Cerebral White Matter Alterations Associated With Oligodendrocyte Vulnerability in Organic Acidurias: Insights in Glutaric Aciduria Type I.

The white matter is an important constituent of the central nervous system, containing axons, oligodendrocytes, and its progenitor cells, astrocytes, and microglial cells. Oligodendrocytes are central for myelin synthesis, the insulating envelope that protects axons and allows normal neural conduction. Both, oligodendrocytes and myelin, are highly vulnerable to toxic factors in many neurodevelopmental and neurodegenerative disorders associated with disturbances of myelination. Here we review the main alterations in oligodendrocytes and myelin observed in some organic acidurias/acidemias, which correspond to inherited neurometabolic disorders biochemically characterized by accumulation of potentially neurotoxic organic acids and their derivatives. The yet incompletely understood mechanisms underlying the high vulnerability of OLs and/or myelin in glutaric acidemia type I, the most prototypical cerebral organic aciduria, are particularly discussed.

A multiomics approach reveals evidence for phenylbutyrate as a potential treatment for combined D,L-2- hydroxyglutaric aciduria.

PURPOSE: To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene. METHODS: Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing. RESULTS: In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink. CONCLUSION: Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease.

Update current understanding of neurometabolic disorders related to lysine metabolism.

Lysine, as an essential amino acid, predominantly undergoes metabolic processes through the saccharopine pathway, whereas a smaller fraction follows the pipecolic acid pathway. Although the liver is considered the primary organ for lysine metabolism, it is worth noting that lysine catabolism also takes place in other tissues and organs throughout the body, including the brain. Enzyme deficiency caused by pathogenic variants in its metabolic pathway may lead to a series of neurometabolic diseases, among which glutaric aciduria type 1 and pyridoxine-dependent epilepsy have the most significant clinical manifestations. At present, through research, we have a deeper understanding of the multiple pathophysiological mechanisms related to these diseases, including intracerebral accumulation of neurotoxic metabolites, imbalance between GABAergic and glutamatergic neurotransmission, energy deprivation due to metabolites, and the dysfunction of antiquitin. Because of the complexity of these diseases, their clinical manifestations are also diverse. The early implementation of lysine-restricted diets and supplementation with arginine and carnitine has reported positive impacts on the neurodevelopmental outcomes of patients. Presently, there is more robust evidence supporting the effectiveness of these treatments in glutaric aciduria type 1 compared with pyridoxine-dependent epilepsy.

Exploring genotype-phenotype correlations in glutaric aciduria type 1.

Glutaric aciduria type 1 (GA1) is a rare neurometabolic disease caused by pathogenic variants in the gene encoding the enzyme glutaryl-CoA dehydrogenase (GCDH). We performed an extensive literature search to collect data on GA1 patients, together with unpublished cases, to provide an up-to-date genetic landscape of GCDH pathogenic variants and to investigate potential genotype-phenotype correlation, as this is still poorly understood. From this search, 421 different GCDH pathogenic variants have been identified, including four novel variants; c.179T>C (p.Leu60Pro), c.214C>T (p.Arg72Cys), c.309G>C (p.Leu103Phe), and c.665T>C (p.Phe222Ser).The variants are mostly distributed across the entire gene; although variant frequency in GA1 patients is relatively high in the regions encoding for active domains of GCDH. To investigate potential genotype-phenotype correlations, phenotypic descriptions of 532 patients have been combined and evaluated using novel combinatorial analyses. To do so, various clinical phenotypes were determined for each pathogenic variant by combining the information of all GA1 patients reported with this pathogenic variant, and subsequently mapped onto the 2D and 3D GCDH protein structure. In addition, the predicted pathogenicity of missense variants was analyzed using different in silico prediction score models. Both analyses showed an almost similar distribution of the highly pathogenic variants across the GCDH protein, although some hotspots, including the active domain, were observed. Moreover, it was demonstrated that highly pathogenic variants are significantly correlated with lower residual enzyme activity and the most accurate estimation was achieved by the REVEL score. A clear correlation of the genotype and the clinical phenotype however is still lacking.

Phenotypic prediction in glutaric aciduria type 1 combining in silico and in vitro modeling with real-world data.

Glutaric aciduria type 1 (GA1) is caused by inherited deficiency of glutaryl-CoA dehydrogenase (GCDH). To further understand the unclear genotype-phenotype correlation, we transfected mutated GCDH into COS-7 cells resembling known biallelic GCDH variants of 47 individuals with GA1. In total, we modeled 36 genotypes with 32 missense variants. Spectrophotometry demonstrated an inverse correlation between residual enzyme activity and the urinary concentration of glutaric acid and 3-hydroxyglutaric acid, confirming previous studies (Pearson correlation, r = -0.34 and r = -0.49, p = 0.045 and p = 0.002, respectively). In silico modeling predicted high pathogenicity for all genotypes, which caused a low enzyme activity. Western blotting revealed a 2.6-times higher GCDH protein amount in patients with an acute encephalopathic crisis (t-test, p = 0.015), and high protein expression correlated with high in silico protein stability (Pearson correlation, r = -0.42, p = 0.011). The protein amount was not correlated with the enzyme activity (Pearson correlation, r = 0.09, p = 0.59). To further assess protein stability, proteolysis was performed, showing that the p.Arg88Cys variant stabilized a heterozygous less stable variant. We conclude that an integration of different data sources helps to predict the complex clinical phenotype in individuals with GA1.

Generation of an induced pluripotent stem cell line carrying a biallelic deletion (SCTCi019-A) in GCDH using CRISPR/Cas9.

GCDH encodes for the enzyme catalyzing the sixth step of the lysine catabolism pathway. Biallelic pathogenic variants in GCDH have been associated with glutaric aciduria type 1 (GA1). In this study CRISPR/Cas9 technology was used to create an isogenic GCDH knock-out human iPSC line. One clone with a biallelic deletion (SCTCi019-A) in GCDH was obtained and fully characterized, revealing a normal karyotype, no off-targets detected and expression of pluripotency markers. This iPSC line can contribute to gain insights in the molecular mechanism of disease.

Optimized integration of metabolomics and lipidomics reveals brain region-specific changes of oxidative stress and neuroinflammation in type 1 diabetic mice with cognitive decline.

INTRODUCTION: Type 1 diabetes (T1D) causes cognitive decline and has been associated with brain metabolic disorders, but its potential molecular mechanisms remain unclear. OBJECTIVES: The purpose of this study was to explore the molecular mechanisms underlying T1D-induced cognitive impairment using metabolomics and lipidomics. METHODS: We developed an optimized integration approach of metabolomics and lipidomics for brain tissue based on UPLC-Q-TOF-MS and analyzed a comprehensive characterization of metabolite and lipid profiles in the hippocampus and frontal cortex of T1D male mice with cognitive decline (T1DCD) and age-matched control (CONT) mice. RESULTS: The results show that T1DCD mice had brain metabolic disorders in a region-specific manner relative to CONT mice, and the frontal cortex exhibited a higher lipid peroxidation than the hippocampus in T1DCD mice. Based on metabolic changes, we found that microglia was activated under diabetic condition and thereby promoted oxidative stress and neuroinflammation, leading to neuronal injury, and this event was more pronounced in the frontal cortex than the hippocampus. CONCLUSION: Our results suggest that brain region-specific shifts in oxidative stress and neuroinflammation may contribute to diabetic cognitive decline, and the frontal cortex could be the more vulnerable brain region than the hippocampus.

NRF2 mediates melanoma addiction to GCDH by modulating apoptotic signalling.

Tumour dependency on specific metabolic signals has been demonstrated and often guided numerous therapeutic approaches. We identify melanoma addiction to the mitochondrial protein glutaryl-CoA dehydrogenase (GCDH), which functions in lysine metabolism and controls protein glutarylation. GCDH knockdown induced cell death programmes in melanoma cells, an activity blocked by inhibition of the upstream lysine catabolism enzyme DHTKD1. The transcription factor NRF2 mediates GCDH-dependent melanoma cell death programmes. Mechanistically, GCDH knockdown induces NRF2 glutarylation, increasing its stability and DNA binding activity, with a concomitant transcriptional upregulation of ATF4, ATF3, DDIT3 and CHAC1, resulting in cell death. In vivo, inducible inactivation of GCDH effectively inhibited melanoma tumour growth. Correspondingly, reduced GCDH expression correlated with improved survival of patients with melanoma. These findings identify melanoma cell addiction to GCDH, limiting apoptotic signalling by controlling NRF2 glutarylation. Inhibiting the GCDH pathway could thus represent a therapeutic approach to treat melanoma.

Identifying encephalopathies from acute metabolic derangements.

Metabolic derangements, when acute and severe, affect brain function. This presents mostly with a marked decline in the level of consciousness, resulting in impaired responsiveness, abnormal receptivity, impaired content, and loss of memory retention. The term metabolic encephalopathy has been used but is conjecture that can be challenged in the age of modern neuroimaging. We now recognize that many metabolic encephalopathies may involve structural lesions and at an early stage. Common clinical conundrums are the evaluation of the degree of brain injury and its recoverability. This review discusses the appropriate terminology for these conditions, the diagnostic approach, therapy recommendations, and prediction of recovery potential. In evaluating a presumed metabolic cause for encephalopathy, we must (1) search for and rule out structural injury to the brain despite an obvious explanatory metabolic derangement, (2) recognize that several confounding conditions often co-exist, and (3) acknowledge that resolution of brain dysfunction may be protracted despite normalization of laboratory values.

Intersections in Neuropsychiatric and Metabolic Disorders: Possible Role of TRPA1 Channels.

Neuropsychiatric disorders (NPDs) are a huge burden to the patient, their family, and society. NPDs have been greatly associated with cardio-metabolic comorbidities such as obesity, type-2 diabetes mellitus, dysglycaemia, insulin resistance, dyslipidemia, atherosclerosis, and other cardiovascular disorders. Antipsychotics, which are frontline drugs in the treatment of schizophrenia and off-label use in other NPDs, also add to this burden by causing severe metabolic perturbations. Despite decades of research, the mechanism deciphering the link between neuropsychiatric and metabolic disorders is still unclear. In recent years, transient receptor potential Ankyrin 1 (TRPA1) channel has emerged as a potential therapeutic target for modulators. TRPA1 agonists/antagonists have shown efficacy in both neuropsychiatric disorders and appetite regulation and thus provide a crucial link between both. TRPA1 channels are activated by compounds such as cinnamaldehyde, allyl isothiocyanate, allicin and methyl syringate, which are present naturally in food items such as cinnamon, wasabi, mustard, garlic, etc. As these are present in many daily food items, it could also improve patient compliance and reduce the patients' monetary burden. In this review, we have tried to present evidence of the possible involvement of TRPA1 channels in neuropsychiatric and metabolic disorders and a possible hint towards using TRPA1 modulators to target appetite, lipid metabolism, glucose and insulin homeostasis and inflammation associated with NPDs.

Characterization of Novel Pathogenic Variants Causing Pyridox(am)ine 5'-Phosphate Oxidase-Dependent Epilepsy.

Several variants of the enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), responsible for a rare form of vitamin B6-dependent neonatal epileptic encephalopathy known as PNPO deficiency (PNPOD), have been reported. However, only a few of them have been characterised with respect to their structural and functional properties, despite the fact that the knowledge of how variants affect the enzyme may clarify the disease mechanism and improve treatment. Here, we report the characterisation of the catalytic, allosteric and structural properties of recombinantly expressed D33V, R161C, P213S, and E50K variants, among which D33V (present in approximately 10% of affected patients) is one of the more common variants responsible for PNPOD. The D33V and E50K variants have only mildly altered catalytic properties. In particular, the E50K variant, given that it has been found on the same chromosome with other known pathogenic variants, may be considered non-pathogenic. The P213S variant has lower thermal stability and reduced capability to bind the FMN cofactor. The variant involving Arg161 (R161C) largely decreases the affinity for the pyridoxine 5'-phosphate substrate and completely abolishes the allosteric feedback inhibition exerted by the pyridoxal 5'-phosphate product.

The biochemical subtype is a predictor for cognitive function in glutaric aciduria type 1: a national prospective follow-up study.

The aim of the study was a systematic evaluation of cognitive development in individuals with glutaric aciduria type 1 (GA1), a rare neurometabolic disorder, identified by newborn screening in Germany. This national, prospective, observational, multi-centre study includes 107 individuals with confirmed GA1 identified by newborn screening between 1999 and 2020 in Germany. Clinical status, development, and IQ were assessed using standardized tests. Impact of interventional and non-interventional parameters on cognitive outcome was evaluated. The majority of tested individuals (n = 72) showed stable IQ values with age (n = 56 with IQ test; median test age 11 years) but a significantly lower performance (median [IQR] IQ 87 [78-98]) than in general population, particularly in individuals with a biochemical high excreter phenotype (84 [75-96]) compared to the low excreter group (98 [92-105]; p = 0.0164). For all patients, IQ results were homogenous on subscale levels. Sex, clinical motor phenotype and quality of metabolic treatment had no impact on cognitive functions. Long-term neurologic outcome in GA1 involves both motor and cognitive functions. The biochemical high excreter phenotype is the major risk factor for cognitive impairment while cognitive functions do not appear to be impacted by current therapy and striatal damage. These findings implicate the necessity of new treatment concepts.

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 in the vitamin B6 salvage enzymes associated with neonatal epileptic encephalopathy and other pathologies.

Pyridoxal 5'-phosphate (PLP), the active cofactor form of vitamin B6 is required by over 160 PLP-dependent (vitamin B6) enzymes serving diverse biological roles, such as carbohydrates, amino acids, hemes, and neurotransmitters metabolism. Three key enzymes, pyridoxal kinase (PL kinase), pyridoxine 5'-phosphate oxidase (PNPO), and phosphatases metabolize and supply PLP to PLP-dependent enzymes through the salvage pathway. In born errors in the salvage enzymes are known to cause inadequate levels of PLP in the cell, particularly in neuronal cells. The resulting PLP deficiency is known to cause or implicated in several pathologies, most notably seizures. One such disorder, PNPO-dependent neonatal epileptic encephalopathy (NEE) results from natural mutations in PNPO and leads to null or reduced enzymatic activity. NEE does not respond to conventional antiepileptic drugs but may respond to treatment with the B6 vitamers PLP and/or pyridoxine (PN). In born errors that lead to PLP deficiency in cells have also been reported in PL kinase, however, to date none has been associated with epilepsy or seizure. One such pathology is polyneuropathy that responds to PLP therapy. Phosphatase deficiency or hypophosphatasia disorder due to pathogenic mutations in alkaline phosphatase is known to cause seizures that respond to PN therapy. In this article, we review the biochemical features of in born errors pertaining to the salvage enzyme's deficiency that leads to NEE and other pathologies. We also present perspective on vitamin B6 treatment for these disorders, along with attempts to develop zebrafish model to study the NEE syndrome in vivo.

Phenotypic and molecular spectrum of pyridoxamine-5'-phosphate oxidase deficiency: A scoping review of 87 cases of pyridoxamine-5'-phosphate oxidase deficiency.

Pyridoxamine-5'-phosphate oxidase (PNPO) deficiency is an autosomal recessive pyridoxal 5'-phosphate (PLP)-vitamin-responsive epileptic encephalopathy. The emerging feature of PNPO deficiency is the occurrence of refractory seizures in the first year of life. Pre-maturity and fetal distress, combined with neonatal seizures, are other associated key characteristics. The phenotype results from a dependency of PLP which regulates several enzymes in the body. We present the phenotypic and genotypic spectrum of (PNPO) deficiency based on a literature review (2002-2020) of reports (n = 33) of patients with confirmed PNPO deficiency (n = 87). All patients who received PLP (n = 36) showed a clinical response, with a complete dramatic PLP response with seizure cessation observed in 61% of patients. In spite of effective seizure control with PLP, approximately 56% of patients affected with PLP-dependent epilepsy suffer developmental delay/intellectual disability. There is no diagnostic biomarker, and molecular testing required for diagnosis. However, we noted that cerebrospinal fluid (CSF) PLP was low in 81%, CSF glycine was high in 80% and urinary vanillactic acid was high in 91% of the cases. We observed only a weak correlation between the severity of PNPO protein disruption and disease outcomes, indicating the importance of other factors, including seizure onset and time of therapy initiation. We found that pre-maturity, the delay in initiation of PLP therapy and early onset of seizures correlate with a poor neurocognitive outcome. Given the amenability of PNPO to PLP therapy for seizure control, early diagnosis is essential.

Inherited Disorders of Lysine Metabolism: A Review.

Lysine is an essential amino acid, and inherited diseases of its metabolism therefore represent defects of lysine catabolism. Although some of these enzyme defects are not well described yet, glutaric aciduria type I (GA1) and antiquitin (2-aminoadipic-6-semialdehyde dehydrogenase) deficiency represent the most well-characterized diseases. GA1 is an autosomal recessive disorder due to a deficiency of glutaryl-CoA dehydrogenase. Untreated patients exhibit early onset macrocephaly and may present a neurological deterioration with regression and movement disorder at the time of a presumably "benign" infection most often during the first year of life. This is associated with a characteristic neuroimaging pattern with frontotemporal atrophy and striatal injuries. Diagnosis relies on the identification of glutaric and 3-hydroxyglutaric acid in urine along with plasma glutarylcarnitine. Treatment consists of a low-lysine diet aiming at reducing the putatively neurotoxic glutaric and 3-hydroxyglutaric acids. Additional therapeutic measures include administration of l-carnitine associated with emergency measures at the time of intercurrent illnesses aiming at preventing brain injury. Early treated (ideally through newborn screening) patients exhibit a favorable long-term neurocognitive outcome, whereas late-treated or untreated patients may present severe neurocognitive irreversible disabilities. Antiquitin deficiency is the most common form of pyridoxine-dependent epilepsy. α-Aminoadipic acid semialdehyde (AASA) and Δ-1-piperideine-6-carboxylate (P6C) accumulate proximal to the enzymatic block. P6C forms a complex with pyridoxal phosphate (PLP), a key vitamer of pyridoxine, thereby reducing PLP bioavailability and subsequently causing epilepsy. Urinary AASA is a biomarker of antiquitin deficiency. Despite seizure control, only 25% of the pyridoxine-treated patients show normal neurodevelopment. Low-lysine diet and arginine supplementation are proposed in some patients with decrease of AASA, but the impact on neurodevelopment is unclear. In summary, GA1 and antiquitin deficiency are the 2 main human defects of lysine catabolism. Both include neurological impairment. Lysine dietary restriction is a key therapy for GA1, whereas its benefits in antiquitin deficiency appear less clear.

Glutaric acidemia type 1: Treatment and outcome of 168 patients over three decades.

Glutaric acidemia type 1 (GA1) is a disorder of cerebral organic acid metabolism resulting from biallelic mutations of GCDH. Without treatment, GA1 causes striatal degeneration in >80% of affected children before two years of age. We analyzed clinical, biochemical, and developmental outcomes for 168 genotypically diverse GA1 patients managed at a single center over 31 years, here separated into three treatment cohorts: children in Cohort I (n = 60; DOB 2006-2019) were identified by newborn screening (NBS) and treated prospectively using a standardized protocol that included a lysine-free, arginine-enriched metabolic formula, enteral l-carnitine (100 mg/kg•day), and emergency intravenous (IV) infusions of dextrose, saline, and l-carnitine during illnesses; children in Cohort II (n = 57; DOB 1989-2018) were identified by NBS and treated with natural protein restriction (1.0-1.3 g/kg•day) and emergency IV infusions; children in Cohort III (n = 51; DOB 1973-2016) did not receive NBS or special diet. The incidence of striatal degeneration in Cohorts I, II, and III was 7%, 47%, and 90%, respectively (p < .0001). No neurologic injuries occurred after 19 months of age. Among uninjured children followed prospectively from birth (Cohort I), measures of growth, nutritional sufficiency, motor development, and cognitive function were normal. Adherence to metabolic formula and l-carnitine supplementation in Cohort I declined to 12% and 32%, respectively, by age 7 years. Cessation of strict dietary therapy altered plasma amino acid and carnitine concentrations but resulted in no serious adverse outcomes. In conclusion, neonatal diagnosis of GA1 coupled to management with lysine-free, arginine-enriched metabolic formula and emergency IV infusions during the first two years of life is safe and effective, preventing more than 90% of striatal injuries while supporting normal growth and psychomotor development. The need for dietary interventions and emergency IV therapies beyond early childhood is uncertain.

Functional Recovery of a GCDH Variant Associated to Severe Deflavinylation-Molecular Insights into Potential Beneficial Effects of Riboflavin Supplementation in Glutaric Aciduria-Type I Patients.

Riboflavin is the biological precursor of two important flavin cofactors-flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)-that are critical prosthetic groups in several redox enzymes. While dietary supplementation with riboflavin is a recognized support therapy in several inborn errors of metabolism, it has yet unproven benefits in several other pathologies affecting flavoproteins. This is the case for glutaric aciduria type I (GA-I), a rare neurometabolic disorder associated with mutations in the GCDH gene, which encodes for glutaryl-coenzyme A (CoA) dehydrogenase (GCDH). Although there are a few reported clinical cases that have responded to riboflavin intake, there is still not enough molecular evidence supporting therapeutic recommendation. Hence, it is necessary to elucidate the molecular basis in favor of riboflavin supplementation in GA-I patients. Here, using a combination of biochemical and biophysical methodologies, we investigate the clinical variant GCDH-p.Val400Met as a model for a phenotype associated with severe deflavinylation. Through a systematic analysis, we establish that recombinant human GCDH-p.Val400Met is expressed in a nonfunctional apo form, which is mainly monomeric rather than tetrameric. However, we show that exogenous FAD is a driver for structural reorganization of the mutant enzyme with concomitant functional recovery, improved thermolability, and resistance to trypsin digestion. Overall, these results establish proof of principle for the beneficial effects of riboflavin supplementation in GA-I patients.

Encephalopathy in cystic fibrosis.

Cystic fibrosis liver disease (CFLD) affects a large proportion of cystic fibrosis (CF) patients; however encephalopathy is a rare complication. While classical hepatic encephalopathy can be a feature of end-stage liver disease, "hyperammonemic encephalopathy" can be precipitated in previously stable CFLD by various triggers including systemic corticosteroids. We describe one such case and review the relevant literature.

The lysine degradation pathway: Subcellular compartmentalization and enzyme deficiencies.

Lysine degradation via formation of saccharopine is a pathway confined to the mitochondria. The second pathway for lysine degradation, the pipecolic acid pathway, is not yet fully elucidated and known enzymes are localized in the mitochondria, cytosol and peroxisome. The tissue-specific roles of these two pathways are still under investigation. The lysine degradation pathway is clinically relevant due to the occurrence of two severe neurometabolic disorders, pyridoxine-dependent epilepsy (PDE) and glutaric aciduria type 1 (GA1). The existence of three other disorders affecting lysine degradation without apparent clinical consequences opens up the possibility to find alternative therapeutic strategies for PDE and GA1 through pathway modulation. A better understanding of the mechanisms, compartmentalization and interplay between the different enzymes and metabolites involved in lysine degradation is of utmost importance.