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

Abstract

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 94C>A and IVS2 ؉ 21A>C. An association between ITPA 94C>A 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 C 18 column using 20 mmol/L phosphate buffer, pH 2.5. We also genotyped samples for ITPA 94C>A and IVS2 ؉ 21A>C by real-time fluorescence PCR. Results: The assay was linear to 3 mmol/L IMP [ϳ500 mol/(g Hb ⅐ h)] with a lower limit of quantification of 4 mol/L [ϳ0.5 mol/(g Hb ⅐ 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 betweenday imprecision was 3.8% and 7.5%, respectively. ITPA

Association of ITPA Gene Polymorphisms and the Risk of Ribavirin-Induced Anemia in HIV/Hepatitis C Virus (HCV)-Coinfected Patients Receiving HCV Combination Therapy

“…Recent evidence indicates that ITP protects against RBV-induced anemia by substituting for GTP (depleted by RBV) in the biosynthesis of ATP (13). Since ITP intracellular levels are dependent on ITPA activity, which in turn is modulated by functional polymorphisms in the ITPA gene (26), these polymorphisms may in the end determine the degree of protection against RBV-induced hemolytic anemia in HIV/HCVcoinfected patients, as has been recently reported (19,23) and our work suggests.…”

“…Among them, mutation at rs1127354 of the ITPA gene causes a substantial reduction in ITPA activity, and homozygosity for the P32T mutation causes nondetectable ITPA activity (9). Altogether these data indicate that there is a good correlation between ITPA gene polymorphisms, ITPA functional activity, and, therefore, ITP accumulation in red blood cells (15,26). Third, the number of patients included in the present study is relatively small.…”

Association of ITPA polymorphisms rs6051702/rs1127354 instead of rs7270101/rs1127354 as predictor of ribavirin-associated anemia in chronic hepatitis C treated patients

“…The association signal was accounted for by two functional variants in the ITPA gene on chromosome 20: a missense variant in exon 2 (rs1127354, P32T) and a splice-altering single nucleotide polymorphism in intron 2 (rs7270101). Both polymorphisms had previously been well characterized and validated as functional variants in studies of patients with ITPase deficiency, a benign inherited enzymopathy in which inosine triphosphate (ITP, the substrate for ITPase) accumulates in red blood cells (Shipkova et al, 2006;Sumi et al, 2002).…”

Evolving Trends in the Hepatitis C Virus Molecular Epidemiology Studies: From the Viral Sequences to the Human Genome

“…The association was explained by two known functional variants in the ITPA gene, located on chromosome 20 and encoding for inosine triphosphatase (ITPase). The two variants, a missense polymorphism in exon 2 (g.3141842C>A, P32T; rs1127354) and a splicealtering SNP located in the second intron (g.8838A>C, rs7270101), result in reduced enzyme activity: homozygosity for the P32T mutation leads to undetectable ITPase activity, accumulation of its substrate ITP in erythrocytes, and increased toxicity of purine analogue drugs [125,[127][128][129][130][131]. Conversely, reduced ITPase activity may be protective from RBV-induced hemolysis through the competition of ITP with RBV-TP [127,132].…”