Strong functional data for pathogenicity or neutrality classify BRCA2 DNA-binding-domain variants of uncertain significance.

Determination of the clinical relevance of rare germline variants of uncertain significance (VUSs) in the BRCA2 cancer predisposition gene remains a challenge as a result of limited availability of data for use in classification models. However, laboratory-based functional data derived from validated functional assays of known sensitivity and specificity may influence the interpretation of VUSs. We evaluated 252 missense VUSs from the BRCA2 DNA-binding domain by using a homology-directed DNA repair (HDR) assay and identified 90 as non-functional and 162 as functional. The functional assay results were integrated with other available data sources into an ACMG/AMP rules-based classification framework used by a hereditary cancer testing laboratory. Of the 186 missense variants observed by the testing laboratory, 154 were classified as VUSs without functional data. However, after applying protein functional data, 86% (132/154) of the VUSs were reclassified as either likely pathogenic/pathogenic (39/132) or likely benign/benign (93/132), which impacted testing results for 1,900 individuals. These results indicate that validated functional assay data can have a substantial impact on VUS classification and associated clinical management for many individuals with inherited alterations in BRCA2.

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.

Search for genetic modifiers of IRF6 and genotype-phenotype correlations in Van der Woude and popliteal pterygium syndromes.

Van der Woude syndrome is the most common form of syndromic orofacial clefting, accounting for 1-2% of all patients with cleft lip and/or cleft palate. Van der Woude and popliteal pterygium syndromes are caused by mutations in IRF6, but phenotypic variability within and among families with either syndrome suggests that other genetic factors contribute to the phenotypes. The aim of this study was to identify common variants acting as genetic modifiers of IRF6 as well as genotype-phenotype correlations based on mutation type and location. We identified an association between mutations in the DNA-binding domain of IRF6 and limb defects (including pterygia). Although we did not detect formally significant associations with the genes tested, borderline associations suggest several genes that could modify the VWS phenotype, including FOXE1, TGFB3, and TFAP2A. Some of these genes are hypothesized to be part of the IRF6 gene regulatory network and may suggest additional genes for future study when larger sample sizes are also available. We also show that families with the Van de Woude phenotype but in whom no mutations have been identified have a lower frequency of cleft lip, suggesting there may be locus and/or mutation class differences in Van de Woude syndrome.

Universal Germline Panel Testing for Individuals With Pheochromocytoma and Paraganglioma Produces High Diagnostic Yield.

BACKGROUND: Practice guidelines to identify individuals with hereditary pheochromocytomas and paragangliomas (PPGLs) advocate for sequential gene testing strategy guided by specific clinical features and predate the routine use of multigene panel testing (MGPT). OBJECTIVE: To describe results of MGPT for hereditary PPGL in a clinically and ancestrally diverse cohort. SETTING: Commercial laboratory based in the United States. METHODS: Clinical data and test results were retrospectively reviewed in 1727 individuals who had targeted MGPT from August 2013 through December 2019 because of a suspicion of hereditary PPGL. RESULTS: Overall, 27.5% of individuals had a pathogenic or likely pathogenic variant (PV), 9.0% had a variant of uncertain significance, and 63.1% had a negative result. Most PVs were identified in SDHB (40.4%), followed by SDHD (21.1%), SDHA (10.1%), VHL (7.8%), SDHC (6.7%), RET (3.7%), and MAX (3.6%). PVs in FH, MEN1, NF1, SDHAF2, and TMEM127 collectively accounted for 6.5% of PVs. Clinical predictors of a PV included extra-adrenal location, early age of onset, multiple tumors, and positive family history of PPGL. Individuals with extra-adrenal PGL and a positive family history were the most likely to have a PV (85.9%). Restricting genetic testing to SDHB/C/D misses one-third (32.8%) of individuals with PVs. CONCLUSION: Our data demonstrate a high diagnostic yield in individuals with and without established risk factors, a low inconclusive result rate, and a substantial contribution to diagnostic yield from rare genes. These findings support universal testing of all individuals with PPGL and the use of concurrent MGPT as the ideal platform.

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.

Healing the heart: a randomized pilot study of a spiritual retreat for depression in acute coronary syndrome patients.

BACKGROUND: Depression is associated with increased risk of cardiovascular morbidity and mortality in coronary heart disease. Numerous conventional and complementary therapies may address depression. Few involving spirituality have been tested. OBJECTIVE: The aim of this study was to compare the effects of a nondenominational spiritual retreat, Medicine for the Earth (MFTE), on depression and other measures of well-being six- to 18-months post acute coronary syndrome (ACS). DESIGN/SETTING: A randomized controlled pilot study of MFTE, Lifestyle Change Program (LCP), or usual cardiac care (control) was conducted in Southeastern Michigan. PARTICIPANTS: ACS patients were recruited via local and national advertising (n = 58 enrolled, 41 completed). INTERVENTIONS: The four-day MFTE intervention included guided imagery, meditation, drumming, journal writing, and nature-based activities. The four-day LCP included nutrition education, exercise, and stress management. Both retreat groups received follow-up phone coaching biweekly for three months. MAIN OUTCOME MEASURES: Validated self-report scales of depression, spiritual well-being, perceived stress, and hope were collected at baseline, immediately post-retreat, and at three and six months. RESULTS: Depression was not significantly different among groups (P = .21). However, the MFTE group had the highest depression scores at baseline and had significantly lower scores at all postintervention time points (P ≤ .002). Hope significantly improved among MFTE participants, an effect that persisted at three- and six-month follow-up (P = .014). Although several measures showed improvement in all groups by six months, the MFTE group had immediate improvement post-retreat, which was maintained. CONCLUSIONS: This pilot study shows that a nondenominational spiritual retreat, MFTE, can be used to increase hope while reducing depression in patients with ACS.