OR34-6 A Novel Mechanism of SDH-Deficient Tumorigenesis and Implications for Genetic Testing in Patients with Pheochromocytoma-Paraganglioma

Abstract

Introduction: Germline mutations in the succinate dehydrogenase genes (SDHA/B/C/D, SDHAF2 - collectively, SDHx) have been implicated in paraganglioma (PGL), renal cell carcinoma (RCC), gastrointestinal stromal tumor (GIST) and pituitary adenoma (PA). Negative SDHB tumor staining is indicative of SDH-deficient tumors, and thereby germline SDHx mutations (Evenepoel et al Genet Med 2015; De Sousa et al Eur J Endocrinol 2017). As for most Sanger and next generation sequencing (NGS) tests, SDHx genetic testing targets exons and ≤20bp of flanking intronic regions. Deep intronic mutations thus represent a potentially missed cause of various heritable disorders. Methods: We investigated a family with novel co-occurrence of all four SDH-related tumors: PGL in two siblings; GIST in a third sibling; PA in the fourth sibling; and RCC in their deceased mother. Despite negative SDHB staining of the PGLs, no germline mutations were found in SDHx or other PGL genes after 12 yr of extensive testing. We proceeded to whole exome sequencing (WES; Illumina NextSeq 500) using germline DNA from the four siblings and tumor DNA from their available formalin-fixed operative specimens. Transcriptome analysis (RNAseq; Illumina TruSeq LT) was performed using whole blood from the proband. Local genomic pathology and clinical genetic databases were searched for similar cases. Results: Amongst 130 germline WES variants of interest, we found a novel, highly conserved, deep intronic variant in a known PGL gene, SDHC, in all four affected siblings. RNAseq revealed aberrant SDHC splicing with 71% of mRNA reads extending 75bp into intron 1 and terminating at the site of the familial variant. The retained intronic segment introduced a premature stop codon immediately after exon 1. Tumor WES of the available PGL specimen demonstrated Chr1 deletion with loss of the wild-type SDHC allele, consistent with the two-hit model of tumor suppressor genes. Amongst other local patients with SDH-deficient tumors, 9/27 (33%) also lacked SDHx mutations by standard testing. Re-analysis of five patients with available raw data from prior NGS tests revealed another patient with the same SDHC variant as the index family. NGS haplotype analysis showed that 7% of Chr1 was identical between this patient and the index family, suggesting cryptic relatedness. WGS is underway in another 12 unsolved cases of SDH-deficient tumors to identify other SDHx deep intronic mutations. Conclusions: This is the first report of a deep intronic SDHx mutation, highlighting a unique mechanism of SDH-related tumorigenesis and explaining at least some previously unsolved cases of SDH-deficient tumors. More broadly, this study demonstrates how deep intronic mutations may be a cause of false negative genetic test results and illustrates the expanding utility of NGS methodologies across heritable disorders.