R.A. de Abreu

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.

A point mutation in an invariant splice donor site leads to exon skipping in two unrelated Dutch patients with dihydropyrimidine dehydrogenase deficiency

SummaryDihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive disease characterized by thymine-uraciluria and associated with a variable clinical phenotype. In order to identify the molecular defect underlying complete DPD deficiency in a Dutch patient previously shown to have a 165 base pair deletion in the mature DPD mRNA, we cloned the genomic region encompassing the skipped exon and its flanking intron sequences. Sequence analysis revealed that the patient was homozygous for a single G→A point mutation in the invariant GT dinucleotide splice donor site downstream of the skipped exon. The same mutation was identified in another, unrelated, Dutch patient. Because this mutation destroys a uniqueMaeII restriction site, rapid screening using restriction enzyme cleavage of the amplified genomic region encompassing this mutation is possible. Analysis of 50 controls revealed no individuals heterozygous for this mutation

Dihydropyrimidinase deficiency : structural organization, chromosomal localization, and mutation analysis of the human dihydropyrimidinase gene

Dihydropyrimidinase (DHP) deficiency (MIM 222748) is characterized by dihydropyrimidinuria and is associated with a variable clinical phenotype. This disease might be associated with a risk of 5-fluorouracil toxicity, although no cases have been reported. We present here both the molecular characterization of the human DHP gene and, for the first time, the mutations causing DHP deficiency. The human DHP gene spans >80 kb and consists of 10 exons. It has been assigned to 8q22, by FISH. We performed mutation analysis of genomic DNA in one symptomatic and five asymptomatic individuals presenting with dihydropyrimidinuria. We identified one frameshift mutation and five missense mutations. Two related Japanese adult subjects were homozygous for the Q334R substitution, whereas two other, unrelated Japanese infant subjects were heterozygous for the same mutation, but this mutation is not common in the Japanese population. A Caucasian pediatric patient exhibiting epileptic attacks, dysmorphic features, and severe developmental delay was homozygous for W360R. Using a eukaryotic expression system, we showed that all mutations reduced enzyme activity significantly, indicating that these are crucial DHP deficiency-causing mutations. There was no significant difference, in residual activity, between mutations observed in the symptomatic and those observed in the asymptomatic individuals.

Purine and pyrimidine metabolism in man

The important role of purines and pyrimidines in man is evident. It has been recognized fnr a ltmg time that any disturbance in the synthesis of these building blocks of D N A and RNA, can lead to serious clinical symptoms. Classical in this aspect is the deficiency in hypoxanthine-guanine phosphoribosyltransferase leading to the Lesch-Nyhan syndrome, while some forms of gout are associated with a partial deficiency of this enzyme. Other classical examples of inborn errors of lnetabolism are deficiencies of :ldenosme deaminuse, purinr nucleoside phosphoLylase and 5-nuclentidase, which are associated wuh immunodeficiency. The recognition of these and other rare inborn errors of metabolism has led to new concepts with respect to the regulation of purine and pyrimidine illetaholism. In turn. tills knowledge has resulted in new approaches for the treatment of leukenl[as, other malignancies, und viral infections. More recently, correction of adenosine dc;lnlinase deficiency became o n e of tile first targets for gene therapy. Ftlllowmg the ln[tlU[ design of nlIopurinol f~,u tile treatment of gout, some O[ tile most ~,ucce.,,sfu[ untiltletabolite~, have emerged from the purine and pyrimidine field. These agents include uzuthloprinc (also used R)r gout), 6nlercaptopurhle, lind cytosine ;(rubinosidc. used in the treatment of Icukemi;.l, 5fluoro-urucil in (hut ~,lf stllid tumors, and more recently acyclovir for the treutntcnt of herpes ntfections and Y-uzido-Ydec~xythymidine (AZT) R~r the care of AIDS patient.,. Several of the ntost prln)llslng new anticancer and anti-HIV agents which are contlnl.nlusly intlt*duccci in the c[inic, such as 2-chlorodeoxyadenoxine in the treuunem tit huu)~ cell leukcntkl, find dide~lxyinoxine (ddI) and dMeox_vcytldme (ddC} Ior the ileHtnlent tit AIDS. rentum analogues of niltural purlne fllttl pr This 4th syntpoMuln ill the Etlrcllleiln Sociely hn tile Study Im Purine find Pyrimidine Metabohsm in Man (ESSPPMM} lbcuses on all the above-mentioned aspects of the t~eld, h ha,, been recognized that it is e.,~,ential to have reliable ntcthods Ik)r detection tlf inborn errors of metaholism, hut a thorough understanding of tile bk)chenlicu[ and molecular hum5 is equully necessary both in model systems and even niobe importantly in tile clinic. The program of this meeting reveals the contnming interest in tile hasic and clinical aspects cff purine und pyrinlidine nletuboJisnl. {Hid the uni1 [o transhlte new acquisitions into therapeutic progress.

Hemodynamic and neurohumoral effects of various grades of selective adenosine transport inhibition in humans. Implications for its future role in cardioprotection.

In 12 healthy male volunteers (27-53 yr), a placebo-controlled randomized double blind cross-over trial was performed to study the effect of the intravenous injection of 0.25, 0.5, 1, 2, 4, and 6 mg draflazine (a selective nucleoside transport inhibitor) on hemodynamic and neurohumoral parameters and ex vivo nucleoside transport inhibition. We hypothesized that an intravenous draflazine dosage without effect on hemodynamic and neurohumoral parameters would still be able to augment the forearm vasodilator response to intraarterially infused adenosine. Heart rate (electrocardiography), systolic blood pressure (Dinamap 1846 SX; Critikon, Portanje Electronica BV, Utrecht, The Netherlands) plasma norepinephrine and epinephrine increased dose-dependently and could almost totally be abolished by caffeine pretreatment indicating the involvement of adenosine receptors. Draflazine did not affect forearm blood flow (venous occlusion plethysmography). Intravenous injection of 0.5 mg draflazine did not affect any of the measured hemodynamic parameters but still induced a significant ex vivo nucleoside-transport inhibition of 31.5 +/- 4.1% (P < 0.05 vs placebo). In a subgroup of 10 subjects the brachial artery was cannulated to infuse adenosine (0.15, 0.5, 1.5, 5, 15, and 50 micrograms/100 ml forearm per min) before and after intravenous injection of 0.5 mg draflazine. Forearm blood flow amounted 1.9 +/- 0.3 ml/100 ml forearm per min for placebo and 1.8 +/- 0.2, 2.0 +/- 0.3, 3.8 +/- 0.9, 6.3 +/- 1.2, 11.3 +/- 2.2, and 19.3 +/- 3.9 ml/100 ml forearm per min for the six incremental adenosine dosages, respectively. After the intravenous draflazine infusion, these values were 1.6 +/- 0.2 ml/100 ml forearm per min for placebo and 2.1 +/- 0.3, 3.3 +/- 0.6, 5.8 +/- 1.1, 6.9 +/- 1.4, 14.4 +/- 2.9, and 23.5 +/- 4.0 ml/100 ml forearm per min, respectively (Friedman ANOVA: P < 0.05 before vs after draflazine infusion). In conclusion, a 30-50% inhibition of adenosine transport significantly augments the forearm vasodilator response to adenosine without significant systemic effects. These results suggest that draflazine is a feasible tool to potentiate adenosine-mediated cardioprotection in man.

Purine enzymes in patients with rheumatoid arthritis treated with methotrexate

Objectives: To study (a) purine metabolism during treatment with methotrexate (MTX) in patients with rheumatoid arthritis (RA) and (b) the relation of purine metabolism with efficacy and toxicity of MTX treatment. Methods: One hundred and three patients with active RA who started treatment with MTX were included. The initial MTX dosage was 7.5 mg/week and raised to a maximum of 25 mg weekly if necessary. The purine enzymes 5`-nucleotidase (5`NT), purine-nucleoside-phosphorylase (PNP), hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), and adenosine-deaminase (ADA) were measured before the start, after six weeks, and after 48 weeks or at study withdrawal. The laboratory results were related to measures of efficacy and toxicity of MTX treatment. Results: Baseline values of 5`NT and PNP (16.9 and 206.8 nmol/106 mononuclear cells/h, respectively) were similar to those in former studies. Activities of HGPRT and ADA were relatively low at the start (8.7 and 80.3 nmol/106 mononuclear cells/h, respectively). After six weeks purine enzyme activities showed no important changes from baseline. After 48 weeks of MTX treatment a decrease of the enzyme activities of ADA (−21.6 nmol/106 mononuclear cells/h; 95% CI −28.6 to −14.7), PNP (−78.9 nmol/106 mononuclear cells/h; 95% CI −109.0 to −48.7), and HGPRT (−2.0 nmol/106 mononuclear cells/h; 95% CI −3.1 to −0.9) was found. No association was shown between the enzyme activities of ADA, PNP, and HGPRT, and the efficacy or toxicity of MTX treatment. In contrast, enzyme activity of 5`NT showed a decrease in the subgroup of patients discontinuing MTX treatment because of hepatotoxicity. Conclusion: MTX treatment in patients with RA leads to a significant decrease of the purine enzyme activities of ADA, PNP, and HGPRT that is not related to the anti-inflammatory efficacy or toxicity of MTX. Hepatotoxicity was related to a decrease in the enzyme activity of 5`NT. These changes may be explained by direct or indirect (via purine de novo and salvage metabolism and the homocysteine pathway) effects of MTX.

Dihydropyrimidinase deficiency: Confirmation of the enzyme defect in dihydropyrimidinuria

Dihydropyrimidinase (DHP, EC 3.5.2.2) is the second enzyme in the degradation pathway of uracil and thymine. It catalyses the degradation of both dihydrouracil and dihydrothymine to N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid, respectively. So far, four cases of dihydropyrimidinuria (McKusick 222748) have been reported (Duran et al 1991; Henderson et al 1993; Bakkeren et al, personal communication, 1994; Ohba et al 1994). The patients show a variable clinical phenotype comprising seizures or epileptic attacks (3 out of 4 patients) mental retardation (2 patients), growth retardation (1 patient) and dysmorphic features (1 patient). Since these patients excrete large amounts of dihydrouracil and dihydrothymine and moderate amounts of uracil and thymine in their urine, they can easily be detected (Van Gennip et al 1993). On the basis of the characteristic urinary metabolite profile it is assumed that the disease is caused by a deficiency of DHP. The direct measurement of the activity of DHP in patients has been hampered by the fact that the enzyme is almost exclusively expressed in liver tissue. Here, we provide for the first time direct evidence at the enzyme level for a deficient activity of DHP in liver in a patient with dihydropyrimidinuria.

DNA methylation patterns in the calcitonin gene region at first diagnosis and at relapse of acute lymphoblastic leukemia (ALL)

Aberrant DNA methylation can occur early in neoplastic transformation and may lead to the development of cancer. We describe the alterations of methylation patterns at the DNA sequence level which occurred in the 5′ region of the calcitonin gene in lymphoblasts from 14 pediatric patients with acute lymphoblastic leukemia (ALL). The DNA methylation status of 25 CpG sites was determined by sequence analysis after bisulfite treatment of the DNA. This method showed that 13 out of 14 patients had increased numbers of methylated CpG sites in the calcitonin gene region at initial diagnosis when compared to control DNA from healthy individuals. The 5′ region of the calcitonin gene appears to be methylated to a significantly higher degree in T lineage ALL compared to B lineage ALL (Pu2009<u20090.01). each of six all patients who were investigated at initial diagnosis and at relapse showed alterations in dna methylation between the two stages. these six cases were also investigated by southern blot analysis with methylcytosine-sensitive restriction enzymes and this method showed an increase in dna methylation in only four of the six cases. the dna sequencing method thus appears to be better suited to assess alterations of dna methylation than southern blot analysis. there are marked regional differences in the frequency of methylation of individual cpg sites and in the frequency of alterations between the two stages. our results show that alterations in dna methylation continue to occur from the initial stage to the relapse stage of all, suggesting that aberrant dna methylation may play a role in tumor progression.

Identification of a four-base deletion (delTCAT296-299) in the dihydropyrimidine dehydrogenase gene with variable clinical expression

Abstract Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting step in the breakdown of thymine, uracil, and the widely used antineoplastic drug, 5-fluorouracil. Sequence analysis of the dihydropyrimidine dehydrogenase cDNA in a Dutch consanguineous family identified a novel four-base deletion (delTCAT296–299) leading to premature termination of translation. The deletion is located in a TCAT tandem-repeat sequence and most likely results from unequal crossing-over or slipped mispairing. In this family we identified three homozygous individuals for this mutation. Two of these showed convulsive disorders but one was clinically normal. This observation suggests that, at least in this family, there is no clear correlation between the dihydropyrimidine dehydrogenase genotype and phenotype.

1H NMR spectroscopy of body fluids in patients with inborn errors of purine and pyrimidine metabolism

1H NMR spectroscopy of body fluids has been used in the diagnosis of many inborn errors of metabolism (Lehnert and Hunkler 1986; Iles and Chalmers 1988). To our knowledge there is no systematic study available of body fluids from patients with inborn errors in the purine or pyrimidine metabolism. The main advantage of the technique over others that are used diagnostically in the screening for inborn errors of metabolism is the minimal sample pretreatment required for NMR spectroscopy. Fractionation, extraction or derivatization of metabolites is not required. A further advantage is the overall view of proton-containing metabolites. Quantification of metabolites is possible. An obvious disadvantage is the substantial cost of high-field NMR spectrometers that are required for this work. This paper demonstrates that NMR spectroscopy can be used in diagnosing inborn errors in purine and pyrimidine metabolism. Examples are given of NMR spectra of body fluids from patients with dihydropyrimidine dehydrogenase deficiency (McKusick 274270) and with dihydropyrimidinase deficiency (McKusick 222748).