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

作者：Xiaomei Luo, Bing Xiao, Lili Liang, Kaichuang Zhang, Ting Xu, Huili Liu, Yi Liu, Yongguo Yu, Yanjie Fan · 发表于：Journal of Translational Medicine · 年份：2025 · DOI：10.1186/s12967-025-06609-w · 被引用次数：10 · 研究领域：Genomics and Rare Diseases、Hereditary Neurological Disorders、Genetic Neurodegenerative Diseases

BACKGROUND: Approximately 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. METHODS: 128 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. RESULTS: The 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% (3/1...