Transition from clinically fully validated panels to medically relevant exome. L. Wong, V. W. Zhang, E. S. Schmitt, J. Wang Molecular and Human Genetics, Baylor College of Medecine.

   Introduction: Whole exome next generation sequencing (NGS) technology has been widely applied to clinical diagnosis. However, the diagnostic yield is only about 25%. Fully validated gene panels focusing on specific diseases with consistently deep coverage for individual exons and the ability to detect copy number changes (CNVs) on the other hand provides much higher diagnostic yields. Methods: Thirty NGS based panels have been developed using SeqCap EZ capture for the enrichment of target genes followed by NGS on Illumina HiSeq2000. All coding exons and 20bp flanking intron regions were sequenced at an average depth of ~1000X and validated by Sanger sequencing. Any exons containing any insufficiently covered bases (<20X) are sequenced separately by PCR/Sanger method. Results: The diagnostic yields of pathway driven panels are usually high due to the indication of abnormal metabolites. These panels include cobalamin and related pathway, MSUD, fatty acid oxidation, congenital deficiency of glycosylation, and glycogen storage disease (GSD). Using GSD as an example, all exons in this panel are sufficiently covered at >20X. The diagnostic yield is about 64%, which is less than expected due to the phenotype overlap between GSD and other metabolic disorders. The clinically defined Usher syndrome panel contains 9 known large genes and has a high diagnostic yield of 92%. Analysis of 66 genes associated with nonsyndromic RP was able to make confirmatory diagnosis in about 84% of RP patients. On the contrary, analysis of 200 genes known to cause the most genetically and clinically heterogeneous mitochondrial disorders only reaches a diagnostic yield of about 25%. Further analysis indicates that many patients labeled with mitochondrial disorders harbor deleterious mutations in genes unrelated to mitochondrial structure, function, or energy metabolism. Conclusion: Our experience in NGS based target gene analysis suggests that unbiased capture and enrichment of the target genes with 100% consistently deep coverage of individual exons are essential to high diagnostic yields, which are achieved by the simultaneous detection of copy number variations (CNVs) using the same set of NGS data. With the clinical availability of NGS based panels of several other diseases, including hereditary cancer, severe combined immunodeficiency, Leigh disease, bone disorders, and all metabolic disorders, transition to a medically relevant exome is anticipated.

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