Effect of cytochrome P450 3A5*3 genotype on the stereoselective pharmacokinetics of amlodipine in healthy subjects

Amlodipine is a racemic mixture composed of S- and R-form and metabolized stereoselectively. Cytochrome P450 3A (CYP3A) including CYP3A5 are involved in the metabolism of amlodipine and it was reported that polymorphic CYP3A5 genotype modulates the plasma levels of amlodipine and thus affect its pharmacokinetics. This study was conducted to find whether stereoselective pharmacokinetics of amlodipine was affected by the polymorphic CYP3A5 genotype. Seventeen healthy subjects were genotyped for CYP3A5*3 variant. After a single dose of 10-mg amlodipine, enantiomers of amlodipine were analyzed using HPLC-MS/MS equipped with an AGP column. Amlodipine showed stereoselective pharmacokinetics. S-amlodipine exhibited higher plasma levels than R-amlodipine in both genotype groups. S-amlodipine showed 15% higher mean peak plasma concentrations (Cmax) in CYP3A5*1/*3 carriers (3.28 ng/ml) than CYP3A5*3/*3 carriers (2.85 ng/ml) (P = 0.194) and R-amlodipine also showed 21% higher Cmax in CYP3A5*1/*3 carriers (3.33 ng/ml) than CYP3A5*3/*3 carriers (2.75 ng/ml) (P = 0.114). CYP3A5*1/*3 carriers also have 23 and 12% higher mean area under the time versus concentration curve of R-amlodipine and S-amlodipine than CYP3A5*3/*3 carriers, respectively (for R-amlodipine, 147.1 ng*h/ml for CYP3A5*1/*3 carriers versus 121.8 ng*h/ml for CYP3A5*3/*3 carriers, P = 0.234; for S-amlodipine, 161.6 ng*h/ml for CYP3A5*1/*3 carriers vs. 144.2 ng*h/ml for CYP3A5*3/*3 carriers, P = 0.353). Other pharmacokinetic parameters also showed no significant difference between them. In conclusion, the present study showed that despite the evidence that amlodipine is stereoselectively metabolized, CYP3A5*3 genotype did not affect stereoselective disposition of amlodipine. It provides the evidence that CYP3A5*3genotype plays a minor role in the interindividual variability of stereoselective disposition of amlodipine in humans.     

Evaluation of a microarray for genotyping polymorphisms related to xenobiotic metabolism and DNA repair

We present an oligonucleotide microarray ("MetaboChip") based on the arrayed primer extension (APEX) technique, allowing genotyping of single nucleotide polymorphisms (SNPs) in genes of interest for cancer susceptibility and pharmacogenetics. APEX consists of a sequencing reaction primed by an oligonucleotide anchored with its 5' end to a glass slide and terminating one nucleotide before the polymorphic site. The extension with one fluorescently labeled dideoxynucleotide complementary to the template reveals the polymorphism. Ninety-three SNPs in 42 genes were selected among those resequenced in the context of the SNP500 project, using a set of 102 reference DNA samples from the Coriell Biorepository. Selected SNPs belong to the following genes: ADH1B, ALDH2, APEX, CDKN2A, COMT, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C19, CYP2C9, CYP2E1, CYP3A4, DRD2, DRD4, EPHX1, ERCC1, ERCC2, ERCC4, ERCC5, GRPR, GSTA4, GSTM3, GSTP1, GSTT2, LIG3, MDM2, MGMT, MPO, NAT1, NAT2, NQO1, OGG1, PCNA, POLB, SLC6A3, SOD2, TP53, XRCC1, XRCC2, XRCC3, and XRCC9. We assessed the performance of APEX by comparing the results obtained with MetaboChip against those reported by the SNP500. Among 88 SNPs that yielded signals, 6 showed less than 99% of concordance, whereas 82 performed accurately, showing that APEX is a reliable and sensitive genotyping method.     

用基因芯片检测DPYD等位基因在受试人群中的发生频率

二氢嘧啶脱氢酶基因（DPYD基因）所编码的二氢嘧啶脱氢酶（DPD酶）是氟化嘧啶类抗肿瘤药物代谢的主要限速酶,其活性存在显著的个体差异,并因此影响药物的疗效和毒副作用.大部分编码低/无活性酶的突变型等位基因是由于基因中的单核苷酸多态性（single nucleotide polymorphism,SNP）造成的,检测这些SNPs是预测患者对药物的反应和实现个体化给药方案的基础.制备并优化了用于检测DPYD基因中6个已知SNPs所编码的等位基因（DPYD^*2,^*3,^*4,^*5,^*9,^*12）的基因芯片,建立了该芯片的基因分型标准.并利用该芯片检测了肿瘤患者（112例）、肾病患者（83例）和健康者（45例）中DPYD突变型等位基因的发生频率.在受试人群中,突变型等位基因DPYD^*5和DPYD^*9平均发生率分别为32.08%和11.25%,未发现DPYD^*2,^*3,^*4,^*12突变型等位基因.而且以上单碱基突变的发生率在肿瘤患者、肾病患者和健康者间以及男性、女性肿瘤患者间无显著性差异,表明其与疾病的发生或性别无显著性关联.对20例标本的基因分型结果采用直接测序法进行验证,19例基因芯片分型结果与直接测序法结果相一致.DPYD^*5、DPYD^*9突变型等位基因在受试人群中具有较高的发生率.利用基因芯片能够对其实现快速准确的检测.     

Influence of CYP3A4, CYP3A5 and MDR-1 polymorphisms on tacrolimus pharmacokinetics and early renal dysfunction in liver transplant recipients

Objectives

Tacrolimus is a widely used immunosuppressive drug in organ transplantation. The oral bioavailability of tacrolimus varies greatly between individuals and depends largely on the activity of both the cytochrome P450 3A (CYP3A) subfamily and P-glycoprotein (P-gp). The possible influence of single nucleotide polymorphisms (SNPs) of CYP3A subfamily and P-gp (MDR-1) in liver transplant recipients has recently been indicated as one of the most important variables affecting the pharmacokinetics of tacrolimus and the renal injury induced by tacrolimus.

Methods

A total of 216 liver transplant recipients were enrolled in this study. The recipients' mean follow-up time was 52 mo (range from 16 to 96 mo). All liver transplant recipients were all in a stable stage with normal serum creatinine (SCr). All liver transplant recipients treated with tacrolimus were genotyped for CYP3A5 (6986A>G), CYP3A4 intron 6 (CYP3A4*22), MDR-1 exon 26 (3435C>T) and exon 12 (1236 C>T) SNPs by HRM analysis (high-resolution melting curve analysis). Recipients were defined as the early renal injury by the elevation of different microproteins in the urine including microalbumin (MA), urine immunoglobulin G (IGU), urine transferrin (TRU) and α1-microglobulin (A1M).

Results

The daily dose of tacrolimus was higher for recipients with CYP3A5*1/*1 (AA) genotype than those with CYP3A5*3/*3 (GG) genotype [3.0 (2.0–4.0) versus 2.0 (1.5–2.5) mg/d, P < 0.05]. The concentration/dose ratio of recipients with CYP3A5*1 homozygotes was lowest compared to recipients with CYP3A5*3/*3 and CYP3A5*1/*3 genotypes. Furthermore, the recipients carrying CYP3A5*3 allele were associated with increased risk of early renal glomerular injury compared to the recipients carrying CYP3A5*1 allele (P = 0.01). MDR-1 polymorphisms were not related with tacrolimus pharmacokinetics and early renal injury.

Conclusion

CYP3A5 6986A>G genetic polymorphism affected daily dose requirements, concentration and nephrotoxicity of tacrolimus. Screening for this single nucleotide polymorphism before the transplantation might be helpful for the selection of adequate initial daily dose and to achieve the desired immunosuppression outcome.     

Personalized Tacrolimus Dose Requirement by CYP3A5 but Not ABCB1 or ACE Genotyping in Both Recipient and Donor after Pediatric Liver Transplantation

Tacrolimus (TAC) is the backbone of an immunosuppressive drug used in most solid organ transplant recipients. A single nucleotide polymorphism (SNP) at position 6986G>A in CYP3A5 has been notably involved in the pharmacokinetic variability of TAC. It is hypothesized that CYP3A5 genotyping in patients may provide a guideline for TAC therapeutic regimen. To further evaluate the impact of CYP3A5 variants in donors and recipients, ABCB1 and ACE SNPs in recipients on TAC disposition, clinical and laboratory data were retrospectively reviewed from 90 pediatric patients with liver transplantation and their corresponding donors after 1 year of transplantation. The recipients with CYP3A5 *1/*1 or *1/*3 required more time to achieve TAC therapeutic range during the induction phase, and needed more upward dose during the late induction and the maintained phases, with lower C/D ratio, compared with those with CYP3A5 *3/*3. And donor CYP3A5 genotypes were found to impact on TAC trough concentrations after liver transplantation. No association between ABCB1 or ACE genotypes and TAC disposition post-transplantation was found. These results strongly suggest that CYP3A5 genotyping both in recipient and donor, not ABCB1 or ACE is necessary for establishing a personalized TAC dosage regimen in pediatric liver transplant patients.     

Allele and genotype frequencies of the polymorphic cytochrome P450 genes (CYP1A1, CYP3A4, CYP3A5, CYP2C9 and CYP2C19) in the Jordanian population

Drug metabolizing enzymes participate in the neutralizing of xenobiotics and biotransformation of drugs. Human cytochrome P450, particularly CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, play an important role in drug metabolism. The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function. The goal of this study was to determine the frequencies of important allelic variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1 and m2), CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), CYP3A4*5, CYP3A5 (*3 and *6), was carried out on Jordanian subjects. Different variants allele were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1*1, 0.165 for CYP1A1*2A and 0.071 for CYP1A1*2C. CYP2C9 allele frequencies in 263 subjects were 0.797 for CYP2C9*1, 0.135 for CYP2C9*2 and 0.068 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16?%) were homozygous for *2/*2. Regarding CYP3A4*1B, only 12 subjects out of 173 subjects (6.9?%) were heterozygote with none were mutant for this polymorphism. With respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5*1,*3 and *6 were 0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in several Caucasian, African-American and Asian populations, Jordanians are most similar to Caucasians with regard to allelic frequencies of the tested variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.     

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.     

CYP1A1, CYP2D6, CYP2E1, NAT2, GSTM1 and GSTT1 polymorphisms or their combinations are associated with the increased risk of the laryngeal squamous cell carcinoma

Polymorphisms in the selected genes controlling carcinogen metabolism (CYP1A1, CYP2D6, CYP2E1, NAT2, GSTM1, GSTT1) considered separately or in different combinations, were investigated for an association with tobacco smoke-associated squamous cell carcinoma (SCC) of the larynx. The case-control study was performed in 289 patients with laryngeal SCC and in 316 cancer-free controls; all were Caucasian males from the same region of Poland and current tobacco smokers. The DNA samples were genotyped using PCR-RFLP and multiplex PCR. The variants' frequencies in both groups were compared; odds ratios and their 95% confidence intervals were calculated by logistic regression analyses. The CYP1A1*1/*4, CYP2D6*4/*4, NAT2*4/*6A genotypes, as well as the CYP1A1*4, CYP2D6*4 and NAT2*4 alleles, were found at significantly higher frequencies in cases than in controls indicating their role as "risk-elevating" factors in laryngeal SCC. Combined genotypes, characterized by the presence of the "risk-elevating" variants at more than one locus, often occurred together with the null variant of the GSTM1 gene and homozygous XPD A/A (Lys751Gln, A35931C) genotype. Furthermore, we identified some "protective" variants, found more frequently in controls than in cases, i.e. the NAT2*6A/*6A and NAT2*5B/*6A genotypes. A distribution of "risk" or "protection" genotypes/alleles seems to be connected with age as an occurrence or risk genes was more frequent in the group of "young" cases (< or = 49 years). Accumulation of certain alleles or genotypes of the CYP1A1, NAT2, GSTM1 and XPD seems to be associated with either increased or decreased risk to develop laryngeal SCC. Therefore, polymorphisms in these genes may play a role in the laryngeal cancer etiology.     

Impact of CYP3A5 and MDR-1 gene polymorphisms on the dose and level of tacrolimus among living-donor liver transplanted patients: single center experience

Aim of work: To assess the impact of Cytochrome P450 3A5 (CYP3A5) and multidrug resistance-1 gene (MDR-1) single nucleotide polymorphisms on the dose and blood level of tacrolimus among liver transplanted patients.

Patients and methods: We enrolled a prospective study of 41 liver transplanted patients. Dose-adjusted trough blood concentration (C/D ratio) was calculated. Polymerase chain reaction-restriction fragment length polymorphism followed by sequencing was done for genotyping of CYP3A5*3 (6986A?>?G).

Results: At 1 week, 1 and 3 months C/D ratio were significantly lower in CYP3A5 expressers *1/*1 patients compared to non-expressers *3/*3.

Conclusion: CYP3A5 (6986A?>?G) genotype, rather than MDR-1 (2677G?>?A/T) variant, has an impact on tacrolimus pharmacokinetics.     

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.     

CYP3A5 genotype predicts renal CYP3A activity and blood pressure in healthy adults.

A single-nucleotide polymorphism (A6986G) in the cytochrome p-450 3A5 (CYP3A5) gene distinguishes an expressor (*1) and a reduced-expressor (*3) allele and largely predicts CYP3A5 content in liver and intestine. CYP3A5 is the prevailing CYP3A isoform in kidney. We report that, among renal microsomes from 21 organ donors, those from *1/*3 individuals had at least eightfold higher mean kidney microsomal CYP3A5 content and 18-fold higher mean CYP3A catalytic activity than did those from *3/*3 individuals (P = 0.0001 and P = 0.0137, respectively). We also report significant associations between the A6986G polymorphism and systolic blood pressure (P = 0.0007), mean arterial pressure (P = 0.0075), and creatinine clearance (P = 0.0035) among 25 healthy African-American adults. These associations remained significant when sex, age, and body mass index were taken into account. The mean systolic blood pressure of homozygous CYP3A5 expressors (*1/*1) exceeded that of homozygous nonexpressors (*3/*3) by 19.3 mmHg. We speculate whether a high CYP3A5 expressor allele frequency among African-Americans may contribute to a high prevalence of sodium-sensitive hypertension in this population.     

Comparative analysis of N-acetylation polymorphism in humans as determined by phenotyping and genotyping

The N-acetylation polymorphisms of volunteers from the Moscow population analyzed by phenotyping and genotyping have been compared. The ratios between the proportions of fast acetylators (FAs) and slow acetylators (SAs) estimated by phenotyping and genotyping do not differ significantly from each other (47 and 44%, respectively). The absolute acetylation rate widely varies in both FAs and SAs. The NAT2 genotype and allele frequencies in the population sample have been calculated. The most frequent alleles are NAT2*4 (a "fast" allele), NAT2*5, and NAT2*6 ("slow" alleles); the most frequent genotypes are NAT2*5/*5, NAT2*4/*6, and NAT2*4/*5. Comparative analysis of N-acetylation polymorphism estimated by phenotyping and genotyping in the same subjects has shown a complete concordance between the phenotype and genotype in only 62 out of 75 subjects (87%). Comparative characteristics and presumed applications of the two approaches (quantitative estimation of acetylation rate and qualitative determination of the acetylator genotype) to the identification of individual acetylation status are presented.     

Pharmacogenetic-Based Efavirenz Dose Modification: Suggestions for an African Population and the Different CYP2B6 Genotypes

Background

Pharmacogenetics contributes to inter-individual variability in pharmacokinetics (PK) of efavirenz (EFV), leading to variations in both efficacy and toxicity. The purpose of this study was to assess the effect of genetic factors on EFV pharmacokinetics, treatment outcomes and genotype based EFV dose recommendations for adult HIV-1 infected Ugandans.

Methods

In total, 556 steady-state plasma EFV concentrations from 99 HIV infected patients (64 female) treated with EFV/lamivudine/zidovidine were analyzed. Patient genotypes for CYP2B6 (*6 & *11), CYP3A5 (*3,*6 & *7) and ABCB1 c.4046A>G, baseline biochemistries and CD4 and viral load change from baseline were determined. A one-compartment population PK model with first-order absorption (NONMEM) was used to estimate genotype effects on EFV pharmacokinetics. PK simulations were performed based upon population genotype frequencies. Predicted AUCs were compared between the product label and simulations for doses of 300 mg, 450 mg, and 600 mg.

Results

EFV apparent clearance (CL/F) was 2.2 and 1.74 fold higher in CYP2B6*6 (*1/*1) and CYP2B6*6 (*1/*6) compared CYP2B6*6 (*6/*6) carriers, while a 22% increase in F1 was observed for carriers of ABCB1 c.4046A>G variant allele. Higher mean AUC was attained in CYP2B6 *6/*6 genotypes compared to CYP2B6 *1/*1 (p<0.0001). Simulation based AUCs for 600 mg doses were 1.25 and 2.10 times the product label mean AUC for the Ugandan population in general and CYP2B6*6/*6 genotypes respectively. Simulated exposures for EFV daily doses of 300 mg and 450 mg are comparable to the product label. Viral load fell precipitously on treatment, with only six patients having HIV RNA >40 copies/mL after 84 days of treatment. No trend with exposure was noted for these six patients.

Conclusion

Results of this study suggest that daily doses of 450 mg and 300 mg might meet the EFV treatment needs of HIV-1 infected Ugandans in general and individuals homozygous for CYP2B6*6 mutation, respectively.     

Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism.

The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C-oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re-evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3' flanking region of 30-40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.     

Influence of CYP3A5, ABCB1 and NR1I2 polymorphisms on prednisolone pharmacokinetics in renal transplant recipients

The objective of this study was to evaluate whether genetic polymorphisms of CYP3A5 (A6986G, CYP3A5*3), ABCB1 (C1236T, G2677T/A, C3435T) and NR1I2 (A7635G) significantly impact the pharmacokinetics of prednisolone in renal transplant recipients. Ninety-five recipients were given repeated doses of triple therapy immunosuppression consisting of prednisolone, tacrolimus and mycophenolate mofetil. Twenty-eight days after renal transplantation, plasma prednisolone concentrations were measured by high-performance liquid chromatography. Comparisons of the CYP3A5 and ABCB1 genotypes revealed no significant differences in the prednisolone pharmacokinetics. The mean prednisolone C(max) for recipients (n=14) having both the ABCB1 3435CC genotype and the CYP3A5*3/*3 genotype was significantly higher than those (n=11) having both ABCB1 3435TT and CYP3A5*3/*3 genotypes (180ng/mL versus 129ng/mL, P=0.0392). The plasma concentrations of prednisolone in recipients having both ABCB1 3435CC and CYP3A5*3/*3 genotypes tended to be higher than those having both ABCB1 3435TT and CYP3A5*3/*3 genotypes. The mean AUC(0-24) and C(max) values for prednisolone in recipients having the NR1I2 7635G allele (AG: n=45, GG: n=32) were significantly lower than in patients having the 7635AA allele (n=18) (7635GG versus 7635AA, P=0.0308 for AUC(0-24), P=0.0382 for C(max) of prednisolone). In conclusion, NR1I2 (A7635G) rather than CYP3A5 or ABCB1 allelic variants affected patient variability of plasma prednisolone concentration. Recipients carrying the NR1I2 7635G allele seemed to possess higher metabolic activity for prednisolone in the intestine, greatly reducing its maximal plasma concentration.     

The Differential Effect of NAT2 Variant Alleles Permits Refinement in Phenotype Inference and Identifies a Very Slow Acetylation Genotype

Indirect evidences suggest that acetylation phenotype categories are heterogeneous and that subcategories, related to specific NAT2 variant alleles might exist. We analyzed the in vivo acetylation phenotype and genotype in 504 north-American subjects of Caucasian origin. The analyses of the SNPs rs1801280 and rs1799930 allowed the discrimination of five categories with different acetylation status within the study population. These categories are related to the distinct effect of NAT2 alleles on the acetylation status in vivo and to the occurrence of a gene-dose effect. These five phenotype categories, from higher to lower acetylation capacity, correspond to the genotypes NAT2*4/*4, NAT2*4/*5 or *4/*6, NAT2*5/*5, NAT2*5/*6 and NAT2*6/*6 (p≤0.001 for all comparisons). The NAT2*6/*6 genotype correspond to a phenotype category of very-slow acetylators. The refinement in phenotype prediction may help to identify risks associated to phenotype subcategories, and warrants the re-analysis of previous studies that may have overlooked phenotype subcategory-specific risks.     

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.     

CYP1A1 *2B and *4 polymorphisms are associated with lung cancer susceptibility in Mexican patients

BACKGROUND: CYP1A1 is a gene involved in the high aryl hydrocarbon hydroxylase -inducible phenotype, which is a genetically-determined variation among individuals that has been associated with lung cancer risk. More specifically, CYP1A1 *2B and *4 polymorphisms have been associated with high susceptibility to lung cancer among cigarette smokers. MATERIALS AND METHODS: DNA was obtained from blood samples and we studied by PCR-RFLP the distribution of CYP1A1 *2B (n=248) and *4 (n=222) polymorphisms in healthy controls and 222 lung cancer patients from a Mexican population. RESULTS: Comparisons between groups showed an increased risk for lung cancer patients of *2B/*2B (18%; OR 7.6; 95% CI 3.0-19.2) and *4/ *4 genotypes (15%; OR 11.45; 95% CI 2.19-59.85) compared to the control group (1% for *2B/ *2B and 4.4% for *4/ *4). A significant association between lung cancer and homozygous *2B/ *2B passive smokers and *4/*4 ever (cigarettes) and passive smokers was also observed (p<0.05). Multivariate analysis revealed an increased risk for the *2B/*2B genotype (OR 6.83), as well as for *4/*4 (OR 28.8). CONCLUSION: The results of the study indicate a significant association between *2B/*2B and *4/*4 genotypes and the risk of developing lung cancer among Mexicans.     

A Novel Simple Method for Determining CYP2D6 Gene Copy Number and Identifying Allele(s) with Duplication/Multiplication

Background

Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.

Methods

We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV) genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.

Results

The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.

Conclusions

Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.     

Effects of the CYP3A4*1B Genetic Polymorphism on the Pharmacokinetics of Tacrolimus in Adult Renal Transplant Recipients: A Meta-Analysis

Background and Objective

The association between the CYP3A4*1B single nucleotide polymorphism (SNP) and tacrolimus pharmacokinetics in different studies is controversial. Therefore, a meta-analysis was employed to evaluate the correlation between the CYP3A4*1B genetic polymorphism and tacrolimus pharmacokinetics at different post-transplantation times in adult renal transplant recipients.

Methods

Studies evaluating the CYP3A4*1B genetic polymorphism and tacrolimus pharmacokinetics were retrieved through a systematical search of Embase, PubMed, the Cochrane Library, ClinicalTrials.gov and three Chinese literature databases (up to Sept. 2014). The pharmacokinetic parameters (weight-adjusted tacrolimus daily dose and tacrolimus trough concentration/weight-adjusted tacrolimus daily dose ratio) were extracted, and the meta-analysis was performed using Stata 12.1.

Results

Seven studies (involving 1182 adult renal transplant recipients) were included in this meta-analysis. For the weight-adjusted tacrolimus daily dose, in all included renal transplant recipients (European & Indian populations), CYP3A4*1/*1 recipients required a significantly lower weight-adjusted tacrolimus daily dose than did CYP3A4*1B carriers at 7 days (WMD -0.048; 95% CI -0.083 ~ -0.014), 6 months (WMD -0.058; 95% CI -0.081 ~ -0.036) and 12 months (WMD - 0.061; 95% CI -0.096 ~ -0.027) post-transplantation. In light of the heterogeneity, the analysis was repeated after removing the only study in an Indian population, and CYP3A4*1/*1 European recipients (mostly Caucasian) required a lower weight-adjusted tacrolimus daily dose within the first year post-transplantation. The tacrolimus trough concentration/weight-adjusted tacrolimus daily dose ratio (C₀/Dose ratio) was significantly higher in CYP3A4*1/*1 recipients than in CYP3A4*1B carriers at 6 months (WMD 52.588; 95% CI 22.387 ~ 82.789) and 12 months (WMD 62.219; 95% CI 14.218 ~ 110.221) post-transplantation. When the only study in an Indian population was removed to examine European recipients (mostly Caucasian), the significant difference persisted at 1 month, 6 months and 12 months post-transplantation.

Conclusion

Based on our meta-analysis, the CYP3A4*1B genetic polymorphism affects tacrolimus dose requirements and tacrolimus trough concentration/weight-adjusted tacrolimus daily dose ratio within the first year post-transplantation in adult renal transplant recipients, especially in European recipients (mostly Caucasian).