CONFIRMATION OF HOMOZYGOSITY FOR A SINGLE NUCLEOTIDE SUBSTITUTION MUTATION IN A COCKAYNE-SYNDROME PATIENT USING MONOALLELIC MUTATION ANALYSIS IN SOMATIC-CELL HYBRIDS

The identification of individuals homozygous for a specific mutation o ffers advantages for the elucidation of molecular mechanisms of heredi tary disease states. Cockayne syndrome is a rare autosomal recessive d isorder, the molecular basis of which is complicated by significant ge netic and clinical heterogeneity. The genes associated with both genet ic complementation groups, CSA and CS-B, have been identified. We have previously identified a number of CSA mutations, including a single b ase substitution that introduces a stop codon ((322)Tyr --> Stop) muta tion in the C-terminal region for at least one allele of the CSA gene in a severely affected patient. We now present data confirming the exi stence of homozygosity in this patient using a strategy with general a pplicability. Somatic cell hybrids were established by fusing patient cells with mouse A9 cells. Screening with chromosome 5 specific polymo rphic markers facilitated identification of hybrid clones bearing only one of the distinct CSA alleles. Sequencing of a portion of the human CSA gene in a subset of these hybrids permitted monoallelic mutation analysis and confirmed the presence of the (322)Tyr --> Stop mutation in both alleles. (C) 1997 Wiley-Liss, Inc.