Identification of six pathogenic RAD51C mutations via mutational screening of 1228 Danish individuals with increased risk of hereditary breast and/or ovarian cancer.

Germ-line mutations in the RAD51C gene have recently been identified in families with breast and ovarian cancer and have been associated with an increased risk of ovarian cancer. In this study, we describe the frequency of pathogenic RAD51C mutations identified in Danish breast and/or ovarian cancer families. We screened the RAD51C gene in 1228 Danish hereditary breast and/or ovarian cancer families by next-generation sequencing analysis. The frequency of the identified variants was examined in the exome sequencing project database and in data from 2000 Danish exomes and the presumed significance of missense and intronic variants was predicted by in silico analysis. We identified six families with a pathogenic mutation in RAD51C, including three frameshift mutations, one nonsense mutation, and 2 missense mutations. Overall, pathogenic RAD51C mutations were identified in 0.5 % of Danish families with increased risk of hereditary breast and/or ovarian cancer. Moreover, we identified 24 additional RAD51C variants of which 14 have not been previously reported in the literature. In this study, we determine the prevalence of RAD51C mutations in Danish breast and/or ovarian cancer families. We identified six pathogenic RAD51C mutations as well as 23 variants of uncertain clinical significance and one benign variant. Together, the study extends our knowledge of the RAD51C mutation spectrum and supports that RAD51C should be included in gene panel testing of individuals with high risk of breast and ovarian cancer.

Splicing analysis of 14 BRCA1 missense variants classifies nine variants as pathogenic.

Pathogenic germline mutations in the BRCA1 gene predispose carriers to early onset breast and ovarian cancer. Clinical genetic screening of BRCA1 often reveals variants with uncertain clinical significance, complicating patient and family management. Therefore, functional examinations are urgently needed to classify whether these uncertain variants are pathogenic or benign. In this study, we investigated 14 BRCA1 variants by in silico splicing analysis and mini-gene splicing assay. All 14 alterations were missense variants located within the BRCT domain of BRCA1 and had previously been examined by functional analysis at the protein level. Results from a validated mini-gene splicing assay indicated that nine BRCA1 variants resulted in splicing aberrations leading to truncated transcripts and thus can be considered pathogenic (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5072C>T/p.Thr1691Ile, c.5074G>C/p.Asp1692His, c.5074G>A/p.Asp1692Asn, c.5074G>T/p.Asp1692Tyr, c.5332G>A/p.Asp1778Asn, c.5332G>T/p.Asp1778Tyr, and c.5408G>C/p.Gly1803Ala), whereas five BRCA1 variants had no effect on splicing (c.4985T>C/p.Phe1662Ser, c.5072C>A/p.Thr1691Lys, c.5153G>C/p.Trp1718Ser, c.5154G>T/p.Trp1718Cys, and c.5333A>G/p.Asp1778Gly). Eight of the variants having an effect on splicing (c.4987A>T/p.Met1663Leu, c.4988T>A/p.Met1663Lys, c.5074G>C/p.Asp1692His, c.5074G>A/p.Asp1692Asn, c.5074G>T/p.Asp1692Tyr, c.5332G>A/p.Asp1778Asn, c.5332G>T/p.Asp1778Tyr, and c.5408G>C/p.Gly1803Ala) were previously determined to have no or an uncertain effect on the protein level, whereas one variant (c.5072C>T/p.Thr1691Ile) were shown to have a strong effect on the protein level as well. In conclusion, our study emphasizes that in silico splicing prediction and mini-gene splicing analysis are important for the classification of BRCA1 missense variants located close to exon/intron boundaries.

Characterization of BRCA1 and BRCA2 splicing variants: a collaborative report by ENIGMA consortium members.

Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.

The silent mutation nucleotide 744 G --> A, Lys172Lys, in exon 6 of BRCA2 results in exon skipping.

Germ-line mutations in BRCA2 predispose to breast and ovarian cancer. Mutations are widespread throughout the gene and include disease-causing mutations as frameshift, nonsense, splicing mutations and large genomic rearrangements. However a large number of mutations, including missense, silent and intron variants are of unknown significance. Here, we describe the functional characterization of a silent mutation (nucleotide 744 G --> A/c.516 G --> A, Lys172Lys) in exon 6 of BRCA2 in a Danish family with breast and ovarian cancer. Exon trapping analysis showed that the mutation results in skipping of exon 6 and/or both exon 5 and 6, which was verified by RT-PCR analysis on RNA isolated from whole blood of the affected patient. We therefore conclude that the BRCA2 silent mutation Lys172Lys is a disease-causing mutation.

Identification of a novel BRCA1 nucleotide 4803delCC/c.4684delCC mutation and a nucleotide 249T>A/c.130T>A (p.Cys44Ser) mutation in two Greenlandic Inuit families: implications for genetic screening of Greenlandic Inuit families with high risk for breast and/or ovarian cancer.

Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We have recently identified a Greenlandic Inuit BRCA1 nucleotide 234T>G/c.115T>G (p.Cys39Gly) founder mutation, which at that time was the only disease-causing BRCA1/BRCA2 mutation identified in this population. Here, we describe the identification of a novel disease-causing BRCA1 nucleotide 4803delCC/c.4684delCC mutation in a Greenlandic Inuit with ovarian cancer. The mutation introduces a frameshift and a premature stop at codon 1572. We have also identified a BRCA1 nucleotide 249T>A/c.130T>A (p.Cys44Ser) mutation in another Greenlandic individual with ovarian cancer. This patient share a 1-2 Mb genomic fragment, containing the BRCA1 gene, with four Danish families harbouring the same mutation, suggesting that the 249T>A/c.130T>A (p.Cys44Ser) mutation originates from a Danish ancestor. We conclude that screening of Greenlandic Inuits with high risk of breast or ovarian cancer should include sequencing of the entire BRCA1 gene.

Classification of the spliceogenic BRCA1 c.4096+3A>G variant as likely benign based on cosegregation data and identification of a healthy homozygous carrier.

BRCA1, c.4096+3A>G was identified in a consanguineous Danish family with several cases of breast/ovarian cancer. In silico analysis and splicing assays indicated that the variant caused aberrant splicing. However, based on segregation data and the finding of a healthy homozygous carrier, we classify the BRCA1 c.4096+3A>G variant as likely benign.

Identification of a breast cancer family double heterozygote for RAD51C and BRCA2 gene mutations.

Next-generation sequencing has entered routine genetic testing of hereditary breast cancer. It has provided the opportunity to screen multiple genes simultaneously, and consequently has identified new complex genotypes. Here we report the first identification of a woman double heterozygote for mutations in the RAD51C and BRCA2 genes. The RAD51C missense mutation p.Arg258His has previously been identified in a homozygous state in a patient with Fanconi anemia. This mutation is known to affect the DNA repair function of the RAD51C protein. The BRCA2 p.Leu3216Leu synonymous mutation has not been described before and mini-gene splicing experiments revealed that the mutation results in skipping of exon 26 containing a part of the DNA-binding domain. We conclude that the woman has two potential disease-causing mutations and that predictive testing of family members should include both the RAD51C and BRCA2 mutation. This study illustrates the advantage of sequencing gene panels using next-generation sequencing in terms of genetic testing.

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

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.

Screening of 1331 Danish breast and/or ovarian cancer families identified 40 novel BRCA1 and BRCA2 mutations.

Germ-line mutations in the tumour suppressor genes BRCA1 and BRCA2 predispose to breast and ovarian cancer. Since 1999 we have performed mutational screening of breast and/or ovarian cancer patients in East Denmark. During this period we have identified 40 novel sequence variations in BRCA1 and BRCA2 in high risk breast and/or ovarian cancer families. The mutations were detected via pre-screening using dHPLC or high-resolution melting and direct sequencing. We identified 16 variants in BRCA1, including 9 deleterious frame-shift mutations, 2 intronic variants, 4 missense mutations, and 1 synonymous variant. The remaining 24 variants were identified in BRCA2, including 10 deleterious mutants (6 frame-shift and 4 nonsense), 2 intronic variants, 10 missense mutations and 2 synonymous variants. The frequency of the variants of unknown significance was examined in control individuals. Moreover, the presumed significance of the missense mutations was predicted in silico using the align GVGD algorithm. In conclusion, the mutation screening identified 40 novel variants in the BRCA1 and BRCA2 genes and thereby extends the knowledge of the BRCA1/BRCA2 mutation spectrum. Nineteen of the mutations were interpreted as pathogenic, 3 missense mutations were suggested to be pathogenic based on in silico analysis, 6 mutations were suggested to be benign since they were identified in patients together with a well-known disease-causing BRCA1/BRCA2 mutation, while 12 were variants of unknown significance.

Identification of a Danish breast/ovarian cancer family double heterozygote for BRCA1 and BRCA2 mutations.

Mutations in the two breast cancer susceptibility genes BRCA1 and BRCA2 are associated with increased risk of breast and ovarian cancer. Patients with mutations in both genes are rarely reported and often involve Ashkenazi founder mutations. Here we report the first identification of a Danish breast and ovarian cancer family heterozygote for mutations in the BRCA1 and BRCA2 genes. The BRCA1 nucleotide 5215G > A/c.5096G > A mutation results in the missense mutation Arg1699Gln, while the BRCA2 nucleotide 859 + 4A > G/c.631 + 4A > G is novel. Exon trapping experiments and reverse transcriptase (RT)-PCR analysis revealed that the BRCA2 mutation results in skipping of exon 7, thereby introducing a frameshift and a premature stop codon. We therefore classify the mutation as disease causing. Since the BRCA1 Arg1699Gln mutation is also suggested to be disease-causing, we consider this family double heterozygote for BRCA1 and BRCA2 mutations.