Resolving the Functional Significance of BRCA1 RING Domain Missense Substitutions
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Abstract
Part 1
Development and calibration of suitably accurate functional assays for BRCA1 RING domain and BRCT domain missense substitutions could dramatically accelerate clinical classification of rare missense substitutions observed in that gene. Leveraging data from 68,000 full sequence tests of BRCA1 and BRCA2 , plus data from the limited number of already classified BRCA1 RING domain missense substitutions, we used logistic regression and related techniques to evaluate three BRCA1 RING domain assays. These were recently described high throughput yeast 2-hybrid and E3 ubiquitin ligase assays, plus a newly developed mammalian 2-hybrid assay. While there were concerns about the accuracy of the yeast 2-hybrid assay and the indirect nature of the ubiquitin ligase assay, the mammalian 2-hybrid assay had excellent correlation with existing missense substitution classifications. After calibration, this assay contributed to classification of one newly reported BRCA1 missense substitution. In principal, the mammalian 2-hybrid assay could be converted to a high-throughput format that would likely retain suitable accuracy.
Part 2
How does one achieve clinically applicable classification of the vast majority of all possible sequence variants in disease susceptibility genes? BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a Mammalian 2-hybrid (M2H) assay. Downstream of the M2H laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that about 20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of about 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 47 with concordant computational and functional assay evidence in favor of pathogenicity; these are particularly likely to be classified as Likely Pathogenic once human observational data become available.
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SciScore for 10.1101/092619: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Cell lysates were harvested in phospho-protecting lysis buffer (PPLB) 48 hours post-transfection and immunoprecipitation performed from clarified cell lysates using α-FLAG (M2) antibody and Protein G Sepharose beads (Engel et al., 2010). α-FLAGsuggested: NoneM2suggested: NoneExperimental Models: Cell Lines Sentences Resources 1-by-1 mammalian 2-hybrid assay: Co-Immunoprecipitation: HEK293 cells were transfected using Polyethylenimine (PEI) (Polysciences Incorporated). HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Recombinant DNA Sentences Resources 18-24 hours later, 2.5 μg of wt BARD1 (pACT_BARD1 1-200) … SciScore for 10.1101/092619: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
NIH rigor criteria are not applicable to paper type.Table 2: Resources
Antibodies Sentences Resources Cell lysates were harvested in phospho-protecting lysis buffer (PPLB) 48 hours post-transfection and immunoprecipitation performed from clarified cell lysates using α-FLAG (M2) antibody and Protein G Sepharose beads (Engel et al., 2010). α-FLAGsuggested: NoneM2suggested: NoneExperimental Models: Cell Lines Sentences Resources 1-by-1 mammalian 2-hybrid assay: Co-Immunoprecipitation: HEK293 cells were transfected using Polyethylenimine (PEI) (Polysciences Incorporated). HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Recombinant DNA Sentences Resources 18-24 hours later, 2.5 μg of wt BARD1 (pACT_BARD1 1-200) or an empty vector (EV), and either wt BRCA1 or various BRCA1 RING missense substitutions (pBIND_BRCA1 1-184) were co-transfected with 500 ng of pBIG (GFP control plasmid), with PEI (1 mg/ml) being used at a 2:1 ratio. pBIGsuggested: None525 ng of pBIND_BRCA1 1-184 and 469 ng of pACT_BARD1 1-200 were co-transfected at a 1:1 molar ratio, and PEI (1 mg/ml) was used at a 6:1 ratio. pBIND_BRCA1suggested: NonepACT_BARD1suggested: NoneSoftware and Algorithms Sentences Resources Regressions to calibrate the Mammalian 2-hybrid assay results were performed in Stata 11.0 (StataCorp). StataCorpsuggested: (Stata, RRID:SCR_012763)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Limitations and Sources of Error: One may envision that a functional assay should yield perfect results; nonetheless, “Strong” odds of 18.7:1 in fact allow for an appreciable error rate. As the RING M2H experiment involves a multiple test of 604 for the comprehensive set of snMS, and almost 900 when all variant types are included, some stochastic error is expected. Technical errors are also possible. For example, outside of the moderate throughput workflow, we performed 1-by-1 assays on a small number of variants that had unexpected results (not shown). Among these, p.K65R was a unique outlier because it reproducibly showed reduced function in the parallel assay but had aberrant BFP:YFP expression when assayed alone, potentially indicative of interference between the missense substitution and the P2A cleavage signal in its expression construct. Mechanistic error would be possible if there can be separation of function between BRAC1-BARD1 RING domain heterodimerization and another function that is critical to tumor suppression. Here, we note that a reciprocal question applies to other assays: if the M2H-autoubiquitination separation of function variant p.I26A has not been tested in other assays, there is concern that they could produce false pathogenic results to the extent that their readout is dependent on, or susceptible to, ubiquitination activity. In addition, cDNA based assays are liable to mechanistic errors because they are insensitive to mRNA splicing defects. Here, w...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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