BioTechniquesVol. 67, No. 3 Letter to the EditorOpen AccessCaution: Plasmid DNA topology affects luciferase assay reproducibility and outcomesEmma Tudini‡, Lez J Burke‡, Phillip J Whiley, Jan Sevcik, Amanda B Spurdle & Melissa A BrownEmma Tudini‡School of Chemistry & Molecular Biosciences, The University of Queensland, St Lucia, 4067 QLD, AustraliaDepartment of Genetics & Computational Biology, QIMR Berghofer Medical Research Institute, Herston, 4006 QLD, Australia‡These authors contributed equallySearch for more papers by this author, Lez J Burke‡School of Chemistry & Molecular Biosciences, The University of Queensland, St Lucia, 4067 QLD, Australia‡These authors contributed equallySearch for more papers by this author, Phillip J WhileySchool of Chemistry & Molecular Biosciences, The University of Queensland, St Lucia, 4067 QLD, AustraliaDepartment of Genetics & Computational Biology, QIMR Berghofer Medical Research Institute, Herston, 4006 QLD, AustraliaSearch for more papers by this author, Jan SevcikSchool of Chemistry & Molecular Biosciences, The University of Queensland, St Lucia, 4067 QLD, AustraliaInstitute of Biochemistry and Experimental Oncology, First Faculty of Medicine, Charles University, Prague, Czech RepublicSearch for more papers by this author, Amanda B SpurdleDepartment of Genetics & Computational Biology, QIMR Berghofer Medical Research Institute, Herston, 4006 QLD, AustraliaSearch for more papers by this author & Melissa A Brown*Author for correspondence: E-mail Address: melissa.brown@uq.edu.auSchool of Chemistry & Molecular Biosciences, The University of Queensland, St Lucia, 4067 QLD, AustraliaSearch for more papers by this authorPublished Online:1 Aug 2019https://doi.org/10.2144/btn-2019-0060AboutSectionsView ArticleSupplemental MaterialPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit View article Keywords: assayscell culturegene expression analysisin vitro modelstransfectionTransfection of cell lines with purified plasmid DNA is a well-established technique for analysis of gene and protein function, and gene regulation [1]. To ensure reproducibility and sensitivity of transfections, it is essential that assays are well controlled and reproducible. This is achieved by optimizing a number of factors, including the ratio of plasmid DNA and transfection reagents, the number of cells transfected, and the choice of cell line and transfection reagent. In addition, multiple biological and technical replicates and robust statistical analyses are crucial.With the use of next-generation sequencing to identify genetic changes underlying disease risk, the number of potential disease-associated variants residing in noncoding regions of the genome has increased dramatically. Determining the functional and clinical significance of such variants is important in order to evaluate the risk such variants pose to variant carriers and provide accurate genetic counselling. Currently, in vitro reporter assays present the most commonly used analysis method for assessing activity of gene regulatory regions, whereby a regulatory element drives the expression of a reporter gene such as luciferase. The influence of particular DNA sequence variants on this regulatory activity can then be determined. Given that many variants that contribute to disease susceptibility have a subtle effect on gene regulation, it is essential that experimental assays are robust and reproducible. Here we show that results of a reporter assay commonly used for this purpose is significantly impacted by the topology of reporter plasmid DNA.Single-nucleotide DNA sequence variants within the transcriptional promoter of the BRCA1 gene (hg19; chr17:41277186–41278132 plus chr17:41276114–41276753) were analyzed for an effect on regulatory activity, using the pGL3 luciferase reporter system (Promega, WI, USA) [2]. To enable evaluation of the effect of the variants, an internal control (pGL3-promoter; Promega) was analyzed simultaneously. The internal control, a wild-type (WT) reporter plasmid, and reporter plasmids containing the variants, were prepared simultaneously and DNA concentration was determined by Nanodrop (ThermoFisher MA, USA). MCF7 breast cancer-derived cells seeded in 24-well plates were transfected with both the reporter plasmid and a transfection control plasmid containing a Renilla reporter sequence (pRL-TK; Promega) using Lipofectamine 3000 (Life Technologies, CA, USA). Luciferase activity was measured after 48 h [3] and relative activities of the variant BRCA1 promoters were compared to the activity of the WT BRCA1 promoter. Biological replicates were generated using independent batches of cells cultured and transfected at separate times with independently prepared sets of plasmids. All reporter luciferase activities were normalized to Renilla activity, and subsequently to the internal control, which allowed calculation of the variance of the WT reporter. Technical replicates were averaged and treated as a single biological replicate, and statistics performed on the biological replicates [2].During these analyses, we noticed considerable variability in our reporter assay data (Figure 1A). Analysis of the purified plasmid DNA on agarose gels revealed differently migrating DNA species. The electrophoretic properties of the plasmids were consistent with well-established topologies of plasmid DNA, specifically supercoiled DNA (fast migrating) and nonsupercoiled DNA (slow migrating) [4,5,6].Figure 1. Plasmid topology can influence luciferase activity in transient transfections.(A) Gel electrophoresis (left panel) and luciferase activity in MCF7 cells (right panel) of four independent preparations (set 1–4) of plasmid DNA (pGL3-basic backbone) containing an active portion of the BRCA1 promoter driving the luciferase gene with the WT promoter or single nucleotide variants (Var 1–6). Plasmids (200 ng), prepared using Qiagen miniprep kits, were separated on 1% TAE agarose gels. Migration position of plasmids that are SC and NSC is indicated. MW: 1 kb DNA ladder (Bioline, TN, USA). Luciferase activity is shown relative to the activity of the SC WT plasmid. The horizontal dotted line represents relative WT promoter activity set at 1.0-fold. Data represent the average ± standard deviation of four independent transfections of independently plated MCF7 cells using plasmids sets prepared at different times. Data are shown from plasmids that are SC (black bars), NSC (striped bars) or a mixture of topologies (checked bars). (B & C) Gel electrophoresis of representative independent plasmid preparations (left panel) and associated luciferase activity (right panel) for SC WT and single-nucleotide variant-containing plasmids that were classified as SC (B) or NSC (C). Relative luciferase activity was compared with the WT SC plasmid using an ANOVA followed by Tukey's post-hoc test in GraphPad Prism.*p < 0.05; ** p < 0.01; *** p < 0.005.MW: Molecular weight; NSC: Not supercoiled; SC: Supercoiled; WT: Wild-type.To analyze the effect of differences in the topology of plasmid DNA on luciferase reporter activity, multiple sets of plasmids were purified for a number of different reporter constructs, each containing different variants in the BRCA1 promoter. Supercoiled and nonsupercoiled versions of each plasmid were then used to transfect cells, and luciferase activity was determined relative to a supercoiled control plasmid, purified at the same time. When all variant-containing plasmids were supercoiled, none of the variants had an effect on the reporter activity relative to WT (Figure 1B). However, when plasmids that were not supercoiled were compared to the supercoiled WT reporter, four of the variants displayed a significant effect on reporter activity (Figure 1C). Thus, plasmid topology has a significant impact on reporter activity and leads to over-reporting of an impact on regulatory activity.These results are consistent with previous reports showing that the conformation of plasmids impacts the results of other cell-based assays [4,5,6]. As such, it is imperative to ensure that the topology of the transfected plasmid DNA is consistent before interpreting the differences in luciferase activity for different nucleotide sequences. Failure to do so may result in functional activity being undetectable in the case of activating variants, or interpreted as having a significant effect where the variant, in fact, has no effect on reporter activity.The mechanism underlying the impact of plasmid topology on reporter activity is unknown. Comparison of DNA concentration by absorbance at 260 nm and electrophoresis on agarose gels (Figure 1) suggests that is it not caused by changes in the measurable amount of DNA. In addition, all plasmid DNA preparations, regardless of topology, were of similar purity (as determined by Nanodrop) and contained no protein and/or other contaminants as indicated by A260/A280 or A260/A230 ratios, that would contribute to the observed results. A more plausible hypothesis is that DNA topology affects transfection and expression of the plasmid. Consistent with this, previous studies have shown that supercoiled plasmids drive higher levels of expression and thus the activity of expressed products, using other transfection methods such as DEAE dextran [4], liposomal DOTAP/DOPE complexes [5] and cationic polymers [6]. A potential mechanism for this may be that nonsupercoiled plasmids have a reduced transfection rate impacting the availability of plasmid DNA for transcription. While we have shown the relevance of plasmid DNA topology for luciferase assays, it would be prudent to consider the impact of plasmid topology for any cell-based functional assays that rely on transfection. Based on this, we recommend that only plasmids that have been confirmed to have a supercoiled topology be used in transfection assays.The conformation of the plasmid DNA is often retained after transformation and repurification; that is, a nonsupercoiled plasmid DNA will give rise to predominantly nonsupercoiled plasmid DNA after repreparation. This hinders purification of supercoiled plasmids from preparations that are not supercoiled. Here we show that digestion of a nonsupercoiled plasmid containing a 950-base pair BRCA1 promoter (hg19; chr17:41277186–41278132) insert with a restriction enzyme that cuts once in the plasmid and religation, results in purification of supercoiled plasmids in 10/11 cases (Figure 2). This procedure allows purification of supercoiled plasmids from plasmids that were previously not supercoiled, and thus for experiments to be controlled for plasmid topology.Figure 2. Supercoiled conformation can be restored by digestion and religation.A plasmid (NSC) containing an active portion of the BRCA1 promoter was digested with SalI, ligated and used to transform Escherichia coli from which plasmid DNA was repurified. Agarose gel showing original plasmid (200 ng) that is not SC (original) and 1 μl of plasmid minipreps after digestion, ligation and retransformation. Migration position of plasmids that are SC and NSC is indicated, showing restoration on 10/11 occasions. MW: 1 kb DNA ladder (Bioline, TN, USA).MW: Molecular weight; NSC: Not supercoiled; SC: Supercoiled.In summary, we have identified another factor that can affect the outcome of luciferase reporter assays of sequence variants mapping to gene regulatory regions. We recommend routine assessment of plasmid topology and the use of only plasmids that are supercoiled for transfection. Good laboratory practice would further suggest that all reporter plasmids to be assayed are prepared simultaneously, that test and control plasmids are analyzed on the same plate, and that a minimum of three technical and three biological replicates are performed for each experiment.Author contributionsET, LJB, ABS and MAB conceived and designed the study. ET, LJB, JS and PJW performed the experiments. ET, LJB, JS, ABS and MAB wrote and revised the manuscript.Financial & completing interests disclosureThis work was supported by grants from the National Health and Medical Research Council (ID1104808) and Cancer Council Queensland (ID1044008 and ID1026095) to MA Brown. and the Ministry of Health of the Czech Republic (AZV 16–33444A) to J Sevcik. AB Spurdle is supported by an NHMRC Senior Research Fellowship (ID1061779). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Ethical conduct of researchThe authors state that they have obtained Institutional Biosafety committee (IBC) approval to undertake the described experiments.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/References1. Kim TK, Eberwine JH. Mammalian cell transfection: the present and the future. Anal. Bioanal. Chem. 397(8), 3173–3178 (2010).Crossref, Medline, CAS, Google Scholar2. Burke LJ, Sevcik J, Gambino G et al. BRCA1 and BRCA2 5′ noncoding region variants identified in breast cancer patients alter promoter activity and protein binding. Hum. Mutat. 39(12), 2025–2039 (2018).Crossref, Medline, CAS, Google Scholar3. Brewster BL, Rossiello F, French JD et al. Identification of fifteen novel germline variants in the BRCA1 3′UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site. Hum. Mutat. 33(12), 1665–1675 (2012).Crossref, Medline, CAS, Google Scholar4. Weintraub H, Cheng PF, Conrad K. Expression of transfected DNA depends on DNA topology. Cell 46(1), 115–122 (1986).Crossref, Medline, CAS, Google Scholar5. Remaut K, Sanders NN, Fayazpour F, Demeester J, De Smedt SC. Influence of plasmid DNA topology on the transfection properties of DOTAP/DOPE lipoplexes. J. Control Release 115(3), 335–343 (2006).Crossref, Medline, CAS, Google Scholar6. Cherng JY, Schuurmans-Nieuwenbroek NM, Jiskoot W et al. Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes. J. Control Release 60(2–3), 343–353 (1999).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByLipid‐based nucleic acid therapeutics with in vivo efficacy30 September 2022 | WIREs Nanomedicine and Nanobiotechnology, Vol. 2Plasmid multimer status and not DNA topology likely affects luciferase assay reproducibilityMichael G Jobling12 June 2020 | BioTechniques, Vol. 69, No. 3Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!21 February 2020 | Pharmaceutics, Vol. 12, No. 2 Vol. 67, No. 3 Follow us on social media for the latest updates Supplemental MaterialsMetrics History Received 15 May 2019 Accepted 17 June 2019 Published online 1 August 2019 Published in print September 2019 Information© 2019 Melissa BrownKeywordsassayscell culturegene expression analysisin vitro modelstransfectionAuthor contributionsET, LJB, ABS and MAB conceived and designed the study. ET, LJB, JS and PJW performed the experiments. ET, LJB, JS, ABS and MAB wrote and revised the manuscript.Financial & completing interests disclosureThis work was supported by grants from the National Health and Medical Research Council (ID1104808) and Cancer Council Queensland (ID1044008 and ID1026095) to MA Brown. and the Ministry of Health of the Czech Republic (AZV 16–33444A) to J Sevcik. AB Spurdle is supported by an NHMRC Senior Research Fellowship (ID1061779). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.Ethical conduct of researchThe authors state that they have obtained Institutional Biosafety committee (IBC) approval to undertake the described experiments.Open accessThis work is licensed under the Attribution-NonCommercial-NoDerivatives 4.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/PDF download

The widespread use of next generation sequencing for clinical testing is detecting an escalating number of variants in noncoding regions of the genome. The clinical significance of the majority of these variants is currently unknown, which presents a significant clinical challenge. We have screened over 6,000 early-onset and/or familial breast cancer (BC) cases collected by the ENIGMA consortium for sequence variants in the 5' noncoding regions of BC susceptibility genes BRCA1 and BRCA2, and identified 141 rare variants with global minor allele frequency < 0.01, 76 of which have not been reported previously. Bioinformatic analysis identified a set of 21 variants most likely to impact transcriptional regulation, and luciferase reporter assays detected altered promoter activity for four of these variants. Electrophoretic mobility shift assays demonstrated that three of these altered the binding of proteins to the respective BRCA1 or BRCA2 promoter regions, including NFYA binding to BRCA1:c.-287C>T and PAX5 binding to BRCA2:c.-296C>T. Clinical classification of variants affecting promoter activity, using existing prediction models, found no evidence to suggest that these variants confer a high risk of disease. Further studies are required to determine if such variation may be associated with a moderate or low risk of BC.

BACKGROUND:BRCA1 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 sequence variants of uncertain clinical significance, some of which may affect normal splicing patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation or defects in gene function. To understand which novel splicing events are associated with splicing mutations and which are part of the normal BRCA2 splicing repertoire, a study was undertaken by members of the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events.METHODS:mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary or agarose gel electrophoresis, followed by sequencing.RESULTS:We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines.CONCLUSIONS:These naturally occurring alternate-splicing events contribute to the array of cDNA fragments that may be seen in assays for mutation-associated splicing defects. Caution must be observed in assigning alternate-splicing events to potential splicing mutations.

Variants that disrupt the translation initiation sequences in cancer predisposition genes are generally assumed to be deleterious. However, few studies have validated these assumptions with functional and clinical data. Two cancer syndrome gene variants likely to affect native translation initiation were identified by clinical genetic testing: MLH1:c.1A>G p.(Met1?) and BRCA2:c.67+3A>G. In vitro GFP-reporter assays were conducted to assess the consequences of translation initiation disruption on alternative downstream initiation codon usage. Analysis of MLH1:c.1A>G p.(Met1?) showed that translation was mostly initiated at an in-frame position 103 nucleotides downstream, but also at two ATG sequences downstream. The protein product encoded by the in-frame transcript initiating from position c.103 showed loss of in vitro mismatch repair activity comparable to known pathogenic mutations. BRCA2:c.67+3A>G was shown by mRNA analysis to result in an aberrantly spliced transcript deleting exon 2 and the consensus ATG site. In the absence of exon 2, translation initiated mostly at an out-of-frame ATG 323 nucleotides downstream, and to a lesser extent at an in-frame ATG 370 nucleotides downstream. Initiation from any of the downstream alternative sites tested in both genes would lead to loss of protein function, but further clinical data is required to confirm if these variants are associated with a high cancer risk. Importantly, our results highlight the need for caution in interpreting the functional and clinical consequences of variation that leads to disruption of the initiation codon, since translation may not necessarily occur from the first downstream alternative start site, or from a single alternative start site.

Background Inactivating germline mutations in the tumour suppressor gene BRCA1 are associated with a significantly increased risk of developing breast and ovarian cancer. A large number (>1500) of unique BRCA1 variants have been identified in the population and can be classified as pathogenic, non-pathogenic or as variants of unknown significance (VUS). Many VUS are rare missense variants leading to single amino acid changes. Their impact on protein function cannot be directly inferred from sequence information, precluding assessment of their pathogenicity. Thus, functional assays are critical to assess the impact of these VUS on protein activity. BRCA1 is a multifunctional protein and different assays have been used to assess the impact of variants on different biochemical activities and biological processes. Methods and results To facilitate VUS analysis, we have developed a visualisation resource that compiles and displays functional data on all documented BRCA1 missense variants. BRCA1 Circos is a web-based visualisation tool based on the freely available Circos software package. The BRCA1 Circos web tool (http://research.nhgri.nih.gov/bic/circos/) aggregates data from all published BRCA1 missense variants for functional studies, harmonises their results and presents various functionalities to search and interpret individual-level functional information for each BRCA1 missense variant. Conclusions This research visualisation tool will serve as a quick one-stop publically available reference for all the BRCA1 missense variants that have been functionally assessed. It will facilitate meta-analysis of functional data and improve assessment of pathogenicity of VUS.

Loss-of-function germline mutations in BRCA1 (MIM #113705) confer markedly increased risk of breast and ovarian cancer. The full-length transcript codifies for a protein involved in DNA repair pathways and cell-cycle checkpoints. Several BRCA1 splicing isoforms have been described in public domain databases, but the physiological role (if any) of BRCA1 alternative splicing remains to be established. An accurate description of 'naturally occurring' alternative splicing at this locus is a prerequisite to understand its biological significance. However, a systematic analysis of alternative splicing at the BRCA1 locus is yet to be conducted. Here, the Evidence-Based Network for the Interpretation of Germ-Line Mutant Alleles consortium combines RT-PCR, exon scanning, cloning, sequencing and relative semi-quantification to describe naturally occurring BRCA1 alternative splicing with unprecedented resolution. The study has been conducted in blood-related RNA sources, commonly used for clinical splicing assays, as well as in one healthy breast tissue. We have characterized a total of 63 BRCA1 alternative splicing events, including 35 novel findings. A minimum of 10 splicing events (Δ1Aq, Δ5, Δ5q, Δ8p, Δ9, Δ(9,10), Δ9_11, Δ11q, Δ13p and Δ14p) represent a substantial fraction of the full-length expression level (ranging from 5 to 100%). Remarkably, our data indicate that BRCA1 alternative splicing is similar in blood and breast, a finding supporting the clinical relevance of blood-based in vitro splicing assays. Overall, our data suggest an alternative splicing model in which most non-mutually exclusive alternative splicing events are randomly combined into individual mRNA molecules to produce hundreds of different BRCA1 isoforms.

Rare exonic, non-truncating variants in known cancer susceptibility genes such as BRCA1 and BRCA2 are problematic for genetic counseling and clinical management of relevant families. This study used multifactorial likelihood analysis and/or bioinformatically-directed mRNA assays to assess pathogenicity of 19 BRCA1 or BRCA2 variants identified following patient referral to clinical genetic services. Two variants were considered to be pathogenic (Class 5). BRCA1:c.4484G> C(p.Arg1495Thr) was shown to result in aberrant mRNA transcripts predicted to encode truncated proteins. The BRCA1:c.122A>G(p.His41Arg) RING-domain variant was found from multifactorial likelihood analysis to have a posterior probability of pathogenicity of 0.995, a result consistent with existing protein functional assay data indicating lost BARD1 binding and ubiquitin ligase activity. Of the remaining variants, seven were determined to be not clinically significant (Class 1), nine were likely not pathogenic (Class 2), and one was uncertain (Class 3).These results have implications for genetic counseling and medical management of families carrying these specific variants. They also provide additional multifactorial likelihood variant classifications as reference to evaluate the sensitivity and specificity of bioinformatic prediction tools and/or functional assay data in future studies.

Splicing assays are commonly undertaken in the clinical setting to assess the clinical relevance of sequence variants in disease predisposition genes. A 5-tier classification system incorporating both bioinformatic and splicing assay information was previously proposed as a method to provide consistent clinical classification of such variants. Members of the ENIGMA Consortium Splicing Working Group undertook a study to assess the applicability of the scheme to published assay results, and the consistency of classifications across multiple reviewers. Splicing assay data were identified for 235 BRCA1 and 176 BRCA2 unique variants, from 77 publications. At least six independent reviewers from research and/or clinical settings comprehensively examined splicing assay methods and data reported for 22 variant assays of 21 variants in four publications, and classified the variants using the 5-tier classification scheme. Inconsistencies in variant classification occurred between reviewers for 17 of the variant assays. These could be attributed to a combination of ambiguity in presentation of the classification criteria, differences in interpretation of the data provided, nonstandardized reporting of results, and the lack of quantitative data for the aberrant transcripts. We propose suggestions for minimum reporting guidelines for splicing assays, and improvements to the 5-tier splicing classification system to allow future evaluation of its performance as a clinical tool.

BACKGROUND 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. METHODS 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. RESULTS 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). CONCLUSIONS We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

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.

BACKGROUND:Clinical classification of rare sequence changes identified in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. We previously showed that variant BRCA1 c.5096G>A p.Arg1699Gln in the BRCA1 transcriptional transactivation domain demonstrated equivocal results from a series of functional assays, and proposed that this variant may confer low to moderate risk of cancer. METHODS:Measures of genetic risk (report of family history, segregation) were assessed for 68 BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) families recruited through family cancer clinics, comparing results with 34 families carrying the previously classified pathogenic BRCA1 c.5095C>T p.Arg1699Trp (R1699W) mutation at the same residue, and to 243 breast cancer families with no BRCA1 pathogenic mutation (BRCA-X). RESULTS:Comparison of BRCA1 carrier prediction scores of probands using the BOADICEA risk prediction tool revealed that BRCA1 c.5096G>A p.Arg1699Gln variant carriers had family histories that were less 'BRCA1-like' than BRCA1 c.5095C>T p.Arg1699Trp mutation carriers (p<0.00001), but more 'BRCA1-like' than BRCA-X families (p=0.0004). Further, modified segregation analysis of the subset of 30 families with additional genotyping showed that BRCA1 c.5096G >A p.Arg1699Gln had reduced penetrance compared with the average truncating BRCA1 mutation penetrance (p=0.0002), with estimated cumulative risks to age 70 of breast or ovarian cancer of 24%. CONCLUSIONS:Our results provide substantial evidence that the BRCA1 c.5096G>A p.Arg1699Gln (R1699Q) variant, demonstrating ambiguous functional deficiency across multiple assays, is associated with intermediate risk of breast and ovarian cancer, highlighting challenges for risk modelling and clinical management of patients of this and other potential moderate-risk variants.

Clinical management of breast cancer families is complicated by identification of BRCA1 and BRCA2 sequence alterations of unknown significance. Molecular assays evaluating the effect of intronic variants on native splicing can help determine their clinical relevance. Twenty‐six intronic BRCA1/2 variants ranging from the consensus dinucleotides in the splice acceptor or donor to 53 nucleotides into the intron were identified in multiple‐case families. The effect of the variants on splicing was assessed using HSF matrices, MaxEntScan and NNsplice, followed by analysis of mRNA from lymphoblastoid cell lines. A total of 12 variants were associated with splicing aberrations predicted to result in production of truncated proteins, including a variant located 12 nucleotides into the intron. The posterior probability of pathogenicity was estimated using a multifactorial likelihood approach, and provided a pathogenic or likely pathogenic classification for seven of the 12 spliceogenic variants. The apparent disparity between experimental evidence and the multifactorial predictions is likely due to several factors, including a paucity of likelihood information and a nonspecific prior probability applied for intronic variants outside the consensus dinucleotides. Development of prior probabilities of pathogenicity incorporating bioinformatic prediction of splicing aberrations should improve identification of functionally relevant variants and enhance multifactorial likelihood analysis of intronic variants. Hum Mutat 32:1–10, 2011. © 2011 Wiley‐Liss, Inc.

Missense substitutions in high-risk cancer susceptibility genes create clinical uncertainty in the genetic counseling process. Multifactorial likelihood classification approaches and in vitro assays are useful for the classification of exonic sequence variants in BRCA1 and BRCA2, but these currently rely on the assumption that changes in protein function are the major biological mechanism of pathogenicity. This study investigates the potentially pathogenic role of aberrant splicing for exonic variants predicted to encode missense substitutions using patient-derived RNA. No splicing aberrations were identified for BRCA1c.5054C>T and BRCA2c.7336A>G, c.8839G>A, and c.9154C>T. However, RT-PCR analysis identified a major splicing aberration for BRCA1c.4868C>G(p.Ala1623Gly), a variant encoding a missense substitution considered likely to be neutral. Splicing aberrations were also observed for BRCA2c.7988A>T(p.Glu2663Val) and c.8168A>G(p.Asp2723Gly), but both variant and wildtype alleles were shown to be present in full-length mRNA transcripts, suggesting that variant protein may be translated. BRCA2 protein function assays indicated that BRCA2p.Glu2663Val, p.Asp2723Gly and p.Arg3052Trp missense proteins have abrogated function consistent with pathogenicity. Multifactorial likelihood analysis provided evidence for pathogenicity for BRCA1 c.5054C>T(p.Thr1685Ile) and BRCA2c.7988A>T(p.Glu2663Val), c.8168A>G(p.Asp2723Gly) and c.9154C>T(p.Arg3052Trp), supporting experimentally derived evidence. These findings highlight the need for improved bioinformatic prediction of splicing aberrations and to refine multifactorial likelihood models used to assess clinical significance. (C) 2010 Wiley-Liss, Inc.

BACKGROUND:Genetic screening of breast cancer patients and their families have identified a number of variants of unknown clinical significance in the breast cancer susceptibility genes, BRCA1 and BRCA2. Evaluation of such unclassified variants may be assisted by web-based bioinformatic prediction tools, although accurate prediction of aberrant splicing by unclassified variants affecting exonic splice enhancers (ESEs) remains a challenge.METHODS:This study used a combination of RT-PCR analysis and splicing reporter minigene assays to assess five unclassified variants in the BRCA2 gene that we had previously predicted to disrupt an ESE using bioinformatic approaches.RESULTS:Analysis of BRCA2 c.8308 G > A (p.Ala2770Thr) by mRNA analysis, and BRCA2 c.8962A > G (p.Ser2988Gly), BRCA2 c.8972G > A (p.Arg2991His), BRCA2 c.9172A > G (p.Ser3058Gly), and BRCA2 c.9213G > T (p.Glu3071Asp) by a minigene assay, revealed no evidence for aberrant splicing.CONCLUSIONS:These results illustrate the need for improved methods for predicting functional ESEs and the potential consequences of sequence variants contained therein.

Classification of intronic and predicted missense sequence variants in the breast cancer susceptibility genes BRCA1 and BRCA2 is essential for appropriate genetic counselling of individuals carrying these variants. However, it remains a significant challenge to determine whether such sequence changes alter gene function sufficiently to predispose the individual to breast cancer. Unclassified variants located in the splice site recognition sequences and intronic or exonic regulatory sequences may disrupt messenger RNA splicing and lead to an imbalance of alternately spliced transcripts, impaired protein function or complete protein loss. To date in silico prediction algorithms have limited capacity to accurately predict the effect of variants on splicing, in particular the use of cryptic splice sites after disruption of donor and acceptor sites, and also the creation of de novo splice sites. We are conducting ongoing studies assessing the clinical significance of BRCA1/2 sequence variants identified within families from the Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab), using a multifactorial likelihood modelling approach. In addition, mRNA splicing analysis by reverse transcriptase PCR and sequencing is being conducted for the subset of variant carriers with available lymphoblastoid cell lines (LCLs), using a large panel of controls to establish the reference RNA splicing pattern. Functional analyses have also been conducted for a subset of the variants that encode missense substitutions. In vitro mRNA analysis identified aberrant splicing patterns for 8/12 variants under current study: BRCA1 IVS 19-12 G>A and 14+2 ins8 cause out of frame insertions. BRCA1 IVS 23+5 G>C, BRCA2 IVS 6+1 G>T, BRCA2 IVS 25+3, BRCA2 IVS 4-12_IVS 4-8 del5 and BRCA2 8396 A>G (D2723G) create whole or part exon deletions. BRCA2 8216 A>T (E2663V) causes upregulation of truncating splice isoforms. However, sequencing and realtime expression analysis indicates that the wildtype isoform expressed in carriers of BRCA2 8396 A>G ( D2723G) and 8216 A>T (E2663V) would encode both wildtype and missense protein. Assays of protein function indicate that both of these missense proteins have abrogated function consistent with pathogenicity. These results have implications for the application of prior probabilities in multifactorial likelihood analysis. Importantly, the results reinforce the utility of splicing assays as a method for identifying clinically significant variants in high-risk cancer genes, including variants outside the conserved donor and acceptor dinucleotides which are not routinely tested in clinical laboratories. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-252.