Severe bone marrow aplasia following azathioprine therapy in patients with NUDT15 deficiency: A case series

INTRODUCTION

Azathioprine is a prodrug that must first be activated to form thioguanine nucleotides (TGNs), the major active metabolites, which further metabolized and inactivated by the enzyme thiopurine methyltransferase (TPMT) and the enzyme nucleoside diphosphate-linked moiety X-type motif Nudix hydrolase 15 (NUDT15) belongs to family Nudix hydrolase15.^[1] Thiopurine methyltransferase (TPMT) inactivates azathioprine (AZA) resulting in less parent drug available to form 6-TGNs while Nudix hydrolase 15 is involved in the metabolism of azathioprine and other thiopurines, as it catalyzes the conversion of active metabolites thioguanine diphosphate (TdGTP) to less toxic metabolites thioguanine monophosphate (TdGMP) and in doing so, prevents the incorporation of toxic metabolites into deoxyribonucleic acid (DNA) [Supplementary Figure S1].^[1] Genetic polymorphism affects enzyme activity, and thus metabolism of azathioprine. People with enzyme deficiency due to TPMT and NUDT15 polymorphism are at increased risk of prolonged and severe life-threatening myelosuppression following treatment with azathioprine and other thiopurine drugs, and may require long in-hospital stay and supportive care. TPMT polymorphism is well known cause of myelosuppression following azathioprine therapy, but TPMT polymorphism is less commonly reported in south Asian population,^[2] including in our case series all patients that were included in study were negative for TPMT polymorphism and are positive for NUDT15 polymorphism. Loss-of-function allele (Polymorphism: c.415C>T) in nucleoside diphosphate-linked moiety X motif 15 NUDT15 has been recognized to contribute to thiopurine tolerance in patients of Asian ancestry.^[3] This variant frequency has been described commonly in East Asians and Hispanics, but rarer in the Europeans and could be the reason for myelosuppression following thiopurines drugs as reported frequency of TPMT polymorphism lower.^[2] While testing for TPMT mutation in patients developing myelosuppression is well known and available, NUDT15 testing is not widely available. Routine testing for NUDT15 is not recommended before starting thiopurine drugs but it should be done in patients who develop severe myelosuppression following these drugs.^[1] Here, we report our experience of severe myelosuppression with bone marrow aplasia following azathioprine therapy in six patients who were found to have NUDT15C415T variant and TPMT wild type.

CASE SERIES

RESULTS

This is a retrospective case series of six patients, who developed severe myelosuppression while on AZA therapy. Out of all six patients, three patients were taking azathioprine for inflammatory bowel disease, two patients were taking drug for atopic dermatitis, and one patient was post renal transplant. Mean duration between introduction of azathioprine and severe pancytopenia was 26 days. Bone marrow aspiration and biopsy was done in three patients who revealed overall cellularity around 15–20%. In all six patients, TPMT was normal (Wild type*1/*1), while five patients were homozygous for NUDT15C415T mutation and one patient was heterozygous for NUDT15 polymorphism. All the patients received supportive blood component transfusion, colony-stimulating factor in view of severe neutropenia long with empirical antimicrobials. Patients were admitted till blood counts recovery and discharged after mean 12.5 days of in-hospital stay, recovery of blood counts was defined as ANC >500/microlitre for 3 days and platelets counts of >20000/microlitre for 3 days .

DISCUSSION

Azathioprine is a prodrug that must be metabolized to TGNs, the major active metabolites. The active metabolites are metabolized and inactivated by the enzyme thiopurine methyl transferase (TPMT) and the enzyme nucleoside diphosphate-linked moiety X-type motif (NUDT15).^[1] Azathioprine toxicity manifests as hepatotoxicity, alopecia, and bone marrow suppression that may lead to severe neutropenia rendering patient to increased risk of life-threatening infections, severe anemia, and bleeding.^[1] Myelosuppression is dose-dependent and correlates with the level of active metabolite TGN, and hepatotoxicity is related to 6-Methyl mercaptopurine (6-MMP).^[3] Asians are found to tolerate AZA poorly and are more susceptible to toxicity, but paradoxically the common genetic polymorphisms in TPMT gene with reduced enzyme activity (TPMT×2, ×3) are extremely rare in Asians compared to the frequency reported in the European population.^[2] Polymorphisms in thiopurine methyl transferase (TPMT) and NUDT15 have been implicated as the predominant cause of thiopurine induced leukopenia in the Western countries and East Asia, respectively.^[4] Jena et al. in meta-analysis of 26 studies found, high prevalence of NUDT15 (16.5%) compared to TPMT polymorphisms (4.57%). In patients with adverse effects following thiopurine therapy, the pooled prevalence of NUDT15 and TPMT polymorphism was 49.51% and 9.47%, respectively.^[5]

Gene NUDT15 encodes an enzyme that belongs to the Nudix hydrolase superfamily. NUDT15 is directly involved in the metabolism of thiopurines, as it catalyzes the conversion of active metabolites (TdGTP) to less toxic metabolites (TdGMP) and in doing so, prevents the incorporation of toxic metabolites into DNA.^[1] Individual with reduced or absent NUDT15 activity are prone to severe adverse event related to azathioprine and other thiopurines. NUDT15C415T (p.R139C) is the first reported variant with absent enzyme activity and it is associated with both NUDT15 × 2 and NUDT15 × 3 genotype.^[6] Similar to TPMT, PharmaVar consortium has catalogued 26 variants of NUDT15 and NUDT15 × 2 and NUDT15 × 3 genotypes are most commonly reported variants. The same genetic variant was also linked with 6-mercaptopurine (6-MP)-related myelosuppression in patients of East Asian ancestry with acute lymphoblastic leukemia.^[2] In a retrospective study from south India by Babu et al. on 17 adult patients with acute leukemia, the prevalence of NUDT15 polymorphism using allele specific real-time PCR was 36%. They also studied impact of omission of 6-MP from the treatment protocols of the patient who were positive for NUDT15 mutation and found that less treatment discontinuation and interruption was noted in these patients.^[7]

In this case series, the five patients were homozygous for NUDT15 polymorphism and one patient was heterozygous and it can be seen that, cytopenias occurred earlier (within 30 days) and it was more severe. AZA was stopped in all of them and was never re-challenged. All patients required longer in hospital stay and supportive treatment. Surprisingly, all patients were normal for TPMT mutation, so it can be stated that NUDT15 polymorphism is more prevalent in north India also and testing is also cost effective. Furthermore, polymorphisms in NUDT15 can explain and predict the occurrence and severity of myelosuppression seen in Indian patients.

To the best of our knowledge, no study has been published from north India in patients with severe pancytopenia and bone marrow hypoplasia following azathioprine therapy in NUDT15 mutated patients.

CONCLUSION

This report indicates that NUDT15 polymorphism is an important determinant of myelosuppression related to the intake of azathioprine. As compared to TPMT polymorphism, NUDT15 polymorphism is more frequent in north Indian population, and thus, testing for the common variants of NUDT15 associated with thiopurines toxicity is necessary in patients who experience severe myelosuppression following introduction of azathioprine or other thiopurines and this may help to tailor the dose or early switch to alternate therapy to achieve better treatment outcomes without life-threatening morbidity.