Approaches to measuring the frequency of achondroplasia and hypochondroplasia causing FGFR-3 mutations in human sperm

Abstract

Achondroplasia and hypochondroplasia are two forms of skeletal dysplasias caused predominantly by single base mutations in the fibroblast growth factor receptor 3 gene (FGFR-3). The mutation for achondroplasia is a G1138A/C substitution and the mutation for hypochondroplasia (occurring about 50% of the time) is a C1620A/G substitution. Recent genetic studies have shown that spontaneous mutations for achondroplasia and hypochondroplasia occur exclusively on the paternally derived chromosome, suggesting that these mutations occur preferentially during spermatogenesis. For unknown reasons, the mutation rates at these FGFR-3 nucleotides appear to occur at a much higher frequency than nucleotide specific mutation rates observed in other human genetic diseases. The purpose of this study was to develop an assay that could detect the frequencies of achondroplasia and hypochondroplasia causing mutations in human sperm. A Needle-in-a-Haystack PCR/RE/LCR selection technique has been developed that measures single base changes, commonly single base substitution mutations, at sensitivities of one mutant allele in one cell in up to 107 wild-type cells. This technique was modified and designed for the achondroplasia and hypochondroplasia base sites 1138 and 1620 of the FGFR-3 gene. With the development of this technique, future studies could focus on determining the frequencies of the mutations in the sperm of fathers of affected children and the frequencies of the mutations in the sperm of the normal population. These studies will help elucidate the paternal age effect, have important implications in genetic counseling and provide a novel method by which to study genetic disease in humans.