Introduction

Drug-resistant pulmonary tuberculosis is a global public health problem. According to a systematic review, the worldwide prevalence of Mycobacterium tuberculosis (M. tuberculosis) with multidrug resistance to anti-tuberculosis drugs is 11.6% (95% CI: 9.1--14.5%) [1]. In 2024, the incidence of pulmonary tuberculosis with multidrug resistance (form No. 33) was 5.7 per 100,000 population.

Treatment of drug-resistant tuberculosis is a complex process involving long-term and high-cost chemotherapy with not always achievable effectiveness and the risk of adverse reactions. In modern phthisiology, to overcome M. tuberculosis resistance to traditional drugs, anti-tuberculosis drugs with a new mechanism of antimicrobial action are used. Since 2013, following WHO recommendations, the bromine atom-containing diarylquinoline derivative bedaquiline has been used in the complex pharmacotherapy of drug-resistant tuberculosis [2].

Bedaquiline exerts a bactericidal effect on dividing and dormant mycobacteria, suppressing extracellular and intracellular pathogens. It causes the death of drug-sensitive and drug-resistant (including multidrug-resistant and extensively drug-resistant) strains of M. tuberculosis at a minimum inhibitory concentration ≤ 0.008--0.12 μg/L. Bedaquiline selectively binds to subunit C of adenosine-5'-triphosphate synthase (ATP synthase), inhibits this enzyme, disrupts ATP synthesis in M. tuberculosis, and deprives them of energy. ATP synthase is partially embedded in the mycobacterial cell wall and catalyzes ATP production using the proton gradient between the cytoplasm and the cell wall. The human enzyme is 20,000 times less sensitive to bedaquiline [3].

After oral administration, the maximum plasma concentration of bedaquiline (Cmax) is achieved within 5 hours. It is almost completely bound to plasma proteins and oxidized in the liver by the cytochrome P450 isoenzyme CYP3A4 to form N-monodesmethyl (M2), which is 3-6 times pharmacologically less active against M. tuberculosis than bedaquiline. Subsequently, the M2 metabolite is demethylated to inactive didesmethyl (M3). Bedaquiline is eliminated from the body via bile. The terminal half-life (T1/2) of bedaquiline and the M2 metabolite is approximately 5 months (ranging from 2 to 8 months on average). The long half-life is due to its high lipophilicity and tissue deposition [2].

Bedaquiline is added to standard chemotherapy regimens for tuberculosis with multidrug, pre-extensively drug, and extensively drug-resistant M. tuberculosis [4]. Combined chemotherapy including bedaquiline has shown higher efficacy compared to treatment outcomes in tuberculosis patients who did not receive bedaquiline [5, 6]. A meta-analysis conducted in 2024 found that the effectiveness of treatment using regimens containing bedaquiline ranges from 76.9% to 81.7% [7].

Acquired resistance of M. tuberculosis to bedaquiline is a major concern, with its frequency ranging from 2.2--4.4% [8] to 12.2% [9]. When prescribing bedaquiline, factors contributing to the development of resistance to this drug should be considered: delayed bactericidal effect, high plasma protein binding, long half-life [10], as well as adverse reactions requiring treatment discontinuation.

Adverse effects are caused by both bedaquiline itself and its M2 metabolite. Possible effects include dizziness, prolongation of the corrected QT interval (QTc) on the ECG with the risk of torsade de pointes ventricular arrhythmia, joint and muscle pain, liver pathology, and dyspepsia [7]. In 1.7% [0.7--4.2] of patients, bedaquiline discontinuation was required due to the development of adverse reactions [11].

Pharmacogenetic studies have established that polymorphisms in genes encoding phase I and II biotransformation enzymes of anti-tuberculosis drugs affect the efficacy and tolerability of tuberculosis chemotherapy [12].

Bedaquiline is oxidized by the cytochrome P450 isoenzyme CYP3A4. Allelic variants of the genes encoding this isoenzyme could potentially influence the pharmacokinetics, clinical outcomes of tuberculosis treatment, and toxicity of bedaquiline.

Currently, there is no information in the available scientific literature on the prevalence of CYP3A4*22 and CYP3A5*3 allelic variants among patients with drug-resistant pulmonary tuberculosis of Yakut and Russian ethnicity.

Objective of the study: To investigate the carrier frequency of CYP3A4*22 (rs35599367) C>T and CYP3A5*3 (rs776746) A>G gene polymorphisms among Yakuts and Russians with drug-resistant tuberculosis, compared to the variability of allelic variants of the studied genes in East Asian and European populations.

Methods

Study Design: Prospective, single-center. The study involved patients hospitalized in 2021--2023 at the State Budgetary Institution of the Republic of Sakha (Yakutia) "E.N. Andreev Scientific and Practical Center 'Phthisiatry'" for the intensive phase of treatment for pulmonary tuberculosis caused by M. tuberculosis with multidrug resistance. The study was approved by the local Ethics Committee (Protocol No. 1/2020 dated 27.01.2020). The study included 171 Yakuts [males --- 121 (70.76%), females --- 50 (29.24%)] and 84 Russians [males --- 61 (72.62%), females --- 23 (27.38%)]. The mean age of Yakuts was 40.00±0.83 years, and of Russians was 40.00±0.67 years. Ethnicity was determined by patient and parent self-identification, analyzing pedigree up to the second generation.

Inclusion criteria: 1) respiratory tuberculosis with multidrug-resistant M. tuberculosis; 2) age 18 years and older; 3) ethnicity: Yakut, Russian; 4) signed informed consent. Non-inclusion criteria: 1) non-compliance with any inclusion criteria; 2) descendants of interethnic marriages were not included in the study.

Pharmacogenetic testing was performed at the Russian Medical Academy of Continuous Professional Education, Moscow, and the Republican Clinical Hospital No. 3, Yakutsk. For genetic analysis, 3-4 mL of blood was collected from the cubital vein into vacuum tubes using a closed vacuum system (Zhejiang Gongdong Medical Technology Co., Ltd, China). The tubes had a fine spray coating of the anticoagulant K3 EDTA (ethylenediaminetetraacetic acid). DNA was isolated from whole blood using the "S-Sorb" reagent kit ("Syntol", Russia). Genotyping of polymorphisms was performed by real-time polymerase chain reaction on a CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.; USA) using CYP3A4*22 (rs35599367) C>T (TestGen LLC, Russia) and CYP3A5*3 (rs776746) A>G ("Syntol", Russia) reagent kits.

CYP3A phenotype was determined according to the data in Table 1 [13].

Table 1

Correspondence of CYP3A4*22 and CYP3A5*3 genotypes to CYP3A metabolism phenotypes

For comparative analysis, data on the carrier frequency of CYP3A4*22 and CYP3A5*3 allelic variants in East Asian and European populations were used. Comparison groups for Yakuts were formed based on the principle of geographical proximity to East Asia. Data on allele and genotype frequencies of CYP3A4*22 and CYP3A5*3 were obtained from the IGSR database: The International Genome Sample Resource (https://www.internationalgenome.org/).

Results were statistically analyzed using the IBM SPSS Statistics software package, version 23 (North-Eastern Federal University license). When assessing the distribution of allele and genotype frequencies of the CYP3A4 and CYP3A5 genes, compliance with the Hardy-Weinberg equilibrium was checked. The standard χ² test with Yates' correction was used to compare frequencies. Differences between compared indicators were considered statistically significant at *p* <0.05.

Results

Genotypes of the polymorphic genes CYP3A4*22 (rs35599367) and CYP3A5*3 (rs776746) were identified in the populations of Yakuts, Russians, East Asians, and Europeans.

The distribution of genotype frequencies and compliance with the Hardy-Weinberg equilibrium are presented in Table 2.

Table 2

Distribution of allele and genotype frequencies of CYP3A4 and CYP3A5 gene polymorphisms in Yakut, Russian, East Asian, and European populations

Notes: * Data on East Asian and European populations were obtained from the publicly available database -- 1000 Genomes Project; NaN -- Hardy-Weinberg equilibrium calculation is not possible due to the absence of polymorphism in the sample at this locus.

Comparative analysis of CYP isoenzyme allele and genotype distribution between Russians and Caucasians/Europeans.

Comparison of the Russian population with the European group revealed a single significant difference for the heterozygous CYP3A4*22 genotype (Table 3). The distribution of CYP3A5*3 alleles and genotypes in the Russian sample showed no differences from the general European profile (Table 4).

Comparative analysis of CYP isoenzyme allele and genotype distribution between Yakuts and East Asians.

When comparing the distribution of CYP3A4*22 and CYP3A5*3 alleles and genotypes in the Yakut population with the East Asian group, statistically significant differences were found for all studied parameters (Tables 3, 4).

Comparative analysis of CYP isoenzyme allele and genotype distribution between Russians and Yakuts.

The distribution of alleles and genotypes of the CYP3A4*22 and CYP3A5*3 genes showed no statistically significant differences between Russians and Yakuts (Tables 3, 4).

The results of the analysis of the prevalence of metabolic rate phenotypes associated with CYP3A4*22 and CYP3A5*3 genetic variants in the studied groups of Yakut and Russian tuberculosis patients are presented in Table 5.

Comparative analysis of CYP isoenzyme allele and genotype distribution between the combined Siberian populations and the combined Eurasian populations.

The allele and genotype frequencies of the CYP3A4*22 polymorphism differed statistically significantly between the combined group of Siberian populations [Yakuts and Russians residing in the territory of the Republic of Sakha (Yakutia)] and the combined group of reference Eurasian populations (East Asians and Europeans).

The frequency of the T allele of the CYP3A4*22 gene was significantly lower in the Siberian group (0.39%; 2 alleles out of 510) compared to the frequency in the Eurasian population (2.43%; 49 alleles out of 2014) (χ²=8.551; *p*=0.003).

The distribution of CYP3A4*22 genotypes also revealed significant differences. The wild-type genotype (CC) was more common in the Siberian group (99.2%; 253 individuals out of 255) compared to the Eurasian population (95.1%; 958 individuals out of 1007) (χ²=8.734; p=0.003). Conversely, the heterozygous genotype (CT) was significantly less represented in the Siberian population (0.78%; 2 individuals) relative to the Eurasian group (4.77%; 48 individuals) (χ²=8.734; p=0.003). The homozygous genotype for the T allele was not found in any representative of the Siberian group; in the Eurasian group, it was identified in one individual (0.1%). The formal statistical significance for the TT genotype of CYP3A4*22 (χ²=601.662; p≈0) should be interpreted with caution due to the extremely low expected frequency of this genotype in the compared groups; the use of exact statistical tests is preferable to confirm this difference.

Statistical analysis revealed significant differences in the genotype distribution of the CYP3A5*3 polymorphism between the combined Siberian populations and the combined Eurasian populations, despite the absence of a statistically significant difference in the frequency of the A allele. The frequency of the A allele was lower in the Siberian group (8.43%; 43 alleles out of 510) than in the Eurasian group (17.18%; 346 alleles out of 2014). However, this difference did not reach statistical significance (χ²=2.578, p=0.108).

The AA genotype was less common in the Siberian group (1.18%; 3 out of 255 individuals) compared to its frequency in the Eurasian group (4.17%; 42 out of 1007 individuals) (χ²=5.301, p=0.021). The heterozygous AG genotype was much less represented in the Siberian group (14.51%; 37 out of 255 individuals) relative to the Eurasian group (26.02%; 262 out of 1007 individuals) (χ²=14.893, p <0.001). The GG genotype was significantly predominant in the Siberian group (84.31%; 215 out of 255 individuals) and was detected less frequently in the Eurasian group (69.81%; 703 out of 1007 individuals) (χ²=21.565, p <0.001).

Table 3

Comparative analysis of CYP3A4*22 polymorphism allele and genotype distribution in the studied populations

Note: * -- comparison of allele and genotype distribution between Russian and Yakut populations.

Table 4

Comparative analysis of CYP3A5*3 polymorphism allele and genotype distribution in the studied populations

Note: * -- comparison of allele and genotype distribution between Russian and Yakut populations.

Table 5

Prevalence of CYP3A-mediated metabolism rate variants among Yakuts and Russians with tuberculosis

Discussion

Cytochrome P450 plays a key role in the oxidation of xenobiotics. It comprises over 1000 isoenzymes, five of which (CYP3A4, CYP2D6, CYP2C9, CYP2C19, and CYP1A2) metabolize up to 90% of all drugs [14]. The most common isoenzyme is CYP3A4, involved in the biotransformation of 50% of drugs [15, 16].

The genes for the CYP3A4 and CYP3A5 isoenzymes are located in adjacent loci on chromosome 7, have similar catalytic specificity, but the activity of CYP3A5 is lower than that of CYP3A4. The CYP3A4 gene is considered low-polymorphism, while CYP3A5 is considered highly polymorphic [17].

Allelic variants of the CYP3A4*22 gene significantly affect drug metabolism, primarily due to altered expression and activity of the CYP3A4 isoenzyme. This allele, identified as a single nucleotide polymorphism (SNP) rs35599367 C>T in intron 6, is associated with reduced hepatic CYP3A4 expression and activity, leading to variability in drug metabolism. CYP3A4*22 is more common in Caucasians (allele frequency 5--7%), but is rare or absent in other populations, such as the Japanese [18, 19]. This polymorphism, in combination with other CYP3A variants, such as CYP3A5*3, can further exacerbate slow drug metabolism, characteristic of some patient cohorts [20].

CYP3A5 is highly polymorphic, with alleles *1, *3, *6, and *7 affecting enzyme expression and activity. The CYP3A5*3 polymorphism is a genetic variant of the CYP3A5 gene. The CYP3A5*3 allele is characterized by an SNP where adenine (A) is replaced by guanine (G) at position 776746 (rs776746). The CYP3A5*3 allele is the most common and is distinguished by low expression in many populations [21, 22].

The G allele of the CYP3A5*3 gene is also widespread. The distribution of CYP3A5*3 varies significantly between ethnic groups, being more frequently detected in European and Asian populations compared to its prevalence in African populations, where the A allele of the CYP3A5*3 gene is more common [23, 24]. For example, in the Chinese population, the frequency of the CYP3A5*3 allele is 76.1%, with genotype distributions of 5.6% for CYP3A5*1/1, 36.7% for CYP3A5*1/3, and 57.8% for CYP3A5*3/3 [25].

The GG genotype of the CYP3A5*3 gene is associated with a slow rate of drug metabolism, increased drug concentrations in plasma and organs, and a potential increase in the risk of adverse reactions [26]. This variability underscores the importance of considering genetic background in clinical practice and necessitates the use of pharmacogenetic testing to optimize drug dosing and therapeutic outcomes.

The relationship between CYP3A4/5 polymorphisms and bedaquiline concentration in the treatment of multidrug-resistant tuberculosis is an important area of research. Bedaquiline is metabolized by the cytochrome P450 isoenzyme CYP3A4, and genetic variations in this enzyme can significantly influence drug metabolism. Homozygous and heterozygous carriage of the CYP3A5*3 (rs776746) allelic variant is associated with slower clearance of bedaquiline (p = 0.0017), but not of the M2 metabolite (p = 0.25) [27].

In our study, the CYP3A4*22 polymorphism demonstrates pronounced interpopulation differences. These are characterized by a significantly lower frequency of the T allele and the heterozygous CT genotype, as well as an almost fixed frequency of the CC genotype in Siberian populations (Yakuts and Russians) compared to the frequency in the combined populations of East Asia and Europe.

In Siberian populations (Yakuts and Russians), the distribution of CYP3A5*3 genotypes is statistically significantly shifted towards the homozygous GG genotype. The frequencies of both the heterozygous (AG) and homozygous (AA) genotypes are lower than in the combined group of East Asian and European populations, although there are no significant differences in allele frequencies. Consequently, the impact of the CYP3A5*3 GG genotype on drug metabolism and therapeutic outcomes may be more significant in the Siberian population. Carriers of this allele may exhibit variations in drug response, necessitating an individualized approach to treatment.

The vast majority of both Yakut and Russian tuberculosis patients are intermediate metabolizers via CYP3A. The poor metabolizer phenotype was very rare and identified only in Yakuts. The normal metabolizer phenotype was less common than the intermediate one but more common than the poor one, with a trend towards slightly higher prevalence in the Russian patient group.

The fact that the majority of patients with drug-resistant tuberculosis are intermediate metabolizers must be considered when assessing treatment efficacy and the risk of adverse reactions during the use of anti-tuberculosis drugs, particularly bedaquiline.

Conclusion

The study revealed significant differences in the distribution of CYP3A4*22 and CYP3A5*3 polymorphisms in patients with drug-resistant tuberculosis in the Yakut and Russian populations compared to other Eurasian populations. This underscores the importance of considering genetic variability in tuberculosis chemotherapy, especially concerning drugs metabolized by CYP3A isoenzymes.

Understanding the genetic factors influencing drug metabolism can lead to the development of more individualized treatment regimens and improved chemotherapy outcomes for patients suffering from drug-resistant tuberculosis. Further research is needed on the prevalence of the studied genes in different ethnic groups and their impact on the efficacy and safety of bedaquiline treatment in patients with multidrug-resistant tuberculosis.

Study Limitations

The study provides preliminary information on the prevalence of CYP3A4*22 and CYP3A5*3 allelic variants among patients with drug-resistant pulmonary tuberculosis of Yakut and Russian ethnicity. However, it is important to acknowledge the limitations of the study's sample size, which may not fully reflect the genetic variability of the studied genes in broader populations. Furthermore, focusing on specific polymorphisms may overlook other non-genetic and genetic factors that could influence drug metabolism, efficacy, and safety of tuberculosis treatment.

Conclusions