Mutations in HOXD13 underlie syndactyly type V and a novel brachydactyly-syndactyly syndrome

Abstract

HOXD13, the homeobox-containing gene located at the most 5' end of the HOXD cluster, plays a critical role in limb development. It has been shown that mutations in human HOXD13 can give rise to limb malformations, with variable expressivity and a wide spectrum of clinical manifestations. Polyalanine expansions in HOXD13 cause synpolydactyly, whereas amino acid substitutions in the homeodomain are associated with brachydactyly types D and E. We describe two large Han Chinese families with different limb malformations, one with syndactyly type V and the other with limb features overlapping brachydactyly types A4, D, and E and mild syndactyly of toes 2 and 3. Two-point linkage analysis showed LOD scores >3 (theta =0) for markers within and/or flanking the HOXD13 locus in both families. In the family with syndactyly type V, we identified a missense mutation in the HOXD13 homeodomain, c.950A-->G (p.Q317R), which leads to substitution of the highly conserved glutamine that is important for DNA-binding specificity and affinity. In the family with complex brachydactyly and syndactyly, we detected a deletion of 21 bp in the imperfect GCN (where N denotes A, C, G, or T) triplet-containing exon 1 of HOXD13, which results in a polyalanine contraction of seven residues. Moreover, we found that the mutant HOXD13 with the p.Q317R substitution was unable to transactivate the human EPHA7 promoter. Molecular modeling data supported these experimental results. The calculated interactions energies were in agreement with the measured changes of the activity. Our data established the link between HOXD13 and two additional limb phenotypes--syndactyly type V and brachydactyly type A4--and demonstrated that a polyalanine contraction in HOXD13, most likely, led to other digital anomalies but not to synpolydactyly. We suggest the term "HOXD13 limb morphopathies" for the spectrum of limb disorders caused by HOXD13 mutations.

Figure 1.

Pedigrees and disease-haplotype segregation of family 1 (A) and family 2 (B). Blackened symbols represent affected individuals with abnormal limb phenotype, and unblackened symbols represent individuals with a normal limb phenotype. Circles and squares indicate females and males, respectively. The arrows identify the probands. The disease haplotype is boxed.

Figure 2.

Photographs and radiographs of the proband (A–D), individual V-10 (E–H), individual V-13 (I–L), and individual IV-10 (M–P) of family 1.

Figure 3.

Photographs and radiographs of the proband (A–D), individual III-10 (E–H), individual IV-19 (I–L), and individual IV-23 (M–P) of family 2.

Figure 4.

HOXD13 missense and polyalanine contraction mutations in study families. A, Genomic DNA sequencing demonstrating the presence and consequence of the missense mutation c.950A→G (p.Q317R) in the proband of family 1. BamHI restriction analysis demonstrates segregation of the mutation, shown as a 249-bp fragment, with the disease phenotype in the family. B, Genomic DNA sequencing demonstrating the presence and consequence of small deletion c.157-177del (p.A53_A59del) in the proband of family 2.

Figure 5.

Transcriptional activity of wild-type and mutant HOXD13 proteins at the human EPH7A promoter. A, Schematic diagram of the reporter construct used in transfection assays. B, Transcriptional activity mediated by HOXD13^WT, HOXD13^Q50R, HOXD13^I47L, and HOXD13^−7A. Bars represent firefly/Renilla luciferase ratios for the different constructs. Values are the means (±SEM) of eight independent experiments. The significance of differences in expression was calculated using the independent-samples T test. P values are presented above the bars. C, Relative decrease of transcriptional activity mediated by mutant HOXD13 proteins compared with the wild-type HOXD13^WT.

Figure 6.

Interaction between HOXD13 and DNA. A, Models based on the Antp/DNA complex. The DNA is color coded as follow: carbon = cyan; oxygen = red; nitrogen = dark blue; phosphate = gold. For the helix III of the homeodomain, the main chain is represented, and only the side chains of the I47 and Q50 (left panel) and of the I47 and R50 (right panel) are drawn. B, Total (red line), electrostatic (blue line), and van der Waals (green line) interactions energies (in kcal/mol) between Q50 and Thy (wild type) and between R50 and Thy (mutant) (left panel) and between I47 and Thy (wild type) and L47 and Thy (mutant) (right panel). The numbers 47 and 50 represent the position of the amino acid in the homeodomain.