Functional characterization of BRCA1 gene variants by mini-gene splicing assay

Abstract

Mutational screening of the breast cancer susceptibility gene BRCA1 leads to the identification of numerous pathogenic variants such as frameshift and nonsense variants, as well as large genomic rearrangements. The screening moreover identifies a large number of variants, for example, missense, silent, and intron variants, which are classified as variants of unknown clinical significance owing to the lack of causal evidence. Variants of unknown clinical significance can potentially have an impact on splicing and therefore functional examinations are warranted to classify whether these variants are pathogenic or benign. Here we validate a mini-gene splicing assay by comparing the results of 24 variants with previously published data from RT-PCR analysis on RNA from blood samples/lymphoblastoid cell lines. The analysis showed an overall concordance of 100%. In addition, we investigated 13 BRCA1 variants of unknown clinical significance or putative variants affecting splicing by in silico analysis and mini-gene splicing assay. Both the in silico analysis and mini-gene splicing assay classified six BRCA1 variants as pathogenic (c.80+1G>A, c.132C>T (p.=), c.213−1G>A, c.670+1delG, c.4185+1G>A, and c.5075−1G>C), whereas six BRCA1 variants were classified as neutral (c.-19-22_-19-21dupAT, c.302−15C>G, c.547+14delG, c.4676−20A>G, c.4987−21G>T, and c.5278−14C>G) and one BRCA1 variant remained unclassified (c.670+16G>A). In conclusion, our study emphasizes that in silico analysis and mini-gene splicing assays are important for the classification of variants, especially if no RNA is available from the patient. This knowledge is crucial for proper genetic counseling of patients and their family members.

Splicing analysis for exonic and intronic mismatch repair gene variants associated with Lynch syndrome confirms high concordance between minigene assays and patient RNA analyses

“…2011; Steffensen et al. 2014), or the partial versus complete exon skip shown for MLH1 c.793C>T in minigene assay and patient RNA analysis, respectively (this study).…”

“…2011; Steffensen et al. 2014), together for 142 unique MMR gene and BRCA1/2 gene variants (this study included; Table3). Overall, high concordances were found between the two methods, with only a few variants showing differences in aberrant splicing profiles.…”

“…2012; Steffensen et al. 2014; Table3). However, for all 10 variants the observed differences in aberrant transcript profile between both tests did not influence their classification as pathogenic or likely pathogenic.…”

Validation of CFTR intronic variants identified during cystic fibrosis population screening by a minigene splicing assay

“…It does not require patient ' s RNA or particularly sophisticated equipment. The utility of this tool is not restricted to the validation of CFTR variants, but it can be conveniently employed for any gene characterized by high mutational heterogeneity (e.g., BRCA1 ) [15] .…”

Investigating Splicing Variants Uncovered by Next-Generation Sequencing the Alzheimer’s Disease Candidate Genes, CLU, PICALM, CR1, ABCA7, BIN1, the MS4A Locus, CD2AP, EPHA1 and CD33

“…This method has been shown to have complete concordance with RT-PCR analysis of patient RNA and is a validated and reliable method for investigating splicing [30].…”