Isoniazid-induced liver injury risk level in different variants of N-acetyltransferase 2 (NAT2) polymorphisms: A literature review

Individual NAT2 genotype identity data should be enriched to prevent Isoniazid-induced liver injury (IDILI) and optimize the dose of Isoniazid (INH). Therefore, this study aims to present the level of IDILI risk for specific genotype alleles. The data collection involves literature indexed by Google Scholar, Scopus


Introduction
Isoniazid (INH) has been used as an anti-tuberculosis (TB) drug since 1952, and reports of INH-induced liver injury (IDILI) are ongoing and common (Erwin et al. 2019).Previously, different studies were conducted to identify IDILI susceptibility in certain populations, demographic characteristics, and its hepatoprotective ability as a follow-up solution (Zhang et al. 2020).These studies also involve the polymorphisms study of enzymes responsible for INH metabolism (Perwitasari et al. 2015;Erwin et al. 2019;Yang et al. 2019;Zhang et al. 2020).
INH metabolic activity related to IDILI are associated with polymorphisms of several genes including N-acetyltransferase II (NAT2), Cytochrome P450 2E1 (CYP2E1), and glutathione S transferases (GST1) (Perwitasari et al. 2015;Erwin et al. 2019;Yang et al. 2019).Studies involving the NAT2 genotype are very important compared to those involving enzymes triggering (Zhang et al. 2020).The single nucleotide polymorphism (SNP) in NAT2 affects the INH metabolism rate.In addition, it affects the variations in treatment efficacy and frequency of adverse reactions (Swaminathan and Ramachandran 2012).Populations with NAT2 enzymes in the slow acetylator group were shown to be susceptible to IDILI exposure (Swaminathan and Ramachandran 2012; Perwitasari et al. 2015;Yang et al. 2019), and this was increased with the standard dose of INH (Shi et al. 2015).
Further studies on the genetic polymorphism of NAT2 are expected to include different ethnic populations (Perwitasari et al. 2015).The literature enrichment guides clinicians in screening patients to predict and prevent the IDILI through optimal pharmacotherapy determination (Swaminathan and Ramachandran 2012;Wang et al. 2012;Perwitasari et al. 2015).Meanwhile, the screening for NAT2 polymorphisms requires the identity of two haplotypes (diplotypes).This is because the identity of the individual NAT2 genotype is very important in adjusting the INH dose (Wichukchinda et al. 2020).
The study priority for the causative factors of IDILI is based on the NAT2 polymorphism, and the group with slow-acetylator was also susceptible to the exposure.Individual NAT2 genotype identity data should be improved by clinicians to optimize INH dose.This study attempts to present the IDILI risk level for specific genotype alleles since there are limited publications that identify the related topic.Therefore, these results should be the basis for adjusting the INH dose in genotype variants susceptible to IDILI.

Literature search strategy
The study involves literature indexed by Google Scholar, Scopus, and Pubmed databases, and the search uses a combination of the following keyword variants; "isoniazid" OR "INH", "liver injury" OR "hepatotoxicity", "polymorphism" OR "pharmacogenomic", AND "N-acetyltransferase 2" OR "NAT2".This study only used original articles in English language which were peer-reviewed journals published between 2011 and 2020 (Fig. 1).

Literature eligibility and research data extraction
This study only involves results that meet all of the following criteria; (a) Study on subjects with tuberculosis without other comorbidities, (b) Study on subjects receiving INH as part of a standard regimen of 5 mg/Kg/day (maximum dose 300 mg/day), (c) Case study-control on subjects exposed to IDILI, (d) Study outcome involving the distribution of single nucleotide polymorphism allele identity and acetylator phenotype.
Each literature was extracted by identifying the name of the publication journal, the author's name, year, title, antituberculosis drugs combination, population settings, number of respondents, and study outcomes in the total number of cases and controls.It also includes the identity frequency of the single nucleotide allele polymorphism and acetylator phenotype.

Statistic analysis
The main analysis aims to measure the level of IDILI risk in each genotype.Meanwhile, the risk assessment due  to NAT2 polymorphism was based on the odds ratio of 95% confidence interval.The distribution of IDILI cases is compared against the controls, with individual case and control comparisons across all other genotypes combined for each allele.Furthermore, the analysis uses Stata MP software edition 14.

Characteristics of research subjects
The literature search strategy obtained 423 articles, and a total of 11 articles met the inclusion criteria (Fig. 1).A series of studies conducted across diverse regions, including Indonesia, Tunisia, Japan, Brazil, Spain, China, and India, aimed to unravel the intricate relationship between genetic factors and the risk of Isoniazid-Induced Liver Injury (IDILI), a concerning side effect of tuberculosis treatment.These investigations primarily focused on NAT2 gene variants and acetylator status, shedding light on their pivotal role in IDILI susceptibility.Notably, NAT2 slow acetylators consistently emerged as a high-risk group for IDILI, with some studies highlighting ultra-slow acetylators as even more susceptible, while fast and intermediate acetylators showed reduced risk.Furthermore, certain studies explored the influence of additional genetic factors, such as CYP2E1 and GST genes, in hepatotoxicity linked to isoniazid use.Collectively, these findings underscore the importance of genetic profiling in tailoring tuberculosis treatment regimens and enhancing patient safety (Table 1).
Research conducted in Indonesia found a significant association between NAT2 slow and ultra-slow acetylators, which increased the risk of IDILI, while fast and intermediate acetylators decreased the risk (Yuliwulandari et al. 2019).A similar study in Indonesia identified NAT2*5, *6, and *7 polymorphisms, revealing distinct genotypes and genetic susceptibility (Suhuyanly et al. 2017).Similarly, in Thailand, slow NAT2 acetylators were significantly associated with an increased risk of IDILI in tuberculosis patients.These findings underscore the crucial role of NAT2 gene variants and acetylator status in understanding and predicting IDILI risk across diverse populations (Wattanapokayakit et al. 2016).
Japan developed a predictive IDILI risk system, showing a significant association between NAT2 slow acetylators and increased risk.A logistic regression model with age and NAT2 genotype predicted effectively (Mushiroda et al. 2016).In Tunisia, high isoniazid serum levels, combined with NAT2/CYP2E1 gene polymorphisms, posed an IDILI risk (Ben Fredj et al. 2017).Slow acetylators in Tunisia had a higher hepatotoxicity risk, with specific NAT2 variants increasing the risk (Ben Mahmoud et al. 2012).
China identified NAT2 slow acetylator genotypes, like NAT26A/7B and NAT26A/6A, as hepatotoxicity risk factors, with combined NAT2/CYP2E1 genotypes amplifying this risk (An et al. 2012).In India, slow acetylators and specific NAT2 genotypes were linked to higher hepatotoxicity risk, with the NAT24 haplotype providing protection protection (Gupta et al. 2013).Additionally, India found specific NAT2 genotypes more prevalent among hepatotoxicity patients, along with lower C1/C1 allele occurrence in the CYP2E1 gene and higher GSTM1 levels (Rana et al. 2014).Slow acetylators had a higher risk of hepatotoxicity.Specific NAT2 variant diplotypes were associated with increased risk.9. (Gupta et al. 2013) India Assessing NAT2 and CYP2E1 gene polymorphisms in IDILI.
Slow acetylators and specific NAT2 genotypes were associated with a higher risk of hepatotoxicity.NAT2*4 haplotype provided protection.10. (Rana et al. 2014) India Elucidating NAT2, CYP2E1, and GST gene polymorphisms in IDILI.Specific NAT2 genotypes were significantly higher in hepatotoxicity patients.C1/C1 allele of CYP2E1 gene was lower in hepatotoxicity patients.GSTM1 was significantly higher in hepatotoxicity patients.11. (Wattanapokayakit et al. 2016) Thailand Investigating NAT2 genotype status in TB patients with IDILI Slow NAT2 acetylators had a significant association with IDILI risk.

IDILI risk in the rapid acetylator of NAT2 enzyme group
Generally, the rapid acetylator NAT2 group has OR (CI) value of 0.61 (0.48-0.77) with a significance of <0.0001, and the identification obtained one type of genotype with significant analysis results.Meanwhile, allele *4/*4 has OR (CI) value; 0.66 (0.52-0.85), and this result showed almost no risk of IDILI due to INH (Table 2).

Significant frequency of cases and controls of six variants triggers IDILI
The NAT2*6A/*6A group had an IDILI risk of 5.76 times that of other populations, and are spread over five countries, with a total frequency of 4%.Furthermore, the doubled cases from controls occurred in the populations of China, Thailand, Indonesia, and Japan.The distribution of cases and controls in Tunisia was balanced, and the NAT2*6A/*7B group was exposed to 5.54 times.They were spread over four countries, with a total frequency of 4%.In addition, the ratio of the total cases in China and Thailand was higher than the controls.On the contrary, the case reports in Indonesia and Japan were lower than the controls even though the numbers were almost equal.The NAT2*6/*7 group had an IDILI risk of 4 times that of the other population, and they were spread over five countries with a total frequency of 1%.More cases than controls were found in India, Indonesia, and Spain.Meanwhile, all populations in Tunisia and Brazil acted as the controls (Fig. 2).
Previous reports suggested the IDILI risk for both Asian and non-Asian is evenly distributed across alleles and ethnic variants of the population (Wang et al. 2012).Moreover, other reports suggested that Asian races are more susceptible to IDILI cases, unlike Caucasians (Cho et al. 2007;Cai et al. 2012;Swaminathan and Ramachandran 2012).However, the development of pharmacogenomic research related to tuberculosis is more involved in the countries of Asia compared to others.Similarly, the cases predominance of this study involved reports from the Asian region (Leiro-Fernandez et al. 2011;An et al. 2012;Ben Mahmoud et al. 2012;Gupta et al. 2013;Santos et al. 2013;Rana et al. 2014;Mushiroda et al. 2016;Wattanapokayakit et al. 2016;Ben Fredj et al. 2017;Suhuyanly et al. 2017;Yuliwulandari et al. 2019), even though Tunisian population caught attention.Zero cases were recorded when various populations showed high cases of NAT2*5/*7, -*6/*7, and -*5B/*7B variants.In the ultra-slow NAT2*6A/*6A variant, the number of cases and controls were balanced.Further studies should be conducted to show that the incidence of IDILI in the slow acetylator variant is not greater than the control (Ben Mahmoud et al. 2012;Ben Fredj et al. 2017).The identity variants of the NAT2 genotype have consistently coded for the INH metabolic phenotype (Zabost et al. 2013;Seng et al. 2015;Huerta-García et al. 2020).This is because genotype identity is not a single trigger for IDILI.The risk also depends on the configuration of cooperation with other enzymes related to the metabolic activity of INH.Furthermore, it encodes the late phenotype, such as CYP2E1*c1/c2, and the null genotype GSTM1 (Cai et al. 2012;Santoso et al. 2021) to consider the variance of the population weight factor (Huerta-García et al. 2020).For every 10 kg increase in body weight, the concentration of INH is reduced by 1 mg/L [12]-[15], [17] (Jung et al. 2015).Therefore, these variants should be considered in the new era of INH dose personalization.
Several investigations consistently affirm the association between NAT2 slow acetylator status and an elevated susceptibility to (Leiro-Fernandez et al. 2011;Ben Mahmoud et al. 2012;Gupta et al. 2013;Mushiroda et al. 2016;Wattanapokayakit et al. 2016;Ben Fredj et al. 2017;Yuliwulandari et al. 2019).Consequently, when prescribing anti-tuberculosis medications, this genetic factor should be taken into consideration.Research also suggests that NAT2 genotyping has the potential to personalize drug dosing, particularly for isoniazid, based on genetic information, which could optimize treatment outcomes and reduce the risk of hepatotoxicity (Ben Mahmoud et al. 2012;Santos et al. 2013;Suhuyanly et al. 2017).Furthermore, combining NAT2 and CYP2E1 genotype analysis can provide valuable insights into the risk assessment of IILI.It has been observed that slow NAT2 acetylator status, especially when coupled with specific CYP2E1 genotypes, may heighten susceptibility to IILI (An et al. 2012;Ben Fredj et al. 2017).Therefore, monitoring isoniazid serum concentrations is vital, as elevated levels are associated with a greater risk of IDILI (An et al. 2012).Therapeutic drug monitoring (TDM) for isoniazid can help identify individuals at risk and facilitate necessary treatment adjustments.
Moreover, there is a call to develop predictive models that integrate both genetic and clinical risk factors to evaluate the risk of IDILI (Mushiroda et al. 2016).Prospective clinical trials are essential to validate the clinical utility of these models and guide INH dosage adjustments.Largescale screening for NAT2 and CYP2E1 genotypes, as proposed by studies (Santos et al. 2013;Rana et al. 2014), can effectively predict adverse effects and enhance clinical management.Routine genotyping tests may be warranted for populations at risk of IILI.Recognizing population-specific genetic variations in NAT2 is crucial (Santos et al. 2013;Wattanapokayakit et al. 2016).Clinicians should consider the genetic diversity within their patient populations when making treatment decisions.Finally, we should highlight the feasibility and cost-effectiveness of genetic testing for preventing severe adverse drug reactions within healthcare systems (Wattanapokayakit et al. 2016).
Recently, some reports have followed up on the NAT2 variant phenotype impact by offering a pharmacokinetic model, to establish a new era of INH dose personalization (Huerta-García et al. 2020;Mthiyane et al. 2020).
The studies included diverse TB and healthy populations (Chen et al. 2009;Rodriguez et al. 2019;Huerta-García et al. 2020;Jing et al. 2020) co-infected with the human immunodeficiency virus (Bhatt et al. 2014) (Kubota et al. 2011;Seng et al. 2015).However, the development of a pharmacokinetic study involving the identity of the specific genotype is still limited.
The results showed that the genotypes NAT2*5/*7, -*7B/*7B, -*6/*7, -*5B/*7B, -*6A/*7B, and -*6A/*6A, have a risk range of IDILI 2.74 to 5.76 times.Meanwhile, the case finding is dominant in the Asian population, due to the pharmacogenomic study development related to tuberculosis involving more populations from Asian countries.Furthermore, personalization of INH dosage is a solution to reduce the IDILI rate.However, the pharmacokinetic development model should consider the synergistic configuration factor of slow acetylator NAT2 with other enzymes related to the INH metabolic activity.These enzymes also encode the slow and null phenotype, such as CYP2E1*c1/c2 and GSTM1 respectively.The six variants are expected to become priorities for future studies, given the limited database on the IDILI susceptible types.

Table 1 .
Summary of literature objectives and results.

Table 2 .
IDILI risk level in the NAT2 phenotype of the rapid acetylator group.

Table 3 .
IDILI risk level in the NAT2 phenotype of the intermediate acetylator group.