M Arenas

Genetic basis of inosine triphosphate pyrophosphohydrolase deficiency.

Abstract. Inosine triphosphate pyrophosphohydrolase (ITPase) deficiency is a common inherited condition characterized by the abnormal accumulation of inosine triphosphate (ITP) in erythrocytes. The genetic basis and pathological consequences of ITPase deficiency are unknown. We have characterized the genomic structure of the ITPA gene, showing that it has eight exons. Five single nucleotide polymorphisms were identified, three silent (138G→A, 561G→A, 708G→A) and two associated with ITPase deficiency (94C→A, IVS2+21A→C). Homozygotes for the 94C→A missense mutation (Pro32 to Thr) had zero erythrocyte ITPase activity, whereas 94C→A heterozygotes averaged 22.5% of the control mean, a level of activity consistent with impaired subunit association of a dimeric enzyme. ITPase activity of IVS2+21A→C homozygotes averaged 60% of the control mean. In order to explore further the relationship between mutations and enzyme activity, we examined the association between genotype and ITPase activity in 100 healthy controls. Ten subjects were heterozygous for 94C→A (allele frequency: 0.06), 24 were heterozygotes for IVS2+21A→C (allele frequency: 0.13) and two were compound heterozygous for these mutations. The activities of IVS2+21A→C heterozygotes and 94C→A/IVS2+21A→C compound heterozygotes were 60% and 10%, respectively, of the normal control mean, suggesting that the intron mutation affects enzyme activity. In all cases when ITPase activity was below the normal range, one or both mutations were found. The ITPA genotype did not correspond to any identifiable red cell phenotype. A possible relationship between ITPase deficiency and increased drug toxicity of purine analogue drugs is proposed.

Prospective evaluation of the pharmacogenetics of azathioprine in the treatment of inflammatory bowel disease

Background  One‐third of patients with inflammatory bowel disease (IBD) receiving azathioprine (AZA) withdraw treatment due to side effects or lack of clinical response.

Mutation in the ITPA Gene Predicts Intolerance to Azathioprine

Inosine triphosphate pyrophosphatase (ITPase) deficiency occurs with polymorphic frequencies in Caucasians and results in the benign accumulation of the inosine nucleotide ITP. In 62 patients treated with azathioprine for inflammatory bowel disease, the ITPA 94C > A deficiency‐associated allele was significantly associated with adverse drug reactions (OR 4.2, 95% CI 1.6–11.5, p = 0.0034). Significant associations were found for flu‐like symptoms (OR 4.7, 95% CI 1.2–18.1, p = 0.0308), rash (OR 10.3, 95% CI 4.7–62.9, p = 0.0213) and pancreatitis (OR 6.2,CI 1.1–32.6, p = 0.0485). Polymorphism in the ITPA gene thus predicts AZA intolerance. Alternative immunosuppressive drugs, particularly 6‐thioguanine, should be considered for AZA‐intolerant patients with ITPase deficiency.

Genetic determinants of the pre- and post-azathioprine therapy thiopurine methyltransferase activity phenotype

Thiopurine drug therapy has been reported to lead to a variable increase in red cell TPMT activity that may alter effective dose and therapeutic outcome. The aim of this study was to correlate Variable Number Tandem Repeat (VNTR) in the promoter region of the TPMT gene with induction of red cell TPMT activity in patients treated with azathioprine (AZA). In 58 patients, TPMT activity measured at 3 months was not significantly induced on average above pre‐therapy levels. Individual patients showed variation in TPMT activity pre‐ and post‐AZA therapy, however changes in TPMT activity were not predicted by VNTR configuration. In conclusion, TPMT promoter VNTRs are unlikely to play a significant role in changes in TPMT activity in response to AZA therapy.

Purine Nucleoside Phosphorylase Deficiency: A Mutation Update

Purine nucleoside phosphorylase (PNPase) deficiency is an autosomal recessive disorder affecting purine degradation and salvage pathways. Clinically, patients typically present with severe immunodeficiency, neurological dysfunction, and autoimmunity. Biochemically, PNPase deficiency may be suspected in the presence of hypouricemia. We report biochemical and genetic data on a cohort of seven patients from six families identified as PNPase deficient. In all patients, inosine, deoxyinosine, guanosine, and deoxyguanosine were elevated in urine, and mutation analysis revealed seven different mutations of which three were novel. The mutation c.770A>G resulted in the substitution p.His257Arg. A second novel mutation c.257A>G (p.His86Arg) was identified in two siblings and a third novel mutation, c.199C>T (p.Arg67X), was found in a 2-year-old female with delayed motor milestones and recurrent respiratory infections. A review of the literature identified 67 cases of PNPase deficiency from 49 families, including the cases from our own laboratory. PNPase deficiency was confirmed in 30 patients by genotyping and 24 disease causing mutations, including the three novel mutations described in this paper, have been reported to date. In five of the seven patients, plasma uric acid was found to be within the pediatric normal range, suggesting that PNPase deficiency should not be ruled out in the absence of hypouricemia.

Allele frequency of inosine triphosphate pyrophosphatase gene polymorphisms in a Japanese population.

The enzyme inosine triphosphate pyrophosphatase (ITPase) catalyses the pyrophosphohydrolysis of ITP to IMP. ITPase deficiency is a clinically benign autosomal recessive condition characterised by the abnormal accumulation of ITP in erythrocytes. A deficiency of ITPase may predict adverse reactions to therapy with the thiopurine drug 6‐mercaptopurine and its prodrug azathioprine. In this study, we examine the frequencies of ITPA polymorphisms in 100 healthy Japanese individuals. The allele frequency of the 94C > A variant in the Japanese sample was 0.135 (Caucasian allele frequency 0.06). The IV2 + 21A > C polymorphism was not found in Japanese (Caucasian allele frequency 0.130). Allele frequencies of the 138G > A, 561G > A and 708G > A polymorphisms were 0.57, 0.18 and 0.06 respectively in the Japanese population, and with the exception of the 138G > A polymorphism, similar to allele frequencies in Caucasians.

A Biochemical Mechanism for the Role of Allopurinol in TPMT Inhibition

Introduction Hypermethylation of thiopurines has been associated with drug toxicity and non-response to treatment. In such patients the use of low dose thiopurines with concomitant allopurinol is advocated12. Allopurinol is observed to cause a reduction in methylated metabolites of thiopurines; however the biochemical mechanisms remain incompletely understood. Using an intact erythrocyte model we propose a novel pathway of allopurinol mediated thiopurine- S- methyltransferase (TPMT) inhibition, through the production of 2-hydroxy-6-thiopurine (2OH6MP). Methods EDTA whole blood was obtained from healthy volunteers; the plasma and top 5 th were removed and the red cells washed with 0.9% saline. 150 μL of Earl9s balanced salt solution was added to 100 μL of packed red cells. Cells were incubated with 250 μM of 6-mercaptopurine (6-MP) for 0, 2, 4 and 6 h at 37°C. In the second experiment cells were pre-incubated for 2 h with 280 μM of 2OH6MP prior to the addition of 250 μM 6-MP for either 2 or 4 h. At the end of the incubation period, the media was removed and the red cells lysed with 15% perchloric acid after the addition of dithiotreitol. Methylated thiopurine-metabolites were reduced to the base by boiling at 100°C for 1 h. 75 μL of the red cell lysates and supernatant media were separated by reverse phase HPLC to detect the methylated metabolites of 6-MP. Results In packed red cells there was an increase in the concentration of 6-methylmercaptopurine (6-MeMP) detected at each time point. However, the rate of 6-MeMP production remained constant (mean 0.825 pmol L −1 h −1 , SEM ± 0.038). The concentration of 6-MeMP observed in the media was up to 7-fold lower than the concentration in red cells (mean 0.133 pmol L −1 h −1 , SEM ± 0.009). In red cells pre-incubated with 2OH6MP prior to the addition of 6-MP there was a significant reduction in the rate of 6-MeMP production at both 2 (0.878 pmol L −1 h −1 vs 0.135 pmol L −1 h −1 , p −1 h −1 vs 0.096 pmol L −1 h −1 , p Conclusion The data suggests that 6-MP enters red blood cells, where it undergoes methylation to 6-MeMP. The presence of 2OH6MP leads to a reduction in the rate of 6-MeMP production, most likely through direct inhibition of TPMT. We propose that 6-MP undergoes preferential oxidation via aldehyde oxidase, producing 2OH6MP, which leads to feed-back inhibition of TMPT and thereby a reduction in methylated thiopurine-metabolites. This mechanism may explain why patients treated with a combination of thiopurine and allopurinol have dramatically decreased methylated metabolites.

HPRT Deficiency: Identification of Twenty-Four Novel Variants Including an Unusual Deep Intronic Mutation

Hypoxanthine phosphoribosyltranferase (HPRT) deficiency is an X-linked disorder of purine salvage that ranges phenotypically from hyperuricaemia to Lesch–Nyhan Syndrome. Molecular testing is necessary to identify female carriers within families as a prelude to prenatal diagnosis. During the period 1999–2010 the Purine Research Laboratory studied 106 patients from 68 different families. Genomic sequencing revealed mutations in 88% of these families, 24 of which were novel. In eight patients, exon sequencing was not informative. Copy-DNA analysis in one patient revealed an insertion derived from a deep intronic sequence with a genomic mutation flanking this region, resulting in the creation of a false exon. Carrier testing was performed in 21 mothers of affected patients, out of these, 81% (17) were found to be carriers of the disease-associated mutation. Our results confirm the extraordinary variety and complexity of mutations in HPRT deficiency. A combination of genomic and cDNA sequencing may be necessary to define mutations.