scholarly journals Beyond BRCA1 and BRCA2: Deleterious Variants in DNA Repair Pathway Genes in Italian Families with Breast/Ovarian and Pancreatic Cancers

2020 ◽  
Vol 9 (9) ◽  
pp. 3003
Author(s):  
Aldo Germani ◽  
Simona Petrucci ◽  
Laura De Marchis ◽  
Fabio Libi ◽  
Camilla Savio ◽  
...  
Keyword(s):  
Therapeutic Approaches ◽  
Brca1 And Brca2 ◽  
Pathogenic Variants ◽  
Pancreatic Cancers ◽  
Damage Repair ◽  
High Incidence ◽  
Uncertain Significance ◽  
Generation Sequencing ◽  
Index Cases ◽  
Multigene Panel

The 5–10% of breast/ovarian cancers (BC and OC) are inherited, and germline pathogenic (P) variants in DNA damage repair (DDR) genes BRCA1 and BRCA2 explain only 10–20% of these cases. Currently, new DDR genes have been related to BC/OC and to pancreatic (PC) cancers, but the prevalence of P variants remains to be explored. The purpose of this study was to investigate the spectrum and the prevalence of pathogenic variants in DDR pathway genes other than BRCA1/2 and to correlate the genotype with the clinical phenotype. A cohort of 113 non-BRCA patients was analyzed by next-generation sequencing using a multigene panel of the 25 DDR pathways genes related to BC, OC, and PC. We found 43 unique variants in 18 of 25 analyzed genes, 14 classified as P/likely pathogenic (LP) and 28 as variants of uncertain significance (VUS). Deleterious variants were identified in 14% of index cases, whereas a VUS was identified in 20% of the probands. We observed a high incidence of deleterious variants in the CHEK2 gene, and a new pathogenic variant was detected in the RECQL gene. These results supported the clinical utility of multigene panel to increase the detection of P/LP carriers and to identify new actionable pathogenic gene variants useful for preventive and therapeutic approaches.

scholarly journals Spectrum of Germline BRCA1 and BRCA2 Variants Identified in 2351 Ovarian and Breast Cancer Patients Referring to a Reference Cancer Hospital of Rome

Cancers ◽  
2020 ◽  
Vol 12 (5) ◽  
pp. 1286 ◽  
Author(s):  
Concetta Santonocito ◽  
Roberta Rizza ◽  
Ida Paris ◽  
Laura De Marchis ◽  
Carmela Paolillo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Breast Cancer Patients ◽  
Brca1 And Brca2 ◽  
Pathogenic Variants ◽  
Uncertain Significance ◽  
In Vitro Diagnostic ◽  
Variant Frequency ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Pathogenic variants (PVs) carriers in BRCA1 or BRCA2 are associated with an elevated lifetime risk of developing breast cancer (BC) and/or ovarian cancer (OC). The prevalence of BRCA1 and BRCA2 germline alterations is extremely variable among different ethnic groups. Particularly, the rate of variants in Italian BC and/or OC families is rather controversial and ranges from 8% to 37%, according to different reports. By In Vitro Diagnostic (IVD) next generation sequencing (NGS)-based pipelines, we routinely screened thousands of patients with either sporadic or cancer family history. By NGS, we identified new PVs and some variants of uncertain significance (VUS) which were also evaluated in silico using dedicated tools. We report in detail data regarding BRCA1/2 variants identified in 517 out of 2351 BC and OC patients. The aim of this study was to report the incidence and spectrum of BRCA1/2 variants observed in BC and/or OC patients, tested in at Policlinico Gemelli Foundation Hospital, the origin of which is mainly from Central and Southern Italy. This study provides an overview of the variant frequency in these geographic areas of Italy and provides data that could be used in the clinical management of patients.

scholarly journals Assessing BRCA1 activity in DNA damage repair using human induced pluripotent stem cells as an approach to assist classification of BRCA1 variants of uncertain significance

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0260852
Author(s):  
Meryem Ozgencil ◽  
Julian Barwell ◽  
Marc Tischkowitz ◽  
Louise Izatt ◽  
Ian Kesterton ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Repair ◽  
Ips Cells ◽  
Variants Of Uncertain Significance ◽  
Cellular Models ◽  
Pathogenic Variants ◽  
Damage Repair ◽  
Uncertain Significance ◽  
Induced Pluripotent ◽  
The Impact

Establishing a universally applicable protocol to assess the impact of BRCA1 variants of uncertain significance (VUS) expression is a problem which has yet to be resolved despite major progresses have been made. The numerous difficulties which must be overcome include the choices of cellular models and functional assays. We hypothesised that the use of induced pluripotent stem (iPS) cells might facilitate the standardisation of protocols for classification, and could better model the disease process. We generated eight iPS cell lines from patient samples expressing either BRCA1 pathogenic variants, non-pathogenic variants, or BRCA1 VUSs. The impact of these variants on DNA damage repair was examined using a ɣH2AX foci formation assay, a Homologous Repair (HR) reporter assay, and a chromosome abnormality assay. Finally, all lines were tested for their ability to differentiate into mammary lineages in vitro. While the results obtained from the two BRCA1 pathogenic variants were consistent with published data, some other variants exhibited differences. The most striking of these was the BRCA1 variant Y856H (classified as benign), which was unexpectedly found to present a faulty HR repair pathway, a finding linked to the presence of an additional variant in the ATM gene. Finally, all lines were able to differentiate first into mammospheres, and then into more advanced mammary lineages expressing luminal- or basal-specific markers. This study stresses that BRCA1 genetic analysis alone is insufficient to establish a reliable and functional classification for assessment of clinical risk, and that it cannot be performed without considering the other genetic aberrations which may be present in patients. The study also provides promising opportunities for elucidating the physiopathology and clinical evolution of breast cancer, by using iPS cells.

Reclassification of BRCA1 and BRCA2 variants of uncertain significance: a multifactorial analysis of multicentre prospective cohort

2018 ◽  
Vol 55 (12) ◽  
pp. 794-802 ◽  
Author(s):  
Jee-Soo Lee ◽  
Sohee Oh ◽  
Sue Kyung Park ◽  
Min-Hyuk Lee ◽  
Jong Won Lee ◽  
...  
Keyword(s):  
Breast Cancer ◽  
External Validation ◽  
Brca1 And Brca2 ◽  
Clinical Genetic ◽  
Validation Data ◽  
Data Set ◽  
Variants Of Uncertain Significance ◽  
Pathogenic Variants ◽  
Uncertain Significance ◽  
Highly Correlated

BackgroundBRCA1 and BRCA2 (BRCA1/2) variants classified ambiguously as variants of uncertain significance (VUS) are a major challenge for clinical genetic testing in breast cancer; their relevance to the cancer risk is unclear and the association with the response to specific BRCA1/2-targeted agents is uncertain. To minimise the proportion of VUS in BRCA1/2, we performed the multifactorial likelihood analysis and validated this method using an independent cohort of patients with breast cancer.MethodsWe used a data set of 2115 patients with breast cancer from the nationwide multicentre prospective Korean Hereditary Breast Cancer study. In total, 83 BRCA1/2 VUSs (BRCA1, n=26; BRCA2, n=57) were analysed. The multifactorial probability was estimated by combining the prior probability with the overall likelihood ratio derived from co-occurrence of each VUS with pathogenic variants, personal and family history, and tumour characteristics. The classification was compared with the interpretation according to the American College of Medical Genetics and Genomics–Association for Molecular Pathology (ACMG/AMP) guidelines. An external validation was conducted using independent data set of 810 patients.ResultsWe were able to redefine 38 VUSs (BRCA1, n=10; BRCA2, n=28). The revised classification was highly correlated with the ACMG/AMP guideline-based interpretation (BRCA1, p for trend=0.015; BRCA2, p=0.001). Our approach reduced the proportion of VUS from 19% (154/810) to 8.9% (72/810) in the retrospective validation data set.ConclusionThe classification in this study would minimise the ‘uncertainty’ in clinical interpretation, and this validated multifactorial model can be used for the reliable annotation of BRCA1/2 VUSs.

scholarly journals Analysis of the pathogenic variants of BRCA1 and BRCA2 using next-generation sequencing in women with familial breast cancer: a case–control study

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Omar Alejandro Zayas-Villanueva ◽  
Luis Daniel Campos-Acevedo ◽  
José de Jesús Lugo-Trampe ◽  
David Hernández-Barajas ◽  
Juan Francisco González-Guerrero ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Next Generation Sequencing ◽  
Familial Breast Cancer ◽  
Case Control Study ◽  
Case Control ◽  
Next Generation ◽  
Brca1 And Brca2 ◽  
Pathogenic Variants ◽  
Generation Sequencing ◽  
Control Study

scholarly journals The variability of SMCHD1 gene in FSHD patients: evidence of new mutations

2019 ◽  
Vol 28 (23) ◽  
pp. 3912-3920 ◽  
Author(s):  
Claudia Strafella ◽  
Valerio Caputo ◽  
Rosaria Maria Galota ◽  
Giulia Campoli ◽  
Cristina Bax ◽  
...  
Keyword(s):  
Fragment Size ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Clinical Severity ◽  
Pathogenic Variants ◽  
Uncertain Significance ◽  
Next Generation Sequencing Ngs ◽  
Structural Maintenance Of Chromosomes ◽  
Generation Sequencing ◽  
Hinge Domain ◽  
New Mutations

Abstract In this study, we investigated the sequence of (Structural Maintenance of Chromosomes flexible Hinge Domain containing 1) SMCHD1 gene in a cohort of clinically defined FSHD (facioscapulohumeral muscular dystrophy) patients in order to assess the distribution of SMCHD1 variants, considering the D4Z4 fragment size in terms of repeated units (RUs; short fragment: 1–7 RU, borderline: 8-10RU and normal fragment: >11RU). The analysis of SMCHD1 revealed the presence of 82 variants scattered throughout the introns, exons and 3’untranslated region (3′UTR) of the gene. Among them, 64 were classified as benign polymorphisms and 6 as VUS (variants of uncertain significance). Interestingly, seven pathogenic/likely pathogenic variants were identified in patients carrying a borderline or normal D4Z4 fragment size, namely c.182_183dupGT (p.Q62Vfs*48), c.2129dupC (p.A711Cfs*11), c.3469G>T (p.G1157*), c.5150_5151delAA (p.K1717Rfs*16) and c.1131+2_1131+5delTAAG, c.3010A>T (p.K1004*), c.853G>C (p.G285R). All of them were predicted to disrupt the structure and conformation of SMCHD1, resulting in the loss of GHKL-ATPase and SMC hinge essential domains. These results are consistent with the FSHD symptomatology and the Clinical Severity Score (CSS) of patients. In addition, five variants (c.*1376A>C, rs7238459; c.*1579G>A, rs559994; c.*1397A>G, rs150573037; c.*1631C>T, rs193227855; c.*1889G>C, rs149259359) were identified in the 3′UTR region of SMCHD1, suggesting a possible miRNA-dependent regulatory effect on FSHD-related pathways. The present study highlights the clinical utility of next-generation sequencing (NGS) platforms for the molecular diagnosis of FSHD and the importance of integrating molecular findings and clinical data in order to improve the accuracy of genotype–phenotype correlations.

scholarly journals Novel candidates of pathogenic variants of the BRCA1 and BRCA2 genes in a 3,552 Japanese whole-genome sequence dataset (3.5KJPNv2)

2020 ◽  
Author(s):  
Hideki Tokunaga ◽  
Keita Iida ◽  
Atsushi Hozawa ◽  
Soichi Ogishima ◽  
Yoh Watanabe ◽  
...  
Keyword(s):  
Prospective Cohort ◽  
Clinical Evidence ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Cancer Syndrome ◽  
Brca1 And Brca2 ◽  
Cancer History ◽  
Pathogenic Variants ◽  
Uncertain Significance

AbstractIdentification of pathogenic germline variants yet no clinical evidence in BRCA genes has become important in patient care of hereditary breast and ovarian cancer syndrome (HBOC). Computational scoring and prospective cohort studies may help to identify such pathogenic variants. We annotated the variants in the BRCA1 and BRCA2 genes from a dataset of 3,552 whole-genome sequences obtained from members of the genome cohorts by Tohoku Medical Megabank Project (TMM) with the InterVar software. Computational impact scores (CADD_phred and Eigen_raw) and minor allele frequencies (MAF) of pathogenic (P) and likely pathogenic (LP) variants in ClinVar are used for filtration criteria. Familial predispositions in cancers among the 35,000 TMM genome cohort participants are analyzed to verify the pathogenicity. Seven potentially pathogenic variants were newly identified. Carriers of these potential pathogenic variants and definite P and LP variants among participants of the TMM prospective cohort show a statistically significant preponderance in cancer onset in sisters in the self-reported cancer history. Filtering by computational scoring and MAF is useful to identify potential pathogenic variants in BRCA genes for Japanese population. These results will be helpful to follow up the carriers of variants of uncertain significance in the HBOC genes.

scholarly journals Prevalence and Spectrum of Germline BRCA1 and BRCA2 Variants of Uncertain Significance in Breast/Ovarian Cancer: Mysterious Signals From the Genome

2021 ◽  
Vol 11 ◽  
Author(s):  
Daniele Fanale ◽  
Alessia Fiorino ◽  
Lorena Incorvaia ◽  
Alessandra Dimino ◽  
Clarissa Filorizzo ◽  
...  
Keyword(s):  
High Risk ◽  
Clinical Significance ◽  
Clinical Management ◽  
University Hospital ◽  
Sequencing Analysis ◽  
Brca1 And Brca2 ◽  
Variants Of Uncertain Significance ◽  
Pathogenic Variants ◽  
Risk Variants ◽  
Uncertain Significance

About 10–20% of breast/ovarian (BC/OC) cancer patients undergoing germline BRCA1/2 genetic testing have been shown to harbor Variants of Uncertain Significance (VUSs). Since little is known about the prevalence of germline BRCA1/2 VUS in Southern Italy, our study aimed at describing the spectrum of these variants detected in BC/OC patients in order to improve the identification of potentially high-risk BRCA variants helpful in patient clinical management. Eight hundred and seventy-four BC or OC patients, enrolled from October 2016 to December 2020 at the “Sicilian Regional Center for the Prevention, Diagnosis and Treatment of Rare and Heredo-Familial Tumors” of University Hospital Policlinico “P. Giaccone” of Palermo, were genetically tested for germline BRCA1/2 variants through Next-Generation Sequencing analysis. The mutational screening showed that 639 (73.1%) out of 874 patients were BRCA-w.t., whereas 67 (7.7%) were carriers of germline BRCA1/2 VUSs, and 168 (19.2%) harbored germline BRCA1/2 pathogenic/likely pathogenic variants. Our analysis revealed the presence of 59 different VUSs detected in 67 patients, 46 of which were affected by BC and 21 by OC. Twenty-one (35.6%) out of 59 variants were located on BRCA1 gene, whereas 38 (64.4%) on BRCA2. We detected six alterations in BRCA1 and two in BRCA2 with unclear interpretation of clinical significance. Familial anamnesis of a patient harboring the BRCA1-c.3367G>T suggests for this variant a potential of pathogenicity, therefore it should be carefully investigated. Understanding clinical significance of germline BRCA1/2 VUS could improve, in future, the identification of potentially high-risk variants useful for clinical management of BC or OC patients and family members.

scholarly journals GFP-Fragment Reassembly Screens for the Functional Characterization of Variants of Uncertain Significance in Protein Interaction Domains of the BRCA1 and BRCA2 Genes

Cancers ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 151 ◽  
Author(s):  
Laura Caleca ◽  
Mara Colombo ◽  
Thomas van Overeem Hansen ◽  
Conxi Lázaro ◽  
Siranoush Manoukian ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Functional Characterization ◽  
Brca1 And Brca2 ◽  
Vitro Assay ◽  
Variants Of Uncertain Significance ◽  
Pathogenic Variants ◽  
Brca1 And Brca2 Genes ◽  
Uncertain Significance ◽  
Genome Protection

Genetic testing for BRCA1 and BRCA2 genes has led to the identification of many unique variants of uncertain significance (VUS). Multifactorial likelihood models that predict the odds ratio for VUS in favor or against cancer causality, have been developed, but their use is conditioned by the amount of necessary data, which are difficult to obtain if a variant is rare. As an alternative, variants mapping to the coding regions can be examined using in vitro functional assays. BRCA1 and BRCA2 proteins promote genome protection by interacting with different proteins. In this study, we assessed the functional effect of two sets of variants in BRCA genes by exploiting the green fluorescent protein (GFP)-reassembly in vitro assay, which was set-up to test the BRCA1/BARD1, BRCA1/UbcH5a, and BRCA2/DSS1 interactions. Based on the findings observed for the validation panels of previously classified variants, BRCA1/UbcH5a and BRCA2/DSS1 binding assays showed 100% sensitivity and specificity in identifying pathogenic and non-pathogenic variants. While the actual efficiency of these assays in assessing the clinical significance of BRCA VUS has to be verified using larger validation panels, our results suggest that the GFP-reassembly assay is a robust method to identify variants affecting normal protein functioning and contributes to the classification of VUS.

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