Introduction:
Splice variants represent a significant portion of variants of uncertain significance (VUS) in clinical whole exome and genome sequencing (ES/GS). However, it is challenging to confirm the impact of these variants by DNA analysis alone due to a lack of functional evidence in RNA splicing. This especially impacts patients with rare disease, where novel variants are more likely to be classified as VUS. To address this concern, we integrated clinically validated RNA sequencing (RNAseq) into the ES/GS clinical workflow as a reflex test to enhance classification of variants with potential impact on splicing.  

Methods:
DNA and RNA were collected from a single EDTA tube of peripheral blood for clinical ES/GS cases where additional consent for reflex RNAseq was obtained from patients. Variants classified as likely pathogenic or VUS that were highly associated with the patient’s phenotype and predicted to significantly impact splicing by prediction algorithms underwent targeted reflex RNA sequencing using NovaSeq. Splicing events were detected through manual IGV review utilizing Sashimi plots. Reclassification of these variants using RNAseq functional evidence was performed via internally developed guidelines for evaluating the aberrant transcript and its degree of splicing impact paired with ACMG guidelines for variant classification.  

Results:
Here, we report 10 cases where reflex RNAseq was performed on variants identified by ES/GS. All variants were detected at the DNA level with an initial classification of VUS. Transcripts Per kilobase Million (TPM) analysis using the GTEx database showed that these genes exhibited a wide range of expression levels (median TPM values from 0.08 to 38) in whole blood. RNAseq data revealed exon skipping, exon deletion, or intron retention as sources of additional functional evidence for 5 variants, which were all upgraded from VUS to likely pathogenic (5/10, 50%). Notably, a synonymous variant, c.156G>A (p.L52=), was found to induce exon deletion in PHF6. Additionally, in CHD3 a c.3547+5G>C caused the retention of 24 nucleotides, leading to an in-frame insertion. Reclassification of FOXP4 splicing variant c.1357+2T>G in a child with complex neurological presentation allowed for resolution of the patient's lengthy diagnostic odyssey which may lead to personalized management or treatment for the patient.     

Conclusion:
The addition of reflex RNAseq to clinical ES/GS enabled the robust detection of splicing variants in genes with very low-expression RNA in whole blood. This additional testing significantly enhanced the reclassification of qualified VUS and improved treatment management for the patients. These results highlight the utility of RNAseq in the rare disease setting.