Mutation analysis of BMPR1B
Initially, sequence analysis of the known BDA2 causing gene, BMPR1B, in the first identified affected family member, VI:1, did not identify any mutations.
Hands and feet were clinically examined in 37 family members, 19 of whom had a short second middle finger as compared to the fourth middle finger. In most affected cases this was obvious, even though the trait was not always recognised by the person themselves. Affected individuals were then included in the linkage analysis.
As GDF5 is the primary ligand for BMPR1B, we subsequently focused on this gene. Linkage analysis with STS markers D20S847 and D20S106 spanning a ~1.35 Mb region containing GDF5 showed a shared haplotype in all affected individuals, and a two point LOD score of 3.98 was calculated in affected subjects only.
Molecular analysis of GDF5
Bidirectional sequencing of GDF5 demonstrated heterozygosity for a single base pair substitution, c.1322T>C (fig 1B), causing a change in amino acid residue 441 from a leucine to a proline. The mutation induced a novel cleavage site for the restriction enzyme HpaII, and digestion confirmed the mutation in 22 subjects. Three of these (VI:3, VIII:2, VII:23; fig 2C) were clinically unaffected (fig 1C). Furthermore, the same mutation was identified in the Danish subject. The mutation was not observed in a panel of 100 Danish control alleles.
Figure 2(A−F) Different degrees of BDA2 observed in the family (A, D: VI:8; B, E: VI:10; C, F: VI:15). (G, H). Foot phenotype with shortened or absence of middle phalanges. (Photographs are reproduced with consent) (A colour version of (more ...)
Phenotypic spectrum in mutation carriers
Hands were more commonly affected than the feet (table 2). Relative shortening of the second mesophalanx was the predominant trait in all affected individuals. In two of nine radiographically evaluated cases this bone was absent, and in five other individuals it was shortened by more than 2 SD. In two cases (VI:15, VII:23) the second mesophalanx was of normal length. Clinodactyly occurred in four cases. Occasionally, other bones were also affected. A short third, fourth, or fifth mesophalanx, or first proximal phalanx was observed in three, two, two, and two cases, respectively. Metacarpal 1 and distal phalanx 1 and 2 were each short in one of the nine cases. Three mutation carriers appeared clinically normal. Radiographs were only available for one of these (VII:23), and confirmed normal bone length.
Table 2Phenotype in 14 patients, determined by clinical examination, and an additional radiological examination in nine of the 14
The MCPP demonstrated that the second mesophalanx was constantly (eight out of nine subjects) relatively shorter than mesophalanges 3, 4, and 5, and the fourth was relatively longer than the others (table 3, fig 3). The third mesophalanx was shorter than the fifth mesophalanx in half the cases. Another common finding was a relatively short first proximal phalanx. The characteristic MCPP was evident in VI:15 who otherwise had bones of normal length, suggesting that this analysis is the most certain method for determining carrier status. The other phenotypically normal mutation carrier (VII:23) had an uncharacteristic profile pattern (fig 3), demonstrating reduced penetrance.
Table 3Measurements of hand bone length in nine mutation carriers, given by standard deviations (SD) from normal6 in 19 hand bones
Figure 3Metacarpal‐phalangeal profile analysis in nine mutation carriers.
Structure of GDF5 and localisation of the mutation
The amino acid L441 in GDF5 is conserved in human, chimp, mouse, and chicken (fig 4A) and resides in the active signalling domain of GDF5 (fig 4B). Furthermore, a leucine corresponding to amino acid residue 441 is conserved in the paralogous proteins GDF−5/−6/−7 and BMP−2/−4 (fig 4C), which suggests a crucial function for this residue. The corresponding leucine in Bmp2 is located in the receptor binding site as revealed by the crystal structure of Bmp2 bound to the Bmpr1a receptor (fig 4D). This suggests that the L441 residue in GDF5 is also located in the receptor binding site and may therefore interfere with the normal binding of BMP type 1 receptors. This was supported by the very recently reported crystal structure of GDF5.10
Figure 4(A) Conservation of L441 in human, chimp, mouse, and chicken. (B) GDF5 monomer contains a signal peptide (black), a prodomain (grey), and an active signalling domain (white). The mutations reported in the active signalling domain are (more ...)