P461 - LBA_ECE_1060 - Functional characterization of a non-canonical SDHD splice-site variant (c.314+4A > G) in a family with thyroid and carotid paragangliomas
Thyroid paragangliomas (TPGLs) are exceptionally rare (<0.1% of thyroid neoplasms, ~0.5% of all paragangliomas) and frequently occur within hereditary head and neck paraganglioma syndromes. While hereditary paragangliomas are frequently linked to SDHD germline variants, intronic splice-site alterations are scarcely reported in TPGLs. We describe the clinical and functional characterization of a TPGL associated with a novel intronic SDHD variant. We investigated a family of a 36-year-old woman with intrathyroidal TPGL and bilateral carotid body paragangliomas in her father and brother, all carrying a heterozygous intronic SDHD variant, c.314+4A > G (NM_003002.5). Germline testing was performed by next-generation sequencing, followed by in-depth RNA sequencing on whole blood to assess splicing and allele-specific expression. RNA-seq libraries were prepared with a directional protocol and sequenced on an Illumina NextSeq 2000 (~80 M 150-bp paired-end reads/sample), aligned with HISAT2, quantified with HTSeq, and analyzed with DESeq2; splice junctions were visualized by sashimi plots in IGV. The SDHD c.314+4A > G variant lies within the intron 3 donor site and is absent from gnomAD and the Leiden University Medical Center SDHD gene variant database; SpliceAI predicts splice disruption (Δ score 0.81). RNA-seq demonstrated two competing exon 3→4 junctions in both carriers: a canonical junction from the wild-type allele and a cryptic junction 4 bp downstream, attributable to the mutant allele. In the proband and her brother, aberrant junctions accounted for approximately half of all exon 3→4 splice events, consistent with a “leaky” splice-site defect and predicted frameshift with premature termination. Despite sharing the same variant, overall SDHD expression was lower in the proband than in her brother, consistent with allele-specific transcript imbalance and possible haploinsufficiency. Co-segregation of c.314+4A > G with thyroid and bilateral carotid body paragangliomas across three paternally related relatives supports its role in a hereditary SDHD-associated paraganglioma syndrome. This study provides direct RNA-level evidence that the intronic SDHD c.314+4A > G variant causes aberrant splicing and supports reclassification from VUS to likely pathogenic under ACMG/AMP criteria (PS3, PM2, PP3, segregation). These findings expand the spectrum of splice-disrupting SDHD c.314 variants implicated in hereditary head and neck paragangliomas, highlight TPGL as part of this spectrum, and underscore the value of RNA-based assays in interpreting non-canonical splice-site changes.