1.Signe Mosegaard, MSc, Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus, Denmark.
Phone: +45 20835507
2. Mirjana Gusic, Institute of Human Genetics, Helmholtz Zentrum München, Germany
3. Sarah Stenton, Institute of Human Genetics, Helmholtz Zentrum München, Germany
4. Simon Olpin, Department of Clinical Chemistry, Sheffield Children’s Hospital, Sheffield, UK
5. Helle Nygaard, Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus, Denmark.
6. Mark Sharrard, Department of Pediatrics, Sheffield Children’s Hospital, Sheffield, UK.
7. Stanley H. Korman, Department of Pediatrics, Shaare Zedek Medical Center, Jerusalem, Israel
8. Jolanta Sykut-Cegielska, Department of Inborn Errors of Metabolism and Paediatrics, The Institute of Mother and Child, Warsaw, Poland
9. Anibh Martin Das, Department of Pediatrics, Hannover Medical School, Hannover, Germany
10.Yusof Rahman, Centre for Inherited Metabolic Disorders, Guy’s & St Thomas’ Hospital NHS Foundation Trust, London, UK
11. Skadi Beblo, University Hospital for Children and Adolescents, University of Leipzig, Leipzig, Germany
12. Eva Morava, Dept of Clin Genomics, Mayo Clinic, Rochester, Minnesota, USA
13. Leo A. J. Kluijtmans, Translational Metabolic Laboratory, Dept of Laboratory Medicine, Radboud University Medical Center, Nijmegen, the Netherlands.
14. Niels Gregersen, Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus, Denmark.
15. Holger Prokisch, Institute of Human Genetics, Helmholtz Zentrum München, Germany
16. Rikke Olsen, Research Unit for Molecular Medicine, Aarhus University and Aarhus University Hospital, Aarhus, Denmark
Background: MADD is a heterogeneous inborn error of fatty acid and amino acid oxidation that is potentially treatable with riboflavin. The disease can be caused by genetic defects of the electron transfer flavoproteins, linking multiple acyl-CoA dehydrogenation reactions to the respiratory chain, or by genetic defects in the synthesis of riboflavin-derived catalyzing co-factors. Whole exome sequencing (WES) proved highly successful in expanding the genetic knowledge of MADD, with RNA-sequencing (RNA-seq) emerging as its complementary tool. However, there are still MADD patients that remain genetically unresolved, suggesting that the genetics of MADD is much more complex than previously assumed.
Methods: WES was performed on DNA from 14 patients with MADD-like acylcarnitine and organic acid patterns, but without identified bi-allelic variants in known MADD candidate genes. RNA-seq was performed on RNA from cultured fibroblasts from 8 of 14 patients.
Results; In 5 patients, we identified likely disease-causing variants and implementation of RNA-seq led to the confirmation of variants’ pathogenicity in 2 of 5 cases. We detected a large copy number variant in ETFDH, which was not detected previously due to limitations of Sanger sequencing. Additionally, we detected pathogenic variants in genes that previously have not been associated with diagnostic MADD metabolites; KCNA2 encoding potassium voltage-gated channel subfamily A member 2, MRPL44 encoding mitochondrial ribosomal protein L44 and HMGCS2 encoding mitochondrial 3- hydroxy-3-methylglutaryl CoA synthase.
Discussion: The combination of WES and RNA-seq revealed a previously unidentified variant in a known MADD gene and enabled the detection of likely pathogenic variants in genes that until now have not been associated with MADD. Since early detection and initiation of riboflavin treatment is essential in clinical management of MADD patients, we propose multi-omics approach as the strategy of choice for genetic diagnosis.