Autosomal dominant craniometaphyseal dysplasia is caused by mutations in the transmembrane protein ANK

Abstract

Craniometaphyseal dysplasia (CMD) is a rare skeletal disorder characterized by progressive thickening and increased mineral density of craniofacial bones and abnormally developed metaphyses in long bones. Linkage studies mapped the locus for the autosomal dominant form of CMD to an approximately 5-cM interval on chromosome 5p, which is defined by recombinations between loci D5S810 and D5S1954. Mutational analysis of positional candidate genes was performed, and we describe herein three different mutations, in five different families and in isolated cases, in ANK, a multipass transmembrane protein involved in the transport of intracellular pyrophosphate into extracellular matrix. The mutations are two in-frame deletions and one in-frame insertion caused by a splicing defect. All mutations cluster within seven amino acids in one of the six possible cytosolic domains of ANK. These results suggest that the mutated protein has a dominant negative effect on the function of ANK, since reduced levels of pyrophosphate in bone matrix are known to increase mineralization.

Figure 1

A and B, Proband of pedigree A, at age 12, showing typical features of CMD—for example, hypertelorism, wide nasal bridge, and paranasal bossing. C, Metaphyseal flaring in a 2-year-old child from family C.

Figure 2

Pedigree of family A shows haplotypes for selected markers in the CMD locus on chromosome 5p, defining an initial 30-cM disease-gene interval. Recombinations in family C reduced the size of the locus to 5 cM, with breakpoints between markers D5S810 (telomeric) and D5S1954 (centromeric). The most likely haplotypes for the linked allele are boxed.

Figure 3

Electropherograms of partial sequences of ANK showing deletions and an insertion in families with CMD. Sequence variations are indicated by arrows (↓). Capital letters next to electropherograms indicate wild-type sequence (WT) and families and sporadic cases for which the respective mutation was tested. Electropherograms for families C, F and D, S show the sequence of the disease allele after clonal selection of PCR fragments from ANK exon 9 amplified from genomic DNA. The electropherograms for family G show wild-type (WT) and mutant allele carrying the insertion from cDNA after clonal selection, and the heterozygous A→G transition in intron 9 from genomic DNA.

Figure 4

Locations of ANK mutations in patients with CMD (model of ANK according to Ho et al. 2000). The mutations cluster in a cytoplasmic domain between two putative transmembrane domains (mutations are indicated by arrows [→]).

Figure 5

Exon-intron boundaries of ANK exons 9 and 10. Capital letters indicate exon sequences; lowercase letters indicate intron sequences. Three nucleotides on the splice-acceptor site of intron 9, which are retained in the cDNA sequence of family G, are marked in boldface type. Exon 9 ends with a split codon, which contributes to the codon (GCA) for the extra alanine. The new splice-acceptor site in the disease allele is created by the heterozygous point mutation (a→g) in position −4 of the splice-donor site of intron 9 (marked by an arrow (↓).

Figure 6

Upper panel, Expression of ANK by RT-PCR from cultured osteoblasts from mandibular (lane 1) and iliac (lane 2) bone, and from osteoclasts from osteoclastoma (lane 3). Lower panel, GAPDH fragments were amplified as internal controls.