Adding In Silico Assessment of Potential Splice Aberration to the Integrated Evaluation ofBRCAGene Unclassified Variants

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Clinical mutation screening of the cancer susceptibility genes BRCA1 and BRCA2 generates many unclassified variants (UVs). Most of these UVs are either rare missense substitutions or nucleotide substitutions near the splice junctions of the protein coding exons. Previously, we developed a quantitative method for evaluation of BRCA gene UVs—the “integrated evaluation”—that combines a sequence analysis-based prior probability of pathogenicity with patient and/or tumor observational data to arrive at a posterior probability of pathogenicity. One limitation of the sequence analysis-based prior has been that it evaluates UVs from the perspective of missense substitution severity but not probability to disrupt normal mRNA splicing. Here, we calibrated output from the splice-site fitness program MaxEntScan to generate spliceogenicity-based prior probabilities of pathogenicity for BRCA gene variants; these range from 0.97 for variants with high probability to damage a donor or acceptor to 0.02 for exonic variants that do not impact a splice junction and are unlikely to create a de novo donor. We created a database provides the combined missense substitution severity and spliceogenicity-based probability of pathogenicity for BRCA gene single-nucleotide substitutions. We also updated the BRCA gene Ex-UV LOVD, available at, with 77 re-evaluable variants.

We have re-calibrated the native output from a splice site analysis program, MaxEntScan, to generate probabilities that sequence variants inBRCA1orBRCA2will be pathogenic because they damage mRNA splicing. Combined with our previous calibration of missense substitution severity (Tavtigian et al., Human Mutation 29: 1342–1354, 2008), we can now provide integrated prior probabilities in favor of pathogenicity that serve as the starting point for clinical classification of variants of unknown significance observed in these genes.

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