High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.     

The decreased in vivo clearance of CYP2D6 substrates by CYP2D6*10 might be caused not only by the low-expression but also by low affinity of CYP2D6

CYP2D6 exhibits genetic polymorphism with interindividual differences in metabolic activity. We have found a significant influence on the pharmacokinetics of venlafaxine by the CYP2D6*10 allele in a Japanese population. CYP2D6.10, which is translated from CYP2D6*10, has two amino acid substitutions: Pro34 --> Ser and Ser486 --> Thr. In this study, CYP2D6.10 was expressed in Saccharomyces cerevisiae and its catalytic activity for CYP2D6 substrates was investigated. The CYP2D6*10B- and *10C-associated cDNA were isolated from human lymphocyte genotyped as CYP2D6*10. In addition, three forms of CYP2D6, Pro34/Thr486 (PT), Ser34/Ser486 (SS), and Pro34/Ser486 (wild type, CYP2D6.1), were constructed by PCR-site mutagenesis to clarify the effects of the two amino-acid substitutions. The expression of CYP2D6 protein was confirmed by immunoblotting using CYP2D antibody. The absorbance at 450 nm was measured by CO-reduced difference spectra from five all microsome preparations. The CYP2D6 forms with Pro34 --> Ser amino acid substitution were at a lower expression than CYP2D6.1 from the findings of immunoblotting and spectral analysis. The apparent K(m) values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 microM, respectively, for bufuralol 1'-hydroxylation, and 9.0, 51.9, and 117.4 microM, respectively, for venlafaxine O-demethylation, respectively. The V(max) values were not significantly different among the three variants. These findings suggest that the decreased in vivo clearance by CYP2D6*10 was caused not only by low expression of but also the increased K(m) value of CYP2D6.     

Characterization of variant alleles of cytochrome CYP2D6 in a Spanish population

The CYP2D6 gene codes for a P450 monooxygenase which is involved in the biotransformation of a large number of commonly prescribed drugs. Adverse drug effects and therapeutic failure can be related to abnormal CYP2D6 activity. We investigated the allele and genotype frequencies of cytochrome P4502D6 in a Spanish population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes in our population and to design a feasible CYP2D6 genotyping protocol. The study included 105 healthy unrelated Spanish Caucasian volunteers. CYP2D6 genotyping was performed by a combination of long-PCR, direct sequencing and allele-specific real-time PCR. The frequency of the wild-type CYP2D6*1 allele was 31%. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity showed frequencies of 40.47, 2.38 and 1.90%, respectively. Frequencies of defective alleles *3, *4, *5 and *6 were 0.95, 13.8, 3.33 and 0.95%, respectively. The defective CYP2D6 alleles *7, *8, *12, *14, *15 and *21 were not found. Duplicated CYP2D6 alleles were detected at a frequency of 4.27%. Our protocol allows the identification of the four inactive CYP2D6 alleles (*3, *4, *5 and *6) and the detection of alleles with CYP2D6 *1, CYP2D6 *2 and CYP2D6*4 gene duplications. Testing for this reduced CYP2D6 allele set would facilitate its use in clinical practice by assisting in the development of individualized pharmacotherapy.     

CYP2A6 polymorphism reveals differences in Japan and the existence of a specific variant in Ovambo and Turk populations

CYP2A6 is a polymorphic enzyme, and CYP2A6 genotype has been shown to be associated with smoking habits and lung cancer. We investigated CYP2A6 polymorphism in Japanese from four different geographic areas of Japan and in the Ovambo and Turk populations. Using two polymerase chain reaction restriction fragment length polymorphisms (PCR-RFLPs), we identified the functionally important variants of CYP2A6: *1A, *1B, *1F, *1G, *4A, and *4D. In the Japanese population the highest frequencies of the CYP2A6*1A allele were observed in subjects from the Fukuoka (Kyushu Island) and Ehime (Shikoku Island) prefectures, whereas subjects in Shimane and Tottori (both located on the Japan Sea side of Honshu Island) showed the highest frequencies of the CYP2A6*1B allele. In the Tottori and Shimane groups no subject was homozygous for the CYP2A6*4A allele, a whole gene deletion type that is prevalent among Asians. In the Ovambo and Turk populations the CYP2A6*1A allele was predominant. Furthermore, two alleles undetected in the Japanese were observed in these latter two ethnic groups: CYP2A6*1G was found solely in the Ovambos, and CYP2A6*1F was found solely in the Turks. The present study is the first to show interprefecture differences in CYP2A6 polymorphism in Japanese who live in relatively close but distinct geographic areas; this is also the first study to evaluate CYP2A6 variations among these Japanese and the Ovambo and Turk populations. The distribution results of these alleles could help to define the true significance of CYP2A6 polymorphism as a genetic susceptibility marker in worldwide populations.     

Genetic variants in the cytochrome P450 2D6 gene in the Sri Lankan population

INTRODUCTION:

Cytochrome P450 2D6 (CYP2D6) enzymes are involved in the metabolism of a large number of commonly prescribed drugs such as antidepressants and cardiovascular drugs. The CYP2D6 *3, *4 and *14 variants associated with the loss of enzyme function; CYP2D6 *10 and *17 variants with reduced enzyme function; and CYP2D6 *2 variant with no effect on enzyme function. Establishing the frequency of these variant alleles in Sri Lankan population would be useful for optimizing pharmacotherapy with CYP2D6-substrate drugs.

OBJECTIVE:

The objective of this study was to determine the prevalence of CYP2D6 *2, *3, *4, *10, *14 and *17 variants in the main ethnic groups in the Sri Lankan population.

MATERIALS AND METHODS:

A total of 90 deoxyribonucleic acid (DNA) samples (30 each from Sinhalese, Tamils and Moors) were selected from a DNA resource at the Human Genetic Unit, Faculty of Medicine, University of Colombo. This collection had been made for population genetic studies from a random population based volunteers. Genotyping was performed using published polymerase chain reaction/restriction fragment length polymorphism methods.

RESULTS:

The prevalence of the CYP2D6 variants in Sinhalese, Sri Lankan Tamils and Moors respectively were CYP2D6 *2: 37%, 41.6% and 37.9%; CYP2D6 *3: 60.3%, 45% and 30%; CYP2D6 *4: 21.6%, 6.6% and 8.3%; CYP2D6 *10: 40%, 35% and 44%. CYP2D6 *14 and *17 variants were not identified.

CONCLUSION:

CYP2D6*3, *4 and *10 variants, which are associated with reduced or loss of CYP2D6 enzyme function were found in our population in significant frequencies. CYP2D6*4, which is reported to be a Caucasian variant was also found in all three ethnic groups.     

Frequencies of four genetic polymorphisms in the CYP1A2 gene in Turkish population

Cytochrome P450 (CYP) 1A2 gene is involved in the metabolic activation of several carcinogens and altered metabolization of some clinically used drugs. We aimed to investigate the distributions of genetic polymorphisms -3860 (G/A)(CYP1A2*1C) and -2467 (T/del)(CYP1A2*1D) in the 5'-flanking region and -739 (T/G)(CYP1A2*1E) and -163(C/A)(CYP1A2*1F) in the first intron of the CYP1A2 gene in 110 unrelated healthy Turkish volunteers by PCR-RFLP technique. The frequencies of each polymorphism in Turkish population were found as 0.04, 0.92, 0.01, 0.27 for CYP1A2*1C, CYP1A2*1D, CYP1A2*1E, CYP1A2*1F, respectively. Compared with other populations, CYP1A2*1D has been found to be significantly increased in Turkish population. On the other hand, in general, the frequencies of the other polymorphisms were concordant with those in the Egyptian and Caucasian populations, and were different from those in the Japanese, Chinese and Ethiopian populations. Our results suggest that due to increased frequency of CYP1A2*1D in Turkish population, functional significance of CYP1A2*1D should be evaluated. It might be screened to determine the relationship between CYP1A2*1D and CYP1A2 related drug metabolisms in associated groups.     

Polymorphisms and phenotypic analysis of cytochrome P450 2D6 in the Tibetan population

The cytochrome P450 2D6 enzyme (CYP2D6) metabolizes about 25% of prescribed drugs in the endoplasmic reticulum, and genetic polymorphisms in CYP2D6 can greatly affect its activity and lead to differences among individuals in drug efficacy and adverse drug reactions. To investigate genetic polymorphisms in CYP2D6 among Tibetan Chinese, we directly sequenced the whole gene in 96 unrelated, healthy Tibetans from The Tibet Autonomous Region of China and screened for genetic variants in the promoter, exons, introns, and 3′UTR. We detected fifty-one genetic polymorphisms in CYP2D6, and 16 of them are novel. The allele frequencies of CYP2D6*1, *2, *5, *10, *41, and *49 were 0.25, 0.43, 0.02, 0.29, 0.02, and 0.01, respectively. The frequency of CYP2D6*10, a putative poor-metabolizer allele, was lower in our sample population compared with that in the Han Chinese population (p < 0.001). In addition, haplotype analysis allowed 15 CYP2D6 haplotypes to be classified into three groups. In conclusion, our results provide basic information about CPY2D6 alleles in Tibetans and suggest that the enzymatic activities of CYP2D6 may differ among the diverse ethnic populations of China. Our results provide a basis for safer drug administration and better therapeutic treatment among Tibetans.     

Frequency of single nucleotide polymorphisms of CYP2D6 in the Czech population

CYP2D6 is a member of cytochrome P450 enzymes that metabolise over 25% of commonly used drugs. Genetic polymorphisms can cause insufficient drug efficacy at usually administered doses or can be the cause of adverse drug reaction. CYP2D6 genotyping can be used to predict CYP2D6 phenotype and thereby explain some abnormalities in drug response and thus optimize pharmacotherapy. The aim of this study was to investigate the frequency of functionally important variant alleles of the CYP2D6 gene throughout the Czech population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes. The DNA of 223 unrelated, healthy volunteers was analysed to detect the presence of CYP2D6*6, *5, *4, *3 and gene duplication. The variant allele frequencies in our population were 0.22%, 3.14%, 22.87%, 1.12% and 3.14% for CYP2D6*6, CYP2D6*5, CYP2D6*4, CYP2D6*3 and CYP2D6*MxN, respectively. Fifteen subjects carried two variant alleles leading to predicted poor type of metabolism, 84 subjects were heterozygous extensive metabolizers (het-EM). The full-text contains detailed comparison with European white populations. The distribution of variant alleles complies with the Hardy-Weinberg equilibrium. The frequencies of functional variant alleles of CYP2D6 in Czech population are in concordance with other Caucasian populations.     

Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences.

Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of .005 (*1x2), .013 (*2x2), and .001 (*4x2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T1957C), *2B (additional C2558T), and *4E (additional C2938T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EM/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment.     

Polymorphism of CYP1A1 and CYP2D6 genes in tundra nentses and European populations of western Siberia

Data on the first examination of the CYP1A1 and CYP2D6 genes' polymorphism in the populations of Tundra Nentsis (Yamalo-Nenetskii Autonomous District) and migrant population of Western Siberia (Novosibirsk oblast and Altaiskii krai) are presented. The frequency of the 2D6*4 mutant allele in Tundra Nentsis, characterized by a two-component Caucasoid and Mongoloid origin, was shown to be intermediate in Caucasoid and Mongoloid populations. The frequencies of the 2D6*4 and 1A1Val* mutant alleles across migrant inhabitants of Western Siberia (Caucasoid populations) were similar to that reported for the Caucasoid populations overall. Distribution of the CYP1A1 genotypes (Ile/Ile, Ile/Val*, and Val*/Val*) in Tundra Nentsis was similar to that found in Mongoloid groups. However, the frequency of the 1A1Val* allele in Tundra Nentsis was 1.5 times higher than that in the Southern Mongoloid populations (Chinese, Koreans, and Japanese).     

CYP2D6 polymorphism and the presence of anti-LKM-1 in patients with chronic hepatitis C

Anti-LKM-1 autoantibodies are directed mostly at cytochrome P450 2D6 (CYP2D6) autoantigen, whose activity ranges from "complete deficiency" to "extensive metabolism" due to genetic polymorphism. We aimed to find any relevance of CYP2D6 alleles to the presence/absence of anti-LKM-1 in Japanese patients with chronic hepatitis C. The frequency of an extensive metabolizer-type allele (CYP2D6*1) in anti-LKM-1-positive patients was higher than that in anti-LKM-1-negative patients (0.800 vs 0.431; P = 0.0035), while the CYP2D6*10 allele with moderately reduced activity was less frequent in the former than the latter (0.050 vs 0.389; P = 0.0069). Moreover, the rate of homozygosity for CYP2D6*1 showed a striking difference between the two groups (70% vs 19%; P = 0.0021). These findings suggest that a genetic predisposition to produce the enzyme CYP2D6 of extensive metabolizer-type is associated with the induction of anti-LKM-1 in chronic hepatitis C patients.     

CYP2D6.10 present in human liver microsomes shows low catalytic activity and thermal stability

Comparing bufuralol 1'-hydroxylase activity among liver microsomes prepared from individuals whose CYP2D6 genotypes had been determined, we found that the activity tended to decrease depending on the number of the CYP2D6*10 allele. Pre-incubation of liver microsomes from individuals homozygous for the CYP2D6*10 allele resulted in a decrease in the enzyme activity more rapidly than those from individuals homozygous for the CYP2D6*1, suggesting that not only the catalytic activity but also the thermal stability of the enzyme appeared to be affected by the genetic polymorphism. To confirm this hypothesis, the kinetic parameters of CYP2D6.1 and CYP2D6.10 were compared for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation using microsomes prepared from yeast transformed with plasmids carrying CYP2D6 cDNAs (*1A and *10B). Kinetic studies of these CYP2D6 forms indicated clear differences in the metabolic activities between the wild (CYP2D6.1) and the mutant enzymes (CYP2D6.10). Bufuralol 1(')-hydroxylase activity in microsomes of yeast expressing CYP2D6.10 was rapidly decreased by heat treatment, supporting the idea that the thermal stability of the enzyme was reduced by amino acid replacement from Pro (CYP2D6.1) to Ser (CYP2D6.10). These data strongly suggest that the thermal instability together with the reduced intrinsic clearance of CYP2D6.10 is one of the causes responsible for the known fact that Orientals show lower metabolic activities than Caucasians for drugs metabolized mainly by CYP2D6, because of a high frequency of CYP2D6*10 in Orientals.     

Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay.

Drugs and carcinogens are excreted from the body after metabolic conversion involving enzymes mediating oxidative metabolism and conjugation. Many of the corresponding genes exhibit functional polymorphisms that contribute to individual cancer susceptibility. To increase the efficiency and to facilitate genotyping, we developed a combined approach (PCR-ASO) which includes multiplex PCR and allele-specific oligonucleotide (ASO) hybridization. PCR primer pairs were used to amplify the following alleles/variants: CYP1A1*1, *2A, *2B; CYP2D6*3, *4; NAT1*4, *3, *10, *11, *14, *15; and NAT2*4, *5A, *5B, *5C, *6A, *7B. The products were dot-blotted and polymorphisms were detected by hybridization with ASO probes for both wild-type and variant sites in parallel. This approach was validated by genotyping DNA samples from a French-Canadian population that was previously analyzed by PCR-RFLP. The variants frequencies were compared with the data on other populations available in the literature. The PCR-ASO assay appears to be simple, efficient, and cost-effective, particularly if a large number of samples are to be screened for several DNA variants. This approach has potential for automation with microplates and robotic workstations for high throughput.     

Identification of new alleles and the determination of alleles and genotypes frequencies at the CYP2D6 gene in Emiratis

CYP2D6 belongs to the cytochrome P450 superfamily of enzymes and plays an important role in the metabolism of 20-25% of clinically used drugs including antidepressants. It displays inter-individual and inter-ethnic variability in activity ranging from complete absence to excessive activity which causes adverse drug reactions and toxicity or therapy failure even at normal drug doses. This variability is due to genetic polymorphisms which form poor, intermediate, extensive or ultrarapid metaboliser phenotypes. This study aimed to determine CYP2D6 alleles and their frequencies in the United Arab Emirates (UAE) local population. CYP2D6 alleles and genotypes were determined by direct DNA sequencing in 151 Emiratis with the majority being psychiatric patients on antidepressants. Several new alleles have been identified and in total we identified seventeen alleles and 49 genotypes. CYP2D6*1 (wild type) and CYP2D6*2 alleles (extensive metaboliser phenotype) were found with frequencies of 39.1% and 12.2%, respectively. CYP2D6*41 (intermediate metaboliser) occurred in 15.2%. Homozygous CYP2D6*4 allele (poor metaboliser) was found with a frequency of 2% while homozygous and heterozygous CYP2D6*4 occurred with a frequency of 9%. CYP2D6*2xn, caused by gene duplication (ultrarapid metaboliser) had a frequency of 4.3%. CYP2D6 gene duplication/multiduplication occurred in 16% but only 11.2% who carried more than 2 active functional alleles were considered ultrarapid metabolisers. CYP2D6 gene deletion in one copy occurred in 7.5% of the study group. In conclusion, CYP2D6 gene locus is heterogeneous in the UAE national population and no significant differences have been identified between the psychiatric patients and controls.     

CYP2D6 and CYP1A1 mutations in the Turkish population

Drugs and carcinogens are substrates of a group of metabolic enzymes including cytochrome p450 enzymes and gluthatione S-transferases. Many of the genes encoding these enzymes exhibit functional polymorphisms that contribute individual cancer susceptibility and drug response. Molecular studies based on these polymorphic enzymes also explain the aetiology of cancer and therapeutic management in clinics. We analysed the cytochrome p4501A1 (CYP1A1) and 2D6 (CYP2D6) variant genotype and allele frequencies by PCR-RFLP in Turkish individuals (n=140). The frequency of the CYP1A1*2A mutant allele was found to be 15.4%, and the CYP2D6*3 and *4 mutant allele (poor metabolizer) frequencies were 2.5% and 13.9%, respectively. This study presents the first results of CYP1A1 and CYP2D6 mutant allele distributions in the Turkish population and these data provide an understanding of epidemiological studies that correlate therapeutic approaches and aetiology of several types of malignancy in Turkish patients.     

Population analysis of xenobiotic metabolizing genes in South Brazilian Euro and Afro-descendants

Individual variability in xenobiotic metabolism has been associated with susceptibility to developing complex diseases. Genes involved in xenobiotic metabolism have been evaluated in association studies; the difficulty of obtaining accurate gene frequencies in mixed populations makes interpretation of the results difficult. We sought to estimate population parameters for the cytochrome P450 and glutathione S-transferase gene families, thus contributing to studies using these genes as markers. We describe the frequencies of six genes (CYP1A1, CYP2D6, CYP2E1, GSTM1, GSTT1, and GSTP1) and estimate population parameters in 115 Euro-descendants and 196 Afro-descendants from Curitiba, South of Brazil. PCR-based methods were used for genotyping, and statistical analysis were performed by AMOVA with ARLEQUIN software. The mutant allele frequencies in the Afro-descendants and Euro-descendants, respectively, were: CYP1A1*2A = 30.1% and 15.2%; CYP2D6*4 = 14.5% and 21.5%; CYP2E1*5B = 7.9% and 5%; GSTP1*B = 37.8% and 28.3%. The null genotype frequencies were: GSTM1*0 = 36.8% and 46.1%; GSTT1*0 = 24.2% and 17.4%.     

Determination of CYP2D6 gene copy number by multiplex polymerase chain reaction analysis

Cytochrome P450 2D6 (CYP2D6) copy number variation (CNV) influences the metabolism of 15-25% of clinical drugs. Here we describe a novel multiplex polymerase chain reaction (PCR) analysis method that accurately detects CYP2D6 CNV and CYP2D6*9 allele. It includes the amplification of 2 CYP2D6 and 7 control (AQP1, CYP3A4, MDR1, and SDHB) fluorescent PCR products that are separated on a capillary sequencer and normalized using reference samples. The technique was validated using 27 PCR-restriction fragment length polymorphism (RFLP) pregenotyped samples and further tested in 75 Caucasian samples. The method assigns the correct CYP2D6 copy number, independent of already characterized CYP2D6 single nucleotide polymorphisms (SNPs), and could easily be applied to clinical samples.     

CYP2A6*6, a novel polymorphism in cytochrome p450 2A6, has a single amino acid substitution (R128Q) that inactivates enzymatic activity

By using the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP), a novel polymorphism of CYP2A6, CYP2A6*6, was detected in 0.4% of the Japanese population. To study the enzymatic properties of the CYP2A6.6 protein with a single amino acid substitution of arginine 128 to glutamine, both this isozyme and the CYP2A6.1 protein (wild-type) were produced in insect cells using a baculovirus system. Coumarin 7-hydroxylation, which reflects CYP2A6 activity, was significantly reduced (one-eighth of normal) in cell lysate from CYP2A6*6-transfected Sf9 cells compared with that lysate from CYP2A6*1-transfected cells. To clarify the mechanism of inactivation of the CYP2A6.6 enzyme, the heme content and reduced CO difference spectrum were examined. Although CYP2A6.6 retained about one-half the heme content of CYP2A6.1, the reduced CO-bound Soret peak was completely lost. These results suggest that the inactivation of CYP2A6.6 is mainly due to disordering of the holoprotein structure rather than a failure of heme incorporation.     

An in vivo pilot study characterizing the new CYP2A6*7, *8, and *10 alleles.

We developed genotyping assays for CYP2A6*7 (Ile471Thr) and CYP2A6*8 (Arg485Leu). We found higher allelic frequencies in Japanese and Chinese versus Caucasians and identified an allele in which both substitutions occur together (CYP2A6*10). We created a homology model for predicting the impact of allelic variants on enzymatic activity and subsequently tested this in vivo in a pilot kinetic study. Consistent with our homology model predictions, we found (i) that CYP2A6*7 produces an enzyme that has decreased (not inactive) activity for metabolizing nicotine and coumarin; (ii) that CYP2A6*8 is unlikely to affect catalytic activity in vivo; and (iii) that having both substitutions together on an allele (CYP2A6*10) dramatically reduces function and may be fully inactive for some substrates. In conclusion, this study identifies, at relatively high frequency in Asians, an allele with decreased activity (may be substrate selective), a fully functional allele, and an allele containing both substitutions in which function is dramatically reduced.