Isolated sulfite oxidase deficiency

Isolated Sulfite Oxidase Deficiency Review of Two Cases in One Family Marianne C. Edwards, MD,1 Jean L. Johnson, PhD,2 Barbara Marriage, MS,1 Tyler N. Graf, BSc,2 Katharine E. Coyne, MS,2 K. V. Rajagopalan, PhD,2 Ian M. MacDonald, MDCM1 Objective: The authors describe two cases of isolated sulfite oxidase deficiency found in one family. This is a rare autosomal-recessive disorder presenting at birth with seizures, severe neurologic disease, and ectopia lentis. It can be easily missed with metabolic screening; however, the finding of lens subluxation stresses the importance of ophthalmic assessment in making the diagnosis. Design: Two observational case reports. Intervention/Methods: Ophthalmic assessment, biochemical assay for specific urinary and plasma metabolites, magnetic resonance imaging, and gene sequencing were used to make the diagnosis of the disease in the proband. The diagnosis was subsequently recognized in a previously affected sibling after the postmortem neuropathology was reviewed. Mutation analysis was performed on cultured fibroblasts from the proband to identify and categorize the specific mutation responsible for the disease in the family. From this, future prenatal detection of sulfite oxidase deficiency is possible. Main Outcome Measures: The diagnosis of sulfite oxidase deficiency was established in this family, enabling appropriate genetic counseling and recurrence risk estimation. Results: Point mutations were found in both alleles of the sulfite oxidase gene in the proband. The first is a 623C 3 A mutation, which predicts an A208D substitution, and the second is a 1109C 3 A, which predicts an S370Y substitution. Both residues A208D and S370Y are critical for sulfite oxidase activity. Conclusions: Isolated sulfite oxidase deficiency is a rare heritable disease for which mutation analysis can allow accurate prenatal screening. It often is difficult to diagnose by clinical presentation alone, but the critical finding of lens subluxation accompanying seizures and diffuse neurologic disease in an infant should alert the physician to the diagnosis. Ophthalmology 1999;106:1957–1961 Sulfite oxidase deficiency is an autosomal-recessive condition that manifests in infancy with severe and progressive neurologic disease, seizures, and lens subluxation. It occurs in two different biochemical forms: as isolated sulfite oxidase deficiency1,2 or as molybdenum cofactor deficiency.3,4 This cofactor, containing the trace element molybdenum and a unique pterin species, molybdopterin (Fig 1), is an essential component of sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Deficiencies of the latter two enzymes produce few clinical signs.5 It is the lack of sulfite oxidase that leads to the severe neurologic manifestations and lens subluxation in both forms.1,2,6,7 Molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are rare diseases, each identified in fewer than 100 cases. Of the two variants, isolated sulfite oxidase

Originally received: March 30, 1999. Revision accepted: June 17, 1999. Manuscript no. 99169. 1 Department of Ophthalmology, University of Alberta, Edmonton, Alberta, Canada. 2 Department of Biochemistry, Duke University Medical Center, Durham, North Carolina. Supported by grant GM44283 from the National Institutes of Health, Duke University Medical Center, Durham, North Carolina. Reprint requests to Ian M. MacDonald, MDCM, 2-129 Clinical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2G3 Canada.

deficiency appears to be much less common. We report two additional cases of isolated sulfite oxidase deficiency in one family. Because the disorder could easily be missed on routine screening, our account emphasizes the importance of early ophthalmic assessment.

Case Report The proband, a male infant, was born in July 1996 after an uncomplicated pregnancy. There had been no bleeding, infection, or drug exposure during gestation. Spontaneous vaginal delivery occurred at term with Apgar scores of 6 and 8. The weight was reported to be in the 50th percentile, the length in the 25th percentile, and the head circumference in the 90th percentile. During the first 24 hours postnatally, the infant developed difficulty breathing and subsequently started to have seizures. Feeding was also a problem and early bronchoscopy revealed tracheomalacia. The child was soon transferred to the intensive care unit because of respiratory distress, feeding difficulties, and intractable seizures. On physical examination, the child did not appear dysmorphic, although he did have slight micrognathia and a chubby appearance over the zygoma. The cardiovascular, abdominal, genitourinary, musculoskeletal, and integumentary examinations were all normal. The respiratory examination was also essentially normal aside from the mild tracheomalacia. The neurologic examination, however, was grossly abnormal, with the infant exhibiting marked

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Ophthalmology Volume 106, Number 10, October 1999

Figure 1. Structure of the molybdenum–molybdopterin complex that comprises the molybdenum cofactor.

hypertonicity, opisthotonic posturing, fist clenching, myoclonus, and repetitive cycling movements interpreted as seizures (Fig 2). After the onset of seizures, a magnetic resonance imaging was performed that showed diffuse white matter abnormalities and extensive macrocystic changes. The basal ganglia were abnormally small, and the cerebral peduncles showed some calcification. The brainstem and cerebellum appeared hypoplastic with significant surrounding cisternal fluid. The overall appearance was diffusely abnormal (Fig 3). An electroencephalogram confirmed the presence of seizures, which were controlled with phenobarbital. As shown in the pedigree (Fig 4), the parents were healthy, unrelated, and had three other normal children: two boys and one girl. However, their first-born son presented to a different institution with a disorder clinically similar to the case just described. He was born in 1990 after an uncomplicated pregnancy and delivery as well, with Apgar scores of 8 and 9. Poor feeding, respiratory distress, and protracted seizure episodes with opisthotonus occurred in the newborn period. A computed tomographic scan showed white matter abnormalities very similar to those described in the proband. As well, an electroretinogram suggested diffuse encephalopathy. He died at 10 months of age. An autopsy showed severe hydrocephalus with neuronal loss and gliosis of the thalamus, basal ganglia, corpus striatum, and periventricular areas.

Figure 2. Photograph of proband.

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Figure 3. Sagittal magnetic resonance imaging of proband showing diffuse white matter abnormality. The brainstem and cerebellum are hypoplastic with extensive surrounding cisternal fluid.

There was also macrocystic degeneration of the white matter. Although there was no clear history, the autopsy report suggested that the brain pathology may have been secondary to prenatal or perinatal hypoxia. An ischemic insult was suggested from in utero infarcts in the distribution of the internal carotid arteries. All routine biochemical screens had been normal (i.e., amino acids, reducing substances, uronic acids, organic acids, and urinary screens). With normal laboratory results and no family history of congenital metabolic disease, a genetic etiology was not considered likely, and sulfite oxidase deficiency was not considered.

Figure 4. Family pedigree of sulfite oxidase deficiency (affected, f).

Edwards et al 䡠 Sulfite Oxidase Deficiency

Figure 5. Bilateral nasal lens subluxation shown in right and left eyes.

Routine blood work and biochemical screening performed on the proband yielded normal results. The karyotype was 46XY. The amino acid analysis, serum pyruvate, lactate, and biotinidase were within normal limits, and the urine screen for reducing substance was nonspecific. A fibroblast culture showed no defect in mitochondrial function. At this time, it was surmised that the affected sibling probably had the same disorder and that it was heritable. Primary hereditary metabolic problems, disorders of energy metabolism, and hereditary leukodystrophies were considered, but the diagnosis was not apparent after initial screening for the more common genetic disorders. Because eye findings may assist in the diagnosis of many heritable conditions, the ophthalmology department was consulted. The infant was 3 months old when bilateral nasal subluxation of the lenses was discovered (Fig 5). In addition, it was noted that the eyes were exotropic and the child was unable to fix and follow. The pupillary reactions were sluggish with no afferent defect, and the anterior segments were normal. His fundus examination showed slight optic disc pallor in keeping with the cortical changes. There was no cherry-red spot; however, there were poor foveal and nerve fiber layer reflexes.

Figure 7. Mutation detection by sequence analysis. The 623C ⬎ A transversion present on one allele is shown on the left; the 1109C ⬎ A mutation in the second allele is shown on the right. In both cases, the normal sequence is listed on top and that of the mutant is below. The asterisks (*) indicate the position of the C ⬎ A substitution in each allele. The 1019G ⬎ A transition, also present on the second allele, is not shown. Mutations are numbered by considering the adenine of the first ATG of the sulfite oxidase cDNA as nucleotide position ⫹1. This triplet encodes the amino terminal methionine (residue ⫹1) of the 22-residue leader sequence of sulfite oxidase.

The discovery of lens subluxation prompted review of the differential diagnosis of ectopia lentis (Table 1).8 From this, it was recognized that seizures coupled with lens subluxation and a previous sibling with an identical presentation strongly suggested sulfite oxidase deficiency. Sulfite oxidase is responsible for the degradation of sulfur-containing amino acids and is essential for the detoxification of sulfite. Lack of the enzyme leads to blood accumulation and urinary excretion of inorganic sulfite, S-sulfocysteine, and thiosulfate (Fig 6).7,9 Tests were performed to detect these metabolites. A Merck Sulfite Ion Dipstick Test (Merckoquant) was used on fresh urine, and urine sulfite was detected at a level of 80 to 100 mg/l (normal, barely detectable or undetectable). Urinary S-sulfocysteine10 was 690 ␮mol/l (normal, 6 –30 ␮mol/l). The values for affected patients with sulfite oxidase deficiency range from 35 to 700 ␮mol/l. An oxypurine quantitation11 was also performed, which showed normal urinary and plasma levels of urate, hypoxanthine, and xanthine, thus confirming our case as isolated sulfite oxidase deficiency and not molybdenum cofactor deficiency (Fig 6).

Figure 6. (Left) Pathway of degradation of sulfur amino acids showing the reaction catalyzed by sulfite oxidase. (Right) Reactions catalyzed by xanthine dehydrogenase. Metabolites that accumulate in cases of molybdenum cofactor deficiency are underlined. Hypoxanthine and xanthine are elevated in molybdenum cofactor deficiency but are present at normal levels in cases of isolated sulfite oxidase deficiency.

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Ophthalmology Volume 106, Number 10, October 1999 Table 1. Hereditary Conditions Associated with Ectopia Lentis Condition Marfan syndrome Homocystinuria Weill-Marchesani Dominant spherophakia Simple ectopia lentis et pupillae Hyperlysinemia Sulfite oxidase deficiency Aniridia* Conradi syndrome* Crouzon syndrome* Ehler’s–Danlos syndrome* Kniest syndrome* Mandibulofacial dysostosis* Megalophthalmos* Pierre Robin syndrome* Oxycephaly* Refsum disease* Retinitis pigmentosa* Wildervanck syndrome*

Inheritance AD AR AD/AR AD AR AR AR AD XL/AD AD AD/AR AR

AR AD/AR/XL

AD ⫽ autosomal dominant; AR ⫽ autosomal recessive; XL ⫽ X-linked. * Less frequent.

Methods and Results of DNA Analysis Total RNA was isolated from the patient’s cultured fibroblasts, and the cDNA was obtained as described earlier.12 Automated DNA sequencing was performed at the Duke University Comprehensive Cancer Center facility using a Perkin Elmer/ABI 377 DNA Sequencer and dRhodamine sequencing chemistry. Sequence analysis revealed a 623C ⬎ A transversion on one allele and an 1109C ⬎ A mutation on the second, predicting A208D and S370Y substitutions, respectively, in the protein (Fig 7). A 1019G ⬎ A transition was also observed consistently in clones exhibiting the 1109C ⬎ A mutation. This would result in an R340Q substitution but is unlikely to impair sulfite oxidase activity since this residue is not conserved in other species. In fact, the amino acid in the corresponding position in the native chicken enzyme is a glutamine.13 The A208D and S370Y mutations, conversely, are predicted to have dramatic effects on sulfite oxidase activity. The crystal structure of the chicken enzyme shows that the protein is folded into three domains: an amino terminal heme domain, a central molybdenum-containing domain, and a carboxyl terminal third domain, which contains most of the residues involved in dimer formation.13 Ala208 is found in the second domain next to a cysteine (207) in the molybdenum active site. The sulfur of Cys207 is the sole protein ligand to the molybdenum, and disruption of the metal ligand field by replacing this cysteine with serine leads to severe attenuation of sulfite oxidase activity.14 It is likely that altering the adjacent residue would also affect the ligation of Cys207 to molybdenum. Substitution of an aspartic acid for Ala208 could also disrupt an active site hydrophobic pocket, evident from the crystal structure, and interfere with the binding of a water ligand to an essential tyrosine. The S370Y mutation falls in the core of the third domain of sulfite oxidase. The presence of the bulky tyrosine would be predicted to cause serious steric interference and possibly prevent dimerization by impairing the normal folding of the third domain.

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Discussion The neuropathologic features of sulfite oxidase deficiency are dramatic but generally nonspecific. The salient features are gross cerebral atrophy and ventricular dilation. The cerebral white matter is greatly diminished and has cystic spaces. The basal ganglia, brainstem, and cerebellum are also atrophic and cystic.7 Microscopic examination shows loss of neurons, extensive demyelination, proliferation of astrocytes, and areas of calcification.7 Neuroradiologic investigation correlates well with the neuropathologic findings. Computed tomographic and magnetic resonance imaging scans initially show cerebral edema followed by gross cerebral and cerebellar atrophy. Although computed tomography will show areas of focal calcification, both imaging methods will show cystic spaces and abnormal gray–white matter interfaces.6 These changes are not diagnostic but are helpful in evaluating the progression of the disease. The cases described in this report displayed many of the classic neuroradiologic and neuropathologic features described in the literature. There is a limited understanding of the biochemical link between sulfite oxidase deficiency and the neuropathologic findings. It is unknown whether the white matter damage arises from the accumulation of a toxic metabolite such as sulfite or from a deficit of the product sulfate.6 The biochemical toxicity of sulfite has been described in the past.15,16 It is suggested that it may destroy thiamine, resulting in abnormal pyruvate metabolism in the central nervous system.6,17 The central nervous system may be more sensitive to the accumulation of sulfite, perhaps because of a disturbance of neurotransmitter function or because of an alteration in the supply of cysteine to certain parts of the brain.6 Alternatively, disturbed brain development may also occur in this disorder because of a lack of sulfate production necessary for the formation of the sulfatides of neural tissue.6 A recent review by Leuder and Steiner5 discusses lens subluxation as well as other ophthalmic abnormalities apparent in the condition. The natural history of ectopia lentis is difficult to delineate because not all cases display lens subluxation in the first year of life, when usually examined, and not all previously reported cases had an ophthalmologic assessment.5 The direction of lens subluxation is unpredictable, and the etiology is poorly defined. The lens zonule has a high concentration of cysteine.18 Speculation exists that the abnormal sulfur metabolism in sulfite oxidase deficiency decreases the levels of cysteine, thereby influencing the integrity of the zonule. Lens subluxation may also arise as a result of disruption of cysteine disulfide bonds by excess sulfite.1 Other ocular abnormalities in the isolated form of the disease are nystagmus, myopia, strabismus, nonreactive pupils, and optic atrophy.2,5,7 Our proband case also demonstrated strabismus, sluggish pupils, and optic atrophy. Both molybdenum cofactor deficiency and isolated sulfite oxidase deficiency are inherited as autosomal-recessive conditions. The obligate heterozygotes display no symptoms. Isolated sulfite oxidase deficiency occurs in white and nonwhite patients from Europe, North America, northern

Edwards et al 䡠 Sulfite Oxidase Deficiency Africa, Turkey, and Asia.6 The condition generally is severe and often fatal, so prevention of new cases by proper screening and genetic counseling is paramount. Prenatal diagnosis of either molybdenum cofactor deficiency or isolated sulfite oxidase deficiency can be easily accomplished with an assay of sulfite oxidase activity in uncultured chorionic villus tissue.19,20 S-sulfocysteine can also be detected in amniotic fluid.21 It is unfortunate that the diagnosis was not recognized in the first affected infant, as prenatal screening could have detected the condition in the second affected sibling in utero. The patients described here were both very severely affected infants. However, individuals with milder clinical symptoms and later onset have been identified, most often among those with isolated sulfite oxidase deficiency.22 It is possible that some patients, especially those with milder forms of sulfite oxidase deficiency, will respond to diets lower in sulfur amino acids, further emphasizing the potential benefits of early screening and intervention. In conclusion, sulfite oxidase deficiency is an inborn error of metabolism with unique clinical features and biochemical abnormalities. It can be diagnosed with the appropriate assays yet can be easily missed on routine screening as exemplified by the first affected sibling in our case family. In the second sibling, the diagnosis was considered after discovery of lens subluxation. Although a rare disease, sulfite oxidase deficiency cases are being reported more frequently now, perhaps because of better clinical recognition and metabolic screening. If physicians are more aware of this disorder, then additional cases, as occurred in this family, will not be missed. Our cases add to the growing body of literature on this condition, reaffirming the critical role of ophthalmic examination in the investigation of infants with seizures of unknown etiology.

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