Bridging the functional gap by synergy of exome/genome and RNA sequencing: A systematic semi-quantitative review demonstrating enhanced diagnostic yield in genetic diagnostics

PURPOSE: Although exome and/or genome sequencing (ES/GS) is highly effective in identifying pathogenic variants, they may not capture the functional consequences of variants. RNA sequencing (RNA-seq), when used alongside ES/GS, addresses this limitation. METHODS: A systematic semi-quantitative review that included 200 studies reporting ES/GS or that were followed by RNA-seq diagnostic rates from 2014 to 2024 was conducted. RESULTS: Although the weighted average diagnostic rate for ES/GS followed by RNA-seq was 0.44 (ranging from 0.34 to 0.53), studies with ES/GS alone reported a pooled diagnostic rate of 0.37 (95% CI: 0.35-0.39; P = .18). The subset of studies with ES/GS as the first-tier test followed by RNA-seq revealed a trend towards improved diagnostic yields with the addition of RNA-seq (odds ratio: 1.31, 95% CI: 1.08-1.58; P = .01). For participants undiagnosed by ES/GS, RNA-seq contributed an incremental diagnostic rate of 5.4% (95% CI: 0.006-0.60, P = .018), achieved through confirming or reclassifying candidate variants as pathogenic/likely pathogenic, and an additional diagnostic rate of 3.3% (95% CI: 0.021-0.088; P = .002) through discovering novel variants. Our results also highlight the advantages of tissue samples over blood samples in improving the diagnostic yield, particularly of RNA-seq, in which an additional 6.2% for confirming ES/GS-identified candidates (95% CI: 0.028-0.97; P < .001) and another addition of 6.2% for new variant discovery (95% CI: 0.025-0.099; P = .001) were found. CONCLUSION: Our findings emphasize the synergistic value of hypothesis-driven and hypothesis-free approaches in transcriptomic data analysis. The integration of RNA-seq into ES/GS workflows has shown promise in improving the molecular diagnosis of genetic diseases.