A(-2)-]G TRANSITION AT THE 3'-ACCEPTOR SPLICE-SITE OF IVS17 CHARACTERIZES THE COL2A1 GENE MUTATION IN THE ORIGINAL STICKLER SYNDROME KINDRED

Hereditary progressive arthro-ophthalmopathy, or ''Stickler syndrome,' ' is an autosomal dominant osteochondrodysplasia characterized by a va riety of ocular and skeletal anomalies which frequently lead to retina l detachment and precocious osteoarthritis. A variety of mutations in the COL2A1 gene have been identified in ''Stickler'' families; in most cases studied thus far, the consequence of mutation is the premature generation of a stop codon, We report here the characterization of a C OL2A1 gene mutation in the original kindred described by Stickler et a l. [1965]. Conformational sensitive gel electrophoresis (CSGE) [Gangul y et al., 1993] was used to screen for mutations in the entire COL2A1 gene in an affected member from the kindred, A prominent heteroduplex species was noted in the polymerase chain reaction (PCR) product from a region of the gene including exons 17 to 20. Direct sequencing of PC R-amplified genomic DNA resulted in the identification of a base subst itution at the A(-2) position of the 3' splice acceptor site of IVS17. Sequencing of DNA from affected and unaffected family members confirm ed that the mutation segregated with the disease phenotype, Reverse tr anscriptase-PCR analysis of poly Af RNA demonstrated that the mutant a llele utilized a cryptic splice site in exon 18 of the gene, eliminati ng 16 bp at the start of exon 18, This frameshift eventually results i n a premature termination codon. These findings are the first report o f a splice site mutation in classical Stickler syndrome and they provi de a satisfying historical context in which to view COL2A1 mutations i n this dysplasia. (C) 1996 Wiley-Liss, Inc.