Molecular diversity at the CYP2D6 locus in the Mediterranean region

Abstract

Despite the importance of cytochrome P450 in the metabolism of many drugs, several aspects of molecular variation at one of the main loci coding for it, CYP2D6, have never been analysed so far. Here we show that it is possible to rapidly and efficiently genotype the main European allelic variants at this locus by a SNaPshot method identifying chromosomal rearrangements and nine single-nucleotide polymorphisms. Haplotypes could be reconstructed from data on 494 chromosomes in six populations of the Mediterranean region. High levels of linkage disequilibrium were found within the chromosome region screened, suggesting that CYP2D6 may be part of a genomic recombination block, and hence that, aside from unequal crossingover that led to large chromosomal rearrangements, its haplotype diversity essentially originated through the accumulation of mutations. With the only, albeit statistically insignificant, exception of Syria, haplotype frequencies do not differ among the populations studied, despite the presence among them of three well-known genetic outliers, which could be the result of common selective pressures playing a role in shaping CYP2D6 variation over the area of Europe that we surveyed.

Analysis of CYP2D6 Allele Frequencies and Identification of Novel SNPs and Sequence Variations in Sardinians

“…Only sub-variant CYP2D6 * 6A was found. CYP2D6 * 9 frequency is similar to that found by Fuselli et al (0.6%) [12], even though this study did not �nd CYP2D6 * 9 in Sardinians.…”

“…Allele frequencies, detected from the 250 Sardinian individuals (Table 5), were compared with frequencies previously reported for Caucasians [11,12,18,[23][24][25][26] and reported in Table 6. Particularly, differences were found in CYP2D6 * 2 and * 41 alleles.…”

“…Isolated populations, such as that found in Sardinia [9,10], could be extremely useful for mapping novel SNPs and haplotypes for speci�c genes. ere are very few studies on CYP450 subfamily genes in Sardinian people [8,11,12]; therefore, we analyzed the CYP2D6 gene for sequence variations in Sardinians and also compared resulting allele frequencies with those observed in Caucasian population. …”

CYP2D6 in the brain: genotype effects on resting brain perfusion

“…Genotyping was performed with a combination of long PCR and primerextension method using the SNaPshot-Kit (Applied Biosystems, Mannheim, Germany) as described previously. 35,36 The sequence between exon 9 of the upstream and intron 2 of the downstream CYP2D6 gene copy of the duplicated CYP2D6 allele was amplified using duplication-specific long PCR. The PCR reaction was performed using primer set CYP2D6-F:…”

Pharmacokinetics of codeine and its metabolite morphine in ultra-rapid metabolizers due to CYP2D6 duplication

“…18 Identification of allele CYP2D6*41 was based on the À1584 G4C and the 4180 G4C polymorphism as described earlier. 19 In addition, we have analyzed which of the single nucleotide polymorphisms was in the duplication allele and which was in the other not duplicated allele by using a modification of the single-base primer extension method described by Fuselli et al 20,21 Briefly, the sequence between the exon 9 of the upstream and the intron 2 of the downstream CYP2D6 gene copy of the CYP2D6 duplication allele was amplified using duplication-specific long PCR with the primers 5 0 -GCC ACC ATG GTG TCT TTG CTT TC-3 0 and 5 0 -ACC GGA TTC CAG CTG GGA AAT G-3 0 22 and Expand Long Template PCR System (Roche, Mannheim, Germany) as follows: 27 ml reactions containing buffer-1 of the long PCR kit were supplemented with additional MgCl 2 to a final concentration of 3.5, 0.35 mM dNTPs, 0.2 mM primers and 1.5 U polymerase mix, and were incubated for 2 min at 941C followed by 35 cycles (10 s at 961C, 20 s at 601C and for 7 min at 681C) and 7 min at 681C. The obtained PCR product was used as a template for the single-base primer extension reaction (SNaPshot, Applied Biosystems) designed to genotype the polymorphic position 4180G4C of the upstream gene-copy and positions À1584G4C and 100C4T of the downstream gene-copy.…”