Oxidase Deficiency Research Articles

Computational analysis of functional single nucleotide polymorphisms associated with the CYP11B2 gene.

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variations in humans and play a major role in the genetics of human phenotype variation and the genetic basis of human complex diseases. Recently, there is considerable interest in understanding the possible role of the CYP11B2 gene with corticosterone methyl oxidase deficiency, primary aldosteronism, and cardio-cerebro-vascular diseases. Hence, the elucidation of the function and molecular dynamic behavior of CYP11B2 mutations is crucial in current genomics. In this study, we investigated the pathogenic effect of 51 nsSNPs and 26 UTR SNPs in the CYP11B2 gene through computational platforms. Using a combination of SIFT, PolyPhen, I-Mutant Suite, and ConSurf server, four nsSNPs (F487V, V129M, T498A, and V403E) were identified to potentially affect the structure, function, and activity of the CYP11B2 protein. Furthermore, molecular dynamics simulation and structure analyses also confirmed the impact of these nsSNPs on the stability and secondary properties of the CYP11B2 protein. Additionally, utilizing the UTRscan, MirSNP, PolymiRTS and miRNASNP, three SNPs in the 3′UTR region were predicted to exhibit a pattern change in the upstream open reading frames (uORF), and eight microRNA binding sites were found to be highly affected due to 3′UTR SNPs. This cataloguing of deleterious SNPs is essential for narrowing down the number of CYP11B2 mutations to be screened in genetic association studies and for a better understanding of the functional and structural aspects of the CYP11B2 protein.

Mitochondrial encephalomyopathy with cytochrome c oxidase deficiency caused by a novel mutation in the MTCO1 gene

Cytochrome c oxidase (COX) deficiency is one of the most common respiratory chain deficiencies. A woman was presented at the age of 18y with acute loss of consciousness, non-convulsive status epilepticus, slow neurological deterioration, transient cortical blindness, exercise intolerance, muscle weakness, hearing loss, cataract and cognitive decline. Muscle biopsy revealed ragged-red fibers, COX negative fibers and a significant decreased activity of complex IV in a homogenate. Using next generation massive parallel sequencing of the mtDNA, a novel heteroplasmic mutation was identified in MTCO1, m.7402delC, causing frameshift and a premature termination codon. Single fiber PCR showed co-segregation of high mutant load in COX negative fibers. Mutation in mitochondrially encoded complex IV subunits should be considered in mitochondrial encephalomyopathies and COX negative fibers after the common mtDNA mutations have been excluded.

A nutritional conditional lethal mutant due to pyridoxine 5'-phosphate oxidase deficiency in Drosophila melanogaster.

The concept of auxotrophic complementation has been proposed as an approach to identify genes in essential metabolic pathways in Drosophila melanogaster. However, it has achieved limited success to date, possibly due to the low probability of finding mutations fit with the chemically defined profile. Instead of using the chemically defined culture media lacking specific nutrients, we used bare minimum culture medium, i.e., 4% sucrose, for adult Drosophila. We identified a nutritional conditional lethal mutant and localized a c.95C > A mutation in the Drosophila pyridoxine 5′-phosphate oxidase gene [dPNPO or sugarlethal (sgll)] using meiotic recombination mapping, deficiency mapping, and whole genome sequencing. PNPO converts dietary vitamin B6 such as pyridoxine to its active form pyridoxal 5′-phosphate (PLP). The missense mutation (sgll95) results in the substitution of alanine to aspartate (p.Ala32Asp). The sgll95 flies survive well on complete medium but all die within 6 d on 4% sucrose only diet, which can be rescued by pyridoxine or PLP supplement, suggesting that the mutation does not cause the complete loss of PNPO activity. The sgll knockdown further confirms its function as the Drosophila PNPO. Because better tools for positional cloning and cheaper whole genome sequencing have made the identification of point mutations much easier than before, alleviating the necessity to pinpoint specific metabolic pathways before gene identification, we propose that nutritional conditional screens based on bare minimum growth media like ours represent promising approaches for discovering important genes and mutations in metabolic pathways, thereby accelerating the establishment of in vivo models that recapitulate human metabolic diseases.

Sulfite disrupts brain mitochondrial energy homeostasis and induces mitochondrial permeability transition pore opening via thiol group modification

Sulfite oxidase (SO) deficiency is biochemically characterized by the accumulation of sulfite, thiosulfate and S-sulfocysteine in tissues and biological fluids of the affected patients. The main clinical symptoms include severe neurological dysfunction and brain abnormalities, whose pathophysiology is still unknown. The present study investigated the in vitro effects of sulfite and thiosulfate on mitochondrial homeostasis in rat brain mitochondria. It was verified that sulfite per se, but not thiosulfate, decreased state 3, CCCP-stimulated state and respiratory control ratio in mitochondria respiring with glutamate plus malate. In line with this, we found that sulfite inhibited the activities of glutamate and malate (MDH) dehydrogenases. In addition, sulfite decreased the activity of a commercial solution of MDH, that was prevented by antioxidants and dithiothreitol. Sulfite also induced mitochondrial swelling and reduced mitochondrial membrane potential, Ca2+ retention capacity, NAD(P)H pool and cytochrome c immunocontent when Ca2+ was present in the medium. These alterations were prevented by ruthenium red, cyclosporine A (CsA) and ADP, supporting the involvement of mitochondrial permeability transition (MPT) in these effects. We further observed that N-ethylmaleimide prevented the sulfite-elicited swelling and that sulfite decreased free thiol group content in brain mitochondria. These findings indicate that sulfite acts directly on MPT pore containing thiol groups. Finally, we verified that sulfite reduced cell viability in cerebral cortex slices and that this effect was prevented by CsA. Therefore, it may be presumed that disturbance of mitochondrial energy homeostasis and MPT induced by sulfite could be involved in the neuronal damage characteristic of SO deficiency.

Molybdenum Trioxide Nanoparticles with Intrinsic Sulfite Oxidase Activity

Sulfite oxidase is a mitochondria-located molybdenum-containing enzyme catalyzing the oxidation of sulfite to sulfate in the amino acid and lipid metabolism. Therefore, it plays a major role in detoxification processes, where defects in the enzyme cause a severe infant disease leading to early death with no efficient or cost-effective therapy in sight. Here we report that molybdenum trioxide (MoO3) nanoparticles display an intrinsic biomimetic sulfite oxidase activity under physiological conditions, and, functionalized with a customized bifunctional ligand containing dopamine as anchor group and triphenylphosphonium ion as targeting agent, they selectively target the mitochondria while being highly dispersible in aqueous solutions. Chemically induced sulfite oxidase knockdown cells treated with MoO3 nanoparticles recovered their sulfite oxidase activity in vitro, which makes MoO3 nanoparticles a potential therapeutic for sulfite oxidase deficiency and opens new avenues for cost-effective therapies for gene-induced deficiencies.

Neurologic Injury in Isolated Sulfite Oxidase Deficiency (P4.328)

Neurologic Injury in Isolated Sulfite Oxidase Deficiency (P4.328)

Prenatal Multicystic Encephalopathy in Isolated Sulfite Oxidase Deficiency With a Novel Mutaion

FIGURE 1. Ultrasonography via the anterior fontanelle revealed cystic leukoencephalopathy (asterisks) in coronal view. This term female baby was the first child of the nonconsanguineous parents. Her mother reported sustained abdominal trembling, like the vibration of mobile phones, during the third trimester. Prenatal ultrasonography revealed an enlarged cisterna magna. She was delivered by Ceasarian section because of decelerating fetal heartbeats. Initial activity was appropriate, with a one minute Apgar score of 7 and a five minute Apgar score of 9, but she became progressive tachypneic. When 14 hours old, clonic seizures with upward gaze were noted. Brain ultrasonography revealed multicystic leukoencephalopathy of the parietal areas (Fig 1). Brain magnetic resonance imaging confirmed extensive frontal and parietal whitematter cystic encephalomalacia (Fig 2). Themother denied trauma during pregnancy. A survey of congenital infections was negative. Metabolic screening revealed positive urine sulfite (80 mg/ L) with a normal serum uric acid (5.5 mg/dL). Isolated sulfite oxidase deficiency was confirmed by the demonstration of a maternal nonsense mutation (c.1200C>G; p.Y400X) and a paternal novel missense mutation (c.1355C>A; p.G452D) in the SUOX genes.1 The differential diagnosis of neonatal multicystic encephalopathy includes gestational vascular insults, hypoxicischemic events, congenital infections, and severe metabolic diseases.2-4 In isolated sulfite oxidase deficiency, several reports document the temporal development of neuroradiological abnormalities, with cerebral edema early in the course of the disease followed by cerebral and cerebellar atrophy and subsequent cystic changes.2-4 Rapid progression of cavitary white matter changes is a striking neuroimaging finding in isolated sulfite oxidase deficiency, because multicystic leukoencephalopathy is often more

Pyridoxine responsiveness in novel mutations of the PNPO gene

To determine whether patients with pyridoxine-responsive seizures but normal biomarkers for antiquitin deficiency and normal sequencing of the ALDH7A1 gene may have PNPO mutations. We sequenced the PNPO gene in 31 patients who fulfilled the above-mentioned criteria. We were able to identify 11 patients carrying 3 novel mutations of the PNPO gene. In 6 families, a homozygous missense mutation p.Arg225His in exon 7 was identified, while 1 family was compound heterozygous for a novel missense mutation p.Arg141Cys in exon 5 and a deletion c.279_290del in exon 3. Pathogenicity of the respective mutations was proven by absence in 100 control alleles and expression studies in CHO-K1 cell lines. The response to pyridoxine was prompt in 4, delayed in 2, on EEG only in 2, and initially absent in another 2 patients. Two unrelated patients homozygous for the p.Arg225His mutation experienced status epilepticus when switched to pyridoxal 5'-phosphate (PLP). This study challenges the paradigm of exclusive PLP responsiveness in patients with pyridoxal 5'-phosphate oxidase deficiency and underlines the importance of consecutive testing of pyridoxine and PLP in neonates with antiepileptic drug-resistant seizures. Patients with pyridoxine response but normal biomarkers for antiquitin deficiency should undergo PNPO mutation analysis.

Epilepsy due to PNPO mutations: genotype, environment and treatment affect presentation and outcome

The first described patients with pyridox(am)ine 5'-phosphate oxidase deficiency all had neonatal onset seizures that did not respond to treatment with pyridoxine but responded to treatment with pyridoxal 5'-phosphate. Our data suggest, however, that the clinical spectrum of pyridox(am)ine 5'-phosphate oxidase deficiency is much broader than has been reported in the literature. Sequencing of the PNPO gene was undertaken for a cohort of 82 individuals who had shown a reduction in frequency and severity of seizures in response to pyridoxine or pyridoxal 5'-phosphate. Novel sequence changes were studied using a new cell-free expression system and a mass spectrometry-based assay for pyridoxamine phosphate oxidase. Three groups of patients with PNPO mutations that had reduced enzyme activity were identified: (i) patients with neonatal onset seizures responding to pyridoxal 5'-phosphate (n = 6); (ii) a patient with infantile spasms (onset 5 months) responsive to pyridoxal 5'-phosphate (n = 1); and (iii) patients with seizures starting under 3 months of age responding to pyridoxine (n = 8). Data suggest that certain genotypes (R225H/C and D33V) are more likely to result in seizures that to respond to treatment with pyridoxine. Other mutations seem to be associated with infertility, miscarriage and prematurity. However, the situation is clearly complex with the same combination of mutations being seen in patients who responded and did not respond to pyridoxine. It is possible that pyridoxine responsiveness in PNPO deficiency is affected by prematurity and age at the time of the therapeutic trial. Other additional factors that are likely to influence treatment response and outcome include riboflavin status and how well the foetus has been supplied with vitamin B6 by the mother. For some patients there was a worsening of symptoms on changing from pyridoxine to pyridoxal 5'-phosphate. Many of the mutations in PNPO affected residues involved in binding flavin mononucleotide or pyridoxal 5'-phosphate and many of them showed residual enzyme activity. One sequence change (R116Q), predicted to affect flavin mononucleotide binding and binding of the two PNPO dimers, and with high residual activity was found in Groups (ii) and (iii). This sequence change has been reported in the 1000 Genomes project suggesting it could be a polymorphism but alternatively it could be a common mutation, perhaps responsible for the susceptibility locus for genetic generalized epilepsy on 17q21.32 (close to rs72823592). We believe the reduction in PNPO activity and B6-responsive epilepsy in the patients reported here indicates that it contributes to the pathogenesis of epilepsy.

Effects of hematopoietic stem cell transplantation on acyl‐CoA oxidase deficiency: a sibling comparison study

Acyl-CoA oxidase (ACOX1) deficiency is a rare disorder of peroxisomal very-long chain fatty acid oxidation. No reports detailing attempted treatment, longitudinal imaging, or neuropathology exist. We describe the natural history of clinical symptoms and brain imaging in two siblings with ACOX1 deficiency, including the younger sibling's response to allogeneic unrelated donor hematopoietic stem cell transplantation (HSCT). We conducted retrospective chart review to obtain clinical history, neuro-imaging, and neuropathology data. ACOX1 genotyping were performed to confirm the disease. In vitro fibroblast and neural stem cell fatty acid oxidation assays were also performed. Both patients experienced a fatal neurodegenerative course, with late-stage cerebellar and cerebral gray matter atrophy. Serial brain magnetic resonance imaging in the younger sibling indicated demyelination began in the medulla and progressed rostrally to include the white matter of the cerebellum, pons, midbrain, and eventually subcortical white matter. The successfully engrafted younger sibling had less brain inflammation, cortical atrophy, and neuronal loss on neuro-imaging and neuropathology compared to the untreated older sister. Fibroblasts and stem cells demonstrated deficient very long chain fatty acid oxidation. Although HSCT did not halt the course of ACOX1 deficiency, it reduced the extent of white matter inflammation in the brain. Demyelination continued because of ongoing neuronal loss, which may be due to inability of transplant to prevent progression of gray matter disease, adverse effects of chronic corticosteroid use to control graft-versus-host disease, or intervention occurring beyond a critical point for therapeutic efficacy.

P30 Does a physiological COX isoform switch contribute to the clinical presentation of infantile reversible cytochrome c oxidase deficiency?

P30 Does a physiological COX isoform switch contribute to the clinical presentation of infantile reversible cytochrome c oxidase deficiency?

A Founder Mutation in PET100 Causes Isolated Complex IV Deficiency in Lebanese Individuals with Leigh Syndrome

Leigh syndrome (LS) is a severe neurodegenerative disorder with characteristic bilateral lesions, typically in the brainstem and basal ganglia. It usually presents in infancy and is genetically heterogeneous, but most individuals with mitochondrial complex IV (or cytochrome c oxidase) deficiency have mutations in the biogenesis factor SURF1. We studied eight complex IV-deficient LS individuals from six families of Lebanese origin. They differed from individuals with SURF1 mutations in having seizures as a prominent feature. Complementation analysis suggested they had mutation(s) in the same gene but targeted massively parallel sequencing (MPS) of 1,034 genes encoding known mitochondrial proteins failed to identify a likely candidate. Linkage and haplotype analyses mapped the location of the gene to chromosome 19 and targeted MPS of the linkage region identified a homozygous c.3G>C (p.Met1?) mutation in C19orf79. Abolishing the initiation codon could potentially still allow initiation at a downstream methionine residue but we showed that this would not result in a functional protein. We confirmed that mutation of this gene was causative by lentiviral-mediated phenotypic correction. C19orf79 was recently renamed PET100 and predicted to encode a complex IV biogenesis factor. We showed that it is located in the mitochondrial inner membrane and forms a ∼300 kDa subcomplex with complex IV subunits. Previous proteomic analyses of mitochondria had overlooked PET100 because its small size was below the cutoff for annotating bona fide proteins. The mutation was estimated to have arisen at least 520 years ago, explaining how the families could have different religions and different geographic origins within Lebanon.

Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase

Cytochrome c oxidase (CIV) deficiency is one of the most common respiratory chain defects in patients presenting with mitochondrial encephalocardiomyopathies. CIV biogenesis is complicated by the dual genetic origin of its structural subunits, and assembly of a functional holoenzyme complex requires a large number of nucleus-encoded assembly factors. In general, the functions of these assembly factors remain poorly understood, and mechanistic investigations of human CIV biogenesis have been limited by the availability of model cell lines. Here, we have used small interference RNA and transcription activator-like effector nucleases (TALENs) technology to create knockdown and knockout human cell lines, respectively, to study the function of the CIV assembly factor COX20 (FAM36A). These cell lines exhibit a severe, isolated CIV deficiency due to instability of COX2, a mitochondrion-encoded CIV subunit. Mitochondria lacking COX20 accumulate CIV subassemblies containing COX1 and COX4, similar to those detected in fibroblasts from patients carrying mutations in the COX2 copper chaperones SCO1 and SCO2. These results imply that in the absence of COX20, COX2 is inefficiently incorporated into early CIV subassemblies. Immunoprecipitation assays using a stable COX20 knockout cell line expressing functional COX20-FLAG allowed us to identify an interaction between COX20 and newly synthesized COX2. Additionally, we show that SCO1 and SCO2 act on COX20-bound COX2. We propose that COX20 acts as a chaperone in the early steps of COX2 maturation, stabilizing the newly synthesized protein and presenting COX2 to its metallochaperone module, which in turn facilitates the incorporation of mature COX2 into the CIV assembly line.

Non-Invasive Screening of Cytochrome c Oxidase Deficiency in Children Using a Dipstick Immunocapture Assay

Cytochrome c oxidase (CIV) deficiency is among the most common childhood mitochondrial disorders. The diagnosis of this deficiency is complex, and muscle biopsy is used as the gold standard of diagnosis. Our aim was to minimize the patient burden and to test the use of a dipstick immunocapture assay (DIA) to determine the amount of CIV in non-invasively obtained buccal epithelial cells. Buccal smears were obtained from five children with Leigh syndrome including three children exhibiting a previously confirmed CIV deficiency in muscle and fibroblasts and two children who were clinical suspects for CIV deficiency; the smear samples were analysed using CI and CIV human protein quantity dipstick assay kits. Samples from five children of similar age and five adults were used as controls. Analysis of the controls demonstrated that only samples of buccal cells that were frozen for a maximum of 4 h after collection provide accurate results. All three patients with confirmed CIV deficiency due to mutations in the SURF1 gene exhibited significantly lower amounts of CIV than the similarly aged controls; significantly lower amounts were also observed in two new patients, for whom later molecular analysis also confirmed pathologic mutations in the SURF1 gene. We conclude that DIA is a simple, fast and sensitive method for the determination of CIV in buccal cells and is suitable for the screening of CIV deficiency in non-invasively obtained material from children who are suspected of having mitochondrial disease.

Cirrhosis associated with pyridoxal 5'-phosphate treatment of pyridoxamine 5'-phosphate oxidase deficiency.

We report the case of an 8-year-old boy with pyridoxamine 5'-phosphate oxidase (PNPO) deficiency. He developed seizures at 24h of age that were refractory to standard anticonvulsant therapy and a trial of pyridoxine but responded to pyridoxal phosphate (PLP) at 28days of life. Genetic testing identified compound heterozygous mutations in the PNPO gene. Management of encephalopathic episodes required escalation of PLP dose to 100 mg/kg/day by 2 years of age. Routine blood tests at this time showed significantly deranged liver function tests (LFTs). A wedge liver biopsy showed early cirrhosis with marked elevation of pyridoxal and pyridoxic acid levels in the liver sample. Despite extensive investigation, no cause other than PLP therapy could be identified for the cirrhosis. The PLP dose was weaned to 50mg/kg/day before episodes of encephalopathy recurred. Concurrent with the reduction of his PLP dose, LFTs showed improvement. However, at 8 years of age, there is persistent evidence of hepatic fibrosis and early portal hypertension. We hypothesise that hepatic toxicity due to PLP or its degradation products is the cause of cirrhosis in this boy. Until further evidence becomes available, we would suggest that people with PNPO deficiency are treated with the minimum dose of PLP required to prevent episodes of encephalopathy.

Neurologic Injury in Isolated Sulfite Oxidase Deficiency

We review clinical, neuroimaging, and genetic information on six individuals with isolated sulfite oxidase deficiency (ISOD). All patients were examined, and clinical records, biochemistry, neuroimaging, and sulfite oxidase gene (SUOX) sequencing were reviewed. Data was available on six individuals from four nuclear families affected by ISOD. Each individual began to seize within the first week of life. neurologic development was arrested at brainstem reflexes, and severe microcephaly developed rapidly. neuroimaging within days of birth revealed hypoplasia of the cerebellum and corpus callosum and damage to the supratentorial brain looking like severe hypoxic-ischemic injury that evolved into cystic hemispheric white matter changes. Affected individuals all had elevated urinary S-sulfocysteine and normal urinary xanthine and hypoxanthine levels diagnostic of ISOD. Genetic studies confirmed SUOX mutations in four patients. ISOD impairs systemic sulfite metabolism, and yet this genetic disease affects only the brain with damage that is commonly confused with the clinical and radiologic features of severe hypoxic-ischemic encephalopathy.

Neonatal molybdenum cofactor deficiency and ectopia lentis in a Saudi Arabian patient

Molybdenum cofactor deficiency (MCD) is a rare and ultimately fatal metabolic dis-ease that results in extensive neurodegeneration in early infancy. The causal association of MCD with sulfite oxidase deficiency, a known cause of subluxed lenses (ectopia lentis), has only been recently defined. We report a 16-month-old Saudi Arabian male product of a consanguin-eous union, who presented as a neonate with intractable seizures and a failure to thrive. Sub-sequent examinations revealed hypotonia, laryngomalacia, global developmental delay, progressive neurodegeneration and ectopia lentis. Urine analysis revealed elevated sulfocys- tiene, xanthine and hypoxanthine, which suggested MCD. This diagnosis was confirmed by sub-sequent genetic analysis, which disclosed a homozygous MOCS1 mutation. A significant family history of sibling death, prior to diagnosis, from intractable seizures and respiratory distress at three months of age, most likely represents the same affliction and suggests an underestima-tion of MCD worldwide. This case underscores the need to consider and investigate MCD in all cases of intractable infantile seizures. Thus, early diagnosis and confirmatory gene analysis before permanent neurodegeneration is imperative for potential therapeutic intervention.

Kinetic results for mutations of conserved residues H304 and R309 of human sulfite oxidase point to mechanistic complexities.

Several point mutations in the gene of human sulfite oxidase (hSO) result in isolated sulfite oxidase deficiency, an inherited metabolic disorder. Three conserved residues (H304, R309, K322) are hydrogen bonded to the phosphate group of the molybdenum cofactor, and the R309H and K322R mutations are responsible for isolated sulfite oxidase deficiency. The kinetic effects of the K322R mutation have been previously reported (Rajapakshe et al., Chem. Biodiversity, 2012, 9, 1621-1634); here we investigate several mutants of H304 and R309 by steady-state kinetics, laser flash photolysis studies of intramolecular electron transfer (IET), and spectroelectrochemistry. An unexpected result is that all of the mutants show decreased rates of IET but increased steady-state rates of catalysis. However, in all cases the rate of IET is greater than the overall turnover rate, showing that IET is not the rate determining step for any of the mutations.

Letter to the Editor: Two Major Phenotypes of Sulfite Hypersensitivity: Asthma and Urticaria

The sulfites are widely used as anti-browning agents and preservatives in food, cosmetics and medicine. The use and amount of sulfites in products have recently been regulated by the Food and Drug Administration (FDA) and the Korean FDA; however, sulfite hypersensitivity reactions, ranging from bronchoconstriction,1 urticaria,2 contact dermatitis3 to life threatening anaphylaxis,4 are still being reported.5 To date, this is the first study to compare two major phenotypes of sulfite hypersensitivity, asthma and urticaria, in this country. Moreover, we carefully analyzed the clinical features of sulfite sensitive asthma, and compared them with those of sulfite tolerant asthma. We retrospectively analyzed the medical records of 26 sulfite hypersensitivity subjects confirmed by a sulfite oral provocation test (OPT). As a control group, 61 asthmatic patients negative to sulfite OPT were enrolled from Ajou University Hospital, Suwon, Korea. We performed sulfite oral provocation test on the subjects who were resistant to conventional treatment of asthma/urticaria or who had histories of allergy to sulfite containing food such as wine, dried fruits, or salads from buffet. To confirm sulfite sensitivity, we conducted single blind placebo controlled provocation test. After the asthmatic patients showed no response to gelatin capsule (EMBO CAPS lot. 051A51-22306, Suheung Capsule, Seoul, Korea) with 2 hours observation, they were administered sodium metabisulfite (Na2S2O5; Sigma Co., St. Louis, MO, USA) starting from 40 mg and increasing to 100-200 mg via gelatin capsule vehicle. The reactions were observed for 2 hours each until the hypersensitivity reactions developed,1 maximally 6 hours. Especially for bronchoconstriction reaction, 1) a decline in FEV1 of 20% or greater, or 2) a decline in FEV1 of 15% to 20% compared to the baseline with definite symptoms/signs of sulfite hypersensitivities (such as wheezing, cough or dyspnea) were considered positive. Pulmonary function tests were repeated at 30 minute intervals.6 In the case of urticarial reaction, sulfite OPT was defined as positive when hives arise; however, it was classified as negative if subjective pruritus developed. Subjects were classified into group I: sulfite sensitive asthma, group II: sulfite sensitive urticaria and group III: sulfite tolerant asthma according to their sulfite OPT reaction. In addition, group I included two patients with concurrent sulfite anaphylaxis. Sulfite sensitive asthma was a more common phenotype of sulfite hypersensitivity than sulfite sensitive urticaria (69.2% vs. 30.8%). Chronic asthma was more common in group I, as previously reported;6 however, chronic urticaria was more common in group II. Group II subjects required significantly higher provocative dosage of sulfite (187.5±35.4 mg vs. 114.4±60.0 mg, p=0.006) and a longer time (100±37.4 min vs. 54.2±34.8 min, p=0.008) to induce hypersensitivity reactions compared with group I subjects. A previous history of adverse reactions was found in 44.4% of group I and 62.5% of group II, being different from previous studies that enrolled subjects with a history of sulfite hypersensitivities (Table 1).1,7 Table 1 Comparison of Clinical Characteristics between Group I and II There were no significant differences in asthma-related parameters-such as atopic status, total IgE level, peripheral eosinophil count, FEV1% and metacholine PC20 level when the clinical characteristics were compared between group I and group III. The prevalence of severe asthma according to American Thoracic Society guidelines was significantly higher in group I than as previously reported in group III (44% vs. 16%, p=0.023).1,8 The asthma exacerbation related hospitalization rate (≥1 times/yr), emergency room visit (≥1 times/yr) and oral steroid burst (≥3 times/yr) were significantly higher in group I than in group III (66.7% vs. 24.6%, p=0.001; 66.7% vs. 24.6%, p=0.001; 55.6% vs. 17.3%, p=0.003). Many potential mechanisms of sulfite hypersensitivity have been postulated. Sulfite oxidase deficiency has been suggested as one of the mechanisms.5 Most asthmatic patients could show a sensitivity to SO2 exposure due to bronchial hypersensitivity.9 However, not all asthmatic patients with severe clinical symptoms presented sulfite hypersensitivity in this study, due to their higher prevalence of severe asthma and more frequent episodes of acute exacerbation compared to sulfite tolerant asthma. We speculate that these findings are due to individual variability of sulfite oxidase inactivity.1 It would, therefore, be helpful to investigate individual factors, including genetic factors, in order to better understand the mechanism of sulfite hypersensitivity. We report herein two major phenotypes of sulfite hypersensitivity, asthma and urticaria in Korea. Considering high prevalence of severe asthma and frequent health care utilization rate in patients with sulfite sensitive asthma, sulfite OPT can be considered as a screening test to identify exacerbating factors for severe asthma patients regardless of history of hypersensitivity reaction.

Typical and atypical phenotypes of PNPO deficiency with elevated CSF and plasma pyridoxamine on treatment

Pyridox(am)ine phosphate oxidase (PNPO) deficiency causes severe early infantile epileptic encephalopathy and has been characterized as responding to pyridoxal-5'-phosphate but not to pyridoxine. Two males with PNPO deficiency and novel PNPO mutations are reported and their clinical, metabolic, and video-electroencephalographic (EEG) findings described. The first child showed electro-clinical responses to pyridoxine and deterioration when pyridoxine was withheld. At last review, he has well-controlled epilepsy with pyridoxal-5'-phosphate monotherapy and an autism spectrum disorder. The second child had a perinatal middle cerebral artery infarct and a myoclonic encephalopathy. He failed to respond to pyridoxine but responded well to pyridoxal-5'-phosphate. At the age of 21months he has global developmental delay and hemiparesis but is seizure-free with pyridoxal-5'-phosphate monotherapy. Plasma and cerebrospinal fluid pyridoxamine levels were increased in both children during treatment with pyridoxine or pyridoxal-5'-phosphate. These observations indicate that differential responses to pyridoxine and pyridoxal-5'-phosphate treatment cannot be relied upon to diagnose PNPO deficiency.