Shipkova Maria, Lorenz Kristin, Oellerich Michael, Wieland Eberhard, von Ahsen Nicolas
Central Institute of Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Stuttgart, Germany.
Clin Chem. 2006 Feb;52(2):240-7. doi: 10.1373/clinchem.2005.059501. Epub 2005 Dec 29.
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.
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 94C>A and IVS2 + 21A>C by real-time fluorescence PCR.
The assay was linear to 3 mmol/L IMP [approximately 500 micromol/(g Hb x h)] with a lower limit of quantification of 4 micromol/L [approximately 0.5 micromol/(g Hb x h)]. With IMP-enriched samples, within- and between-day imprecision was < or = 3.6% and < or = 4.9%, respectively, and the inaccuracy was < or = 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 micromol IMP/(g Hb x h). Mutant allele frequencies were 0.062 (94C>A) and 0.131 (IVS2 + 21A>C). When we used a cutoff of 125 micromol IMP/(g Hb x h), phenotyping detected all 94C>A mutant cases, all 94C>A and IVS2 + 21A>C compound heterozygotes, all IVS2 + 21A>C homozygotes, and 6 of 24 IVS2 + 21A>C heterozygote-only cases. A novel IVS2 + 68T>C mutation was also found.
The HPLC procedure provides an excellent ITPA phenotype-genotype correlation and led to the discovery of a novel IVS2 + 68T>C mutation. The method could facilitate investigation of the role of ITPA activity for drug toxicity during thiopurine therapy.
肌苷三磷酸（ITP）焦磷酸水解酶（ITPA）催化ITP/dITP和三磷酸黄苷的焦磷酸水解，以防止异常核苷酸掺入RNA和DNA。导致酶缺乏的重要突变是94C>A和IVS2 + 21A>C。已显示ITPA 94C>A与硫唑嘌呤治疗期间的不良反应之间存在关联。为了研究ITPA表型，开发了一种高效液相色谱（HPLC）方法并评估了表型-基因型相关性。
用高氯酸和饱和磷酸氢二钾终止ITP向肌苷单磷酸（IMP）的酶促转化。使用20 mmol/L pH 2.5的磷酸盐缓冲液在Aqua perfect C18柱上分离后，于262 nm处对IMP进行定量。我们还通过实时荧光PCR对样本进行ITPA 94C>A和IVS2 + 21A>C基因分型。
该测定法在IMP浓度达3 mmol/L [约500 μmol/（g血红蛋白×小时）]时呈线性，定量下限为4 μmol/L [约0.5 μmol/（g血红蛋白×小时）]。对于富含IMP的样本，日内和日间不精密度分别≤3.6%和≤4.9%，误差率≤5.2%。对于混合红细胞，日内和日间不精密度分别为3.8%和7.5%。130名健康对照者的ITPA活性在<0.5至408 μmol IMP/（g血红蛋白×小时）之间。突变等位基因频率分别为0.062（94C>A）和0.131（IVS2 + 21A>C）。当我们采用125 μmol IMP/（g血红蛋白×小时）的临界值时，表型分析检测到了所有94C>A突变病例、所有94C>A和IVS2 + 21A>C复合杂合子、所有IVS2 + 21A>C纯合子以及24例仅为IVS2 + 21A>C杂合子病例中的6例。还发现了一种新的IVS2 + 68T>C突变。
HPLC方法提供了出色的ITPA表型-基因型相关性，并导致发现了一种新的IVS2 + 68T>C突变。该方法有助于研究ITPA活性在硫嘌呤治疗期间对药物毒性的作用。
