Succinylaminoimidazole Carboxamide Riboside Research Articles

Identification of pathognomonic purine synthesis biomarkers by metabolomic profiling of adolescents with obesity and type 2 diabetes

The incidence of type 2 diabetes is increasing more rapidly in adolescents than in any other age group. We identified and compared metabolite signatures in obese children with type 2 diabetes (T2D), obese children without diabetes (OB), and healthy, age- and gender-matched normal weight controls (NW) by measuring 273 analytes in fasting plasma and 24-hour urine samples from 90 subjects by targeted LC-MS/MS. Diabetic subjects were within 2 years of diagnosis in an attempt to capture early-stage disease prior to declining renal function. We found 22 urine metabolites that were uniquely associated with T2D when compared to OB and NW groups. The metabolites most significantly elevated in T2D youth included members of the betaine pathway, nucleic acid metabolism, and branched-chain amino acids (BCAAs) and their catabolites. Notably, the metabolite pattern in OB and T2D groups differed between urine and plasma, suggesting that urinary BCAAs and their intermediates behaved as a more specific biomarker for T2D, while plasma BCAAs associated with the obese, insulin resistant state independent of diabetes status. Correlative analysis of metabolites in the T2D signature indicated that betaine metabolites, BCAAs, and aromatic amino acids were associated with hyperglycemia, but BCAA acylglycine derivatives and nucleic acid metabolites were linked to insulin resistance. Of major interest, we found that urine levels of succinylaminoimidazole carboxamide riboside (SAICA-riboside) were increased in diabetic youth, identifying urine SAICA-riboside as a potential biomarker for T2D.

Identification of pathognomonic purine synthesis biomarkers by metabolomic profiling of adolescents with obesity and type 2 diabetes.

The incidence of type 2 diabetes is increasing more rapidly in adolescents than in any other age group. We identified and compared metabolite signatures in obese children with type 2 diabetes (T2D), obese children without diabetes (OB), and healthy, age- and gender-matched normal weight controls (NW) by measuring 273 analytes in fasting plasma and 24-hour urine samples from 90 subjects by targeted LC-MS/MS. Diabetic subjects were within 2 years of diagnosis in an attempt to capture early-stage disease prior to declining renal function. We found 22 urine metabolites that were uniquely associated with T2D when compared to OB and NW groups. The metabolites most significantly elevated in T2D youth included members of the betaine pathway, nucleic acid metabolism, and branched-chain amino acids (BCAAs) and their catabolites. Notably, the metabolite pattern in OB and T2D groups differed between urine and plasma, suggesting that urinary BCAAs and their intermediates behaved as a more specific biomarker for T2D, while plasma BCAAs associated with the obese, insulin resistant state independent of diabetes status. Correlative analysis of metabolites in the T2D signature indicated that betaine metabolites, BCAAs, and aromatic amino acids were associated with hyperglycemia, but BCAA acylglycine derivatives and nucleic acid metabolites were linked to insulin resistance. Of major interest, we found that urine levels of succinylaminoimidazole carboxamide riboside (SAICA-riboside) were increased in diabetic youth, identifying urine SAICA-riboside as a potential biomarker for T2D.

A mild form of adenylosuccinate lyase deficiency in absence of typical brain MRI features diagnosed by whole exome sequencing

BackgroundAdenylosuccinate 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. The disorder shows a wide spectrum of symptoms from slowly to rapidly progressing forms. The most severe form is characterized by neonatal encephalopathy, absence of spontaneous movement, respiratory failure, intractable seizures, and early death within the first weeks of life. More commonly, ADSL presents purely neurologic clinical picture characterized by severe psychomotor retardation, microcephaly, early onset of seizures, and autistic features (type I) or a more slowly progressing form with later onset, and major features including slight to moderate psychomotor retardation, and transient contact disturbances (type II). Diagnostic markers are the presence of succinylaminoimidazole carboxamide riboside (SAICAr) and succinyladenosine (SAdo) in extracellular fluids. ADSL is a rare disorder, although its prevalence remains unknown. Of note, the wide range of essentially nonspecific manifestations and lack of awareness of the condition often prevent diagnosis.Case presentationWe present here the case of particularly mild, late onset ADSL that has been unsuccessfully investigated until whole exome sequencing (WES) was performed.ConclusionsBesides emphasizing the valuable diagnostic value of WES, this report provides new data further documenting the relatively wide clinical manifestation of ADSL.

Screening for adenylosuccinate lyase deficiency using tandem mass spectrometry analysis of succinylpurines in neonatal dried blood spots

ObjectivesStable isotope dilution coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS) is the sensitive method for screening for various inherited metabolic disorders using dried blood spots (DBSs). We present a method for LC–MS/MS determination of succinyladenosine (SAdo) and succinylaminoimidazole carboxamide riboside (SAICAr), biomarkers for adenylosuccinate lyase deficiency (dADSL), in DBS. Design and methodsSAICAr and SAdo were separated on a Symmetry-C18 column and detected using positive electrospray ionisation in selected reaction monitoring mode. The quantification was performed using the isotopically labelled internal standards SAdo-13C4 and SAICAr-13C4, which were prepared via ADSL-catalysed reactions of fumarate-13C4 with adenosine monophosphate and aminoimidazole carboxamide ribotide, respectively, and subsequent alkaline phosphatase-catalysed dephosphorylation of the resulting products. ResultsThe detection of SAICAr and SAdo in DBS was linear over the range of 0–25μmol/L. The respective intra-assay and inter-assay imprecision values were less than 10.7% and 15.2% for SAICAr and 4.7% and 5.7% for SAdo. The recoveries from DBS spiked with different concentrations of SAICAr and SAdo were between 94% and 117%. The concentrations of SAICAr and SAdo were higher in the archived DBS from dADSL patients (SAICAr, 0.03–4.7μmol/L; SAdo, 1.5–21.3μmol/L; n=5) compared to those of the control subjects (SAICAr, 0–0.026μmol/L; SAdo, 0.06–0.14μmol/L; n=31), even after DBSs from dADSL patients were stored for 2–23years. ConclusionsWe developed and validated a method of succinylpurine analysis in DBS that improves selective screening for dADSL in the paediatric population and may be used for retrospective diagnosis to aid the genetic counselling of affected families.

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.

The need for vigilance: False-negative screening for adenylosuccinate lyase deficiency caused by deribosylation of urinary biomarkers

ObjectivesAdenylosuccinate lyase deficiency (dADSL) is a rare inherited metabolic disorder. Biochemical diagnosis of the disease is based on the determination of enormously elevated urinary levels of succinylaminoimidazole carboxamide riboside (SAICA-riboside) and succinyladenosine (SAdo). We report a case of false negative screening for dADSL caused by deribosylation of the urinary biomarkers SAICA-riboside and SAdo. Design and methodsA thin-layer chromatography (TLC) method with Pauly reagent detection of SAICA-riboside was used as a screening method. High-performance liquid chromatography with diode-array detection (HPLC-DAD) and LC–MS/MS methods were used for the identification and quantitative determination of SAICA-riboside, SAdo, succinylaminoimidazole carboxamide (SAICA) and succinyladenine (SA). ResultsFollowing a negative TLC screening in a known case of dADSL, we analyzed urine using HPLC-DAD. The concentration of SAICA-riboside was 2.7mmol/mol creatinine (below the TLC detection limit), and we detected the two abnormal metabolites identified by LC–MS/MS as SAICA and SA. We showed that SAICA and SA were produced by deribosylation of SAICA-riboside and SAdo in the patient's urine. Studies performed by monitoring the production of SAICA and SA after the addition of SAICA-riboside and SAdo to the patient's urine and to urine samples from patients with urinary tract infections suggested that deribosylation is facilitated by bacterial enzymes. ConclusionsScreening methods for the diagnosis of dADSL may be falsely negative due to bacteria-mediated deribosylation of SAICA-riboside and SAdo. HPLC-DAD or LC–MS/MS analyses allowing for simultaneous detection of SAICA-riboside, SAdo and their deribosylation products SAICA and SA should be preferentially used for the diagnosis of dADSL in urine.

Inherent properties of adenylosuccinate lyase could explain S-Ado/SAICAr ratio due to homozygous R426H and R303C mutations

Adenylosuccinate lyase (ADSL) is a homotetrameric enzyme involved in the de novo purine biosynthesis pathway and purine nucleotide cycle. Missense mutations in the protein lead to ADSL deficiency, an inborn error of purine metabolism characterized by neurological and physiological symptoms. ADSL deficiency is biochemically diagnosed by elevated levels of succinylaminoimidazolecarboxamide riboside (SAICAr) and succinyladenosine (S-Ado), the dephosphorylated derivatives of the substrates. S-Ado/SAICAr ratios have been associated with three phenotypic groups. Different hypotheses to explain these ratios have been proposed. Recent studies have focused on measuring activity on the substrates independently. However, it is important to examine mixtures of the substrates to determine if mutations affect enzyme activity on both substrates similarly in these conditions. The two substrates may experience an indirect communication due to being acted upon by the same enzyme, altering their activities from the non-competitive case. In this study, we investigate this hidden coupling between the two substrates. We chose two mutations that represent extremes of the phenotype, R426H and R303C. We describe a novel electrochemical-detection method of measuring the kinetic activity of ADSL in solution with its two substrates at varying concentration ratios. Furthermore, we develop an enzyme kinetic model to predict substrate activity from a given ratio of substrate concentrations. Our findings indicate a non-linear dependence of the activities on the substrate ratios due to competitive binding, distinct differences in the behaviors of the different mutations, and S-Ado/SAICAr ratios in patients could be explained by inherent properties of the mutant enzyme.

Attenuated Adenylosuccinate Lyase Deficiency: A Report of One Case and a Review of the Literature

We present a 9-year follow-up of a patient with an attenuated (type II) adenylosuccinate lyase deficiency with no obvious signs of disease progression and degradation. We also review the literature, focusing on attenuated phenotype, and we report a positive effect of a ketogenic diet on seizure control. The patient presented at the age of 5 months with a history of global developmental delay. Screening of urinary purine metabolites revealed elevation of succinyladenosine and succinylaminoimidazolecarboxamide riboside (a ratio of 2:1). Mutation analysis revealed a compound heterozygosity for missense mutations: p.R426H and p.D268H. She began to walk independently at the age of 3 years. From the age of 4 years, her communication skills improved and she presented fewer autistic features. Due to poor results in seizure control, the ketogenic diet was introduced at the age of 7 years, resulting in reduction of seizure frequency. Currently, at the age of 9 years, the girl is attending a special kindergarten and is functioning very well in her preschool group. She began to make statements that form a logical continuity and make progress in simple manual operations. The patient participates in therapies such as pet therapy, hippotherapy, speech therapy, physiotherapy, hydrotherapy, and music therapy.

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.

Isolated Neonatal Seizures: Adenylosuccinase Deficiency is Another Cause!

I read with great interest the very useful review “Isolated Neonatal Seizures: When to Suspect Inborn Errors of Metabolism” by Ficicioglu and Bearden [ [1] Ficicioglu C. Bearden D. Isolated neonatal seizures: When to suspect inborn errors of metabolism. Pediatr Neurol. 2011; 45: 283-291 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar ]. I would like to add adenylosuccinase deficiency to their causes for suspicion. This genetic, mainly neurologic disorder of the de novo purine synthesis was first reported in 1984 [ [2] Jaeken J. van den Berghe G. An infantile autistic syndrome characterized by the presence of succinylpurines in body fluids. Lancet. 1984; 2: 1058-1061 PubMed Google Scholar ]. Most of the approximately 60 reported patients demonstrate variable associations of moderate to severe psychomotor retardation, epilepsy after their first years, autistic features, and growth retardation [ [3] van den Berghe G. Vincent M.-F. Marie S. Disorders of purine and pyrimidine metabolism. in: Saudubray J.-M. van den Berghe G. Walter J.H. Inborn metabolic diseases: Diagnosis and treatment. 5th ed. Springer-Verlag, Berlin2012: 499-518 Crossref Scopus (11) Google Scholar ]. However, a subgroup of patients presents with intractable seizures beginning within the first days or weeks of age [ [4] Mouchegh K. Zikánová M. Hoffmann G.F. et al. Lethal fetal and early neonatal presentation of adenylosuccinate lyase deficiency: Observation of 6 patients in 4 families. J Pediatr. 2007; 150: 57-61 Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar ]. Often severe hypotonia is associated such as seen, e.g., in nonketotic hyperglycinemia and γ-aminobutyric acid transaminase deficiency, as mentioned by Ficicioglu and Bearden [ [1] Ficicioglu C. Bearden D. Isolated neonatal seizures: When to suspect inborn errors of metabolism. Pediatr Neurol. 2011; 45: 283-291 Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar ]. These seizures can lead to death or severe mental retardation. In some patients, intrauterine growth retardation, microcephaly, fetal hypokinesia, and a lack of fetal heart rate variability have also been observed. Adenylosuccinase catalyzes two steps in purine synthesis: the conversion of succinyl aminoimidazole carboxamide ribotide to aminoimidazole carboxamide ribotide (in the de novo pathway), and the conversion of adenylosuccinate to adenosine monophosphate. Its deficiency results in the accumulation in body fluids of succinyl purines, i.e., succinyl aminoimidazole carboxamide riboside and succinyl adenosine (the dephosphorylated substrates of the enzyme). A modified Bratton-Marshall (colorimetric) test performed on urine can be used as a screening test, but some medications such as sulfonamides cause a false-positive result. A final diagnosis requires high-pressure liquid chromatography with ultraviolet light detection, or high-pressure liquid chromatography with electrospray ionization tandem mass spectroscopy. Isolated Neonatal Seizures: When to Suspect Inborn Errors of MetabolismPediatric NeurologyVol. 45Issue 5PreviewNeonatal seizures are common, and often comprise the first clinical indicator of central nervous system dysfunction. Although most neonatal seizures are secondary to processes such as hypoxic-ischemic injury, infection, or cortical malformations (which are readily identifiable through routine testing and imaging), seizures secondary to inborn errors of metabolism can be much more difficult to diagnose, and thus a high index of suspicion is required. The early diagnosis of inborn errors of metabolism is crucial, considering that many can receive effective treatment (e.g., dietary supplementation or restriction) with favorable long-term outcomes. Full-Text PDF ResponsePediatric NeurologyVol. 47Issue 3PreviewWe thank Dr. Jaaken for his insightful discussion of adenylosuccinase deficiency as a cause of neonatal seizures. Our review was intended to offer an overview of metabolic disorders presenting with isolated neonatal seizures, i.e., neonatal seizures not associated with other characteristic abnormalities. Dr. Jaaken is correct in observing that neonatal seizures may constitute an atypical presentation of this rare disease, with six cases reported in the literature [1]. However, the severe form of adenylosuccinase deficiency that presents with neonatal seizures is often accompanied by severe hypotonia, intrauterine growth retardation, and magnetic resonance imaging abnormalities that are variable but that generally include dysmyelination [2]. Full-Text PDF

Novel features in the evolution of adenylosuccinate lyase deficiency

BackgroundAdenylosuccinate lyase (ADSL) deficiency is an autosomal recessive disorder of the purine synthesis which results in accumulation of succinylpurines (succinyladenosine (S-Ado) and succinylamino-imidazole carboxamide riboside (SAICAr)) in body fluids. Patients present developmental delay, often accompanied by epilepsy and autistic spectrum disorders. ObjectivesTo describe atypical neurological features in the evolution of three novel unrelated cases of ADSL deficiency. PatientsA 9-year-old boy with severe cognitive impairment and autistic behaviour received d-ribose therapy for one year. Drug withdrawal was associated with acute neurological deterioration, severe brain atrophy and demyelination on MRI. The second patient is a 5.5-year-old girl with mild developmental delay who presented a benign course with moderate cognitive impairment as the only feature in her evolution. The final patient is a 14-year-old boy with severe cognitive impairment who developed drug-resistant epilepsy and bathing reflex seizures, progressive spasticity in the lower limbs and thoracic deformity. MethodsSAICAr and S-Ado in urine were analysed by HPLC with diode array detection. Diagnosis was confirmed by molecular analysis of the ADSL gene. ResultsAn elevation of S-Ado and SAICAr excretion in urine was detected in all three patients. The patients were homozygous for the missence change p.I369L and for the novel change p.M389V. ConclusionDrug-resistant epilepsy and specific therapeutic interventions may modify the neurological outcome in ADSL deficiency. d-ribose must be considered with caution as, in our experience, it returns no clinical benefit and drug withdrawal can precipitate status epilepticus and acute neurological deterioration.

Progress Towards Simple and Direct Detection of Adenylosuccinate Lyase Deficiency in Human Urine

A rhodamine based boronic acid linearly responds to increasing 5-aminoimidazole-4-carboxamide riboside (AICAr) concentrations in human urine. This method is thus an advance in detecting adenylosuccinate lyase (ADSL) deficiency as AICAr is a model riboside for the ADSL substrates succinyladenosine (S-Ado) and succinylaminoimidazolecarboxamide riboside (SAICAr). ADSL deficiency is a rare but devastating disease of de novo purine synthesis in infants. Its diagnosis is also significant as it is one of the autism spectrum disorders.

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.

Misleading behavioural phenotype with adenylosuccinate lyase deficiency

Adenylosuccinate lyase deficiency is a rare autosomal disorder of de novo purine synthesis, which results in the accumulation of succinylpurines in body fluids. Patients with adenylosuccinate lyase deficiency show a variable combination of mental retardation, epilepsy and autistic features and are usually discovered during screens for unexplained encephalopathy using the Bratton-Marshall assay that reveals the excretion of the succinylaminoimidazolecarboxamide riboside (SAICAr). Here, we report on two sisters aged 11 and 12 years presented with global developmental delay, motor apraxia, severe speech deficits, seizures and behavioural features, which combined excessive laughter, a very happy disposition, hyperactivity, a short attention span, the mouthing of objects, tantrums and stereotyped movements that gave a behavioural profile mimicking Angelman syndrome. Both patients had an increased succinyladenosine/SAICAr ratio of 1.6, and exhibited a novel homozygous missense mutation (c.674T>C; p.Met225Thr) in the exon 6 of the ADSL gene. We suggest that these clinical features might be a new presentation of adenylosuccinate lyase deficiency. On the basis of this observation, although adenylosuccinate lyase deficiency is a rare disorder, this diagnosis should be considered in patients with mental retardation and a behavioural profile suggestive of Angelman syndrome.

Adenylosuccinate lyase deficiency: The first identified polish patient

Adenylosuccinate lyase (ADSL) deficiency is a rare disease of de novo purine synthesis. The main symptoms are psychomotor retardation, epilepsy, autistic features, occasionally associated with muscular hypotonia. Diagnosis is made by detection of abnormal purine metabolites (succinyladenosine – S-Ado and succinylaminoimidazole carboxamide riboside – SAICAr) in body fluids. The severity of the clinical features correlates with low S-Ado/SAICAr ratio. We report clinical, biochemical and brain MRI findings of a female infant with severe early epilepsy and hypotonia, who died at the age of 10 weeks.

Mutation of a Nuclear Respiratory Factor 2 Binding Site in the 5′ Untranslated Region of the ADSL Gene in Three Patients with Adenylosuccinate Lyase Deficiency

Adenylosuccinate lyase (ADSL; also called "adenylosuccinase") catalyzes two steps in the synthesis of purine nucleotides: (1) the conversion of succinylaminoimidazolecarboxamide ribotide into aminoimidazolecarboxamide ribotide and (2) the conversion of adenylosuccinate into adenosine monophosphate. ADSL deficiency, a recessively inherited disorder, causes variable-but most often severe-mental retardation, frequently accompanied by epilepsy and/or autism. It is characterized by the accumulation, in body fluids, of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Analysis of the ADSL gene of three unrelated patients with ADSL deficiency, in whom one of the ADSL alleles displayed a normal coding sequence, revealed a -49T-->C mutation in the 5' untranslated region of this allele. Measurements of the amount of mRNA transcribed from the latter allele showed that it was reduced to approximately 33% of that transcribed from the alleles mutated in their coding sequence. Further investigations showed that the -49T-->C mutation provokes a reduction to 25% of wild-type control of promoter function, as evaluated by luciferase activity and mRNA level in transfection experiments. The mutation also affects the binding of nuclear respiratory factor 2 (NRF-2), a known activator of transcription, as assessed by gel-shift studies. Our findings indicate that a mutation of a regulatory region of the ADSL gene might be an unusually frequent cause of ADSL deficiency, and they suggest a role for NRF-2 in the gene regulation of the purine biosynthetic pathway.

Clinical, biochemical and molecular genetic correlations in adenylosuccinate lyase deficiency.

Adenylosuccinate lyase (ADSL) deficiency (MIM 103050) is an autosomal recessive inborn error of purine synthesis characterized by the accumulation in body fluids of succinylaminoimidazolecarboxamide (SAICA) riboside and succinyladenosine (S-Ado), the dephosphorylated derivatives of the two substrates of the enzyme. Because ADSL-deficient patients display widely variable degrees of psychomotor retardation, we have expressed eight mutated ADSL enzymes as thioredoxin fusions and compared their properties with the clinical and biochemical characteristics of 10 patients. Three expressed mutated ADSL enzymes (M26L, R426H and T450S) were thermolabile, four (A2V, R141W, R303C and S395R) were thermostable and one (del206-218), was inactive. Thermolabile mutations decreased activities with SAICA ribotide (SAICAR) and adenylosuccinate (S-AMP) in parallel, or more with SAICAR than with S-AMP. Patients homozygous for one of these mutations, R426H, displayed similarly decreased ADSL activities in their fibroblasts, S-Ado:SAICA riboside ratios of approximately 1 in their cerebrospinal fluid and were profoundly retarded. With the exception of A2V, thermostable mutations decreased activity with S-AMP to a much more marked extent than with SAICAR. Two unrelated patients homozygous for one of the thermostable mutations, R303C, also displayed a much more marked decrease in the activity of fibroblast ADSL with S-AMP than with SAICAR, had S-Ado:SAICA riboside ratios between 3 and 4 in their cerebrospinal fluid and were mildly retarded. These results suggest that, in some cases, the genetic lesion of ADSL determines the ratio of its activities with S-AMP versus SAICAR, which in turn defines the S-Ado:SAICA riboside ratio and the patients' mental status.

Human adenylosuccinate lyase (ADSL), cloning and characterization of full-length cDNA and its isoform, gene structure and molecular basis for ADSL deficiency in six patients.

Adenylosuccinate lyase (ADSL) is a bifunctional enzyme acting in de novo purine synthesis and purine nucleotide recycling. ADSL deficiency is a selectively neuronopathic disorder with psychomotor retardation and epilepsy as leading traits. Both dephosphorylated enzyme substrates, succinylaminoimidazole-carboxamide riboside (SAICAr) and succinyladenosine (S-Ado), accumulate in the cerebrospinal fluid (CSF) of affected individuals with S-Ado/SAICAr concentration ratios proportional to the phenotype severity. We studied the disorder at various levels in a group of six patients with ADSL deficiency. We identified the complete ADSL cDNA and its alternatively spliced isoform resulting from exon 12 skipping. Both mRNA isoforms were expressed in all the tissues studied with the non-spliced form 10-fold more abundant. Both cDNAs were expressed in Escherichia coli and functionally characterized at the protein level. The results showed only the unspliced ADSL to be active. The gene consists of 13 exons spanning 23 kb. The promotor region shows typical features of the housekeeping gene. Eight mutations were identified in a group of six patients. The expression studies of the mutant proteins carried out in an attempt to study genotype-phenotype correlation showed that the level of residual enzyme activity correlates with the severity of the clinical phenotype. All the mutant enzymes studied in vitro displayed a proportional decrease in activity against both of their substrates. However, this was not concordant with strikingly different concentration ratios in the CSF of individual patients. This suggests either different in vivo enzyme activities against each of the substrates and/or their different turnover across the CSF-blood barrier, which may be decisive in determining disease severity.

Prenatal diagnosis in adenylosuccinate lyase deficiency.

Adenylosuccinate lyase deficiency, an autosomal recessive inborn error of purine synthesis, provokes accumulation in body fluids of succinylaminoimidazolecarboxamide riboside and succinyladenosine, the dephosphorylated derivatives of the two substrates of the enzyme. Most patients display severe psychomotor retardation, often accompanied by epilepsy and/or autistic features, although some are only mildly retarded. About 20 mutations are known. Prenatal diagnosis was performed twice on chorion villi of the mother of a previously diagnosed patient with a C5T mutation (exon 1) on the maternal allele, and a C1185A mutation (exon 11) on the paternal allele. Both suppress a Fnu4HI restriction site. In a first fetus, incubation of PCR products generated from genomic DNA of exon 1 with Fnu4HI yielded a 113 bp fragment from a control and the father's gene, and both a 113 bp and 170 bp fragment from the mother, affected sibling and fetus. Incubation of PCR products of exons 11-12 with Fnu4HI yielded a 550 bp fragment from a control and the mother's gene, and a 550 bp and 600 bp fragment from the father, affected sibling and fetus. Assay of adenylosuccinate lyase on the aborted fetal liver confirmed the enzyme deficiency. A second fetus displayed only the maternal mutation.

Adenylosuccinase deficiency: possibly underdiagnosed encephalopathy with variable clinical features

Adenylosuccinase deficiency is an autosomal recessive inherited defect of purine synthesis. In enzyme deficient patients, two normally undetectable compounds, succinylaminoimidazole carboxamide riboside and succinyladenosine, accumulate in urine, cerebrospinal fluid and, to a minor extent, in plasma. Analysing 150 highly selected urine specimens from patients with unidentified neurogenerative disorders we discovered the first two German cases of adenylosuccinase deficiency. The deficiency causes moderate to severe mental retardation, often accompanied by epileptic seizures and/or autistic features, and is occasionally associated with growth retardation and muscular hypotonia. Of the two German cases we present here, one patient fits into the clinical picture outlined by previous reports. The other patient, however, shows a pattern of symptoms so far undescribed: severe early infantile epileptic encephalopathy with reduced myelination. On mutation analysis this patient is the first to reveal a 39 base pair deletion in the adenylosuccinase gene in contrast to the point mutations detected in previous cases. Adenylosuccinase deficiency may be an underdiagnosed metabolic disorder with variable expression. This should be taken into consideration in patients with unclassified neurological conditions.