An LC-MS/MS-Based Method for the Quantification of Pyridox(am)ine 5'-Phosphate Oxidase Activity in Dried Blood Spots from Patients with Epilepsy

We report the development of a rapid, simple, and robust LC-MS/MS-based enzyme assay using dried blood spots (DBS) for the diagnosis of pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency (OMIM 610090). PNPO deficiency leads to potentially fatal early infantile epileptic encephalopathy, severe developmental delay, and other features of neurological dysfunction. However, upon prompt treatment with high doses of vitamin B6, affected patients can have a normal developmental outcome. Prognosis of these patients is therefore reliant upon a rapid diagnosis. PNPO activity was quantified by measuring pyridoxal 5'-phosphate (PLP) concentrations in a DBS before and after a 30 min incubation with pyridoxine 5'-phosphate (PNP). Samples from 18 PNPO deficient patients (1 day-25 years), 13 children with other seizure disorders receiving B6 supplementation (1 month-16 years), and 37 child hospital controls (5 days-15 years) were analyzed. DBS from the PNPO-deficient samples showed enzyme activity levels lower than all samples from these two other groups as well as seven adult controls; no false positives or negatives were identified. The method was fully validated and is suitable for translation into the clinical diagnostic arena

Smoking-induced expression of the GPR15 gene indicates its potential role in chronic inflammatory pathologies

Despite the described clear epigenetic effects of smoking, the effect of smoking on genome-wide gene expression in the blood is obscure. We therefore studied the smoking-induced changes in the gene-expression profile of the peripheral blood. RNA was extracted from the whole blood of 48 individuals with a detailed smoking history (24 never-smokers, 16 smokers, and 8 ex-smokers). Gene-expression profiles were evaluated with RNA sequencing, and results were analyzed separately in 24 men and 24 women. In the male smokers, 13 genes were statistically significantly (false-discovery rate <0.1) differentially expressed; in female smokers, 5 genes. Although most of the differentially expressed genes were different between the male and female smokers, the G-protein–coupled receptor 15 gene (GPR15) was differentially expressed in both male and female smokers compared with never-smokers. Analysis of GPR15 methylation identified significantly greater hypomethylation in smokers compared with that in never-smokers. GPR15 is the chemoattractant receptor that regulates T-cell migration and immunity. Up-regulation of GPR15 could explain to some extent the health hazards of smoking with regard to chronic inflammatory diseases

The incidence of movement disorder increases with age and contrasts with subtle and limited neuroimaging abnormalities in argininosuccinic aciduria.

Publication status: PublishedFunder: NIHR Great Ormond Street Hospital Biomedical Research Centre; doi: http://dx.doi.org/10.13039/501100019256Funder: Moderna Therapeutics; doi: http://dx.doi.org/10.13039/100019533Argininosuccinate lyase (ASL) is integral to the urea cycle detoxifying neurotoxic ammonia and the nitric oxide (NO) biosynthesis cycle. Inherited ASL deficiency causes argininosuccinic aciduria (ASA), a rare disease with hyperammonemia and NO deficiency. Patients present with developmental delay, epilepsy and movement disorder, associated with NO-mediated downregulation of central catecholamine biosynthesis. A neurodegenerative phenotype has been proposed in ASA. To better characterise this neurodegenerative phenotype in ASA, we conducted a retrospective study in six paediatric and adult metabolic centres in the UK in 2022. We identified 60 patients and specifically looked for neurodegeneration-related symptoms: movement disorder such as ataxia, tremor and dystonia, hypotonia/fatigue and abnormal behaviour. We analysed neuroimaging with diffusion tensor imaging (DTI) magnetic resonance imaging (MRI) in an individual with ASA with movement disorders. We assessed conventional and DTI MRI alongside single photon emission computer tomography (SPECT) with dopamine analogue radionuclide 123 I-ioflupane, in Asl-deficient mice treated by hASL mRNA with normalised ureagenesis. Movement disorders in ASA appear in the second and third decades of life, becoming more prevalent with ageing and independent from the age of onset of hyperammonemia. Neuroimaging can show abnormal DTI features affecting both grey and white matter, preferentially basal ganglia. ASA mouse model with normalised ureagenesis did not recapitulate these DTI findings and showed normal 123 I-ioflupane SPECT and cerebral dopamine metabolomics. Altogether these findings support the pathophysiology of a late-onset movement disorder with cell-autonomous functional central catecholamine dysregulation but without or limited neurodegeneration of dopaminergic neurons, making these symptoms amenable to targeted therapy