Distal limb malformations: underlying mechanisms and clinical associations.

Congenital malformations of the extremities are conspicuous and have been described through the ages. Over the past decade, a wealth of knowledge has been generated regarding the genetic regulation of limb development and the underlying molecular mechanisms. Recent studies have identified several of the signaling molecules, growth factors, and transcriptional regulators involved in the initiation and maintenance of the apical ectodermal ridge (AER) as well as the molecular markers defining the three axes of the developing limb. Studies of abnormal murine phenotypes have uncovered the role played by genes such as p63 and Dactylin in the maintenance of AER activity. These phenotypes resemble human malformations and in this review we describe the underlying mechanisms and clinical associations of split hand/foot malformation and ectrodactyly-ectodermal dysplasia-cleft lip/palate syndrome, which have both been associated with mutations in the p63 gene.

Acheiropodia is caused by a genomic deletion in C7orf2, the human orthologue of the Lmbr1 gene.

Acheiropodia is an autosomal recessive developmental disorder presenting with bilateral congenital amputations of the upper and lower extremities and aplasia of the hands and feet. This severely handicapping condition appears to affect only the extremities, with no other systemic manifestations reported. Recently, a locus for acheiropodia was mapped on chromosome 7q36. Herein we report the narrowing of the critical region for the acheiropodia gene and the subsequent identification of a common mutation in C7orf2-the human orthologue of the mouse Lmbr1 gene-that is responsible for the disease. Analysis of five families with acheiropodia, by means of 15 polymorphic markers, narrowed the critical region to 1.3 cM, on the basis of identity by descent, and to <0.5 Mb, on the basis of physical mapping. Analysis of C7orf2, the human orthologue of the mouse Lmbr1 gene, identified a deletion in all five families, thus identifying a common acheiropodia mutation. The deletion was identified at both the genomic-DNA and mRNA level. It leads to the production of a C7orf2 transcript lacking exon 4 and introduces a premature stop codon downstream of exon 3. Given the nature of the acheiropodia phenotype, it appears likely that the Lmbr1 gene plays an important role in limb development.

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

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.

Localization of an acromesomelic dysplasia on chromosome 9 by homozygosity mapping.

The acromesomelic dysplasias (AMDs) are a group of genetic disorders that primarily affect the middle and distal segments of the extremities. A form of AMD is present on the isolated island of St Helena in the South Atlantic, which has a population of approximately 5500 derived from a number of founder individuals. DNA from four affected individuals and 11 first-degree relatives in four related nuclear families segregating an AMD was collected for gene mapping studies. Six consecutive markers on chromosome 9, spanning an approximately 5 cM region, showed identical homozygosity in all affected individuals, thus identifying a region of homozygosity by descent. Multipoint analysis generated a maximum lod score of Z = 2.85. These data localize the gene for this dysplasia to the pericentromeric region of chromosome 9 where the gene for the Maroteaux form of AMD is situated. The identification of the gene responsible for this disorder may shed further light on the complex processes involved in limb morphogenesis.

A novel human gene encoding an F-box/WD40 containing protein maps in the SHFM3 critical region on 10q24.

We report the cloning and characterization of a new human gene, Dactylin, encoding a novel member of the F-box/WD40 protein family. The Dactylin gene comprises nine exons distributed in more than 85 kb of genomic DNA and encoding a protein with four WD40 repeats and an F-box motif. Northern blot analysis demonstrates a single 2.8 kb transcript in brain, kidney, lung and liver. FISH hybridization localized Dactylin to 10q24.3. Using an Msc I SNP identified in the first exon of the gene, we were able to assign Dactylin within the critical region for Split Hand Split Foot malformation (SHFM3) that has been mapped to 10q24. The SHFM3 phenotype includes absence or hypoplasia of the central digital rays, a deep median cleft and syndactyly of the remaining digits. Recent studies have demonstrated the importance of F-box/WD40 proteins in the regulation of developmental processes, by a mechanism of specific ubiquitinization and subsequent proteolysis of target proteins belonging to the Wnt, Hh and NF-kappaB signaling pathways. The chromosomal location of Dactylin and its putative function as an F-box/WD40 repeat protein, likely to be involved in key signaling pathways crucial for normal limb development, make it a promising candidate gene for SHFM3.