Minigene-based characterization and classification of splice-associated variants in succinate dehydrogenase B
TL;DR
It is demonstrated that targeted sequencing of minigene-derived transcripts provides a scalable approach to evaluate splice-associated SDHB variants and improve variant classification.
Abstract
Pathogenic germline variants in SDHB predispose to pheochromocytoma and paraganglioma, but limited functional evidence challenges clinical interpretation. To investigate splice-associated SDHB variants, we developed a minigene spanning exons 2-5 and assessed derived SDHB transcripts in HEK293T cells using targeted RNA sequencing. We evaluated 48 variants prioritized by SpliceAI (Δ≥0.42), two negative controls and endogenous SDHB, and compared findings with tumor data (n = 2). Nineteen variants (38%) showed ≥90% wildtype splicing, whereas 17 (34%) exhibited ≥90% aberrant splicing. Across all variants, 73 aberrant transcripts were observed (average of 2.3 per variant, 22 unique transcripts). Using a customized decision framework, RNA-based evidence strengths were assigned to 64 aberrant transcripts (88%). Among 26 classified variants, 10 received PVS1_Strong (RNA) (38%), including eight canonical splice-site variants, one missense variant and one stop-gain variant; two received PVS1_Moderate (RNA) and 14 received BP7_Strong (RNA). Integration of minigene RNA data changed ACMG scores by a mean of 2.7 points and led to reclassification of 13 variants (50%), including 12 downgrades from VUS to likely benign and one downgrade from likely pathogenic to VUS. These findings demonstrate that targeted sequencing of minigene-derived transcripts provides a scalable approach to evaluate splice-associated SDHB variants and improve variant classification.