Antiquitin Deficiency Research Articles

Urinary AASA excretion is elevated in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency

Analysis of α-aminoadipic semialdehyde is an important tool in the diagnosis of antiquitin deficiency (pyridoxine-dependent epilepsy). However continuing use of this test has revealed that elevated urinary excretion of α-aminoadipic semialdehyde is not only found in patients with pyridoxine-dependent epilepsy but is also seen in patients with molybdenum cofactor deficiency and isolated sulphite oxidase deficiency. This should be taken into account when interpreting the laboratory data. Sulphite was shown to inhibit α-aminoadipic semialdehyde dehydrogenase in vitro.

The measurement of urinary Δ1‐piperideine‐6‐carboxylate, the alter ego of α‐aminoadipic semialdehyde, in Antiquitin deficiency

The assessment of urinary α-aminoadipic semialdehyde (α-AASA) has become the diagnostic laboratory test for pyridoxine dependent seizures (PDS). α-AASA is in spontaneous equilibrium with its cyclic form Δ1-piperideine-6-carboxylate (P6C); a molecule with a heterocyclic ring structure. Ongoing diagnostic screening and monitoring revealed that in some individuals with milder ALDH7A1 variants, and patients co-treated with a lysine restricted diet, α-AASA was only modestly increased. This prompted us to investigate the diagnostic power and added value of the assessment of urinary P6C compared to α-AASA. Urine samples were diluted to a creatinine content of 0.1 mmol/L, followed by the addition of 0.01 nmol [2H9]pipecolic acid as internal standard (IS) and 5 μL was injected onto a Waters C18 T3 HPLC column. Chromatography was performed using water/methanol 97/3 (v/v) including 0.03 % formic acid by volume with a flow rate of 150 μL/min and detection was accomplished in the multiple reaction monitoring mode: P6C m/z 128.1 > 82.1; [2H9]pipecolic acid m/z 139.1 > 93.1. Due to the dualistic nature of α-AASA/P6C, and the lack of a proper internal standard, the method is semi quantitative. The intra-assay CVs (n = 10) for two urine samples of proven PDS patients with only modest P6C increases were 4.7% and 8.1%, whereas their inter-assay CVs (n = 10) were 16 and 18% respectively. In all 40 urine samples from 35 individuals with proven PDS, we detected increased levels of P6C. Therefore, we conclude that the diagnostic power of the assessments of urinary P6C and α-AASA is comparable.

Status epilepticus in a neonate treated with pyridoxine because of a familial recurrence risk for antiquitin deficiency: pyridoxine toxicity?

Pyridoxine-dependent epilepsy (PDE) is a treatable inborn error of metabolism with autosomal recessive inheritance. Antenatal and postnatal prophylactic administration of pyridoxine has been recommended to improve the developmental outcome in possible future pregnancies. We report on a male offspring of a second pregnancy at risk for PDE. While on prophylactic treatment with oral pyridoxine, the newborn developed encephalopathy and status epilepticus at age 14 days. Seizures did not respond to parenteral pyridoxine and additional treatment with folinic acid. After treatment was changed to pyridoxal 5'-phosphate, the infant's condition improved. Antiquitin deficiency was excluded by biochemical and molecular genetic testing, and cofactor treatment was stopped on day 26. He has since remained seizure-free with normal psychomotor development. In healthy newborns, high-dose treatment with pyridoxine may result in increased rather than decreased neuroexcitability. Postnatal prophylactic pyridoxine treatment of fetuses and neonates at risk for PDE should be limited to the shortest possible time, by either prenatal diagnosis or immediate postnatal biochemical and genetic testing.

Pyridoxal 5'‐phosphate in cerebrospinal fluid; factors affecting concentration

Analysis of pyridoxal 5'-phosphate (PLP) concentration in 256 cerebrospinal fluid (CSF) samples from patients with neurological symptoms showed that the variance is greater than indicated by previous studies. The age-related lower reference limit has been revised to detect inborn errors of metabolism that lead to PLP depletion without a high false positive rate: < 30 days, 26 nmol/L; 30 days to 12 months, 14 nmol/L; 1-2 years, 11 nmol/L; > 3 years, 10 nmol/L. Inborn errors leading to PLP concentrations below these values include pyridoxine-dependent epilepsy due to antiquitin deficiency, and molybdenum cofactor deficiency that leads to the accumulation of sulfite, a nucleophile capable of reacting with PLP. Low PLP levels were also seen in a group of children with transiently elevated urinary excretion of sulfite and/or sulfocysteine, suggesting that there may be other situations in which sulfite accumulates and inactivates PLP. There was no evidence that seizures or the anticonvulsant drugs prescribed for patients in this study led to significant lowering of CSF PLP. A small proportion of patients receiving L-dopa therapy were found to have a CSF PLP concentration below the appropriate reference range. This may have implications for monitoring and treatment. A positive correlation was seen between the CSF PLP and 5-methyl-tetrahydrofolate (5-MTHF) and tetrahydrobiopterin (BH(4)) concentrations. All are susceptible to attack by nucleophiles and oxygen-derived free-radicals, and CSF has relatively low concentrations of other molecules that can react with these compounds. Further studies of CSF PLP levels in a wide range of neurological diseases might lead to improved understanding of pathogenesis and possibilities for treatment.

Genotypic and phenotypic spectrum of pyridoxine-dependent epilepsy (ALDH7A1 deficiency)

Pyridoxine-dependent epilepsy was recently shown to be due to mutations in the ALDH7A1 gene, which encodes antiquitin, an enzyme that catalyses the nicotinamide adenine dinucleotide-dependent dehydrogenation of l-α-aminoadipic semialdehyde/l-Δ1-piperideine 6-carboxylate. However, whilst this is a highly treatable disorder, there is general uncertainty about when to consider this diagnosis and how to test for it. This study aimed to evaluate the use of measurement of urine l-α-aminoadipic semialdehyde/creatinine ratio and mutation analysis of ALDH7A1 (antiquitin) in investigation of patients with suspected or clinically proven pyridoxine-dependent epilepsy and to characterize further the phenotypic spectrum of antiquitin deficiency. Urinary l-α-aminoadipic semialdehyde concentration was determined by liquid chromatography tandem mass spectrometry. When this was above the normal range, DNA sequencing of the ALDH7A1 gene was performed. Clinicians were asked to complete questionnaires on clinical, biochemical, magnetic resonance imaging and electroencephalography features of patients. The clinical spectrum of antiquitin deficiency extended from ventriculomegaly detected on foetal ultrasound, through abnormal foetal movements and a multisystem neonatal disorder, to the onset of seizures and autistic features after the first year of life. Our relatively large series suggested that clinical diagnosis of pyridoxine dependent epilepsy can be challenging because: (i) there may be some response to antiepileptic drugs; (ii) in infants with multisystem pathology, the response to pyridoxine may not be instant and obvious; and (iii) structural brain abnormalities may co-exist and be considered sufficient cause of epilepsy, whereas the fits may be a consequence of antiquitin deficiency and are then responsive to pyridoxine. These findings support the use of biochemical and DNA tests for antiquitin deficiency and a clinical trial of pyridoxine in infants and children with epilepsy across a broad range of clinical scenarios.

Simultaneous determination of alpha-aminoadipic semialdehyde, piperideine-6-carboxylate and pipecolic acid by LC–MS/MS for pyridoxine-dependent seizures and folinic acid-responsive seizures

Pyridoxine-dependent seizures (PDS) is an autosomal recessive disorder characterized by seizures presenting in neonates or infants up to 3 years of age which respond to pharmacological doses of pyridoxine. Alpha-aminoadipic semialdehyde dehydrogenase (antiquitin) deficiency was identified as an underlying defect in PDS characterized by accumulation of alpha-aminoadipic semialdehyde (α-AASA) as a specific marker and recently folinic acid-responsive seizures (FRS) were found to be allelic to PDS as the putative mutations were identified in the antiquitin gene ( ALDH7A1). α-AASA is known to be in reversible equilibrium with its cyclic Shiff base, δ 1-piperideine-6-carboxylate (P6C). Pipecolic acid (PA) is another biomarker often elevated but is not specific to PDS. Here, we developed the liquid chromatography–mass spectrometry (LC–MS/MS) method to determine the analytes of α-AASA, P6C and PA simultaneously in plasma and validated the assay using samples from confirmed cases. This approach eliminates the extra time and expense of running multiple assays and provides valuable information for the rapid diagnosis and treatment of patients with PDS and FRS which potentially could lead to a better outcome with improved quality of life. The stability study showed that α-AASA and P6C were unstable even at −20 °C. A careful sample handling with immediate freezing and testing is required for reliable result.

Pyridoxine-dependent Epilepsy: Normal Outcome in a Patient with Late Diagnosis after Prolonged Status Epilepticus Causing Cortical Blindness

We report on a male proband with pyridoxine-dependent epilepsy (PDE) and neonatal seizure onset. At the age of 31 months, a prolonged status epilepticus led to severe neurological regression with cortical blindness, loss of speech and muscular hypotonia with slow recovery over the following 3 months. At 33 months of age pyridoxine therapy was initiated with excellent response and the boy remained seizure-free on pyridoxine monotherapy, except for two occasions with seizure recurrence 10 days after accidental pyridoxine withdrawal. alpha-aminoadipic semialdehyde dehydrogenase (antiquitin) deficiency was indicated by elevated pipecolic acid concentrations in plasma and alpha-aminoadipic semialdehyde excretion in urine. Molecular analysis of the antiquitin gene revealed a novel missense mutation c.57insA, while the mutation of the other allele remained unidentified so far. Despite the delay in diagnosis and prolonged status epilepticus, neuropsychological evaluations at the ages of 11 and 18 years demonstrated full-scale IQ of 93 and 92, respectively, with better verbal IQ (103 and 101) than performance IQ (85 and 82).

Inborn errors affecting vitamin B 6 metabolism

Vitamin B6is an important vitamin for normal brain function. The metabolism of dietary vitamin B6to its active cofactor pyridoxal 5´-phosphate is described. The mechanism of action of pyridoxal 5´-phosphate is described, as are some important functions in the brain. The clinical features and biochemistry of three inborn errors of metabolism affecting brain pyridoxal 5´-phosphate concentrations are described, each of which cause early-onset epilepsy of variable severity. These are pyridoxine phosphate oxidase deficiency, hyperprolinemia Type 2 and pyridoxine-dependent epilepsy caused by antiquitin deficiency. Hypophosphatasia is also discussed briefly, as the epilepsy that can complicate this disorder appears to be due to pyridoxal phosphate deficiency. Lastly, the antiepileptic properties of pyridoxine and pyridoxal phosphate are discussed.