Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27

Abstract

Split-hand/split-foot malformation (SHFM), a limb malformation involving the central rays of the autopod and presenting with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals, and metatarsals, is phenotypically analogous to the naturally occurring murine Dactylaplasia mutant (Dac). Results of recent studies have shown that, in heterozygous Dac embryos, the central segment of the apical ectodermal ridge (AER) degenerates, leaving the anterior and posterior segments intact; this finding suggests that localized failure of ridge maintenance activity is the fundamental developmental defect in Dac and, by inference, in SHFM. Results of gene-targeting studies have demonstrated that p63, a homologue of the cell-cycle regulator TP53, plays a critically important role in regulation of the formation and differentiation of the AER. Two missense mutations, 724A-->G, which predicts amino acid substitution K194E, and 982T-->C, which predicts amino acid substitution R280C, were identified in exons 5 and 7, respectively, of the p63 gene in two families with SHFM. Two additional mutations (279R-->H and 304R-->Q) were identified in families with EEC (ectrodactyly, ectodermal dysplasia, and facial cleft) syndrome. All four mutations are found in exons that fall within the DNA-binding domain of p63. The two amino acids mutated in the families with SHFM appear to be primarily involved in maintenance of the overall structure of the domain, in contrast to the p63 mutations responsible for EEC syndrome, which reside in amino acid residues that directly interact with the DNA.

Figure 1

A, Family R (left upper and lower panels). Note the “lobster-claw” anomaly of the hands and feet. Family A (right upper and lower panels). Note the median clefts and syndactyly. B, Previously reported (Spranger and Schapera 1988) radiograph of family R (left). Note monodactyly with triphalangeal thumb and duplication of the distal phalanx. Family A (center and right) shows absence of the phalanges of the second and third digits in both foot and hand.

Figure 2

Extended pedigrees of family A (top) and family R (bottom). Individuals whose DNA was tested are indicated by an asterisk. The phenotypic status of individuals in generations I and II of family R is unknown.

Figure 3

A, Schematic representation of exons 4–8 of the p63 gene, the region that contains the DNA-binding domain. Exons 4–8 were amplified individually from genomic DNA samples, and the resulting fragments were analyzed by means of direct sequence analysis. The K194E and R280C mutations, present in exons 5 and 7, respectively, are shown in comparison with the wild-type sequence. B, Identification of the K194E and R280C mutations in individuals with SHFM. The individual with the K194E mutation is heterozygous A/G at nucleotide 724, compared with the normal individual, who is homozygous A at this position. The individual with the R280C mutation is heterozygous T/C at nucleotide 982, compared with the normal individual, who is homozygous C at this position.

Figure 4

A, Segregation of the K194E mutation in family R. The presence of the K194E mutation was detected by SSCP analysis of a 284-bp PCR fragment encompassing exon 5. Normal individuals show two bands corresponding to the two DNA strands, whereas affected individuals show two doublets corresponding to the mutant and normal DNA strands. B, Segregation of the R280C mutation in family A. The presence of the R280C mutation was detected by its creation of a CCGCTG recognition site for the restriction enzyme MspA1-I. Digestion of a 245-bp PCR fragment encompassing exon 7 produces subfragments of 138 bp and 107 bp, in addition to the undigested 245-bp fragment, in affected individuals. In normal individuals, only the full-length 245-bp fragment is seen. In both cases, the mutation segregated with the disease in all family members tested. A representative section of each family is shown.

Figure 5

Ribbon diagram of the p63 DNA-binding domain, based on the structure of its homologue, p53. The narrow arrowheads denote side chains of the amino acid residues mutated in SHFM (K194 and R280 [red]), and the broad arrowheads denote residues mutated in EEC (R279 and R304 [green]).