The male-to-female ratio in late-onset multiple acyl-CoA dehydrogenase deficiency: a systematic review and meta-analysis.

BACKGROUND: Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common lipid storage myopathy. There are sex differences in fat metabolism and it is not known whether late-onset MADD affects men and women equally. METHODS: In this systematic review and meta-analysis, the PubMed, Embase, Web of Science, CNKI, CBM, and Wanfang databases were searched until 01/08/2023. Studies reporting sex distribution in patients with late-onset MADD were included. Two authors independently screened studies for eligibility, extracted data, and assessed risk of bias. Pre-specified outcomes of interest were the male-to-female ratio (MFR) of patients with late-onset MADD, the differences of clinical characteristics between the sexes, and factors influencing the MFR. RESULTS: Of 3379 identified studies, 34 met inclusion criteria, yielding a total of 609 late-onset MADD patients. The overall pooled percentage of males was 58% (95% CI, 54-63%) with low heterogeneity across studies (I2 = 2.99%; P = 0.42). The mean onset ages, diagnostic delay, serum creatine kinase (CK), and allelic frequencies of 3 hotspot variants in ETFDH gene were similar between male and female patients (P > 0.05). Meta-regressions revealed that ethnic group was associated with the MFR in late-onset MADD, and subgroup meta-analyses demonstrated that East-Asian patients had a higher percentage of male, lower CK, and higher proportion of hotspot variants in ETFDH gene than non-East-Asian patients (P < 0.05). CONCLUSIONS: Male patients with late-onset MADD were more common than female patients. Ethnicity was proved to be a factor influencing the MFR in late-onset MADD. These findings suggest that male sex may be a risk factor for the disease.

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.

Retrograde response to mitochondrial dysfunctions associated to LOF variations in FLAD1 exon 2: unraveling the importance of RFVT2.

Flavin adenine dinucleotide (FAD) synthase (EC 2.7.7.2), encoded by human flavin adenine dinucleotide synthetase 1 (FLAD1), catalyzes the last step of the pathway converting riboflavin (Rf) into FAD. FLAD1 variations were identified as a cause of LSMFLAD (lipid storage myopathy due to FAD synthase deficiency, OMIM #255100), resembling Multiple Acyl-CoA Dehydrogenase Deficiency, sometimes treatable with high doses of Rf; no alternative therapeutic strategies are available. We describe here cell morphological and mitochondrial alterations in dermal fibroblasts derived from a LSMFLAD patient carrying a homozygous truncating FLAD1 variant (c.745C > T) in exon 2. Despite a severe decrease in FAD synthesis rate, the patient had decreased cellular levels of Rf and flavin mononucleotide and responded to Rf treatment. We hypothesized that disturbed flavin homeostasis and Rf-responsiveness could be due to a secondary impairment in the expression of the Rf transporter 2 (RFVT2), encoded by SLC52A2, in the frame of an adaptive retrograde signaling to mitochondrial dysfunction. Interestingly, an antioxidant response element (ARE) is found in the region upstream of the transcriptional start site of SLC52A2. Accordingly, we found that abnormal mitochondrial morphology and impairments in bioenergetics were accompanied by increased cellular reactive oxygen species content and mtDNA oxidative damage. Concomitantly, an active response to mitochondrial stress is suggested by increased levels of PPARγ-co-activator-1α and Peroxiredoxin III. In this scenario, the treatment with high doses of Rf might compensate for the secondary RFVT2 molecular defect, providing a molecular rationale for the Rf responsiveness in patients with loss of function variants in FLAD1 exon 2.HIGHLIGHTSFAD synthase deficiency alters mitochondrial morphology and bioenergetics;FAD synthase deficiency triggers a mitochondrial retrograde response;FAD synthase deficiency evokes nuclear signals that adapt the expression of RFVT2.

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.

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.

Novel variant of ETFDH leading to multiple acyl-CoA dehydrogenase deficiency by promoting protein degradation via ubiquitin proteasome pathway.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare autosomal recessive metabolic disease. Patients present with metabolic decompensation, muscle weakness, respiratory failure, and cardiomyopathy. Late-onset MADD is primarily caused by mutations in the ETFDH gene. Here, we report a patient who has been diagnosed with Down syndrome after birth following karyotype analysis and simultaneously carrying compound heterozygous variants of ETFDH (c.3G > C (p. M1?); c.725C > T (p. T242I), which is novel). Further molecular analyses revealed that the novel c.725C > T (p. T242I) mutation enhances the degradation of electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) via the ubiquitin proteasome pathway. Five ubiquitin E3 ligases (STUB1, RNF40, UBE3C, CUL3, and CUL1) and one ubiquitin modification site (Cystein, C101) of the ETF-QO were reported in this study. Our study not only expanded the pathogenic variant spectrum of ETFDH gene but also proved that the c.725C > T (p. T242I) will promote protein degradation through ubiquitin proteasome pathway.

Multiple acyl-CoA dehydrogenase deficiency kills Mycobacterium tuberculosis in vitro and during infection.

The human pathogen Mycobacterium tuberculosis depends on host fatty acids as a carbon source. However, fatty acid ß-oxidation is mediated by redundant enzymes, which hampers the development of antitubercular drugs targeting this pathway. Here, we show that rv0338c, which we refer to as etfD, encodes a membrane oxidoreductase essential for ß-oxidation in M. tuberculosis. An etfD deletion mutant is incapable of growing on fatty acids or cholesterol, with long-chain fatty acids being bactericidal, and fails to grow and survive in mice. Analysis of the mutant's metabolome reveals a block in ß-oxidation at the step catalyzed by acyl-CoA dehydrogenases (ACADs), which in other organisms are functionally dependent on an electron transfer flavoprotein (ETF) and its cognate oxidoreductase. We use immunoprecipitation to show that M. tuberculosis EtfD interacts with FixA (EtfB), a protein that is homologous to the human ETF subunit ß and is encoded in an operon with fixB, encoding a homologue of human ETF subunit α. We thus refer to FixA and FixB as EtfB and EtfA, respectively. Our results indicate that EtfBA and EtfD (which is not homologous to human EtfD) function as the ETF and oxidoreductase for ß-oxidation in M. tuberculosis and support this pathway as a potential target for tuberculosis drug development.

Exploring the contribution of mitochondrial dynamics to multiple acyl-CoA dehydrogenase deficiency-related phenotype.

Mitochondrial fatty acid ß-oxidation disorders (FAOD) are among the diseases detected by newborn screening in most developed countries. Alterations of mitochondrial functionality are characteristic of these metabolic disorders. However, many questions remain to be clarified, namely how the interplay between the signaling pathways harbored in mitochondria contributes to the disease-related phenotype. Herein, we overview the role of mitochondria on the regulation of cell homeostasis through the production of ROS, mitophagy, apoptosis, and mitochondrial biogenesis. Emphasis is given to the signaling pathways involving MnSOD, sirtuins and PGC-1α, which seem to contribute to FAOD phenotype, namely to multiple acyl-CoA dehydrogenase deficiency (MADD). The association between phenotype and genotype is not straightforward, suggesting that specific molecular mechanisms may contribute to MADD pathogenesis, making MADD an interesting model to better understand this interplay. However, more work needs to be done envisioning the development of novel therapeutic strategies.

Disorders of flavin adenine dinucleotide metabolism: MADD and related deficiencies.

Multiple acyl-coenzyme A dehydrogenase deficiency (MADD), or glutaric aciduria type II (GAII), is a group of clinically heterogeneous disorders caused by mutations in electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) - the two enzymes responsible for the re-oxidation of enzyme-bound flavin adenine dinucleotide (FADH2) via electron transfer to the respiratory chain at the level of coenzyme Q10. Over the past decade, an increasing body of evidence has further coupled mutations in FAD metabolism (including intercellular riboflavin transport, FAD biosynthesis and FAD transport) to MADD-like phenotypes. In this review we provide a detailed description of the overarching and specific metabolic pathways involved in MADD. We examine the eight associated genes (ETFA, ETFB, ETFDH, FLAD1, SLC25A32 and SLC52A1-3) and clinical phenotypes, and report ∼436 causative mutations following a systematic literature review. Finally, we focus attention on the value and shortcomings of current diagnostic approaches, as well as current and future therapeutic options for MADD and its phenotypic disorders.

Mitochondrial energetic impairment in a patient with late-onset glutaric acidemia Type 2.

Glutaric acidemia type 2 (GA2), also called multiple acyl-CoA dehydrogenase deficiency, is an autosomal recessive disorder of fatty acid, amino acid, and choline metabolism resulting in excretion of multiple organic acids and glycine conjugates as well as elevation of various plasma acylcarnitine species (C4-C18). It is caused by mutations in the ETFA, ETFB, or ETFDH genes which are involved in the transfer of electrons from 11 flavin-containing dehydrogenases to Coenzyme Q10 (CoQ10 ) of the mitochondrial electron transport chain (ETC). We report a patient who was originally reported as the first case with primary myopathic CoQ10 deficiency when he presented at 11.5 years with exercise intolerance and myopathy that improved after treatment with ubiquinone and carnitine. At age 23, his symptoms relapsed despite increasing doses of ubiquinone and he was shown to have biallelic mutations in the ETFDH gene. Treatment with riboflavin was started and ubiquinone was changed to ubiquinol. After 4 months, the patient recovered his muscle strength with normalization of laboratory exams and exercise tolerance. Functional studies on fibroblasts revealed decreased levels of ETFDH as well as of very long-chain acyl-CoA dehydrogenase and trifunctional protein α. In addition, the mitochondrial mass was decreased, with increased formation of reactive oxygen species and oxygen consumption rate, but with a decreased spared respiratory capacity, and decreased adenosine triphosphate level. These findings of widespread dysfunction of fatty acid oxidation and ETC enzymes support the impairment of a larger mitochondrial ETC supercomplex in our patient.

Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy.

Inborn errors of Riboflavin (Rf) transport and metabolism have been recently related to severe human neuromuscular disorders, as resulting in profound alteration of human flavoproteome and, therefore, of cellular bioenergetics. This explains why the interest in studying the "flavin world", a topic which has not been intensively investigated before, has increased much over the last few years. This also prompts basic questions concerning how Rf transporters and FAD (flavin adenine dinucleotide) -forming enzymes work in humans, and how they can create a coordinated network ensuring the maintenance of intracellular flavoproteome. The concept of a coordinated cellular "flavin network", introduced long ago studying humans suffering for Multiple Acyl-CoA Dehydrogenase Deficiency (MADD), has been, later on, addressed in model organisms and more recently in cell models. In the frame of the underlying relevance of a correct supply of Rf in humans and of a better understanding of the molecular rationale of Rf therapy in patients, this review wants to deal with theories and existing experimental models in the aim to potentiate possible therapeutic interventions in Rf-related neuromuscular diseases.

Conformational analysis of the riboflavin-responsive ETF:QO-p.Pro456Leu variant associated with mild multiple acyl-CoA dehydrogenase deficiency.

Multiple-CoA dehydrogenase deficiency (MADD) is an inborn disorder of fatty acid and amino acid metabolism caused by mutations in the genes encoding for human electron transfer flavoprotein (ETF) and its partner electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). Albeit a rare disease, extensive newborn screening programs contributed to a wider coverage of MADD genotypes. However, the impact of non-lethal mutations on ETF:QO function remains scarcely understood from a structural perspective. To this end, we here revisit the relatively common MADD mutation ETF:QO-p.Pro456Leu, in order to clarify how it affects enzyme structure and folding. Given the limitation in recombinant expression of human ETF:QO, we resort to its bacterial homologue from Rhodobacter sphaeroides (Rs), in which the corresponding mutation (p.Pro389Leu) was inserted. The in vitro biochemical and biophysical investigations of the Rs ETF:QO-p.Pro389Leu variant showed that, while the mutation does not significantly affect the protein α/ß fold, it introduces some plasticity on the tertiary structure and within flavin interactions. Indeed, in the p.Pro389Leu variant, FAD exhibits a higher thermolability during thermal denaturation and a faster rate of release in temperature-induced dissociation experiments, in comparison to the wild type. Therefore, although this clinical mutation occurs in the ubiquinone domain, its effect likely propagates to the nearby FAD binding domain, probably influencing electron transfer and redox potentials. Overall, our results provide a molecular rational for the decreased enzyme activity observed in patients and suggest that compromised FAD interactions in ETF:QO might account for the known riboflavin responsiveness of this mutation.

Hepatic neddylation targets and stabilizes electron transfer flavoproteins to facilitate fatty acid ß-oxidation.

Neddylation is a ubiquitination-like pathway that controls cell survival and proliferation by covalently conjugating NEDD8 to lysines in specific substrate proteins. However, the physiological role of neddylation in mammalian metabolism remains elusive, and no mitochondrial targets have been identified. Here, we report that mouse models with liver-specific deficiency of NEDD8 or ubiquitin-like modifier activating enzyme 3 (UBA3), the catalytic subunit of the NEDD8-activating enzyme, exhibit neonatal death with spontaneous fatty liver as well as hepatic cellular senescence. In particular, liver-specific UBA3 deficiency leads to systemic abnormalities similar to glutaric aciduria type II (GA-II), a rare autosomal recessive inherited fatty acid oxidation disorder resulting from defects in mitochondrial electron transfer flavoproteins (ETFs: ETFA and ETFB) or the corresponding ubiquinone oxidoreductase. Neddylation inhibition by various strategies results in decreased protein levels of ETFs in neonatal livers and embryonic hepatocytes. Hepatic neddylation also enhances ETF expression in adult mice and prevents fasting-induced steatosis and mortality. Interestingly, neddylation is active in hepatic mitochondria. ETFs are neddylation substrates, and neddylation stabilizes ETFs by inhibiting their ubiquitination and degradation. Moreover, certain mutations of ETFs found in GA-II patients hinder the neddylation of these substrates. Taken together, our results reveal substrates for neddylation and add insight into GA-II.

Determinants of Riboflavin Responsiveness in Multiple Acyl-CoA Dehydrogenase Deficiency.

BACKGROUND: Multiple acyl-CoA dehydrogenase (MADD) deficiency, which is a rare metabolic disorder involving electron transport flavoproteins, has a wide array of clinical phenotypes. In this article, we describe 25 patients with MADD deficiency and present the clinical and laboratory characteristics and diagnostic challenges associated with riboflavin-responsive MADD deficiency. METHODS: Hospital records of patients with biallelic mutations in ETFA, ETFB, or ETFDH genes diagnosed in a single center were analyzed retrospectively. Demographic, clinical, and laboratory characteristics of patients with riboflavin-responsive and riboflavin-unresponsive MADD deficiency were compared using Mann-Whitney U and Fisher's exact tests. RESULTS: Respiratory distress and depressed consciousness were significantly more common in patients with riboflavin-unresponsive MADD deficiency (P = 0.015 and P < 0.001), who presented at a younger age (P < 0.001). Patients with riboflavin-responsive MADD deficiency had favorable outcomes but also had life-threatening complications, longer diagnostic delay (median of two years versus 30 days; P < 0.001), and multiple differential diagnoses, resulting in unnecessary investigations and maltreatment. Biopsies showed lipid storage, and complete autopsy was performed in one newborn with riboflavin-unresponsive MADD deficiency, revealing multiple abnormalities. Metabolic profiles were not distinguishable between riboflavin-responsive and riboflavin-unresponsive MADD deficiency (P > 0.05). Four novel variants were detected in ETFDH, one of which (c.1790C>T) may confer riboflavin responsiveness. Siblings with the common myopathic ETFDH c.1130T>C mutation presented with a new phenotype dominated by chronic fatigue without apparent myopathy. CONCLUSIONS: Symptoms and outcomes significantly differed between riboflavin-responsive and unresponsive MADD deficiency, but metabolic profiles did not. Functional studies are needed to better characterize the novel ETFDH variants. As treatment is available for riboflavin-responsive MADD deficiency, physicians should maintain a high index of suspicion for MADD deficiency in all age groups.

Nucleotide exchange factor Rab3GEP requires DENN and non-DENN elements for activation and targeting of Rab27a.

Rab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3GKO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3GKO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.

Two cases of glutaric aciduria type II: how to differentiate from inflammatory myopathies?

Muscle weakness is a nonspecific finding of myopathy of any etiology that include iatrogenic, toxic, endocrinological, infectious, immunologic, and metabolic disorders. Among the metabolic myopathies glutaric aciduria type II (GAII) is an autosomal recessively inherited rare disorder of fatty acid and amino acid metabolisms. The late onset form is heterogeneous in terms of symptomatology and severity and for the cases that chronic manifestations of lipid storage myopathy are the only clues for the disease, differential diagnosis can be challenging. Here we report two cases of GAII: the first one was 18-year old boy who presented with proximal muscle weakness and in another center, he was diagnosed as polymyositis and treated with immunosuppressive therapies. He admitted to our clinic with ongoing muscle weakness and symptoms that were related to the side effects of immunosuppressive therapies. The second case was also presented with muscle weakness. For both cases, muscle biopsies and urinary organic acid analyses were consistent with the diagnosis of GAII. To differentiate inflammatory myositis from non-inflammatory myopathies; rheumatic symptoms, accompanying complaints of the patient and autoantibody positivity can be helpful. To our knowledge this is the first report to underline the differential diagnosis of inflammatory myopathies from metabolic myopathies.

Cofactors revisited - Predicting the impact of flavoprotein-related diseases on a genome scale.

Flavin adenine dinucleotide (FAD) and its precursor flavin mononucleotide (FMN) are redox cofactors that are required for the activity of more than hundred human enzymes. Mutations in the genes encoding these proteins cause severe phenotypes, including a lack of energy supply and accumulation of toxic intermediates. Ideally, patients should be diagnosed before they show symptoms so that treatment and/or preventive care can start immediately. This can be achieved by standardized newborn screening tests. However, many of the flavin-related diseases lack appropriate biomarker profiles. Genome-scale metabolic models can aid in biomarker research by predicting altered profiles of potential biomarkers. Unfortunately, current models, including the most recent human metabolic reconstructions Recon and HMR, typically treat enzyme-bound flavins incorrectly as free metabolites. This in turn leads to artificial degrees of freedom in pathways that are strictly coupled. Here, we present a reconstruction of human metabolism with a curated and extended flavoproteome. To illustrate the functional consequences, we show that simulations with the curated model - unlike simulations with earlier Recon versions - correctly predict the metabolic impact of multiple-acyl-CoA-dehydrogenase deficiency as well as of systemic flavin-depletion. Moreover, simulations with the new model allowed us to identify a larger number of biomarkers in flavoproteome-related diseases, without loss of accuracy. We conclude that adequate inclusion of cofactors in constraint-based modelling contributes to higher precision in computational predictions.

ETFDH Mutations and Flavin Adenine Dinucleotide Homeostasis Disturbance Are Essential for Developing Riboflavin-Responsive Multiple Acyl-Coenzyme A Dehydrogenation Deficiency.

OBJECTIVE: Riboflavin-responsive multiple acyl-coenzyme A dehydrogenation deficiency (RR-MADD) is an inherited fatty acid metabolism disorder mainly caused by genetic defects in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF:QO). The variant ETF:QO protein folding deficiency, which can be corrected by therapeutic dosage of riboflavin supplement, has been identified in HEK-293 cells and is believed to be the molecular mechanism of this disease. To verify this hypothesis in vivo, we generated Etfdh (h)A84T knockin (KI) mice. METHODS: Tissues from these mice as well as muscle biopsies and fibroblasts from 7 RR-MADD patients were used to examine the flavin adenine dinucleotide (FAD) concentration and ETF:QO protein amount. RESULTS: All of the homozygous KI mice (Etfdh (h)A84T/(h)A84T , KI/KI) were initially normal. After being given a high-fat and vitamin B2 -deficient (HF-B2 D) diet for 5 weeks, they developed weight loss, movement ability defects, lipid storage in muscle and liver, and elevated serum acyl-carnitine levels, which are clinically and biochemically similar to RR-MADD patients. Both ETF:QO protein and FAD concentrations were significantly decreased in tissues of HF-B2 D-KI/KI mice and in cultured fibroblasts from RR-MADD patients. After riboflavin treatment, ETF:QO protein increased in proportion to elevated FAD concentrations, but not related to mRNA levels. These results were further confirmed in cultured fibroblasts from RR-MADD patients. INTERPRETATION: For the first time, we successfully developed a RR-MADD mice model and confirmed that FAD homeostasis disturbances played a crucial role on the pathomechanism of RR-MADD in this mouse model and culture cells from patients. Supplementation of riboflavin may stabilize variant ETF:QO protein by rebuilding FAD homeostasis. Ann Neurol 2018;84:667-681.

An intronic variation in SLC52A1 causes exon skipping and transient riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency.

Vitamin B2, riboflavin is essential for cellular function, as it participates in a diversity of redox reactions central to human metabolism, through its role as precursor for the cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are electron carriers. The electron transfer flavoprotein (ETF) and its dehydrogenase (ETFDH), uses FAD as cofactor. The ETF and ETFDH are forming the electron transport pathway for many mitochondrial flavoprotein dehydrogenases involved in fatty acid, amino acid and choline metabolism. A variation in either ETF or ETFDH causes multiple acyl-CoA dehydrogenation deficiency (MADD), but genetic variations in the riboflavin metabolism or transportation of riboflavin can also cause MADD. The most common variations are located in the riboflavin transporter 2 (RFVT2) and 3 (RFVT3), that are highly expressed in brain and intestinal tissues, respectively. Deficiency of riboflavin transporter 1 (RFVT1), encoded by the SLC52A1 gene, highly expressed in the placenta, has only been reported once. We here report a case of transient MADD, caused by a heterozygous intronic variation, c.1134+11G>A, in the SLC52A1 gene encoding RFVT1. This variation creates a binding site for the splice inhibitory hnRNP A1 protein and causes exon 4 skipping. Riboflavin deficiency and maternal malnutrition during pregnancy might have been the determining factor in the outcome of this case.