Paired DNA/RNA genetic testing to uncover cryptic pathogenic variants (PV) associated with PTEN hamartoma tumor syndrome (PHTS) and hereditary cancer (CA).

e22598 Background: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PTHS) is a family of related genetic disorders including Cowden Syndrome (CS), Bannayan-Riley-Ruvalcaba Syndrome (BRRS), and Proteus Syndrome (PS) associated with distinct phenotypes and increased CA risk. Germline PTEN PVs remain unidentified in many patients (pts), including 40% w/BRRS, 80% w/PS, and 15% w/CS. Standard DNA testing may fail to identify clinically relevant deep intronic PTEN PVs detectable by comprehensive testing that includes RNA analysis. Using a commercial laboratory database, we identify pts tested by DNA/RNA and found to harbor deep intronic PVs associated with alternative RNA splicing of PTEN and predicted to cause PHTS. Methods: Pts tested by paired DNA/RNA testing (2019-2024, Ambry Genetics) and found to have a deep intronic PV leading to a splice site variant were identified. DNA/RNA results were linked to clinical data from a test requisition form. RNA was isolated using standard methodology and converted to cDNA by RT-PCR. Sequence enrichment of the targeted coding exons and adjacent intronic nucleotides is carried out by a bait-capture methodology using long biotinylated oligonucleotide probes followed by PCR and NGS. Absence or presence of RNA transcripts meeting quality thresholds is incorporated as evidence towards classification of DNA variants. Study was IRB reviewed (FCCC IRB #24-9030/Ambry WCG IRB exempt). Results: Index case: 64 yo F w/hx of breast CA (42 & 53) and thyroidectomy for nodules and papillary thyroid CA was referred for genetic counseling following colonoscopy showing extensive ganglioneuromatosis. Exam found macrocephaly (59.5 cm), tongue papules, likely facial trichilemmomas, and axillary acrochordons. RNA testing identified a deep intronic PV at c.209+2047A>G resulting in alternative PTEN splicing. In the combined DNA/RNA testing database (n=489,960), 17 additional individuals/families undergoing PTEN testing were identified with a deep intronic PV/LPV (n=14) or suspicious VUS (n=3). In total, 12 probands reported CA (9 <50 yrs) including cases of early breast (n=4, 38-63 yrs), thyroid (n=1, 25 yrs), salivary (n=1, 31 yrs), uterine (n=1, 46 yrs), bladder (n=1, 29 yrs), prostate (n=1, 66 yrs) and ovary CA (n=3, 28-32 yrs). Other PHTS features reported in probands/families: macrocephaly (n=7), colon ganglioneuromas and/or polyposis (n=7), goiter/thyroid nodules (n=6), penile freckling (n=2), skin findings (n=3), and intellectual disability (n=2). Potentially clinically relevant co-occurring variants were detected in NF1 (x3), GALNT12 (x2), BRCA2 and SDHC . Conclusions: Deep intronic PTEN PVs missed by DNA sequencing but detectable by RNA testing and associated with early-onset CA explain a portion of negative results in pts meeting clinical criteria for CS and/or manifesting phenotypic features of PHTS.