NAT2 Acetylation Phenotypes in India: A Narrative Review of Personalized TB Therapy

Introduction

What is the Acetylation Process and How is it Important for Health in Humans?

Acetylation is a basic biochemical mechanism of the human body whereby an acetyl group (CH₃CO) from acetyl-CoA is transferred to a target molecule, particularly to lysine residues of proteins with the help of a covalent bond.¹ This process plays a significant role in controlling various biochemical mechanisms within an organism. Regulation of the chromatin structure through histone modifications, regulation of enzymatic activity by controlling the transcription process, and stability of various protein molecules are examples of this process. The acetylation process is reversible and highly controlled by two enzymes: acetyltransferases that initiate this process by adding acetyl groups, and deacetylases that terminate this process by removing this molecule.² Acetylation has a detoxifying function in conjugation processes such as glucuronidation, sulfation, and methylation.³ Acetylation is a crucial process that plays a key role in metabolic balance, neurological function, cardiovascular health, cancer, and neurodegenerative disease pathophysiology;^4–7 For example, the progression of tumors is linked to abnormal histone deacetylase (HDAC) activity.⁸ Genetic and epigenetic variations in acetylation pathways, such as differences in enzymes like NAT2 and changes in histone acetylation profiles, greatly affect drug metabolism and disease progression in humans.^9,10 These variations provide potential biomarkers to improve personalized treatment strategies.

Functionality of the acetylation process is observed through two molecular pathways: (i) acetylation of proteins (acetylation of histones) and (ii) acetylation of xenobiotic compounds (drug metabolism).¹¹ Activation of the transcription process occurs with the help of the acetylation of chromatin remodelling, while non-histone acetylation influences various cellular processes, specifically in epigenetic regulation such as DNA repair and regulation of the cell cycle and apoptosis.¹² On the other hand, acetylation of xenobiotic compounds plays a crucial role in Phase II metabolism containing metabolizing enzymes, that is, N-acetyltransferases (NAT1 and NAT2).¹³ The NAT gene family mainly contains three enzymes: the non-functional NATP and functional NAT1 and NAT2. While NAT1 helps in the metabolism of xenobiotic compounds, NAT2 catalyses important Phase II metabolizing acetylation reactions that impact drug response and susceptibility.¹⁴ Both functional genes are highly polymorphic in human populations due to single nucleotide polymorphisms (SNPs) present in coding (CDS) regions.⁹

Genotypic Approaches to NAT2 Polymorphism Analysis for Acetylation Phenotype Classification

India accounted for approximately 27% of the global TB burden in 2023, with an estimated incidence of 176–195 cases per 100,000 population, which emphasizes the urgent need for improved and personalized treatment strategies.^15,16 Acetylation profiles are becoming promising biomarkers for the anticipation of drug responses or exogenous compound metabolisms. A combination of acetylation profiling with genomic and transcriptomic information could enhance patient stratification. The NAT2 gene, located on chromosome 8p22, possesses more than 100 allele variants, as listed in the PharmVar database (2024). These are the result of different combinations of seven globally known SNPs, 191G>A, 282C>T, 341T>C, 481C>T, 590G>A, 803A>G and 857G>A, which are present in the coding region of exon-2. Updated allele information was cross-checked with global pharmacogenomic resources, including PharmVar, PharmGKB, and dbSNP, to ensure accurate representation of NAT2 variant diversity. These different alleles are assigned to two broad metabolization phenotypes (slow or fast) with varying abilities to metabolize drug/exogenous compounds that characterize an individual broadly into slow, intermediate or fast acetylator phenotypes.¹⁷ These NAT2 polymorphisms have been directly linked with clinically important outcomes, including isoniazid-induced hepatotoxicity in slow acetylators and increased risk of treatment failure or emergence of drug-resistant TB in fast acetylators.¹⁸

Several molecular strategies are available for NAT2 genotyping, each with unique principles, advantages, and applications in research and clinical settings (Table 1). Polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) is an old, cost-effective and reliable technique for detecting known variants but is limited to SNPs that alter restriction to enzyme sites (by first amplifying SNPs sequences in the NAT2 gene followed by digestion by specific restriction enzymes to produce SNP-specific sequences), making it unsuitable for high-resolution or large-scale screening of indigenous communities.¹⁹ Allele-specific PCR (AS-PCR) offers high specificity and accuracy to detect known SNPs but requires separate primer sets for each SNP (to hybridize with the wild-type or mutant allele), limiting its utility in multiplex or large-scale studies.²⁰ TaqMan real-time PCR (TaqMan qPCR) is rapid and well-suited for clinical and public health use for screening and diagnosis, providing high-throughput genotyping for known SNPs using NAT2 SNP-specific fluorescent probes; however, it cannot detect novel variants.²¹ Sanger sequencing and next-generation sequencing (NGS) deliver comprehensive genotypic data and enable haplotype construction, essential for determining acetylator status by identifying both previously known and novel SNPs. While accurate, Sanger is low-throughput (cannot sequence more than 1200 bp) and targets specific genomic regions, whereas NGS that can provide whole genome information is relatively expensive and technically demanding, limiting its use in resource-limited settings.^9,22 Current rapid and decentralised platforms in India such as TrueNat and CBNAAT (a cartridge-based nucleic acid amplification test, eg, GeneXpert) are in use for screening of TB and drug resistance status.²³ These tools offer a potential model to incorporate NAT2 genotyping either directly or through platform-compatible point-of-care extensions that can facilitate scalable implementation of pharmacogenetic testing within the existing TB care infrastructure.²⁴

Table 1 Comparative Overview of Molecular Approaches for NAT2 Genotyping. The Table Summarizes Sample Types, Processing Time, Analytic Performance, and Practical Advantages and Limitations Associated with Each Method Used to Determine NAT2 Acetylation Phenotypes

It is therefore established that each method has its own purpose depending upon the size of sequence, setting, and purpose of genotyping methods. PCR-RFLP and AS-PCR are best for targeted and budget-friendly environments, whereas sequencing methods are oriented towards haplotype diversity or in locating novel variants. Moreover, TaqMan assays are best suited for population-scale pharmacogenomic studies in resource-limited settings. Additionally, CBNAAT (eg, GeneXpert) and TrueNat platforms could be adapted to deliver point-of-care pharmacogenetic testing, helping bridge the gap between precision medicine and real-world implementation in high-burden, low-resource areas.

How Do Different Acetylation Phenotypes of the NAT2 Gene Help Individuals Tackle Tuberculosis Treatment?

Genetic polymorphisms of the NAT2 gene are significant in the treatment of TB, particularly related to isoniazid (INH), a first line pro drug administered during TB treatment. NAT2 is a hepatic and intestinal enzyme that acetylates and deactivates INH. Polymorphisms in the 873bp coding region of exon-2 of the NAT2 gene give rise to unique acetylation phenotypes (slow, intermediate, or fast) that significantly impact the pharmacokinetics and pharmacodynamics of INH metabolism. This process subsequently affects the effectiveness of therapy and risk of development of adverse drug reactions. Slow metabolism and weak drug clearance from the body have been associated with the slow acetylator phenotype due to reduction in the capacity of the functional enzyme.²⁵ On the other hand, the fast acetylator phenotype increases the metabolism and clearance rate of INH due to its highly effective enzymatic capacity.²⁵ High concentrations of the drug in plasma and blood streams increase the threat of hepatotoxicity leading to liver failure and also make them prone to antitubercular drug-induced liver injury (AT-DILI) and peripheral neuropathy, collectively challenging treatment outcomes.²⁶ Conversely, in individuals having the fast type, inadequate availability of INH in the system (shorter exposures of the drug) provides an enriching environment for the pathogen causing tuberculosis in humans, Mycobacterium tuberculosis (Mtb), allowing it to develop resistance strains.²⁷ For example, infection with drug-resistant Mtb, defined by multiple drug-resistant (MDR) or extremely drug-resistant (XDR) infections that, in the worst-case scenario, turn into treatment failure.²⁸ Resistant Mtb, adverse drug reactions and unsuccessful treatment results are a few of the most important global concerns in general and in TB endemic countries like India, in particular. Ongoing widespread usage of INH in preventive management of tuberculosis preventive treatment (PMTPT) as preventive therapy may represent a silent but critical risk of emergence of MDR-TB in fast acetylators due to insufficient INH dosing.²⁹

TB treatment under the INH-NAT2 model has huge potential to significantly lower risks and improve treatment outcomes. For example, adjusted (reduced) INH dosage along with monitoring of liver function per month during the course of first-line treatment for six months continuously may reduce adverse drug reactions and toxicity levels in slow acetylators.³⁰ On the other hand, in fast acetylators, alternative (increased) INH dosage may reduce drug resistance cases and treatment failures. Perfectly optimized dosage of INH based on NAT2 genotyping status before starting TB treatment would allow for the development of personalized treatment.³⁰ These dosages will be clinically relevant to each patient’s genotype and help reinforce adherence by maintaining a simplified care route. Integration of NAT2 genotyping-based INH dosing in first-line TB treatment can start a move towards personalized medicine-based TB treatment in general and for genetically diverse populations like India, in particular. A recent meta-analysis study showed that slow NAT2 acetylators involve a substantially increased risk of anti-TB drug-related hepatotoxicity (OR ≈ 2.5).¹⁸ Moreover, randomized and pilot pharmacogenetic studies indicated that genotype-guided INH dosing can reduce hepatotoxicity and early treatment failure,³¹ although large multicentre RCTs in diverse populations (including India) remain limited. Recent cohort and clinical evaluations, however, do support the association between NAT2 status, hepatotoxicity, and treatment outcomes.³² Therefore, prospective clinical validation and cost-effectiveness studies are required before implementation of routine NAT2-guided dosing.

Genetic Diversity in the Indian Population with a Focus on NAT2 Polymorphisms

The population of India is one of the most genetically diverse in the world, shaped by decades of migration, endogamy and socio-religiously segregation on the basis of caste/religion, tribe, and language.³³ This high level of diversity and overall population structure significantly influence NAT2 allele frequency patterns. NAT2 is a broad-spectrum drug metabolizing gene that metabolises various drugs used in the treatment of infectious diseases such as isoniazid (tuberculosis), procainamide (cardiac arrhythmia and systemic lupus erythematous), hydralazine (hypertension), dapsone (leprosy and malaria) and sulfadoxine (malaria). The NAT2 gene is highly polymorphic in nature due to seven globally known SNPs located in the 873bp long CDS region of exon-2. Although, Indian populations largely possesses the slow acetylator type, the distribution pattern of NAT2 alleles is highly variable among Indian subpopulations, leading to a range of acetylation phenotypes (slow, intermediate or fast) significantly correlating with geographic and ethnic diversity within the country itself.⁹ However, the central and eastern Indian tribal populations have been reported to possess the slow acetylator type predominantly (70–80% frequency), whereas north Indian populations display an equal distribution pattern of the slow and fast types.^9,34 The slow acetylator types NAT2 *5 and *6 and the *7 haplotypes have been reported as the most prevalent alleles in Indian populations. They are the result of different combinations of SNP found on 341T>C, 590G>A, and 857G>A locations in CDS. This type of genotype (slow) significantly reduces enzymatic capacity at the phenotypic level, which slows down drug metabolization.³⁵ This population-wise diversity in acetylation possession demands subpopulation-specific approaches in pharmacogenetic screening before initiating TB treatment. Despite several studies describing NAT2 allele distribution in India, there remains a lack of integrated, population-based synthesis connecting genetic diversity, acetylator phenotype, and implications for personalized TB therapy and this review aims to address this gap.

As previously discussed, the NAT2 acetylation type is highly influential in relation to isoniazid efficacy and toxicity levels, the generalised status of India. This is because slow acetylators are directly associated with increased risks of adverse drug reactions such as hepatotoxicity. On the other hand, fast acetylator subpopulations are under risks of subtherapeutic drug levels and emergence of drug resistant Mtb stains.³⁶ Furthermore, this variable distribution of allele type also highlights limitations of conventional TB treatment based on a “one-size-fits-all” model, whereby the drug (INH) is given to patients based on their age and weight without considering pharmaco-genotyping.³⁷ Therefore, in a country where endogamous traditions, genetic drift and founder effects can shape unique haplotypes of the NAT2 gene within closed communities, incorporation of regional and ethnic diversities into national-level pharmacogenomic policies and NAT2 genotyping into TB control programs is recommended, particularly under the revised National TB Elimination Programme (NTEP). This might significantly reduce the burden of adverse drug reactions, drug-induced toxicity, and drug resistance and improve treatment outcomes. Incorporating these into national pharmacogenomic approaches will be crucial for providing healthcare to Indians in an equitable manner and may pave a new way to transitioning from generalized treatment protocols to precision medicine in India for tuberculosis.

Translational Potential of Population-Based NAT2 Phenotyping in Developing Precision Medicine for Tuberculosis in India

Since no effective vaccine for lethal disease-like TB is yet part of the TB elimination program, treatment is solely dependent on chemotherapy. However, as already discussed above, not everyone can metabolize INH equally due to the influence of the NAT2 genotype. Therefore, population-scale NAT2 genotyping before the start of TB treatment could positvely influence both the economy and public health. An optimized dosage of INH can enhance drug adherence, bacterial clearance and treatment success rates in all TB patients. This can further reduce development of MDR-TB, the requirement of second-line TB treatment and hospitalization costs since second-line TB treatment is very expensive.³⁷

Apart from cost reduction, incorporation of an INH-NAT2 pharmacogenomic-guided therapy model into the Indian healthcare system with the help of new and old techniques like TaqMan PCR assays and Sanger or next generation sequencing in laboratory settings and microfluidic-based point-of-care tests in the field may make NAT2 genotyping faster, less expensive, and more scalable.^9,22,26 These technologies may be established at centralized diagnostic laboratories or mobile genomic testing facilities, especially in highly endemic remote or tribal areas where healthcare facilities are very limited. NTEP provides an already established structure for clinicians and health workers to include pharmacogenetic screening both before starting TB treatment and in routine TB care.²² Under this model, pilot studies in endemic regions scaled-up according to epidemiological necessity could operate. NAT2 genotyping together with other important significant molecular markers such as SLCO1B1, CYP2E1, GSTT1, and UGT1A1 and machine learning approaches creates an opportunity for India to move toward comprehensive, genotype-guided TB therapy and extensive precision-based public health efforts in the future. Inclusion of diverse genetic backgrounds within national treatment guidelines would surely improve treatment failure rates. More importantly, INH-NAT2 based pharmacogenomic-guided therapy to TB would initiate a realization of personalized medicine in TB treatment and serve as a model for the management of other infectious diseases in other low- and middle-income countries with similar public health needs.

Conclusion and Future Directions in Acetylation-Driven Pharmacogenomics

The implementation of an INH-NAT2 pharmacogenomic model is needed to achieve personalized TB treatment in a country where immense genetic diversity is present across more than 4500 anthropologically recognized ethno-linguistic groups, as documented by the People of India project (Anthropological Survey of India)³⁸ and exhibits extremely high genetic diversity^39,40 (Ref, GenomeIndia Consortium, 2023; IndiGenomes, 2021). Population-based NAT2 genotyping is necessary to classify individuals as slow, intermediate, or fast acetylators, which will enable more precise and safer INH dosing (Figure 1). To make personalized medicine for TB a reality, a national mandate should focus on allele frequency mapping across populations and development of an affordable, point-of-care NAT2 genotyping platform.⁴¹ Integrating already existing diagnostic technologies (eg, GeneXpert) and digital healthcare platforms with this approach may provide immediate, individualized guidance on treatment, though robust clinical trials are essential to justify this approach. However, NAT2 testing has not yet been formally validated on platforms such as TrueNat or GeneXpert but these systems do hold strong potential for future integration, and dedicated pilot studies could help in the assessment of their feasibility for NAT2 genotyping in TB programs. Practical implementation will also depend on addressing key challenges such as cost considerations, laboratory infrastructure, workforce training, and quality-assurance mechanisms in decentralized settings. Importantly, robust clinical validation studies, dose–response trials, and cost–benefit analyses are required to establish the clinical impact and health-system feasibility of NAT2-guided therapy. Aligning this approach with national (NTEP) strategies and the World Health Organization’s pharmacogenomics-informed TB guidelines will help ensure policy relevance and programmatic uptake.

Figure 1 Framework for NAT2-Guided Isoniazid Dosing in Personalized Tuberculosis Therapy in India. (This approach aims to reduce toxicity, prevent resistance, and improve treatment outcomes within diverse Indian population).

In summary, integrating NAT2 pharmacogenomics into TB care is consistent with Indian health priorities and international goals to eliminate TB.⁴² This approach could provide a clearer path toward more equitable, effective, and evidence-based treatment, and will be a significant step toward precision public health in the field of infectious diseases.