Metabolomics for Investigating Physiological and Pathophysiological Processes.

Metabolomics uses advanced analytical chemistry techniques to enable the high-throughput characterization of metabolites from cells, organs, tissues, or biofluids. The rapid growth in metabolomics is leading to a renewed interest in metabolism and the role that small molecule metabolites play in many biological processes. As a result, traditional views of metabolites as being simply the "bricks and mortar" of cells or just the fuel for cellular energetics are being upended. Indeed, metabolites appear to have much more varied and far more important roles as signaling molecules, immune modulators, endogenous toxins, and environmental sensors. This review explores how metabolomics is yielding important new insights into a number of important biological and physiological processes. In particular, a major focus is on illustrating how metabolomics and discoveries made through metabolomics are improving our understanding of both normal physiology and the pathophysiology of many diseases. These discoveries are yielding new insights into how metabolites influence organ function, immune function, nutrient sensing, and gut physiology. Collectively, this work is leading to a much more unified and system-wide perspective of biology wherein metabolites, proteins, and genes are understood to interact synergistically to modify the actions and functions of organelles, organs, and organisms.

An evaluation of untargeted metabolomics methods to characterize inborn errors of metabolism.

Inborn errors of metabolism (IEMs) encompass a diverse group of disorders that can be difficult to classify due to heterogenous clinical, molecular, and biochemical manifestations. Untargeted metabolomics platforms have become a popular approach to analyze IEM patient samples because of their ability to detect many metabolites at once, accelerating discovery of novel biomarkers, and metabolic mechanisms of disease. However, there are concerns about the reproducibility of untargeted metabolomics research due to the absence of uniform reporting practices, data analyses, and experimental design guidelines. Therefore, we critically evaluated published untargeted metabolomic platforms used to characterize IEMs to summarize the strengths and areas for improvement of this technology as it progresses towards the clinical laboratory. A total of 96 distinct IEMs were collectively evaluated by the included studies. However, most of these IEMs were evaluated by a single untargeted metabolomic method, in a single study, with a limited cohort size (55/96, 57%). The goals of the included studies generally fell into two, often overlapping, categories: detecting known biomarkers from many biochemically distinct IEMs using a single platform, and detecting novel metabolites or metabolic pathways. There was notable diversity in the design of the untargeted metabolomic platforms. Importantly, the majority of studies reported adherence to quality metrics, including the use of quality control samples and internal standards in their experiments, as well as confirmation of at least some of their feature annotations with commercial reference standards. Future applications of untargeted metabolomics platforms to the study of IEMs should move beyond single-subject analyses, and evaluate reproducibility using a prospective, or validation cohort.

The Mendelian disorders of chromatin machinery: Harnessing metabolic pathways and therapies for treatment.

The Mendelian disorders of chromatin machinery (MDCMs) represent a distinct subgroup of disorders that present with neurodevelopmental disability. The chromatin machinery regulates gene expression by a range of mechanisms, including by post-translational modification of histones, responding to histone marks, and remodelling nucleosomes. Some of the MDCMs that impact on histone modification may have potential therapeutic interventions. Two potential treatment strategies are to enhance the intracellular pool of metabolites that can act as substrates for histone modifiers and the use of medications that may inhibit or promote the modification of histone residues to influence gene expression. In this article we discuss the influence and potential treatments of histone modifications involving histone acetylation and histone methylation. Genomic technologies are facilitating earlier diagnosis of many Mendelian disorders, providing potential opportunities for early treatment from infancy. This has parallels with how inborn errors of metabolism have been afforded early treatment with newborn screening. Before this promise can be fulfilled, we require greater understanding of the biochemical fingerprint of these conditions, which may provide opportunities to supplement metabolites that can act as substrates for chromatin modifying enzymes. Importantly, understanding the metabolomic profile of affected individuals may also provide disorder-specific biomarkers that will be critical for demonstrating efficacy of treatment, as treatment response may not be able to be accurately assessed by clinical measures.

Inborn errors of the malate aspartate shuttle - Update on patients and cellular models.

The malate aspartate shuttle (MAS) plays a pivotal role in transporting cytosolic reducing equivalents - electrons - into the mitochondria for energy conversion at the electron transport chain (ETC) and in the process of oxidative phosphorylation. The MAS consists of two pairs of cytosolic and mitochondrial isoenzymes (malate dehydrogenases 1 and 2; and glutamate oxaloacetate transaminases 1 and 2) and two transporters (malate-2-oxoglutarate carrier and aspartate glutamate carrier (AGC), the latter of which has two tissue-dependent isoforms AGC1 and AGC2). While the inner mitochondrial membrane is impermeable to NADH, the MAS forms one of the main routes for mitochondrial electron uptake by promoting uptake of malate. Inherited bi-allelic pathogenic variants in five of the seven components of the MAS have been described hitherto and cause a wide spectrum of symptoms including early-onset epileptic encephalopathy. This review provides an overview of reported patients suffering from MAS deficiencies. In addition, we give an overview of diagnostic procedures and research performed on patient-derived cellular models and tissues. Current cellular models are briefly discussed and novel ways to achieve a better understanding of MAS deficiencies are highlighted.

PXE, a Mysterious Inborn Error Clarified.

Ever since Garrod deduced the existence of inborn errors in 1901, a vast array of metabolic diseases has been identified and characterized in molecular terms. In 2018 it is difficult to imagine that there is any uncharted backyard left in the metabolic disease landscape. Nevertheless, it took until 2013 to identify the cause of a relatively frequent inborn error, pseudoxanthoma elasticum (PXE), a disorder resulting in aberrant calcification. The mechanism found was not only biochemically interesting but also points to possible new treatments for PXE, a disease that has remained untreatable. In this review we sketch the tortuous road that led to the biochemical understanding of PXE and to new ideas for treatment. We also discuss some of the controversies still haunting the field.

Ex vivo proton spectroscopy (1 H-NMR) analysis of inborn errors of metabolism: Automatic and computer-assisted analyses.

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy (1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism.

Dysfunctional mitochondrial translation and combined oxidative phosphorylation deficiency in a mouse model of hepatoencephalopathy due to Gfm1 mutations.

Hepatoencephalopathy due to combined oxidative phosphorylation deficiency type 1 (COXPD1) is a recessive mitochondrial translation disorder caused by mutations in GFM1, a nuclear gene encoding mitochondrial elongation factor G1 (EFG1). Patients with COXPD1 typically present hepatoencephalopathy early after birth with rapid disease progression, and usually die within the first few weeks or years of life. We have generated two different mouse models: a Gfm1 knock-in (KI) harboring the p.R671C missense mutation, found in at least 10 patients who survived more than 1 year, and a Gfm1 knock-out (KO) model. Homozygous KO mice (Gfm1-/- ) were embryonically lethal, whereas homozygous KI (Gfm1R671C/R671C ) mice were viable and showed normal growth. R671C mutation in Gfm1 caused drastic reductions in the mitochondrial EFG1 protein content in different organs. Six- to eight-week-old Gfm1R671C/R671C mice showed partial reductions of in organello mitochondrial translation and respiratory complex IV enzyme activity in the liver. Compound heterozygous Gfm1R671C/- showed a more pronounced decrease of EFG1 protein in liver and brain mitochondria, as compared with Gfm1R671C/R671C mice. At 8 weeks of age, their mitochondrial translation rates were significantly reduced in both tissues. Additionally, Gfm1R671C/- mice showed combined oxidative phosphorylation deficiency (reduced complex I and IV enzyme activities in liver and brain), and blue native polyacrylamide gel electrophoresis analysis revealed lower amounts of both affected complexes. We conclude that the compound heterozygous Gfm1R671C/- mouse presents a clear dysfunctional molecular phenotype, showing impaired mitochondrial translation and combined respiratory chain dysfunction, making it a suitable animal model for the study of COXPD1.

Diagnosis and management of metabolic myopathies.

Metabolic myopathies are a set of rare inborn errors of metabolism leading to disruption in energy production. Relevant to skeletal muscle, glycogen storage disease and fatty acid oxidation defects can lead to exercise intolerance, rhabdomyolysis, and weakness in children and adults, distinct from the severe forms that involve multiple-organ systems. These nonspecific, dynamic symptoms along with conditions that mimic metabolic myopathies can make diagnosis challenging. Clinicians can shorten the time to diagnosis by recognizing the typical clinical phenotypes and performing next generation sequencing. With improved access and affordability of molecular testing, clinicians need to be well-versed in resolving variants of uncertain significance relevant to metabolic myopathies. Once identified, patients can improve quality of life, safely engage in exercise, and reduce episodes of rhabdomyolysis by modifying diet and lifestyle habits.

Adiponectin, Leptin and Cardiovascular Disorders.

The landmark discoveries of leptin and adiponectin firmly established adipose tissue as a sophisticated and highly active endocrine organ, opening a new era of investigating adipose-mediated tissue crosstalk. Both obesity-associated hyperleptinemia and hypoadiponectinemia are important biomarkers to predict cardiovascular outcomes, suggesting a crucial role for adiponectin and leptin in obesity-associated cardiovascular disorders. Normal physiological levels of adiponectin and leptin are indeed essential to maintain proper cardiovascular function. Insufficient adiponectin and leptin signaling results in cardiovascular dysfunction. However, a paradox of high levels of both leptin and adiponectin is emerging in the pathogenesis of cardiovascular disorders. Here, we (1) summarize the recent progress in the field of adiponectin and leptin and its association with cardiovascular disorders, (2) further discuss the underlying mechanisms for this new paradox of leptin and adiponectin action, and (3) explore the possible application of partial leptin reduction, in addition to increasing the adiponectin/leptin ratio as a means to prevent or reverse cardiovascular disorders.

Exercise testing and prescription in patients with inborn errors of muscle energy metabolism.

Skeletal muscle is a dynamic organ requiring tight regulation of energy metabolism in order to provide bursts of energy for effective function. Several inborn errors of muscle energy metabolism (IEMEM) affect skeletal muscle function and therefore the ability to initiate and sustain physical activity. Exercise testing can be valuable in supporting diagnosis, however its use remains limited due to the inconsistency in data to inform its application in IEMEM populations. While exercise testing is often used in adults with IEMEM, its use in children is far more limited. Once a physiological limitation has been identified and the aetiology defined, habitual exercise can assist with improving functional capacity, with reports supporting favourable adaptations in adult patients with IEMEM. Despite the potential benefits of structured exercise programs, data in paediatric populations remain limited. This review will focus on the utilisation and limitations of exercise testing and prescription for both adults and children, in the management of McArdle Disease, long chain fatty acid oxidation disorders, and primary mitochondrial myopathies.

Hereditary Disorders of Cardiovascular Calcification.

Arterial calcification is a common phenomenon in the elderly, in patients with atherosclerosis or renal failure and in diabetes. However, when present in very young individuals, it is likely to be associated with an underlying hereditary disorder of arterial calcification. Here, we present an overview of the few monogenic disorders presenting with early-onset cardiovascular calcification. These disorders can be classified according to the function of the respective disease gene into (1) disorders caused by an altered purine and phosphate/pyrophosphate metabolism, (2) interferonopathies, and (3) Gaucher disease. The finding of arterial calcification in early life should alert the clinician and prompt further genetic work-up to define the underlying genetic defect, to establish the correct diagnosis, and to enable appropriate therapy.

SPINT2 (HAI-2) missense variants identified in congenital sodium diarrhea/tufting enteropathy affect the ability of HAI-2 to inhibit prostasin but not matriptase.

The syndromic form of congenital sodium diarrhea (SCSD) is caused by bi-allelic mutations in SPINT2, which encodes a Kunitz-type serine protease inhibitor (HAI-2). We report three novel SCSD patients, two novel SPINT2 mutations and review published cases. The most common findings in SCSD patients were choanal atresia (20/34) and keratitis of infantile onset (26/34). Characteristic epithelial tufts on intestinal histology were reported in 13/34 patients. Of 13 different SPINT2 variants identified in SCSD, 4 are missense variants and localize to the second Kunitz domain (KD2) of HAI-2. HAI-2 has been implicated in the regulation of the activities of several serine proteases including prostasin and matriptase, which are both important for epithelial barrier formation. No patient with bi-allelic stop mutations was identified, suggesting that at least one SPINT2 allele encoding a protein with residual HAI-2 function is necessary for survival. We show that the SCSD-associated HAI-2 variants p.Phe161Val, p.Tyr163Cys and p.Gly168Ser all display decreased ability to inhibit prostasin-catalyzed cleavage. However, the SCSD-associated HAI-2 variants inhibited matriptase as efficiently as the wild-type HAI-2. Homology modeling indicated limited solvent exposure of the mutated amino acids, suggesting that they induce misfolding of KD2. This suggests that prostasin needs to engage with an exosite motif located on KD2 in addition to the binding loop (Cys47/Arg48) located on the first Kunitz domain in order to inhibit prostasin. In conclusion our data suggests that SCSD is caused by lack of inhibition of prostasin or a similar protease in the secretory pathway or on the plasma membrane.

Dysbiosis of the intestinal microbiome as a component of pathophysiology in the inborn errors of metabolism.

Inborn errors of metabolism (IEMs) represent monogenic disorders in which specific enzyme deficiencies, or a group of enzyme deficiencies (e.g., peroxisomal biogenesis disorders) result in either toxic accumulation of metabolic intermediates or deficiency in the production of key end-products (e.g., low cholesterol in Smith-Lemli-Opitz syndrome (Gedam et al., 2012 [1]); low creatine in guanidinoacetic acid methyltransferase deficiency (Stromberger, 2003 [2])). Some IEMs can be effectively treated by dietary restrictions (e.g., phenylketonuria (PKU), maple syrup urine disease (MSUD)), and/or dietary intervention to remove offending compounds (e.g., acylcarnitine excretion with the oral intake of l-carnitine in the disorders of fatty acid oxidation). While the IEMs are predominantly monogenic disorders, their phenotypic presentation is complex and pleiotropic, impacting multiple physiological systems (hepatic and neurological function, renal and musculoskeletal impairment, cardiovascular and pulmonary activity, etc.). The metabolic dysfunction induced by the IEMs, as well as the dietary interventions used to treat them, are predicted to impact the gut microbiome in patients, and it is highly likely that microbiome dysbiosis leads to further exacerbation of the clinical phenotype. That said, only recently has the gut microbiome been considered as a potential pathomechanistic consideration in the IEMs. In this review, we overview the function of the gut-brain axis, the crosstalk between these compartments, and the expanding reports of dysbiosis in the IEMs recently reported. The potential use of pre- and probiotics to improve clinical outcomes in IEMs is also highlighted.

Clinical and biochemical footprints of inherited metabolic diseases. IV. Metabolic cardiovascular disease.

Inherited metabolic diseases account for 15-20% of all cases of pediatric cardiomyopathy, with a high mortality of 15-47%. Metabolic diseases can also commonly be associated with other types of cardiovascular involvement such as arrhythmias, valvulopathy or vasculopathy. We reviewed and updated the list of known metabolic etiologies associated with cardiovascular involvement, and found 246 relevant inborn errors of metabolism. This represents the fourth of a series of articles attempting to create and maintain a comprehensive list of clinical and metabolic differential diagnoses according to system involvement.

Molecular and metabolic bases of tetrahydrobiopterin (BH4) deficiencies.

Tetrahydrobiopterin (BH4) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH4 metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH4 deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine.

Successful orthotopic heart transplantation in CPTII deficiency.

Carnitine palmitoyl transferase II (CPT II) catalyzes the release of activated long-chain fatty acids from acylcarnitines into mitochondria for subsequent fatty acid oxidation. Depending on residual enzyme activity, deficiency of this enzyme leads to a spectrum of symptoms from early onset hypoglycemia, hyperammonemia, cardiomyopathy and death to onset of recurrent rhabdomyolysis in adolescents and young adults. We present a case of successful orthotopic heart transplantation in a patient with severe infantile onset cardiomyopathy due to CPT II deficiency identified through newborn screening. Excellent cardiac function is preserved 12 years post-transplantation; however, the patient has developed intermittent episodes of hyperammonemia and rhabdomyolysis later in childhood and early adolescence readily resolved with intravenous glucose. Successful heart transplant in this patient demonstrates the feasibility of this management option in patients with even severe forms of long chain fatty acid oxidation disorders.

The first knock-in rat model for glutaric aciduria type I allows further insights into pathophysiology in brain and periphery.

Glutaric aciduria type I (GA-I, OMIM # 231670) is an inborn error of metabolism caused by a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients develop acute encephalopathic crises (AEC) with striatal injury most often triggered by catabolic stress. The pathophysiology of GA-I, particularly in brain, is still not fully understood. We generated the first knock-in rat model for GA-I by introduction of the mutation p.R411W, the rat sequence homologue of the most common Caucasian mutation p.R402W, into the Gcdh gene of Sprague Dawley rats by CRISPR/CAS9 technology. Homozygous Gcdhki/ki rats revealed a high excretor phenotype, but did not present any signs of AEC under normal diet (ND). Exposure to a high lysine diet (HLD, 4.7%) after weaning resulted in clinical and biochemical signs of AEC. A significant increase of plasmatic ammonium concentrations was found in Gcdhki/ki rats under HLD, accompanied by a decrease of urea concentrations and a concomitant increase of arginine excretion. This might indicate an inhibition of the urea cycle. Gcdhki/ki rats exposed to HLD showed highly diminished food intake resulting in severely decreased weight gain and moderate reduction of body mass index (BMI). This constellation suggests a loss of appetite. Under HLD, pipecolic acid increased significantly in cerebral and extra-cerebral liquids and tissues of Gcdhki/ki rats, but not in WT rats. It seems that Gcdhki/ki rats under HLD activate the pipecolate pathway for lysine degradation. Gcdhki/ki rat brains revealed depletion of free carnitine, microglial activation, astroglyosis, astrocytic death by apoptosis, increased vacuole numbers, impaired OXPHOS activities and neuronal damage. Under HLD, Gcdhki/ki rats showed imbalance of intra- and extracellular creatine concentrations and indirect signs of an intracerebral ammonium accumulation. We successfully created the first rat model for GA-I. Characterization of this Gcdhki/ki strain confirmed that it is a suitable model not only for the study of pathophysiological processes, but also for the development of new therapeutic interventions. We further brought up interesting new insights into the pathophysiology of GA-I in brain and periphery.

Inborn errors of metabolism: Lessons from iPSC models.

The possibility of reprogramming human somatic cells to pluripotency has opened unprecedented opportunities for creating genuinely human experimental models of disease. Inborn errors of metabolism (IEMs) constitute a greatly heterogeneous class of diseases that appear, in principle, especially suited to be modeled by iPSC-based technology. Indeed, dozens of IEMs have already been modeled to some extent using patient-specific iPSCs. Here, we review the advantages and disadvantages of iPSC-based disease modeling in the context of IEMs, as well as particular challenges associated to this approach, together with solutions researchers have proposed to tackle them. We have structured this review around six lessons that we have learnt from those previous modeling efforts, and that we believe should be carefully considered by researchers wishing to embark in future iPSC-based models of IEMs.

Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

Metabolic impact of pathogenic variants in the mitochondrial glutamyl-tRNA synthetase EARS2.

Glutamyl-tRNA synthetase 2 (encoded by EARS2) is a mitochondrial aminoacyl-tRNA synthetase required to translate the 13 subunits of the electron transport chain encoded by the mitochondrial DNA. Pathogenic EARS2 variants cause combined oxidative phosphorylation deficiency, subtype 12 (COXPD12), an autosomal recessive disorder involving lactic acidosis, intellectual disability, and other features of mitochondrial compromise. Patients with EARS2 deficiency present with variable phenotypes ranging from neonatal lethality to a mitigated disease with clinical improvement in early childhood. Here, we report a neonate homozygous for a rare pathogenic variant in EARS2 (c.949G>T; p.G317C). Metabolomics in primary fibroblasts from this patient revealed expected abnormalities in TCA cycle metabolites, as well as numerous changes in purine, pyrimidine, and fatty acid metabolism. To examine genotype-phenotype correlations in COXPD12, we compared the metabolic impact of reconstituting these fibroblasts with wild-type EARS2 versus four additional EARS2 variants from COXPD12 patients with varying clinical severity. Metabolomics identified a group of signature metabolites, mostly from the TCA cycle and amino acid metabolism, that discriminate between EARS2 variants causing relatively mild and severe COXPD12. Taken together, these findings indicate that metabolomics in patient-derived fibroblasts may help establish genotype-phenotype correlations in EARS2 deficiency and likely other mitochondrial disorders.