Objective: We used single-strand conformational polymorphism (SSCP) to screen for mutations at nucleotides 833 and 919 of the cystathionine β- synthase (CBS) gene in 13 patients with homocystinuria and 11 of their relatives. Methods: Exon 8 of genomic DNA was selectively amplified by PCR using primers derived from intronic sequences of the human CBS gene. SSCP analysis was performed on the amplified products Genotypes identified by SSCP were confirmed by DNA sequencing and an allele-specific PCR method. Results: SSCP identified 5 patterns corresponding to five genotypes. We confirmed that the different genotypes result from mutations at nucleotides 833 and 919 of the CBS gene, and that these 2 mutations account for approximately 50% of affected alleles in homocystinuria patients Conclusion: Our recent elucidation of intron-exon borders and intronic sequences of the CBS gene has made possible the use of SSCP to screen for known/unknown mutations in the CBS gene. Because T833 C and G919A represent the two most common mutations and both are located within exon 8 of the CBS gene, SSCP of exon 8 allows screening of the heterozygous carrier state of these mutations in a large population, to determine the importance of heterozygosity of CBS mutations as the cause of mild hyperhomocyst(e)inemia associated with premature vascular diseases.
- amplification refractory mutation system
- cystathione β-synthase
- genetic variants
- single- strand conformational polymorphism