Pyridox(AM)ine 5'-phosphate oxidase activity in patients with vitamin b6 responsive epilepsy: Findings and the effect of the common p.r116q variant upon enzymatic activity

Abstract

Objective: Determination of PNPO activity in patients with variants identified in the PNPO gene, child hospital controls and those with other B6-responsive seizure disorders. Methods: LC-MS/MS was used to quantify Pyridox(am)ine 5'-phosphate oxidase (PNPO) activity in dried blood spots (DBS) using a method that we have recently developed. Results: PNPO deficiency is an autosomal recessive disorder classically characterized by early onset epileptic encephalopathy responsive to high doses of vitamin B6. Control PNPO activity ranges were established using DBS from 37 hospital controls (5 d-15 y) and 18 children (1 m-16 y) receiving B6 supplementation for seizure control in whom PNPO deficiency had been excluded. These cohorts had PNPO activities of 10.0-95.0 pmol DBS-1 h-1 (mean = 41.7 pmol DBS-1 h-1) and 23.0-85.9 pmol DBS-1 h-1 (mean = 56.0 pmol DBS-1 h-1), respectively. No correlation of PNPO activity with age was found. Patients receiving B6 supplementation had mildly increased PNPO activity compared to those not receiving supplements (P < .05). Patients with mutations in PNPO (n = 19) had PNPO activities significantly lower (nd: 4.6 pmol DBS-1 h-1; mean = 1.1 pmol DBS-1 h-1) (P < .0001) than those measured in control cohorts with all of the 14 potentially pathogenic variants investigated having a dramatic effect on activity. Of these individuals, 3/19 (including one asymptomatic sibling who is not on B6 supplementation) were homozygous for c.347G>A (p.R116Q), present in the general population with an allele frequency of 0.0558 (ExAC) suggesting that it could be a polymorphism. Whilst previous in vitro studies have shown that this variant has high residual activity (80% of WT), in our study all 3 individuals were found to have low DBS PNPO activity (< 0.12 pmol DBS-1 h-1). Conclusion: PNPO activity in DBS is dramatically decreased in patients homozygous for the p.R116Q variant, providing further evidence for p.R116Q as an epilepsy susceptibility locus. However, questions remain with regards to its variable pathogenicity. This variability is likely dependent upon other genetic or environmental factors. These could include variable tissue-specific expression of the PNPO protein, variants in another gene affecting B6 homeostasis or at another epilepsy susceptibility locus, dietary vitamin B6 intake and maternal B2/B6 status during pregnancy or breastfeeding.L6236785442018-09-04 <br />