Early diagnosis of adenylosuccinate lyase deficiency using a high-throughput screening method and a trial of oral S-adenosyl-l-methionine as a treatment method


Correspondence to Dr James J Pitt, Victorian Clinical Genetics Services, Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, Melbourne, Vic., 3052, Australia. E-mail: james.pitt@vcgs.org.au



The aim of this study was to develop a high-throughput urine screening technique for adenylosuccinate lyase (ADSL) deficiency and to evaluate S-adenosyl-l-methionine (SAMe) as a potential treatment for this disorder.


Testing for succinyladenosine (S-Ado), a marker of ADSL deficiency, was incorporated into a screening panel for urine biomarkers for inborn errors of metabolism using electrospray tandem mass spectrometry. Liquid chromatography–mass spectrometry and high-performance liquid chromatography were used to confirm and monitor the response of metabolites to oral SAMe treatment.


Increased levels of S-Ado were detected in a 3-month-old male infant with hypotonia and seizures. ADSL gene sequencing revealed a previously described c.–49T>C mutation and a novel c.889_891dupAAT mutation, which was likely to disrupt enzyme function. After 9 months of SAMe treatment, there was no clear response evidenced in urine metabolite levels or clinical parameters.


These results demonstrate proof of the principle for the high-throughput urine screening technique, allowing earlier diagnosis of patients with ADSL deficiency. However, early treatment with SAMe does not appear to be effective in ADSL deficiency. It is suggested that although SAMe treatment may ameliorate purine nucleotide deficiency, it cannot correct metabolic syndromes in which a toxic nucleotide is present, in this case presumed to be succinylaminoimidazole carboxamide ribotide.


Adenylosuccinate lyase


Adenosine triphosphate


High-performance liquid chromatography


Liquid chromatography–mass spectrometry




Succinylaminoimidazole carboxamide riboside



What this paper adds

  • An improved high-throughput screening technique for ADSL deficiency is described.
  • S-adenosyl-l-methionine treatment for ADSL deficiency does not appear to be efficacious.

The de novo purine biosynthesis pathway involves multiple steps that lead from 5-phosphoribosylpyrophosphate to inosine monophosphate, from which the adenine and guanine nucleotides are formed (see Fig. S1, online supporting information). Adenylosuccinate lyase (ADSL) catalyses two steps in purine biosynthesis. First, it is involved in converting the ribotide of succinylaminoimidazole carboxamide into the ribotide of aminoimidazole carboxamide as part of the de novo synthesis pathway, and second, it catalyses the conversion of adenylosuccinate (the ribotide of succinyladenosine [S-Ado]) into adenosine monophosphate in the nucleotide cycle pathway.[1]

Adenylosuccinate lyase deficiency (OMIM 608222) is a rare inborn error of purine metabolism whose symptoms include psychomotor delay, epilepsy, and autism, and which is occasionally associated with muscular hypotonia. The diagnosis is made by the detection of elevated levels of the dephosphorylated forms of adenylosuccinate (the ribotide of S-Ado) and succinylaminoimidazole carboxamide ribotide (namely S-Ado and succinylaminoimidazole carboxamide riboside [SAICAr] respectively) in urine or plasma.[1, 2] The gene for ADSL has been mapped to chromosome 22q13.1–q13.2[3] and several mutations have been identified.[1] Treatment options are limited and are aimed at controlling seizures.

To date, most urine screening tests for ADSL deficiency have relied on the detection of SAICAr using the colorimetric Bratton–Marshall test[4] or the detection of S-Ado and/or SAICAr using high-performance liquid chromatography (HPLC),[5] HPLC–mass spectrometry (LC-MS),[6] or nuclear magnetic resonance imaging[7] to screen for inborn errors of purine and pyrimidine metabolism. ADSL deficiency, or a defect in purine or pyrimidine metabolism, must, therefore, be clinically suspected in order to prompt the ordering of these tests. Furthermore, these tests are not routinely performed on all samples in urine screening programmes. Given the relatively non-specific nature of the symptoms in ADSL deficiency, it is likely that some patients are not diagnosed when tested using routine urine screening tests for an inborn error of metabolism, such as amino acids or organic acids.

In this report we describe a high-throughput urine screening technique for the detection of ADSL deficiency that can be applied in an unbiased fashion to all urine samples submitted for screening for inborn errors of metabolism. Its effectiveness is illustrated by the early diagnosis of a child with unsuspected ADSL deficiency, who presented with early symptoms. We also provide a report on the effectiveness of S-adenosyl-l-methionine (SAMe), as a new, and experimental, treatment for the disorder.



The patient was a Caucasian male infant. From the first week of life he exhibited focal seizures, with eye deviation and unresponsiveness. In the week before referral at 3 months of age he developed epileptic spasms with some focal clinical features. On examination, visual fixation was absent and motor development was delayed. Initial electroencephalography showed frequent independent bilateral epileptiform discharges and poorly developed background activity, and a cluster of epileptic spasms was recorded. Urine metabolite screening was requested. At the time, ADSL deficiency was not specifically suspected and purine/pyrimidine screening was not requested. Treatment with high-dose oral prednisolone was ineffective. After the diagnosis of ADSL deficiency, the child was treated from the age of 5 months with SAMe at a dose of 75mg twice daily (approximately 20mg/kg/d) until 7 months of age, when the dose was increased to 140mg twice daily (approximately 35mg/kg/d). Epileptic spasms continued and development remained static. Topiramate was trialled, and at the age 7.5 months vigabatrin 250mg twice daily (65mg/kg/d) was commenced and was immediately effective in controlling all seizures. Treatment with SAMe was continued, together with vigabatrin, until the family ran out of SAMe when the infant reached 9 months of age. The parents reported poorer head control and visual fixation after discontinuation of the SAMe treatment, and so treatment was recommenced at 9.5 months of age at a dose of 160mg twice daily. However, it was not proven that SAMe treatment had a beneficial effect and the supplement was discontinued at 25 months of age. At the child's last follow-up (at 5y), he was diagnosed as having autism, with normal motor development but no verbal communication. Electroencephalography showed a slow posterior rhythm and infrequent generalized interictal epileptiform activity. Currently, the child experiences brief absences associated with generalized 4 Hz spike-and-wave bursts; these are partly controlled with valproate monotherapy, with vigabatrin having been discontinued at 15 months of age.

Metabolite measurements

Qualitative urine screening

Our method for batched urine metabolite screening was performed using electrospray tandem mass spectrometry. In brief, using our method, samples were corrected for creatinine concentration and mixed with internal standards and analysed in negative (underivatized) and positive (butyl derivatives) ion mode, and a panel of 110 metabolites was targeted.[8] The panel was modified to include a transition for S-Ado (−382.1 to −206.1m/z, dwell time 0.050s, cone voltage 35V and a collision energy of 22eV). S-Ado was synthesized as has been previously described.[9] The S-Ado response was measured as a ratio relative to an added internal standard (hexanoyl-13C2-glycine). The S-Ado response ratios for each sample were converted to multiples of batch medians.

For the LC-MS analysis, an Atlantis dC18 2.1 × 100 mm column (Waters, Milford, MA, USA) with 3 μm particles was used. Solvent A was 0.02 mol/L ammonium acetate pH 5.0, while solvent B was 90% methanol containing 0.02 mol/L ammonium acetate pH 5.0. A linear gradient of 0% to 50% solvent B was run over 6 minutes at a flow rate of 0.2 mL/minute. The column outlet was interfaced with a Quattro-LC electrospray tandem mass spectrometer (Waters) operating in single MS negative ion mode. The mass spectrometer was scanned from −100 to −400 m/z every 1 second.

Quantitative urine screening

Urine purines and pyrimidines were measured by reversed-phase HPLC using multiple-wavelength ultraviolet detection using a previously described method,[5] with slight modifications. SAICAr is not commercially available, therefore, a standard of aminoimidazole carboxamide riboside was used as a surrogate calibrator. Peak areas from the 254 and 280 nm traces were used to quantitate SAICAr and S-Ado levels respectively.

ADSL gene sequencing

The 5′ untranslated region (UTR) and coding region of the ADSL gene were amplified using previously described methods.[10, 11] The polymerase chain reaction (PCR) products were purified using a Roche High Pure Purification kit (Roche Diagnostics GmbH, Penzberg, Germany) and bidirectionally sequenced using an Applied Biosystems Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Austin, TX, USA), according to the manufacturer's instructions. Sequences were read on an ABI 3130xl Genetic Analyser (Applied Biosystems) and were compared with reference sequence NM_000026.1.


Urine Screening

Qualitative electrospray tandem mass spectrometry urine metabolic screening showed consistent, grossly elevated levels of S-Ado in the urine of the patient (Fig. 1). The S-Ado level was greater than 100 multiples of batch median in the presenting urine and in three subsequent urine samples. In comparison participants, some variation in S-Ado excretion with age was apparent, but the levels were much lower than for the patient, regardless of age (the 99th centile was 6.2, n=2362). LC-MS urine analysis confirmed the presence of large chromatographic peaks with M-H ions of −382 m/z and −373 m/z, consistent with S-Ado and SAICAr respectively (data not shown). These peaks were almost undetectable in the urine of comparison samples, with peak areas at least 100 times lower than those of the patient. The levels of S-Ado, and also of SAICAr, were more accurately determined by HPLC, which gave concentrations of S-Ado of 421 μmol per mmol of creatinine (comparison samples <4; n=9) and of SAICAr of 324 μmol per mmol of creatinine (comparison samples <7; n=9) in the first tested urine of this patient with an S-Ado/SAICAr ratio of 1.3.

Figure 1.

Qualitative urine screening succinyladenosine (S-Ado) levels in adenylosuccinate lyase deficiency (log–log scale). The vertical axis is the multiple of batch median for the S-Ado response ratio measured by tandem mass spectrometry. Comparison group (n=2362) samples were submitted for urine metabolic screening in the same year that the patient was diagnosed.


Sequencing of the patient's ADSL gene revealed two mutations, c.–49T>C and c.889_891dupAAT (p.Asn297dup). Sequencing of the parents' ADSL genes showed that these mutations were maternally and paternally inherited respectively. The c.–49T>C mutation has previously been described.[11] The c.889_891dupAAT variant results in an in-frame duplication of an asparagine residue at codon 297. It has not previously been described and is located in a sequence motif that is believed to be critical for enzyme function. ADSL is a member of the fumarase superfamily of enzymes and this motif, which encompasses the active site, is well conserved among members of this superfamily.[1] In particular, Asn297 is highly conserved within this motif and inspection of the crystal structure of human ADSL complexed with adenosine monophosphate (Protein Data Bank accession number 2VD6) indicates that Asn297 is in close proximity to adenosine monophosphate.

Trial of SAMe

Urine S-Ado and SAICAr levels monitored during the trial of SAMe are shown in Figure 2. There was an apparent decrease in levels in one sample collected concurrently with the commencement of SAMe, but we believe that this is not significant and merely reflects the normal variability of random urine samples. Urine S-Ado and SAICAr levels did not alter in response to SAMe withdrawal at around 9 months of age. The S-Ado/SAICAr ratio did not respond significantly throughout, ranging from 1.0 to 1.4 (mean 1.2). Amino acids, folate, total homocysteine, and vitamin B12 levels remained within normal limits during SAMe treatment. The patient was clinically reassessed at 9.5 months of age, when his head circumference was 45 cm (50th centile). Gross motor function and visual behaviour had not advanced significantly compared with that noted at 3 months of age.

Figure 2.

Urine succinyladenosine (S-AD) and succinylaminoimidazole carboxamide riboside (SAICAr)levels during the trial of oral S-adenosyl-l-methionine (SAMe) treatment.


Patients with ADSL deficiency have a range of symptoms including psychomotor delay, epilepsy, autism, and occasionally hypotonia. Biochemically, they are diagnosed by elevated levels of SAICAr and S-Ado in their urine. In this report, a patient who presented with epileptic seizures and global developmental delay has been described. Urine screening by the tandem mass spectrometry procedure showed elevated levels of S-Ado: an unexpected initial finding. In combination with the clinical presentation, this led to the diagnosis of ADSL deficiency. LC-MS and HPLC urine analysis confirmed the elevated levels of S-Ado, and also showed the expected elevated levels of SAICAr.

Our patient exhibited seizure symptoms from the first week of life, suggesting a relatively severe form of ADSL deficiency. Other clinical studies report similar symptoms in patients with ADSL deficiency and also an early age at onset, although very severely affected patients may die in the first year of life.[1] The relatively low S–Ado/SAICAr ratio of approximately 1.2 was also suggestive of a more severe phenotype,[12] as was the patient's genotype. Sequencing of the ADSL gene revealed a c.–49T>C mutation and a c.889_891dupAAT mutation. The first mutation is in the regulatory 5′-UTR of the ADSL gene; it results in significant reductions in transcribed mRNA[11] and has been reported only in patients with a severe form of ADSL deficiency.[1] The second mutation predicted a duplication of the Asn297 residue at the active site of the enzyme, which is also likely to be a severe mutation.

There is no effective treatment for ADSL deficiency and current treatment is mainly supportive and aimed at controlling seizures. Trials of d-ribose[13] and uridine[14] therapy have not shown any benefit. Our patient was given a trial of oral SAMe, which is proposed to act as an alternative adenosine donor to form adenosine monophosphate,[15, 16] and could provide a theoretical exogenous source of adenosine triphosphate (ATP). Maintenance of full mitochondrial function and sufficient levels of ATP are essential for tissues with high energy requirements, such as central nervous tissues, muscles (including smooth muscle in the gut for peristalsis), and the retina. These tissues are significantly affected in mitochondrial disorders, in which oxidative phosphorylation, coupled to ATP production, is impaired. Alternatively, disorders affecting nucleotide production may also affect ATP pools. This may provide an explanation for why central nervous system tissues and muscle are mainly affected in disorders of purine nucleotide synthesis such as hypoxanthine–guanine phosphoribosyl transferase and phosphoribosyl-pyrophosphate synthetase deficiencies. Muscle symptoms are common in ADSL deficiency,[1] and there is evidence that muscle ATP levels are decreased in ADSL patients,[17] although in vitro studies have been unable to demonstrate changes in purine nucleotide and ATP levels in ADSL-deficient cell lines.[18] SAMe treatment is currently being trialled in patients with Arts syndrome (OMIM 301835),[15] caused by mutations in the PRPS1 gene. The enzyme that is encoded by this gene is needed for the synthesis of phosphoribosyl-pyrophosphate as the first step of the de novo purine nucleotide synthesis pathway (see Fig. S1, online supporting information). SAMe has also been reported to have benefitted a patient with hypoxanthine–guanine phosphoribosyl transferase deficiency (Lesch–Nyhan disease).[19]

We reasoned that any response to SAMe, particularly in the central nervous system, would be particularly evident in our patient, given the early diagnosis at a young age. However, the trial of SAMe showed no significant biochemical or clinical effect, although withdrawal of the drug was anecdotally reported by the parents as detrimental to the patient's muscle tone. No significant changes in urine S-Ado, SAICAr, or the S–Ado/SAICAr ratio were found when compared with baseline samples (Fig. 2). Various reasons can be put forward for this lack of response and may also provide an insight into the pathogenesis of ADSL deficiency. The fact that SAMe, at least partially, corrects ‘nucleotide depletion’ in disorders such as phosphoribosyl pyrophosphate synthetase and hypoxanthine–guanine phosphoribosyl transferase deficiencies, but not in ADSL deficiency, is indirect evidence that the pathogenesis of ADSL deficiency arises from accumulation of a toxic metabolite. In particular, trials of SAICAr injections into rat central nervous system have shown that this metabolite is neurotoxic.[20] This occurs for some other nucleotide disorders, such as adenosine deaminase and purine nucleoside phosphorylase deficiencies, in which the toxic metabolites are deoxy-ATP/deoxyadenosine and deoxy-guanosine triphosphate respectively.[21] Replenishment of the adenosine pool by SAMe did not appear to alter SAICAr and S-Ado production, suggesting an absence of feedback mechanisms to decrease the flux through the pathway. A lack of inhibition of purine synthesis in ADSL deficiency is also evidenced by the total purine overproduction seen in patients with the deficiency.[22] Measurement of metabolite levels in cerebrospinal fluid would have been a more rigorous way of detecting a response to SAMe treatment, given that the central nervous system is the main tissue that is affected. SAMe is capable of crossing the blood–brain barrier and it is thought to work in the brain as an adenosine donor.[15] However, it was unethical to obtain multiple CSF samples for this study, and it was considered that any subtle alteration in the CSF purine profile was unlikely to be reflected in the urine.

S-adenosyl-l-methionine can be converted to methionine in vivo, which may theoretically raise homocysteine levels, producing possible vascular toxicity, and this may represent a potential risk of SAMe treatment. However, our patient's plasma amino acids, including methionine and plasma total homocysteine, were monitored and found to be normal before and during SAMe treatment. Folate and vitamin B12, which are cofactors needed to convert homocysteine back to methionine, were also investigated during SAMe treatment and levels of these were found to be normal. At the age of 3 years 8 months the patient was walking but had no verbal communication and continued to have seizures. He was not being treated with SAMe, but was being treated with valproate monotherapy.

There is anecdotal evidence that ADSL deficiency is underdiagnosed; for example, there is an apparently lower prevalence in the UK than elsewhere in Europe, and there are delays between initial presentation and diagnosis.[23] This is understandable given the non-specific and wide-ranging nature of the symptoms[1] and a relative lack of awareness of inborn errors of purine and pyrimidine metabolism and of the laboratory tests required for their diagnosis. However, the symptoms of ADSL deficiency overlap with many other inborn errors of metabolism and they are sufficiently serious to prompt testing for these disorders with a ‘urine metabolic screen’. In most centres this comprises a series of discrete tests for amino acids, organic acids, etc., but purine and pyrimidine testing is not routinely performed on all samples. This may be partly because purine and pyrimidine testing is performed in only a few centres worldwide.

The advantage of our tandem mass spectrometry screening approach is that it targets a much wider range of metabolic diseases, including ADSL deficiency, and it can be applied to all urine samples sent for metabolic testing. This results in diagnoses that would be missed using traditional screening approaches, as illustrated by our patient. Before the introduction of our screening technique in 2004, no cases of ADSL deficiency were diagnosed in the state of Victoria, Australia despite the availability of the Bratton–Marshall test and HPLC-based purine and pyrimidine screening. During the preparation of this manuscript, three further cases (in two families) of clinically unsuspected ADSL deficiency were diagnosed using our screening technique. More widespread use of tandem mass spectrometry based metabolic screening may eventually provide a more accurate estimate of the incidence of this serious metabolic disorder.


Diagnosis of ADSL deficiency is feasible using electrospray tandem mass spectrometry urine screening. The symptoms of this disorder are non-specific and may not prompt specific testing for purine metabolites. Therefore, the ability of the screening technique to analyse large numbers of samples as part of a broader metabolic screen represents an important advance in the diagnosis of this disorder. Oral treatment with SAMe was not effective in our patient.


We thank the following staff from the Murdoch Childrens Research Institute: Dr Joy Lee for initial discussions regarding the patient and Mary Eggington and Avantika Mishra for performing the initial urine screening. The Victorian Government's Operational Infrastructure Support Program supported sections of this work.