Utility of Targeted RNA Analysis in Neurogenetic Disorders

Background and Objectives: predictive tools have improved, accurate classification of potentially spliceogenic variants still requires a demonstrated splicing impact. One major challenge for RNA studies for neurogenetic conditions is the access to disease-relevant tissues for analysis. In this study, we sought to determine how RNA studies of whole-blood RNA can effectively reclassify variants in genes associated with neurologic disorders. Methods: predictions were identified in patients who underwent genetic testing (single gene, multigene panel, or exome) for a neurologic phenotype. These included 26 intronic (4 canonical and 22 noncanonical) and 12 exonic (8 missense, 2 nonsense, and 2 synonymous) variants. RNA isolated from the whole blood of the patients and healthy controls underwent targeted RT-PCR sequencing. Variants were assessed pre-RNA and post-RNA analysis based on the ACMG/AMP standards and guidelines. In addition, we reviewed the whole blood expression levels, based on the Genotype Tissue Expression (GTEx) Portal, for 212 genes commonly ordered for neurodevelopmental clinical genetic testing. Results: , VUS to pathogenic or likely pathogenic), and one likely pathogenic was upgraded to pathogenic. Eleven variants were downgraded to likely benign. The remaining variants did not change classification due to inconclusive RNA data (N = 3) or availability and/or strength of other lines of evidence (N = 5). Based on the GTEx expression data, 77% (164/212) of commonly ordered neurodevelopmental genes had expression in whole blood that is sufficient for targeted RNA analysis. Discussion: RNA studies provided molecular evidence enabling reclassification for the majority of variants. While diagnostic reclassifications provided a genetic answer for patients, downgrades also had an important impact by resolving VUS. Our data indicate that targeted RNA analysis using whole blood clarify the pathogenicity of potentially spliceogenic variants identified in clinical genetic testing and improve the accuracy of genetic diagnoses in neurologic disorders.