G C A T T A C G G C A T genes Article Multiple Acyl-CoA Dehydrogenase Deficiency with Variable Presentation Due to a Homozygous Mutation in a Bedouin Tribe Orna Staretz-Chacham 1,2,* , Shirly Amar 3, Shlomo Almashanu 4, Ben Pode-Shakked 5,6, Ann Saada 7,8 , Ohad Wormser 9 and Eli Hershkovitz 2,10 1 Metabolic Clinic, Pediatric Division, Soroka University Medical Center, Beer Sheva 84101, Israel 2 Faculty of Health Sciences, Ben-Gurion University, Beer Sheva 84101, Israel; [email protected] 3 Genetic Lab, Soroka University Medical Center, Beer Sheva 84101, Israel; [email protected] 4 National Newborn Screening Program, Ministry of Health, Tel-HaShomer, Ramat Gan 52621, Israel; [email protected] 5 Metabolic Disease Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel-Hashomer, Ramat Gan 52621, Israel; [email protected] 6 Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 39040, Israel 7 Hadassah Medical Center, Department of Genetics, Jerusalem 911201, Israel; [email protected] 8 Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem 911201, Israel 9 The Morris Kahn Laboratory of Human Genetics, National Institute for Biotechnology in the Negev and Faculty of Health Sciences, Ben Gurion University of the Negev, Beer Sheva 84101, Israel; [email protected] 10 Department of Pediatrics D, Soroka Medical Center, Beer Sheva 84101, Israel * Correspondence: [email protected]; Tel.: +972-545-713-191 Abstract: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a fatty acid and amino acid Citation: Staretz-Chacham, O.; Amar, oxidation defect caused by a deficiency of the electron-transfer flavoprotein (ETF) or the electron- S.; Almashanu, S.; Pode-Shakked, B.; transfer flavoprotein dehydrogenase (ETFDH). There are three phenotypes of the disease, two Saada, A.; Wormser, O.; Hershkovitz, neonatal forms and one late-onset. Previous studies have suggested that there is a phenotype– E. Multiple Acyl-CoA Dehydrogenase genotype correlation. We report on six patients from a single Bedouin tribe, five of whom were Deficiency with Variable Presentation sequenced and found to be homozygous to the same variant in the ETFDH gene, with variable severity Due to a Homozygous Mutation in a and age of presentation. The variant, NM_004453.3 (ETFDH): c.524G>A, p.(R175H), was previously Bedouin Tribe. Genes 2021, 12, 1140. recognized as pathogenic, although it has not been reported in the literature in a homozygous state https://doi.org/10.3390/genes12081140 before. R175H is located near the FAD binding site, likely affecting the affinity of FAD for EFT:QO. Academic Editors: Michele Cioffi and The single homozygous ETFDH pathogenic variant was found to be causing MADD in this cohort Maria Teresa Vietri with an unexpectedly variable severity of presentation. The difference in severity could partly be explained by early diagnosis via newborn screening and early treatment with the FAD precursor Received: 30 June 2021 riboflavin, highlighting the importance of early detection by newborn screening. Accepted: 23 July 2021 Published: 28 July 2021 Keywords: multiple acyl-CoA dehydrogenase deficiency (MADD); electron-transfer flavoprotein (ETF); electron-transfer flavoprotein dehydrogenase (ETFDH); genotype; phenotype Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Multiple acyl-CoA dehydrogenase deficiency (MADD) is a fatty acid and amino acid oxidation defect also known as glutaric aciduria type II. Its estimated prevalence is 1 in 750,000–2,000,000 newborns [1] with a much higher prevalence in China [2]. Copyright: © 2021 by the authors. MADD affects mitochondrial fatty acid oxidation as well as the catabolism of branched Licensee MDPI, Basel, Switzerland. amino acids, lysine and tryptophan [3]. The disease is caused by a deficiency of ei- This article is an open access article ther electron-transfer flavoprotein (ETF) or electron-transfer flavoprotein dehydrogenase distributed under the terms and (ETFDH). ETF is composed of α and β subunits, encoded by the ETFA and ETFB genes, conditions of the Creative Commons respectively, as well as electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF- Attribution (CC BY) license (https:// QO) [4,5], encoded by ETFDH [4,6]. MADD is inherited in an autosomal recessive manner. creativecommons.org/licenses/by/ 4.0/). Genes 2021, 12, 1140. https://doi.org/10.3390/genes12081140 https://www.mdpi.com/journal/genes Genes 2021, 12, x FOR PEER REVIEW 2 of 9 Genes 2021, 12, 1140 respectively, as well as electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-2 of 9 QO) [4,5], encoded by ETFDH [4,6]. MADD is inherited in an autosomal recessive manner. There are three types of the disease based on clinical symptomatology: a neonatal- onset formThere with are threecongenital types anomalies of the disease (type based I), a neonatal-onset on clinical symptomatology: form without congenital a neonatal- anomaliesonset form (type with II) congenital and a late-onset anomalies form (type (type I), III). a neonatal-onset form without congenital anomaliesThe two (type neonatal-onset II) and a late-onset forms are form usually (type fa III).tal, and affected patients develop severe non-ketoticThe two hypoglycemia neonatal-onset and forms metabolic are usually acidosis fatal, with and multisystem affected patients involvement develop accom- severe paniednon-ketotic by the hypoglycemia excretion of large and metabolicamounts of acidosis fatty acids with and multisystem branched-chain involvement amino accom- acid metabolitespanied by the[6]. excretionThose with of congenital large amounts anomalies of fatty usually acids and succumb branched-chain during the amino neonatal acid period,metabolites while [those6]. Those without with anom congenitalalies die anomalies during the usually first months succumb of during life due the to neonatal severe cardiomyopathyperiod, while those or, if without they survive anomalies longer, die tend during to develop the first episodes months of Reye life due syndrome- to severe likecardiomyopathy decompensations or, if [3]. they survive longer, tend to develop episodes of Reye syndrome-like decompensationsThe third or late-onset [3]. form has a wide spectrum of presentation, with either recur- rent episodes,The third triggered or late-onset by metabolic form has astress, wide of spectrum non-ketotic of presentation, hypoglycemia, with vomiting, either recurrent leth- argy,episodes, metabolic triggered acidosis by metabolicand hepatomegaly, stress, of non-ketoticor recurrent hypoglycemia, episodes of muscle vomiting, involvement lethargy, presentingmetabolic as acidosis weakness and and hepatomegaly, pain [3,6]. or recurrent episodes of muscle involvement pre- sentingOlsen as et weakness al., among and others, pain [3 have,6]. previously reported a clear genotype–phenotype correlationOlsen with et al., respect among to disease others, haveseverity previously [6,7]. reported a clear genotype–phenotype correlationWe present with here respect six topatients disease from severity an extended [6,7]. Bedouin tribe, of which five were found Weto be present homozygous here six patientsto the same from path an extendedogenic variant Bedouin c.524G>A, tribe, of p.(R175H) which five werein the found ET- FDHto be gene, homozygous with variable to the severity same and pathogenic age at presentation. variant c.524G>A, p.(R175H) in the ETFDH gene, with variable severity and age at presentation. 2. Materials and Methods 2. Materials and Methods Clinical and biochemical details were collected from six patients, all from the same Clinical and biochemical details were collected from six patients, all from the same extended Bedouin family (Figure 1). Five of them were found to be homozygous to the extended Bedouin family (Figure1). Five of them were found to be homozygous to the same mutation in the ETFDH gene. same mutation in the ETFDH gene. Figure 1. Pedigree of the consanguineous extended Bedouin family studied. Individuals affected Figure 1. Pedigree of the consanguineous extended Bedouin family studied. Individuals with MADD are represented by full symbols. Patient V1 succumbed before molecular investigations affected with MADD are represented by full symbols. Patient V1 succumbed before mo- were performed. lecular investigations were performed. ® TheThe automated automated extraction extraction of of DNA DNA was was performed performed using using the the QIAsymphony QIAsymphony® DSPDSP DNADNA Midi Midi Kit Kit (Qiagen, (Qiagen, Hilden, Germany)Germany) following following the the manufacturer’s manufacturer’s recommendations. recommenda- µ tions.DNA DNA was dilutedwas diluted in approximately in approximately 120 L120 ofμ bufferL of buffer ATE. DNAATE. DNA concentration concentration and quality and qualitywas quantified was quantified using ausing Nanodrop a Nanodrop ND-2000 ND spectrophotometer-2000 spectrophotometer (Nanodrop (Nanodrop Technologies, Tech- Wilmington, DE, USA). nologies, Wilmington, DE, USA). Primer3 [8] was used for designing primers FW-AAAGTGTGACCATCAATGTAGCA Primer3 [8] was used for designing primers FW-AAAGTGTGAC- REV-AAACAAAACTATACAAACCTCAGCAG for amplification of a 263 bp fragment CATCAATGTAGCA REV-AAACAAAACTATACAAACCTCAGCAG for amplification of the ETFDH gene (NM_004453.3) containing the mutation c.524G>A; p.(R175H). PCR optimal annealing temperature was determined. Cycle sequencing was performed in both directions using amplification primers