CYP3A5 and NAT2 gene polymorphisms: role in childhood acute lymphoblastic leukemia risk and treatment outcome

Susceptibility to acute lymphoblastic leukemia can be highly influenced by genetic polymorphisms in metabolizing enzyme genes of environmental carcinogens. This study aimed to evaluate the impact of the CYP3A5 and NAT2 metabolizing enzyme polymorphisms on the risk of childhood acute lymphoblastic leukemia. The analysis was conducted on 204 ALL patients and in 364 controls from a Brazilian population, using PCR-RFLP. The CYP3A5*3 polymorphic homozygous genotype was more frequent among ALL patients and the *3 allele variant was significantly associated with increased risk of childhood ALL (OR = 0.29; 95% CI, 0.14-0.60). The homozygous polymorphic genotype for the *6 allele variant was extremely rare and found in only two individuals. The heterozygous frequencies were similar for the ALL group and the control group. No significant differences were observed between the groups analyzed regarding NAT2 variant polymorphisms. None of the polymorphisms analyzed was related to treatment outcome. The results suggest that CYP3A5*3 polymorphism may play an important role in the risk of childhood ALL.

GWAS identifies an NAT2 acetylator status tag single nucleotide polymorphism to be a major locus for skin fluorescence

Skin fluorescence (SF) is a non-invasive marker of AGEs and is associated with the long-term complications of diabetes. SF increases with age and is also greater among individuals with diabetes. A familial correlation of SF suggests that genetics may play a role. We therefore performed parallel genome-wide association studies of SF in two cohorts.

Localization of polymorphic N-acetyltransferase (NAT2) in tissues of inbred mice

Like humans, mice exhibit polymorphism in the N-acetylation of aromatic amines, many of which are toxic and/or carcinogenic. Mice have three N-acetyltransferase (Nat) genes, Nat1, Nat2 and Nat3, and Nat2 is known to be polymorphic. There is a dramatic difference in the acetylation of NAT2 substrates by blood from fast (C57BL/6J) compared with slow acetylator (A/J) mice. However, the acetylation of these substrates by liver cytosols from the two strains is very similar. In order to determine whether the expression of the NAT2 protein corresponded with the activities measured, a polyclonal antipeptide antisera was raised against the C-terminal decapeptide of NAT2 and characterized using recombinant murine NAT2 antigen. Enzyme-linked immunosorbent assays (ELISAs) demonstrated that the anti-NAT2 antiserum bound in a concentration-dependent fashion to recombinant NAT2. Immunochemical analysis of mouse liver cytosols from C57BL/6J or A/J livers indicated that the level of NAT2 protein expressed in the two strains was similar. Immunohistochemical staining of C57BL/6J liver with anti-NAT2 antiserum showed that NAT2 was expressed in hepatocytes throughout the liver although the intensity of staining in the perivenous (centrilobular) region was higher than that in the periportal region. NAT2 was also detected in epithelial cells in the lung, kidney, bladder, small intestine and skin as well as in erythrocytes and lymphocytes in the spleen and hair follicles and sebaceous glands in the skin. Characterization of the distribution of NAT2 will be of value in elucidating the role of polymorphic N-acetylation in protecting the organism from environmental insults as well as in endogenous metabolism.

Effect of single nucleotide polymorphisms in cytochrome P450 isoenzyme and N-acetyltransferase 2 genes on the metabolism of artemisinin-based combination therapies in malaria patients from Cambodia and Tanzania.

The pharmacogenetics of antimalarial agents are poorly known, although the application of pharmacogenetics might be critical in optimizing treatment. This population pharmacokinetic-pharmacogenetic study aimed at assessing the effects of single nucleotide polymorphisms (SNPs) in cytochrome P450 isoenzyme genes (CYP, namely, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) and the N-acetyltransferase 2 gene (NAT2) on the pharmacokinetics of artemisinin-based combination therapies in 150 Tanzanian patients treated with artemether-lumefantrine, 64 Cambodian patients treated with artesunate-mefloquine, and 61 Cambodian patients treated with dihydroartemisinin-piperaquine. The frequency of SNPs varied with the enzyme and the population. Higher frequencies of mutant alleles were found in Cambodians than Tanzanians for CYP2C9*3, CYP2D6*10 (100C → T), CYP3A5*3, NAT2*6, and NAT2*7. In contrast, higher frequencies of mutant alleles were found in Tanzanians for CYP2D6*17 (1023C → T and 2850C → T), CYP3A4*1B, NAT2*5, and NAT2*14. For 8 SNPs, no significant differences in frequencies were observed. In the genetic-based population pharmacokinetic analyses, none of the SNPs improved model fit. This suggests that pharmacogenetic data need not be included in appropriate first-line treatments with the current artemisinin derivatives and quinolines for uncomplicated malaria in specific populations. However, it cannot be ruled out that our results represent isolated findings, and therefore more studies in different populations, ideally with the same artemisinin-based combination therapies, are needed to evaluate the influence of pharmacogenetic factors on the clearance of antimalarials.

Caracterización del genotipo N-ACETIL Transferasa 2 (NAT2) en una muestra de la población del noreste de México

Tesis (Maestría en Ciencias con Especialidad en Biología Molecular e Ingeniería Genética) UANL

CYP3A5*3A allele is associated with reduced lowering-lipid response to atorvastatin in individuals with hypercholesterolemia

Background: The cytochrome P450 isoenzyme 3A5 (CYP3A5) has an important role on biotransformation of xenobiotics. CYP3A5 SNPs have been associated with variations on enzyme activity that can modify the metabolism of several drugs. Methods: In order to evaluate the influence of CYP3A5 variants on response to lowering-cholesterol drugs, 139 individuals with hypercholesterolemia were selected. After a wash-out period of 4 weeks, individuals were treated with atorvastatin (10 mg/day/4 weeks). Genomic DNA was extracted by a salting-out procedure. CYP3A5*3C, CYP3A5*6 and CYP3A5*1D were analyzed by PCR-RFLP and DNA sequencing. Results: >Frequencies of the CYP3A5*3C and CYP3A5*1D alleles were lower in individuals of African descent (*3C: 47.8% and *1D: 55.2%) than in non-Africans (*3C: 84.9% and *1D 84.8%, p<0.01). Non-Africans carrying *3A allele (*3C and *1D combined alleles) had lower total and LDL-cholesterol response to atorvastatin than non-*3A allele carriers (p<0.05). Conclusion: CYP3A5*3A allele is associated with reduced cholesterol-lowering response to atorvastatin in non-African individuals. (C) 2008 Elsevier B.V. All rights reserved.

Associação do perfil de acetilação lenta do gene NAT2 na susceptibilidade ao câncer, na Região Norte do Brasil

Objetivos: O gene N-acetiltransferase 2 (NAT2) é um marcador para o estudo da susceptibilidade interindividual ao desenvolvimento de neoplasias malignas, visto que a enzima NAT2 participa da metabolização de agentes carcinogênicos e os polimorfismos de base única (SNP) do seu gene produzem enzimas com diferentes atividades, levando a acetilação lenta ou rápida de xenobióticos. O objetivo do presente estudo foi verificar uma possível associação entre os SNPS do gene NAT2 e a susceptibilidade ao acometimento de Adenocarcinoma gástrico ou Carcinoma ductal invasivo da mama em pacientes da região norte do Brasil. Material e Métodos: Os cinco polimorfismos de grande importância para a determinação do perfil de metabolização da enzima NAT2 (C282T, T341 C, C481 T, A803G e G857A) foram investigados por sequenciamento direto de 986 pares de bases, amplificados em duas reações de PCR, no total de 133 pacientes com câncer (63 com Câncer Gástrico e 70 com Câncer de Mama) e 89 indivíduos Controles. Para evitar interpretações espúrias decorrentes do subestruturamento populacional, empregamos um painel de 48 marcadores informativos de ancestralidade (IAM). Resultados: Encontramos diferenças estatísticas para a contribuição parental Africana e Européia, quando comparadas entre os grupos com Câncer e Controles, uma contribuição maior do grupo Africano foi detectada no grupo de estudo com câncer e, no grupo controle, foi detectada uma maior contribuição do grupo Europeu (p<0,001). Os genótipos do polimorfismo C282T dominante (TT + CT) apresentaram associação significativa (p<0,001; OR 3,076; Cl 95% 1,664-5,687) para a susceptibilidade as diferentes formas de Câncer investigadas. Foi observada uma associação significante do perfil de acetilação lenta e rápida com a susceptibilidade ao desenvolvimento das neoplasias investigadas (p=0,010; OR 3,054; Cl 95% 1,303-7,159) e (p= 0,041; OR 0,527 Cl 95% 0,280-0,973) evidenciando que indivíduos com o perfil acetilador lento apresentaram um risco aumentado em até três vezes no desenvolvimento de neoplasias quando comparado com os indivíduos controles. Conclusão: O controle genômico da ancestralidade foi efetivamente importante para a presente investigação possibilitando controlar o efeito da ancestralidade na associação do gene NAT2 para susceptibilidade ao câncer. Neste trabalho foi possivel evidenciar a forte influência do perfil de acetilação lenta do gene NAT2 de xenobióticos na susceptibilidade ao Câncer Gástrico e de Mama.

Interethnic diversity of NAT2 polymorphisms in Brazilian admixed populations

Abstract Background N-acetyltransferase type 2 (Nat2) is a phase II drug- metabolizing enzyme that plays a key role in the bioactivation of aromatic and heterocyclic amines. Its relevance in drug metabolism and disease susceptibility remains a central theme for pharmacogenetic research, mainly because of its genetic variability among human populations. In fact, the evolutionary and ethnic-specific SNPs on the NAT2 gene remain a focus for the potential discoveries in personalized drug therapy and genetic markers of diseases. Despite the wide characterization of NAT2 SNPs frequency in established ethnic groups, little data are available for highly admixed populations. In this context, five common NAT2 SNPs (G191A, C481T, G590A, A803G and G857A) were investigated in a highly admixed population comprised of Afro-Brazilians, Whites, and Amerindians in northeastern Brazil. Thus, we sought to determine whether the distribution of NAT2 polymorphism is different among these three ethnic groups. Results Overall, there were no statistically significant differences in the distribution of NAT2 polymorphism when Afro-Brazilian and White groups were compared. Even the allele frequency of 191A, relatively common in African descendents, was not different between the Afro-Brazilian and White groups. However, allele and genotype frequencies of G590A were significantly higher in the Amerindian group than either in the Afro-Brazilian or White groups. Interestingly, a haplotype block between G590A and A803G was verified exclusively among Amerindians. Conclusions Our results indicate that ethnic admixture might contribute to a particular pattern of genetic diversity in the NAT2 gene and also offer new insights for the investigation of possible new NAT2 gene-environment effects in admixed populations.

In vivo and in vitro investigation of the tissue-specific activity of the human CYP3A4 and CYP3A5 promoters

The human cytochrome P450 3A4 (CYP3A4), the predominant but variably expressed cytochrome P450 in adult liver and small intestine is involved in the metabolism of over 50% of currently used drugs. Its paralog CYP3A5 plays a crucial role in the disposition of several drugs with low therapeutic index, including tacrolimus. Limited information is available for the CYP3A5 transcriptional regulation and its induction by xenobiotics remains controversial. In the first part of this study, we analysed the CYP3A5 transcriptional regulation and its induction by xenobiotics in vivo using transgenic mice. To this end, two transgenic strains were established by pronuclear injection of a plasmid, expressing firefly luciferase driven by a 6.2 kb of the human CYP3A5 promoter. A detailed analysis of both strains shows a tissue distribution largely reflecting that of CYP3A5 transcripts in humans. Thus, the highest luciferase activity was detected in the small intestine, followed by oesophagus, testis, lung, adrenal gland, ovary, prostate and kidney. However, no activity was observed in the liver. CYP3A5-luc transgenic mice were similarly induced in both sexes with either PCN or TCPOBOP in small intestine in a dose-dependent manner. Thus, the 6.2 kb upstream promoter of CYP3A5 mediates the broad tissue activity in transgenic mice. CYP3A5 promoter is inducible in the small intestine in vivo, which may contribute to the variable expression of CYP3A in this organ. rnThe hepato-intestinal level of the detoxifying oxidases CYP3A4 and CYP3A5 is adjusted to the xenobiotic exposure mainly via the xenosensor and transcriptional factor PXR. CYP3A5 is additionally expressed in several other organs lacking PXR, including kidney. In the second part of this study, we investigated the mechanism of the differential expression of CYP3A5 and CYP3A4 and its evolutionary origin using renal and intestinal cells, and comparative genomics. For this examination, we established a two-cell line models reflecting the expression relationships of CYP3A4 and CYP3A5 in the kidney and small intestine in vivo. Our data demonstrate that the CYP3A5 expression in renal cells was enabled by the loss of a suppressing Yin Yang 1 (YY1)-binding site from the CYP3A5 promoter. This allowed for a renal CYP3A5 expression in a PXR-independent manner. The YY1 element is retained in the CYP3A4 gene, leading to its suppression, perhaps via interference with the NF1 activity in renal cells. In intestinal cells, the inhibition of CYP3A4 expression by YY1 is abrogated by a combined activating effect of PXR and NF1 acting on their respective response elements located adjacent to the YY1-binding site on CYP3A4 proximal promoter. CYP3A4 expression is further facilitated by a point mutation attenuating the suppressing effect of YY1 binding site. The differential expression of CYP3A4 and CYP3A5 in these organs results from the loss of the YY1 binding element from the CYP3A5 promoter, acting in concert with the differential organ expression of PXR, and with the higher accumulation of PXR response elements in CYP3A4. rn

CYP3A5*3 and POR*28 genetic variants influence the required dose of tacrolimus in heart transplant recipients

BACKGROUND After heart transplantation (HTx), the interindividual pharmacokinetic variability of immunosuppressive drugs represents a major therapeutic challenge due to the narrow therapeutic window between over-immunosuppression causing toxicity and under-immunosuppression leading to graft rejection. Although genetic polymorphisms have been shown to influence pharmacokinetics of immunosuppressants, data in the context of HTx are scarce. We thus assessed the role of genetic variation in CYP3A4, CYP3A5, POR, NR1I2, and ABCB1 acting jointly in immunosuppressive drug pathways in tacrolimus (TAC) and ciclosporin (CSA) dose requirement in HTx recipients. METHODS Associations between 7 functional genetic variants and blood dose-adjusted trough (C0) concentrations of TAC and CSA at 1, 3, 6, and 12 months after HTx were evaluated in cohorts of 52 and 45 patients, respectively. RESULTS Compared with CYP3A5 nonexpressors (*3/*3 genotype), CYP3A5 expressors (*1/*3 or *1/*1 genotype) required around 2.2- to 2.6-fold higher daily TAC doses to reach the targeted C0 concentration at all studied time points (P ≤ 0.003). Additionally, the POR*28 variant carriers showed higher dose-adjusted TAC-C0 concentrations at all time points resulting in significant differences at 3 (P = 0.025) and 6 months (P = 0.047) after HTx. No significant associations were observed between the genetic variants and the CSA dose requirement. CONCLUSIONS The CYP3A5*3 variant has a major influence on the required TAC dose in HTx recipients, whereas the POR*28 may additionally contribute to the observed variability. These results support the importance of genetic markers in TAC dose optimization after HTx.

Influence of CYP3A5 genetic variation on everolimus maintenance dosing after cardiac transplantation

BACKGROUND Everolimus (ERL) has become an alternative to calcineurin inhibitors (CNIs) due to its renal-sparing properties, especially in heart transplant (HTx) recipients with kidney dysfunction. However, ERL dosing is challenging due to its narrow therapeutic window combined with high inter-individual pharmacokinetic variability. Our aim was to evaluate the effect of clinical and genetic factors on ERL dosing in a pilot cohort of 37 HTx recipients. METHODS Variants in CYP3A5, CYP3A4, CYP2C8, POR, NR1I2, and ABCB1 were genotyped and clinical data were retrieved from patient charts. RESULTS While ERL trough concentration (C0 ) was within the targeted range for most patients, over 30-fold variability in the dose-adjusted ERL C0 was observed. Regression analysis revealed a significant effect of the non-functional CYP3A5*3 variant on the dose-adjusted ERL C0 (P = 0.031). ERL dose requirement was 0.02 mg/kg/day higher in patients with CYP3A5*1/*3 genotype compared to patients with CYP3A5*3/*3 to reach the targeted C0 (P = 0.041). ERL therapy substantially improved estimated glomerular filtration rate (28.6 ± 6.6 ml/min/1.73m(2) ) in patients with baseline kidney dysfunction. CONCLUSION ERL pharmacokinetics in HTx recipients is highly variable. Our preliminary data on patients on a CNI-free therapy regimen suggest that CYP3A5 genetic variation may contribute to this variability. This article is protected by copyright. All rights reserved.

The impact of interindividual variation in NAT2 activity on benzidine urinary metabolites and urothelial DNA adducts in exposed workers.

Several epidemiologic studies indicate that NAT2-related slow N-acetylation increases bladder cancer risk among workers exposed to aromatic amines, presumably because N-acetylation is important for the detoxification of these compounds. Previously, we showed that NAT2 polymorphisms did not influence bladder cancer risk among Chinese workers exposed exclusively to benzidine (BZ), suggesting that NAT2 N-acetylation is not a critical detoxifying pathway for this aromatic amine. To evaluate the biologic plausibility of this finding, we carried out a cross-sectional study of 33 workers exposed to BZ and 15 unexposed controls in Ahmedabad, India, to evaluate the presence of BZ-related DNA adducts in exfoliated urothelial cells, the excretion pattern of BZ metabolites, and the impact of NAT2 activity on these outcomes. Four DNA adducts were significantly elevated in exposed workers compared to controls; of these, the predominant adduct cochromatographed with a synthetic N-(3'- phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine standard and was the only adduct that was significantly associated with total BZ urinary metabolites (r = 0.68, P < 0.0001). To our knowledge this is the first report to show that BZ forms DNA adducts in exfoliated urothelial cells of exposed humans and that the predominant adduct formed is N-acetylated, supporting the concept that monofunctional acetylation is an activation, rather than a detoxification, step for BZ. However, because almost all BZ-related metabolites measured in the urine of exposed workers were acetylated among slow, as well as rapid, acetylators (mean +/- SD 95 +/- 1.9% vs. 97 +/- 1.6%, respectively) and NAT2 activity did not affect the levels of any DNA adduct measured, it is unlikely that interindividual variation in NAT2 function is relevant for BZ-associated bladder carcinogenesis.

L'utilisation de la dompéridone comme substrat marqueur de l'activité in vitro des CYP3A4 et CYP3A5

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

L'utilisation de la dompéridone comme substrat marqueur de l'activité in vitro des CYP3A4 et CYP3A5

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

CYP3A5 genetic polymorphism in HIV-patients

Poster presented at the From Basic Sciences to Clinical Research - First International Congress of CiiEM. Egas Moniz, Caparica, Portugal, 27-28 November 2015