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

Abstract

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.

Figure 1

Pedigrees of panel of families with acheiropodia that were used in this study

Figure 2

Affected individual (IV-18) from family 4. Note the small fingerlike appendages at the end of the arm, which are present bilaterally (A and B). C, Radiograph showing tapered amputation of the distal tibia. The proximal tibial epiphysis is well preserved. D, Radiograph showing dysplastic distal humerus articulating with a rudimentary forearm composed of three dysplastic long bones.

Figure 3

Haplotypes derived by analysis of the five nuclear families. The putative ancestral haplotype shared by all affected individuals is boxed.

Figure 4

Map of the 7q36 region containing the acheiropodia locus. Distances (in cM) between markers are shown. The acheiropodia critical region derived by Escamilla et al. (2000) is represented the white vertical bar, and the critical region identified in the present study is represented by the hatched vertical bar. A physical map of the D7S550-D7S2465 minimal critical region is shown, including the c655a11 YAC that spans the region and the positions of three BAC clones (RP11 332e22, RP11 580k21, and RP5 982e9) that contain the C7orf2 gene. Graphic representations of both the C7orf2 gene and the Δexon4 acheiropodia mutation are shown below the physical map; sizes (in kb) of introns are shown above representation of the C7orf2 gene, and exon designations are shown below the representation of the Δexon4 acheiropodia mutation.

Figure 5

A, Results of multiplex PCR analysis of genomic DNA. For each of the five families with acheiropodia, results of analysis of exons 3–5 of the C7orf2 gene, both in affected individuals (lanes A) and in one of their obligate-carrier parents (lanes C), are shown. Lane M, Size markers and positions of bands from each of the three exons (which are identified to the right of the gel). B, Results of RT-PCR analysis of total RNA, using primers spanning exons 1–6 from an affected individual and obligate-carrier parents in family 4 and from a normal control individual. Lane M, Size markers and sizes of RT-PCR fragments (which are denoted to the right of the gel). C, Results of sequence analysis of cDNA from an affected individual in family 4 and from a normal control individual. The sequence from the normal individual shows the exon 3/exon 4 and exon 4/exon 5 boundaries. The sequence from the affected individual shows the exon 3/exon 5 boundary created by the Δexon4 mutation.