A comparative study on riboflavin responsive multiple acyl-CoA dehydrogenation deficiency due to variants in FLAD1 and ETFDH gene.

Lipid storage myopathy (LSM) is a heterogeneous group of lipid metabolism disorders predominantly affecting skeletal muscle by triglyceride accumulation in muscle fibers. Riboflavin therapy has been shown to ameliorate symptoms in some LSM patients who are essentially concerned with multiple acyl-CoA dehydrogenation deficiency (MADD). It is proved that riboflavin responsive LSM caused by MADD is mainly due to ETFDH gene variant (ETFDH-RRMADD). We described here a case with riboflavin responsive LSM and MADD resulting from FLAD1 gene variants (c.1588 C > T p.Arg530Cys and c.1589 G > C p.Arg530Pro, FLAD1-RRMADD). And we compared our patient together with 9 FLAD1-RRMADD cases from literature to 106 ETFDH-RRMADD cases in our neuromuscular center on clinical history, laboratory investigations and pathological features. Furthermore, the transcriptomics study on FLAD1-RRMADD and ETFDH-RRMADD were carried out. On muscle pathology, both FLAD1-RRMADD and ETFDH-RRMADD were proved with lipid storage myopathy in which atypical ragged red fibers were more frequent in ETFDH-RRMADD, while fibers with faint COX staining were more common in FLAD1-RRMADD. Molecular study revealed that the expression of GDF15 gene in muscle and GDF15 protein in both serum and muscle was significantly increased in FLAD1-RRMADD and ETFDH-RRMADD groups. Our data revealed that FLAD1-RRMADD (p.Arg530) has similar clinical, biochemical, and fatty acid metabolism changes to ETFDH-RRMADD except for muscle pathological features.

An ETFDH-driven metabolon supports OXPHOS efficiency in skeletal muscle by regulating coenzyme Q homeostasis.

Coenzyme Q (Q) is a key lipid electron transporter, but several aspects of its biosynthesis and redox homeostasis remain undefined. Various flavoproteins reduce ubiquinone (oxidized form of Q) to ubiquinol (QH2); however, in eukaryotes, only oxidative phosphorylation (OXPHOS) complex III (CIII) oxidizes QH2 to Q. The mechanism of action of CIII is still debated. Herein, we show that the Q reductase electron-transfer flavoprotein dehydrogenase (ETFDH) is essential for CIII activity in skeletal muscle. We identify a complex (comprising ETFDH, CIII and the Q-biosynthesis regulator COQ2) that directs electrons from lipid substrates to the respiratory chain, thereby reducing electron leaks and reactive oxygen species production. This metabolon maintains total Q levels, minimizes QH2-reductive stress and improves OXPHOS efficiency. Muscle-specific Etfdh-/- mice develop myopathy due to CIII dysfunction, indicating that ETFDH is a required OXPHOS component and a potential therapeutic target for mitochondrial redox medicine.

A case report on multiple acyl-CoA dehydrogenase deficiency with severe myopathy and osteoporosis.

A 35-year-old male patient presented fluctuating bilateral lower extremity weakness for 3 years. Physical examination showed grade 4 proximal muscle weakness in both lower extremities and grade 5 distal muscle weakness. Laboratory data revealed elevated creatine kinase, triglycerides, and cholesterol. Muscle pathology showed deposition of lipid droplet under the sarcolemma. Bone densitometry indicated severe osteoporosis. Next-generation sequencing revealed a pathogenic mutation in the ETFDH gene. The patient was diagnosed with late-onset multiple acyl-CoA dehydrogenase deficiency. After riboflavin treatment, symptoms of the patient were relieved, physical endurance was restored, and bone mineral density was improved.

High-fat diet increases electron transfer flavoprotein synthesis and lipid respiration in skeletal muscle during exercise training in female mice.

High-fat diet (HFD) and exercise remodel skeletal muscle mitochondria. The electron transfer flavoproteins (ETF) transfer reducing equivalents from ß-oxidation into the electron transfer system. Exercise may stimulate the synthesis of ETF proteins to increase lipid respiration. We determined mitochondrial remodeling for lipid respiration through ETF in the context of higher mitochondrial abundance/capacity seen in female mice. We hypothesized HFD would be a greater stimulus than exercise to remodel ETF and lipid pathways through increased protein synthesis alongside increased lipid respiration. Female C57BL/6J mice (n = 15 per group) consumed HFD or low-fat diet (LFD) for 4 weeks then remained sedentary (SED) or completed 8 weeks of treadmill training (EX). We determined mitochondrial lipid respiration, RNA abundance, individual protein synthesis, and abundance for ETFα, ETFß, and ETF dehydrogenase (ETFDH). HFD increased absolute and relative lipid respiration (p = 0.018 and p = 0.034) and RNA abundance for ETFα (p = 0.026), ETFß (p = 0.003), and ETFDH (p = 0.0003). HFD increased synthesis for ETFα and ETFDH (p = 0.0007 and p = 0.002). EX increased synthesis of ETFß and ETFDH (p = 0.008 and p = 0.006). Higher synthesis rates of ETF were not always reflected in greater protein abundance. Greater synthesis of ETF during HFD indicates mitochondrial remodeling which may contribute higher mitochondrial lipid respiration through enhanced ETF function.

Generation of an induced pluripotent stem cell (iPSC) line (INNDSUi002-A) from a patient with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency.

Mutations in the ETFDH gene, encoding electron transfer flavoprotein dehydrogenase, have been identified to cause riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD) (Wen et al., 2010). We performed the generation and characterization of human induced pluripotent stem cell (iPSC) line from skin fibroblasts of a patient with RR-MADD carrying two heterozygous ETFDH mutations (p.D130V and p.A84V). Their pluripotency was verified by the expression of several pluripotency markers on RNA and protein levels and the capability to differentiate into all three germ layers.

Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein.

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.

The clinical, pathological, and genetic characteristics of lipid storage myopathy in northern China.

BACKGROUND: The lipid storage myopathy (LSM) diagnosis is based on the patient's clinical manifestations and muscle pathology. However, when genetic testing is lacking, there is a high rate of misdiagnosis of the disease. This study aimed to investigate the clinical and pathological features of genetically diagnosed LSM in northern China, analyze genetic mutations' characteristics, and improve the LSM diagnostic rate. METHODS: Twenty patients with LSM diagnosed were collected; meanwhile, the clinical data, muscle samples, and routine pathological staining of muscle specimens were collected. The morphological changes of muscle fibers were observed under an optical microscope. RESULTS: Among the included patients, 18 cases had ETFDH (HGNC ID: 3483) mutations, and two had PNPLA2 mutations. Family pedigree verification was performed on three patients with heterozygous mutations in the ETFDH gene complex. Histopathological staining showed that all patients had fine vacuoles in the muscle fibers, and some of them merged to form fissures, and the lipid droplets increased in cells. After therapy, 18 patients were associated with a favorable prognosis, and two patients were ineffective with the treatment of neutral lipid storage myopathy (NLSDM) caused by PNPLA2 mutation. DISCUSSION: The clinical manifestations of LSM are complex and diverse, mainly manifested by proximal muscle weakness and exercise intolerance in the extremities. The pathological images of LSM muscles are abnormal storage of lipid droplets in muscle fibers, primarily involving type I fibers. The LSM patients were mainly multiple acyl-CoA dehydrogenase deficiency (MADD) caused by the ETFDH gene mutation. It is necessary to perform an accurate typing diagnosis of LSM.

An unusual presentation of type III late onset multiple-acyl-CoA dehydrogenase deficiency leading to a review of its classification system.

Multiple-acyl-CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive disorder which can be split into three types. Type III MADD is associated with acute or subacute proximal muscle weakness and other variable non-specific features making it a challenging diagnosis for the clinician. This case report describes MADD in a 64 year-old lady, thought to be one of the latest first presentations of the disease. Unusually for this condition, the initial presentation was with dyspnoea. Furthermore, since this case provides further evidence that gene variants can predict age of onset, we advocate for further subclassification of type III MADD into late onset MADD (LO-MADD) when homozygous gene variants are present and very LO-MADD when heterozygous gene variants are found.

Incorporating second-tier genetic screening for multiple acyl-CoA dehydrogenase deficiency.

BACKGROUND: Newborn screening (NBS) for multiple acyl-CoA dehydrogenase deficiency (MADD) has poor sensitivity. This study aimed to evaluate the feasibility of incorporating second-tier genetic screening for MADD. METHODS: A total of 453,390 newborns were screened for inherited metabolic disorders using tandem mass spectrometry from January 2017 to May 2022. A matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assay was developed to identify 23 common ETFDH variants and used for second-tier screening of MADD. RESULTS: Overall, 185 newborns with suspected MADD received second-tier genetic screening. Thirty-three (17.8 %) newborns with positive results, of which 7 were homozygotes, 5 were compound heterozygotes, 21 were heterozygotes. Further genetic analysis revealed that 6 of the 21 newborns had a second ETFDH variant. Therefore, 18 patients were finally diagnosed with MADD, with a positive predictive value of 9.73 %. The detection rate and diagnostic rate of MALDI-TOF MS assay were 83.33 % and 66.67 %, respectively. Thus the incidence of MADD in our population was estimated at 1:25,188. Nine different ETFDH variants were identified in MADD patients. The most common ETFDH variant being c.250G > A with an allelic frequency of 47.22 %, followed by c.524G > A (13.89 %) and c.998A > G (13.89 %). All patients had elevation of multiple acylcarnitines at NBS. However, seven patients had normal acylcarnitine levels and two patients showed mild elevation of only two acylcarnitines during the recall review. CONCLUSION: We have established a high throughput MALDI-TOF MS assay for MADD screening. Half of the MADD patients would not be detected under conventional screening protocols. Incorporating second-tier genetic screening into the current NBS could improve the performance of MADD NBS.

Unusual reactivity of a flavin in a bifurcating electron-transferring flavoprotein leads to flavin modification and a charge-transfer complex.

From the outset, canonical electron transferring flavoproteins (ETFs) earned a reputation for containing modified flavin. We now show that modification occurs in the recently recognized bifurcating (Bf) ETFs as well. In Bf ETFs, the 'electron transfer' (ET) flavin mediates single electron transfer via a stable anionic semiquinone state, akin to the FAD of canonical ETFs, whereas a second flavin mediates bifurcation (the Bf FAD). We demonstrate that the ET FAD undergoes transformation to two different modified flavins by a sequence of protein-catalyzed reactions that occurs specifically in the ET site, when the enzyme is maintained at pH 9 in an amine-based buffer. Our optical and mass spectrometric characterizations identify 8-formyl flavin early in the process and 8-amino flavins (8AFs) at later times. The latter have not previously been documented in an ETF to our knowledge. Mass spectrometry of flavin products formed in Tris or bis-tris-aminopropane solutions demonstrates that the source of the amine adduct is the buffer. Stepwise reduction of the 8AF demonstrates that it can explain a charge transfer band observed near 726 nm in Bf ETF, as a complex involving the hydroquinone state of the 8AF in the ET site with the oxidized state of unmodified flavin in the Bf site. This supports the possibility that Bf ETF can populate a conformation enabling direct electron transfer between its two flavins, as has been proposed for cofactors brought together in complexes between ETF and its partner proteins.

Generation of a human induced pluripotent stem cell line (LZUSHi002-A) from a MADD patient with ETFDH mutation.

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is an inborn metabolic disorder that affects fatty acid oxidation and the catabolism of branched-chain amino acids, vitamins B and energy metabolism. In this study, the induced pluripotent stem cell (iPSC) line LZUSHi002-A from PBMCs of a 10-year-old male patient with ETFDH mutations using the episomal plasmids was established, which is an ideal in vitro model to understand the exact pathogenesis of MADD.

A fatal case of neonatal onset multiple acyl-CoA dehydrogenase deficiency caused by novel mutation of ETFDH gene: case report.

BACKGROUND: Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II is an extremely rare autosomal recessive inborn error of fatty acid beta oxidation and branched-chain amino acids, secondary to mutations in the genes encoding the electron transfer flavoproteins A and B (ETFs; ETFA or ETFB) or ETF dehydrogenase (ETFDH). The clinical manifestation of MADD are heterogeneous, from severe neonatal forms to mild late-onset forms. CASE PRESENTATION: We report the case of a preterm newborn who died a few days after birth for a severe picture of untreatable metabolic acidosis. The diagnosis of neonatal onset MADD was suggested on the basis of clinical features displaying congenital abnormalities and confirmed by the results of expanded newborn screening, which arrived the day the newborn died. Molecular genetic test revealed a homozygous indel variant c.606 + 1 _606 + 2insT in the ETFDH gene, localized in a canonical splite site. This variant, segregated from the two heterozygous parents, is not present in the general population frequency database and has never been reported in the literature. DISCUSSION AND CONCLUSION: Recently introduced Expanded Newborn Screening is very important for a timely diagnosis of Inherited Metabolic Disorders like MADD. In some cases which are the most severe, diagnosis may arrive after symptoms are already present or may be the neonate already died. This stress the importance of collecting all possible samples to give parents a proper diagnosis and a genetic counselling for future pregnacies.

Features and diagnostic value of body composition in patients with late-onset multiple acyl-CoA dehydrogenase deficiency.

OBJECTIVE: This study aims to analyse the body composition features and its changes after treatment in patients with late-onset multiple acyl-CoA dehydrogenase deficiency (MADD). METHODS: Body composition was measured in patients with late-onset MADD, inflammatory myopathies, mitochondrial myopathy, and healthy controls. The correlation analyses between body composition and traditional parameters were performed. Comparisons between groups and receiver operating characteristic curve analyses were performed. RESULTS: A total of 42 participants included 13 patients with late-onset MADD, 13 healthy controls, 10 with inflammatory myopathy, and 6 with mitochondrial myopathy. Bilateral grip strength and forced vital capacity (FVC) were moderate-strong correlated with skeletal muscle mass (right hand grip strength: r = 0.728, P < 0.001; left hand grip strength: r = 0.676, P < 0.001; FVC: r = 0.754, P < 0.001). Serum CK was moderately and negatively correlated with right hand grip strength (r = - 0.618, P = 0.005), left hand grip strength (r = - 0.630, P = 0.004), FVC (r = - 0.665, P = 0.002), manual muscle testing (MMT) (r = - 0.729, P = 0.000), and lean body mass skeletal muscle percentage (r = - 0.501, P = 0.029). Body composition features in patients with late-onset MADD were as follows: (1) obvious fat accumulation, (2) reduction of muscle mass, and (3) reduction of body water and intracellular water ratio. Some indicators of body composition were found to be valuable in diagnosis and eliminating differential diagnoses, such as visceral fat area (sensitivity 84.62%; specificity 92.31%; AUC 0.905) and fat mass (sensitivity 84.62%; specificity 75.00%; AUC 0.837). Seven patients were followed-up (2-9 months). Prior to treatment, the changes in body composition in these patients were conflicting. CONCLUSIONS: Hand grip strength and FVC were strongly associated with body composition. Body composition features in late-onset MADD are fat accumulation, muscle loss, decrease in total body water, and intracellular water ratio. Body composition features are valuable for diagnosis and assessment.

S1P defects cause a new entity of cataract, alopecia, oral mucosal disorder, and psoriasis-like syndrome.

In this report, we discovered a new entity named cataract, alopecia, oral mucosal disorder, and psoriasis-like (CAOP) syndrome in two unrelated and ethnically diverse patients. Furthermore, patient 1 failed to respond to regular treatment. We found that CAOP syndrome was caused by an autosomal recessive defect in the mitochondrial membrane-bound transcription factor peptidase/site-1 protease (MBTPS1, S1P). Mitochondrial abnormalities were observed in patient 1 with CAOP syndrome. Furthermore, we found that S1P is a novel mitochondrial protein that forms a trimeric complex with ETFA/ETFB. S1P enhances ETFA/ETFB flavination and maintains its stability. Patient S1P variants destabilize ETFA/ETFB, impair mitochondrial respiration, decrease fatty acid ß-oxidation activity, and shift mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. Mitochondrial dysfunction and inflammatory lesions in patient 1 were significantly ameliorated by riboflavin supplementation, which restored the stability of ETFA/ETFB. Our study discovered that mutations in MBTPS1 resulted in a new entity of CAOP syndrome and elucidated the mechanism of the mutations in the new disease.

The reductive half-reaction of two bifurcating electron-transferring flavoproteins: Evidence for changes in flavin reduction potentials mediated by specific conformational changes.

The EtfAB components of two bifurcating flavoprotein systems, the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase from the bacterium Megasphaera elsdenii and the menaquinone-dependent NADH:ferredoxin oxidoreductase from the archaeon Pyrobaculum aerophilum, have been investigated. With both proteins, we find that removal of the electron-transferring flavin adenine dinucleotide (FAD) moiety from both proteins results in an uncrossing of the reduction potentials of the remaining bifurcating FAD; this significantly stabilizes the otherwise very unstable semiquinone state, which accumulates over the course of reductive titrations with sodium dithionite. Furthermore, reduction of both EtfABs depleted of their electron-transferring FAD by NADH was monophasic with a hyperbolic dependence of reaction rate on the concentration of NADH. On the other hand, NADH reduction of the replete proteins containing the electron-transferring FAD was multiphasic, consisting of a fast phase comparable to that seen with the depleted proteins followed by an intermediate phase that involves significant accumulation of FAD⋅-, again reflecting uncrossing of the half-potentials of the bifurcating FAD. This is then followed by a slow phase that represents the slow reduction of the electron-transferring FAD to FADH-, with reduction of the now fully reoxidized bifurcating FAD by a second equivalent of NADH. We suggest that the crossing and uncrossing of the reduction half-potentials of the bifurcating FAD is due to specific conformational changes that have been structurally characterized.

Electron Transfer Flavoprotein (ETF) α Controls Blood Vessel Development by Regulating Endothelial Mitochondrial Bioenergetics and Oxygen Consumption.

While impairment of vascular homeostasis induced by hypercholesterolemia is the first step of cardiovascular diseases, the molecular mechanism behind such impairment is not well known. Here, we reported that high-cholesterol diet (HCD) induced defective vessel sprouting in zebrafish larvae. Electron transfer flavoprotein subunit α (ETFα) (encoded by the ETFA gene), a protein that mediates transfer of electrons from a series of mitochondrial flavoenzymes to the respiratory chain, was downregulated in HCD-fed zebrafish and in endothelial cells treated with oxidized low-density lipoprotein. Knockdown of ETFα with morpholino antisense oligonucleotides reproduced vascular sprouting defects in zebrafish larvae, while replenishing with exogeneous ETFA mRNA could successfully rescue these defects. ETFA knockdown in endothelial cells reduces cell migration, proliferation, and tube formation in vitro. Finally, knockdown of ETFA in endothelial cells also reduced fatty acid oxidation, oxygen consumption rate, and hypoxia-inducible factor-1α (HIF1α) protein levels. Taken together, we demonstrate that downregulation of ETFα is involved in hypercholesterolemia-induced defective vessel sprouting in zebrafish larvae via inhibition of endothelial proliferation and migration. The molecular mechanism behind this phenomenon is the decrease of HIF1α induced by downregulation of ETFα in endothelial cells. This work suggests that disturbance of ETFα-mediated oxygen homeostasis is one of the mechanisms behind hypercholesterolemia-induced vascular dysfunction.

Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins.

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.

Nitrogen nutrition contributes to plant fertility by affecting meiosis initiation.

Nitrogen (N), one of the most important plant nutrients, plays crucial roles in multiple plant developmental processes. Spikelets are the primary sink tissues during reproductive growth, and N deficiency can cause floral abortion. However, the roles of N nutrition in meiosis, the crucial step in plant sexual reproduction, are poorly understood. Here, we identified an N-dependent meiotic entrance mutant with loss of function of ELECTRON TRANSFER FLAVOPROTEIN SUBUNIT ß (ETFß) in rice (Oryza sativa). etfß displayed meiosis initiation defects, excessive accumulation of branched-chain amino acids (BCAAs) and decrease in total N contents in spikelets under N starvation, which were rescued by applying excess exogenous inorganic N. Under N starvation, ETFß, through its involvement in BCAA catabolism, promotes N reutilization and contributes to meeting N demands of spikelets, highlighting the impact of N nutrition on meiosis initiation. We conclude that N nutrition contributes to plant fertility by affecting meiosis initiation.

The role of the electron-transfer flavoprotein: ubiquinone oxidoreductase following carbohydrate starvation in Arabidopsis cell cultures.

KEY MESSAGE: The functional absence of the electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) directly impacts electrons donation to the mitochondrial electron transport chain under carbohydrate-limiting conditions without major impacts on the respiration of cell cultures. Alternative substrates (e.g., amino acids) can directly feed electrons into the mitochondrial electron transport chain (mETC) via the electron transfer flavoprotein/electron-transfer flavoprotein: ubiquinone oxidoreductase (ETF/ETFQO) complex, which supports plant respiration during stress situations. By using a cell culture system, here we investigated the responses of Arabidopsis thaliana mutants deficient in the expression of ETFQO (etfqo-1) following carbon limitation and supplied with amino acids. Our results demonstrate that isovaleryl-CoA dehydrogenase (IVDH) activity was induced during carbon limitation only in wild-type and that these changes occurred concomit with enhanced protein content. By contrast, neither the activity nor the total amount of IVDH was altered in etfqo-1 mutants. We also demonstrate that the activities of mitochondrial complexes in etfqo-1 mutants, display a similar pattern as in wild-type cells. Our findings suggest that the defect of ETFQO protein culminates with an impaired functioning of the IVDH, since no induction of IVDH activity was observed. However, the functional absence of the ETFQO seems not to cause major impacts on plant respiration under carbon limiting conditions, most likely due to other alternative electron entry pathways.

Hypoketotic hypoglycemia without neuromuscular complications in patients with SLC25A32 deficiency.

Mitochondrial flavin adenine dinucleotide (FAD) transporter deficiencies are new entities recently reported to cause a neuro-myopathic phenotype. We report three patients from two unrelated families who presented primarily with hypoketotic hypoglycemia. They all had acylcarnitine profiles suggestive of multiple acyl-CoA dehydrogenase deficiency (MADD) with negative next-generation sequencing of electron-transfer flavoprotein genes (ETFA, ETFB, and ETFDH). Whole exome sequencing revealed a homozygous c.272 G > T (p.Gly91Val) variant in exon 2 of the SLC25A32 gene. The three patients shared the same variant, and they all demonstrated similar clinical and biochemical improvement with riboflavin supplementation. To date, these are the first patients to be reported with hypoketotic hypoglycemia without the neuromuscular phenotype previously reported in patients with SLC25A32 deficiency.