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Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li-Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

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Lynch syndrome (LS) is the main hereditary colorectal cancer syndrome. There have been few reports regarding the clinical and molecular characteristics of LS patients in Latin America; this is particularly true in the Mexican population, where no information is available. The present study aims to describe the clinical and molecular spectrum of variants in a cohort of patients diagnosed with LS in Mexico. We present a retrospective analysis of 412 patients with suspected LS, whose main site of cancer diagnosis was the colon (58.25%), followed by the endometrium (18.93%). Next-generation sequencing analysis, with an extensive multigene panel, showed that 27.1% (112/414) had a variant in one of the genes of the mismatch repair pathway (MMR); 30.4% (126/414) had a variant in non-MMR genes such as CHEK2, APC, MUTYH, BRCA1, and BRCA2; and 42.5% (176/414) had no genetic variants. Most of the variants were found in MLH1. Pathogenic variants (PVs) in MMR genes were identified in 65.7% (96/146) of the total PVs, and 34.24% (45/146) were in non-MMR genes. Molecular and clinical characterization of patients with LS in specific populations allowed personalized follow-up, with the option for targeted treatment with immune checkpoint inhibitors and the development of public health policies. Moreover, such characterization allows for family cascade testing and consequent prevention strategies.

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The growing use of next-generation sequencing technologies on genetic diagnosis has produced an exponential increase in the number of variants of uncertain significance (VUS). In this manuscript, we compare three machine learning methods to classify VUS as Pathogenic or No pathogenic, implementing a Random Forest (RF), a Support Vector Machine (SVM), and a Multilayer Perceptron. To train the models, we extracted high-quality variants from ClinVar that were previously classified as VUS. For each variant, we retrieved nine conservation scores, the loss-of-function tool, and allele frequencies. For the RF and SVM models, hyperparameters were tuned using cross-validation with a grid search. The three models were tested on a nonoverlapping set of variants that had been classified as VUS over the last 3 years, but had been reclassified in August 2020. The three models yielded superior accuracy on this set compared to the benchmarked tools. The RF-based model yielded the best performance across different variant types and was used to create VusPrize, an open-source software tool for prioritization of VUS. We believe that our model can improve the process of genetic diagnosis in research and clinical settings.

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PURPOSE: BRCA1 pathogenic variant heterozygotes are at a substantially increased risk for breast and ovarian cancer. The widespread uptake of testing has led to a significant increase in the detection of missense variants in BRCA1, the vast majority of which are variants of uncertain clinical significance (VUS), posing a challenge to genetic counseling. Here, we harness a wealth of functional data for thousands of variants to aid in variant classification. METHODS: We have collected, curated, and harmonized functional data for 2701 missense variants representing 24.5% of possible missense variants in BRCA1. Results were harmonized across studies by converting data into binary categorical variables (functional impact versus no functional impact). Using a panel of reference variants we identified a subset of assays with high sensitivity and specificity (≥80%) and apply the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant interpretation guidelines to assign evidence criteria for classification. RESULTS: Integration of data from validated assays provided ACMG/AMP evidence criteria in favor of pathogenicity for 297 variants or against pathogenicity for 2058 representing 96.2% of current VUS functionally assessed. We also explore discordant results and identify limitations in the approach. CONCLUSION: High quality functional data are available for BRCA1 missense variants and provide evidence for classification of 2355 VUS according to their pathogenicity.

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Since its discovery, partner and localizer of breast cancer 2 (BRCA2) (PALB2) has emerged as a major tumor suppressor gene linked to breast cancer (BC), pancreatic cancer (PC), and ovarian cancer (OC) susceptibility. Its protein product plays a pivotal role in the maintenance of genome integrity. Here we discuss the first functional evaluation of a large set of PALB2 missense variants of uncertain significance (VUSs). Assessment of 136 VUSs interrogating a range of PALB2 biological functions resulted in the identification of 15 variants with consistent loss of function across different assays. All loss-of-function variants are located at the PALB2 coiled coil (CC) or at the WD40 domain, highlighting the importance of modular domains mechanistically involved in the DNA damage response (DDR) and pinpointing their roles in tumor suppression.

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Li-Fraumeni syndrome (LFS) and Li-Fraumeni Like (LFL) are autosomal dominant cancer predisposition syndromes caused by pathogenic germline variants in the TP53 gene. Recent studies have shown that the incorporation of next-generation sequencing by using multigene panels in clinical practice has resulted in the frequent identification of variants of uncertain significance (VUS). Given that there is no established medical management for VUS carriers, the identification of these variants may cause confusion and anxiety for both patients and practitioners. Herein, we aimed to verify VUS frequency and review VUS classification and interpretation in 1844 patients submitted for comprehensive germline TP53 testing independent of clinical criteria. Variant characterization was done assessing clinical information whenever available, variant frequency in population databases, pathogenicity predictions using in silico tools and previous functional studies. All variants were classified based on the guidelines proposed by the American College of Medical Genetics and Genomics (2015) and by the Sherloc framework (2017). Of the twelve VUS (0.65%) identified in TP53, two were classified as likely pathogenic and two were classified as likely benign after re-evaluation, potentially resulting in significant management modification for the proband and relatives. This report cases highlights the challenges and impact of TP53 variant interpretation especially when there is no clear LFS/LFL phenotype.

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Objective: We aimed to improve the classification of SCN1A missense variants in patients with Dravet syndrome (DS) by combining and modifying the current variants classification criteria to minimize inconclusive test results. Methods: We established a score classification workflow based on evidence of pathogenicity to adapt the classification of DS-related SCN1A missense variants. In addition, we compiled the variants reported in the literature and our cohort and assessed the proposed pathogenic classification criteria. We combined information regarding previously established pathogenic amino acid changes, mode of inheritance, population-specific allele frequencies, localization within protein domains, and deleterious effect prediction analysis. Results: Our meta-analysis showed that 46% (506/1,101) of DS-associated SCN1A variants are missense. We applied the score classification workflow and 56.5% (286/506) of the variants had their classification changed from VUS: 17.8% (90/506) into "pathogenic" and 38.7% (196/506) as "likely pathogenic." Conclusion: Our results indicate that using multimodal analysis seems to be the best approach to interpret the pathogenic impact of SCN1A missense changes for the molecular diagnosis of patients with DS. By applying the proposed workflow, most DS related SCN1A variants had their classification improved.