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Objective The purpose of this study was to recontact individuals with clinically actionable test results identified through a retrospective research study and to provide a framework for laboratories to recontact patients. Methods Genetic testing was conducted on 2977 individuals originally referred for BRCA1 and BRCA2 hereditary breast and ovarian cancer testing that had a negative genetic test result. A gene panel was used to identify pathogenic variants in known or newly discovered genes that could explain the underlying cause of disease; however, analysis was restricted to PALB2 for the purposes of this study. A patient recontact decision tree was developed to assist in the returning of updated genetic test results to clinics and patients. Results Novel clinically actionable pathogenic variants were identified in the PALB2 gene in 18 participants (0.6%), the majority of whom were recontacted with their new or updated genetic test results. Eight individuals were unable to be recontacted; five individuals had already learnt about their new or updated findings from genetic testing outside the context of this study; three individuals prompted cascade testing in family members; two individuals were deceased. Conclusion Novel pathogenic variants in PALB2 were identified in 18 individuals through retrospective gene panel testing. Recontacting these individuals regarding these new or updated findings had a range of outcomes. The process of conveying genomic results within this framework can be effectively accomplished while upholding patient autonomy, potentially leading to advantageous outcomes for patients and their families. [ABSTRACT FROM AUTHOR]

Background This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. Methods Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. Results Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. Conclusions The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care. [ABSTRACT FROM AUTHOR]

Abstract Background β-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with β-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the β-catenin/TCF transcription factor complex. Results We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the β-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and β-catenin. Furthermore, endogenous Tiam1 associated with endogenous β-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner. Conclusion Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active β-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.

Background The value of retesting women who previously tested negative for a pathogenic variant (mutation) in BRCA1 and BRCA2 using an expanded panel of breast and ovarian cancer genes is unclear. Methods We studied 110 BRCA1/2-negative women who were retested using a panel of 20 breast and/or ovarian cancer susceptibility genes at the Advanced Molecular Diagnostics Laboratory (AMDL) at Mount Sinai Hospital in Toronto between March 2017 and March 2019. All patients had previously tested negative for BRCA pathogenic variants at the AMDL between January 2012 and March 2018 and were subsequently referred for retesting by their physician. Results Overall, six pathogenic variants in genes other than BRCA1 and BRCA2 were found (prevalence 5.5%). There were two pathogenic variants found in RAD51C, and one found in each of BRIP1, PALB2, PMS2 and PTEN. The prevalence of pathogenic variants was 6.5% for women affected with cancer (6 of 93), including 4.9% for women with breast cancer (4 of 82) and 22.2% for women with ovarian cancer (2 of 9). None of the 17 unaffected women had a clinically significant or pathogenic variant. There were 44 women (40%) for whom the result of the panel test was inconclusive due to the detection of a variant of uncertain significance. Conclusions Our findings indicate that the retesting of BRCA1/2-negative individuals with an expanded panel of 20 breast and ovarian cancer genes can produce clinically relevant results, with a yield of 5.5% for pathogenic variants in genes other than BRCA1 and BRCA2. [ABSTRACT FROM AUTHOR]

The new 2017 diagnostic criteria for hypermobile Ehlers–Danlos Syndrome (hEDS) provide a framework for diagnosing hEDS but are more stringent than the previous Villefranche criteria. Our clinical experience at the GoodHope EDS clinic was that the 2017 criteria left many highly symptomatic patients without a diagnosis of hEDS. We conducted a retrospective cohort study to confirm our clinic experience and assess the accuracy of the 2017 diagnostic criteria for hEDS in patients who had a previous hEDS diagnosis based on the Villefranche criteria. Our study found that 15% (n = 20 of 131) of patients with a prior diagnosis of hEDS met the 2017 diagnostic criteria, and many of the traits used to distinguish hEDS were not significantly more frequent in patients who met 2017 criteria versus those who did not. In both groups objective systemic manifestations were found less frequently than subjective systemic manifestations. Beighton score (BS) as assessed by primary care practitioner was found to be higher than assessment by EDS practitioner in 81% (n = 74 of 91) of cases. Generalized joint hypermobility was confirmed in only 46% (n = 51 of 111) of patients who had a previous diagnosis of hEDS. Higher BS did not correlate with increased number of systemic manifestations in our cohort. Common comorbidities of hEDS were found with similar frequency in those who met 2017 criteria and those who did not. Based on our cohort, the 2017 hEDS diagnostic criteria require refinement to improve its diagnostic accuracy. [ABSTRACT FROM AUTHOR]

Background: Proteogenomics is an emerging field at the intersection of genomics and proteomics. Many variant peptides corresponding to single nucleotide variations (SNVs) are associated with specific diseases. The aim of this study was to demonstrate the feasibility of proteogenomic-based variant peptide detection in disease models and clinical specimens. Methods: We sought to detect p53 single amino acid variant (SAAV) peptides in breast cancer tumor samples that have been previously subjected to sequencing analysis. Initially, two cancer cell lines having a cellular tumor antigen p53 (TP53) mutation and one wild type for TP53 were analyzed by selected reaction monitoring (SRM) assays as controls. One pool of wild type and one pool of mutated for TP53 cytosolic extracts were assayed with a shotgun proteogenomic workflow. Furthermore, 18 individual samples having a mutation in TP53 were assayed by SRM. Results: Two mutant p53 peptides were successfully detected in two cancer cell lines as expected from their DNA sequence. Wild type p53 peptides were detected in both cytosolic pools, however, none of the mutant p53 peptides were identified. Mutations at the protein level were detected in two cytosolic extracts and whole tumor lysates from the same patients by SRM analysis. Six thousand and six hundred and twenty eight non-redundant proteins were identified in the two cytosolic pools, thus greatly improving a previously reported cytosolic proteome. Conclusions: In the current study we show the great potential of using proteogenomics for the direct identification of cancer-associated mutations in clinical samples and we discuss current limitations and future perspectives. [ABSTRACT FROM AUTHOR]