The biochemical investigation of genetic disorders responsive to vitamin B6 supplementation

The active form of vitamin B6, pyridoxal 5’-phosphate (PLP), is a cofactor required for many essential functions such as the metabolism of amino acids and neurotransmitters, the one-carbon cycle, haem biosynthesis, glycogenolysis, and sphingolipid metabolism. Humans are not capable of de novo PLP synthesis but do have a pathway for the interconversion of B6 vitamers. Several inborn errors of metabolism (IEMs) can lead to an insufficient supply of available PLP (e.g. pyridox(am)ine 5’-phosphate oxidase [PNPO], aldehyde dehydrogenase 7 family member A1 [ALDH7A1] and pyridoxal 5’-phosphate homeostasis protein [PLPHP] deficiencies). These disorders are typically characterised by neonatal/infantile-onset seizures refractive to standard anti-epileptic drugs but responsive to vitamin B6 supplementation. This thesis describes the investigation of B6 vitamer measurement from dried blood spots (DBS) as a diagnostic method for the B6-responsive epilepsies with a focus on PNPO deficiency. In addition, a diagnostic LC-MS/MS-based enzyme assay was developed for the measurement of PNPO activity from DBS. The biochemical effect of a novel IEM leading to pyridoxal kinase deficiency was also characterised using LC-MS/MS-based enzyme assays. Some PNPO deficient individuals receiving high-dose PLP for seizure treatment develop signs of liver damage leading eventually to cirrhosis. The photodegradation profile of PLP was characterised in order to help elucidate the mechanism causing liver damage in these patients; hypotheses as to the cause of this phenomenon are discussed. Vitamin B6 has been reported as an effective anticonvulsant in genetic epilepsies other than those known to directly affect vitamin B6 metabolism. Whole exome and whole genome sequencing data was used in order to investigate a genetic basis for vitamin B6-responsive seizures in 5 children. In two of these individuals, variants affecting the ion channel KCNQ2 were identified. A response to B6 supplementation in cases of KCNQ2-related epilepsy has also been documented in the literature. The mechanism behind this was investigated using electrophysiological techniques