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.
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 and several mutations have been identified. 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 or the detection of S-Ado and/or SAICAr using high-performance liquid chromatography (HPLC), HPLC–mass spectrometry (LC-MS), or nuclear magnetic resonance imaging 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.
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- Supporting Information
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. The relatively low S–Ado/SAICAr ratio of approximately 1.2 was also suggestive of a more severe phenotype, 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 and has been reported only in patients with a severe form of ADSL deficiency. 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 and uridine 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, and there is evidence that muscle ATP levels are decreased in ADSL patients, although in vitro studies have been unable to demonstrate changes in purine nucleotide and ATP levels in ADSL-deficient cell lines. SAMe treatment is currently being trialled in patients with Arts syndrome (OMIM 301835), 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).
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. 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. 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. 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. 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. This is understandable given the non-specific and wide-ranging nature of the symptoms 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.