Next-generation sequencing (NGS) is changing genetic diagnosis due to its huge sequencing capacity and cost-effectiveness. The aim of this study was to develop an NGS-based workflow for routine ...diagnostics for hereditary breast and ovarian cancer syndrome (HBOCS), to improve genetic testing for BRCA1 and BRCA2. A NGS-based workflow was designed using BRCA MASTR kit amplicon libraries followed by GS Junior pyrosequencing. Data analysis combined Variant Identification Pipeline freely available software and ad hoc R scripts, including a cascade of filters to generate coverage and variant calling reports. A BRCA homopolymer assay was performed in parallel. A research scheme was designed in two parts. A Training Set of 28 DNA samples containing 23 unique pathogenic mutations and 213 other variants (33 unique) was used. The workflow was validated in a set of 14 samples from HBOCS families in parallel with the current diagnostic workflow (Validation Set). The NGS-based workflow developed permitted the identification of all pathogenic mutations and genetic variants, including those located in or close to homopolymers. The use of NGS for detecting copy-number alterations was also investigated. The workflow meets the sensitivity and specificity requirements for the genetic diagnosis of HBOCS and improves on the cost-effectiveness of current approaches.
Understanding the mechanisms of cancer therapeutic resistance is fundamental to improving cancer care. There is clear benefit from chemotherapy in different breast cancer settings; however, knowledge ...of the mutations and genes that mediate resistance is incomplete. In this study, by modeling chemoresistance in patient-derived xenografts (PDXs), we show that adaptation to therapy is genetically complex and identify that loss of transcription factor 4 (TCF4; also known as ITF2) is associated with this process. A triple-negative
-mutated PDX was used to study the genetics of chemoresistance. The PDX was treated in parallel with four chemotherapies for five iterative cycles. Exome sequencing identified few genes with
or enriched mutations in common among the different therapies, whereas many common depleted mutations/genes were observed. Analysis of somatic mutations from The Cancer Genome Atlas (TCGA) supported the prognostic relevance of the identified genes. A mutation in
was found
in all treatments, and analysis of drug sensitivity profiles across cancer cell lines supported the link to chemoresistance. Loss of TCF4 conferred chemoresistance in breast cancer cell models, possibly by altering cell cycle regulation. Targeted sequencing in chemoresistant tumors identified an intronic variant of
that may represent an expression quantitative trait locus associated with relapse outcome in TCGA. Immunohistochemical studies suggest a common loss of nuclear TCF4 expression post-chemotherapy. Together, these results from tumor xenograft modeling depict a link between altered TCF4 expression and breast cancer chemoresistance.
Germline inactivating mutations in BRCA1 and BRCA2 genes are responsible for Hereditary Breast and Ovarian Cancer Syndrome (HBOCS). Genetic testing of these genes is available, although approximately ...15% of tests identify variants of uncertain significance (VUS). Classification of these variants into pathogenic or non-pathogenic type is an important challenge in genetic diagnosis and counseling. The aim of the present study is to functionally assess a set of 7 missense VUS (Q1409L, S1473P, E1586G, R1589H, Y1703S, W1718L and G1770V) located in the C-terminal region of BRCA1 by combining in silico prediction tools and structural analysis with a transcription activation (TA) assay. The in silico prediction programs gave discrepant results making its interpretation difficult. Structural analysis of the three variants located in the BRCT domains (Y1703S, W1718L and G1770V) reveals significant alterations of BRCT structure. The TA assay shows that variants Y1703S, W1718L and G1770V dramatically compromise the transcriptional activity of BRCA1, while variants Q1409L, S1473P, E1586G and R1589H behave like wild-type BRCA1. In conclusion, our results suggest that variants Y1703S, W1718L and G1770V can be classified as likely pathogenic BRCA1 mutations.
Abstract
Motivation
Germline variant classification allows accurate genetic diagnosis and risk assessment. However, it is a tedious iterative process integrating information from several sources and ...types of evidence. It should follow gene-specific (if available) or general updated international guidelines. Thus, it is the main burden of the incorporation of next-generation sequencing into the clinical setting.
Results
We created the vaRiants in HC (vaRHC) R package to assist the process of variant classification in hereditary cancer by: (i) collecting information from diverse databases; (ii) assigning or denying different types of evidence according to updated American College of Molecular Genetics and Genomics/Association of Molecular Pathologist gene-specific criteria for ATM, CDH1, CHEK2, MLH1, MSH2, MSH6, PMS2, PTEN, and TP53 and general criteria for other genes; (iii) providing an automated classification of variants using a Bayesian metastructure and considering CanVIG-UK recommendations; and (iv) optionally printing the output to an .xlsx file. A validation using 659 classified variants demonstrated the robustness of vaRHC, presenting a better criteria assignment than Cancer SIGVAR, an available similar tool.
Availability and implementation
The source code can be consulted in the GitHub repository (https://github.com/emunte/vaRHC) Additionally, it will be submitted to CRAN soon.
Multigene panels provide a powerful tool for analyzing several genes simultaneously. We evaluated the frequency of pathogenic variants (PV) in customized predefined panels according to clinical ...suspicion by phenotype and compared it to the yield obtained in the analysis of our clinical research gene panel. We also investigated mutational yield of opportunistic testing of BRCA1/2 and mismatch repair (MMR) genes in all patients. A total of 1,205 unrelated probands with clinical suspicion of hereditary cancer were screened for germline mutations using panel testing. Overall, 1,048 females and 157 males were analyzed, mean age at cancer diagnosis was 48; 883 had hereditary breast/ovarian cancer‐suspicion, 205 hereditary nonpolyposis colorectal cancer (HNPCC)‐suspicion, 73 adenomatous‐polyposis‐suspicion and 44 with other/multiple clinical criteria. At least one PV was found in 150 probands (12%) analyzed by our customized phenotype‐driven panel. Tumoral MMR deficiency predicted for the presence of germline MMR gene mutations in patients with HNPCC‐suspicion (46/136 vs. 0/56 in patients with and without MMR deficiency, respectively). Opportunistic testing additionally identified five MSH6, one BRCA1 and one BRCA2 carriers (0.6%). The analysis of the extended 24‐gene panel provided 25 additional PVs (2%), including in 4 out of 51 individuals harboring MMR‐proficient colorectal tumors (2 CHEK2 and 2 ATM). Phenotype‐based panels provide a notable rate of PVs with clinical actionability. Opportunistic testing of MMR and BRCA genes leads to a significant straightforward identification of MSH6, BRCA1 and BRCA2 mutation carriers, and endorses the model of opportunistic testing of genes with clinical utility within a standard genetic counseling framework.
What's new?
Multigene panels offer a powerful tool for analyzing several cancer‐related genes with a single test. But which genes are actually useful in guiding medical decisions in the clinic? In this study, the authors analyzed several customized, phenotype‐driven diagnostic gene panels. These yielded a notable rate of pathogenic variants with clear clinical actionability. The study also found that opportunistic testing of MMR and BRCA genes leads to a significant, straightforward identification of MSH6, BRCA1 and BRCA2 mutation carriers. This approach could be applied within a standard genetic counseling framework.
RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that ...represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.
RNA analyses are a potent tool to identify spliceogenic effects of DNA variants, although they are time-consuming and cannot always be performed. We present splicing assays of 20 variants that ...represent a variety of mutation types in 10 hereditary cancer genes and attempt to incorporate these results into American College of Medical Genetics and Genomics (ACMG) classification guidelines. Sixteen single-nucleotide variants, 3 exon duplications, and 1 single-exon deletion were selected and prioritized by in silico algorithms. RNA was extracted from short-term lymphocyte cultures to perform RT-PCR and Sanger sequencing, and allele-specific expression was assessed whenever possible. Aberrant transcripts were detected in 14 variants (70%). Variant interpretation was difficult, especially comparing old classification standards to generic ACMG guidelines and a proposal was devised to weigh functional analyses at RNA level. According to the ACMG guidelines, only 12 variants were reclassified as pathogenic/likely pathogenic because the other two variants did not gather enough evidence. This study highlights the importance of RNA studies to improve variant classification. However, it also indicates the challenge of incorporating these results into generic ACMG guidelines and the need to refine these criteria gene specifically. Nevertheless, 60% of variants were reclassified, thus improving genetic counseling and surveillance for carriers of these variants.
We wanted to implement an NGS strategy to globally analyze hereditary cancer with diagnostic quality while retaining the same degree of understanding and control we had in pre-NGS strategies. To do ...this, we developed the I2HCP panel, a custom bait library covering 122 hereditary cancer genes. We improved bait design, tested different NGS platforms and created a clinically driven custom data analysis pipeline. The I2HCP panel was developed using a training set of hereditary colorectal cancer, hereditary breast and ovarian cancer and neurofibromatosis patients and reached an accuracy, analytical sensitivity and specificity greater than 99%, which was maintained in a validation set. I2HCP changed our diagnostic approach, involving clinicians and a genetic diagnostics team from panel design to reporting. The new strategy improved diagnostic sensitivity, solved uncertain clinical diagnoses and identified mutations in new genes. We assessed the genetic variation in the complete set of hereditary cancer genes, revealing a complex variation landscape that coexists with the disease-causing mutation. We developed, validated and implemented a custom NGS-based strategy for hereditary cancer diagnostics that improved our previous workflows. Additionally, the existence of a rich genetic variation in hereditary cancer genes favors the use of this panel to investigate their role in cancer risk.
CHEK2 variants are associated with intermediate breast cancer risk, among other cancers. We aimed to comprehensively describe CHEK2 variants in a Spanish hereditary cancer (HC) cohort and adjust the ...American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG‐AMP) guidelines for their classification. First, three CHEK2 frequent variants were screened in a retrospective Hereditary Breast and Ovarian Cancer cohort of 516 patients. After, the whole CHEK2 coding region was analyzed by next‐generation sequencing in 1848 prospective patients with HC suspicion. We refined ACMG‐AMP criteria and applied different combined rules to classify CHEK2 variants and define risk alleles. We identified 10 CHEK2 null variants, 6 missense variants with discordant interpretation in ClinVar database, and 35 additional variants of unknown significance. Twelve variants were classified as (likely)‐pathogenic; two can also be considered “established risk‐alleles” and one as “likely risk‐allele.” The prevalence of (likely)‐pathogenic variants in the HC cohort was 0.8% (1.3% in breast cancer patients and 1.0% in hereditary nonpolyposis colorectal cancer patients). Here, we provide ACMG adjustment guidelines to classify CHEK2 variants. We hope that this study would be useful for variant classification of other genes with low effect variants.
Comprehensive analysis of CHEK2 variants found in 2346 hereditary cancer patients and adjustment of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG‐AMP) guidelines for their classification.
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