A mutation in CTSK gene in an autosomal recessive pycnodysostosis family of Pakistani origin

Abstract

Background: Pycnodysostosis is a rare autosomal recessive skeletal dysplasia characterized by short stature, osteosclerosis, acro-osteolysis, frequent fractures and skull deformities. Mutations in the gene encoding cathepsin K (CTSK), a lysosomal cysteine protease, have been found to be responsible for this disease.

Objectives: To identify pathogenic mutation in a consanguineous Pakistani family with 3 affected individuals demonstrating autosomal recessive pycnodysostosis.

Methods: Genotyping of 10 members of the family, including three affected and seven unaffected individuals was carried out by using polymorphic markers D1S442, D1S498, and D1S305, which are closely linked to the CTSK gene on chromosome 1q21. To screen for mutations in the CTSK gene, all of its exons and splice junctions were PCR amplified from genomic DNA and sequenced directly in an ABI Prism 310 automated sequencer.

Results: Genotyping results showed linkage of the pycnodysostosis Pakistani family to the CTSK locus. Sequence analysis of the CTSK gene revealed homozygosity for a missense mutation (A277V) in the affected individuals.

Conclusion: We describe a missense mutation in the CTSK gene in a Pakistani family affected with autosomal recessive pycnodysostosis. Our study strengthens the role of this particular mutation in the pathogenesis of pycnodysostosis and suggests its prevalence in Pakistani patients.

Figure 1

Pedigree of the individuals affected with Pycnodysostosis. Filled symbols identify affected subjects. Consanguineous marriages are represented with double lines. Haplotypes for the most closely linked markers are shown below each symbol.

Figure 2

Mutation analysis of the CTSK gene. DNA sequences of exon 7 of the CTSK gene from (a) a control individual, (b) a heterozygous carrier and (c) a homozygous (affected) individual are shown. The arrow denotes nucleotide position 935, which is mutated from C to T, resulting in A277V missense mutation.

Figure 3

An ethidium bromide-stained agarose gel containing 331-bp amplified product that was digested with AciI. The digested PCR products from a normal control individual (N), an affected individual (P) and a heterozygous carrier (C) are shown. The normal PCR product was digested into 188-bp and 143-bp fragments, whereas the 331-bp products with the A277V defect were not digested. A 100-bp DNA ladder is shown, left.

Figure 4

Alignment of conserved motif from the mature region of several members of the papain family of cysteine proteases and various species. The fourteen-residue motif from four human cysteine cathepsins (cathepsin K, cathepsin L, cathepsin S and cathepsin H) and cathepsin K of various species are aligned. The site of the cathepsin K A277V mutation is indicated by the arrow. * indicates highly conserved amino acids while. and: indicate polar → polar and polar → non polar amino acid change, respectively.