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 B 2 –deficient (HF‐B 2 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‐B 2 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