PYCNODYSOSTOSIS: MAPPING AND EVALUATION OF THREE CANDIDATE GENES.† 854

Abstract

Pycnodysostosis (PYCNO) is an autosomal recessive sclerosing skeletal dysplasia of unknown etiology. Initially, we demonstrated linkage of PYCNO to chromosome 1q21 in a large Arab family in which only marker D1S498 failed to recombine with the disease (Nature Genetics,10:235). Recently, the PYCNO critical region was refined to a 2 cM interval from D1S2344 to D1S2343/D1S2347. We report the evaluation of three candidate genes for PYCNO which had been mapped to 1q21: the interleukin-6 receptor (IL6R), MCL1 which is a BCL2 homolog, and cathepsin S (CTSS). Sequence-tagged sites were developed for these three PYCNO candidate genes and tested against the Stanford G3 diploid radiation hybrid panel. Analysis of the data with the RHMAP software package revealed that IL6R was tightly linked to D1S305, excluding it from the PYCNO critical region. MCL1 and CTSS were linked to D1S498 and D1S2347, placing them within the critical region. In order to search for disease-causing mutations, PCR amplification and sequencing of MCL1 and CTSS exons and their exon-intron boundaries from genomic DNA of three PYCNO families were carried out. A polymorphism resulting from a single base pair substitution for CTSS was found but no putative disease-causing mutations were detected in either MCL1 or CTSS. CTSS protease activity in cultured lymphoblasts from unrelated PYCNO patients also was determined and was normal. The absence of coding region mutations and the presence of normal protease activity for CTSS effectively excludes its candidacy. While promoter and other mutations have not been excluded for MCL1, the lack of coding region mutations in three separate PYCNO families makes it an unlikely candidate. Current efforts are directed towards further refining the PYCNO critical region with additional families and developing the physical map of that region.