Blood RNA-seq in rare disease diagnostics: a comparative study of cases with and without candidate variants.

backgroundApproximately 60% of rare disease cases remain unsolved after exome and genome sequencing (ES/GS). Blood RNA sequencing (RNA-seq) complements DNA-level diagnosis by revealing the functional impact of variants on gene expression and splicing, but to what extent RNA-driven approaches offer diagnostic benefits across different scenarios-with and without pre-existing candidate variants-remains uncertain.

methods128 unrelated probands with suspected Mendelian disorders who had previously undergone ES/GS were recruited. A validation cohort (n = 7, with variants expected to alter RNA) and a test cohort (n = 121, including 10 with variants of uncertain significance (VUS) and 111 with no previously identified candidate variants) were analyzed. Blood RNA-seq was performed, and aberrant splicing (AS) and aberrant expression (AE) were detected using the DROP pipeline. SpliceAI predictions were compared with RNA-seq results for splicing-related VUS variants, and pathogenicity was re-evaluated. AS/AE outliers were evaluated for diagnostic potential in cases without candidate variants. The feasibility of an RNA-driven approach was assessed by ranking causal variant-associated aberrant events.

resultsThe pipeline correctly identified all expected AS/AE events in the validation cohort. In the test cohort with candidate VUS, RNA-seq provided a 60% (6/10) diagnostic uplift. Notably, SpliceAI predictions matched RNA-seq observations perfectly only in 40% of these VUS. A 2.7% (3/111) diagnostic uplift was achieved in the test cohort with no prior candidates. Overall, target AS and AE events ranked among the top eight in 14 of the 16 diagnosed cases using a purely RNA-driven approach; however, two cases would have been missed without prior candidate identification from DNA sequencing.

conclusionBlood RNA-seq is highly effective in refining the interpretation of splicing VUS, frequently leading to reclassification and diagnosis. Meanwhile, RNA-driven identification of causal variants shows a more modest yield in cases without prior candidates. This study supports an RNA-complementary approach as the preferred strategy for clinical utility.