Single-nucleotide polymorphisms in intron 2 of CYPZ1P: Evidence for a higher rate of mutation at CpG dinucleotides in the functional steroid 21-hydroxylase gene and application to segregation analysis in congenital adrenal hyperplasia

Background: Intron 2 of CYP21, the functional steroid al-hydroxylase gene c ontains several single-nucleotide polymorphisms (SNPs). We tested the hypot hesis that intron 2 of the pseudogene, CYP21P, might also be polymorphic an d provide markers for segregation analysis of this region of the genome, in cluding observable markers for segregation analysis of CYP21 gene deletions . A comparison of SNPs in both genes might provide insights into the rates of mutation in these duplicated genes. Methods: After amplification with PCR, we examined restriction site polymor phisms in intron 2 of CYP21P in 24 members of the parental generation of th e Centre d'Etude du Polymorphisme Humain families and selected offspring. Results: Intron 2 of CYP21P contains frequent SNPs around nucleotide 398 an d nucleotide 509, which can be typed by PCR/restriction enzyme digestion wi th HaeIII. Of the 48 CYP21P alleles examined, 44 could be characterized una mbiguously. Of these 44 alleles, 4 were deleted, and the frequencies of res triction at the polymorphic HaeIII sites were 20 of 40 at nucleotide 398 an d 30 of 40 at nucleotide 509. Both polymorphisms result from C-->T transiti ons that occur at CpG dinucleotides. The frequencies of C at these nucleoti des in CYP21P are significantly higher than at the corresponding nucleotide s in CYP21 of the same individuals (P <0.01). Conclusion: These data suggest that these CpG dinucleotides are more freque ntly mutated in CYP21 than in CYP21P, and that several mutations at CpG din ucleotides in the coding regions of CYP21 might result from CpG instability rather than the more usually proposed mechanism of gene conversion. These frequent SNPs provide useful markers for studying both allelic segregation of CYP21, particularly for chromosomes with known CYP21 deletions, and for investigating the origin of these polymorphisms. (C) 1999 American Associat ion for Clinical Chemistry.