Solving Missing Heritability in Patients With Familial Adenomatous Polyposis With DNA-RNA Paired Testing.

PURPOSE: Patients with germline pathogenic variants (PVs) in APC develop tens (attenuated familial adenomatous polyposis [AFAP]) to innumerable (classic FAP) adenomatous polyps in their colon and are at significantly increased lifetime risk of colorectal cancer. Up to 10% of FAP and up to 50% of patients with AFAP who have undergone DNA-only multigene panel testing (MGPT) do not have an identified PV in APC. We seek to demonstrate how the addition of RNA sequencing run concurrently with DNA can improve detection of germline PVs in individuals with a clinical presentation of AFAP/FAP. METHODS: We performed a retrospective query of individuals tested with paired DNA-RNA MGPT from 2021 to 2022 at a single laboratory and included those with a novel APC PV located in intronic regions infrequently covered by MGPT, a personal history of polyposis, and family medical history provided. All clinical data were deidentified in this institutional review board-exempt study. RESULTS: Three novel APC variants were identified in six families and were shown to cause aberrant splicing because of the creation of a deep intronic cryptic splice site that leads to an RNA transcript subject nonsense-mediated decay. Several carriers had previously undergone DNA-only genetic testing and had received a negative result. CONCLUSION: Here, we describe how paired DNA-RNA MGPT can be used to solve missing heritability in FAP families, which can have important implications in family planning and treatment decisions for patients and their families.

Diagnostic Outcomes of Concurrent DNA and RNA Sequencing in Individuals Undergoing Hereditary Cancer Testing.

Importance: Personalized surveillance, prophylaxis, and cancer treatment options for individuals with hereditary cancer predisposition are informed by results of germline genetic testing. Improvements to genomic technology, such as the availability of RNA sequencing, may increase identification of individuals eligible for personalized interventions by improving the accuracy and yield of germline testing. Objective: To assess the cumulative association of paired DNA and RNA testing with detection of disease-causing germline genetic variants and resolution of variants of uncertain significance (VUS). Design, Setting, and Participants: Paired DNA and RNA sequencing was performed on individuals undergoing germline testing for hereditary cancer indication at a single diagnostic laboratory from March 2019 through April 2020. Demographic characteristics, clinical data, and test results were curated as samples were received, and changes to variant classification were assessed over time. Data analysis was performed from May 2020 to June 2023. Main Outcomes and Measures: Main outcomes were increase in diagnostic yield, decrease in VUS rate, the overall results by variant type, the association of RNA evidence with variant classification, and the corresponding predicted effect on cancer risk management. Results: A total of 43 524 individuals were included (median [range] age at testing, 54 [2-101] years; 37 373 female individuals [85.7%], 6224 male individuals [14.3%], and 2 individuals of unknown sex [<0.1%]), with 43 599 tests. A total of 2197 (5.0%) were Ashkenazi Jewish, 1539 (3.5%) were Asian, 3077 (7.1%) were Black, 2437 (5.6%) were Hispanic, 27 793 (63.7%) were White, and 2049 (4.7%) were other race, and for 4507 individuals (10.3%), race and ethnicity were unknown. Variant classification was impacted in 549 individuals (1.3%). Medically significant upgrades were made in 97 individuals, including 70 individuals who had a variant reclassified from VUS to pathogenic/likely pathogenic (P/LP) and 27 individuals who had a novel deep intronic P/LP variant that would not have been detected using DNA sequencing alone. A total of 93 of 545 P/LP splicing variants (17.1%) were dependent on RNA evidence for classification, and 312 of 439 existing splicing VUS (71.1%) were resolved by RNA evidence. Notably, the increase in positive rate (3.1%) and decrease in VUS rate (-3.9%) was higher in Asian, Black, and Hispanic individuals combined compared to White individuals (1.6%; P = .02; and -2.5%; P < .001). Conclusions and Relevance: Findings of this diagnostic study demonstrate that the ability to perform RNA sequencing concurrently with DNA sequencing represents an important advancement in germline genetic testing by improving detection of novel variants and classification of existing variants. This expands the identification of individuals with hereditary cancer predisposition and increases opportunities for personalization of therapeutics and surveillance.

Mutational and splicing landscape in a cohort of 43,000 patients tested for hereditary cancer.

DNA germline genetic testing can identify individuals with cancer susceptibility. However, DNA sequencing alone is limited in its detection and classification of mRNA splicing variants, particularly those located far from coding sequences. Here we address the limitations of splicing variant identification and interpretation by pairing DNA and RNA sequencing and describe the mutational and splicing landscape in a clinical cohort of 43,524 individuals undergoing genetic testing for hereditary cancer predisposition.

VHL mosaicism: the added value of multi-tissue analysis.

Von Hippel-Lindau disease (VHL) is an autosomal dominant, inherited syndrome with variants in the VHL gene causing predisposition to multi-organ benign and malignant neoplasms. A germline VHL variant is identified in 95-100% of individuals with a clinical diagnosis of VHL. Here, we present the case of an individual with a clinical diagnosis of VHL disease where peripheral blood DNA analysis did not detect a VHL variant. Sequencing of four tumor tissues (ccRCC, pheochromocytoma, lung via sputum, liver) revealed a VHL c.593 T > C (p.Leu198Pro) variant at varying allele fractions (range: 10-55%) in all tissues. Re-examination of the peripheral blood sequencing data identified this variant at 6% allele fraction. Tumor analysis revealed characteristic cytomorphological, immunohistochemical reactivity for alpha-inhibin, and CAIX, and reduced pVHL reactivity supported VHL-related pseudohypoxia. This report of a rare case of VHL mosaicism highlights the value of tissue testing in VHL variant negative cases.

Maternal methylmercury from a wild-caught walleye diet induces developmental abnormalities in zebrafish.

Maternal methylmercury (MeHg) exposure from a contaminated diet causes adverse effects in offspring, but the underlying mechanism(s) remains unclear. In the present study, we investigated the effects of maternal dietary MeHg-exposure on the offspring, using the zebrafish (Danio rerio) as a model system. Female zebrafish were exposed to MeHg (0.88-3.10ppm) by consuming a diet made from wild-caught walleye originally intended for human consumption. While dietary MeHg exposure did not significantly influence fecundity, offspring showed increases in morphologic alterations and mortality, neurobehavioral dysfunction, and dysregulation of global gene expression. Gene expression analysis suggested that MeHg might affect neuronal and muscular development via dysregulation of genes related to transcriptional regulation (such as supt5h) and cell cycle (such as ccnb1). Results from this study provide evidence that food intended for human consumption, with relatively modest levels of MeHg, may induce adverse effects in offspring.

Challenges to clinical utilization of hereditary cancer gene panel testing: perspectives from the front lines.

Next generation sequencing (NGS) technology is rapidly being implemented into clinical practice. Qualitative research was performed to gain an improved understanding of the landscape surrounding the use of NGS in cancer genetics. A focus group was conducted at the Wisconsin Cancer Risk Programs Network biannual meeting. Free flowing discussion with occasional open-ended questions provided insights into the use of NGS. 19 genetic counselors and medical professionals participated. Three major themes were identified with respect to NGS and its use in cancer genetics: knowledge gaps, the evolving clinician role, and uncertain utility. Several corresponding subthemes were identified. With respect to knowledge gaps, participants expressed concern regarding unexpected results and variants of unknown significance, lack of data about NGS findings, absence of standardization regarding use of NGS and guidelines for interpretation, and discomfort with new technology. Regarding the evolving clinician role, necessary changes to the roles of genetic counselors and physicians were noted, as was the resultant impact on care received by patients and their families. Finally, the clinical and economic utility of NGS was questioned. While a shift from traditional Sanger sequencing to NGS is occurring in molecular genetic testing for disease susceptibility, there are several obstacles that need to be overcome before widespread adoption of this technology can occur. Furthermore, key aspects of NGS and it utility remain unexplored. Continued investigation into these subjects is necessary before this technology will consistently be of benefit to patients and their families.

Regulation of colorectal cancer cell apoptosis by the n-3 polyunsaturated fatty acids Docosahexaenoic and Eicosapentaenoic.

Several studies have suggested that the n-3 fatty acids Docosahexaenoic (DHA) and Eicosapentaenoic (EPA) have an important protective effect on colorectal cancer, and this could be at least partly due to their proapoptotic activity. It is unclear, however, how this phenomenon is triggered and what mechanisms are implicated. Here, we show that both DHA and EPA have an important proapoptotic effect on colorectal cancer cells with different molecular phenotypes but not in noncancerous cells. Apoptosis is caspase dependent, and both intrinsic and extrinsic pathways are implicated. The dimerization of Bax and Bak, the depolarization of the mitochondrial membrane, and the subsequent release of cytochrome c and Smac/Diablo to the cytosol evidence the activation of the intrinsic pathway. The implication of the extrinsic pathway is shown by the activation of caspase-8, along with the down-regulation of FLIP. The timing of caspase-8 activation, and the oligomerization of Bid with Bax, suggest a cross-talk with the intrinsic pathway. None of the death receptors that commonly initiate the extrinsic pathway: FAS, TNF-R1, and TRAIL-R2 are found to be responsible for triggering the apoptosis cascade induced by DHA and EPA. Neither PPARgamma nor cyclooxygenase-2, two likely candidates to regulate this process, play a significant role. Our findings suggest that the down-regulation of two key regulatory elements of the extrinsic and intrinsic pathways, FLIP and XIAP, respectively, is determinant in the induction of apoptosis by DHA and EPA. These fatty acids could potentially be useful adjuvant anticancer agents in combination with other chemotherapeutic elements.