Detection and allele-frequencies of the 833T>C, 844ins68 and a novel mutation in the cystathionine β-synthase gene

Background: The most common 833T>C/844ins68 in cis double mutation in the cystathionine beta synthase (CBS) gene probably is non-pathogenic because the 68-bp insertion eliminates the 833T>C mutation due to alternative splicing. However, allele frequency and effects of the isolated 833T>C mutation are unclear. Method: DNA was isolated from 500 volunteers and used directly for PCR-RFLP of CBS gene exon-8. A new primer design was developed to create annealing sites upstream and downstream of exon-8 for forward and reverse primers, respectively. The design was made to exclude sequence homology of the forward primer with the insert fragment and to introduce an internal Bsr1 digestion site. Results: A new 9276G>A mutation was found in intron 8. Because of this mutation, an extra Bsr1 digestion site is created in intron 8. In Caucasian volunteers, the following allele frequencies were found: 833T>C=0.2%, 833T>C/844ins68=10.2%, and 9276G>A=0.2%. Conclusion: The developed PCR-RFLP method is able to detect the 833T>C mutation, the 833T>C/844ins68 polymorphism as well as a new 9276G>A mutation in intron 8. Further study should explore the effect of the isolated 9276G>A mutation.