Riboflavin-Responsive and -Non-responsive Mutations in FAD Synthase Cause Multiple Acyl-CoA Dehydrogenase and Combined Respiratory-Chain Deficiency

Abstract

Multiple acyl-CoA dehydrogenase deficiencies (MADDs) are a heterogeneous group of metabolic disorders with combined respiratory-chain deficiency and a neuromuscular phenotype. Despite recent advances in understanding the genetic basis of MADD, a number of cases remain unexplained. Here, we report clinically relevant variants in FLAD1, which encodes FAD synthase (FADS), as the cause of MADD and respiratory-chain dysfunction in nine individuals recruited from metabolic centers in six countries. In most individuals, we identified biallelic frameshift variants in the molybdopterin binding (MPTb) domain, located upstream of the FADS domain. Inasmuch as FADS is essential for cellular supply of FAD cofactors, the finding of biallelic frameshift variants was unexpected. Using RNA sequencing analysis combined with protein mass spectrometry, we discovered FLAD1 isoforms, which only encode the FADS domain. The existence of these isoforms might explain why affected individuals with biallelic FLAD1 frameshift variants still harbor substantial FADS activity. Another group of individuals with a milder phenotype responsive to riboflavin were shown to have single amino acid changes in the FADS domain. When produced in E. coli, these mutant FADS proteins resulted in impaired but detectable FADS activity; for one of the variant proteins, the addition of FAD significantly improved protein stability, arguing for a chaperone-like action similar to what has been reported in other riboflavin-responsive inborn errors of metabolism. In conclusion, our studies identify FLAD1 variants as a cause of potentially treatable inborn errors of metabolism manifesting with MADD and shed light on the mechanisms by which FADS ensures cellular FAD homeostasis.

Disorders of riboflavin metabolism

“…Blood and urine biochemistry in FADS deficiency are typical of riboflavin deficiency or MADD, with increased acylcarnitines and urinary organic acids in all cases tested (Table ). Elevations of C4‐C14 even chain acylcarnitines, C5 and C5‐DC and C10:1, C14:1, C16:1, C18:1, and C18:2 have all been reported . In one infant only C4 was elevated, and in another the acylcarnitine profile was normal on some occasions and abnormal on others .…”

“…Another had cardiomyopathy in the first year of life and recurrent supraventricular tachycardias necessitating implantable cardioverter defibrillator placement. This infant had a dramatic response to riboflavin supplementation and was still alive at 22 years . Another infant who needed a pacemaker because of recurrent episodes of sudden cardiac arrest died of multiorgan failure at 7 months .…”

“…The most abundant of these isoforms, that is, the cytosolic enzyme or isoform 2, has been shown to be a bifunctional enzyme with both FADS and hydrolase activity . The enzyme contains an N‐terminal molybdopterin binding (MPTb) domain which has FAD hydrolase activity, and a C‐terminal 3′‐phosphoadenosine‐5′‐phosphosulphate (PAPS) reductase domain, which is sufficient to catalyse FAD synthesis and so has been renamed the FADS domain . Very recently, a novel FADS6 isoform containing only the PAPS reductase domain was discovered in patients harboring frameshift mutations of FLAD1 .…”

“…Presenting features in almost all of these infants included hypotonia and severe muscle weakness leading to feeding difficulties (poor suck and swallow, frequently necessitating tube feeding) and respiratory insufficiency. One patient presented with cardiorespiratory collapse at 32 hours of age and died at 3 days . Another had cardiomyopathy in the first year of life and recurrent supraventricular tachycardias necessitating implantable cardioverter defibrillator placement.…”

Mechanistic Bases of Neurotoxicity Provoked by Fatty Acids Accumulating in MCAD and LCHAD Deficiencies

“…12 On the other hand, newborn screening of MCAD deficiency has dramatically improved the clinical course of the disease, significantly preventing mortality and morbidity. 13 Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency Long-chain 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.211) deficiency (OMIM # 609016) is FAOD with an approximate prevalence of 1:50 000 newborns.…”