Molecular genetic analysis of candidate genes for glutaric aciduria type II in a cohort of patients from Queensland, Australia.

Glutaric aciduria type II (GAII) is a heterogeneous genetic disorder affecting mitochondrial fatty acid, amino acid and choline oxidation. Clinical manifestations vary across the lifespan and onset may occur at any time from the early neonatal period to advanced adulthood. Historically, some patients, in particular those with late onset disease, have experienced significant benefit from riboflavin supplementation. GAII has been considered an autosomal recessive condition caused by pathogenic variants in the gene encoding electron-transfer flavoprotein ubiquinone-oxidoreductase (ETFDH) or in the genes encoding electron-transfer flavoprotein subunits A and B (ETFA and ETFB respectively). Variants in genes involved in riboflavin metabolism have also been reported. However, in some patients, molecular analysis has failed to reveal diagnostic molecular results. In this study, we report the outcome of molecular analysis in 28 Australian patients across the lifespan, 10 paediatric and 18 adult, who had a diagnosis of glutaric aciduria type II based on both clinical and biochemical parameters. Whole genome sequencing was performed on 26 of the patients and two neonatal onset patients had targeted sequencing of candidate genes. The two patients who had targeted sequencing had biallelic pathogenic variants (in ETFA and ETFDH). None of the 26 patients whose whole genome was sequenced had biallelic variants in any of the primary candidate genes. Interestingly, nine of these patients (34.6%) had a monoallelic pathogenic or likely pathogenic variant in a single primary candidate gene and one patient (3.9%) had a monoallelic pathogenic or likely pathogenic variant in two separate genes within the same pathway. The frequencies of the damaging variants within ETFDH and FAD transporter gene SLC25A32 were significantly higher than expected when compared to the corresponding allele frequencies in the general population. The remaining 16 patients (61.5%) had no pathogenic or likely pathogenic variants in the candidate genes. Ten (56%) of the 18 adult patients were taking the selective serotonin reuptake inhibitor antidepressant sertraline, which has been shown to produce a GAII phenotype, and another two adults (11%) were taking a serotonin-norepinephrine reuptake inhibitor antidepressant, venlafaxine or duloxetine, which have a mechanism of action overlapping that of sertraline. Riboflavin deficiency can also mimic both the clinical and biochemical phenotype of GAII. Several patients on these antidepressants showed an initial response to riboflavin but then that response waned. These results suggest that the GAII phenotype can result from a complex interaction between monoallelic variants and the cellular environment. Whole genome or targeted gene panel analysis may not provide a clear molecular diagnosis.

Modified turbidometric microassay for the measurement of sulfate in plasma and urine.

Sulfate is the fourth most abundant anion in circulation. Despite being an essential nutrient for healthy growth and development, sulfate is not routinely measured in clinical settings. In research settings, animal studies have shown that hyposulfatemia and hypersulfaturia are associated with adverse developmental outcomes. Those findings have increased interest in measuring plasma and urine sulfate levels. In this study, we describe a modified assay to measure sulfate in low volumes of plasma and urine. •A streamlined microassay to measure sulfate levels using a microtiter plate format was developed.•To determine the robustness of the assay, this method assessed reagent stability and concentrations, as well as absorbance at different wavelengths and following a range of incubation times.•The optimized microassay was used to measure sulfate level in pig plasma and urine samples, which were compared to a validated ion chromatography method.

Rapid amino acid quantitation with pre-column derivatization; ultra-performance reverse phase liquid chromatography and single quadrupole mass spectrometry.

BACKGROUND: We optimized a quantitative amino acid method with pre-column derivatization, norvaline (nva) internal standard and reverse phase ultra-performance liquid chromatography by replacing the ultraviolet detector with a single quadrupole mass spectrometer (MSnva). METHOD: We used 13C15N isotopically labeled amino acid internal standards and a C18 column with 1.6µm particles to optimize the chromatography and to confirm separation of isobaric compounds (MSlis). We compared the analytical performance of MSnva with MSlis and the original method (UVnva) with clinical samples. RESULTS: The chromatography time per sample of MSnva was 8min, detection capabilities were <1µmol/L for most components, intermediate imprecisions at low concentrations were <10% and there was negligible carryover. MSnva was linear up to a total amino acid concentration in a sample of approximately 9500µmol/L. The agreements between most individual amino acids were satisfactory compared to UVnva with the latter prone to outliers and suboptimal quantitation of urinary arginine, aspartate, glutamate and methionine. MSnva reliably detected argnininosuccinate, ß-alanine, citrulline and cysteine-s-sulfate. CONCLUSION: MSnva resulted in a more than fivefold increase in operational efficiency with accurate detection of amino acids and metabolic intermediates in clinical samples.

Reference intervals for plasma sulfate and urinary sulfate excretion in pregnancy.

BACKGROUND: Sulfate is important for fetal growth and development. During pregnancy, the fetus relies on sulfate from the maternal circulation. We report reference intervals for maternal plasma sulfate levels and fractional excretion index (FEI) for sulfate in pregnancy, as well as sulfate levels in cord blood from term pregnancies. METHODS: Plasma and urine were collected from 103 pregnant women of 10-20 weeks gestation and 106 pregnant women of 30-37 weeks gestation. Venous cord plasma was collected from 80 healthy term babies. Sulfate levels were measured by ion chromatography. Plasma and urinary creatinine levels were used to calculate FEI sulfate in pregnant women. Analyses provide reference intervals, and explored the relationship between maternal sulfate data with several prenatal factors. RESULTS: Median maternal plasma sulfate levels were 452 µmol/L and 502 µmol/L at 10-20 and 30-37 weeks gestation, respectively, and inversely correlated with FEI sulfate median values of 0.15 and 0.11. Overall reference intervals were 305-710 and 335-701 µmol/L (2.5th; 97.5th percentile; for 10-20 and 30-37 weeks gestation, respectively) for maternal plasma sulfate, and 0.06-0.31 and 0.05-0.28 for maternal FEI sulfate. Term venous cord plasma sulfate median levels were significantly (p = 0.038) higher in female babies (375 µmol/L) when compared to male babies (342 µmol/L), with an overall reference interval of 175-603 µmol/L. CONCLUSIONS: We provide the first reference intervals for maternal plasma sulfate levels and FEI sulfate, as well as cord plasma sulfate levels. These findings provide reference data for further studies of sulfate levels in both mother and child.

Resuscitation of a neonate with medium chain acyl-coenzyme a dehydrogenase deficiency using extracorporeal life support.

We report a neonate with medium chain acyl-coenzyme A dehydrogenase deficiency (MCAD) who had a cardiac arrest due to ventricular tachycardia and fibrillation. Extracorporeal life support (ECLS) was deployed, from which the baby was subsequently separated and discharged from hospital. This case was a rare neonatal presentation of MCAD and an uncommon indication for ECLS. We discuss the presentations of patients with MCAD and the use of ECLS for patients with possible inborn errors of metabolism and other unknown primary diagnoses.

Plasma and urinary sulfate determination in a cohort with autism.

Sulfate is important for mammalian development but is not routinely measured in clinical settings. The renal NaS1 sulfate transporter maintains circulating sulfate levels and is linked to renal sulfate wasting in mice. Some autistic individuals exhibit renal sulfate wasting, but the etiology is yet unknown. We measured plasma and urinary sulfate levels, calculated the fractional excretion index (FEI) of sulfate, and screened for two loss-of-function NaS1 sequence variants (R12X and N174S) in 23 autistic individuals. The FEI sulfate values ranged from 0.13 to 0.50. NaS1 variants were detected in 18 of the 23 individuals (11 heterozygous N174S, four homozygous N174S, two heterozygous R12X, and one individual carried both R12X and N174S). Those individuals with neither sequence variant had FEI sulfate ≤ 0.34, whereas FEI sulfate ≥ 0.35 was found in about 60 % (11 of 18) of individuals that had R12X and/or N174S. This study links renal sulfate wasting with loss-of-function NaS1 sequence variants in humans.