Recent advances in understanding and managing adenosine deaminase and purine nucleoside phosphorylase deficiencies.

PURPOSE OF THE REVIEW: To review the recent advances in the understanding and management of the immune and nonimmune effects of inherited adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies. RECENT FINDINGS: Abnormal thymocyte development and peripheral T-cell activation in ADA-deficient and PNP-deficient patients cause increased susceptibility to infections and immune dysregulation. The impaired purine homeostasis also damages many other cell types and tissues. Animal studies suggest that defects in surfactant metabolism by alveolar macrophages cause the pulmonary alveolar proteinosis commonly seen in ADA-deficient infants, while toxicity of purine metabolites to cerebellar Purkinje cells may lead to the ataxia frequently observed in PNP deficiency. Patients' outcome with current treatments including enzyme replacement and stem cell transplantations are inferior to those achieved in most severe immunodeficiency conditions. New strategies, including intracellular enzyme replacement, gene therapy and innovative protocols for stem cell transplantations hold great promise for improved outcomes in ADA and PNP deficiency. Moreover, newborn screening and early diagnosis will allow prompt application of these novel treatment strategies, further improving survival and reducing morbidity. SUMMARY: Better understanding of the complex immune and nonimmune effects of ADA and PNP deficiency holds great promise for improved patients' outcome.

Novel proton MR spectroscopy findings in adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency is a rare inborn error of metabolism resulting in accumulation of metabolites including succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado) in the brain and other tissues. Patients with ADSL have progressive psychomotor retardation, neonatal seizures, global developmental delay, hypotonia, and autistic features, although variable clinical manifestations may make the initial diagnosis challenging. Two cases of the severe form of the disease are reported here: an 18-month-old boy with global developmental delay, intractable neonatal seizures, progressive cerebral atrophy, and marked hypomyelination, and a 3-month-old girl presenting with microcephaly, neonatal seizures, and marked psychomotor retardation. In both patients in vivo proton magnetic resonance spectroscopy (MRS) showed the presence of S-Ado signal at 8.3 ppm, consistent with a prior report. Interestingly, SAICAr signal was also detectable at 7.5 ppm in affected white matter, which has not been reported in vivo before. A novel splice-site mutation, c.IVS12 + 1/G > C, in the ADSL gene was identified in the second patient. Our findings confirm the utility of in vivo proton MRS in suggesting a specific diagnosis of ADSL deficiency, and also demonstrate an additional in vivo resonance (7.5 ppm) of SAICAr in the cases of severe disease.

Establishment of thiopurine S-methyltransferase gene knockdown in jurkat T-lymphocytes: an in vitro model of TPMT polymorphism.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) is an excellent example of an enzyme whose pharmacogenetic polymorphisms affect efficacy and toxicity of a drug. The association between TPMT activity and thiopurine-related myelosuppression is well recognized. To study the significance of TPMT deficiency in thiopurine metabolism and immunosuppressive activity in vitro, we established RNA interference-based TPMT knockdown (kd) in a Jurkat cell line. RESULTS: In Jurkat TPMT kd cells, TPMT expression was reduced to 73% at the RNA level and 83% at the protein level. TPMT kd cells were more sensitive to 6-mercaptopurine (6-MP) (10 µmol/L) and 6-thioguanine (6-TG) (8 µmol/L) than wild-type (wt) cells, (32% versus 20%) and (18% versus 9%), respectively. Both Jurkat wt and kd cells were more sensitive to 6-TG-induced apoptosis than to 6-MP. 6-TG activity was also more affected by TPMT levels than was 6-MP as reflected by IC60, concentrations that is, 6-MP [4.6 µmol/L (wt) and 4.7 µmol/L (kd)], 6-TG [2.7 µmol/L (wt) and 0.8 µmol/L (kd)]. IC60 concentrations induced significant apoptosis in both Jurkat wt and kd cells (257%, versus 314%) with 6-MP and (323% versus 306%) with 6-TG, respectively. At IC60 (6-MP) 6-thioguanine nucleotides (6-TGN) accumulation in cells was 518 versus 447 pmol/million cells in wt and kd cells, respectively. On the other hand 6-TGN accumulation at IC60 (6-TG) was 477 versus 570 pmol/million cells in wt and kd cells, respectively. 6-Methylated mercaptopurine (6-MeMP) concentrations were more affected than 6-TGN by TPMT kd (194 versus 10 pmol/million cells) in wt and kd cells, respectively. CONCLUSION: We conclude that TPMT kd cells are an appropriate in vitro model to investigate the significance of TPMT deficiency with thiopurine therapy and could be helpful in understanding possible clinical consequences of TPMT polymorphism.

Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency.

The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway.

Molecular analysis of X-linked inborn errors of purine metabolism: HPRT1 and PRPS1 mutations.

Mutations of two enzyme genes, HPRT1 encoding hypoxanthine guanine phosphoribosyltransferase (HPRT) and PRPS1 encoding a catalytic subunit (PRS-I) of phosphoribosylpyrophosphate synthetase, cause X-linked inborn errors of purine metabolism. Analyzing these two genes, we have identified three HPRT1 mutations in Lesch-Nyhan families following our last report. One of them, a new mutation involving the deletion of 4224 bp from intron 4 to intron 5 and the insertion of an unknown 28 bp, has been identified. This mutation resulted in an enzyme polypeptide with six amino acids deleted due to abnormal mRNA skipping exon 5. The other HPRT1 mutations, a single base deletion (548delT, 183fs189X), and a point mutation causing a splicing error (532+1G>A, 163fs165X) were detected first in Japanese patients but have been reported in European families. On the other hand, in the analysis of PRPS1, no mutation was identified in any patient.

In vivo proton MR spectroscopy findings specific for adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency is an inherited metabolic disorder affecting predominantly the central nervous system. The disease is characterized by the accumulation of succinylaminoimidazolecarboxamide riboside and succinyladenosine (S-Ado) in tissue and body fluids. Three children presented with muscular hypotonia, psychomotor delay, behavioral abnormalities, and white matter changes on brain MRI. Two of them were affected by seizures. Screening for inborn errors of metabolism including in vitro high resolution proton MRS revealed an ADSL deficiency that was confirmed genetically in all cases. All patients were studied by in vivo proton MRS. In vitro high resolution proton MRS of patient cerebrospinal fluid showed singlet resonances at 8.27 and 8.29 ppm that correspond to accumulated S-Ado. In vivo proton MRS measurements also revealed a prominent signal at 8.3 ppm in gray and white matter brain regions of all patients. The resonance was undetectable in healthy human brain. In vivo proton MRS provides a conclusive finding in ADSL deficiency and represents a reliable noninvasive diagnostic tool for this neurometabolic disorder.

Adenylosuccinate lyase deficiency in a Malaysian patient, with novel adenylosuccinate lyase gene mutations.

Most cases of adenylosuccinate lyase (ADSL OMIM 103050) deficiency reported to date are confined to the various European ethnic groups. We report on the first Malaysian case of ADSL deficiency, which appears also to be the first reported Asian case. The case was diagnosed among a cohort of 450 patients with clinical features of psychomotor retardation, global developmental delay, seizures, microcephaly and/or autistic behaviour. The patient presented with frequent convulsions and severe myoclonic jerk within the first few days of life and severe psychomotor retardation. The high performance liquid chromatography (HPLC) profile of the urine revealed the characteristic biochemical markers of succinyladenosine (S-Ado) and succinyl-aminoimidazole carboximide riboside (SAICAr). The urinary S-Ado/SAICAr ratio was found to be 1.02 (type I ADSL deficiency). The patient was compound heterozygous for two novel mutations, c.445C > G (p.R149G) and c.774_778insG (p.A260GfsX24).

D-ribose therapy in four Polish patients with adenylosuccinate lyase deficiency: absence of positive effect.

Deficiency of adenylosuccinate lyase (ADSL) (OMIM 103050) is an autosomal recessive disorder of the purine de novo synthesis pathway and purine nucleotide cycle, diagnosed so far in approximately 50 patients. The clinical presentation is characterized by severe neurological involvement including hypotonia, seizures, developmental delay and autistic features. Epilepsy in ADSL deficiency is frequent and occurs in approximately two-thirds of patients, beginning either early in the neonatal period or after the first year of life. At present there is no treatment of proven clinical efficacy. Despite of the increasing number of ADSL-deficient patients reported, there are only a few communications of therapeutic considerations or efforts. Among them only two showed some beneficial effects in ADSL-deficient patients. D-ribose, a simple and relatively cheap therapy, has been associated with improvement of behaviour and progressive reduction of the seizure frequency in one 13-year-old patient with ADSL deficiency. In this study we have re-examined D-ribose treatment in four ADSL-deficient patients. Assessments consisted of biochemical markers and neurological outcome. The 12-month trial of D-ribose failed to show any clinical benefit in ADSL patients with both milder and severe phenotype. D-ribose administration was accompanied by neither reduction in seizure frequency nor growth enhancement. Additionally, patients with milder type II presented the first seizure after 4 and 8 months of the D-ribose treatment. Therefore, we could not confirm a positive effect of D-ribose as previously reported.

beta-Ureidopropionase deficiency: an inborn error of pyrimidine degradation associated with neurological abnormalities.

beta-Ureidopropionase deficiency is an inborn error of the pyrimidine degradation pathway, affecting the cleavage of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid. In this study, we report the elucidation of the genetic basis underlying a beta-ureidopropionase deficiency in four patients presenting with neurological abnormalities and strongly elevated levels of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyric acid in plasma, cerebrospinal fluid and urine. No beta-ureidopropionase activity could be detected in a liver biopsy obtained from one of the patients, which reflected the complete absence of the beta-ureidopropionase protein. Analysis of the beta-ureidopropionase gene (UPB1) of these patients revealed the presence of two splice-site mutations (IVS1-2A>G and IVS8-1G>A) and one missense mutation (A85E). Heterologous expression of the mutant enzyme in Escherichia coli showed that the A85E mutation resulted in a mutant beta-ureidopropionase enzyme without residual activity. Our results demonstrate that the N-carbamyl-beta-amino aciduria in these patients is due to a deficiency of beta-ureidopropionase, which is caused by mutations in the UPB1 gene. Furthermore, an altered homeostasis of beta-aminoisobutyric acid and/or increased oxidative stress might contribute to some of the clinical abnormalities encountered in patients with a beta-ureidopropionase deficiency. An analysis of the presence of the two splice site mutations and the missense mutation in 95 controls identified one individual who proved to be heterozygous for the IVS8-1G>A mutation. Thus, a beta-ureidopropionase deficiency might not be as rare as is generally considered.

Mutational analysis of the human dihydropyrimidine dehydrogenase gene by denaturing high-performance liquid chromatography.

Mutations in the DPYD gene, which encodes dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme in the catabolism of pyrimidines, are responsible for an inborn error of metabolism associated with thymine-uraciluria and neurological symptoms. Because the antimetabolite 5-fluorouracil (5-FU) is metabolized by the same enzyme, deficient DPYD alleles may also constitute a risk factor for severe toxicity following treatment with this anticancer drug. The aim of this study was to develop a comprehensive and rapid method to detect sequence variations within the DPYD gene. Using polymerase chain reaction (PCR) amplification and denaturing high-performance liquid chromatography (DHPLC), we established a protocol that makes it possible to screen all 23 exons of the DPYD gene and their exon-intron boundaries for both known and unknown mutations under identical conditions. A novel one-step PCR mutagenesis procedure was developed to generate heterozygous mutant amplicons as positive controls to optimize DHPLC detection of any sequence variation. DHPLC analysis was shown to result in mutation-specific elution profiles and to be able to distinguish different base changes within the same exon or different heterozygous combinations of mutations within the same exon. By analyzing the DPYD gene in 16 affected individuals, a total of 47 base changes were detected, representing eight known mutations and three novel intronic base changes. Sequence analysis confirmed all base changes detected. This method will be useful in identifying patients at risk for toxicity prior to 5-FU treatment, as well as in the analysis of individual patients with thymine-uraciluria.

Screening and diagnosis of beta-ureidopropionase deficiency by gas chromatographic/mass spectrometric analysis of urine.

Dihydropyrimidine dehydrogenase (DHPDase), dihydropyrimidinase (DHPase) and beta-ureidopropionase (betaUPase) are the enzymes that catalyze the first, second, and third steps of the degradation of pyrimidines, respectively. beta-Ureidopropionate (betaUP) and beta-ureidoisobutyrate (betaUIB) are increased in the urine of patients with betaUPase deficiency. The original case in which betaUPase deficiency was discovered by NMR spectroscopy was an 11-month-old patient who presented with hypotonia and dystonic movement. We detected a second but asymptomatic case during a pilot study of neonatal screening with filter-paper urine, urease pretreatment and gas chromatography/mass spectrometry (GC/MS). The urease pretreatment of urine without fractionation resulted in a high recovery of these polar ureide compounds and allowed the highly sensitive GC/MS detection and diagnosis of betaUPase deficiency. betaUP and betaUIB were identified using GC/MS techniques. In the urine of the neonate with betaUPase deficiency, betaUP and betaUIB were persistently increased. Thymine, 5,6-dihydrothymine and 5,6-dihydrouracil were increased only moderately but significantly. It is known that thymine and uracil increase markedly in DHPDase deficiency, and 5,6-dihydrothymine and 5,6-dihydrouracil increase in DHPase deficiency. Therefore, betaUPase deficiency can be differentially diagnosed from the first and second enzyme deficiencies. Application of this specific and sensitive diagnostic procedure will lead to an understanding of the clinical heterogeneity of betaUPase deficiency. Furthermore, the identification of patients with defects in pyrimidine metabolism will enable doctors to avoid cancer chemotherapy with pyrimidine analogues such as 5-fluorouracil, which could be dangerous for these patients.

Capillary electrophoresis for detection of inherited disorders of purine and pyrimidine metabolism: a selective approach.

We developed a capillary electrophoresis method as a diagnostic tool for purine and pyrimidine metabolic disorders. Optimal conditions allowed the separation of the major diagnostic metabolites in urine samples within an analysis time of 10 min and with a separation efficiency of about 350,000 theoretical plates/m. The diagnostically important metabolites (adenine, adenosine, 2-deoxyadenosine, 2-deoxyguanosine, 2,8-dihydroxyadenine, guanosine, hypoxanthine, orotidine, orotic acid, and creatinine) were detectable at concentrations of 1.0-5.7 micromol/L. The method gives a linear calibration curve for tested purine and pyrimidine derivatives within the range of 5-500 micromol/L (r > 0.996) The coefficients of variation for the within- and between-day imprecisions were less than 3.2 and 5.8%, respectively. Characteristic abnormalities were detected in the electropherograms of urine samples from patients with purine and pyrimidine enzyme deficiencies. We provide electrophoretic and spectral characteristics of intermediates in purine and pyrimidine metabolism and possible artifacts from medication and their UV-absorbing compounds. Our method allows the detection of the majority of inborn errors of purine and pyrimidine metabolism.

Identification of a new point mutation in the human xanthine dehydrogenase gene responsible for a case of classical type I xanthinuria.

A 60-year-old Japanese man was diagnosed as having hypouricemia at an annual health check-up. The routine laboratory data was not remarkable except that the patient's hypouricemia and plasma levels of xanthine and hypoxanthine were much higher than those of normal subjects. Furthermore, the patient's daily urinary excretion of xanthine and hypoxanthine was markedly increased compared with reference values. The xanthine dehyrogenase activity of the duodenal mucosa was below the limits of detection. Nevertheless, allopurinol was metabolized to oxypurinol in vivo. Based on these findings, a subtype of classical xanthinuria (type I) was diagnosed. The xanthine dehyrogenase protein was detected by Western blotting analysis. Sequencing of the cDNA of the xanthine dehyrogenase obtained from the duodenal mucosa revealed that a point mutation of C to T had occurred in nucleotide 445. This changed codon 149 from CGC (Arg) to TGC (Cys), a finding that has not been previously reported in patients with classical xanthinuria type I.

A deoxyribonucleotidase in mitochondria: involvement in regulation of dNTP pools and possible link to genetic disease.

Three cytosolic and one plasma membrane-bound 5'-nucleotidases have been cloned and characterized. Their various substrate specificities suggest widely different functions in nucleotide metabolism. We now describe a 5'-nucleotidase in mitochondria. The enzyme, named dNT-2, dephosphorylates specifically the 5'- and 2'(3')-phosphates of uracil and thymine deoxyribonucleotides. The cDNA of human dNT-2 codes for a 25.9-kDa polypeptide with a typical mitochondrial leader peptide, providing the structural basis for two-step processing during import into the mitochondrial matrix. The deduced amino acid sequence is 52% identical to that of a recently described cytosolic deoxyribonucleotidase (dNT-1). The two enzymes share many catalytic properties, but dNT-2 shows a narrower substrate specificity. Mitochondrial localization of dNT-2 was demonstrated by the mitochondrial fluorescence of 293 cells expressing a dNT-2-green fluorescent protein (GFP) fusion protein. 293 cells expressing fusion proteins without leader peptide or with dNT-1 showed a cytosolic fluorescence. During in vitro import into mitochondria, the preprotein lost the leader peptide. We suggest that dNT-2 protects mitochondrial DNA replication from overproduction of dTTP, in particular in resting cells. Mitochondrial toxicity of dTTP can be inferred from a severe inborn error of metabolism in which the loss of thymidine phosphorylase led to dTTP accumulation and aberrant mitochondrial DNA replication. We localized the gene for dNT-2 on chromosome 17p11.2 in the Smith-Magenis syndrome-critical region, raising the possibility that dNT-2 is involved in the etiology of this genetic disease.

Nucleotide pool imbalance and adenosine deaminase deficiency induce alterations of N-region insertions during V(D)J recombination.

Template-independent nucleotide additions (N regions) generated at sites of V(D)J recombination by terminal deoxynucleotidyl transferase (TdT) increase the diversity of antigen receptors. Two inborn errors of purine metabolism, deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP), result in defective lymphoid development and aberrant pools of 2'-deoxynucleotides that are substrates for TdT in lymphoid precursors. We have asked whether selective increases in dATP or dGTP pools result in altered N regions in an extrachromosomal substrate transfected into T-cell or pre-B-cell lines. Exposure of the transfected cells to 2'-deoxyadenosine and an ADA inhibitor increased the dATP pool and resulted in a marked increase in A-T insertions at recombination junctions, with an overall decreased frequency of V(D)J recombination. Sequence analysis of VH-DH-JH junctions from the IgM locus in B-cell lines from ADA-deficient patients demonstrated an increase in A-T insertions equivalent to that found in the transfected cells. In contrast, elevation of dGTP pools, as would occur in PNP deficiency, did not alter the already rich G-C content of N regions. We conclude that the frequency of V(D)J recombination and the composition of N-insertions are influenced by increases in dATP levels, potentially leading to alterations in antigen receptors and aberrant lymphoid development. Alterations in N-region insertions may contribute to the B-cell dysfunction associated with ADA deficiency.

Adenosine deaminase-deficient mice generated using a two-stage genetic engineering strategy exhibit a combined immunodeficiency.

Adenosine deaminase (ADA) deficiency in humans leads to a combined immunodeficiency. The mechanisms involved in the lymphoid specificity of the disease are not fully understood due to the inaccessibility of human tissues for detailed analysis and the absence of an adequate animal model for the disease. We report the use of a two-stage genetic engineering strategy to generate ADA-deficient mice that retain many features associated with ADA deficiency in humans, including a combined immunodeficiency. Severe T and B cell lymphopenia was accompanied by a pronounced accumulation of 2'-deoxyadenosine and dATP in the thymus and spleen, and a marked inhibition of S-adenosylhomocysteine hydrolase in these organs. Accumulation of adenosine was widespread among all tissues examined. ADA-deficient mice also exhibited severe pulmonary insufficiency, bone abnormalities, and kidney pathogenesis. These mice have provided in vivo information into the metabolic basis for the immune phenotype associated with ADA deficiency.