Abstract
Thiopurines are clinically useful in the management of diverse immunological and malignant conditions. Nevertheless, these purine analogues can cause lethal myelosuppression, which may be prevented by prospective testing for variants in the thiopurine S-methyltransferase (TPMT) and, in East Asians, Nudix hydrolase 15 (NUDT15) genes. Two single-tube, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) assays were developed to genotype the common loss-of-function variants NUDT15 c.415C>T (rs116855232) and TPMT*3C c.719A>G (rs1142345). In a group of 60 unselected patients, one and seven were found to be homozygous and heterozygous, respectively, for NUDT15 c.415C>T; one was found to be heterozygous for TPMT*3C c.719A>G. There was no non-specific amplification, and the genotypes were 100% concordant with Sanger sequencing. Limit-of-detection for both assays was below 1 ng of heterozygous template per reaction. Time- and cost-effective ARMS-PCR assays, suitable for genotyping East-Asian patients for thiopurine intolerance, were successfully developed and validated.
Highlights
Thiopurines are purine analogues with cytotoxic effect upon conversion to thioguanine nucleotides.Clinically, the thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG)are used in treatment of hematological malignancy (6-TG, 6-MP) [1,2,3,4], as steroid-sparing agents in ulcerative colitis and Crohn’s disease (AZA, 6-MP) [5,6,7,8] and, less commonly, as immunosuppressants in organ transplant recipients (AZA) [9]
To further streamline the workflow and reduce proprietary reagent costs, we developed two single-tube, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS-PCR) assays for genotyping the variants and validated our new assays against Sanger sequencing using 60 patient samples
As only PCR reagents and standard oligonucleotide primers are needed, suited to molecular laboratories which may find the cost and shelf-life of special such assays are suited to molecular laboratories which may find the cost and shelf-life fluorescent dyes and special restriction enzymes prohibitive [29,30,31]
Summary
Thiopurines are purine analogues with cytotoxic effect upon conversion to thioguanine nucleotides.Clinically, the thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG)are used in treatment of hematological malignancy (6-TG, 6-MP) [1,2,3,4], as steroid-sparing agents in ulcerative colitis and Crohn’s disease (AZA, 6-MP) [5,6,7,8] and, less commonly, as immunosuppressants in organ transplant recipients (AZA) [9]. Thiopurines are purine analogues with cytotoxic effect upon conversion to thioguanine nucleotides. The thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Thiopurine treatment is limited by myelosuppression, which may lead to prematurely terminated or suboptimal treatment, and can complicate as severe neutropenia, sepsis and death [10]. The possible role of genetic variation in thiopurine metabolism and sensitivity has been noted since the 1980s [11], and testing of azathioprine intolerance due to non-functional thiopurine S-methyltransferase (TPMT) variants has been recommended by the US Food and Drug Administration since 2003 [12]. Prospective genotyping would allow the clinician to reduce thiopurine dosage for heterozygous patients, who may be unable to tolerate the full dose, and avoid administration of the drug in homozygotes, who are otherwise at fatal risk of profound myelosuppression.
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