Thiopurine S-methyltransferase deficiency associated with a novel mutation

Thiopurine S-Methyltransferase Deficiency Associated with a Novel Mutation

To the Editor:

We report a case of a Caucasian patient with Crohn's disease in whom a novel mutation in the thiopurine S-methyltransferase (TPMT) gene TPMT was detected following a genotype/phenotype discrepancy. A 28-year-old man with a 5-month history of Crohn's disease involving the stomach and small bowel was treated with mesalamine 2g/day and varenicline (Champix) for smoking addiction. Since symptoms had not improved, prednisolone (60 mg/day, in a tapering regimen) was introduced. Considering diffuse extension of the disease, active smoking, young age at onset, and need for steroid at diagnosis, treatment with azathioprine was decided. Pretherapeutic TMPT genotyping, performed using mutation-specific real-time polymerase chain reaction (PCR) assays (Taqman SNP Genotyping Assays ready to use, Applied Biosystems, Courtaboeuf, France), revealed heterozygosity for the c.460 G>A and c.719 A>G mutations, characteristic of the nonfunctional TPMT variant TPMT*3A, predicting a TPMT intermediate activity. Azathioprine treatment was thus started at 1 mg/kg/daily and hematological weekly monitoring was performed. We observed a rapid decrease in white blood cells (WBC) that did not respond to dose reduction and that eventually resulted in the interruption of azathioprine. After 5 weeks of treatment WBC counts had decreased from 13.7 to 4.2 × 10⁹/L, absolute neutrophils count from 9.06 to 1.42 × 10⁹/L, lymphocytes from 3.49 to 2.28 × 10⁹/L, and platelets from 290 to 171 × 10^9/L. Active metabolites, 6-thioguanine nucleotides (6-TGNs), measured by a high-performance liquid chromatography (HPLC) assay¹ were 7026 pmol/8 × 10⁸ RBC (therapeutic range: 235–400). Since this 6-TGN level was exceptionally elevated, a TPMT deficiency was suspected and TPMT activity was measured in erythrocyte lysates using an HPLC assay.² Results revealed a very low TPMT activity at 2.56 nmol/ml RBC/h characteristic of a deficient methylator (deficient activity: <5.0; intermediate: 5.0–8.5; high activity: >8.5). To further elucidate this genotype/phenotype discrepancy, sequencing of the entire coding sequence of the TPMT gene was performed as previously described.³ Comparison of amplified DNA sequences with the wildtype reference confirmed the presence of the c.460 G>A and c.719 A>G mutations and in addition revealed a novel mutation (c.291delA) which had not yet been described. This new mutation, located in exon 5 of the gene, results in a shift in the translational reading frame starting at codon 97, which introduces a premature stop codon and creates a highly truncated mutant protein (147 amino acids versus 245 for the wildtype human TPMT) which cannot correctly function. To support this finding, sequencing of the TPMT gene was performed in both parents after informed consent was obtained. Data revealed that the mother was heterozygous for the c.460 G>A and c.719 A>G (TPMT*3A) and that the father was heterozygous for c.291delA. TPMT activity was also measured in both parents; data showed that the mother displayed an intermediate activity (8.0 nmol/mlm RBC/h) and that heterozygosity for c.291delA in the father also resulted in an intermediate activity (7.5 nmol/ml RBC/h). The patient was thus clearly identified as compound heterozygous for TPMT*3A and for the novel deleterious mutation c.291delA. On follow-up a gradual increase in total WBC in parallel with a decrease of 6-TGN levels was observed with normalization within 10 weeks (Fig. 1). Despite 6-TGN high levels, no infectious complications or other adverse events occurred. The patient was well while 6-TGNs levels remained elevated, but his clinical condition deteriorated when they decreased below the therapeutic range.