broad, slightly adducted forefoot. Since our initial report, we have ascertained an additional 15 family members including nine affected subjects. The phenotype of these additional family members is similar to those subjects in the family already described, and no additional clinical findings were associated with BDA1 in this family. We now report linkage of BDA1 in this kindred to a novel locus on chromosome 5. METHODS The linkage study comprised 34 members including 20 affected subjects and was conducted after approval by the Children’s Hospital of Eastern Ontario Ethics Review Committee. Peripheral blood samples were taken with informed consent from all participating family members, and a standard protocol was used to isolate DNA. A genome wide scan was initiated using 36 primer sets from the MAPPAIRSTM microsatellite markers (Research Genetics, Huntsville, Alabama), encompassing markers from 16 chromosomes. Particular emphasis was placed on markers from chromosome 5 and 17, based on the report by Fukushima et al describing a translocation between 5q11.2 and 17q23 in a girl with Klippel-Feil anomaly and BDA1. Additional markers from Marshfield’s sex averaged genetic map were examined. These included three markers from chromosome 17 (D17S1301, D17S1290, and D17S1303) and 14 markers from chromosome 5 (table 1). Each of the loci examined were individually amplified in 10 μl PCR containing 10 mmol/l Tris HCl (pH 8.3), 1.5 mmol/l MgCl2, 100-200 ng genomic DNA, 0.2 mmol/l dNTP, 0.12 μmol/l M13 tailed forward primer, 0.12 μmol/l reverse primer, 0.12 μmol/l IRD-700 labelled M13 forward(-29) primer (LI-COR, Lincoln, NE), and 1 Unit Taq enzyme. DNA was amplified over 16 cycles at 94°C for 30 seconds, 66-50°C for 30 seconds (−1°C per cycle), and 72°C for 30 seconds, followed by 23 cycles at 94°C for 45 seconds, 50°C for 30 seconds, and 72°C for 30 seconds. PCR products were size separated on 6% acrylamide gels, using the LI-COR DNA sequencer Model 4000 (LI-COR, Lincoln, NE) and analysed using RFLPscan software (version 3.0). Haplotypes were subsequently generated using Cyrillic software (version 2.1). Two point lod scores were calculated using MLINK, ILINK, and LODSCORE from the FASTLINK version 13 of the LINKAGE software package. The LINKMAP program from the same software package was used to calculate location scores for multipoint linkage analyses. All lod and location scores were calculated using an autosomal dominant model, penetrance of 100%, and a disease frequency of 0.000001. Equal recombination frequencies between males and females were assumed.