Clinical and Laboratory Barriers to the Timely Diagnosis of Sulphite Oxidase Deficiency

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1 Clinical and Laboratory Barriers to the Timely Diagnosis of Sulphite Oxidase Deficiency Chew Thye Choong 1, MBBS, MMED (Paed), Claire Hart 2, MSc Clinical Biochemistry, FRCPath, Ee Shien Tan 3, MBBS, MMED (Paed), Denise Li Meng Goh 4, MBBS, MMED (Paed) 1 Neurology Service, Division of Paediatric Medicine, KK Women s and Children s Hospital, Singapore 2 Biochemical Genetics Laboratory, Department of Pathology and Laboratory Medicine, KK Women s and Children s Hospital, Singapore 3 Genetics Service, Division of Paediatric Medicine, KK Women s and Children s Hospital, Singapore 4 Genetics Service, Department of Paediatric Medicine, National University Hospital, Singapore Abstract Isolated sulphite oxidase deficiency (SOD) is a rare genetic neurometabolic disorder characterised by intractable seizures and progressive severe neurological dysfunction. Affected children commonly present at or soon after birth with seizures and neurological deficits. In the newborn period, diagnosis may be missed as clinical features may mimic perinatal hypoxia-ischaemia, a more common entity. We report an infant with SOD whose clinical presentation of seizures and lethargy on the 4th day of life had suggested hypoxic-ischaemic encephalopathy, and discuss the challenges in the diagnostic process. The infant s progressive severe neurological deterioration suggested a metabolic disorder. His brain magnetic resonance imaging at 10 weeks showed severe cerebral atrophy and cystic changes. Biochemical findings of a very low plasma cystine concentration (<5µmol/L, reference range ) and elevated urinary sulphocysteine in the presence of a normal plasma uric acid pointed towards SOD. The diagnosis was confirmed by mutational analysis. Isolated SOD can be difficult to diagnose on the basis of clinical features and routine metabolic tests. Nonetheless heightened awareness of the SOD entity coupled with selective biochemical testing will help towards a more accurate diagnosis. The disorder should be considered in all infants with unexplained hypoxic-ischaemic encephalopathy or progressive neurological dysfunction. Keywords: encephalopathy, molybdenum cofactor, urinary sulphocysteine INTRODUCTION Isolated sulphite oxidase deficiency (SOD) is a rare genetic disorder affecting the metabolism of sulphated amino acids. Methionine and cysteine are normally metabolised to sulphite and then are oxidised to sulphate by the enzyme sulphite oxidase. When sulphite oxidase is deficient, alternative metabolic pathways lead to the formation of metabolites sulphocysteine and thiosulphate. The clinical features of isolated SOD are similar to a related variant, molybdenum cofactor deficiency (MoCD), a combined deficiency of sulphite oxidase, xanthine oxidase and aldehyde oxidase. Molybdenum co-factor is necessary for the function of all three enzymes. The 2 disorders can be differentiated by their biochemical findings. Fig. 1 shows the metabolic pathways affected by SOD and MoCD. Affected children commonly present at or soon after birth with seizures, abnormal tone, irritability and other neurological abnormalities. SOD is likely an under-diagnosed disorder. Diagnosis can be difficult in the young infant as early clinical features and neuro-imaging changes may mimic hypoxic-ischaemic insult, a more common 94

2 Clinical and Laboratory Barriers to the Timely Diagnosis of Sulphite Oxidase Deficiency Legend: Increase in SOD / MoCD Decrease in SOD / MoCD Increase in MoCD only Methionine Decrease in MoCD only Homocysteine Purine bases from DNA / RNA Sulphocysteine Cysteine Hypoxanthine Mercatopyruvate Cysteine Sulphonate Taurine Xanthine Oxidase Xanthine Sulphite Thiosulphate Sulphite Oxidase Uric Acid Sulphate Molybdenum cofactor requiring enzyme Fig. 1. Metabolic pathways affected by sulphite oxidase and MoCD, with metabolite abnormalities shown. Although cysteine and homocysteine are before the block in the pathway, and would therefore be expected to be raised, they are low because they react with the excess sulphite to produce other species for example, sulphocysteine. entity 1. Furthermore, isolated SOD does not have a simple and readily available diagnostic laboratory test. Non-specific abnormalities in the plasma amino acid profile (low cystine, high taurine) may provide a pointer, as can the finding of a very low total homocysteine. However, the significance of results like these can be overlooked as abnormalities may not be marked, and these tests are not likely to be requested unless the possibility of an inborn error of metabolism is considered. Nonetheless, heightened awareness of the SOD entity coupled with selective biochemical testing will help towards a more accurate diagnosis. Progressive neurological dysfunction should alert the clinician to a neurometabolic disorder, particularly where the etiology of the encephalopathy is unclear. Craniofacial anomalies have been described in children with SOD, including narrow bifrontal diameter and deep-set eyes. Subluxation of lenses has been observed at a later age 2. Accurate diagnosis is important for genetic counselling, access to prenatal testing, and may open a window for therapy. It has been reported that early intervention with a special diet low in 95

3 Fig. 2A (left) and 2B (right). Cranial magnetic resonance imaging at age 10 weeks. Fig. 2A is an axial T2-weighted MR which shows severe diffuse white matter atrophy, lateral ventricles dilatation and cystic changes at lentiform nuclei. Fig. 2B is a sagittal T1-weighted MR that shows thinning of corpus callosum and widened sulci. sulphur amino acids may be effective to reduce the neurological and mental deterioration in mild cases of SOD 3,4. We report a young infant with isolated SOD whose neonatal presentation of seizures, poor feeding and lethargy had suggested an initial diagnosis of hypoxic-ischaemic encephalopathy. We discuss the challenges in the diagnostic process and suggest an algorithm for laboratory testing. CASE REPORT The male infant, the first born of nonconsanguineous parents, was delivered at a private medical facility at 37 weeks gestation via emergency caesarean section for fetal tachycardia. The infant s birth weight was 2,850gm and his head circumference was 35cm. Apgar scores were 9 and 10 at one and five minutes respectively. The mother had gestational diabetes which had been managed with dietary control. On Day 1, the infant sucked slowly but feeding improved over the next day. Blood glucose monitoring revealed normal levels concentrations ( mmol/l). At 4 days of age, the infant became lethargic with poor suck and developed seizures. Blood counts, blood culture, blood gases and routine biochemistry were normal, including normal levels of blood glucose, urea and electrolytes, serum calcium, magnesium and ammonia. An urgent computer tomography brain scan disclosed bilateral small subdural haematoma, which was managed conservatively. The infant was treated for suspected hypoxic ischaemia and received phenobarbitone which controlled the seizures. He was discharged from hospital after 2 weeks. However, over the ensuing weeks, the seizures returned and the infant became increasingly irritable with inconsolable crying episodes and feeding difficulties. The infant first presented to our institution at 10 weeks of age with seizures, clusters of stiffening and inconsolable crying episodes. Clinical examination revealed a fretful infant who was episodically opisthotonic. He was hypertonic with hyperreflexia in all extremities. He had subtle dysmorphic features of bi-temporal narrowing and deep-set eyes. Developmentally, he was globally delayed with significant head lag, poor visual fixation and demonstrated little awareness of his surroundings. The liver function tests, serum urea and electrolytes, plasma ammonia and lactate were within normal concentrations. Blood and cerebrospinal fluid cultures were negative for bacteria growth. The urine cultures revealed infection with enterobacter aerogene. The electroencephalogram was abnormal with focal epileptiform discharges over the temporo-parietal regions bilaterally. The cranial MR imaging showed severe diffuse white matter atrophy, bilateral cystic changes in the region of the lentiform nuclei and dilated lateral ventricles 96

4 Clinical and Laboratory Barriers to the Timely Diagnosis of Sulphite Oxidase Deficiency Table 1. Metabolic studies at age ten weeks. Test Result Reference Range Plasma Cystine < µmol/l Plasma Homocysteine < 0.7 (below detection level) 1-18 µmol/l Plasma Uric Acid µmol/l Plasma Taurine µmol/l Plasma Ammonia µmol/l Plasma Lactate mmol/l Urine Sulphocysteine 0.15 <0.01 mol/mol creatinine (Fig. 2a, 2b). Further metabolic tests (Table 1) revealed a very low plasma cystine concentration of <5µmol/L, which first raised the suspicion of sulphite oxidase or MoCD. A plasma total homocysteine below the detection limit of the assay (<0.7µmol/L) supported this suspicion. The plasma uric acid level was normal making MoCD unlikely. Urinary sulphocysteine was sent for analysis and found to be elevated at 0.15mol/mol creatinine (Table 1), consistent with SOD. Genetic analysis of the sulphite oxidase SUOX gene was performed. The patient was found to be compound heterozygous for p.r160q and p.y343x mutations. Both are known disease causing mutations and further analysis of parental samples confirmed that both parents are carriers of the above mutations. The infant was started on a diet low in sulphur amino acids and continued on phenobarbitone. At 20 months of age he remained dystonic and globally delayed developmentally. Ophthalmologic examination confirmed absence of lens dislocation. Seizures were controlled. DISCUSSION Clinical Diagnosis of isolated SOD can be easily missed unless clinical features are obvious and classical. Our patient illustrates some of the diagnostic challenges. The neonatal presentation of seizures, lethargy and poor feeding on the 4th day of life suggested hypoxic-ischaemic encephalopathy. Progressive neurological dysfunction together with severely abnormal magnetic resonance imaging findings raised the suspicion of inborn errors of metabolism. The findings of a very low plasma cystine concentration and an undetectable level of plasma homocysteine set off a chain of diagnostic testing that ultimately led to confirmation of the diagnosis of SOD. Laboratory Diagnosis The laboratory diagnosis of isolated SOD is particularly problematic as there is no simple and readily available diagnostic test. There are a number of approaches to diagnosis but all have their problems. Traditionally urine has been tested for the presence of sulphite using dipsticks. However, sulphite is very unstable and rapidly oxidises to sulphate at room temperature; therefore urine is best tested while it is still warm from the patient, which is logistically difficult. Even when the dipsticks are used correctly it is possible to get both false negative 5 and false positive 6 results secondary to drug interactions, hence the test is of little practical value. Thiosulphate is a more stable metabolite of sulphite and analysis may be done on stored frozen samples. However, the spectrophotometric method is very susceptible to interference from common antibiotics such as cefotaxime and ampicillin, and so false positives are extremely common as many neonates presenting with seizures are often empirically treated with antibiotics 7. False negatives have also been reported 8. Urinary sulphocysteine is a stable and specific metabolite but this test is not widely available. Plasma and urine amino acids assay adds value to diagnostic evaluation. Patients with SOD or MoCD may have a low plasma cystine concentration and high plasma taurine concentration. However, these findings are far from specific and not universal. Taurine may be within the reference range in some patients (as it was in our patient), and cystine is frequently low in unaffected patients if samples are not 97

5 immediately separated and deproteinised once sampled. Therefore unless the concentration of cystine is very low, the significance may be missed. Although sulphocysteine is an amino acid, it is not reliably detected by the same methodology used to measure the standard plasma amino acid profile. Quantitative amino acid analysis is currently most often carried out by ion exchange chromatography systems. However, there are several different systems available with slightly different column and buffer conditions. Not all of these systems are capable of separating sulphocysteine from other amino acids that run close by in the chromatogram. Even in those systems that can do so, the peak appears at the very beginning of the chromatogram where interference from other drugs often makes a positive identification impossible. Therefore the presence of sulphocysteine can only be suspected. It has also been observed that patients with SOD or MoCD have very low plasma total homocysteine results 9,10. Disorders of homocysteine metabolism are a differential diagnosis in the investigation of seizures and therefore it is possible that such a finding could be the first clue. Such a finding should always be followed up with urinary sulphocysteine (specific method). These approaches to laboratory diagnosis apply equally to the diagnosis of isolated SOD and MoCD, but in the latter the typically very low plasma urate concentration serves to identify patients needing further follow up, and therefore these patients are less likely to be missed. However, a patient with partial MoCD with a normal plasma urate concentration but increased urinary xanthine and hypoxanthine excretion has been described and therefore the diagnosis should not be ruled out without measuring urinary xanthine and hypoxanthine concentrations, particularly in patients with milder presentations or those presenting with symptoms later 11. Molecular Diagnosis SOD is an autosomal recessive disorder. Molecular diagnosis is available 12. The human sulphite oxidase gene (SUOX) is located on chromosome 12q At least 16 different pathogenic mutations and 1 polymorphism have been reported for the gene, with most mutations being private. Genotypephenotype correlation is still unclear. Analysis of the sulphite oxidase gene revealed that our patient was compound heterozygous for the known diseasecausing mutations p.r1 60Q and p.y343x. Both his parents were carriers of the same mutations. Prenatal Diagnosis Prenatal diagnosis for SOD was first performed using sulphocysteine concentrations in amniotic fluid. However, this was found to be unreliable for making a diagnosis. Although sulphite oxidase activity can be measured for greater accuracy, this is still not ideal as the enzyme concentration in amniocytes is low. It is therefore important to determine exact genetic mutation in the proband to allow for accurate mutation analysis prenatally. We propose an algorithm to aid in the diagnostic evaluation of SOD (Fig. 3). Since it is neither possible nor desirable to isolate diagnostic testing for SOD from testing for other inborn errors of metabolism that may present in a related fashion, consideration of these is included at first line testing level. Relatively readily available tests, such as plasma amino acids, total homocysteine and plasma uric acid, should be included as first line tests. Where diagnosis remains elusive, or results are suggestive of sulphite oxidase or MoCD, urinary sulphocysteine should be requested. The analytical problems involved with the diagnosis of SOD illustrate the value of good communication between clinicians and laboratory staff. Knowledge on locally available tests allows clinicians to request the appropriate investigations while understanding the limitations of the tests ordered. For the laboratory, knowledge of the patient, such as clinical status and drug history, facilitates interpretation of results and allows prompt guidance on the necessary follow up tests. CONCLUSIONS SOD is a severe genetic disorder with no known effective cure. It is possibly under-diagnosed as there is no specific, simple and readily available diagnostic laboratory tests and clinical features mimic hypoxic-ischaemic insults. However, heightened awareness of the disorder together with selected laboratory testing will help towards a more timely diagnosis. The disorder should be part of the differential diagnoses for all young children with unexplained encephalopathy. Making a diagnosis of SOD enables affected families access to prenatal testing in subsequent pregnancies and provides more accurate information on management and prognosis for the proband. 98

6 Clinical and Laboratory Barriers to the Timely Diagnosis of Sulphite Oxidase Deficiency HIE with no clear aetiology Request: Basic biochemistry tests and ammonia, lactate, glucose Urine organic acids Plasma amino acids Total homocysteine (plasma) Plasma uric acid CSF lactate, amino acids Raised ammonia, lactate, or total homocysteine Low glucose Abnormal organic acids Low cystine Or high taurine Or low total homocysteine Or low plasma uric acid No abnormal results but clinical suspicion of SOD / MoCD remains Evaluate in accordance with specific abnormality documented Request: Urinary sulphocysteine Urinary xanthine / hypoxanthine No abnormality. Consider other disorders, consult with metabolic specialist Increased sulphocysteine, normal xanthine / hypoxanthine Increased sulphocysteine, increased xanthine / hypoxanthine Gene or enzyme testing for SOD Gene testing for MoCD Fig. 3. Suggested diagnostic algorithm for SOD / MoCD. (HIE = Hypoxic ischaemic encephalopathy; MoCD = Molybdenum cofactor deficiency, SOD = Sulphite oxidase deficiency). 99

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