Allele Nomenclature Committee Research Articles

Prediction of the CYP2D6 enzymatic activity based on investigating of the CYP2D6 genotypes around the vivax malaria patients in Yunnan Province, China

BackgroundIn recent years, the incidence rate of vivax malaria recurrence still had 3.1% in Yunnan Province population after eradication therapy using primaquine (PQ). In order to understand the specific failure reasons for preventing vivax malaria relapses, a preliminary exploration on the CYP2D6 enzyme activity was carried out in the vivax malaria patients in Yunnan Province population by analysing mutational polymorphism in the coding region of CYP2D6 gene.MethodsBlood samples were collected from vivax malaria patients with suspected relapse (SR) and non-relapsed (NR) malaria in Yunnan Province. The DNA fragments containing 9 exons regions of human CYP2D6 gene were amplified by performing PCR and sequenced. The sequencing results were aligned by using DNAStar 11.0 to obtain the coding DNA sequence (CDS) of CYP2D6 gene. DnaSP 6.11.01 software was used to identify mutant polymorphisms and haplotypes of the CDS chain. The waterfall function of GenVisR package in R was utilized to visualize the mutational landscape. The alleles of CYP2D6 gene were identified according to the criteria prescribed by Human Cytochrome P450 (CYP) Allele Nomenclature Committee Database and the CYP2D6 enzyme activity was predicted based on diploid genotype.ResultsA total of 320 maternal CDS chains, including 63 from SR group and 257 from NR group, were obtained. Twelve mutant loci, including c.31 (rs769259), c.100 (rs1065852), c.271 (rs28371703), c.281 (rs28371704), c.294 (rs28371705), c.297 (rs200269944), c.336 (rs1081003), c.408 (rs1058164), c.505 (rs5030865), c.801 (rs28371718), c.886 (rs16947), and c.1,457 (rs1135840) were observed on the 640 CDS chains (including 320 maternal and 320 paternal chains). The high-frequency mutation at rs1135840 (0.703) and low-frequency mutation, such as rs28371703, were detected only in the SR group. The frequency of mutant rs1058164 and rs1135840 were significantly increased in the SR group ({x}^{2}= 4.468, 5.889, P < 0.05), as opposed to the NR group. Of the 23 haplotypes (from Hap_1 to Hap_23), the nomenclatures of 11 allelic forms could be found: Hap_3 was non-mutant, Hap_2 accounted for the highest frequency (36.9%, 236/640), and Hap_9 had the most complex sequence structure, containing 7 loci mutations. Allele *10 was the most frequent among these genotypes (0.423). Among the allele *10 standard named genotypes, *1/*10, *1/*1 and *2/*10 were significantly more frequent in the NR group ({x}^{2}= 3.911, P < 0.05) and all showed uncompromised enzyme activity; the impaired genotype *10/*39 was more frequent in the SR group ({x}^{2}= 10.050, P < 0.05), and genotype *4/*4was detected only in the SR group.ConclusionIn the patients receiving PQ dosage in Yunnan Province population, both rs1135840 single nucleotide polymorphism and *10 allele form was common in the CYP2D6 gene. Low-frequency mutation sites, such as rs28371703, were only presented in patients with vivax malaria relapse.

Identification and Functional Assessment of a New CYP2C9 Allelic Variant CYP2C9*59.

CYP2C9, one of the most important drug-metabolizing enzymes, is responsible for metabolizing approximately 15% of clinically important drugs, including warfarin, diclofenac, and losartan. Similar to other CYP members, human CYP2C9 exhibits marked genetic polymorphisms among individuals of different ethnicities. In this study, a novel missense mutation (1300A>T) was identified in a warfarin-sensitive patient after a genetic screen of three candidate genes related to high variability in response to warfarin doses. This base transversion leads to an Ile-to-Phe amino acid substitution at codon 434 within the CYP2C9 protein, and this new variant has been named a novel allele, CYP2C9*59, by the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki.se/cyp2c9.htm). The exogenous expression of CYP2C9.59 in insect cell microsomes revealed that, despite a similar protein expression level as wild-type CYP2C9, variant CYP2C9.59 exhibited significantly reduced maximal velocity, Vmax, and/or increased Michaelis constant, Km, values toward three CYP2C9-specific substrates. Our data suggest that the 1300A>T mutation can greatly decrease the enzymatic activity of the CYP2C9 protein both in vitro and in vivo.

Analysis of genetic polymorphism and biochemical characterization of a functionally decreased variant in prostacyclin synthase gene (CYP8A1) in humans

Prostacyclin synthase (CYP8A1) is an enzyme responsible for the biosynthesis of prostacyclin (PGI2) which inhibits platelet activation and exhibits anti-inflammatory effect. The objectives of this study were to identify CYP8A1 genetic variants and characterize functional consequences of CYP8A1 variants. In total, 27 variants including four previously unidentified single-nucleotide polymorphisms (SNPs) were identified by direct DNA sequencing in Koreans (n=48). Among them, CYP8A1 A447T and E314Stop were newly assigned as CYP8A1∗5 and CYP8A1∗6 by the Human Cytochrome P450 Allele Nomenclature Committee, respectively. CYP8A1∗5 was found in the heme binding area in three individuals as a heterozygous mutation. To investigate the functional change of CYP8A1∗5, CYP8A1∗5 and wild-type CYP8A1 protein were overexpressed in an Escherichia coli expression system and purified. Metabolism of PGH2 by the CYP8A1∗5 protein exhibited significantly decreased activity, resulting in a 45% decrease in Vmax and a 1.8-fold decrease in intrinsic clearance compared to the wild-type. Based on the predicted crystal structure of CYP8A1∗5 using the Molecular Operating Environment platform, the distance from CYP8A1 Cys441 to the heme was altered with a significantly changed binding free energy for the mutant protein. Further studies would be needed to determine the effect of CYP8A1∗5 on PGI2 levels in humans.

Genetic Variations of Human CYP2D6 in the Chinese Han Population

The purpose of this study was to determine the genetic polymorphisms of the CYP2D6 gene and to elucidate the allele distribution pattern in the Chinese Han population. We used PCR and bidirectional sequencing methods to analyze all nine exons of the CYP2D6 gene in 2129 unrelated, healthy Chinese Han subjects from two geographical locations in China: the northern and southern regions. In total, 165 mutated sites were detected in 2129 participants, of which 67 sites were reported for the first time. Among these novel mutation sites, 22 were nonsynonymous and 12 were named as novel alleles (*87-*93, *94A, *94B and *95-*98) by the Human CYP Allele Nomenclature Committee. In addition, 29 previously reported alleles and 84 genotypes were also detected in 1954 volunteers. Functional prediction of novel variants revealed that eight variants might have a deleterious effect on CYP2D6. Linkage disequilibrium analysis and tagSNP selection were performed separately. By using these methods, distinct differences were found between the two regions. This study provides the most comprehensive data concerning CYP2D6 polymorphisms in the Chinese Han population to date and increases the number of known alleles; these findings may greatly contribute to the development of personalized medicine for the Chinese Han population.

CYP21A2 p.E238 Deletion as Result of Multiple Microconversion Events

More than 90% of congenital adrenal hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macroconversion or microconversion events. Only around 5% of all disease-causing CYP21A2 alleles harbor rare mutations that do not originate from the pseudogene. A complete list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.imm.Ki.se/CYPalleles/cyp21.htm). In this report, we describe clinical and genetic findings regarding an Italian woman suffering from a classic salt-wasting form of CAH due to a severe 21-hydroxylase deficiency. A complex genetic family study was performed including a prenatal diagnosis. The patient was found to be heterozygous for p.I172N (exon 4), p.E238del (exon 6), p.M239K (exon 6), and p.F306insT (exon 7) mutations and homozygous for p.I236N (exon 6) and p.V237E (exon 6) mutations. The deletion of glutamic acid 238 is a new mutation not reported before in the literature. CYP21A2 genotyping has become a valuable complement to biochemical CAH investigation. We highlight the contribution of molecular genetic advancements to the clinical management of patients with 21-hydroxylase deficiency.

CYP2C9 polymorphism analysis in Han Chinese populations: building the largest allele frequency database

Genetic polymorphisms of CYP2C9 significantly influence the pharmacokinetics and pharmacodynamics of some drugs, which might result in adverse drug effects and therapeutic failure. Several studies have been performed on CYP2C9 genetic polymorphisms in Han Chinese populations. However, these studies only focused on two commonly investigated alleles, *2 and *3, in relatively small sample sizes. To scale up the gene-scanning region and determine relatively precise data on the genetic distribution pattern in Chinese populations, unrelated healthy Han Chinese volunteers from Zhejiang Province (n=1127) and Hebei (n=1000) Province were recruited as subjects for the direct sequencing of all exons of CYP2C9. As a result, 14 previously reported alleles were detected in this work, and 8 of these alleles (*14, *16, *19, *23, *27, *29, *33 and *34) were described for the first time in Chinese populations. In addition, 37 novel mutations were also detected, of which 22 variants were non-synonymous, and 21 new alleles, *36-*56, were designated by the Human CYP Allele Nomenclature Committee. In vitro functional analysis of these 22 novel CYP2C9 variants revealed that 17 mutations had a significant influence on the protein's catalytic activity. Our study provides the most accurate data on CYP2C9 polymorphisms in Han Chinese populations and detects the largest number of novel allelic variants existing to date. These new alleles will greatly enrich the current knowledge of naturally occurring CYP2C9 variants in Chinese populations.

CYP2D6*11 and Challenges in Clinical Genotyping of the Highly Polymorphic CYP2D6 Gene

CYP2D6 is genotyped clinically for prediction of response to tamoxifen, psychotropic drugs and other medications. Phenotype prediction is dependent upon accurate genotyping. The CYP Allele Nomenclature Committee maintains the allelic nomenclature for CYP2D6; however, in some cases, the list of polymorphisms associated with a given allele is incomplete. Clinical laboratories and in vitro diagnostic manufacturers rely upon this nomenclature, in addition to the literature, to infer allelic function and haplotypes and when they design CYP2D6-testing platforms. This article provides more complete sequencing data for the CYP2D6*11 allele and describes the difficulties encountered in genotyping CYP2D6 when incomplete data are available. The CYP Allele Nomenclature Committee should provide clear information about the completeness of the original data used to define each allele.

Resequencing, Haplotype Construction and Identification of Novel variants of CYP2D6 in Mexican Mestizos

The CYP2D6 enzyme participates in the metabolism of commonly prescribed drugs: antidepressants, antipsychotics and antihypertensives. The CYP2D6 gene shows a high degree of interindividual and interethnic variability that influences its expression and function. Mexican Mestizos are a recently admixed population resulting from the combination of Amerindian, European and, to a lesser extent, African populations. This study aimed to comprehensively characterize the CYP2D6 gene in Mexican Mestizos. We performed linkage disequilibrium and network analyses in resequencing data of 96 individuals from two regions within Mexico with a different history of admixture and particular population dynamics, the Northwestern state of Sonora and the Central-Pacific state of Guerrero. We identified 64 polymorphisms, including 14 novel variants: 13 SNPs and a CYP2D7 exon 2 conversion, that was assigned CYP2D6*82 by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee. Three novel SNPs were predicted to have functional effects. For CYP2D6*82 we hypothesize an Amerindian origin that is supported by its identification in three Mexican Amerindian groups (Mayas, Tepehuanos and Mixtecos). Frequencies of CYP2D6*1, *2, *4, *5, *10, *29, *53, *82 and its duplications were 50.0, 25.5, 14.1, 2.0, 2.6, 1.0, 0.5, 2.1 and 3.6%, respectively. We found significant frequency differences in CYP2D6*1 and *2 between Mexican Mestizos and in CYP2D6*1, *2, *4, *5, *10 and *29 between Mexicans and at least one other population. We observed strong linkage disequilibrium and phylogenetic relationships between haplotypes. To our knowledge, this study is the first comprehensive resequencing analysis of CYP2D6 in Mexicans or any other Latin American population, providing information about genetic diversity relevant in the development of pharmacogenomics in this region.

Discovery of a Novel Allelic Variant of CYP2C8, CYP2C8*11, in Asian Populations and Its Clinical Effect on the Rosiglitazone Disposition In Vivo

The objectives of this study were to identify the genetic variants of CYP2C8, analyze CYP2C8 single nucleotide polymorphisms (SNPs), and characterize their functional consequences in the CYP2C8 substrate drug rosiglitazone in humans. The direct full sequencing of CYP2C8 genomic DNA was performed in a Korean population (n = 50). A total of 17 CYP2C8 variants including a novel coding variant (E274Stop) were identified. The novel CYP2C8 E274Stop variant was assigned as CYP2C8*11 by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee. Seventeen SNPs were used to characterize linkage disequilibrium, haplotype structures, and haplotype tagging SNPs. Genotyping for CYP2C8*11 in an extended set of Koreans (n = 400), whites (n = 100), Han Chinese (n = 348), Vietnamese (n = 100), and African Americans (n = 93) was performed by a newly developed pyrosequencing method. The frequency of CYP2C8*11 was 0.3% in Koreans, 1% in Vietnamese, and 0.14% in Chinese. However, none of the whites or African Americans contained the CYP2C8*11 allele. Subjects with CYP2C8*1/*11 exhibited higher plasma concentration-time profiles of rosiglitazone than those of nine control subjects carrying CYP2C8*1/*1. The area under the concentration-time curve and peak plasma concentration of rosiglitazone in individuals carrying CYP2C8*1/*11 (n = 5) were 54 and 34% higher than the mean values observed in the control subjects carrying CYP2C8*1/*1 (P = 0.015 and P = 0.025, respectively). In summary, this is the first report to characterize the allele frequency and haplotype distribution of CYP2C8 in a Korean population, and it provides functional analysis of a new variant CYP2C8*11. Our findings suggest that individuals carrying CYP2C8*11, a null allele found in Asians only, may have lower activity for metabolizing CYP2C8 substrate drugs.

Papel del citocromo P450 en la farmacocinética y en la farmacogenética de los fármacos antihipertensivos

Introduction Antihypertensive drugs are principally metabolised by enzymes of the P450 (CYP450) cytochrome family. Response to hypertensive treatment whose genetic basis is beginning to be known presents inter-individual differences among patients. Objective The aim of this review is to document the role of cytochrome enzymes P450 in the metabolising process of antihypertensive drugs, and to identify the most relevant polymorphisms in genes that code for these enzymes in order to facilitate future studies in hypertension pharmacogenetics. Methods: A keyword search was performed in the following literature databases: Pubmed, Rxlist and Medscape. Genetic polymorphism public databases were also consulted (PharmGKB, NCBI and the CYP450 allele nomenclature committee web page . Results Enzymes CYP2D6, CYP2C9, CYP2D19 and CYP3A4 participate in the metabolising process of most antihypertensive drugs. Considering the allelic frequency in the population and the variability in the clinical response associated with genetic polymorphism, we find the study of the following alleles CYP2D6 *2, *4, deletion and duplication; CYP2C9*2 and *3, CYP2C19 *2 and CYP3A4*1B to be of crucial importance. Conclusions The study of polymorphisms in P450 cytochrome genes may contribute to an individualised therapy in the treatment against hypertension.

Molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency: an update of new CYP21A2 mutations

Steroid 21-hydroxylase deficiency is present in more than 90% of patients with congenital adrenal hyperplasia, an inherited metabolic disorder of adrenal steroidogenesis. Impaired enzymatic activity leads to the accumulation of metabolic intermediates (progesterone and 17-hydroxyprogesterone), which results in excessive androgen production and varied signs of virilisation. CYP21A2 is an active gene and encodes for the steroid 21-hydroxylase enzyme, whereas CYP21A1P is an inactive pseudogene that contains a series of deleterious mutations. The major part of disease-causing mutations in CYP21A2 alleles are CYP21A1P-derived sequence transferred to the active gene by macro or microconversion events. Approximately 5% of all disease-causing CYP21A2 alleles harbour rare mutations that do not originate from the pseudogene. A list of all reported CYP21A2 mutations can be found in the CYP21A2 database created by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (http:www.imm.Ki.se/CYPalleles/cyp21.htm). Unfortunately, the last update of this database was in 2006. However, over the last 4 years many other novel CYP21A2 mutations have been reported in PubMed. The aim of this review is to provide a focus on the molecular and genetic aspects of the diagnosis of 21-hydroxylase deficiency. In addition, an updated list of the last new CYP21A2 mutations is included.

Identification of a null allele of cytochrome P450 3A7: CYP3A7 polymorphism in a Korean population

Cytochrome P450 3A7 (CYP3A7) is expressed in the human fetal liver and plays a role in the metabolism of hormones, drugs, and toxic compounds. Genetic variants of CYP3A7 are associated with serum estrone level, bone density, and hepatic CYP3A activity in adults. We analyzed the genetic variations of CYP3A7 in a Korean population. From direct sequencing of all exons and flanking regions of the CYP3A7 gene in 48 Koreans, we found five genetic variants, including three novel variants. One variant, a thymidine insertion in exon 2 (4011insT), causes premature termination of CYP3A7 translation, which may result in a null phenotype. The novel variant was assigned to the CYP3A7*3 allele by the CYP allele nomenclature committee. For further screen of this novel variant in other ethnic populations, we used pyrosequencing to analyze an additional 185 Koreans, 100 African Americans, 100 Caucasians, and 159 Vietnamese for the presence of this variant. The variant was not found in any other individuals, except for one Korean subject. The frequencies of two known functional alleles, CYP3A7*2 and CYP3A7*1C, were 26 and 0%, respectively, in Koreans. The frequencies of the functional CYP3A7 polymorphisms in Koreans were significantly different from those in Caucasians and African Americans. This is the first report of a null-type allele of the CYP3A7 gene. It also provides population-level genetic data on CYP3A7 in Koreans to reveal the wide ethnic variation in CYP3A7 polymorphism.

Nomenclature for alleles of the cytochrome P450 oxidoreductase gene

Recent focus on the cytochrome P450 oxidoreductase (POR) gene has resulted in the discovery of numerous new polymorphic alleles. Many of these were found [1–6] because of their association with steroidogenic disorders and congenital skeletal malformations resembling the phenotype of Antley-Bixler syndrome [7], whereas other alleles have been found as a consequence of sequencing the POR gene in normal unrelated individuals [8,9]. The association of POR variants with clinical phenotypes is the result of POR serving as the major electron donor for cytochrome P450 (CYP) enzymes with important endogenous functions in hormone biosynthesis. Consequently, defective POR alleles can be the cause of abnormal glucocorticoid, mineralocorticoid, and sex steroid synthesis [10], thus leading to a form of congenital adrenal hyperplasia. In addition, POR deficiency can cause skeletal defects, the mechanism of which is yet unknown but has been suggested to result from impaired sterol synthesis [11] because of decreased electron flow from POR to lanosterol 14-alpha-demethylase (CYP51A1) and squalene monooxygenase (SQLE). In addition, as POR is equally important as an electron donor to CYP enzymes involved in the metabolism of drugs, POR variants may affect drug bioavailability. The effect of POR mutations on the activity of some drug-metabolizing CYP enzymes has been documented in vitro [12–14], but not yet in vivo. In addition, POR is an electron donor for heme oxygenase, cytochrome b5, and several additional small molecules that can be directly metabolized by POR without CYP enzymes. Thus, an increasing focus on the importance of POR in drug response and adverse drug reactions is to be expected. Until now, no systematic guidelines have been proposed for the naming of POR alleles. To standardize POR allelic nomenclature, the Human CYP Allele Nomenclature Chair and Committee have taken the initiative to devise a system for the designation of POR alleles that follows the guidelines for CYP allelic star (CYP*) nomenclature (http://www.cypalleles.ki.se/criteria.htm). The POR allele nomenclature web page (http://www.cypalleles.ki.se/por.htm) was launched in September 2008, listing 35 different alleles. On this POR web page, the alleles are presented together with their corresponding nucleotide and amino acid changes, and the phenotypic consequences observed by in vitro and in vivo studies. Among the more important POR variants are POR*2 and *5 (Arg457His and Ala287Pro, respectively), the former being the most frequent mutation in Japanese and Chinese POR-deficient patients [5,15], whereas the latter is the POR mutation most frequently found in Caucasians. Alleles with frameshift mutations (POR*9, *10, and *20–24), deletions, insertions, and several of the alleles that result in amino acid substitutions are also associated with in vivo phenotypes, as is a splice defect in the POR*3 allele. To maintain a common nomenclature system within the field, fellow scientists investigating POR polymorphisms are highly recommended to submit novel POR allelic variants to the Human CYP Allele Nomenclature Committee (http://www.cypalleles.ki.se/criteria.htm) by contacting the Webmaster for designation and reservation of novel POR allele names. The authors of this Letter, a number of whom have identified the novel POR alleles, are supportive of this new nomenclature system, and will use this system in their future work.

Discovery of Novel Functional Variants and Extensive Evaluation ofCYP2D6Genetic Polymorphisms in Koreans

Our objectives were to identify CYP2D6 genetic polymorphisms in a Korean population, to compare the allele frequencies with those of other ethnic groups, and to evaluate variant-induced functional variations in dextromethorphan (DM) metabolism in vitro and in vivo. Thirty-eight single nucleotide polymorphisms of CYP2D6 were identified by direct DNA sequencing in 51 Koreans. An extended set of 707 subjects were screened for the identified variants. A group of 202 healthy subjects was subjected to phenotypic analysis on DM metabolism. CYP2D6*10 was found to be the most frequent allele (45.6%), followed by CYP2D6*1 (32.3%), *2 (9.9%), *5 (5.6%), *41 (2.2%), *49 (1.4%), and some other rare alleles (<1%). The newly identified E418K and S183Stop were assigned as CYP2D6*52 and CYP2D6*60, respectively, by the Human P450 (CYP) Allele Nomenclature Committee. Individuals having the CYP2D6*10/*49 genotype (n = 5) exhibited a significant decrease in CYP2D6 metabolic activity compared with those with the CYP2D6*1/*1 genotype (n = 31) (P < 0.019). Variations in CYP2D6 protein levels in liver tissues (n = 49) were observed with CYP2D6 genotypes, and correlation between the CYP2D6 protein content and the activity was significant (r(2) = 0.7). Given the importance of CYP2D6 in drug metabolism, subjects with the CYP2D6*10/*49 genotype may benefit from genotype analysis to achieve optimal drug therapy.

The discovery of new coding alleles of human CYP26A1 that are potentially defective in the metabolism of all-trans retinoic acid and their assessment in a recombinant cDNA expression system

Retinoic acid (RA) is a critical regulator of gene expression during embryonic development and in the maintenance of adult epithelial tissues. This study was undertaken to identify genetic polymorphisms of CYP26A1 which might affect these processes. We sequenced CYP26A1 in racially diverse individuals and assessed the metabolism of retinoic acid by newly identified coding alleles of CYP26A1 in a recombinant system. CYP26A1 was sequenced in 24 Caucasians, 24 African-Americans, 24 Asians, and 20 individuals of unknown racial origin. cDNA constructs for wild-type and coding alleles of CYP26A1 were constructed in a pcDNA3.1 expression vector and expressed in Cos-1 cells. A FLAG tag at the C-terminal end of the cDNA was used to quantitate the recombinant CYP26A1 proteins. A total of 13 single nucleotide polymorphisms (SNPs) were identified in CYP26A1. Three SNPs produced coding changes: R173S, F186L, and C358R. These alleles were termed as CYP26A1*2, CYP26A1*3, and CYP26A1*4, respectively, by the Human Cytochrome P450 (CYP) Allele Nomenclature Committee at http://www.cypalleles.ki.se/. Wild type CYP26A1 protein metabolized all-trans-retinoic acid (at-RA) to 4-oxo-RA, 4-OH-RA as well as water-soluble metabolites. CYP26A1.3 (F186L) and CYP26A1.4 (C358R) allelic proteins exhibited significantly lower metabolism (40-80%) of at-RA than wild-type CYP26A1.1 protein. This is the first study to identify coding alleles of CYP26A1. Two coding alleles, CYP26A1*3 and CYP26A1*4, are predicted to be defective based on the metabolism of at-RA by the recombinant proteins. These studies suggest the need for future clinical studies of polymorphisms of CYP26A1 in embryonic development.

Clopidogrel Pharmacogenetics

Clopidogrel is a pro-drug that requires oxidation to its active metabolite, 2-oxoclopidogrel, by CYP3A4 and other CYP enzymes. This active thiol metabolite inhibits adenosine diphosphate (ADP)-induced platelet aggregation by blocking the platelet P2Y12 receptor (Figure), resulting in ≈50% reduction in ADP-mediated platelet aggregation. Clopidogrel is standard of care in many patients undergoing percutaneous coronary intervention (PCI) and those experiencing acute coronary syndromes. However, it has been suggested that response to clopidogrel varies widely with nonresponse rates ranging from 4% to 30% at 24 hours.1,2 Suggested mechanisms for this variability have included under-dosing, drug interactions with CYP3A4 substrates and inhibitors,3 and intrinsic interindividual differences resulting from genetic polymorphisms in the pathways of clopidogrel pharmacokinetics and pharmacodynamics. Clopidogrel metabolism and mechanism of action. Clopidogrel is metabolized to its active metabolite by the cytochrome P450 (CYP) 3A enzyme family. The CYP3A gene family is located on chromosome 7q21-q22. Genes are shown as block arrows. Select CYP3A4 and CYP3A5 polymorphisms (named according to the Human CYP Allele Nomenclature Committee) are shown with vertical black lines in relation to their position in the genes. The proposed effect of the polymorphisms on protein expression or function is shown below. CYP3A4 IVS10+12G>A is indicated as CYP3A4*1G. The clopidogrel active metabolite antagonizes the GI protein–coupled PY212 receptor resulting in irreversible blockade of ADP binding on platelets. See page 1895 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Angiolillo and colleagues report on the influence of CYP3A4 genotype on interpatient variability in clopidogrel responsiveness.4 The authors find that an intronic single nucleotide polymorphism (SNP) in the CYP3A4 gene, IVS10+12G>A (also called CYP3A4*1G), influences platelet reactivity …

The Human Cytochrome P450 Allele Nomenclature Committee Web Site : Submission Criteria, Procedures, and Objectives.

Interindividual variability in xenobiotic metabolism and drug response is extensive. Genetic factors are predicted to account for 15-30% of this variability in general, but for certain drugs the genetic factor is the major determinant for outcome of drug therapy. Of particular importance for drug metabolism, drug response, and adverse drug reactions are the cytochrome P450 (CYP) enzymes, many of which are polymorphic. An essential basis for research and applications regarding interindividual variability in xenobiotic metabolism and toxicity by polymorphic CYPs is to have a common nomenclature for genetic variants and a system that allows researchers to be rapidly updated within the field. Since 1999 this has been achieved by the operation of the Human Cytochrome P450 Allele Nomenclature Committee Web site ( http://www.imm.ki.se/CYPalleles/ ), where novel allelic variants are published after peer review. Currently, this Web site covers the nomenclature for polymorphic alleles of 22 CYP isoforms including more than 200 functionally different variants. Each CYP has its own Web page, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications where the allele has been identified and characterized. The CYP allele Web site offers a rapid on-line publication of new alleles, provides an overview of peer-reviewed data, and serves as a form of quality control on research on new alleles.

Functionally Defective or Altered CYP3A4 and CYP3A5 Single Nucleotide Polymorphisms and Their Detection with Genotyping Tests

Among the four cytochrome P450 (CYP)3A genes, CYP3A4 and CYP3A5 are the most abundantly expressed in the human liver. Eighty single nucleotide polymorphisms (SNPs) of CYP3A4/5 have been reported to the Human P450 Allele Nomenclature Committee. CYP3A4 alleles with minimal function compared with wild type include the CYP3A4*6 and CYP3A4*17. Alleles with moderately decreased or altered activity include: CYP3A4*2, *8, *11, *12, *13, *16, and *18. CYP3A5 alleles with minimal function include the splice variants CYP3A5*3, *5, *6 and CYP3A5* 10, as well as the null allele CYP3A5*7. Alleles with moderately decreased catalytic activity include CYP3A5*8 and CYP3A5*9. This report reviews the current progress in the functional characterization of CYP3A4 and CYP3A5 SNPs and provides genotyping tests for possible defective variants. A combination of genotyping tests for defective CYP3A4/CYP3A5 haplotypes will be necessary to understand the variations in the metabolism and clinical toxicity of a wide variety of clinical drugs, since these two CYP proteins have overlapping substrate specificities.

Polymorphism of cytochrome P450 and xenobiotic toxicity

The majority of human P450-dependent xenobiotic metabolism is carried out by polymorphic enzymes which can cause abolished, quantitatively or qualitatively altered or enhanced metabolism. The latter situation is due to stable duplication, multiduplication or amplification of active genes, most likely in response to dietary components that have resulted in a selection of alleles with multiple non-inducible genes. An updated list of variant CYP alleles is present at the Home Page of the Human Cytochrome P450 (CYP) Allele Nomenclature Committee ( http://www.imm.ki.se/CYPalleles/). Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultrarapid metabolism or, alternatively, adverse effects from the drug treatment due to the presence of defective alleles. Dosage requirements for several commonly used drugs that have a narrow therapeutic range can differ more than 20-fold dependent on the genotype or the enzyme expression status. By contrast, carcinogen metabolising cytochrome P450s are less polymorphic and no firm relationships have been established linking increased risk for cancer with any specific P450 polymorphism. In the present overview recent aspects of cytochrome P450 polymorphism and xenobiotic toxicity are discussed.