Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants
Abstract
:1. Introduction
2. Results
2.1. BRCA1 Missense Variants’ Classification
2.2. BRCA1 Pathogenic Variants Increased HR and GR in Yeast
2.3. Effect of VUS on HR and GR in Yeast
2.4. Development of yBRCA1: A Classifier Combination Approach for Functional Characterization
2.5. VUS Functional Characterization by the Classifier Combination Method yBRCA1
3. Discussion
4. Materials and Methods
4.1. BRCA1 Variant Selection
4.2. Yeast Strains
4.3. Protein Extraction and Western Blotting
4.4. Homologous Recombination and Gene Reversion Assays
4.5. Small Colony Phenotype Assay
4.6. Best Cut-Off Calculation and Integration of Datasets
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Assay | Sensitivity | Specificity | Accuracy | AUROC | YI | Best Cut-off | FP | FN |
---|---|---|---|---|---|---|---|---|
Intra-HR | 0.957 | 0.783 | 0.87 | 0.914 | 0.739 | 1.535 | Y179C I1275V R1347G S1512I P1776H | R71K |
Inter-HR | 0.87 | 0.913 | 0.891 | 0.929 | 0.783 | 1.32 | Y179C I1275V | R71K R1699W A1708E |
GR | 0.913 | 0.87 | 0.891 | 0.867 | 0.783 | 1.077 | N132K I1275V T1675I | R71K R1495M |
SCP | 1.00 | 0.913 | 0.957 | 0.951 | 0.913 | 25.833 | I1275V S1512I |
yBRCA1 Method | |
---|---|
Accuracy (CI 95%) | 0.9565 (0.8516–0.9947) |
Sensitivity | 0.9565 |
Specificity | 0.9565 |
AUROC | 0.9855 |
Cohen’s Kappa | 0.9575 |
MCC | 0.9583 |
Inter-HR | Intra-HR | GR | SCP | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Variant | PS | FI | PS | FI | PS | FI | PS | FI | PV | PPS | yBRCA1 |
A1669S | 0.63575 | Pathogenic | 0.00675 | Benign | 0.00075 | Benign | 0.00025 | Benign | 1 | 0.25 | Benign |
A1789T | 0.00025 | Benign | 0.00025 | Benign | 0.00025 | Benign | 0.99975 | Pathogenic | 1 | 0.25 | Benign |
E1352K | 0.00025 | Benign | 0.56025 | Pathogenic | 0.99925 | Pathogenic | 0.99975 | Pathogenic | 3 | 0.75 | Pathogenic |
N1730I | 0.00025 | Benign | 0.43825 | Pathogenic | 0.99925 | Pathogenic | 0.88025 | Pathogenic | 3 | 0.75 | Pathogenic |
N1819S | 0.00125 | Benign | 0.01725 | Benign | 0.99975 | Pathogenic | 0.93575 | Pathogenic | 2 | 0.5 | Benign |
P1010S | 0.40675 | Pathogenic | 0.00025 | Benign | 0.54025 | Pathogenic | 0.00025 | Benign | 2 | 0.5 | Benign |
S1164I | 0.99975 | Pathogenic | 0.90575 | Pathogenic | 0.75425 | Pathogenic | 0.00025 | Benign | 3 | 0.75 | Pathogenic |
S592N | 0.00025 | Benign | 0.89925 | Pathogenic | 0.99975 | Pathogenic | 0.99975 | Pathogenic | 3 | 0.75 | Pathogenic |
V1791L | 0.99975 | Pathogenic | 0.30975 | Pathogenic | 0.00275 | Benign | 0.99975 | Pathogenic | 3 | 0.75 | Pathogenic |
Y1703C | 0.99975 | Pathogenic | 0.32425 | Pathogenic | 0.54275 | Pathogenic | 0.99975 | Pathogenic | 4 | 1 | Pathogenic |
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Bellè, F.; Mercatanti, A.; Lodovichi, S.; Congregati, C.; Guglielmi, C.; Tancredi, M.; Caligo, M.A.; Cervelli, T.; Galli, A. Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants. Int. J. Mol. Sci. 2022, 23, 4049. https://doi.org/10.3390/ijms23074049
Bellè F, Mercatanti A, Lodovichi S, Congregati C, Guglielmi C, Tancredi M, Caligo MA, Cervelli T, Galli A. Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants. International Journal of Molecular Sciences. 2022; 23(7):4049. https://doi.org/10.3390/ijms23074049
Chicago/Turabian StyleBellè, Francesca, Alberto Mercatanti, Samuele Lodovichi, Caterina Congregati, Chiara Guglielmi, Mariella Tancredi, Maria Adelaide Caligo, Tiziana Cervelli, and Alvaro Galli. 2022. "Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants" International Journal of Molecular Sciences 23, no. 7: 4049. https://doi.org/10.3390/ijms23074049