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Incorporation of semi‐quantitative analysis of splicing alterations for the clinical interpretation of variants in BRCA1 and BRCA2 genes
Author(s) -
Montalban Gemma,
Bonache Sandra,
MolesFernández Alejandro,
Gadea Neus,
Tenés Anna,
TorresEsquius Sara,
Carrasco Estela,
Balmaña Judith,
Diez Orland,
GutiérrezEnríquez Sara
Publication year - 2019
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.23882
Subject(s) - biology , rna splicing , sanger sequencing , genetics , alternative splicing , gene , allele , germline , missense mutation , computational biology , dna sequencing , mutation , messenger rna , rna
BRCA1 and BRCA2 ( BRCA1/2 ) genetic variants that disrupt messenger RNA splicing are commonly associated with increased risks of developing breast/ovarian cancer. The majority of splicing studies published to date rely on qualitative methodologies (i.e., Sanger sequencing), but it is necessary to incorporate semi‐quantitative or quantitative approaches to accurately interpret the clinical significance of spliceogenic variants. Here, we characterize the splicing impact of 31 BRCA1/2 variants using semi‐quantitative capillary electrophoresis of fluorescent amplicons (CE), Sanger sequencing and allele‐specific assays. A total of 14 variants were found to disrupt splicing. Allelic‐specific assays could be performed for BRCA1 c.302−1G>A and BRCA2 c.516+2T>A, c.1909+1G>A, c.8332–13T>G, c.8332−2A>G, c.8954−2A>T variants, showing a monoallelic contribution to full‐length transcript expression that was concordant with semi‐quantitative data. The splicing fraction of alternative and aberrant transcripts was also measured by CE, facilitating variant interpretation. Following Evidence‐based Network for the Interpretation of Germline Mutant Alleles criteria, we successfully classified eight variants as pathogenic (Class 5), five variants as likely pathogenic (Class 4), and 14 variants as benign (Class 1). We also provide splicing data for four variants classified as uncertain (Class 3), which produced a “leaky” splicing effect or introduced a missense change in the protein sequence, that will require further assessment to determine their clinical significance.