Measurement of Erythrocyte Inosine Triphosphate Pyrophosphohydrolase (ITPA) Activity by HPLC and Correlation of ITPA Genotype-Phenotype in a Caucasian Population

摘要

Background: Inosine triphosphate (ITP) pyrophosphohydrolase (ITPA) catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate to prevent incorporation of unusual nucleotides into RNA and DNA. Important mutations leading to enzyme deficiency are 94CA and IVS2 + 21AC. An association between ITPA 94CA and adverse reactions during azathioprine treatment has been shown. To investigate the ITPA phenotype, an HPLC procedure was developed and phenotype-genotype correlations were assessed.Methods: The enzymatic conversion of ITP to inosine monophosphate (IMP) was terminated by perchloric acid and saturated dipotassium hydrogen phosphate. We quantified the IMP at 262 nm after separation on an Aqua perfect C18 column using 20 mmol/L phosphate buffer, pH 2.5. We also genotyped samples for ITPA 94CA and IVS2 + 21AC by real-time fluorescence PCR.Results: The assay was linear to 3 mmol/L IMP [～500 μmol/(g Hb · h)] with a lower limit of quantification of 4 μmol/L [～0.5 μmol/(g Hb · h)]. With IMP-enriched samples, within- and between-day imprecision was ≤3.6% and ≤4.9%, respectively, and the inaccuracy was ≤5.2%. With pooled erythrocytes, within- and between-day imprecision was 3.8% and 7.5%, respectively. ITPA activity in 130 healthy controls was between 0.5 and 408 μmol IMP/(g Hb · h). Mutant allele frequencies were 0.062 (94CA) and 0.131 (IVS2 + 21AC). When we used a cutoff of 125 μmol IMP/(g Hb · h), phenotyping detected all 94CA mutant cases, all 94CA and IVS2 + 21AC compound heterozygotes, all IVS2 + 21AC homozygotes, and 6 of 24 IVS2 + 21AC heterozygote-only cases. A novel IVS2 + 68TC mutation was also found.Conclusions: The HPLC procedure provides an excellent ITPA phenotype-genotype correlation and led to the discovery of a novel IVS2 + 68TC mutation. The method could facilitate investigation of the role of ITPA activity for drug toxicity during thiopurine therapy.