Effect of drugs on renal development

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Abnormal VLCADD newborn screening resembling MADD in four neonates with decreased riboflavin levels and VLCAD activity

Early detection of congenital disorders by newborn screening (NBS) programs is essential to prevent or limit disease manifestation in affected neonates. These programs balance between the detection of the highest number of true cases and the lowest number of false-positives. In this case report, we describe four unrelated cases with a false-positive NBS result for very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Three neonates presented with decreased but not deficient VLCAD enzyme activity and two of them carried a single heterozygous ACADVL c.1844G>A mutation. Initial biochemical investigations after positive NBS referral in these infants revealed acylcarnitine and organic acid profiles resembling those seen in multiple acyl-CoA dehydrogenase deficiency (MADD). Genetic analysis did not reveal any pathogenic mutations in the genes encoding the electron transfer flavoprotein (ETF alpha and beta subunits) nor in ETF dehydrogenase. Subsequent further diagnostics revealed decreased levels of riboflavin in the newborns and oral riboflavin administration normalized the MADD-like biochemical profiles. During pregnancy, the mothers followed a vegan, vegetarian or lactose-free diet which probably caused alimentary riboflavin deficiency in the neonates. This report demonstrates that a secondary (alimentary) maternal riboflavin deficiency in combination with reduced VLCAD activity in the newborns can result in an abnormal VLCADD/MADD acylcarnitine profile and can cause false-positive NBS. We hypothesize that maternal riboflavin deficiency contributed to the false-positive VLCADD neonatal screening results

Organic anion transporter 1 and 3 influence cellular energy metabolism in renal proximal tubule cells

Organic anion transporter (OAT) 1 and 3 are, besides uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype

The role of clinical response to treatment in determining pathogenicity of genomic variants

Purpose: The 2015 American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants provide a framework to standardize terminology in the classification of variants uncovered through genetic testing. We aimed to assess the validity of utilizing clinical response to therapies specifically targeted to a suspected disease in clarifying variant pathogenicity. Methods: Five families with disparate clinical presentations and different genetic diseases evaluated and treated in multiple diagnostic settings are summarized. Results: Extended evaluations indicated possible genetic diagnoses and assigned candidate causal variants, but the cumulative clinical, biochemical, and molecular information in each instance was not completely consistent with the identified disease. Initiation of treatment specific to the suspected diagnoses in the affected individuals led to clinical improvement in all five families. Conclusion: We propose that the effect of therapies that are specific and targeted to treatable genetic diseases embodies an in vivo physiological response and could be considered as additional criteria within the 2015 ACMG/AMP guidelines in determining genomic variant pathogenicity

Organic anion transporters 1 and 3 influence cellular energy metabolism in renal proximal tubule cells

Organic anion transporter (OAT) 1 and 3 are, besides uptake transporters, key in several cellular metabolic pathways. The underlying mechanisms are largely unknown. Hence, we used human conditionally immortalized proximal tubule epithelial cells (ciPTEC) overexpressing OAT1 or OAT3 to gain insight into these mechanisms. In ciPTEC-OAT1 and -OAT3, extracellular lactate levels were decreased (by 77% and 71%, respectively), while intracellular ATP levels remained unchanged, suggesting a shift towards an oxidative phenotype upon OAT1 or OAT3 overexpression. This was confirmed by increased respiration of ciPTEC-OAT1 and -OAT3 (1.4-fold), a decreased sensitivity to respiratory inhibition, and characterized by a higher demand on mitochondrial oxidative capacity. In-depth profiling of tricarboxylic acid (TCA) cycle metabolites revealed reduced levels of intermediates converging into α-ketoglutarate in ciPTEC-OAT1 and -OAT3, which via 2-hydroxyglutarate metabolism explains the increased respiration. These interactions with TCA cycle metabolites were in agreement with metabolomic network modeling studies published earlier. Further studies using OAT or oxidative phosphorylation (OXPHOS) inhibitors confirmed our idea that OATs are responsible for increased use and synthesis of α-ketoglutarate. In conclusion, our results indicate an increased α-ketoglutarate efflux by OAT1 and OAT3, resulting in a metabolic shift towards an oxidative phenotype

Identification of Δ-1-pyrroline-5-carboxylate derived biomarkers for hyperprolinemia type II

Hyperprolinemia type II (HPII) is an inborn error of metabolism due to genetic variants in ALDH4A1, leading to a deficiency in Δ-1-pyrroline-5-carboxylate (P5C) dehydrogenase. This leads to an accumulation of toxic levels of P5C, an intermediate in proline catabolism. The accumulating P5C spontaneously reacts with, and inactivates, pyridoxal 5'-phosphate, a crucial cofactor for many enzymatic processes, which is thought to be the pathophysiological mechanism for HPII. Here, we describe the use of a combination of LC-QTOF untargeted metabolomics, NMR spectroscopy and infrared ion spectroscopy (IRIS) to identify and characterize biomarkers for HPII that result of the spontaneous reaction of P5C with malonic acid and acetoacetic acid. We show that these biomarkers can differentiate between HPI, caused by a deficiency of proline oxidase activity, and HPII. The elucidation of their molecular structures yields insights into the disease pathophysiology of HPII

Metabolite Identification Using Infrared Ion Spectroscopy – Novel Biomarkers for Pyridoxine-Dependent Epilepsy

Untargeted LC-MS based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing “grand challenge” in the utilization of this approach is metabolite identification – confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids, and are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly rendered novel insights in the pathophysiology of PDE-ALDH7A1

Metabolite Identification Using Infrared Ion Spectroscopy-Novel Biomarkers for Pyridoxine-Dependent Epilepsy

Untargeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics strategies are being increasingly applied in metabolite screening for a wide variety of medical conditions. The long-standing "grand challenge"in the utilization of this approach is metabolite identification-confidently determining the chemical structures of m/z-detected unknowns. Here, we use a novel workflow based on the detection of molecular features of interest by high-throughput untargeted LC-MS analysis of patient body fluids combined with targeted molecular identification of those features using infrared ion spectroscopy (IRIS), effectively providing diagnostic IR fingerprints for mass-isolated targets. A significant advantage of this approach is that in silico-predicted IR spectra of candidate chemical structures can be used to suggest the molecular structure of unknown features, thus mitigating the need for the synthesis of a broad range of physical reference standards. Pyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine metabolism, resulting from a mutation in the ALDH7A1 gene that leads to an accumulation of toxic levels of α-aminoadipic semialdehyde (α-AASA), piperideine-6-carboxylate (P6C), and pipecolic acid in body fluids. While α-AASA and P6C are known biomarkers for PDE in urine, their instability makes them poor candidates for diagnostic analysis from blood, which would be required for application in newborn screening protocols. Here, we use combined untargeted metabolomics-IRIS to identify several new biomarkers for PDE-ALDH7A1 that can be used for diagnostic analysis in urine, plasma, and cerebrospinal fluids and that are compatible with analysis in dried blood spots for newborn screening. The identification of these novel metabolites has directly provided novel insights into the pathophysiology of PDE-ALDH7A1