INTRODUCTION
The thiopurine drugs azathioprine (AZA) and 6-mercaptopurine (6-MP) have been used for many years for the treatment of numerous disorders, including UC, CD, acute lymphoblastic leukemia (ALL), and rheumatoid arthritis. In IBD (either UC or CD), thiopurines are used together with 5-aminosalicylic acid or the anti-tumor necrosis factor α antibodies infliximab and adalimumab to induce or maintain remission.
1,2
Although thiopurine therapy has obvious clinical benefits in IBD, AZA and 6-MP occasionally induce severe adverse events such as leukopenia and alopecia. Thiopurine-induced severe leukopenia can result in infectious diseases with life-threatening consequences. Therefore, careful monitoring of white blood cell (WBC) count is required during treatment with these agents. Severe hair loss causes considerable stress to patients, and recovery may take more than 6 months following discontinuation of thiopurine therapy. These adverse events contribute to the interruption or discontinuation of thiopurine treatment. Therefore, genetic risk factors predictive of such thiopurine-induced effects have been studied in patients with IBD.
3 Certain polymorphisms of thiopurine methyltransferase (
TPMT) are well-characterized genetic markers of risk of thiopurine-induced adverse events, and
TPMT genotyping prior to initiation of thiopurine treatment is recommended by the U.S. Food and Drug Administration. However, it is believed that other factors affecting the risk of such adverse events are present in the Japanese population, since
TPMT risk allele frequencies in this country are lower than those in Western countries, and the incidence of adverse reactions to thiopurines is high in Japan, even among carriers of wild-type
TPMT.
3,4,5
In 2014, Yang et al.
6 reported that a missense variant in exon 3 of the
NUDT15 gene (p.Arg139Cys or R139C; single nucleotide polymorphism [SNP] ID: rs116855232) strongly correlates with thiopurine-induced leukopenia among Korean patients with CD. The high specificity and sensitivity (89.4% and 93.2%, respectively) of
NUDT15 p.Arg139Cys recorded by these authors indicate that this variant, rather than
TPMT polymorphisms, may be an effective genetic marker for predicting thiopurine-induced adverse events, at least in East Asian populations. To date, this strong association has been confirmed in numerous studies involving subjects with IBD or ALL.
7,8,9,10,11,12,13,14,15,16,17,18 In addition, a further investigation identified 4
NUDT15 coding variants, including p.Arg139Cys, that influence both nucleotide diphosphatase activity and levels of thiopurine active metabolites, and found loss-of-function
NUDT15 variants to be associated with thiopurine intolerance.
13 Moreover, a more recent genome-wide association study conducted by Kim et al.
19 found an
FTO coding variant and a proximal region of
RUNX1 to be associated with thiopurine-induced leukopenia among East Asian patients with IBD.
These findings suggest that the p.Arg139Cys coding variant of NUDT15 may be a highly reliable marker of thiopurine cytotoxicity risk, especially in Asian populations. However, in patients lacking a risk-associated TPMT variant and NUDT15 p.Arg139Cys, thiopurine cytotoxicity may be explained by the other abovementioned NUDT15 coding sequence variations or FTO or RUNX1 variants. Therefore, it is important to examine correlations between genes recently identified using genome-wide analysis and adverse clinical events.
Thus, in the present study, we investigated the associations between 3 coding variants in NUDT15 and thiopurine-induced adverse events among Japanese subjects. Furthermore, we examined potential relationships between single sequence variations in FTO and RUNX1 with such events in a Japanese population.
DISCUSSION
AZA and 6-MP have been widely used as immunomodulators and chemotherapeutic agents for the treatment of various diseases. At our hospital, more than 100 patients with IBD commence thiopurine therapy each year, and this number is increasing due to the growing incidence of IBD in Japan. Every year, several patients present with early leukopenia or severe hair loss after initiating thiopurine treatment. To date, for such patients carrying the wild-type TPMT genotype, the cause of these adverse events could not be determined.
Discovery of the association between
NUDT15 p.Arg139Cys and thiopurine-induced leukopenia among Korean CD patients may enable unprecedented progress in the management of adverse thiopurine effects in the East Asian population.
6 Importantly, many investigators have been able to replicate these results in studies of thiopurine-treated patients with IBD or ALL.
7,8,9,10,11,12,13,14,15,16,17,18 In the current study, we successfully established a significant association between
NUDT15 p.Arg139Cys and thiopurine-induced leukopenia and severe hair loss.
We obtained evidence that this variant correlates with the time interval between AZA initiation and GI intolerance to this drug (
Fig. 1). The abnormal accumulation of thiopurine metabolites in patients carrying p.Arg139Cys may accelerate development of strong nausea. However, our results in this respect may be limited by certain arbitrary aspects of the analysis, given the subjectivity of nausea and that the decision to discontinue thiopurine therapy is dependent on physicians and patients.
As evidence of the role of
NUDT15 in thiopurine treatment accumulates, the demand for
NUDT15 genotyping prior to such therapy is increasing in Japan. Although these replicable results should be translated into clinical practice, further evaluation of
NUDT15 variants is necessary before clinical application. Consequently, we investigated three coding sequence variations in
NUDT15 identified by Moriyama et al.
13 No association between the p.Val18_Val19insGlyVal and p.Val18Ile variants and leukopenia or severe hair loss was evident in our study. However, it should be noted that these adverse events did occur more frequently among subjects heterozygous for these coding variants (
Table 4); therefore, it is likely that they are significantly more common among homozygous risk allele carriers, as observed in relation to the p.Arg139Cys mutation. The strong linkage disequilibrium between p.Val18_Val19insGlyVal and p.Arg139Cys should be taken into account however.
13 These coding variants result in 74.4% to 100% loss of NUDT15 enzymatic activity.
13 Therefore, carriers of the exon 1 variation may need to be followed-up carefully to monitor for signs of leukopenia. Future studies are expected to shed light on the importance of p.Val18_Val19insGlyVal and p.Val18Ile as predictive markers of thiopurine-induced adverse events.
In our study sample, 8 subjects homozygous for the T allele of
NUDT15 p.Arg139Cys were identified. One of these individuals discontinued AZA therapy within 7 days following its initiation due to intolerable GI symptoms (
Table 3,
Fig. 1). Six of the 8 subjects were able to continue AZA treatment for more than 2 weeks, but discontinued it because of early or late leukopenia, the latter being detected at 9 weeks (
Table 4). All of the homozygous T/T subjects with leukopenia exhibited grade 4 leukopenia (defined as a WBC <1,000 cells/µL) and required both granulocyte-colony stimulating factor and antibiotics. Only 1 patient carrying this genotype did not develop leukopenia. However, all 7 patients who continued thiopurine therapy for more than 2 weeks experienced severe hair loss. Therefore, administration of thiopurines to patients with the
NUDT15 p.Arg139Cys T/T genotype caused drastic adverse events. We currently do not use thiopurine therapy for patients with IBD and this genotype because it is contraindicated. It remains unclear whether an extremely low dose of 6-MP would be tolerable or clinically efficacious for Japanese carriers of this genotype suffering IBD.
The physiological function of NUDT15 is considered to be the hydrolysis of 8-oxo-dGTP generated from reactive oxygen species.
23 Recent studies indicate that NUDT15 preferentially inactivates thiopurine metabolites over 8-oxo-dGTP,
24 converting those such as TGTP and TdGTP to TGMP and TdGMP, respectively.
13 NUDT15 is a negative regulator that decreases levels of bioactive thiopurine metabolites, the cytotoxicity of which is therefore increased by loss-of-function variants. Our data obtained from analyzing adverse events among thiopurine-treated patients indicate that the NUDT15 Arg139Cys site may be more critical to thiopurine metabolism than those loci affected by the other coding variants examined. The precise molecular mechanisms by which the p.Arg139Cys mutation leads to thiopurine-induced cell death remain unclear.
The site of the Arg139Cys mutation is reported to be within the TGTP-binding pocket of the NUDT15 protein. Substitution of arginine with cysteine at this position may affect the enzyme's structure, and thereby, reduce cellular TGTP hydrolysis.
24 One study has shown that NUDT15 proteins harboring the Arg139Cys mutation demonstrate enzyme activity comparable to that of the wild-type form, but are degraded rapidly in cells.
25 Therefore, NUDT15 protein level may be implicated in the cytotoxicity of and sensitivity to thiopurines. The development of biochemical tests of NUDT15 enzymatic activity would be beneficial.
Sequence variations in the genes
FTO and
RUNX1 identified using a genome-wide association study are also promising candidates as predictive markers of thiopurine cytotoxicity.
19 However, we could not verify the association between thiopurine-induced adverse events and
FTO p.Ala134Thr and a noncoding variant in the proximal region of
RUNX1 among Japanese patients with IBD. This may be due to differences between Korean and Japanese patients with this condition in terms of the thiopurine doses administered. We usually initiate AZA and 6-MP treatment at relatively low doses (25 and 10-20 mg/day, respectively), and sometimes gradually increase the dose while paying attention to the development of adverse events such as GI symptoms and leukopenia. For Japanese patients with IBD, even low doses of thiopurines are clinically effective and result in therapeutic concentrations of the thiopurine metabolite 6-thioguanine nucleotide;
26,27 therefore, the AZA maintenance dose of 25 to 100 mg/day (equivalent to 0.5-2.0 mg/kg/day for a patient weighing 50 kg) is commonly used, as shown in
Fig. 2B. We did not identify any homozygous
FTO p.Ala134Thr risk allele carriers in this study, who are expected to exhibit more prominent effects of thiopurine exposure. As our sample size was not large enough, further analysis will be required to elucidate the association between these genes and thiopurine-induced adverse events in the Japanese population.
In summary, we verified the strong association between NUDT15 p.Arg139Cys and thiopurine-induced early and late leukopenia and severe hair loss among Japanese patients with IBD. However, NUDT15 p.Val18_Val19insGlyVal, NUDT15 p.Val18Ile, FTO p.Ala134Thr, and a noncoding variant in the proximal region of RUNX1 (rs2834826) were not associated with these adverse events. Thus, NUDT15 p.Arg139Cys genotyping should currently be prioritized for the prediction of thiopurine-induced adverse events among Japanese patients with IBD, and its application in precision medicine should be considered in the future.