Distribution of Genetic Polymorphisms of Genes Implicated in Thiopurine Drugs Metabolism.

Thiopurine-S-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) are crucial enzymes involved in the metabolism of thiopurine drugs. Significant interethnic variation in the expression of TPMT and ITPA is caused by single nucleotide polymorphisms of genes encoding these proteins. The aim of this study was to describe the distribution of TPMT and ITPA polymorphisms in healthy Tunisian subjects and to establish the metabolizer status of thiopurine drugs in this population. A total of 309 healthy Tunisian subjects were recruited among blood donors of Fattouma Bourguiba Hospital of Monastir. A written informed consent was obtained from all subjects. Whole blood samples were collected from every subject in ethylenediaminetetraacetic acid tubes. TPMT (c.238 G > C, c.460 G > A and c.719A > G) and ITPA (c.94C > A and IVS2+21A > C) mutations were genotyped using polymerase chain reaction-restriction fragment length polymorphism. The observed frequencies of TPMT*3A and TPMT*3C alleles were both 0.8%. The phenotype distribution of TPMT was bimodal: 96.8% of subjects were extensive metabolizers and 3.2% were intermediate metabolizers. Genotyping of ITPA revealed frequencies of 9% and 3% for IVS2+21A > C and c.94C > A mutations, respectively. Accordingly, a trimodal phenotype distribution was found: 75.4% of the subjects were extensive metabolizers, 23.4% were intermediate metabolizers, and 1.2% wereslow metabolizers. Combination of TPMT and ITPA genotyping has revealed that a quarter of the Tunisian Population carries polymorphisms that reduce the metabolic activities of these enzymes.

Thiopurine S-methyltransferase testing for averting drug toxicity: a meta-analysis of diagnostic test accuracy.

Thiopurine S-methyltransferase (TPMT) deficiency increases the risk of serious adverse events in persons receiving thiopurines. The objective was to synthesize reported sensitivity and specificity of TPMT phenotyping and genotyping using a latent class hierarchical summary receiver operating characteristic meta-analysis. In 27 studies, pooled sensitivity and specificity of phenotyping for deficient individuals was 75.9% (95% credible interval (CrI), 58.3-87.0%) and 98.9% (96.3-100%), respectively. For genotype tests evaluating TPMT*2 and TPMT*3, sensitivity and specificity was 90.4% (79.1-99.4%) and 100.0% (99.9-100%), respectively. For individuals with deficient or intermediate activity, phenotype sensitivity and specificity was 91.3% (86.4-95.5%) and 92.6% (86.5-96.6%), respectively. For genotype tests evaluating TPMT*2 and TPMT*3, sensitivity and specificity was 88.9% (81.6-97.5%) and 99.2% (98.4-99.9%), respectively. Genotyping has higher sensitivity as long as TPMT*2 and TPMT*3 are tested. Both approaches display high specificity. Latent class meta-analysis is a useful method for synthesizing diagnostic test performance data for clinical practice guidelines.The Pharmacogenomics Journal advance online publication, 24 May 2016; doi:10.1038/tpj.2016.37.

Adenylosuccinate lyase deficiency.

Adenylosuccinate lyase ADSL) deficiency is a defect of purine metabolism affecting purinosome assembly and reducing metabolite fluxes through purine de novo synthesis and purine nucleotide recycling pathways. Biochemically this defect manifests by the presence in the biologic fluids of two dephosphorylated substrates of ADSL enzyme: succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (S-Ado). More than 80 individuals with ADSL deficiency have been identified, but incidence of the disease remains unknown. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The fatal neonatal form has onset from birth and presents with fatal neonatal encephalopathy with a lack of spontaneous movement, respiratory failure, and intractable seizures resulting in early death within the first weeks of life. Patients with type I (severe form) present with a purely neurologic clinical picture characterized by severe psychomotor retardation, microcephaly, early onset of seizures, and autistic features. A more slowly progressing form has also been described (type II, moderate or mild form), as having later onset, usually within the first years of life, slight to moderate psychomotor retardation and transient contact disturbances. Diagnosis is facilitated by demonstration of SAICAr and S-Ado in extracellular fluids such as plasma, cerebrospinal fluid and/or followed by genomic and/or cDNA sequencing and characterization of mutant proteins. Over 50 ADSL mutations have been identified and their effects on protein biogenesis, structural stability and activity as well as on purinosome assembly were characterized. To date there is no specific and effective therapy for ADSL deficiency.

Thirteen years experience with selective screening for disorders in purine and pyrimidine metabolism.

Purine and pyrimidine disorders represent a heterogeneous group with variable clinical symptoms and low prevalence rate. In the last thirteen years, we have studied urine/plasma specimens from about 1600 patients and we have identified 35 patients: eight patients with adenylosuccinate lyase deficiency, eight patients with hypoxanthine-guanine phosphoribosyltransferase deficiency, one patient with purine nucleoside phosphorylase deficiency, ten patients with xanthine dehydrogenase deficiency, six patients with molybdenum cofactor deficiency and two patients with dihydropyrimidine dehydrogenase deficiency. Despite low incidence of these diseases, our findings highlight the importance of including the purine and pyrimidine analysis in the selective screening for inborn errors of metabolism in specialized laboratories, where amino acid and organic acid disorders are simultaneously investigated.

Association of thiopurine methyltransferase status with azathioprine side effects in Chinese patients with systemic lupus erythematosus.

Azathioprine (AZA) is indicated for the treatment of systemic lupus erythematosus (SLE). Thiopurine methyltransferase (TPMT) is the rate-limiting enzyme in the steps of AZA metabolization. Heritable deficiency of TPMT enzyme activity and polymorphisms may lead to leukopenia. This study aims to detect TPMT polymorphisms and TPMT enzyme activity in Chinese SLE patients and to describe the association between TPMT genotypes and adverse effects of AZA. One hundred and twenty-six SLE patients with present or previous thiopurine therapy were identified from a local database. Adverse effects were documented. No TPMT*2, TPMT*3A, or TPMT*3B mutant alleles were detected. TPMT*3C was detected in four patients (3.17 %). The heterozygotes had significantly lower mean TPMT activity as compared to the homozygotes (2.38 ± 1.24 vs. 12.56 ± 7.02 U/mL, P < 0.001). Twenty-seven cases (21.42 %) exhibited adverse effects. All of the heterozygotes (4/4, 100 %) developed severe leukopenia, and three cases (3/4, 75 %) of whom exhibited alopecia simultaneously. The specificity of TPMT*3C for predicting leukopenia and alopecia was 100 and 99.17 %, respectively, and the sensitivity was 28.57 and 60.00 %, respectively. The mean value of TPMT activity with leukopenia (4.67 ± 3.01 vs. 13.2 ± 6.94 U/mL RBC, P < 0.001) or alopecia (2.31 ± 1.16 vs. 12.65 ± 6.98 U/mL RBC, P < 0.001) was significantly lower than those without. TPMT*3C was the most common mutant polymorphism found in the study group. TPMT activity is reduced in TPMT*3C mutant. AZA-induced leukopenia and alopecia were partly correlated to TPMT*3C heterozygotes and low TPMT activity. The results of this study suggest that the value of TPMT genotyping before AZA therapy was limited in Chinese SLE patients, considering the low sensitivity. Routine monitoring of TPMT activity before prescribing and continuous hematological monitoring dose were recommended.

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.

Mutations in ALDH6A1 encoding methylmalonate semialdehyde dehydrogenase are associated with dysmyelination and transient methylmalonic aciduria.

BACKGROUND: Methylmalonate semialdehyde dehydrogenase (MMSDH) deficiency is a rare autosomal recessive disorder with varied metabolite abnormalities, including accumulation of 3-hydroxyisobutyric, 3-hydroxypropionic, 3-aminoisobutyric and methylmalonic acids, as well as ß-alanine. Existing reports describe a highly variable clinical and biochemical phenotype, which can make diagnosis a challenge. To date, only three reported cases have been confirmed at the molecular level, through identification of homozygous mutations in ALDH6A1, the gene encoding MMSDH. Confirmation by enzyme assay has until now not been possible, due to the extreme instability of the enzyme substrate. METHODS AND RESULTS: We report a child with severe developmental delays, abnormal myelination on brain MRI, and transient/variable elevations in lactate, methylmalonic acid, 3-hydroxyisobutyric and 3-aminoisobutyric acids. Compound heterozygous mutations were identified by exome sequencing and confirmed by Sanger sequencing within exon 6 (c.514 T > C; p. Tyr172His) and exon 12 (c.1603C > T; p. Arg535Cys) of ALDH6A1. The resulting amino acid changes, both occurring in residues conserved among mammals, are predicted to be damaging at the protein level. Subsequent MMSDH enzyme assay demonstrated reduced activity in patient fibroblasts, measuring 2.5 standard deviations below the mean. CONCLUSIONS: We present the fourth reported case of MMSDH deficiency with confirmation at the molecular level, and expand on what is already an extremely variable clinical and biochemical phenotype. Furthermore, this is the first report to demonstrate a corresponding reduction in MMSDH enzyme activity. This report illustrates the emerging utilization of whole exome sequencing and variant data filtering using clinical data as an early tool in the diagnosis of rare and variable conditions.

[Purine nucleoside phosphorylase].

Purine nucleoside phosphorylase (PNP) is one of the most important enzymes of the purine metabolism, wich promotes the recycling of purine bases. Nowadays is the actual to search for effective inhibitors of this enzyme which is necessary for creation T-cell immunodeficient status of the organism in the organs and tissues transplantation, and chemotherapy of a number pathologies as well. For their successful practical application necessary to conduct in-depth and comprehensive study of the enzyme, namely a structure, functions, and an affinity of the reaction mechanism. In the review the contemporary achievements in the study of PNP from various biological objects are presented. New data describing the structure of PNP are summarised and analysed. The physiological role of the enzyme is discussed. The enzyme basic reaction mechanisms and actions are considered. The studies on enzyme physicochemical, kinetic, and catalytic research are presented.

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.

Errores innatos del metabolismo de las purinas y otras enfermedades relacionadas / Inborn purine metabolism errors and other related diseases

Los errores innatos en el metabolismo de las purinas son trastornos hereditarios complejos de gran impacto clínico, que presentan síntomas variables de acuerdo con el tipo de enfermedad. Pueden presentarse problemas renales de origen desconocido, retardo mental con manifestaciones neurológicas, retardo del crecimiento, infecciones recurrentes, automutilación, inmunodeficiencias, anemia hemolítica inexplicable, artritis gotosa, historia familiar, consanguinidad y reacciones adversas a fármacos que son análogos de las purinas. Las investigaciones de estas enfermedades comienzan generalmente con la cuantificación del ácido úrico en suero y en orina, por ser el producto final del metabolismo de las purinas en humanos. La dieta y el consumo de medicamentos, entre otras condiciones patológicas, fisiológicas y clínicas, también pueden modificar los niveles de este compuesto. Esta revisión pretende divulgar información de los errores innatos en el metabolismo de las purinas, y facilitar la interpretación de los niveles del ácido úrico y otros marcadores bioquímicos útiles en el diagnóstico de estas enfermedades. Se incluyen tablas que relacionan estas enfermedades con los niveles de excreción de ácido úrico y otros marcadores bioquímicos, las enzimas alteradas, los síntomas clínicos, el modo de herencia y, en algunos casos, el tratamiento propuesto. Este trabajo nos permite afirmar que las variaciones en los niveles del ácido úrico y la presencia de otros marcadores bioquímicos en orina, constituyen una herramienta importante en la pesquisa de algunos errores innatos en el metabolismo de las purinas, así como de otras condiciones patológicas relacionadas(AU)

Inborn purine metabolism errors are complex inherited disorders of great clinical impact that present with variable symptoms according to the type of disease. It might occur renal problems of unknown origin, metal retardation with neurological manifestations, retarded growth, recurrent infections, self-mutilation, immunodeficiencies, unexplainable haemolytic anemia, gout-related arthritis, family history, consanguinity and adverse reactions to those drugs that are analogous of purines. The study of these diseases generally begins by quantifying serum uric acid and uric acid present in the urine which is the final product of purine metabolism in human beings. Diet and drug consumption are among the pathological, physiological and clinical conditions capable of changing the level of this compound. This review was intended to disseminate information on the inborn purine metabolism errors as well as to facilitate the interpretation of the uric acid levels and other biochemical markers making the diagnosis of these diseases possible. The tables relating these diseases to the excretory levels of uric acid and other biochemical markers, the altered enzymes, the clinical symptoms, the model of inheritance, and in some cases, the suggested treatment. This paper allowed us to affirm that variations in the uric acid levels and the presence of other biochemical markers in urine are important tools in screening some inborn purine metabolism errors, and also other related pathological conditions(AU)

ß-ureidopropionase deficiency: phenotype, genotype and protein structural consequences in 16 patients.

ß-ureidopropionase is the third enzyme of the pyrimidine degradation pathway and catalyzes the conversion of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid to ß-alanine and ß-aminoisobutyric acid, ammonia and CO(2). To date, only five genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 11 newly identified ß-ureidopropionase deficient patients as well as the analysis of the mutations in a three-dimensional framework. Patients presented mainly with neurological abnormalities (intellectual disabilities, seizures, abnormal tonus regulation, microcephaly, and malformations on neuro-imaging) and markedly elevated levels of N-carbamyl-ß-alanine and N-carbamyl-ß-aminoisobutyric acid in urine and plasma. Analysis of UPB1, encoding ß-ureidopropionase, showed 6 novel missense mutations and one novel splice-site mutation. Heterologous expression of the 6 mutant enzymes in Escherichia coli showed that all mutations yielded mutant ß-ureidopropionase proteins with significantly decreased activity. Analysis of a homology model of human ß-ureidopropionase generated using the crystal structure of the enzyme from Drosophila melanogaster indicated that the point mutations p.G235R, p.R236W and p.S264R lead to amino acid exchanges in the active site and therefore affect substrate binding and catalysis. The mutations L13S, R326Q and T359M resulted most likely in folding defects and oligomer assembly impairment. Two mutations were identified in several unrelated ß-ureidopropionase patients, indicating that ß-ureidopropionase deficiency may be more common than anticipated.

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.

Inborn errors in purine metabolism: role of 5'-nucleotidases and their involvement in the etiology of neurological impairments.

A number of scientists have been involved for decades in the study of nucleotide metabolism in different species of living beings. We are, therefore, aware of the relevant roles of purine compounds and of the many different ways in which these compounds influence cell life, acting both inside and outside the cells. Nevertheless, the consequences of an alteration (lack of expression, or hypo- or hyperexpression) in the activity of enzymes involved in the metabolism of these compounds are sometimes surprising, and far from being mechanistically explained. Alterations in enzyme activities involved in nucleotide metabolism are frequently associated with syndromes characterized by two different types of problems--one, metabolic, which is expected and can be easily explained, and the second, neurological and behavioral. Neurological and behavioral impairments are more difficult to explain and show a very high degree of individual variability. The molecular bases of the neurological impairment linked to purine metabolism disorders have been extensively studied. These studies have generated a lot of hypotheses but very few certainties. In this short review, neurological and behavioral symptoms linked to the dysfunction of some enzymes involved in purine synthesis, catabolism, and salvage will be briefly described, with particular attention to their metabolic and regulatory consequences. Finally, attention will be focused on the 5'-nucleotidase family members and on their involvement in the regulation of purine and pyrimidine metabolism.

An HPLC-based assay of adenylosuccinate lyase in erythrocytes.

ADSL deficiency is a disorder of purine metabolism with a broad clinical spectrum. A rapid and simple HPLC-based assay to measure ADSL activity in erythrocytes was developed. The suitability of DBSs was assessed. ADSL activity was measured in erythrocyte lysates and DBS using succinyl-AMP as the substrate. Detection and quantification were performed using isocratic ion-pairing reversed-phase HPLC with UV-detection. Reference values in erythrocyte lysates were established. The intra- and interassay variations were 2% and 8%, respectively. ADSL deficiency was easily recognized. ADSL activity in DBS was highly unstable, disqualifying DBS for diagnostic procedures.

HPLC analysis for the clinical-biochemical diagnosis of inborn errors of metabolism of purines and pyrimidines.

The determination of purines and pyrimidines in biofluids is useful for the clinical-biochemical characterization of acute and chronic pathological states that induce transient or permanent alterations of metabolism. In particular, the diagnosis of several inborn errors of metabolism (IEMs) is accomplished by the analysis of circulating and excreted purines and pyrimidines. It is certainly advantageous to simultaneously determine the full purine and pyrimidine profile, as well as to quantify other compounds of relevance (e.g., organic acids, amino acids, sugars) in various metabolic hereditary diseases, in order to screen for a large number of IEMs using a reliable and sensitive analytical method characterized by mild to moderate costs. Toward this end, we have developed an ion-pairing HPLC method with diode array detection for the synchronous separation of several purines and pyrimidines. This method also allows the quantification of additional compounds such as N-acetylated amino acids and dicarboxylic acids, the concentrations of which are profoundly altered in different IEMs. The application of the method in the analysis of biological samples from patients with suspected purine and pyrimidine disorders is presented to illustrate its applicability for the clinical-biochemical diagnosis of IEM.

Molecular characterization of the AdeI mutant of Chinese hamster ovary cells: a cellular model of adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL, E. C. 4.3.2.2) carries out two non-sequential steps in de novo AMP synthesis, the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) to aminoimidazolecarboxamide ribotide (AICAR) and the conversion of succinyl AMP (AMPS) to AMP. In humans, mutations in ADSL lead to an inborn error of metabolism originally characterized by developmental delay, often with autistic features. There is no effective treatment for ADSL deficiency. Hypotheses regarding the pathogenesis include toxicity of high levels of SAICAR, AMPS, or their metabolites, deficiency of the de novo purine biosynthetic pathway, or lack of a completely functional purine cycle in muscle and brain. One important approach to understand ADSL deficiency is to develop cell culture models that allow investigation of the properties of ADSL mutants and the consequences of ADSL deficiency at the cellular level. We previously reported the isolation and initial characterization of mutants of Chinese hamster ovary (CHO-K1) cells (AdeI) that lack detectable ADSL activity, accumulate SAICAR and AMPS, and require adenine for growth. Here we report the cDNA sequences of ADSL from CHO-K1 and AdeI cells and describe a mutation resulting in an alanine to valine amino acid substitution at position 291 (A291V) in AdeI ADSL. This substitution lies in the "signature sequence" of ADSL, inactivates the enzyme, and validates AdeI as a cellular model of ADSL deficiency.