The landscape of reported VUS in multi-gene panel and genomic testing: Time for a change.

PURPOSE: Variants of uncertain significance (VUS) are a common result of diagnostic genetic testing and can be difficult to manage with potential misinterpretation and downstream costs, including time investment by clinicians. We investigated the rate of VUS reported on diagnostic testing via multi-gene panels (MGPs) and exome and genome sequencing (ES/GS) to measure the magnitude of uncertain results and explore ways to reduce their potentially detrimental impact. METHODS: Rates of inconclusive results due to VUS were collected from over 1.5 million sequencing test results from 19 clinical laboratories in North America from 2020 to 2021. RESULTS: We found a lower rate of inconclusive test results due to VUSs from ES/GS (22.5%) compared with MGPs (32.6%; P < .0001). For MGPs, the rate of inconclusive results correlated with panel size. The use of trios reduced inconclusive rates (18.9% vs 27.6%; P < .0001), whereas the use of GS compared with ES had no impact (22.2% vs 22.6%; P = ns). CONCLUSION: The high rate of VUS observed in diagnostic MGP testing warrants examining current variant reporting practices. We propose several approaches to reduce reported VUS rates, while directing clinician resources toward important VUS follow-up.

Prevalence of Germline Pathogenic and Likely Pathogenic Variants in Patients With Second Breast Cancers.

BACKGROUND: Few studies have examined gene-specific associations with contralateral and/or second breast cancer (SBC). METHODS: The frequency of pathogenic and likely pathogenic (P/LP) variants in clinically actionable genes (BRCA1, BRCA2, PTEN, TP53, CHEK2, CDH1, ATM, PALB2, NBN, and NF1) was compared between women with a primary breast cancer (PBC) and SBC who underwent multigene panel testing at a single diagnostic testing laboratory. Race- and ethnicity-specific logistic regression burden tests adjusted for age at diagnosis of first breast cancer, histology, presence of first- or second-degree relatives with breast cancer, and prior testing for BRCA1/2 genes were used to test for associations with SBC. All statistical tests were 2-sided. RESULTS: The study was comprised of 75 550 women with PBC and 7728 with SBC. Median time between breast cancers for SBC was 11 (interquartile range = 6-17) years. Restricting to women tested for all actionable genes (n = 60 310), there were 4231 (7.8%) carriers of P/LP variants in actionable genes among the controls (PBC) compared with 652 (11.1%) women with SBC (P< .001). Among Caucasians, exclusive of Ashkenazi Jewish women, those carrying a P/LP variant in a clinically actionable gene were 1.44 (95% confidence interval [CI] = 1.30 to 1.60) times as likely to have SBC than noncarriers, after accounting for potential confounders. Among African American and Hispanic women, a P/LP variant in a clinically actionable gene was 1.88 (95% CI = 1.36 to 2.56) and 1.66 (9% CI = 1.02 to 2.58) times as likely to be associated with SBC, respectively (P < .001 and P = .03). CONCLUSION: Women with P/LP variants in breast cancer predisposition genes are more likely to have SBC than noncarriers. Prospective studies are needed confirm these findings.

Genotype-phenotype associations among panel-based TP53+ subjects.

PURPOSE: Panel testing has led to the identification of TP53 pathogenic/likely pathogenic (P/LP) variant carriers (TP53+) who exhibit a broad range of phenotypes. We sought to evaluate and compare genotype-phenotype associations among TP53+ panel-ascertained subjects. METHODS: Between 2012 and 2017, 317 TP53+ subjects (279 females and 38 males) identified through panel testing at one testing laboratory were found to have evaluable clinical histories and molecular results. Subject cancer histories were obtained from test requisition forms. P/LP variants were categorized by type and were examined in relation to phenotype. RESULTS: Loss-of-function (LOF) variants were associated with the earliest age at first cancer, with a median age of 30.5 years (P = 0.014); increased frequency of a sarcoma diagnosis (P = 0.016); and more often meeting classic LFS testing and Chompret 2015 criteria (P = 0.004 and 0.002 respectively), as compared with dominant-negative missense, other missense, or miscellaneous (splice or in-frame deletion) P/LP variant categories. CONCLUSION: Loss-of-function variants were more often associated with characteristic LFS cancer histories than other variant categories in TP53+ carriers ascertained through multigene panel testing. These findings require validation in other TP53+ cohorts. Genetic counseling for panel-ascertained TP53+ individuals should reflect the dynamic expansion of the Li-Fraumeni syndrome phenotype.

A Bayesian framework for efficient and accurate variant prediction.

There is a growing need to develop variant prediction tools capable of assessing a wide spectrum of evidence. We present a Bayesian framework that involves aggregating pathogenicity data across multiple in silico scores on a gene-by-gene basis and multiple evidence statistics in both quantitative and qualitative forms, and performs 5-tiered variant classification based on the resulting probability credible interval. When evaluated in 1,161 missense variants, our gene-specific in silico model-based meta-predictor yielded an area under the curve (AUC) of 96.0% and outperformed all other in silico predictors. Multifactorial model analysis incorporating all available evidence yielded 99.7% AUC, with 22.8% predicted as variants of uncertain significance (VUS). Use of only 3 auto-computed evidence statistics yielded 98.6% AUC with 56.0% predicted as VUS, which represented sufficient accuracy to rapidly assign a significant portion of VUS to clinically meaningful classifications. Collectively, our findings support the use of this framework to conduct large-scale variant prioritization using in silico predictors followed by variant prediction and classification with a high degree of predictive accuracy.

PTEN Promoter Variants Are Not Associated With Common Cancers: Implications for Multigene Panel Testing.

PURPOSE: PTEN mutations are associated with breast, colon, endometrial, kidney, and thyroid cancers. Most PTEN promoter alterations, however, are characterized as variants of unknown significance, and their contribution to cancer risk is unclear. MATERIALS AND METHODS: Personal and family histories of 88,333 patients undergoing PTEN analysis as part of multigene panel testing (MGPT) were retrospectively reviewed. Cases (n = 59,784) were individuals with personal history of PTEN-related cancer. Controls (n = 28,549) had no personal history of cancer. Individuals were categorized as positive for one or more mutations (PATHO), without mutations but carrying one or more promoter variant (PROM), or negative for alterations (WT). Multivariable logistic regression was used to assess PTEN associations with phenotypes, adjusted for race/ethnicity, age, sex, and MGPT. RESULTS: Overall, 79 (0.09%) patients were PATHO and 791 (0.9%) were PROM carriers. Compared with WT, PATHOs were 2.30 (95% CI, 1.19 to 4.72) times as likely to have breast, 7.23 (95% CI, 2.74 to 19.14) times as likely to have bilateral/multiple primary breast, and 7.56 (95% CI, 1.97 to 23.98) times as likely to have uterine/endometrial cancer. PROMs were not significantly more likely than WT to have cancer (all 0.84 < odds ratio < 1.15; P > .05). CONCLUSION: PTEN promoter variants were not associated with cancer. These results do not support the inclusion of PTEN promoter sequencing in MGPT.

Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

Conformational sampling of peptides in cellular environments.

Biological systems provide a complex environment that can be understood in terms of its dielectric properties. High concentrations of macromolecules and cosolvents effectively reduce the dielectric constant of cellular environments, thereby affecting the conformational sampling of biomolecules. To examine this effect in more detail, the conformational preference of alanine dipeptide, poly-alanine, and melittin in different dielectric environments is studied with computer simulations based on recently developed generalized Born methodology. Results from these simulations suggest that extended conformations are favored over alpha-helical conformations at the dipeptide level at and below dielectric constants of 5-10. Furthermore, lower-dielectric environments begin to significantly stabilize helical structures in poly-alanine at epsilon = 20. In the more complex peptide melittin, different dielectric environments shift the equilibrium between two main conformations: a nearly fully extended helix that is most stable in low dielectrics and a compact, V-shaped conformation consisting of two helices that is preferred in higher dielectric environments. An additional conformation is only found to be significantly populated at intermediate dielectric constants. Good agreement with previous studies of different peptides in specific, less-polar solvent environments, suggest that helix stabilization and shifts in conformational preferences in such environments are primarily due to a reduced dielectric environment rather than specific molecular details. The findings presented here make predictions of how peptide sampling may be altered in dense cellular environments with reduced dielectric response.