Comparative analysis of N-acetyltransferase 2 genotyping results among patients with newly diagnosed pulmonary tuberculosis residing in the Sakha Republic (Yakutia)

Aim. To assess the variability of the NAT2 gene and to comparatively analyze the prevalence of NAT2 polymorphisms and acetylation types among Yakut and Russian patients newly diagnosed with pulmonary tuberculosis (TB), permanently residing in the Sakha Republic (Yakutia). Materials and methods. The study included 197 patients with newly diagnosed pulmonary TB (132 Yakuts and 65 Russians) aged (43.3 ± 14.4). The following single-nucleotide polymorphisms were analyzed, using re -al-time polymerase chain reaction (PCR): NAT2*5 (rs1801280, Т341С), NAT2*6 (rs1799930, G590A), NAT2*7 (rs1799931, G857A), NAT2*11 (rs1799929, C481T), NAT2*12 (rs1208, A803G), and NAT2*13 (rs1041983, C282T). Genetically determined basal metabolic rates were calculated using the NATpred online tool. Results. 75% of residents, both of Yakut and Russian ethnicity, were identified as carriers of NAT2 polymorphic variants known to be related to isoniazid biotransformation. NAT2*6 and * 13 allelic variants were more frequent in Yakuts (occurring in 40.9% and 64.4%, respectively); variants NAT2*5, *6, *11, *12 , and * 13 were more common in Russians (69.2; 55.4; 67.7; 69.2, and 64.6%, respectively). The NAT2*5, *7, *11 , and * 12 polymorphisms were found to be significantly ethnicity-dependent. The study established substantial prevalence of medium acetylation type (58.3%) in Yakuts and slow acetylation type in Russians (61.5%). Correlations were shown between ethnicity and different prevalence rates of rapid, medium, or slow acetylation types among patients with TB. Conclusion. The observed NAT2 polymorphism distribution patterns and isoniazid acetylation types among Yakut and Russian patients with newly diagnosed pulmonary TB demonstrated that pharmacologic responses can be significantly different between ethnic groups. Findings of pharmacogenetic studies in Yakut and Russian populations should be incorporated in clinical practice for personalized administration of isoniazid.


INTRODUCTION
Conventionally recommended treatment for newly identified drug-sensitive pulmonary tuberculosis consists of a combination of 4 most effective anti-TB drugs, such as isoniazid, rifampicin, pyrazinamide, and ethambutol, administered in standard doses (http://cr.rosminzdrav.ru/#!/schema/943). In reality, individual differences in pharmacologic responses to these drugs, developing quite often, include poor chemotherapy outcomes in some patients, possible development of M. tuberculosis drug resistance followed by disease relapse, and adverse drug reactions [1]. In particular, isoniazid is a drug with a known hepatotoxic effect, which can cause liver damage with clinical manifestations ranging from asymptomatic hyperenzymemia (10-20% of patients) to severe hepatitis or acute hepatic failure (0.5-1%) [2]. Toxic liver effect is produced by highly active isoniazid metabolites, hydrazine and acetylhydrazine [3,4].
NAT2 gene polymorphism distribution is known to vary substantially and has been shown to correlate with race, ethnic origin, and place of residence [9][10][11]. The aim of this study was to assess the variability of NAT2 gene and to comparatively analyze prevalence of NAT2 gene polymorphisms and acetylation types among Yakuts and Russians with newly identified pulmonary tuberculosis (PTB).

MATERIALS AND METHODS
Single-center, one stage, observational sampling study was conducted, including 197 patients with newly identified PTB, selected from representatives of 2 ethnic groups living in the Sakha Republic (Yakutia): 132 Yakuts (77 women, 55 men) and 65 Russians (35 women, 30 men). Patients were hospitalized to Phthisiatry Research-Practice Center in Yakutsk during the intensive chemotherapy phase. Patient's average age was 43.3 ± 14.4 years. Inclusion criteria were PTB diagnosed for the first time, age of 18 years or over, informed consent, and Yakut or Russian ethnicity. Ethnicity was established based on self-definition by patients and their parents; family trees were also analyzed to the second generation. In earlier studies, it was shown that ethnic self-definition corresponded to microsatellite analysis in 99.9% of cases [12]. Descendants from mixed marriages and patients who did not meet any of the inclusion criteria were excluded.
Statistical analysis was performed using IBM SPSS Statistics ver. 23. Pearson's chi-squared test and its modification with Yates's correction were used for analysis. Compliance of genotype distribution with Hardy -Weinberg equilibrium was checked using 95% Clopper -Pearson confidence intervals. The critical significance level p was 0.05.

DISCUSSION
Genetic diversity of the NAT2 gene and acetylation phenotypes developed as a result of human adaption to living environment. Transition from nomadic to sedentary life profoundly changed food choices, re-sulting in the body being exposed to novel pathogens and xenobiotics. Further, due to the need for better survival the activity of detoxifying enzymes had been altered, producing a new heritable phenotype of biotransformation [14].
Comparative analysis of NAT2 genotype distribution showed that Russian patients were more frequent carriers of NAT2*5, *11, and *12 than Yakuts (Table 1). To date, evidence is lacking on the contribution of NAT2*5 and *11 genotypes to severity and frequency of isoniazid-induced liver damage in patients with tuberculosis. There is a known correlation between increased risk of isoniazid-induced hepatotoxicity and minor allele homozygous genotypes, compared with the same risk in carriers of major alleles of NAT*5 and *11 [15,16].
Polymorphic variant NAT2*7 was more frequent in Yakuts (28.8%) than in Russians (13.8%) (p < 0.05). Genotype A/A NAT2*7 was observed in a small number of Yakut patients (2.3%) and in none of the Russian patients (Table 1). Few studies have reported inconclusive data on association between minor allele A NAT2*7 and hepatotoxicity risk. Some authors pointed out a higher risk of hepatotoxic reactions to first-line anti-TB drugs in individuals with A/A genotype, in contrast to carriers of G/G genotype [17,18], while other researchers reported absence of such associations [2,19].
Major alleles of NAT2*5, *6, and NAT2*7 encode synthesis of NAT2 with altered amino acid sequence and, therefore, lower activity. People with NAT2*5 allele in combination with NAT2*6, or *7 polymorphic variant are slow acetylators [8]. Geographic dis-tribution of slow acetylators has been well studied: this phenotype occurs in 60% of the population of Europe, Middle East, North Africa, and South Asia, and in 10% of the population of East Asia and Native Americans [20].
In Yakut population, the most widespread acetylation type was medium type (58.3%), while Russians mostly were characterized by slow acetylation type (61.5%). The proportion of rapid acetylators was much larger among Yakuts, than among Russians (18.9% versus 3.1%). This is consistent with previous comparative studies among Asians and Caucasians.
In clinical practice, NAT2 polymorphism and genetically determined variability in isoniazid acetylation speed can have a considerable impact on the outcome and safety of tuberculosis pharmacotherapy. A link between liver damage rate and slow acetylation type was confirmed in several meta-analyses [21][22][23][24]. Slow acetylators showed high serum concentrations of isoniazid and its toxic metabolites [25]. Rapid acetylators had lower serum isoniazid concentrations, but higher risk of drug resistance to M. tuberculosis [25][26][27][28].

CONCLUSION
Our study results suggest that NAT2 gene polymorphisms linked to isoniazid acetylation have considerable prevalence rates among Yakuts and Russians. Yakuts mostly tended to be carriers of allelic variants NAT2*6 and *13, while Russians mostly carried variants NAT2*5, *6, *11, *12, and *13. Comparative analysis within the study sample showed the presence of statistically significant differences in frequencies of NAT2*5, *7, *11, and *12 genotypes, depending on ethnicity. As a result of NAT2 genotype combinations, Yakuts tended to develop mostly medium acetylation type, while Russians more often developed slow acetylation type. The observed patterns in distributions of NAT2 gene polymorphisms and acetylation types among Yakuts and Russians with newly identified TB can serve as a confirmation that pharmacologic responses can substantially differ depending on patients' ethnicity.