Genetic testing for mitochondrial disorders in the next-generation sequencing era: Targeted multiplex PCR gene panel or capture-based WES? R. Bai1, N. Smaoui1, E. Haverfield1, J. Higgs1, S. F. Suchy1, D. Arjona1, K. Retterer1, A. Shanmugham1, F. D. Kendall2, S. Parikh3, A. L. Gropman4, R. Haas5, A. Goldstein6, J. Panzer7, S. Yum7, M. J. Falk7, R. P. Saneto8, G. M. Enns9, W. K. Chung10, S. Bale1, G. Richard1 1) GeneDx Inc, Gaithersburg, MD; 2) Virtual Medical Practice, LLC, Atlanta, GA; 3) Neurogenetics/Neurometabolism, Neurosciences Institute, Cleveland Clinic, Cleveland, OH; 4) Department of Neurology, Children's National Medical Center, Washington, DC; 5) Department of Neurosciences, University of California San Diego, La Jolla, CA; 6) Division of Child Neurology, Children's Hospital of Pittsburgh, Pittsburgh, PA; 7) Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA; 8) Seattle Children's Hospital/University of Washington, Seattle, WA; 9) Pediatric Genetics, Stanford University School of Medicine, Palo Alto, CA; 10) Columbia University Medical Center, New York, NY.

   Majority of primary mitochondrial disorders (MtD) are caused by mutations in nuclear genes. Current genetic testing for MtD includes different approaches utilizing next generation sequencing. Some diagnostic laboratories use PCR-based target enrichment (PCR-TE) panels for well-established MtD genes, others use hybridization-based target enrichment (capture) of some or all MitoCarta genes, or whole exome sequencing (WES). To determine the clinical utility, we compared results from 201 unrelated patients with a suspected MtD; 155 cases were evaluated using a 101-gene panel by PCR-TE (Mito101) and 50 cases by capture-based WES, including 20 patients previously tested (2 positive, 18 negative) by Mito101. The cases in each group were classified into definite, probable, possible MtD according to published diagnostic criteria for MtD. All identified sequence variants were evaluated and classified as mutation (Mut), likely mutation (VLM), VUS, likely benign (VLB) or polymorphism. Of the 155 cases tested by Mito101, pathogenic mutation(s) were identified in 26 (17%) and thus confirmed a MtD. The positive rate was 38% in patients with a definite/probable MtD and 7% in those with a possible MtD. Of the 50 patients tested by WES, pathogenic mutation(s) were identified in 18 (36%), with a positive rate of 25% for patients with a definitive/probable MtD and 37% for those with a possible MtD. 58% of WES cases had an undetermined result, and 6% were negative. In 61% of positive cases, including 4 that had a previous negative result by Mito101, patients were found to have mutations in non-MitoCarta genes associated with metabolic, movement or neurological disorders, emphasizing that many disorders can have clinical features overlapping those of a MtD, and will be missed if testing is limited to the latter. However, WES technology does not allow complete coverage of all genes due to low coverage and/or pseudogene issues, and about 7% of the reportable variants (Mut/VLM/VUS/VLB) by Mito101 were not detected by WES. When comparing the clinical sensitivity for patients with definite/probable MtD, Mito101 had a greater yield (38%) over WES (25%). However, in patients with a less specific phenotype (possible MtD), WES had a distinct advantage (37%) over Mito101 (7%). Based on our data, a targeted MtD gene panel by PCR-TE and reflex to WES is recommended for patients with definite/probable MtD, whereas WES is recommended as the first-line test for patients with possible MtD.

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