The Pathogenic Mechanism Of Riboflavin-responsive Multiple Acyl-CoAdehydrogenation Deficiency

Riboflavin-responsive forms of multiple acyl-Co A dehydrogenation deficiency(RR-MADD) is an autosomal-recessive inherited, fatty acid, amino acid and choline metabolism disorder caused by defects in electron flavopprotein(ETF) or ETF ubiquinone oxidoreductase(ETFQO), due to dysfunction of multiple mitochondrial flavoprotein dehydrogenases. Clinical manifestations may develop progressive proximal myopathy with heterogeneous symptoms such as intermittent episodes of vomiting, nausea, metabolic acidosis et al. EMG showed diversiform changes including myogenic, neurogenic and both patterns. Most of RR-MADD patients riboflavin treatment strikingly ameliorates the symptoms. Pathology showed lipid droplets were prominently in type I fibers. Light microscopic assessment of histochemical stains filled with numerous lipid droplet vacuoles in fiber.We recruited to investigate 53 RR- MADD patients with ETFDH mutations,ETFDH mutations as a major cause of RR-MADD. ETFDH-c.250 G >A is the homozygous mutations and compound heterozygous mutations, representing a high allelic frequency of 83.3%(80/96).RR-MADD is due to ETFDH that fatty acid β-oxidation disordered. The analysis of acylglycines and urinary organic acid are very important biochemical tool for the diagnosis of inherited disorders. Acylglycine analysis by tandem mass spectrometry. Blood acylcarnitine analysis usually displays elevated concentrations of mainly medium- and long-chain acylcarnitines in RR-MADD patients. Organic acid analysis by tandem mass spectrometry. especially ethylmalonic acid,2-hydroxyglutaric acid, lactic acid and ketone bodies and glycine conjugate was found significant increase. We found Blood acylcarnitine and urinary organic acid usually displays elevated while before and after riboflavin treatment or stability and acute stage of disease most of RR-MADD patients.This is the collection of 53 RR-MADD patients detected, the human ETFDHmutations was screening, to analysis clinical features, a genotype–phenotype relationship has been demonstrated in RR-MADD of the Han ethnic group from southern China. The analysis of acylcarnitine is an important biochemical tool for the diagnosis of RR-MADD, and to provide the help of riboflavin treatment.Methods1. 53 RR-MADD patients were screen in ETFDH gene by PCR, direct sequencing and PCR-RFLP.2. Lipid storage myopathy was confirmed by Hematoxylin and eosin stain, ORO stain and ATPase stain.3. The lipid accumulations in human RR-MADD and normal control fibroblasts by immunocytochemical.4. Blood acylcarnitine analysis by tandem mass spectrometry urinary organic acid assay by gas chromatography/mass spectrometry.5. Immunocytochemical showed cell types.Results1. RR-MADD is a major caused by ETFDH mutations.2. RR-MADD patients, fibers were filled with numerous vacuoles.3. Cultured cells contained myoblasts and fibroblasts.4. Measurement of blood acylcarnitines and urinary organic acid profiles is useful to make a diagnosis in RR-MADD.Multiple acyl-Co A dehydrogenation deficiency(MADD) is a defect of the electron transfer flavoprotein or the electron transfer flavoprotein ubiquinone oxidoreductase, multiple dehydrogenation reactions are impaired because of defective of electrons from a number of flavoprotein dehydrogenates to the mitochondrial respiratory chain. Most RR-MADD patients have mutations in the ETFDH gene encoding ETF-QO. Mutations of ETFDH might lead to ETFDH proteins with decreased stability. The molecular defect is still unknown that mutations of ETFDH is regulated post-translational modifications to ETFDH.In our previous study, our results showed that variant ETFDH protein underwent the ubiquitin proteasomal degradation. When cells were treated with the proteasome inhibitor epoxomicin, ETFDH protein increased. When the proteasome inhibitor by epoxomicin caused accumulation of the poly-ubiquitinated forms of ETFDH. It found ubiquitination that mutations ETFDH more than wild-type. We conformed CHIP of E3 ubiquitin ligases for mutations ETFDH. ETFDH forms a complex with CHIP at endogenous and transient transfection level. CHIP were ubiquitinated ETFDH.Overexpression of CHIP lead to endogenous ETFDH proteasomal degradation in MADD fibroblast cells. On the contrary,knockdown of endogenous CHIP causes an accumulation of level of ETFDH. In summary, we think ETFDH degradation and ubiquitination that mutations ETFDH more than wild-type.Mutations ETFDH often lead to decreased stability.We also tested external FAD increases medium cultured human fibroblast cells,when treated in medium containing 10 u M FAD for 5 h, ETF-QO variant proteins showed almost back to normal when compared with wild-type, our data suggest that FAD Could be stabilize ETFDH protein expression.It is elucidate the mechanism that mutations of ETFDH lead to protein decreased. In order to provide a new perspective with study variant ETFDH gene. To help elucidate the pathogenesis of RR-MADD. To provide scientific basis and theoretical foundation for treatment.Methods1. 293 T cells were transiently co-transfected with HA-ETFDH or HA-ETFDH-A84 T mutant and Myc-CHIP. ETFDH protein amount was detected by western blot analysis of whole cell lysates.2. The immunoprecipitates were analyzed ETFDH forms a complex with CHIP,and CHIP ubiquitnates ETFDH in vivo.3. Fibroblasts were infected with ETFDH shRNA based on lentivirus vector. 7 to 14 days after infection, the specificity of ETFDH antibody was detected by western blot and Immunocytochemical staining.4. Fibroblasts were infected with CHIP sh RNA based on lentivirus vector. 7 to 14 days after infection, CHIP ubiquitnates ETFDH by western blot and Immunocyto-chemical staining.5. FAD could be stabilizes the native conformation of certain types of ETFDH variant proteins, and protein expression of RFK and FADS in human fibroblasts by western blot analysis.Results1. The antibody is specificity human ETFDH protein.2. Skeletal muscle and cultured fibroblasts that ETF-QO variant proteins showed also more than 50% decreased protein when compared with wild-type.3. E3 ubiquition ligase CHIP efficiently promoted ETFDH degradation, especially mutant ETFDH.3.1 We found CHIPand E3 ubiquitin ligase activity and required for promoting ETFDH degradation.3.2 ETFDH forms a complex with CHIP in vivo.3.3 CHIP ubiquitnates ETFDH3.4 Overexpression of CHIP lead to endogenous ETFDH proteasomal degradation. On the contrary, knockdown of endogenous CHIP causes an accumulation of level of ETFDH.4. FAD stabilizes ETFDH variant ETFDH.4.1 RFK and FADS protein level no different RR-MADD pateients and normal control.4.2 FAD could be promoted folding and stabilizes the native conformation of certain types of ETFDH variant proteins.