Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4 C

Abstract

During the course of investigating the frequency of a CYP2A6 whole deletion-type polymorphism (CYP2A6*4C) in Japanese, an unexpectedly large population of heterozygotes for CYP2A6*4C and the wild-type (CYP2A6*1A) was found. Cloning of a cDNA encoding CYP2A6 from the liver of individuals judged as heterozygotes for CYP2A6*4C and the CYP2A6*1A was carried out to identify the causal allele(s) responsible for a possible overestimation. A clone isolated from the liver cDNA library possessed 58 bp sequences in the 3'-untranslated region, which was replaced with the corresponding region of the CYP2A7 gene. The same gene conversion existed in the genomic DNA, indicating that the replacement was not a cloning artifact. Based on the gene structure of the allele (CYP2A6*1B), this variant was thought to be one of the causal alleles responsible for overestimation of heterozygotes for CYP2A6*4C and CYP2A6* A. To investigate this further, we developed a genotyping method which could distinguish the CYP2A6*A, CYP2A6*1B and CYP2A6*4C alleles from each other. The results clearly showed that CYP2A6*1B was the sole allele responsible for the overestimation. We conclude that the new genotyping method allows determination of six genotypes of the CYP2A6 gene, simultaneously and precisely, in both Oriental and Caucasian populations.

Association of CYP2A6*1B genetic variant with the amount of smoking in French adults from the Stanislas cohort

“…4,5 11 observed that subjects with the same reduced-activity alleles were not less likely to smoke. Loriot et al, 12 observed no difference in the smoking status according to the CYP2A6 genotype.…”

“…32 CYP2A6 gene was analysed for the gene conversion in the 3 0 -flanking region adjacent to exon 9 (CYP2A6*1B) and the deletion type (CYP2A6*4A) according to a method reported by Ariyoshi et al 4 In brief, a region ranging from exon 8 of the CYP2A7 gene or CYP2A6 gene to a 3 0 -untranslated region in exon 9 of the CYP2A6 gene was amplified, and then digested by the restriction enzymes, AccII and Eco81I. The CYP2A6*1A genotype was defined according to Ariyoshi et al 4 as 'wild type' but may carry undetected mutations in other regions than the one explored.…”

“…2 The allele frequency of CYP2A6*4 is low in European and particularly French subjects (4%) compared to Japanese (18%). 4,5 A gene conversion of interest is CYP2A6*1B where the 3 0 -flanking region adjacent to exon 9 has been replaced by the corresponding CYP2A7 sequence (72 bp). 6 The subjects who possess the CYP2A6*1B allele appear to show higher capabilities of nicotine metabolism to cotinine.…”

CYP2A7 polymorphic alleles confound the genotyping of CYP2A6*4A allele

“…In the genotyping method by Ariyoshi et al, 15 the primer set of 2A6-B4 and 2A6UTR-AS1 was used (Figure 6a). The 2A6-B4 primer can anneal to exon 8 of both the CYP2A6 and CYP2A7 genes.…”

“…Restriction enzymes were 15 and Nakajima et al 14 The primers of 2A6-B4 15 and 2Aint7F 14 anneal to both the CYP2A6 and CYP2A7 genes. The primers of 2A6UTR-AS1 15 and 2A6R2 14 anneal to the CYP2A7 polymorphic alleles (CYP2A7*1B, CYP2A7*1C and CYP2A7*1D) as well as the CYP2A6 gene, resulting in the misgenotyping of CYP2A6*4. (b) Method by Oscarson et al 8 The primer of 2Aex7F anneals to both the CYP2A6 and CYP2A7 genes.…”