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Genetic diagnosis plays a crucial role in rare diseases, particularly with the increasing availability of emerging and accessible treatments. The International Rare Diseases Research Consortium (IRDiRC) has set its primary goal as: "Ensuring that all patients who present with a suspected rare disease receive a diagnosis within one year if their disorder is documented in the medical literature". Despite significant advances in genomic sequencing technologies, more than half of the patients with suspected Mendelian disorders remain undiagnosed. In response, IRDiRC proposes the establishment of "a globally coordinated diagnostic and research pipeline". To help facilitate this, IRDiRC formed the Task Force on Integrating New Technologies for Rare Disease Diagnosis. This multi-stakeholder Task Force aims to provide an overview of the current state of innovative diagnostic technologies for clinicians and researchers, focusing on the patient's diagnostic journey. Herein, we provide an overview of a broad spectrum of emerging diagnostic technologies involving genomics, epigenomics and multi-omics, functional testing and model systems, data sharing, bioinformatics, and Artificial Intelligence (AI), highlighting their advantages, limitations, and the current state of clinical adaption. We provide expert recommendations outlining the stepwise application of these innovative technologies in the diagnostic pathways while considering global differences in accessibility. The importance of FAIR (Findability, Accessibility, Interoperability, and Reusability) and CARE (Collective benefit, Authority to control, Responsibility, and Ethics) data management is emphasized, along with the need for enhanced and continuing education in medical genomics. We provide a perspective on future technological developments in genome diagnostics and their integration into clinical practice. Lastly, we summarize the challenges related to genomic diversity and accessibility, highlighting the significance of innovative diagnostic technologies, global collaboration, and equitable access to diagnosis and treatment for people living with rare disease. [ABSTRACT FROM AUTHOR]

Direct infusion–high-resolution mass spectrometry (DI-HRMS) allows for rapid profiling of complex mixtures of metabolites in blood, cerebrospinal fluid, tissue samples and cultured cells. Here, we present a DI-HRMS method suitable for the rapid determination of metabolic fluxes of isotopically labeled substrates in cultured cells and organoids. We adapted an automated annotation pipeline by selecting labeled adducts that best represent the majority of 13C and/or 15N-labeled glycolytic and tricarboxylic acid cycle intermediates as well as a number of their derivatives. Furthermore, valine, leucine and several of their degradation products were included. We show that DI-HRMS can determine anticipated and unanticipated alterations in metabolic fluxes along these pathways that result from the genetic alteration of single metabolic enzymes, including pyruvate dehydrogenase (PDHA1) and glutaminase (GLS). In addition, it can precisely pinpoint metabolic adaptations to the loss of methylmalonyl-CoA mutase in patient-derived liver organoids. Our results highlight the power of DI-HRMS in combination with stable isotopically labeled compounds as an efficient screening method for fluxomics. [ABSTRACT FROM AUTHOR]

NAD synthetase 1 (encoded by the gene NADSYN1) is a cytosolic enzyme that catalyzes the final step in the biosynthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan and nicotinic acid. NADSYN1 deficiency has recently been added to the spectrum of congenital NAD+ deficiency disorders. To gain insight into the metabolic consequences of NADSYN1 deficiency, the encoding gene was disrupted in A549 and HEK293T cells, and the metabolome was profiled in the presence of different NAD+ precursors, including tryptophan, nicotinamide and nicotinic acid. We demonstrate that when precursors of the NAD+ salvage pathway in the form of nicotinamide become limiting, NADSYN1 deficiency results in a decline in intracellular NAD+ levels even in the presence of other potential NAD+ sources such as tryptophan and nicotinic acid. As a consequence, alterations in 122 and 69 metabolites are observed in NADSYN1-deficient A549 and HEK293T cells compared to the wild-type cell line (FC > 2 and p < 0.05). We thus show that NADSYN1 deficiency results in a metabolic phenotype characterized by alterations in glycolysis, the TCA cycle, the pentose phosphate pathway, and the polyol pathway. [ABSTRACT FROM AUTHOR]

Two siblings showed increased galactose and galactose-related metabolites in neonatal screening. Diagnostic workup did not reveal abnormalities in any of the known disease-causing enzymes involved in galactose metabolism. Using whole-exome sequencing, we identified a homozygous missense variant in PPA1 encoding the cytosolic pyrophosphatase 1 (PPA1), c.557C>T (p.Thr186Ile). The enzyme activity of PPA1 was determined using a colorimetric assay, and the protein content was visualized via western blotting in skin fibroblasts from one of the affected individuals. The galactolytic activity of the affected fibroblasts was determined by measuring extracellular acidification with a Seahorse XFe96 analyzer. PPA1 activity decreased to 22% of that of controls in the cytosolic fraction of homogenates from patient fibroblasts. PPA1 protein content decreased by 50% according to western blot analysis, indicating a reduced stability of the variant protein. The extracellular acidification rate was reduced in patient fibroblasts when galactose was used as a substrate. Untargeted metabolomics of blood samples revealed an elevation of other metabolites related to pyrophosphate metabolism. Besides hyperbilirubinemia in the neonatal period in one child, both children were clinically unremarkable at the ages of 3 and 14 years, respectively. We hypothesize that the observed metabolic derangement is a possible mild manifestation of PPA1 deficiency. Unresolved abnormalities in galactosemia screening might result in the identification of more individuals with PPA1 deficiency, a newly discovered inborn metabolic disorder (IMD). [ABSTRACT FROM AUTHOR]

In physiological glucose homeostasis, the liver plays a crucial role in the extraction of glucose from the portal circulation and storage as glycogen to enable release through glycogenolysis upon fasting. In addition, insulin secreted by the pancreas is partly eliminated from the systemic circulation by hepatic firstpass. Therefore, patients with a congenital porto-systemic shunt present a unique combination of (a) postabsorptive hyperinsulinemic hypoglycaemia (HH) because of decreased insulin elimination and (b) fasting (ketotic) hypoglycaemia because of decreased glycogenolysis. Patients with portosystemic shunts therefore provide important insight into the role of the portal circulation and hepatic function in different phases of glucose homeostasis. [ABSTRACT FROM AUTHOR]

Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disease, commonly caused by pathogenic MYBPC3 variants, and a significant cause of sudden cardiac death. Severity is highly variable, with incomplete penetrance among genotype-positive family members. Previous studies demonstrated metabolic changes in HCM. We aimed to identify metabolite profiles associated with disease severity in carriers of MYBPC3 founder variants using direct-infusion high-resolution mass spectrometry in plasma of 30 carriers with a severe phenotype (maximum wall thickness ≥20 mm, septal reduction therapy, congestive heart failure, left ventricular ejection fraction <50%, or malignant ventricular arrhythmia) and 30 age- and sex-matched carriers with no or a mild phenotype. Of the top 25 mass spectrometry peaks selected by sparse partial least squares discriminant analysis, XGBoost gradient boosted trees, and Lasso logistic regression (42 total), 36 associated with severe HCM at a p < 0.05, 20 at p < 0.01, and 3 at p < 0.001. These peaks could be clustered to several metabolic pathways, including acylcarnitine, histidine, lysine, purine and steroid hormone metabolism, and proteolysis. In conclusion, this exploratory case-control study identified metabolites associated with severe phenotypes in MYBPC3 founder variant carriers. Future studies should assess whether these biomarkers contribute to HCM pathogenesis and evaluate their contribution to risk stratification. [ABSTRACT FROM AUTHOR]

Introduction: To decrease antibiotic resistance, their use as growth promoters in the agricultural sector has been largely abandoned. This may lead to decreased health due to infectious disease or microbiome changes leading to gut inflammation. Objectives: We aimed to generate a m/z signature classifying chicken health in blood, and obtain biological insights from the resulting m/z signature. Methods: We used direct infusion mass-spectrometry to determine a machine-learned metabolomics signature that classifies chicken health from a blood sample. We then challenged the resulting models by investigating the classification capability of the signature on novel data obtained at poultry houses in previously unseen countries using a Leave-One-Country-Out (LOCO) cross-validation strategy. Additionally, we optimised the number of mass/charge (m/z) values required to maximise the classification capability of Random Forest models, by developing a novel ranking system based on combined univariate t-test and fold-change analyses and building models based on this ranking through forward and reverse feature selection. Results: The multi-country and LOCO models could classify chicken health. Both resulting 25-m/z and 3784-m/z signatures reliably classified chicken health in multiple countries. Through mummichog enrichment analysis on the large m/z signature, we found changes in amino acid metabolism, including branched chain amino acids and polyamines. Conclusion: We reliably classified chicken health from blood, independent of genetic-, farm-, feed- and country-specific confounding factors. The 25-m/z signature can be used to aid development of a per-metabolite panel. The extended 3784-m/z version can be used to gain a deeper understanding of the metabolic causes and consequences of low chicken health. Together, they may facilitate future treatment, prevention and intervention. [ABSTRACT FROM AUTHOR]

Summary: Erythrocytosis is associated with increased red blood cell mass and can be either congenital or acquired. Congenital secondary causes are rare and include germline variants increasing haemoglobin (Hb)‐oxygen affinity (e.g., Hb or bisphosphoglycerate mutase (BPGM) variants) or affecting oxygen‐sensing pathway proteins. Here, we describe five adults from three kindreds with erythrocytosis associated with heterozygosity for BPGM variants, including one novel. Functional analyses showed partial BPGM deficiency, reduced 2,3‐bisphosphoglycerate levels and/or increased Hb‐oxygen affinity. We also review currently known BPGM variants. This study contributes to raising awareness of BPGM variants, and in particular that heterozygosity for BPGM deficiency may already manifest clinically. [ABSTRACT FROM AUTHOR]

Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient‐own liver‐derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient‐derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient‐derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched‐chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient‐specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process. [ABSTRACT FROM AUTHOR]

Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD+/NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox‐dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3‐phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation. [ABSTRACT FROM AUTHOR]

Summary: The diagnostic evaluation of Diamond Blackfan Anaemia (DBA), an inherited bone marrow failure syndrome characterised by erythroid hypoplasia, is challenging because of a broad phenotypic variability and the lack of functional screening tests. In this study, we explored the potential of untargeted metabolomics to diagnose DBA. In dried blood spot samples from 18 DBA patients and 40 healthy controls, a total of 1752 unique metabolite features were identified. This metabolic fingerprint was incorporated into a machine‐learning algorithm, and a binary classification model was constructed using a training set. The model showed high performance characteristics (average accuracy 91·9%), and correct prediction of class was observed for all controls (n = 12) and all but one patient (n = 4/5) from the validation or 'test' set (accuracy 94%). Importantly, in patients with congenital dyserythropoietic anaemia (CDA) – an erythroid disorder with overlapping features – we observed a distinct metabolic profile, indicating the disease specificity of the DBA fingerprint and underlining its diagnostic potential. Furthermore, when exploring phenotypic heterogeneity, DBA treatment subgroups yielded discrete differences in metabolic profiles, which could hold future potential in understanding therapy responses. Our data demonstrate that untargeted metabolomics in dried blood spots is a promising new diagnostic tool for DBA. [ABSTRACT FROM AUTHOR]

Background: Cerebrotendinous xanthomatosis (CTX) is a rare genetic disorder, characterised by chronic diarrhoea, xanthomas, cataracts, and neurological deterioration. CTX is caused by CYP27A1 deficiency, which leads to abnormal cholesterol and bile acid metabolism. Urinary bile acid profiling (increased m/z 627: glucuronide‐5β‐cholestane‐pentol) serves as diagnostic screening for CTX. However, this led to a false positive CTX diagnosis in two patients, who had received total intravenous anaesthesia (TIVA) with propofol. Methods: To determine the influence of propofol on bile acid profiling, 10 urinary samples and 2 blood samples were collected after TIVA with propofol Fresenius 7 to 10 mg/kg/h from 12 subjects undergoing scoliosis correction. Urinary bile acids were analysed using flow injection negative electrospray mass spectrometry. Propofol binding to recombinant CYP27A1, the effects of propofol on recombinant CYP27A1 activity, and CYP27A1 expression in liver organoids were investigated using spectral binding, enzyme activity assays, and qPCR, respectively. Accurate masses were determined with high‐resolution mass spectrometry. Results: Abnormal urinary profiles were identified in all subjects after TIVA, with a trend correlating propofol dose per kilogramme and m/z 627 peak intensity. Propofol only induced a weak CYP27A1 response in the spectral binding assay, minimally affected CYP27A1 activity and did not affect CYP27A1 expression. The accurate mass of m/z 627 induced by propofol differed >10 PPM from m/z 627 observed in CTX. Conclusions: TIVA with propofol invariably led to a urinary profile misleadingly suggestive of CTX, but not through CYP27A1 inhibition. To avoid further misdiagnoses, propofol administration should be considered when interpreting urinary bile acid profiles. [ABSTRACT FROM AUTHOR]

Evidence for effectiveness of newborn screening (NBS) for propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) is scarce. Prior to implementation in the Netherlands, we aim to estimate the expected health gain of NBS for PA and MMA. In this national retrospective cohort study, the clinical course of 76/83 Dutch PA and MMA patients, diagnosed between January 1979 and July 2019, was evaluated. Five clinical outcome parameters were defined: adverse outcome of the first symptomatic phase, frequency of acute metabolic decompensations (AMD), cognitive function, mitochondrial complications, and treatment‐related complications. Outcomes of patients identified by family testing were compared with the outcomes of their index siblings. An adverse outcome due to the first symptomatic phase was recorded in 46% of the clinically diagnosed patients. Outcome of the first symptomatic phase was similar in 5/9 sibling pairs and better in 4/9 pairs. Based on the day of diagnosis of the clinically diagnosed patients and sibling pair analysis, a preliminary estimated reduction of adverse outcome due to the first symptomatic phase from 46% to 36%‐38% was calculated. Among the sibling pairs, AMD frequency, cognitive function, mitochondrial, and treatment‐related complications were comparable. These results suggest that the health gain of NBS for PA and MMA in overall outcome may be limited, as only a modest decrease of adverse outcomes due to the first symptomatic phase is expected. With current clinical practice, no reduced AMD frequency, improved cognitive function, or reduced frequency of mitochondrial or treatment‐related complications can be expected. [ABSTRACT FROM AUTHOR]

Glutamate is involved in a variety of metabolic pathways. We reviewed the literature on genetic defects of enzymes that directly metabolise glutamate, leading to inborn errors of glutamate metabolism. Seventeen genetic defects of glutamate metabolising enzymes have been reported, of which three were only recently identified. These 17 defects affect the inter‐conversion of glutamine and glutamate, amino acid metabolism, ammonia detoxification, and glutathione metabolism. We provide an overview of the clinical and biochemical phenotypes of these rare defects in an effort to ease their recognition. By categorising these by biochemical pathway, we aim to create insight into the contributing role of deviant glutamate and glutamine levels to the pathophysiology. For those disorders involving the inter‐conversion of glutamine and glutamate, these deviant levels are postulated to play a pivotal pathophysiologic role. For the other IEM however—with the exception of urea cycle defects—abnormal glutamate and glutamine concentrations were rarely reported. To create insight into the clinical consequences of disturbed glutamate metabolism—rather than individual glutamate and glutamine levels—the prevalence of phenotypic abnormalities within the 17 IEM was compared to their prevalence within all Mendelian disorders and subsequently all disorders with metabolic abnormalities notated in the Human Phenotype Ontology (HPO) database. For this, a hierarchical database of all phenotypic abnormalities of the 17 defects in glutamate metabolism based on HPO was created. A neurologic phenotypic spectrum of developmental delay, ataxia, seizures, and hypotonia are common in the inborn errors of enzymes in glutamate metabolism. Additionally, ophthalmologic and skin abnormalities are often present, suggesting that disturbed glutamate homeostasis affects tissues of ectodermal origin: brain, eye, and skin. Reporting glutamate and glutamine concentrations in patients with inborn errors of glutamate metabolism would provide additional insight into the pathophysiology. [ABSTRACT FROM AUTHOR]

The reversible oxidation of l-malate to oxaloacetate is catalyzed by NAD(H)-dependent malate dehydrogenase (MDH). MDH plays essential roles in the malate–aspartate shuttle and the tricarboxylic acid cycle. These metabolic processes are important in mitochondrial NADH supply for oxidative phosphorylation. Recently, bi-allelic mutations in mitochondrial MDH2 were identified in patients with global developmental delay, epilepsy and lactic acidosis. We now report two patients from an extended consanguineous family with a deleterious variant in the cytosolic isoenzyme of MDH (MDH1). The homozygous missense variant in the NAD+-binding domain of MDH1 led to severely diminished MDH protein expression. The patients presented with global developmental delay, epilepsy and progressive microcephaly. Both patients had normal concentrations of plasma amino acids, acylcarnitines, lactate, and urine organic acids. To identify the metabolic consequences of MDH1 deficiency, untargeted metabolomics was performed on dried blood spots (DBS) from the patients and in MDH1 knockout HEK293 cells that were generated by Crispr/Cas9. Increased levels of glutamate and glycerol-3-phosphate were found in DBS of both patients. In MDH1 KO HEK293 cells, increased levels of glycerol-3-phosphate were also observed, as well as increased levels of aspartate and decreased levels of fumarate. The consistent finding of increased concentrations of glycerol-3-phosphate may represent a compensatory mechanism to enhance cytosolic oxidation of NADH by the glycerol-P-shuttle. In conclusion, MDH1 deficiency is a new metabolic defect in the malate–aspartate shuttle characterized by a severe neurodevelopmental phenotype with elevated concentrations of glycerol-3-phosphate as a potential biomarker. [ABSTRACT FROM AUTHOR]

Over the last decades, advances in clinical care for patients suffering from propionic acidemia (PA) and isolated methylmalonic acidemia (MMA) have resulted in improved survival. These advances were possible thanks to new pathophysiological insights. However, patients may still suffer from devastating complications which largely determine the unsatisfying overall outcome. To optimize our treatment strategies, better insight in the pathophysiology of complications is needed. Here, we perform a systematic data‐analysis of cohort studies and case‐reports on PA and MMA. For each of the prevalent and rare complications, we summarize the current hypotheses and evidence for the underlying pathophysiology of that complication. A common hypothesis on pathophysiology of many of these complications is that mitochondrial impairment plays a major role. Assuming that complications in which mitochondrial impairment may play a role are overrepresented in monogenic mitochondrial diseases and, conversely, that complications in which mitochondrial impairment does not play a role are underrepresented in mitochondrial disease, we studied the occurrence of the complications in PA and MMA in mitochondrial and other monogenic diseases, using data provided by the Human Phenotype Ontology. Lastly, we combined this with evidence from literature to draw conclusions on the possible role of mitochondrial impairment in each complication. Altogether, this review provides a comprehensive overview on what we, to date, do and do not understand about pathophysiology of complications occurring in PA and MMA and about the role of mitochondrial impairment herein. [ABSTRACT FROM AUTHOR]

Despite realizing increased survival rates for propionic acidemia (PA) and methylmalonic acidemia (MMA) patients, the current therapeutic regimen is inadequate for preventing or treating the devastating complications that still can occur. The elucidation of pathophysiology of these complications allows us to evaluate and rethink treatment strategies. In this review we display and discuss potential therapy targets and we give a systematic overview on current, experimental and unexplored treatment strategies in order to provide insight in what we have to offer PA and MMA patients, now and in the future. Evidence on the effectiveness of treatment strategies is often scarce, since none were tested in randomized clinical trials. This raises concerns, since even the current consensus on best practice treatment for PA and MMA is not without controversy. To attain substantial improvements in overall outcome, gene, mRNA or enzyme replacement therapy is most promising since permanent reduction of toxic metabolites allows for a less strict therapeutic regime. Hereby, both mitochondrial‐associated and therapy induced complications can theoretically be prevented. However, the road from bench to bedside is long, as it is challenging to design a drug that is delivered to the mitochondria of all tissues that require enzymatic activity, including the brain, without inducing any off‐target effects. To improve survival rate and quality of life of PA and MMA patients, there is a need for systematic (re‐)evaluation of accepted and potential treatment strategies, so that we can better determine who will benefit when and how from which treatment strategy. [ABSTRACT FROM AUTHOR]

Pyridoxal 5′-phosphate (PLP), the metabolically active form of vitamin B6, plays an essential role in brain metabolism as a cofactor in numerous enzyme reactions. PLP deficiency in brain, either genetic or acquired, results in severe drug-resistant seizures that respond to vitamin B6 supplementation. The pathogenesis of vitamin B6 deficiency is largely unknown. To shed more light on the metabolic consequences of vitamin B6 deficiency in brain, we performed untargeted metabolomics in vitamin B6-deprived Neuro-2a cells. Significant alterations were observed in a range of metabolites. The most surprising observation was a decrease of serine and glycine, two amino acids that are known to be elevated in the plasma of vitamin B6 deficient patients. To investigate the cause of the low concentrations of serine and glycine, a metabolic flux analysis on serine biosynthesis was performed. The metabolic flux results showed that the de novo synthesis of serine was significantly reduced in vitamin B6-deprived cells. In addition, formation of glycine and 5-methyltetrahydrofolate was decreased. Thus, vitamin B6 is essential for serine de novo biosynthesis in neuronal cells, and serine de novo synthesis is critical to maintain intracellular serine and glycine. These findings suggest that serine and glycine concentrations in brain may be deficient in patients with vitamin B6 responsive epilepsy. The low intracellular 5-mTHF concentrations observed in vitro may explain the favourable but so far unexplained response of some patients with pyridoxine-dependent epilepsy to folinic acid supplementation. [ABSTRACT FROM AUTHOR]

Serine biosynthesis defects can present in a broad phenotypic spectrum ranging from Neu-Laxova syndrome, a lethal disease with multiple congenital anomalies at the severe end, to an infantile disease with severe psychomotor retardation and seizures as an intermediate phenotype, to a childhood disease with intellectual disability at the mild end. In this report we present 6 individuals from 3 families with infantile phosphoglycerate dehydrogenase (PGDH) deficiency who presented with psychomotor delay, growth failure, microcephaly, and spasticity. The phenotype was variable with absence of seizures in 2 sisters in family 1 and 1 infant in family 2 and seizures with pronounced happy affect in 3 sisters in family 3. The initiation of serine treatment had pronounced effect on seizures and spasticity in the sisters in family 3, but minimal developmental effects on the children in families 1 and 2. With such phenotypic variability, the diagnosis of PGDH deficiency can be challenging. [ABSTRACT FROM AUTHOR]

The malate-aspartate shuttle (MAS) is a redox shuttle that transports reducing equivalents across the inner mitochondrial membrane while recycling cytosolic NADH to NAD+. We genetically disrupted each MAS component to generate a panel of MAS-deficient HEK293 cell lines in which we performed [U-13C]-glucose tracing. MAS-deficient cells have reduced serine biosynthesis, which strongly correlates with the lactate M+3/pyruvate M+3 ratio (reflective of the cytosolic NAD+/NADH ratio), consistent with the NAD+ dependency of phosphoglycerate dehydrogenase in the serine synthesis pathway. Among the MAS-deficient cells, those lacking malate dehydrogenase 1 (MDH1) show the most severe metabolic disruptions, whereas oxoglutarate-malate carrier (OGC)- and MDH2-deficient cells are less affected. Increasing the NAD+-regenerating capacity using pyruvate supplementation resolves most of the metabolic disturbances. Overall, we show that the MAS is important for de novo serine biosynthesis, implying that serine supplementation could be used as a therapeutic strategy for MAS defects and possibly other redox disorders. [Display omitted] • Loss of MAS components reduces de novo serine biosynthesis • Loss of MAS components lowers the NAD+/NADH ratio and constrains glycolysis • Losses of MDH1, GOT1, AGC, and GOT2 show the most severe metabolic disruptions • Pyruvate restores NAD+/NADH, glycolysis, and serine biosynthesis in MDH1 KO cells Broeks et al. find that disruption of the malate-aspartate shuttle reduces de novo serine biosynthesis. Using 13C 6 -glucose tracer analysis in a comprehensive panel of knockout cell lines, they show that MAS-deficient cells have a low NAD+/NADH ratio, which affects metabolite fluxes of glycolysis and the TCA cycle. [ABSTRACT FROM AUTHOR]

Background: Over the past years, the essential role of vitamin B6 in brain development and functioning has been recognized and genetic metabolic disorders resulting in functional vitamin B6 deficiency have been identified. However, data on B6 vitamers in children are scarce. Materials and Methods: B6 vitamer concentrations in simultaneously sampled plasma and cerebrospinal fluid (CSF) of 70 children with intellectual disability were determined by ultra performance liquid chromatography-tandem mass spectrometry. For ethical reasons, CSF samples could not be obtained from healthy children. The influence of sex, age, epilepsy and treatment with anti-epileptic drugs, were investigated. Results: The B6 vitamer composition of plasma (pyridoxal phosphate (PLP) > pyridoxic acid > pyridoxal (PL)) differed from that of CSF (PL > PLP > pyridoxic acid > pyridoxamine). Strong correlations were found for B6 vitamers in and between plasma and CSF. Treatment with anti-epileptic drugs resulted in decreased concentrations of PL and PLP in CSF. Conclusion: We provide concentrations of all B6 vitamers in plasma and CSF of children with intellectual disability (±epilepsy), which can be used in the investigation of known and novel disorders associated with vitamin B6 metabolism as well as in monitoring of the biochemical effects of treatment with vitamin B6. [ABSTRACT FROM AUTHOR]

Background: Vitamin B-6 comprises a group of 6 interrelated vitamers and is essential for numerous physiologic processes, including brain functioning. Genetic disorders disrupting vitamin B-6 metabolism have severe clinical consequences. Objective: To adequately diagnose known and novel disorders in vitamin B-6 metabolism, a reference set is required containing information on all vitamin B-6 vitamers in plasma and cerebrospinal fluid (CSF). Design: Concentrations of vitamin B-6 vitamers in the plasma and CSF of 533 adult subjects were measured by ultra high-performance liquid chromatography-tandem mass spectrometry. Results: The relative vitamin B-6 vitamer composition of plasma [pyridoxal phosphate (PLP) > pyridoxic acid (PA) > pyridoxal] differed from that of CSF (pyridoxal > PLP > PA > pyridoxamine). Sex influenced vitamin B-6 vitamer concentrations in plasma and CSF and should therefore be taken into account when interpreting vitamin B-6 vitamer concentrations. The strict ratios and strong correlations between vitamin B-6 vitamers point to a tight regulation of vitamin B-6 vitamer concentrations in blood and CSF. Given the unique design of this study, with simultaneously withdrawn blood and CSF from a large number of subjects, reliable CSF:plasma ratios and correlations of vitamin B-6 vitamers could be established. Conclusions: We provide an extensive reference set of vitamin B-6 vitamer concentrations in plasma and CSF. In addition to providing insight on the regulation of individual vitamers and their intercompartmental distribution, we anticipate that these data will prove to be a valuable reference set for the diagnosis and treatment of conditions associated with altered vitamin B-6 metabolism. [ABSTRACT FROM AUTHOR]

Biallelic mutations of the von Hippel-Lindau ( VHL) gene are the most common cause of sporadic and inherited renal cell carcinoma ( RCC). Loss of VHL has been reported to affect cell proliferation by deregulating cell cycle-associated proteins. We report that the VHL gene product ( pVHL) inhibits E2F1 expression at both mRNA and protein level in zebrafish and human RCC cells, while loss of VHL increases E2F1 expression in patient kidney tumour tissue and RCC cells, resulting in a delay of cell cycle progression. RCCs from von Hippel-Lindau patients with known germline VHL mutations express significantly more E2F1 compared to sporadic RCCs with either clear-cell (cc) or non-cc histology. Analysis of 138 primary RCCs reveals that E2F1 expression is significantly higher in tumours with a diameter ≤7 cm and with a favourable American Joint Committee on Cancer ( AJCC) stage. The expression of E2F1 in RCC significantly correlates with p27 expression, suggesting that increased expression of E2F1 in RCC induces tumour cell senescence via p27. Cox regression analysis shows significant prediction of E2F1 expression for disease-free survival and overall survival, implying that E2F1 expression in kidney tumour is a novel prognostic factor for patients with RCC. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Purpose: The increasing incidence of small renal tumors in an aging population with comorbidities has stimulated the development of minimally invasive treatments. This study aimed to assess the efficacy and demonstrate feasibility of multimodality imaging of intratumoral administration of holmium-166 microspheres (166HoAcAcMS). This new technique locally ablates renal tumors through high-energy beta particles, while the gamma rays allow for nuclear imaging and the paramagnetism of holmium allows for MRI. Methods: 166HoAcAcMS were administered intratumorally in orthotopic renal tumors (Balb/C mice). Post administration CT, SPECT and MRI was performed. At several time points (2 h, 1, 2, 3, 7 and 14 days) after MS administration, tumors were measured and histologically analyzed. Holmium accumulation in organs was measured using inductively coupled plasma mass spectrometry. Results: 166HoAcAcMS were successfully administered to tumor bearing mice. A striking near-complete tumor-control was observed in 166HoAcAcMS treated mice (0.10±0.01 cm3 vs. 4.15±0.3 cm³ for control tumors). Focal necrosis and inflammation was present from 24 h following treatment. Renal parenchyma outside the radiated region showed no histological alterations. Post administration CT, MRI and SPECT imaging revealed clear deposits of 166HoAcAcMS in the kidney. Conclusions: Intratumorally administered 166HoAcAcMS has great potential as a new local treatment of renal tumors for surgically unfit patients. In addition to strong cancer control, it provides powerful multimodality imaging opportunities. [ABSTRACT FROM AUTHOR]

What's known on the subject? and What does the study add? Thermal ablation influences the local tissue microenvironment. Several studies have reported that residual tumour cells may exhibit a more aggressive phenotype. This study shows that incomplete CA and RFA cause an increased proliferation and decreased apptosis of residual renal tumour cells. This may be caused by stimulatory factors such as hypoxia, HSPs and inflammatory cells. OBJECTIVE To compare the effect of incomplete thermal ablation vs partial nephrectomy (PN) on growth stimulation and cellular survival in renal tumours., MATERIALS AND METHODS Renca renal tumours were transplanted under the renal capsule of mice (four to six mice/group) after which incomplete radiofrequency ablation (RFA), cryoablation (CA) or PN was performed., At several time points after treatment, presence of cell proliferation, apoptosis, hypoxic areas, inflammatory factors and the heat-shock proteins (HSPs) 70 and 90 were evaluated using immunohistochemistry., RESULTS At 2 h after thermal ablation residual tumour cells showed increased proliferation. This hyperproliferation was significantly stronger after RFA than CA ( P < 0.05) and not present after PN., Residual cells showed increased apoptosis after 2 h and decreased apoptosis from 2 days after thermal ablation. Apoptotic cells were significantly less evident at 3 days after RFA ( P < 0.001)., Hypoxic areas and HSPs were increasingly present from 2 h up to 7 days after thermal ablation ( P < 0.001)., Inflammatory cells infiltrated mainly the necrotic areas after thermal ablation, and their abundance peaked at 1 week after ablation ( P < 0.05)., The increased cell growth was preceded by hypoxia and presence of HSPs., CONCLUSIONS CA and RFA result in an increased proliferation and decreased apoptosis of residual renal tumour cells., This hyperproliferation may be caused by stimulatory factors, e.g. hypoxia, HSPs and inflammatory cells, and could facilitate recurrences of renal tumours after thermal ablation., This study highlights the importance of achieving complete tumour destruction. [ABSTRACT FROM AUTHOR]

Study Type - Retrospective (cohort) Level of Evidence 2b What's known on the subject? and What does the study add? Tumour characteristics, physical status and comorbidities are considered important for surgical outcome and prognosis. The present study objectively evaluates the association between comorbidity and postoperative complications after nephrectomy for RCC, by using the modified Clavien Classification of Surgical Complications to grade complications after nephrectomy. OBJECTIVE To present a single-centre experience of open nephrectomy for lesions suspected for renal cell carcinoma (RCC), evaluating the association between comorbidity and postoperative complications using a standardized classification system for postoperative complications., PATIENTS AND METHODS Clinicopathological data of 198 patients undergoing open radical or partial nephrectomy for lesions suspected of RCC were retrospectively analysed., Comorbidity scored by the Charlson comorbidity index (CCI), body mass index, age, gender, surgical procedure and surgical history were examined as predictive factors for postoperative complications, which were scored using the modified Clavien Classification of Surgical Complications (CCSC)., RESULTS The overall complication rate was 34%: 7% grade I, 15% grade II, 5% grade III, 3% grade IV and 4% grade V. Preoperative comorbidities were present in 51% of all patients., There were significantly more major complications (CCSC >2) in patients with major comorbidities (CCI >2), at 16% vs 7% ( P= 0.018)., Patients with high-stage RCC had significantly more severe complications than low-stage RCC ( P= 0.018)., In multivariable analysis, comorbidity (odds ratio [OR] 7.55, P= 0.004) and tumour stage 3-4 (OR 6.23, P= 0.007) were independent predictive factors for major complications., CONCLUSIONS Major complications occur significantly more often when major comorbidities are present., Comorbidity scores can be used in risk stratification for complications and should be considered during decision-making and counselling of patients before nephrectomy. [ABSTRACT FROM AUTHOR]

Gene expression in metazoans is regulated not only at the level of individual genes but also in a coordinated manner across large chromosomal domains (for example centromeres, telomeres and imprinted gene clusters) and along entire chromosomes (for example X-chromosome dosage compensation). The primary DNA sequence usually specifies the regulation of individual genes, but the nature of cis-acting information that controls genes over large regions has been elusive: higher-order DNA structure, specific histone modifications, subnuclear compartmentalization and primary DNA sequence are possibilities. One paradigm of chromosome-wide gene regulation is Caenorhabditis elegans dosage compensation in which a large dosage compensation complex (DCC) is targeted to both X chromosomes of hermaphrodites to repress transcript levels by half. This essential process equalizes X-linked gene expression between the sexes (XO males and XX hermaphrodites). Here we report the discovery and dissection of cis-acting sites that mark nematode X chromosomes as targets for gene repression by the DCC. These rex (recruitment element on X) sites are widely dispersed along X and reside in promoters, exons and intergenic regions. rex sites share at least two distinct motifs that act in combination to recruit the DCC. Mutating these motifs severely reduces or abolishes DCC binding in vivo, demonstrating the importance of primary DNA sequence in chromosome-wide regulation. Unexpectedly, the motifs are not enriched on X, but altering motif numbers within rex sites demonstrates that motif co-occurrence in unusually high densities is essential for optimal DCC recruitment. Thus, X-specific repression is established through sequences not specific to X. The distribution of common motifs provides the foundation for repression along an entire chromosome. [ABSTRACT FROM AUTHOR]

Although compound heterozygosity, or the presence of two different mutant alleles of the same gene, is common in human recessive disease, its potential to impact disease outcome has not been well documented. This is most likely because of the inherent difficulty in distinguishing specific biallelic effects from differences in environment or genetic background. We addressed the potential of different recessive alleles to contribute to the enigmatic pleiotropy associated with XPD recessive disorders in compound heterozygous mouse models. Alterations in this essential helicase, with functions in both DNA repair and basal transcription, result in diverse pathologies ranging from elevated UV sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic effects on organismal phenotype attributable to combinations of recessive Xpd alleles, including the following: (i) the ability of homozygous lethal Xpd alleles to ameliorate a variety of disease symptoms when their essential basal transcription function is supplied by a different disease-causing allele, (ii) differential developmental and tissue-specific functions of distinct Xpd allele products, and (iii) interallelic complementation, a phenomenon rarely reported at clinically relevant loci in mammals. Our data suggest a re-evaluation of the contribution of “null” alleles to XPD disorders and highlight the potential of combinations of recessive alleles to affect both normal and pathological phenotypic plasticity in mammals. [ABSTRACT FROM AUTHOR]

Photolyase transgenic mice have opened new avenues to improve our understanding of the cytotoxic effects of ultraviolet (UV) light on skin by providing a means to selectively remove either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone photoproducts. Here, we have taken a genomics approach to delineate pathways through which CPDs might contribute to the harmful effects of UV exposure. We show that CPDs, rather than other DNA lesions or damaged macromolecules, comprise the principal mediator of the cellular transcriptional response to UV. The most prominent pathway induced by CPDs is that associated with DNA double-strand break (DSB) signalling and repair. Moreover, we show that CPDs provoke accumulation of γ-H2AX, P53bp1 and Rad51 foci as well as an increase in the amount of DSBs, which coincides with accumulation of cells in S phase. Thus, conversion of unrepaired CPD lesions into DNA breaks during DNA replication may comprise one of the principal instigators of UV-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

During evolution, placental mammals appear to have lost cyclobutane pyrimidine dimer (CPD) photolyase, an enzyme that efficiently removes UV-induced CPDs from DNA in a tight-dependent manner. As a consequence, they have to rely solely on the more complex, and for this lesion less efficient, nucleotide excision repair pathway. To assess the contribution of poor repair of CPDs to various biological effects of UV, we generated mice expressing a marsupial CPD photolyase transgene. Expression from the ubiquitous β-actin promoter allowed rapid repair of CPDs in epidermis and dermis. UV-exposed cultured dermal fibroblasts from these mice displayed superior survival when treated with photoreactivating tight. Moreover, photoreactivation of CPDs in intact skin dramatically reduced acute UV effects like erythema (sunburn), hyperplasia and apoptosis. Mice expressing the photolyase from keratin 14 promoter photo-reactivate CPDs in basal and early differentiating keratinocytes only. Strikingly, in these animals, the anti-apoptotic effect appears to extend to other skin compartments, suggesting the presence of intercellular apoptotic signals. Thus, providing mice with CPD photolyase significantly improves repair and uncovers the biological effects of CPD lesions. [ABSTRACT FROM AUTHOR]

Background: Allergic diseases seem less prevalent in communities in less developed parts of the world, where parasite infections are highly prevalent. Altogether not much is known about the association between chronic infections with tissue and blood-dwelling parasites and atopy. Methods: In an area in Gabon endemic for blood and tissue parasites, 520 schoolchildren were parasitologically examined and skin prick-tested for a set of common environmental aeroallergens. Levels of allergen-specific IgE and polyclonal IgE were measured. Results: In schoolchildren schistosome and filarial infections increased with age, whereas malaria was more prevalent in younger children. In contrast to allergen sensitization that increased with age, skin test reactivity tended to decline. The number of children with mite-specific IgE antibodies (47%) by far exceeded the number responding to skin prick testing (11%). Mite sensitization was found to be the highest in children infected with schistosomes and/or filariae whereas skin test reactivity was lowest. The multiple logistic regression showed that the risk of a positive skin test was 8-fold higher with increasing levels of mite-specific IgE but was reduced by 72% when infected with blood stage helminths. Conclusions: Chronic blood and tissue parasite infections that are often capable of modulating immune responses in the host are negatively associated with skin test reactivity in a sensitized population.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Hypertrophic Cardiomyopathy (HCM) is a common inherited heart disease with poor risk prediction due to incomplete penetrance and a lack of clear genotype–phenotype correlations. Advanced imaging techniques have shown altered myocardial energetics already in preclinical gene variant carriers. To determine whether disturbed myocardial energetics with the potential to serve as biomarkers are also reflected in the serum metabolome, we analyzed the serum metabolome of asymptomatic carriers in comparison to healthy controls and obstructive HCM patients (HOCM). We performed non-quantitative direct-infusion high-resolution mass spectrometry-based untargeted metabolomics on serum from fasted asymptomatic gene variant carriers, symptomatic HOCM patients and healthy controls (n = 31, 14 and 9, respectively). Biomarker panels that discriminated the groups were identified by performing multivariate modeling with gradient-boosting classifiers. For all three group-wise comparisons we identified a panel of 30 serum metabolites that best discriminated the groups. These metabolite panels performed equally well as advanced cardiac imaging modalities in distinguishing the groups. Seven metabolites were found to be predictive in two different comparisons and may play an important role in defining the disease stage. This study reveals unique metabolic signatures in serum of preclinical carriers and HOCM patients that may potentially be used for HCM risk stratification and precision therapeutics. [ABSTRACT FROM AUTHOR]