Association of Thiopurine S-Methyltransferase and NUDT15 Polymorphisms with Azathioprine-Induced Myelotoxicity in Chinese Patients with Rheumatological Disease
Rheumatic diseases, characterized by joint inflammation and systemic involvement, are prevalent in China. Azathioprine (AZA), a purine analog immunosuppressant, is increasingly used in the treatment of these conditions. However, its clinical application is limited by adverse effects, particularly leukopenia. This study investigates the role of genetic polymorphisms in thiopurine S-methyltransferase (TPMT) and NUDT15 in predicting AZA-induced myelotoxicity in Chinese patients with rheumatological diseases.
Background and Rationale
Thiopurine S-methyltransferase (TPMT) is a critical enzyme in the metabolism of thiopurines, including AZA and 6-mercaptopurine. Mutations in the TPMT gene can lead to reduced enzyme activity, increasing the risk of severe myelosuppression. Previous studies have shown that the frequency of TPMT gene variations in the Chinese population is approximately 0.9%. Despite this low frequency, the incidence of AZA-induced leukopenia in Chinese patients remains high, ranging from 27.0% to 41.3%. This discrepancy suggests that other genetic factors may contribute to AZA toxicity.
Recent research has identified NUDT15 as another gene associated with AZA-induced leukopenia. Specifically, the NUDT15 polymorphism rs116855232 (c.415C>T) has been linked to myelotoxicity in patients with acute lymphoblastic leukemia and inflammatory bowel disease (IBD). However, there is limited data on the role of NUDT15 polymorphisms in AZA-induced leukopenia in Chinese patients with rheumatological diseases. This study aims to fill this gap by examining the allele frequencies of TPMT and NUDT15 polymorphisms and their association with AZA-induced myelotoxicity in this patient population.
Study Design and Methods
This study was conducted at the Quanzhou First Hospital Affiliated to Fujian Medical University in China. The study protocol was approved by the hospital’s ethics committee, and all participants provided written informed consent. A total of 70 patients with rheumatological diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), were enrolled. The patients were diagnosed according to the criteria of the American College of Rheumatology and were treated with AZA (50 mg/day) combined with hydroxychloroquine (0.2 g, twice daily).
Peripheral blood samples (2-3 mL) were collected from each patient, and genomic DNA was extracted using the EZBead Blood DNA Extraction Kit. Genotyping of TPMT p.Tyr240Cys (c.719A>G, rs1142345), NUDT15 p.Arg139Cys (c.415C>T, rs116855232), and NUDT15 p.Arg139His (c.416G>A, rs147390019) was performed using Sanger sequencing. The sequences of the forward and reverse primers for rs1142345 were 5′-GGTTGATGCTTTTGAAGAACGAC-3′ and 5′-TGCAAGACACATAGGCAATAATCT-3′, respectively. For rs116855232 and rs147390019, the primers were 5′-AGCTTACCCAAATAAACACCCT-3′ and 5′-TCAAATCTTCTCGGCCACCT-3′, respectively.
Patients receiving AZA therapy underwent routine blood tests weekly for the first month, followed by monthly tests. Liver function tests were conducted monthly. If leukopenia (white blood cell count <3.5 × 10^9/L) or thrombocytopenia (platelet count <1.5 × 10^9/L) occurred during treatment, the AZA dose was reduced by 50% or discontinued. If cytopenia persisted after dose reduction, AZA was permanently discontinued.
Results
The variant allele frequency of TPMT p.Tyr240Cys (c.719A>G, rs1142345) was 1% in this cohort. Only two patients (2.9%) were heterozygous for TPMT*3C, and no homozygous carriers of the variant allele were identified. For NUDT15 p.Arg139Cys (c.415C>T, rs116855232), 51 patients (72.9%) were homozygous for the wild-type allele (C/C), 18 (25.7%) were heterozygous (C/T), and one (1.4%) was homozygous for the variant allele (T/T). The NUDT15 p.Arg139His (c.416G>A, rs147390019) variant was not observed in this study. The overall mutation rate for TPMT was 1%, while for NUDT15, it was 14%, a statistically significant difference (P < 0.001).
Baseline characteristics of the patients are summarized in Table 1. There were no significant differences in sex, age, body weight, or disease type between patients with and without myelotoxicity. However, the average body mass index (BMI) of patients with myelotoxicity was significantly lower than that of those without myelotoxicity (P < 0.001).
Of the 70 patients, 68 were homozygous for the wild-type TPMT allele (A/A), and 27 (39.7%) of these developed leukopenia after AZA treatment. Only two patients were heterozygous for TPMT (A/G), and one (50.0%) developed myelotoxicity. No significant relationship was observed between TPMT polymorphisms and myelotoxicity (P = 0.973), indicating that TPMT is not an effective predictor of AZA-induced myelotoxicity in this population.
In contrast, NUDT15 polymorphisms showed a strong association with myelotoxicity. Among patients with the NUDT15 C/T genotype, 12 out of 18 (66.7%) developed myelotoxicity, compared to 15 out of 51 (29.4%) with the C/C genotype. The one patient with the T/T genotype developed myelotoxicity. Logistic regression analysis revealed that the NUDT15 c.415C>T polymorphism was associated with a 5.19-fold higher risk of myelotoxicity (odds ratio [OR]: 5.19; 95% confidence interval [CI]: 1.65-16.29).
Discussion
AZA is a prodrug that is metabolized into active thioguanine nucleotides, which exert immunosuppressive effects. The activity of TPMT is crucial in determining the toxicity of AZA, as insufficient enzyme activity leads to the accumulation of thioguanine nucleotides, resulting in myelosuppression. However, the frequency of TPMT mutations is low in Asian populations, including the Chinese cohort in this study, where the variant allele frequency was only 1%. This low frequency explains why TPMT polymorphisms were not predictive of AZA-induced myelotoxicity in this population.
NUDT15, on the other hand, plays a role in the hydrolysis of nucleoside diphosphate derivatives. The c.415C>T polymorphism in NUDT15 has been associated with increased sensitivity to thiopurines, leading to myelotoxicity. This study confirms that the NUDT15 c.415C>T polymorphism is a significant predictor of AZA-induced myelotoxicity in Chinese patients with rheumatological diseases. The mutation frequency of NUDT15 c.415C>T was markedly higher than that of TPMT*3C, and the difference was statistically significant.
Another notable finding was the association between lower BMI and increased risk of myelotoxicity. Patients with lower BMI were more likely to develop myelotoxicity, suggesting that nutritional status may influence the incidence of adverse effects in patients treated with AZA. This finding warrants further investigation to determine whether nutritional interventions could reduce the risk of myelotoxicity in these patients.
Conclusion
This study demonstrates that NUDT15 c.415C>T is a more effective predictor of AZA-induced myelotoxicity than TPMT3C in Chinese patients with rheumatological diseases. The mutation frequency of NUDT15 c.415C>T was significantly higher than that of TPMT3C, and it was strongly associated with an increased risk of myelotoxicity. Additionally, lower BMI was identified as a potential risk factor for myelotoxicity, highlighting the need for further research into the role of nutrition in thiopurine therapy. These findings have important implications for the personalized use of AZA in Chinese patients with rheumatological diseases, potentially improving treatment outcomes and reducing adverse effects.
doi.org/10.1097/CM9.0000000000000756
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