N-acetyltransferase 2 Phenotype Research Articles

Influence of N-acetyltransferase 2 polymorphisms and clinical variables on liver function profile of tuberculosis patients

ABSTRACT Background Single nucleotide polymorphisms (SNPs) in the N-acetyltransferase 2 (NAT2) gene as well as several other clinical factors can contribute to the elevation of liver function test values in tuberculosis (TB) patients receiving antitubercular therapy (ATT). Research design and methods A prospective study involving dynamic monitoring of the liver function tests among 130 TB patients from baseline to 98 days post ATT initiation was undertaken to assess the influence of pharmacogenomic and clinical variables on the elevation of liver function test values. Genomic DNA was extracted from serum samples for the assessment of NAT2 SNPs. Further, within this study population, we conducted a case control study to identify the odds of developing ATT-induced drug-induced liver injury (DILI) based on NAT2 SNPs, genotype and phenotype, and clinical variables. Results NAT2 slow acetylators had higher mean [90%CI] liver function test values for 8–28 days post ATT and higher odds of developing DILI (OR: 2.73, 90%CI: 1.05–7.09) than intermediate acetylators/rapid acetylators. Conclusion The current study findings provide evidence for closer monitoring among TB patients with specific NAT2 SNPs, genotype and phenotype, and clinical variables, particularly between the period of more than a week to one-month post ATT initiation for better treatment outcomes.

Treatment outcomes by stratifying the N-acetyl transferase 2 phenotype in pre-treated patients with advanced refractory biliary tract cancer treated with nanvuranlat, an L-type amino acid transporter inhibitor: An ad-hoc analysis of a randomized, double-blind, placebo-controlled phase 2 study.

483 Background: L-type amino acid transporter 1 (LAT1, SLC7A5) is overexpressed in cancer cells, leading to aggressive proliferation and lymphatic metastases. In a placebo-controlled, randomized phase 2 trial involving patients with pre-treated, advanced, refractory biliary tract cancer (BTC), monotherapy with nanvuranlat (JPH203), a selective LAT1 inhibitor, demonstrated a significant improvement in progression free survival (PFS) as the primary endpoint compared to placebo. The safety profile was comparable between nanvuranlat and placebo treated patients, except for a higher rate of cholangitis in the nanvuranlat group. Nanvuranlat is metabolized to its main inactive, off-target metabolite, Nac-JPH203 by N-acetyl transferase 2 ( NAT2). Since the NAT2 unknown patients mislead the benefit and risk ratio of nanvuranlat treatment, an ad-hoc analysis was conducted in enriched NAT2 non-rapid (NR) phenotype patients who slowly produced Nac-JPH203 in plasma. Methods: Among 104 (full analytical set (FAS) - nanvuranlat:69 & placebo:35) enrolled patients, 25 with unknown NAT2 phenotype were enrolled prior to an amendment to only enroll NAT2-NR phenotype. Subsequently, further analysis was conducted to determine the NAT2 phenotypes of these 25 patients. Results: Among the 25 NAT2 unknown patients, 16 and 9 patients were assigned to nanvuranlat or placebo, respectively. NAT2 ad hoc analysis recorded those 9 patients in nanvuranlat and 5 in placebo as NAT2 NR, and 7 patients in nanvuranlat and 4 in placebo were NAT2 rapid (R). As a result, number of NAT2 NR patients was increased from 81 to 94. Efficacy analysis of this enriched population (FAS-NR-nanvuranlat:63 & placebo:31) revealed a significant improvement in PFS. With respect to cholangitis, 8 all-grade (12.7%) and 7 over grade 3 (11.1%) were documented in the FAS-NR population. In the FAS-R population, 2 all grades (28.5%) and 2 over grade 3 (28.5%) were observed (Table). Conclusions: By classifying BTC patients by NAT2 phenotype in this ad hoc analysis, the LAT1 inhibitor nanvuranlat further improved its clinical efficacy and safety in the NAT2 NR population. Therefore, selecting NAT2 NR for nanvuranlat treatment may improve the risk -benefit ratio. Clinical trial information: UMIN000034080 . [Table: see text]

Risk adjustment model for tuberculosis compared to non-tuberculosis mycobacterium or latent tuberculosis infection: Center for Personalized Precision Medicine of Tuberculosis (cPMTb) cohort database

BackgroundThe Center for Personalized Precision Medicine of Tuberculosis (cPMTb) was constructed to develop personalized pharmacotherapeutic systems for tuberculosis (TB). This study aimed to introduce the cPMTb cohort and compare the distinct characteristics of patients with TB, non-tuberculosis mycobacterium (NTM) infection, or latent TB infection (LTBI). We also determined the prevalence and specific traits of polymorphisms in N-acetyltransferase-2 (NAT2) and solute carrier organic anion transporter family member 1B1 (SLCO1B1) phenotypes using this prospective multinational cohort.MethodsUntil August 2021, 964, 167, and 95 patients with TB, NTM infection, and LTBI, respectively, were included. Clinical, laboratory, and radiographic data were collected. NAT2 and SLCO1B1 phenotypes were classified by genomic DNA analysis.ResultsPatients with TB were older, had lower body mass index (BMI), higher diabetes rate, and higher male proportion than patients with LTBI. Patients with NTM infection were older, had lower BMI, lower diabetes rate, higher previous TB history, and higher female proportion than patients with TB. Patients with TB had the lowest albumin levels, and the prevalence of the rapid, intermediate, and slow/ultra-slow acetylator phenotypes were 39.2%, 48.1%, and 12.7%, respectively. The prevalence of rapid, intermediate, and slow/ultra-slow acetylator phenotypes were 42.0%, 44.6%, and 13.3% for NTM infection, and 42.5%, 48.3%, and 9.1% for LTBI, respectively, which did not differ significantly from TB. The prevalence of the normal, intermediate, and lower transporter SLCO1B1 phenotypes in TB, NTM, and LTBI did not differ significantly; 74.9%, 22.7%, and 2.4% in TB; 72.0%, 26.1%, and 1.9% in NTM; and 80.7%, 19.3%, and 0% in LTBI, respectively.ConclusionsUnderstanding disease characteristics and identifying pharmacokinetic traits are fundamental steps in optimizing treatment. Further longitudinal data are required for personalized precision medicine.Trial registrationThis study registered ClinicalTrials.gov NO. NCT05280886.

Application of a physiologically based pharmacokinetic model to predict isoniazid disposition during pregnancy.

Pregnancy can increase the risk of latent tuberculosis infection (LTBI) progression to tuberculosis (TB) disease. Isoniazid (INH) is the preferred preventative treatment for LTBI in pregnancy. INH is mainly cleared by N-acetyltransferase 2 (NAT2) but the pharmacokinetics (PK) of INH in different NAT2 phenotypes during pregnancy is not well characterized. To address this knowledge gap, we used physiologically based pharmacokinetic (PBPK) modeling to evaluate NAT2 phenotype-specific effects of pregnancy on INH disposition. A whole-body PBPK model for INH was developed and verified for non-pregnant NAT2 fast (FA), intermediate (IA), and slow (SA) acetylators. Model predictive performance was assessed using a drug-specific model acceptance criterion for mean plasma area under the curve (AUC) and peak plasma concentration (Cmax ), and the absolute average fold error (AAFE) for individual plasma concentrations. The verified model was extended to simulate INH disposition during pregnancy in NAT2 SA, IA, and FA populations. A sensitivity analysis was conducted using the verified PBPK model and known changes in INH disposition during pregnancy to determine whether NAT2 activity changes during pregnancy or other INH clearance pathways are altered. This analysis suggested that NAT2 activity is unchanged while other INH clearance pathways increase by ~80% during pregnancy. The model was applied to explore the effect of pregnancy on INH disposition in two ethnic populations with different NAT2 phenotype distributions and with high TB burden. Our PBPK model can be used to predict INH disposition during pregnancy in diverse populations and expanded to other drugs cleared by NAT2 during pregnancy.

Accumulation and distribution of persistent organic pollutants in surface sediment of the estuarine region and correlation between genetic and environmental factors: A case study

In this study, the accumulation and distribution of nine organochlorine pesticides (OCPs) and seven polychlorinated biphenyls (PCBs) in the An Hoa Estuary, Quang Nam Province, Central Vietnam, and the correlation between genetic and environmental factors have been analyzed and assessed. This study analyzed sediment samples from 15 positions in the An Hoa Estuary during the dry season. The OCP concentrations varied between ND (not detected) and 1.82 μ g/kg (S1), mainly consisting of DDE. The total concentration of PCBs ranged between 1.52–19.5 μ g/kg (highest at S13), with PCB-138 and PCB-153 as the congeners most frequently detected in the analyzed samples. The results show that the ecological risk of OCPs and PCBs in the An Hoa Estuary ranges from low to moderate levels. In order to clarify the significance of genetic factors that can be influenced and affected by environmental factors over a certain period, this study also determined the N-acetyltransferase (NAT) enzyme activity in the population residing in the An Hoa Estuary for a period of 30–50 years to show clearly the relationship between weak environmental and genetic factors in determining the risks for environmental health. Based on the results, out of a total of 72 volunteer participants, the phenotype of the N-acetyltransferase system was determined and divided into two groups: rapid (30.6%) and slow (69.4%). Among them, the slow acetylator phenotype is prevalent in two locations, S11 and S13, with 88% and 91.7% of volunteers, respectively. The rapid acetylator phenotype was detected in 17 of 23 participants at location S7, accounting for more than 77% of the total individuals with the rapid acetylator phenotype in this experiment. • The accumulation of nine OCPs and seven PCBs in sediment was determined and assessed. • The highest OCPs (1.82 μ g/kg) and PCBs (19.5 μ g/kg) were detected at sampling sites S1 and S13, respectively. • The activity of xenobiotics biotransformation enzymatic system N-acetyltransferase was determined. • The relationship between weak environmental factors and genetic factors was examined. • The N-acetyltransferase phenotype of citizens was divided into rapid (30.6%) and slow (69.4%).

Functional Characterization of the Effects of N-acetyltransferase 2 Alleles on N-acetylation of Eight Drugs and Worldwide Distribution of Substrate-Specific Diversity.

Variability in the enzymatic activity of N-acetyltransferase 2 (NAT2) is an important contributor to interindividual differences in drug responses. However, there is little information on functional differences in N-acetylation activities according to NAT2 phenotypes, i.e., rapid, intermediate, slow, and ultra-slow acetylators, between different substrate drugs. Here, we estimated NAT2 genotypes in 990 Japanese individuals and compared the frequencies of different genotypes with those of different populations. We then calculated in vitro kinetic parameters of four NAT2 alleles (NAT2∗4, ∗5, ∗6, and ∗7) for N-acetylation of aminoglutethimide, diaminodiphenyl sulfone, hydralazine, isoniazid, phenelzine, procaineamide, sulfamethazine (SMZ), and sulfapyrizine. NAT2∗5, ∗6, and ∗7 exhibited significantly reduced N-acetylation activities with lower Vmax and CLint values of all drugs when compared with NAT2∗4. Hierarchical clustering analysis revealed that 10 NAT2 genotypes were categorized into three or four clusters. According to the results of in vitro metabolic experiments using SMZ as a substrate, the frequencies of ultra-slow acetylators were calculated to be 29.05–54.27% in Europeans, Africans, and South East Asians, whereas Japanese and East Asian populations showed lower frequencies (4.75 and 11.11%, respectively). Our findings will be helpful for prediction of responses to drugs primarily metabolized by NAT2.

Association of NAT2 genetic polymorphism with the efficacy of Neurotropin\xae for the enhancement of aggrecan gene expression in nucleus pulposus cells: a pilot study

BackgroundIntervertebral disc degeneration, one of the major causes of low-back pain, results from altered biosynthesis/turnover of extracellular matrix in the disc. Previously, we reported that the analgesic drug Neurotropin® (NTP) had an anabolic effect on glycosaminoglycan synthesis in cultured nucleus pulposus (NP) cells via the stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1. However, its effect on the aggrecan core protein was not significantly detected, because of the data variance. A microarray analysis suggested that the effect of NTP on aggrecan was correlated with N-acetyltransferase 2 (NAT2), a drug-metabolizing enzyme. Specific NAT2 alleles are known to correlate with rapid, intermediate, and slow acetylation activities and side effects of various drugs. We investigated the association between the efficacy of NTP on aggrecan expression and the NAT2 genotype in cell donors.MethodsNP cells were isolated from intervertebral disc tissues donated by 31 Japanese patients (28–68 years) who underwent discectomy. NTP was added to the primary cell cultures and its effect on the aggrecan mRNA was analyzed using real-time quantitative PCR. To assess acetylator status, genotyping was performed based on the inferred NAT2 haplotypes of five common single-nucleotide polymorphisms using allele-specific PCR.ResultsThe phenotype frequencies of NAT2 in the patients were 0%, 42.0%, and 58.0% for slow, intermediate, and rapid acetylators, respectively. The proportions of responders to NTP treatment (aggrecan upregulation, ≥ 1.1-fold) in the intermediate and rapid acetylators were 76.9% and 38.9%, respectively. The odds ratio of the comparison of the intermediate acetylator status between responders and nonresponders was 5.2 (95% CI 1.06–26.0, P = 0.036), and regarding the 19 male patients, this was 14.0 (95% CI 1.54–127.2, P = 0.012). In the 12 females, the effect was not correlated with NAT2 phenotype but seemed to become weaker along with aging.ConclusionsAn intermediate acetylator status significantly favored the efficacy of NTP treatment to enhance aggrecan production in NP cells. In males, this tendency was detected with higher significance. This study provides suggestive data of the association between NAT2 variants and the efficacy of NTP treatment. Given the small sample size, results should be further confirmed.

Haplotype-specific PCR for NAT2 diplotyping

N-acetyltransferase 2 (NAT2) is an enzyme that acetylates many kinds of drugs, including the antituberculosis drug isoniazid. The NAT2 gene is highly diverse across populations. An individual can be classified as having a slow acetylator (SA), an intermediate acetylator (IA), or a rapid acetylator (RA) phenotype based on its two haplotypes (diplotype) of NAT2. SA individuals are at a higher risk for isoniazid-induced hepatitis, while the RA phenotype contributes to failure in tuberculosis treatment. Being able to predict individual NAT2 phenotypes is important for dose adjustment of isoniazid. NAT2 haplotypes are commonly determined via an indirect method of statistical haplotype inference from SNP genotyping. Here, we report a direct NAT2 haplotyping method using haplotype-specific PCR (HS-PCR) for the 6 most commonly found NAT2 haplotypes: NAT2*4, NAT2*5B, NAT2*6A, NAT2*7B, NAT2*12A, and NAT2*13A. Validation of this HS-PCR method via comparison with a sequencing method in 650 Thai DNA samples (107 RA, 279 IA, and 264 SA samples) showed a concordance rate for diplotype calls of 99.23% (645/650 samples). The discordant results in 5 samples were due to 3 rare NAT2 haplotypes: NAT*5C (n = 3), NAT2*7C (n = 1), and NAT2*11A (n = 1). This novel HS-PCR method allows direct NAT2 diplotyping, enabling the implementation of NAT2 acetylator phenotypes in clinical pharmacogenetic testing.

NAT2 gene polymorphisms and endometriosis risk: A PRISMA-compliant meta-analysis.

ObjectiveEndometriosis is a common chronic, gynecological disease. Despite many studies on the role of N-acetyltransferase 2 (NAT2) in endometriosis, its clinical significance is unclear. In this study, associations between NAT2 phenotypes as well as single nucleotide polymorphisms (SNPs) within NAT2 (i.e. rs1799929, rs1799930, rs1208, and rs1799931) and endometriosis risk were evaluated using a meta-analysis approach.MethodsEmbase, PubMed, ClinicalTrials.gov, CNKI (China National Knowledge Infrastructure), Wanfang databases, Cochrane Library for clinical trials, and Web of Science were searched to identify relevant articles. ORs (odds ratios) and 95% CIs (95% confidence intervals) were used to estimate the associations between NAT2 polymorphisms and endometriosis risk. Heterogeneity among included studies was also assessed. In addition, a subgroup analysis of NAT2 phenotypes and endometriosis risk based on ethnicity was performed.ResultsNine case-control studies met the inclusion criteria. The odds ratio was 2.30 (95% CI: 1.61–3.28) for the NAT2 slow acetylation phenotype versus the intermediate + fast acetylation phenotype in the Asian population. These results suggest that Asian individuals with the NAT2 slow acetylation phenotype have a 130% increased risk of endometriosis. A significant association was also found for rs1799930 (OR = 0.74; 95% CI, 0.59–0.92), suggesting that individuals with this mutant genotype have a 26% decreased risk of endometriosis.ConclusionsThe rs1799930 mutant genotypes are associated with a decreased risk of endometriosis. No statistically significant associations were found between rs1799931, rs1208, or rs1799929 and endometriosis. Based on a subgroup analysis based on ethnicity, the NAT2 slow acetylation phenotype was found to increase the risk of endometriosis in Asians. No statistically significant associations were found between the NAT2 slow acetylation phenotype and endometriosis risk in Caucasians. Accordingly, NAT2 phenotypes and SNPs are potential biomarkers for the diagnosis and treatment of endometriosis.

N-acetyltransferase 2 enzyme genotype-phenotype discordances in both HIV-negative and HIV-positive Nigerians.

The N-acetyltransferase 2 (NAT2) enzyme has been understudied in Nigerians including genotype-phenotype association studies. The aim of this study was NAT2 haplotype identification and genotype-phenotype investigations in HIV-positive and HIV-negative Nigerians. Phenotypes included self-reported sulphonamide hypersensitivity survey, experimental and computational NAT2 phenotyping. The NAT2 gene was amplified by PCR. Gene sequencing used ABI 3730 and Haploview 4.2 for haplotype reconstruction. Genotype-phenotype analyses used the χ P-value and odds ratio with a 95% confidence interval. Self-reported sulphonamide hypersensitivity showed a prevalence of 3.1 and 12.4% in HIV-positive and HIV-negative Nigerians, respectively. NAT2 genetic variants 191G>A, 282C>T, 341T>C, 481C>T, 590G>A, 803A>G and 857G>A were not significantly different between both groups (odds ratio=0.87; 95% confidence interval: 0.54-1.38, P=0.55). Nine haplotypes: NAT2*4, NAT2*12A, NAT2*13A, NAT2*5B, NAT2*6A, NAT2*7B, NAT2*5C, NAT2*14B and NAT2*14A had frequencies more than 1%, whereas NAT2*12B had 1.1% in the HIV-positive and 0.4% in the HIV-negative group. Overall, slow acetylator haplotypes made up 68%. The NAT2*12 signature single-nucleotide polymorphism was in high linkage disequilibrium with signature single-nucleotide polymorphism for NAT2*13 (D'=0.97, r=0.61) and NAT2*5 (D'=0.98, r=0.64). Genotype-phenotype association analysis showed haplotypes NAT2*13A, NAT2*5C, NAT2*7B and NAT2*14A to be associated strongly with the slow metabolic phenotype (P=0.002, 0.029, 0.032 and 0.050, respectively). Computational phenotypes were similar, with 30.9, 66 and 3.1% for slow, intermediate and rapid acetylators, respectively, among HIV-positive Nigerians and 31.2, 66.3 and 2.5% among the HIV-negative group. Overall, slow phenotypes made up 31%. NAT2 haplotype frequencies are similar in Nigerians, irrespective of HIV status, but genotype-phenotype discordances exist.

Establishing the Genotyping Method for NAT2 Polymorphism in Vietnamese Tuberculoma Patients

The metabolism of Isoniazid, one of the first-line antituberculosis drugs for TB treatment and prophylaxis, depends on the acetyltransferase 2 acetylation (NAT2) phenotype. Different phenotypes of NAT2 will lead to differences in drug concentration and the risk of uncontrolled side effects, such as hepatitis, peripheral neuropathy, gastrointestinal disorders (nausea, vomiting, and stomach pain). These risks are related to the presence of mutant NAT2 alleles such as NAT2*5 (c.341T> C), *6 (c.590G> A) and *7 (c.857G> A), that reduce the N- acetyltransferase activity. Therefore, the genotyping method for NAT2 polymorphism using RFLP and Sanger sequencing was established. The method was successfully applied to determine the polymorphism of 84 TB patients. This study provides a better tool for analyzing NAT2 gene to assist clinicians in treating isoniazid.
 Keywords
 Enzyme NAT2, isoniazid, single nucleotide polymorphism, RFLP, Sanger sequencing.
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Relevance of NAT2 genotype to anti-tuberculosis drug-induced hepatotoxicity in a Chinese Han population.

Anti-tuberculosis drug-induced hepatotoxicity (ATDH) is a serious adverse drug reaction. The slow acetylator status of N-acetyl transferase 2 (NAT2) is a well-established risk factor for ATDH. One novel tagging single nucleotide polymorphism (tagging SNP), rs1495741, in NAT2 has been found to be highly predictive of the NAT2 phenotype. The present study aimed to validate the relationships between tagging SNP rs1495741 and ATDH in a Chinese Han population. A 1:2 matched case-control study was conducted using 235 ATDH cases and 470 controls. Conditional or unconditional logistic regression analysis was used to estimate the association between genotypes and the risk of ATDH according to the odds ratio (OR) with a 95% confidence interval (CI). Patients carrying the AA genotype of tagging SNP rs1495741 were at higher risk of ATDH than those carrying the GG genotype (OR=1.653, 95% CI=1.050-2.601; p=0.030). Subgroup analysis suggested that the AA genotype was a risk factor for ATDH in patients aged older than 50years (OR=2.486, 95% CI=1.313-4.706; p=0.005), weighing over 50kg (OR=1.757, 95% CI=1.016-3.038; p=0.044) or using a hepatoprotectant (OR=1.611, 95% CI=1.009-2.572; p=0.046). Tagging SNP rs1495741 was not a significant risk factor for moderate and severe hepatotoxicity but appears to be relevant to risk of mild hepatotoxicity specifically. The present study is the first to validate the relationships between the tagging SNP rs1495741 and ATDH in a Chinese population. Based on this case-control study, the NAT2 rs1495741 polymorphism is a risk factor for mild but not more severe ATDH in Chinese Han patients.

Expression and genotype-dependent catalytic activity of N-acetyltransferase 2 (NAT2) in human peripheral blood mononuclear cells and its modulation by Sirtuin 1

N-acetyltransferase 2 (NAT2) catalyzes the biotransformation of numerous arylamine and hydrazine drugs and carcinogens. Genetic polymorphisms of NAT2 modify drug efficacy and toxicity and susceptibility to diseases such as cancer and type 2 diabetes. Expression of NAT2 has been documented in the liver and gastrointestinal tract but not in other tissues. Deacetylation of cytosolic proteins by sirtuins is a post-translational modification important in regulatory networks of diverse cellular processes. The aim of the present study was to investigate NAT2 expression in peripheral blood mononuclear cells (PBMC) and the effects of NAT2 genotype and Sirtuin 1 (SIRT1). Both NAT2 and SIRT1 proteins were expressed on PBMC. Their expression was more prevalent on CD3+ compared to CD19+ and CD56+ cell populations. N-acetylation capacity of PBMC exhibited a NAT2 gene-dose response toward the N-acetylation of isoniazid. Subjects with rapid NAT2 genotype showed an apparent Vmax of 42.1 ± 2.4; intermediate NAT2 genotypes an apparent Vmax of 22.6 ± 2.2; and slow acetylator NAT2 genotypes an apparent Vmax of 19.9 ± 1.7 nM acetyl-isoniazid/24 h/million cells. The N-acetylation capacity of NAT2 in the presence of SIRT1 enhancer was significantly decreased (p < 0.001), conversely, the transient silencing of SIRT1 resulted in an increase of N-acetylation capacity (p < 0.001). These findings are the first report of NAT2 genotype-dependent expression on PBMC and post-translational modification by SIRT1. These findings constitute a substantial advance in our understanding of human N-acetyltransferase expression and a new much less invasive method for measurement of human NAT2 expression and phenotype.

Retrospective analysis of estrogen receptor1 and N‑acetyltransferase gene expression in normal breast tissue, primary breast tumors, and established breast cancer cell lines.

The expression levels of estrogen receptor 1 (ESR1), arylamine N-acetyltransferase 1 (NAT1), and arylamine N-acetyltransferase 2 (NAT2) are implicated in breast cancer; however, their co-expression profiles in normal breast tissue, primary breast tumors and established breast cancer cell lines are undefined. NAT1 expression is widely reported to be associated with ESR1 expression and is frequently investigated in breast cancer etiology. Furthermore, the NAT2 phenotype has been reported to modify breast cancer risk in molecular epidemiological association studies. Understanding the relationships between the expression levels of these genes is essential to understand their role in breast cancer etiology and treatment. In the present study, NAT1, NAT2 and ESR1 expression data were accessed from repositories of RNA-Seq data covering 57 breast cancer cell lines, 1,043 primary breast tumors and 99 normal breast tissues. The relationships between gene expression, and between NAT1 activity and RNA expression in breast cancer cell lines were evaluated using non-parametric statistical analyses. Differences in gene expression in each dataset, as well as gene expression differences in normal breast tissue compared to primary breast tumors, and stratification by estrogen receptor status were determined. NAT1 and NAT2 mRNA expression were detected in normal and primary breast tumor tissues; NAT1 expression was much higher than NAT2. NAT1 and ESR1 expression were strongly associated, whereas NAT2 and ESR1 expression were not. Although NAT1 and NAT2 expression were associated, the magnitude was moderate. NAT1, NAT2, and ESR1 expression were increased in primary breast tumor tissue compared with normal breast tissue; however, the magnitude and significance of the differences were lower for NAT2. Analysis of NAT1, NAT2, and ESR1 expression in normal and primary breast tissues and breast cancer cell lines suggested that NAT1 and NAT2 expression are regulated by distinctive mechanisms, whereas NAT1 and ESR1 expression may have overlapping regulation. Defining these relationships is important for future investigations into breast cancer prevention.

N-Acetyltransferase-2 (NAT2) phenotype is influenced by genotype-environment interaction in Ethiopians

Background and objectivesN-acetyltransferase 2 (NAT2) metabolize several drugs including isoniazid. We investigated the effect of genotype, geographical difference, and smoking habit on NAT2 phenotype in Ethiopians.MethodsGenotyping for NAT2 191G > A, 341 T > C, 590G > A, and 857G > A was performed in 163 unrelated healthy Ethiopians (85 living in Ethiopia and 78 living in Sweden). The NAT2 phenotype was determined using caffeine as a probe and log AFMU/(AFMU + 1X + 1 U) urinary metabolic ratio (MR) as an index.ResultsThe frequencies of NAT2*4, *5, *6, *7, and *14 haplotypes were 14.1, 48.5, 30.1, 5.5, and 1.8%, respectively. The frequencies of rapid (NAT2*4/*4), intermediate (heterozygous *4), and slow (no *4 allele) acetylator genotypes were 1.8, 24.6, and 73.6%, respectively. The distribution NAT2 MR was bimodal with 70% being phenotypically slow acetylators. NAT2 genotype (p < 0.0001) and country of residence (p = 0.004) independently predicted NAT2 phenotype. Controlling for the effect of genotype, ethnic Ethiopians living in Ethiopia had significantly higher NAT2 MR than those living in Sweden (p = 0.006). NAT2 genotype-phenotype concordance rate was 75%. Distinct country-of-residence-based genotype-phenotype discordance was observed. The proportion of phenotypically determined rapid acetylators was significantly higher and slow acetylators was lower in Ethiopians living in Ethiopia (39% rapid, 61% slow) than in Sweden (20% rapid, 80% slow). Sex and smoking had no significant effect on NAT2 MR.ConclusionsWe report a high prevalence of NAT 2 slow acetylators in Ethiopians and a conditional NAT2 genotype-phenotype discordance implicating a partial phenotype conversion and metabolic adaptation. Gene-environment interactions regulate NAT2 phenotype.

Biomarkers of Presbycusis and Tinnitus in a Portuguese Older Population.

Introduction: Presbycusis or age-related hearing loss (ARHL) is a ubiquitous health problem. It is estimated that it will affect up to 1.5 billion people by 2025. In addition, tinnitus occurs in a large majority of cases with presbycusis. Glutamate metabotropic receptor 7 (GRM7) and N-acetyltransferase 2 (NAT2) are some of the genetic markers for presbycusis.Objectives: To explore patterns of hearing loss and the role of GRM7 and NAT2 as possible markers of presbycusis and tinnitus in a Portuguese population sample.Materials and Methods: Tonal and speech audiometry, tinnitus assessment, clinical interview, and DNA samples were obtained from patients aged from 55 to 75 with or without tinnitus. GRM7 analysis was performed by qPCR. Genotyping of single nucleotide polymorphisms (SNPs) in NAT2 was performed by PCR amplification followed by Sanger sequencing or by qPCR.Results: We screened samples from 78 individuals (33 men and 45 women). T allele at GRM7 gene was the most observed (60.3% T/T and 33.3% A/T). Individuals with a T/T genotype have a higher risk for ARHL and 33% lower risk for tinnitus, compared to individuals with A/A and A/T genotype, respectively. Being a slow acetylator (53%) was the most common NAT2 phenotype, more common in men (55.8%). Intermediate acetylator was the second most common phenotype (35.9%) also more frequent in men (82.6%). Noise exposed individuals and individuals with ‘high frequency’ hearing loss seem to have a higher risk for tinnitus. Our data suggests that allele AT of GRM7 can have a statistically significant influence toward the severity of tinnitus.Conclusion: For each increasing year of age the chance of HL increases by 9%. The risk for ARHL was not significantly associated with GRM7 neither NAT2. However, we cannot conclude from our data whether the presence of T allele at GRM7 increases the odds for ARHL or whether the A allele has a protective effect. Genotype A/T at GRM7 could potentially be considered a biomarker of tinnitus severity. This is the first study evaluating the effect of GRM7 and NAT2 gene in tinnitus.

Algorithm for the Automated Evaluation of NAT2 Genotypes.

N-Acetyltransferase 2 (NAT2) genotyping by PCR and RFLP-based methods provides information on seven single nucleotide polymorphisms (SNPs) without deriving the chromosomal phase (haplotype). So genotyping results must be processed to get all possible NAT2haplotype (or allele) combinations. Here we describe the procedure for genotyping and present a program based on Microsoft® Access® which automatically generates all possible haplotype pairs for a given unphased NAT2 genotype. NAT2haplotypes are important to predict the NAT2 phenotype.

Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B>NAT2*5B/*6A>NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p>0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

Arylamine N-acetyltransferase 2 gene polymorphism in an Algerian population

Background: The arylamine N-acetyltransferase 2 (NAT2) is a key enzyme in the biotransformation of xenobiotics. NAT2 gene polymorphisms have been associated with the risk of isoniazid hepatotoxicity and these polymorphisms change among different populations.Aim: The objective of this study is to investigate NAT2 polymorphisms in order to predict the prevalence of NAT2 phenotype in an Algerian population.Subjects and methods: Genotyping of NAT2 was done using a PCR-RFLP method. Haplotype was analysed using the software package PHASE, version 2.0.Results: The major haplotypes were NAT2*5B (23.72%), NAT2*6 A (18.61%), NAT2*4 (14.60%) and NAT2*5 F (10%). The average of the expected slow acetylator phenotype was 53%.Conclusion: Our results suggest that the high frequency of slow acetylator phenotype requires investigation into its possible association with ATDH.

Single nucleotide polymorphism coverage and inference of N-acetyltransferase-2 acetylator phenotypes in wordwide population groups.

Several algorithms have been proposed to reduce the genotyping effort and cost, while retaining the accuracy of N-acetyltransferase-2 (NAT2) phenotype prediction. Data from the 1000 Genomes (1KG) project and an admixed cohort of Black Brazilians were used to assess the accuracy of NAT2 phenotype prediction using algorithms based on paired single nucleotide polymorphisms (SNPs) (rs1041983 and rs1801280) or a tag SNP (rs1495741). NAT2 haplotypes comprising SNPs rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208 and rs1799931 were assigned according to the arylamine N-acetyltransferases database. Contingency tables were used to visualize the agreement between the NAT2 acetylator phenotypes on the basis of these haplotypes versus phenotypes inferred by the prediction algorithms. The paired and tag SNP algorithms provided more than 96% agreement with the 7-SNP derived phenotypes in Europeans, East Asians, South Asians and Admixed Americans, but discordance of phenotype prediction occurred in 30.2 and 24.8% 1KG Africans and in 14.4 and 18.6% Black Brazilians, respectively. Paired SNP panel misclassification occurs in carriers of NATs haplotypes *13A (282T alone), *12B (282T and 803G), *6B (590A alone) and *14A (191A alone), whereas haplotype *14, defined by the 191A allele, is the major culprit of misclassification by the tag allele. Both the paired SNP and the tag SNP algorithms may be used, with economy of scale, to infer NAT2 acetylator phenotypes, including the ultra-slow phenotype, in European, East Asian, South Asian and American populations represented in the 1KG cohort. Both algorithms, however, perform poorly in populations of predominant African descent, including admixed African-Americans, African Caribbeans and Black Brazilians.