Rapid determination of COL2A1 mutations in individuals with Stickler syndrome: Analysis of potential premature termination codons

Stickler syndrome is one of the milder phenotypes resulting from mutations in the gene that encodes type-II collagen, COL2A1, All COL2A1 mutations kno wn to cause Stickler syndrome result in the formation of a premature termin ation codon within the type-II collagen gene. COL2A1 has 10 inframe CGA cod ons, which can mutate to TGA STOP codons via a methylationdeamination mecha nism. We have analyzed these sites in genomic DNA from a panel of 40 Stickl er syndrome patients to test the hypothesis that mutations that cause Stick ler syndrome preferentially occur at these bases. Polymerase chain reaction (PCR) amplification of genomic DNA containing each of the in-frame CGA cod ons was done by one of two methods: either using primers that amplify DNA t hat includes the CGA codon, or using allele-specific primers that either am plify normal sequence containing a CGA codon or amplify a mutant sequence c ontaining a TGA codon, Analysis of PCR products by restriction endonuclease digestion or sequencing demonstrated the presence of a normal or mutated c odon, TGA mutations were identified in eight patients, at five of the 10 in -frame CGA codons, The identification of these mutations in eight of 40 pat ients demonstrates that these sites are common sites for mutations in indiv iduals with Stickler syndrome and, we propose, should be analyzed as a firs t step in the search for mutations that result in this disorder. Published 2000 Wiley-Liss, Inc.(dagger).