Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila

Abstract

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of β-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4, C8 and C12) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a β-strand connected by a short loop to an α-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as a potential model organism for MADD.

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

“…Some organism models, such as the Xavier and dark Xavier zebrafish, [23][24][25] have made many contributions to a broader understanding of the pathogenesis mechanism in MADD. Some organism models, such as the Xavier and dark Xavier zebrafish, [23][24][25] have made many contributions to a broader understanding of the pathogenesis mechanism in MADD.…”

Riboflavin‐responsive multiple acyl‐CoA dehydrogenase deficiency: A frequent condition in the southern Chinese population

“…More recently, a Drosophila model with etfdh mutation was produced. 35 It also showed similar abnormalities in terms of metabolic profile. However, both models did not fully recapitulate the human manifestations; further establishing an animal model of mammalian species might be still necessary for assessing the therapeutic efficacy of new drugs and elucidating the pathomechanism.…”

“…Interestingly, xavier shows several neural phenotypes including abnormal glial patterning, aberrant neural proliferation, reduced motor axon branching and neuromuscular synaptogenesis, which so far have not been reported in MADD patients. More recently, a Drosophila model with etfdh mutation was produced . It also showed similar abnormalities in terms of metabolic profile.…”

Novel ETFDH mutations in four cases of riboflavin responsive multiple acyl-CoA dehydrogenase deficiency

“…Three of the identified mutations in this study are located in the FAD binding domain (p.Arg175His, p.Gly77Ser, and p.Gly386Asp), and one is located in the UQ binding domain (p.Trp484Arg). Most of the previously reported ETFDH mutations are located in the FAD binding domain, suggesting that this region is a sensitive functional hot spot [ 26 ]. Riboflavin treatment has been suggested to promote FAD binding to ETF-QO mutants with variants in the FAD domain by affecting the kinetics and/or thermodynamics to stabilize the mutant conformations [ 27 ].…”