AZA was a classic maintenance treatment drug for AIH. However, such a drug had large individual differences and severe adverse reactions, which received widespread attention. At present, there was no clear guideline for the individualized medication of AZA in China. In this study, we found that patients with at least one genetic variant in TPMT*3C, ITPA94C > A and NUDT15c.415C > T were more likely to have leukopenia and neutropenia. T allele variation in NUDT15c.415C > T was an independent risk factor leading to leukopenia and neutropenia.
The blood toxicity of AZA is related to the genetic polymorphism of TPMT. Mutation or low enzyme activity leads to a high concentration of 6-TGN, which increases the risk of myelosuppression.6 Therefore, as early as 2005, the FDA had included the pre-administration TPMT genotype test in AZA's drug label.21 In AZA pharmacogenomics research, TPMT (*2, *3A and *3C) is to date the most widely studied single nucleotide polymorphism for AZA metabolism. The incidence is approximately 10–15% in the Caucasian population (commonly TPMT*3A).22 However, the incidence in the Chinese population is lower than that in the Caucasian population. The literature uniformly reports that the TPMT allele (commonly TPMT*3C) accounts for less than 5% prevalence, closer to Japan and South Korea.23,24 In this study, we observed that the C allele's mutation frequency in TPMT*3C was 0.9%. There are only 2 (1.8%) patients with heterozygous mutations of TPMT*3C, and none of them with myelosuppression, but in TPMT*3C wild-type patients, 15 (13.5%) patients suffered leukopenia, 34 (30.6%) patients suffered thrombocytopenia, and 10 (9.0%) patients suffered neutropenia. The above results indicate that TPMT*3C gene test has limited predictive value for AZA-induced myelosuppression in the Chinese population. Therefore, although TPMT*3C has been considered the leading risk factor for AZA-induced myelosuppression, no significant difference was observed in this study, which may be because of its low prevalence and the small sample size of this study.
ITPA polymorphism is another essential enzyme involved in the metabolism of AZA. Studies have shown that ITPA c.94C > A mutation can cause the enzyme activity to decrease, causing the toxic metabolite 6-TITP to accumulate in the body, causing flu-like symptoms, gastrointestinal reactions, skin rash, pancreatitis, and even neutropenia and liver damage, leading to interruption of treatment.9,25 The incidence of ITPA 94C > A mutations we observed in this study was 16.4%, consistent with other studies.8 Only one case of homozygous mutation was observed in 113 subjects. Since he took AZA doses less than 1 mg·kg− 1·d− 1, so did not suffer myelosuppression. Although in this study, the incidences of leukopenia and thrombocytopenia in patients with heterozygous mutations were 22.9% (8/35) and 28.6% (10/35). However, we did not observe significant differences in the different genotypes of ITPA 94C > A for AZA-induced leukopenia, thrombocytopenia and neutropenia. It may be that ITPA 94C > A is mainly related to AZA-induced liver toxicity. On the other hand, the small sample size may also be the reason for the insignificant difference.
The NUDT15 belongs to the Nudix (nucleoside diphosphate linked to x) hydrolase superfamily. It mainly consists of pyrophosphohydrolase, which acts on nucleoside diphosphates linked to other moieties. The NUDT15 hydrolyzes 6-thio-GTP (TGTP) and 6-thio-GDP (TGDP) into 6-thio-GMP (TGMP), which reduces their cytotoxic effects. Once NUDT15 is mutated, it will increase the cytotoxicity of mercaptopurine drugs. Most studies have shown that the incidence of NUDT15 allelic mutations in Asian populations is 8.5%-16%,26,27 while it is less than 1%28 in Caucasians. The frequency of NUDT15 mutations in IBD patients in Japan and South Korea is 12% and 10.4%, respectively, but it can be as high as 32.1% in Chinese patients with autoimmune diseases.29 The frequency we observed in this study was 12.4%, which is the same as the frequency of 9.4% in AIH patients reported by Xiaoli Fan et al..19 Recent studies have found NUDT15 c.415C > T variants were associated with thiopurine-induced leukopenia, particularly in Asian populations.26,30−32 In 2014, Yang et al.11 revealed for the first time that NUDT15 c.415C > T allelic mutation is significantly associated with AZA-induced leukopenia in Korean IBD patients (P = 5.58 × 10− 43, OR = 8.61). Subsequently, it was also confirmed in Japanese IBD patients that NUDT15 c.415C > T allele mutation is closely related to AZA-induced early leukopenia13 (P = 1.92 × 10 − 16, OR = 28.4). The studies by Xiang Fei et al.29 and Xiaoli Fan et al.19 on Chinese autoimmune diseases and AIH patients also showed that NUDT15 c.415C > T SNP is significantly related to AZA-induced early leukopenia (P = 1.79 × 10− 7; OR = 7.59 and P < 0.00001; OR = 20.41, respectively). Our result was concordant with previous studies, which showed NUDT15 c.415C > T allelic mutation was associated with early leukopenia (P = 8.26 × 10− 7; OR = 7.5). At the same time, we also found that NUDT15 c.415C > T mutation is implicated in AZA-induced myelosuppression with neutropenia as the primary manifestation (P = 3.54 × 10− 6; OR = 8.05). Therefore, compared with TPMT*3C and ITPA 94C > A, the detection of NUDT15 c.415C > T in the Chinese population may have a better predictive value for AZA-induced myelosuppression with leukopenia and neutropenia as the primary manifestations. In addition, research has shown that NUDT15 c.415C > T was associated with not only early (< 8 weeks) leukopenia but also middle (8–24weeks) and late (> 24weeks) leukopenia.14 However, these findings could not be fully confirmed in our study because there was a shorter duration of follow-up (12 weeks). This happens to be the limitation of this study.
It is well known that 6-TGN is the active metabolite responsible for AZA's efficacy and cytotoxicity, and one of the side effects of AZA therapy is myelosuppression. Therapeutic drug monitoring (TDM) of the pharmacologically active metabolites of thiopurines, 6-TGN, has proven beneficial.33 However, there is no unified conclusion about the relationship between the concentration of 6-TGN in red blood cells and adverse reactions. In the research of Asada et al.12 and Xiang Fei et al.29, there was no statistically significant concentration difference observed between different NUDT15c.415C > T genotypes. However, Xiaoli Fan et al.19 reported that the 6-TGN concentration in CT genotype patients in NUDT15c.415C > T variants was significantly higher than in CC wild-type patients. The above studies have shown no significant difference in the concentration of 6-TNG between patients with or without leukopenia. This finding was replicated in our research (P = 0.556, Table S1). Among 113 AIH patients included in this study, we did not observe significant differences in 6-TGN concentration and Correction 6-TGN concentration among different genotypes of TPMT*3C, ITPA 94C > A and NUDT15c.415 C > T (P > 0.05, Table S1). The same is true between different gender groups and maintenance dose groups. However, we found significant differences in the corrected concentration of 6-TGN between groups of patients with different BMI (P = 0.026, Table S1). It indicates that obesity may be an important non-genetic factor affecting the concentration of AZA active metabolites. In recent years, studies have proposed that measuring the concentration of peripheral blood mononuclear cells (PBMC) for immunosuppressants is a new valuable biomarker to improve the therapeutic drugs monitoring.34 Combined with the results of this study, we guess that the new therapeutic monitoring method detecting the metabolites of AZA in PBMC may have more clinical value than in whole blood.
Our shortcoming is that this is a single-center study with a limited number of patients and regional limitations, which precludes adequate statistical inference. On the other hand, the follow-up time is short, and the long-term adverse reactions cannot be thoroughly evaluated. Finally, we used commercial kits to detect the most common mutations in Asian populations. The lack of comprehensive testing of AZA metabolism-related genes may lead to biased results.