Periodic Paralysis in a Child With Thermosensitive Mitochondrial Trifunctional Protein Deficiency.

Mitochondrial trifunctional protein (MTP) deficiency is a fatty acid oxidation disorder associated with a spectrum of phenotypes. Patients with high residual enzyme activity tend to have milder phenotypes, and recently, fever-induced episodic myopathy was reported in association with a thermosensitive form of MTP deficiency. We report a 10-year-old male with recurrent episodes of acute flaccid paralysis involving upper and lower extremities in association with bulbar muscle weakness in the context of febrile illness, a phenotype reminiscent of recurrent periodic paralysis. The episodes started at the age of 3 years and have always been followed by full recovery within 1-2 weeks with no residual weakness. Whole exome sequencing revealed a homozygous c.2132C > T, p.(Pro711Leu) variant in HADHA. The variant leads to mildly reduced long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) and long-chain ketoacyl-CoA thiolase (LCKAT) enzyme activities and reduced MTP protein expression in patient's fibroblasts when cultured at 37°C. Enzyme activities and MTP protein expression diminished when fibroblasts were cultured at 40°C. This is the first published report of confirmed recurrent periodic paralysis as a manifestation of a thermosensitive form of MTP deficiency, and it calls for this condition to be considered when evaluating patients with recurrent periodic paralysis given therapeutic implications.

Extending diagnostic practices in gyrate atrophy: Enzymatic characterization and the development of an in vitro pyridoxine responsiveness assay.

Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5'-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [14C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care.

Thermo-sensitive mitochondrial trifunctional protein deficiency presenting with episodic myopathy.

Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.

3-Hydroxyisobutyric acid dehydrogenase deficiency: Expanding the clinical spectrum and quantitation of D- and L-3-Hydroxyisobutyric acid by an LC-MS/MS method.

A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients.

An autosomal dominant neurological disorder caused by de novo variants in FAR1 resulting in uncontrolled synthesis of ether lipids.

PURPOSE: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu). METHODS: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics. RESULTS: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production. CONCLUSION: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

BACKGROUND: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. METHODS: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. RESULTS: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001). CONCLUSIONS: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.

Mutations in PCYT2 disrupt etherlipid biosynthesis and cause a complex hereditary spastic paraplegia.

CTP:phosphoethanolamine cytidylyltransferase (ET), encoded by PCYT2, is the rate-limiting enzyme for phosphatidylethanolamine synthesis via the CDP-ethanolamine pathway. Phosphatidylethanolamine is one of the most abundant membrane lipids and is particularly enriched in the brain. We identified five individuals with biallelic PCYT2 variants clinically characterized by global developmental delay with regression, spastic para- or tetraparesis, epilepsy and progressive cerebral and cerebellar atrophy. Using patient fibroblasts we demonstrated that these variants are hypomorphic, result in altered but residual ET protein levels and concomitant reduced enzyme activity without affecting mRNA levels. The significantly better survival of hypomorphic CRISPR-Cas9 generated pcyt2 zebrafish knockout compared to a complete knockout, in conjunction with previously described data on the Pcyt2 mouse model, indicates that complete loss of ET function may be incompatible with life in vertebrates. Lipidomic analysis revealed profound lipid abnormalities in patient fibroblasts impacting both neutral etherlipid and etherphospholipid metabolism. Plasma lipidomics studies also identified changes in etherlipids that have the potential to be used as biomarkers for ET deficiency. In conclusion, our data establish PCYT2 as a disease gene for a new complex hereditary spastic paraplegia and confirm that etherlipid homeostasis is important for the development and function of the brain.

Prediction of disease severity in multiple acyl-CoA dehydrogenase deficiency: A retrospective and laboratory cohort study.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is an ultra-rare inborn error of mitochondrial fatty acid oxidation (FAO) and amino acid metabolism. Individual phenotypes and treatment response can vary markedly. We aimed to identify markers that predict MADD phenotypes. We performed a retrospective nationwide cohort study; then developed an MADD-disease severity scoring system (MADD-DS3) based on signs and symptoms with weighed expert opinions; and finally correlated phenotypes and MADD-DS3 scores to FAO flux (oleate and myristate oxidation rates) and acylcarnitine profiles after palmitate loading in fibroblasts. Eighteen patients, diagnosed between 1989 and 2014, were identified. The MADD-DS3 entails enumeration of eight domain scores, which are calculated by averaging the relevant symptom scores. Lifetime MADD-DS3 scores of patients in our cohort ranged from 0 to 29. FAO flux and [U-13 C]C2-, C5-, and [U-13 C]C16-acylcarnitines were identified as key variables that discriminated neonatal from later onset patients (all P < .05) and strongly correlated to MADD-DS3 scores (oleate: r = -.86; myristate: r = -.91; [U-13 C]C2-acylcarnitine: r = -.96; C5-acylcarnitine: r = .97; [U-13 C]C16-acylcarnitine: r = .98, all P < .01). Functional studies in fibroblasts were found to differentiate between neonatal and later onset MADD-patients and were correlated to MADD-DS3 scores. Our data may improve early prediction of disease severity in order to start (preventive) and follow-up treatment appropriately. This is especially relevant in view of the inclusion of MADD in population newborn screening programs.

A mutation creating an upstream translation initiation codon in SLC22A5 5'UTR is a frequent cause of primary carnitine deficiency.

Primary carnitine deficiency is caused by a defect in the active cellular uptake of carnitine by Na+ -dependent organic cation transporter novel 2 (OCTN2). Genetic diagnostic yield for this metabolic disorder has been relatively low, suggesting that disease-causing variants are missed. We Sanger sequenced the 5' untranslated region (UTR) of SLC22A5 in individuals with possible primary carnitine deficiency in whom no or only one mutant allele had been found. We identified a novel 5'-UTR c.-149G>A variant which we characterized by expression studies with reporter constructs in HeLa cells and by carnitine-transport measurements in fibroblasts using a newly developed sensitive assay based on tandem mass spectrometry. This variant, which we identified in 57 of 236 individuals of our cohort, introduces a functional upstream out-of-frame translation initiation codon. We show that the codon suppresses translation from the wild-type ATG of SLC22A5, resulting in reduced OCTN2 protein levels and concomitantly lower transport activity. With an allele frequency of 24.2% the c.-149G>A variant is the most frequent cause of primary carnitine deficiency in our cohort and may explain other reported cases with an incomplete genetic diagnosis. Individuals carrying this variant should be clinically re-evaluated and monitored to determine if this variant has clinical consequences.

Overexpression of carbamoyl-phosphate synthase 1 significantly improves ureagenesis of human liver HepaRG cells only when cultured under shaking conditions.

Hyperammonemia is an important contributing factor to hepatic encephalopathy in end-stage liver failure patients. Therefore reducing hyperammonemia is a requisite of bioartificial liver support (BAL). Ammonia elimination by human liver HepaRG cells occurs predominantly through reversible fixation into amino acids, whereas the irreversible conversion into urea is limited. Compared to human liver, the expression and activity of the three urea cycle (UC) enzymes carbamoyl-phosphate synthase1 (CPS1), ornithine transcarbamoylase (OTC) and arginase1, are low. To improve HepaRG cells as BAL biocomponent, its rate limiting factor of the UC was determined under two culture conditions: static and dynamic medium flow (DMF) achieved by shaking. HepaRG cells increasingly converted escalating arginine doses into urea, indicating that arginase activity is not limiting ureagenesis. Neither was OTC activity, as a stable HepaRG line overexpressing OTC exhibited a 90- and 15.7-fold upregulation of OTC transcript and activity levels, without improvement in ureagenesis. However, a stable HepaRG line overexpressing CPS1 showed increased mitochondrial stress and reduced hepatic differentiation without promotion of the CPS1 transcript level or ureagenesis under static-culturing conditions, yet, it exhibited a 4.3-fold increased ureagenesis under DMF. This was associated with increased CPS1 transcript and activity levels amounting to >2-fold, increased mitochondrial abundance and hepatic differentiation. Unexpectedly, the transcript levels of several other UC genes increased up to 6.8-fold. We conclude that ureagenesis can be improved in HepaRG cells by CPS1 overexpression, however, only in combination with DMF-culturing, suggesting that both the low CPS1 level and static-culturing, possibly due to insufficient mitochondria, are limiting UC.

Barth syndrome cells display widespread remodeling of mitochondrial complexes without affecting metabolic flux distribution.

Barth syndrome (BTHS) is a rare X-linked disorder that is characterized by cardiac and skeletal myopathy, neutropenia and growth abnormalities. The disease is caused by mutations in the tafazzin (TAZ) gene encoding an enzyme involved in the acyl chain remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, this leads to decreased levels of mature CL and accumulation of the intermediate monolysocardiolipin (MLCL). At a cellular level, this causes mitochondrial fragmentation and reduced stability of the respiratory chain supercomplexes. However, the exact mechanism through which tafazzin deficiency leads to disease development remains unclear. We therefore aimed to elucidate the pathways affected in BTHS cells by employing proteomic and metabolic profiling assays. Complexome profiling of patient skin fibroblasts revealed significant effects for about 200 different mitochondrial proteins. Prominently, we found a specific destabilization of higher order oxidative phosphorylation (OXPHOS) supercomplexes, as well as changes in complexes involved in cristae organization and CL trafficking. Moreover, the key metabolic complexes 2-oxoglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase (BCKD) were profoundly destabilized in BTHS patient samples. Surprisingly, metabolic flux distribution assays using stable isotope tracer-based metabolomics did not show reduced flux through the TCA cycle. Overall, insights from analyzing the impact of TAZ mutations on the mitochondrial complexome provided a better understanding of the resulting functional and structural consequences and thus the pathological mechanisms leading to Barth syndrome.

Biallelic loss of function variants in COASY cause prenatal onset pontocerebellar hypoplasia, microcephaly, and arthrogryposis.

Pontocerebellar hypoplasia (PCH) is a heterogeneous neurodegenerative disorder with a prenatal onset. Using whole-exome sequencing, we identified variants in the gene Coenzyme A (CoA) synthase (COASY) gene, an enzyme essential in CoA synthesis, in four individuals from two families with PCH, prenatal onset microcephaly, and arthrogryposis. In family 1, compound heterozygous variants were identified in COASY: c.[1549_1550delAG]; [1486-3 C>G]. In family 2, all three affected siblings were homozygous for the c.1486-3 C>G variant. In both families, the variants segregated with the phenotype. RNA analysis showed that the c.1486-3 C>G variant leads to skipping of exon 7 with partial retention of intron 7, disturbing the reading frame and resulting in a premature stop codon (p.(Ala496Ilefs*20)). No CoA synthase protein was detected in patient cells by immunoblot analysis and CoA synthase activity was virtually absent. Partial CoA synthase defects were previously described as a cause of COASY Protein-Associated Neurodegeneration (CoPAN), a type of Neurodegeneration and Brain Iron Accumulation (NBIA). Here we demonstrate that near complete loss of function variants in COASY are associated with lethal PCH and arthrogryposis.

Identification of enzymes involved in oxidation of phenylbutyrate.

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the ß-oxidation of PB. We used cells deficient in specific steps of fatty acid ß-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug.

A novel UPLC-MS/MS based method to determine the activity of N-acetylglutamate synthase in liver tissue.

BACKGROUND: N-acetylglutamate synthase (NAGS) plays a key role in the removal of ammonia via the urea cycle by catalyzing the synthesis of N-acetylglutamate (NAG), the obligatory cofactor in the carbamyl phosphate synthetase 1 reaction. Enzymatic analysis of NAGS in liver homogenates has remained insensitive and inaccurate, which prompted the development of a novel method. METHODS: UPLC-MS/MS was used in conjunction with stable isotope (N-acetylglutamic-2,3,3,4,4-d5 acid) dilution for the quantitative detection of NAG produced by the NAGS enzyme. The assay conditions were optimized using purified human NAGS and the optimized enzyme conditions were used to measure the activity in mouse liver homogenates. RESULTS: A low signal-to-noise ratio in liver tissue samples was observed due to non-enzymatic formation of N-acetylglutamate and low specific activity, which interfered with quantitative analysis. Quenching of acetyl-CoA immediately after the incubation circumvented this analytical difficulty and allowed accurate and sensitive determination of mammalian NAGS activity. The specificity of the assay was validated by demonstrating a complete deficiency of NAGS in liver homogenates from Nags -/- mice. CONCLUSION: The novel NAGS enzyme assay reported herein can be used for the diagnosis of inherited NAGS deficiency and may also be of value in the study of secondary hyperammonemia present in various inborn errors of metabolism as well as drug treatment.

Clinical and biochemical characterization of four patients with mutations in ECHS1.

BACKGROUND: Short-chain enoyl-CoA hydratase (SCEH, encoded by ECHS1) catalyzes hydration of 2-trans-enoyl-CoAs to 3(S)-hydroxy-acyl-CoAs. SCEH has a broad substrate specificity and is believed to play an important role in mitochondrial fatty acid oxidation and in the metabolism of branched-chain amino acids. Recently, the first patients with SCEH deficiency have been reported revealing only a defect in valine catabolism. We investigated the role of SCEH in fatty acid and branched-chain amino acid metabolism in four newly identified patients. In addition, because of the Leigh-like presentation, we studied enzymes involved in bioenergetics. METHODS: Metabolite, enzymatic, protein and genetic analyses were performed in four patients, including two siblings. Palmitate loading studies in fibroblasts were performed to study mitochondrial ß-oxidation. In addition, enoyl-CoA hydratase activity was measured with crotonyl-CoA, methacrylyl-CoA, tiglyl-CoA and 3-methylcrotonyl-CoA both in fibroblasts and liver to further study the role of SCEH in different metabolic pathways. Analyses of pyruvate dehydrogenase and respiratory chain complexes were performed in multiple tissues of two patients. RESULTS: All patients were either homozygous or compound heterozygous for mutations in the ECHS1 gene, had markedly reduced SCEH enzymatic activity and protein level in fibroblasts. All patients presented with lactic acidosis. The first two patients presented with vacuolating leukoencephalopathy and basal ganglia abnormalities. The third patient showed a slow neurodegenerative condition with global brain atrophy and the fourth patient showed Leigh-like lesions with a single episode of metabolic acidosis. Clinical picture and metabolite analysis were not consistent with a mitochondrial fatty acid oxidation disorder, which was supported by the normal palmitate loading test in fibroblasts. Patient fibroblasts displayed deficient hydratase activity with different substrates tested. Pyruvate dehydrogenase activity was markedly reduced in particular in muscle from the most severely affected patients, which was caused by reduced expression of E2 protein, whereas E2 mRNA was increased. CONCLUSIONS: Despite its activity towards substrates from different metabolic pathways, SCEH appears to be only crucial in valine metabolism, but not in isoleucine metabolism, and only of limited importance for mitochondrial fatty acid oxidation. In severely affected patients SCEH deficiency can cause a secondary pyruvate dehydrogenase deficiency contributing to the clinical presentation.

Fatty acid oxidation flux predicts the clinical severity of VLCAD deficiency.

PURPOSE: Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is an inherited disorder of mitochondrial long-chain fatty acid ß-oxidation (LC-FAO) and is included in many newborn screening (NBS) programs worldwide. Patients may present with hypoketotic hypoglycemia, cardiomyopathy, and/or myopathy, but clinical severity varies widely and the clinical outcome is unpredictable. We investigated predictive markers that may determine clinical severity. METHODS: We developed a clinical severity score (CSS), which was determined for 13 Dutch patients with VLCADD, all of whom were diagnosed before the introduction of VLCADD in NBS to prevent bias from early diagnosis. In cultured skin fibroblasts from these patients, we measured LC-FAO flux (the rate of oleate oxidation), VLCAD activity, and acylcarnitine profiles following palmitate loading. RESULTS: The strongest correlation (r = 0.93; P < 0.0001) was observed between LC-FAO flux and the CSS. VLCAD activity measurement and the C14/C16-to-acylcarnitine ratio correlated much less. A median LC-FAO flux of 6% of control values (range 5.6-6.8%) was associated with cardiomyopathy (P < 0.01), and 32.4% (range 5.6-50.5%) was associated with myopathy (P < 0.05). CONCLUSION: Our results demonstrate a very strong correlation between LC-FAO flux in fibroblasts and the clinical severity of VLCADD. LC-FAO flux measurements may thus predict whether patients are likely to develop symptoms.

Monocarboxylate transporter 1 deficiency and ketone utilization.

Ketoacidosis is a potentially lethal condition caused by the imbalance between hepatic production and extrahepatic utilization of ketone bodies. We performed exome sequencing in a patient with recurrent, severe ketoacidosis and identified a homozygous frameshift mutation in the gene encoding monocarboxylate transporter 1 (SLC16A1, also called MCT1). Genetic analysis in 96 patients suspected of having ketolytic defects yielded seven additional inactivating mutations in MCT1, both homozygous and heterozygous. Mutational status was found to be correlated with ketoacidosis severity, MCT1 protein levels, and transport capacity. Thus, MCT1 deficiency is a novel cause of profound ketoacidosis; the present work suggests that MCT1-mediated ketone-body transport is needed to maintain acid-base balance.

Identification and characterization of Eci3, a murine kidney-specific Δ3,Δ2-enoyl-CoA isomerase.

Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ(3),Δ(2)-enoyl-CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl-CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans-3-nonenoyl-CoA to trans-2-nonenoyl-CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E-3) and tissue morphogenesis (P=1.0E-3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.

Impaired amino acid metabolism contributes to fasting-induced hypoglycemia in fatty acid oxidation defects.

The importance of mitochondrial fatty acid ß-oxidation (FAO) as a glucose-sparing process is illustrated by patients with inherited defects in FAO, who may present with life-threatening fasting-induced hypoketotic hypoglycemia. It is unknown why peripheral glucose demand outpaces hepatic gluconeogenesis in these patients. In this study, we have systematically addressed the fasting response in long-chain acyl-CoA dehydrogenase-deficient (LCAD KO) mice. We demonstrate that the fasting-induced hypoglycemia in LCAD KO mice was initiated by an increased glucose requirement in peripheral tissues, leading to rapid hepatic glycogen depletion. Gluconeogenesis did not compensate for the increased glucose demand, which was not due to insufficient hepatic glucogenic capacity but rather caused by a shortage in the supply of glucogenic precursors. This shortage in supply was explained by a suppressed glucose-alanine cycle, decreased branched-chain amino acid metabolism and ultimately impaired protein mobilization. We conclude that during fasting, FAO not only serves to spare glucose but is also indispensable for amino acid metabolism, which is essential for the maintenance of adequate glucose production.