Francesco Baldo

Multiple acyl-CoA dehydrogenase deficiency (MADD) is considered an autosomal recessive disorder; yet, recent findings suggest up to 10% of cases may result from heterozygous electron transfer flavoprotein dehydrogenase (ETFDH) variants exhibiting dominant or dominant-like effects. Here, a novel heterozygous ETFDH variant (c.1798A>C, p.Asn600His) was identified within a three-generation family. The grandfather presented with muscular weakness at age 35, and the father developed similar symptoms at 19 following a tonsillectomy. Both were diagnosed with MADD based on muscle biopsies revealing neutral lipid accumulation and acylcarnitine profiles and responded fully to riboflavin therapy (150 mg/day). The two siblings, aged 8 and 10, carry the same mutation and show increased acyl-carnitine levels but remain asymptomatic due to early riboflavin treatment. Skin fibroblasts from affected individuals were immortalized and subjected to normal and reduced riboflavin levels. Gene expression analysis demonstrated unchanged ETFDH RNA but reduced protein levels in mutant cells, particularly under low riboflavin. Structural modelling suggested the Asn600His substitution destabilizes the protein, diminishing its mitochondrial function. Proximity ligation assays indicated a decreased interaction with mitochondrial complex III, while oxygen consumption via fatty acid oxidation was impaired, especially at reduced riboflavin. The novel ETFDH variant found in this family gives a possible dominant pattern of inheritance for MADD, where a single mutant allele impairs the mitochondrial metabolism, particularly under riboflavin-deficient conditions, and highlights the importance of early riboflavin supplementation in preventing clinical symptoms.