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

Rikke K.J Olsen,Angela Pyle,Dorothea Möslinger,Caterina Terrile,Manuel Schiff,Daniela Mazzà,Johannes A Mayr,Signe Mosegaard,Lodovica Vergani,Holger Prokisch,Annalisa Botta,Adriana Malena,Thomas Meitinger,Niels Gregersen,Marcella Zollino,Samia Pichard,Elisabeth Graf,Elena Pegoraro,Patrick F Chinnery,Avihu Boneh,Marcus Oppenheim,Beril Talim,Tiina Tyni,Tim M Strom,René G Feichtinger,Eliška Koňaříková,Michele Galluccio,Odile Rigal,Tobias B Haack,Johan Palmfeldt,Daniele Ghezzi,Apolline Imbard,Thomas Schwarzmayr,Vassiliki Konstantopoulou,Piero Leone,Lavinija Mataković,Maria Barile,Mari Auranen,Rita Horvath,Veronika Boczonadi,Teresa Anna Giancaspero ,Alice Veauville‐Merllié ,Şafak Güçer ,Turgay Coşkun ,Christine Vianey‐Saban ,Haluk Topaloğlu ,Cécile Acquaviva ,Purificación Gutiérrez-Ríos

doi:10.1016/j.ajhg.2016.04.006

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.

Highlights

Riboflavin, or vitamin B2, is the precursor of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are essential cofactors of numerous dehydrogenases involved in cellular metabolism and a number of other essential cellular pathways, such as antioxidant defense, protein folding, and apoptosis.[1]
Three riboflavin transporters (RFVTs) (RFVT1, RFVT2, and RFVT3), each differing in tissue-specific presence, have been identified far and assigned to a new solute carrier family named SLC52.4 Within cells, riboflavin is converted into FMN by riboflavin kinase (RFK), and FMN is converted into FAD by FAD synthase (FADS), previously known as FAD synthetase or FMN:ATP adenylyltransferase.[7,8]
Riboflavin-responsive forms of Multiple acyl-CoA dehydrogenase deficiencies (MADDs) leading to improvement in biochemical and clinical symptoms after high doses of riboflavin treatment were first explained by missense variants in electron transfer flavoprotein dehydrogenase (ETFDH) (MIM: 231675)[25,26,27,28,29] and more recently by variants in the riboflavin transporter genes (SLC52A1 [MIM: 607883], SLC52A2 [MIM: 607882], and SLC52A3 [MIM: 613350]),[30,31,32] as well as in the mitochondrial FAD transporter gene (SLC25A32 [MIM: 610815]).[33]

Summary

Introduction

Riboflavin, or vitamin B2, is the precursor of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are essential cofactors of numerous dehydrogenases involved in cellular metabolism and a number of other essential cellular pathways, such as antioxidant defense, protein folding, and apoptosis.[1]. All recombinant FADS produced up to now exhibit the ability to bind FAD, the product of their activity, tightly but not covalently.[9] FAD release from FADS is likely to be tightly controlled and presumably requires well-defined conditions, including a correct redox state.[9,12] FAD (or as recently proposed, catalytically active FADS enzyme) binds to apoproteins and chaperones their folding into stable and functional flavoenzymes.[14,15,16,17,18,19] several studies have shown that a number of flavoproteins, and in particular mature mitochondrial acyl-CoA dehydrogenases, undergo fast degradation under depletion of riboflavin or flavin cofactors.[20,21,22,23] The biochemistry of riboflavin deficiency, with elevated multiple acylcarnitines and urine organic acids, resembles that seen in individuals who suffer from multiple acyl-CoA dehydrogenase deficiency (MADD [MIM: 231680]) or glutaric aciduria type 2 and have genetic defects in three genes encoding two enzymes, electron transfer flavoprotein (ETF) and electron transfer flavoprotein dehydrogenase (ETFDH). Riboflavin-responsive forms of MADD leading to improvement in biochemical and clinical symptoms after high doses of riboflavin treatment were first explained by missense variants in ETFDH (MIM: 231675)[25,26,27,28,29] and more recently by variants in the riboflavin transporter genes (SLC52A1 [MIM: 607883], SLC52A2 [MIM: 607882], and SLC52A3 [MIM: 613350]),[30,31,32] as well as in the mitochondrial FAD transporter gene (SLC25A32 [MIM: 610815]).[33]

Methods

Results

Conclusion