Genotype-environment interaction drives the onset of riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency in carriers of single heterozygous ETFDH variants.

backgroundRiboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD) is an autosomal recessive disorder of fatty acid oxidation predominantly caused by variants in the ETFDH gene. However, approximately 10% of patients carry a single heterozygous variant. We hypothesize that ETFDH haploinsufficiency may contribute to the development of RR-MADD, especially under certain environmental stressors.

methodsSkin fibroblasts derived from one RR-MADD patient carrying a heterozygous variant in ETFDH and his asymptomatic father carrying the same variant were cultured. Under varying concentrations of riboflavin, ETFDH gene expression profiles, intracellular free fatty acid (FFA) levels, mitochondrial function, cellular viability, and accumulation of reactive oxygen species (ROS) and lipid droplets were assessed and compared between the two cell lines. These phenotypic comparisons were subsequently extended to HEK293 cell models engineered to carry either heterozygous or homozygous c.917G > A variants in ETFDH. Etfdh knock-in mice carrying c.250G > A (p.A84T) variants were used to evaluate the contribution of single heterozygous variants to disease susceptibility at the organismal level.

resultsUnder identical culture conditions, fibroblasts from the RR-MADD patient and his father exhibited comparable levels of ETFDH gene expression and FFA, along with similar mitochondrial function, cellular viability, ROS production, and accumulation of lipid droplets. This finding suggests that environmental factors rather than genotype cause phenotypic differences between the two individuals. Moreover, exposure to severe riboflavin deficiency induced a similar RR-MADD phenotype in HEK293 cells regardless of whether they carried heterozygous or homozygous variants. Mice harboring the heterozygous Etfdh c.250G > A variant exhibited a classic RR-MADD phenotype and pathological changes following combined dietary interventions consisting of riboflavin deficiency and high-fat diet.

conclusionsThis study demonstrates that single heterozygous ETFDH variants confer increased susceptibility to RR-MADD in the presence of specific environmental stressors, offering new genetic insights into the inheritance pattern of the disease.