Minigene-based characterization and classification of splice-associated variants in succinate dehydrogenase B.

SDHB

Germline SDHB variants affecting splicing, assessed with a minigene spanning exons 2 to 5 and targeted RNA sequencing

The authors built a minigene spanning SDHB exons 2 to 5 and analysed the resulting transcripts in HEK293T cells by targeted RNA sequencing, for 48 variants prioritised by SpliceAI (Δ ≥ 0.42), two negative controls and the endogenous gene, with comparison to tumour data (n = 2). Nineteen variants (38%) retained at least 90% wild-type splicing while 17 (34%) produced at least 90% aberrant splicing; 73 aberrant transcripts were observed overall, averaging 2.3 per variant across 22 unique transcripts. A customised decision framework assigned RNA evidence strength to 64 aberrant transcripts (88%). Among 26 classified variants, 10 received PVS1_Strong (RNA), 2 PVS1_Moderate (RNA) and 14 BP7_Strong (RNA). Integrating minigene RNA data changed ACMG scores by a mean of 2.7 points and reclassified 13 variants (50%), including 12 downgrades from variant of uncertain significance to likely benign and one downgrade from likely pathogenic to variant of uncertain significance.

Synthesis written by Geno'X. For the full original abstract, please refer to the source publication.

The most useful result is the direction of the reclassifications: 12 downgrades out of 13, meaning mostly families who can be released from lifelong pheochromocytoma and paraganglioma surveillance, and a positive predictive value for SpliceAI that looks clearly lower than the threshold used would suggest. The single downgrade from likely pathogenic to variant of uncertain significance is a reminder that functional evidence can also withdraw a diagnosis already delivered — an awkward situation to convey in clinic. A structural limitation of the model: a minigene in HEK293T reproduces neither the tissue context nor distant regulatory elements, and the tumour comparison covers only two cases — the evidence still needs confirmation on patient RNA before being used on its own.

Analysis by Dr Thibaut Benquey