Evidence-based Network For The Interpretation Of Germline Mutant Alleles Research Articles

Clinical implications of VUS reclassification in a single-centre series from application of ACMG/AMP classification rules specified for BRCA1/2

BackgroundBRCA1/2 testing is crucial to guide clinical decisions in patients with hereditary breast/ovarian cancer, but detection of variants of uncertain significance (VUSs) prevents proper management of carriers. The ENIGMA (Evidence-based...

BRCA1-specific machine learning model predicts variant pathogenicity with high accuracy.

Identification of novel BRCA1 variants outpaces their clinical annotation which highlights the importance of developing accurate computational methods for risk assessment. Therefore our aim was to develop a BRCA1-specific machine learning model to predict the pathogenicity of all types of BRCA1 variants and to apply this model and our previous BRCA2-specific model to assess BRCA variants of uncertain significance (VUS) among Qatari patients with breast cancer. We developed an XGBoost model that utilizes variant information such as position frequency and consequence as well as prediction scores from numerous in silico tools. We trained and tested the model with BRCA1 variants that were reviewed and classified by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. In addition we tested the model's performance on an independent set of missense variants of uncertain significance with experimentally determined functional scores. The model performed excellently in predicting the pathogenicity of ENIGMA-classified variants (accuracy: 99.9%) and in predicting the functional consequence of the independent set of missense variants (accuracy: 93.4%). Moreover it predicted 2 115 potentially pathogenic variants among the 31 058 unreviewed BRCA1 variants in the BRCA exchange database. Using two BRCA-specific models we did not identify any pathogenic BRCA1 variants among those found in patients in Qatar but predicted four potentially pathogenic BRCA2 variants, which could be prioritized for functional validation.

Abstract P6-02-15: Don’t get lost in translation: Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) recommendations for reporting germline cancer susceptibility gene variants in 19 languages – breast cancer as a model

Abstract Genetic testing for cancer susceptibility is a cornerstone of precision cancer prevention and care. Major communication hurdles remain for the differently specialized professionals involved in the identification, counselling, and clinical management of at-risk individuals. This may be ascribed to gaps in the genetic/genomic literacy of health care providers and to an ambiguous lexicon used for variant description. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) international consortium endorses controlled terminology and a framework for interpretation and reporting of germline variants in cancer susceptibility genes (PMID: 30962250). However, for most ENIGMA affiliates a language other than English is used for written and verbal communication of genetic test results, potentially confounding local application of the published framework. The ENIGMA Clinical Working Group thus launched a Vocabulary Translation Project (VTP) to translate the ENIGMA recommendations into the various languages spoken by the membership. The VTP involved 65 ENIGMA members from 22 countries organized into 19 language-specific teams, covering Catalan, Chinese, Czech, Danish, Dutch, Finnish, French, Galician, German, Greek, Italian, Japanese, Malay, Norwegian, Polish, Portuguese, Spanish (Castilian), Swedish, and Tagalog. Excerpts from the original publication were selected for translation based on a majority consensus and included a glossary of terms and recommendations for interpreting and reporting germline sequence variants in (breast) cancer susceptibility genes. Using a two-step process, each team conducted the relevant translation followed by independent back-translation to English. The VTP proved useful to reappraise the reference text. It disclosed transnational issues, which prompted revision of the original source to emphasize that risk estimates and actionability were based on breast cancer as an exemplar. It also highlighted country-specific differences with regards to breast cancer risk assessment (e.g. different absolute/relative breast cancer risk cut points) and management. As a secondary outcome, via electronic survey of the participating teams we documented the perceived high value of the translation effort and its expected positive impact on more consistent clinical management of carrier individuals. The identified target audience encompasses medical geneticists, physicians of other specialties participating in multidisciplinary teams, genetic counselors, primary care physicians, as well as non-health care professionals, e.g. journalists and science communicators. The outreach program includes dissemination of the translations via local, regional, and especially national networks and their use for education and training purposes. Because French, Portuguese, and Spanish are widely used as official, co-official, or secondary languages, the reach of the VTP potentially extends to a greater number of countries and territories, mostly in Central and South America, Caribbean, and Africa. By moving a step forward towards terminological coherence across disciplines and borders, we will facilitate more precise delivery and clinical application of genetic test results for breast cancer predisposition. Our translated recommendations will improve interdisciplinary cross-talk and carriers’ awareness of the risks and implications associated with their status, contributing to more informed decision-making. We used breast cancer as a blueprint. Application of the model to other cancer types will require calibration on the cancer-specific absolute and relative risks. Citation Format: Arcangela De Nicolo, Diana M. Eccles, Sarah Louise Ariansen, Michela Biancolella, Miguel de la Hoya, Orland Diez, Hans Ehrencrona, Florentia Fostira, Tiara Hassan, Issei Imoto, Artur Kowalik, Fabienne Lesueur, Arjen R. Mensenkamp, Heli Nevanlinna, Joanne Ngeow, Edenir I. Palmero, Inge Søkilde Pedersen, Frances Que, Jana Soukupová, Yen Tan, Ana Vega, Amanda B. Spurdle, Paolo Radice. Don’t get lost in translation: Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) recommendations for reporting germline cancer susceptibility gene variants in 19 languages – breast cancer as a model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-02-15.

Gene-specific machine learning model to predict the pathogenicity of BRCA2 variants.

Background: Existing BRCA2-specific variant pathogenicity prediction algorithms focus on the prediction of the functional impact of a subtype of variants alone. General variant effect predictors are applicable to all subtypes, but are trained on putative benign and pathogenic variants and do not account for gene-specific information, such as hotspots of pathogenic variants. Local, gene-specific information have been shown to aid variant pathogenicity prediction; therefore, our aim was to develop a BRCA2-specific machine learning model to predict pathogenicity of all types of BRCA2 variants. Methods: We developed an XGBoost-based machine learning model to predict pathogenicity of BRCA2 variants. The model utilizes general variant information such as position, frequency, and consequence for the canonical BRCA2 transcript, as well as deleteriousness prediction scores from several tools. We trained the model on 80% of the expert reviewed variants by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium and tested its performance on the remaining 20%, as well as on an independent set of variants of uncertain significance with experimentally determined functional scores. Results: The novel gene-specific model predicted the pathogenicity of ENIGMA BRCA2 variants with an accuracy of 99.9%. The model also performed excellently on predicting the functional consequence of the independent set of variants (accuracy was up to 91.3%). Conclusion: This new, gene-specific model is an accurate method for interpreting the pathogenicity of variants in the BRCA2 gene. It is a valuable addition for variant classification and can prioritize unreviewed variants for functional analysis or expert review.

BRCA1/2 NGS Somatic Testing in Clinical Practice: A Short Report.

High-grade serous ovarian carcinoma (HGSOC) is the most common subtype of all ovarian carcinomas. HGSOC harboring BRCA1/2 germline or somatic mutations are sensitive to the poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Therefore, detecting these mutations is crucial to identifying patients for PARPi-targeted treatment. In the clinical setting, next generation sequencing (NGS) has proven to be a reliable diagnostic approach BRCA1/2 molecular evaluation. Here, we review the results of our BRCA1/2 NGS analysis obtained in a year and a half of diagnostic routine practice. BRCA1/2 molecular NGS records of HGSOC patients were retrieved from our institutional archive covering the period from January 2020 to September 2021. NGS analysis was performed on the Ion S5™ System (Thermo Fisher Scientific, Waltham, MA, USA) with the Oncomine™ BRCA Research Assay panel (Thermo Fisher Scientific). Variants were classified as pathogenic or likely pathogenic according to the guidelines of the American College of Medical Genetics and Genomics by using the inspection of Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) and ClinVar (NCBI) databases. Sixty-five HGSOC patient samples were successfully analyzed. Overall, 11 (16.9%) out of 65 cases harbored a pathogenic alteration in BRCA1/2, in particular, six BRCA1 and five BRCA2 pathogenic variations. This study confirms the efficiency and high sensitivity of NGS analysis in detecting BRCA1/2 germline or somatic variations in patients with HGSOC.

BRIDGES, Breast Cancer Risk after Diagnostic Gene Sequencing, HORIZON2020

"Breast cancer affects more than 360,000 women per year in the EU and causes more than 90,000 deaths. Identification of women at high risk of the disease can lead to early detection or disease prevention through intensive screening, therapeutic and/or lifestyle preventive measures, or prophylactic surgery. Breast cancer risk is determined by a combination of genetic and lifestyle risk factors. The advent of next generation sequencing has opened the opportunity for testing in many disease genes, and diagnostic gene panel testing is being introduced in many EU countries. However, the cancer risks associated with most variants in most genes are unknown. This leads to a major problem in appropriate counselling and management of women undergoing panel testing. The BRIDGES and B-CAST projects are jointly building a knowledge base that will allow identification of women at high-risk of specific subtypes of breast cancer, through comprehensive evaluation of DNA variants in known and suspected breast cancer genes. The effort exploits the huge resources established through the Breast Cancer Association Consortium (BCAC) and ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles). Existing datasets will be expanded by sequencing all known breast cancer susceptibility genes in >100,000 breast cancer cases and controls from population-based studies. Risk factor and tumour genome data have been collected for 10,000 cases. Jointly, the data will allow us to generate a comprehensive risk model with unprecedented discriminative power, that can provide personalised risk estimates. We will develop online tools to aid the interpretation of gene variants and provide risk estimates in a user-friendly format, to help genetic counsellors and patients worldwide to make informed clinical decisions for risk management. We will evaluate the acceptability and utility of comprehensive gene panel testing in the clinical genetics context."

BRCA Challenge: BRCA Exchange as a global resource for variants in BRCA1 and BRCA2.

The BRCA Challenge is a long-term data-sharing project initiated within the Global Alliance for Genomics and Health (GA4GH) to aggregate BRCA1 and BRCA2 data to support highly collaborative research activities. Its goal is to generate an informed and current understanding of the impact of genetic variation on cancer risk across the iconic cancer predisposition genes, BRCA1 and BRCA2. Initially, reported variants in BRCA1 and BRCA2 available from public databases were integrated into a single, newly created site, www.brcaexchange.org. The purpose of the BRCA Exchange is to provide the community with a reliable and easily accessible record of variants interpreted for a high-penetrance phenotype. More than 20,000 variants have been aggregated, three times the number found in the next-largest public database at the project’s outset, of which approximately 7,250 have expert classifications. The data set is based on shared information from existing clinical databases—Breast Cancer Information Core (BIC), ClinVar, and the Leiden Open Variation Database (LOVD)—as well as population databases, all linked to a single point of access. The BRCA Challenge has brought together the existing international Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium expert panel, along with expert clinicians, diagnosticians, researchers, and database providers, all with a common goal of advancing our understanding of BRCA1 and BRCA2 variation. Ongoing work includes direct contact with national centers with access to BRCA1 and BRCA2 diagnostic data to encourage data sharing, development of methods suitable for extraction of genetic variation at the level of individual laboratory reports, and engagement with participant communities to enable a more comprehensive understanding of the clinical significance of genetic variation in BRCA1 and BRCA2.

Genetic Testing and Clinical Management Practices for Variants in Non-BRCA1/2 Breast (and Breast/Ovarian) Cancer Susceptibility Genes: An International Survey by the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) Clinical Working Group.

To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non-BRCA1/2 genes. Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six (PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome-associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. Currently, a small number of genes beyond BRCA1/2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

Quantitative Analysis of BRCA1 and BRCA2 Germline Splicing Variants Using a Novel RNA-Massively Parallel Sequencing Assay

Clinical genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming widespread. However, the interpretation of variants of unknown significance (VUS) in HBOC genes, such as the clinically actionable genes BRCA1 and BRCA2, remain a challenge. Among the variants that are frequently classified as VUS are those with unclear effects on splicing. In order to address this issue we developed a high-throughput RNA-massively parallel sequencing assay—CloneSeq—capable to perform quantitative and qualitative analysis of transcripts in cell lines and HBOC patients. This assay is based on cloning of RT-PCR products followed by massive parallel sequencing of the cloned transcripts. To validate this assay we compared it to the RNA splicing assays recommended by members of the ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium. This comparison was performed using well-characterized lymphoblastoid cell lines (LCLs) generated from carriers of the BRCA1 or BRCA2 germline variants that have been previously described to be associated with splicing defects. CloneSeq was able to replicate the ENIGMA results, in addition to providing quantitative characterization of BRCA1 and BRCA2 germline splicing alterations in a high-throughput fashion. Furthermore, CloneSeq was used to analyze blood samples obtained from carriers of BRCA1 or BRCA2 germline sequence variants, including the novel uncharacterized alteration BRCA1 c.5152+5G>T, which was identified in a HBOC family. CloneSeq provided a high-resolution picture of all the transcripts induced by BRCA1 c.5152+5G>T, indicating it results in significant levels of exon skipping. This analysis proved to be important for the classification of BRCA1 c.5152+5G>T as a clinically actionable likely pathogenic variant. Reclassifications such as these are fundamental in order to offer preventive measures, targeted treatment, and pre-symptomatic screening to the correct individuals.

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.

Abstract 1450: Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes

Abstract Genetic testing for hereditary breast and ovarian cancer (HBOC) is becoming increasingly widespread in the era of precision medicine. The implementation of next-generation sequencing (NGS) has resulted in an explosion of genetic data. While the majority of patients receive definitive results, germline unclassified variants with unknown function are regularly detected in thousands of patients. In particular, variants of unknown significance (VUS) in the HBOC susceptibility genes BRCA1 and BRCA2 pose a quandary to medical providers and patients because these genes are clinically actionable. A large percentage of VUS in BRCA1/2 are predicted to affect splicing. Previous efforts have focused on interrogating splicing VUS using low-throughput and/or imprecise techniques. Therefore, we developed and validated a method for using RT-PCR NGS to accurately and efficiently characterize germline splicing defects. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium recommends capillary electrophoresis and Sanger sequencing of subcloned transcripts as the gold standard to identify the BRCA1/2 transcripts that are present in patient samples. We followed the recommendations of the ENIGMA consortium and were able to largely replicate their results when we analyzed the cDNA of lymphoblastoid cell lines (LCLs) generated by the kConFab consortium from carriers of BRCA1 or BRCA2 variants known to be associated with splicing defects. In addition to LCLs, we analyzed RNA extracted from normal blood controls and normal breast tissue for the entire BRCA1 gene. We compared our capillary electrophoresis and Sanger sequencing results to our newly developed RT-PCR NGS assay. For this assay, we used RT-PCR NGS coupled to our custom in-house bioinformatics analysis to identify splicing events including exon skipping, novel intron insertion, and alternative 5’ donor and 3’ acceptor splicing sites. Capillary electrophoresis allowed us to roughly visualize the various transcripts present in our samples, though sequencing was needed to confidently identify the exact splicing event. Similar to Sanger sequencing, our RT-PCR NGS assay detected all major splicing events. Interestingly, we found that RT-PCR NGS allowed us to visualize more minor splicing events present in the samples due to our high coverage. We sequenced tens of thousands of reads with RT-PCR NGS, as opposed to Sanger sequencing, which was limited to low-throughput sequencing of single colonies containing subcloned RT-PCR products. In conclusion, RT-PCR NGS is a reliable high-throughput alternative to the gold-standard splicing assays. Our assay will greatly improve the interpretation of splicing VUS detected by clinical genetic tests for BRCA1/2. Citation Format: Suzette Farber-Katz, Vickie Hsuan, Jayne Hoo, Sitao Wu, Huy Vuong, Dong Xu, Hsiao-Mei Lu, Phillip Gray, Aaron Elliott, Rachid Karam. Alternative splicing analysis identifies mutation hotspots in hereditary breast and ovarian cancer genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1450. doi:10.1158/1538-7445.AM2017-1450

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24 MLH1 and MSH2 Gene Variants.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Abstract 1859: Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes

Abstract Many germline DNA sequence variants in gene regulatory elements are associated with an increased risk of cancer. These include large genomic deletions of the promoter of the BRCA1 gene, through to single nucleotide variants in the promoter, UTRs and long-range enhancers of multiple other cancer related genes. Next generation sequence analysis of early onset and familial breast cancer cases is currently identifying an escalating number of variants in non-coding regions of the genome, however for most of these the significance in unknown. This project aims to determine the functional and clinical significance of breast cancer associated variants in regulatory regions of BRCA1 and BRCA2, as part of an international collaborative project arising from the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium. To date over two hundred variants mapping to promoter, UTR and deep intronic regions have been identified in early onset or familial breast cancer patients that have no identifiable coding or splicing changes in BRCA1 or BRCA2. Through a pipeline of bioinformatics analyses using ENCODE datasets, these variants have been prioritized for experimental evaluation. This has lead to the identification of several variants that significantly alter the regulation of BRCA1 or BRCA2 expression, through mechanisms including altered binding of transcription factors and microRNAs. These studies will lay the groundwork for comprehensive statistical analyses, in which bioinformatics and experimental data is combined with clinical and genetic data to establish multifactorial risk prediction models that can ultimately be used in a clinical setting. Citation Format: Jan Sevcik, Leslie Burke, Gaetana Gambino, Brooke L. Brewster, Emma Tudini, Philip J. Whiley, Siranoush Manoukian, ENIGMA Consortium, Thomas van Overeem Hansen, Marta Santamariña Pena, Ana Vega, Maria A. Caligo, Paolo Radice, Paolo Peterlongo, Etienne Rouleau, Amanda B. Spurdle, Melissa A. Brown. Bioinformatic and experimental evaluation of regulatory variants in breast cancer susceptibility genes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1859.

Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

BackgroundBRCA1 and BRCA2 are the two principal tumour suppressor genes associated with inherited high risk of breast and ovarian cancer. Genetic testing of BRCA1/2 will often reveal one or more...

Comparison of mRNA Splicing Assay Protocols across Multiple Laboratories: Recommendations for Best Practice in Standardized Clinical Testing

Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

Variants of Uncertain Significance in BRCA1 and BRCA2 assessment of in silico analysis and a proposal for communication in genetic counselling

BackgroundNearly 15% of BRCA1 and BRCA2 DNA tests lead to the identification of Variants of Uncertain Significance (VUS). VUS are classified in the Netherlands according to the Bell system and...

A multi-centre international quality control study comparing mRNA splicing assay protocols and reporting practices from the ENIGMA consortium

Classification of intronic and predicted missense changes in the breast cancer susceptibility genes BRCA1 and BRCA2 remains a significant challenge for management of patients carrying these variants. Defective mRNA splicing is established as a pathway to disease, and mRNA analysis of unclassified variants has been shown to assist in classification and genetic counselling. However the interpretation of splicing assay results can be difficult, particularly for those variants that give rise to aberrations in a background of naturally occurring isoforms. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) consortium was set up to facilitate research and improve research methods used to classify rare variants in the BRCA1 and BRCA2 (and potentially other) breast cancer predisposition genes. ENIGMA has established a Splicing Working Group, with stated purpose to pool the expertise of different active research groups to conduct large-scale studies that improve the clinical classification of likely spliceogenic variants. An initial project of the Splicing Working Group is to assess the consistency of protocols and results obtained across the multiple participating laboratories from Australia, Europe, UK and the USA. A comparison of mRNA assay protocols in use across 21 labs has identified differences in source material for RNA assays (cultured and uncultured lymphocytes, lymphoblastoid cell lines (LCLs) or constructs), differential use of nonsense-mediated decay inhibitors, and numerous differences in mRNA extraction, DNase treatment and cDNA synthesis methods. A second phase of the project is now underway to determine the impact of the splicing assay methods routinely used by these laboratories on assay data and clinical interpretation of a panel of variants. LCLs were selected from the kConFab repository from carriers of a variant associated with single major aberrant mRNA transcript absent in controls (n=4); carriers of a variant associated with a complicated aberrant mRNA splicing profile involving multiple transcripts including naturally occurring isoforms (n=5); female cancer-free controls (n=11). LCLs have already been distributed to 15 of 20 participating sites, and mRNA assays are underway. Preliminary results indicate that major aberrations associated with several variants mirror results previously observed for mRNA from uncultured lymphocytes. In addition, there is evidence for notable differences in expression of some isoforms compared to results previously observed for RNA from uncultured lymphocytes . This collaborative effort will provide information to inform optimal standardised mRNA splicing assay methodology, and to improve guidelines for clinical interpretation of assay results.

Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members

Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.