Tumor Genetic Testing
Thousands of sequence variants have been so far identified in the BRCA1 and BRCA2 genes through the analysis of breast cancer families and population-based case studies. A total of 19.655 different mutations and polymorphisms are reported in the BRCA1 gene by dal BRCA Exchange, whereas the same database reports 20.734 unique mutations and polymorphisms in the BRCA2 gene (last updated January 2021). ClinVar has also a large dataset, reporting, in 2021, 11.561 and 12.794 variants for BRCA1 and BRCA2 respectively, including point mutations and small indels, variably classified as pathogenic, likely pathogenic, uncertain, disease-associated, likely benign, or benign, and large genomic rearrangements. The mutational spectrum is therefore very large, even if some founder mutations may be found at a relatively higher frequency than other rarer variants in selected populations.
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Variation types
BRCA1 and BRCA2 variants can be subdivided into three broad classes: single-nucleotide variants (SNVs), small insertion or deletion events (indels) and large genomic rearrangements (LGRs). Pathogenic single-nucleotide variants and small indels are widely distributed across the whole coding sequence and intronic regions of both genes. Large genomic rearrangements may be consistent with exonic, multiexonic or whole gene large deletions/duplications.
To get an idea about the type of mutations (and their proportions) occurring within the BRCA1 and BRCA2 sequences, there is another database (HGMD Public; access July 2013), which reports numbers as below:
| Mutation Type | BRCA1 | BRCA2 |
| Missense/nonsense | 696 | 668 |
| Splicing | 199 | 166 |
| Regulatory | 17 | 1 |
| Small deletions | 661 | 780 |
| Small insertions | 234 | 267 |
| Small indels | 41 | 41 |
| Gross deletion | 245 | 57 |
| Gross insertion | 47 | 19 |
| Complex rearrangements | 22 | 9 |
| Repeat variations | 0 | 0 |
There appears to be no mutational ‘hot spots’ in the BRCA1 gene sequence, although a large amount of mutations falls within exons 10 to 12, which are highly important for the tumour suppressor function of BRCA1. This middle region of BRCA1 is known to interact with several proteins involved in a wide range of cellular pathways such as transcription, DNA repair, and cell cycle progression. BRCA1-interacting partners include the retinoblastoma protein (Rb), c-Myc, RAD50, and RAD51. Exon 10 contains two nuclear localization sequences (NLS) and encodes nearly 60% of the BRCA1 protein. The NLS sequences enable the interaction with importin-alpha, which mediates BRCA1 transport from the cytosol to the nucleus. Mutations in the NLSs result in a shift toward cytosolic localization of BRCA1 and a subsequent increase in unrepaired mutations and chromosomal abnormalities in malignancies.
Some recurring mutations have been detected in the BRCA1 and BRCA2 genes, the latter ones mainly deriving from a founder effect. Preservation of a common haplotype has been reported. Common BRCA1 mutations in different families have been found to be often accompanied by a number of polymorphic markers in widely separated geographic regions and communities. This not only suggests founder effects rather than mutational “hot spots” as the basis for the recurrent mutations, but also points to a high degree of genetic stability at this locus, with a relatively low rate of new mutations. Founder mutations have been identified in many populations, such as the Ashkenazi Jewish, and in some countries such as Iceland, Norway, Poland, the Baltic countries, The Netherlands and Belgium. For example, three mutations are commonly found in the Ashkenazi Jewish population:
- c.68_69delAG (or 185delAG in BIC nomenclature) in the BRCA1 gene;
- c.5266dupC (or 5382insC in BIC nomenclature) in the BRCA1 gene;
- c.5946delT (or 6174delT in BIC nomenclature) in the BRCA2 gene.
The mutation c.68_69delAG in BRCA1 has been found in 20% of Ashkenazi Jewish women diagnosed with breast cancer before age 42 years, whereas the mutation c.5946delT in BRCA2 is present in 8% of women diagnosed with breast cancer before age 42 years and in 1.5% of unselected Ashkenazim.
The mutation c.771_775delTCAAA (or 999del5 in BIC nomenclature) in BRCA2 occurs in 0.6% of the Icelandic population and in 10.4% of women and 38% of men with breast cancer from Iceland.
The prevalence of recurrent mutations due to a founder effect may impact the testing strategy in some countries.
Variants of unknown significance (VUS)
The interpretation of the results of BRCA1 and BRCA2 screening is complicated by variants of unknown clinical significance (VUS) are detected. When a VUS is detected, the prediction on the protein function and the disease risk remains elusive. Variants in promoter regions, intronic nucleotide changes close to the exon boundary, small in-frame insertions/deletions, missense changes and synonymous changes may often be classified as VUS. The frequency of VUS is in general lower in well-characterized populations, as databases are frequently updated and previous VUS are often re-interpreted and reclassified. Ways to help to clarify the clinical significance of a VUS include the following:
- familial carrier testing in as many informative relatives as possible. Unveiling the segregation pattern is the first step in understanding a VUS. A variant which tend to be present only in affected patients and absent in the unaffected consanguineous has certainly a higher probability to be pathogenic rather than benign. However, caution is warranted in interpreting segregation data, especially because of incomplete penetrance and odds that co-segregation VUS-disease is casual.
- functional studies, mainly on RNA. Especially in case of putative splice mutations, such as intronic variants or synonymous changes, RNA expression studies may reveal the variant deleterious effect on the messenger (mRNA). Variants impacting the splicing process, as well as nonsense mutations introducing a premature stop codon, may lead to protein truncation or nonsense-mediated messenger decay (NMD). NMD may be unveiled by allele-specific expression studies, which are possible by qPCR or capillary electrophoresis (Sanger) applications (see, for instance, the SNaPshot technology). Caution is warranted also in functional studies, mostly because of marked instability of the RNA molecule and uniqueness of study protocols (every VUS requires an entirely new study design, which remains in principle “experimental” and largely operator-dependent). Not all laboratories are equipped for proceeding to functional studies.
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BRCA1 and BRCA2 multiple mutations
Some families with more than one disease-causing mutation in BRCA genes have been described. In particular, families with one BRCA1 and one BRCA2 mutation or two BRCA1 mutations have been described. Mutations can segregate singularly in different family members, but can also be present in one patient as double heterozygous mutations. Heidemann et al, 2012 (PMID 22535016) stated that females carrying one BRCA1 and one BRCA2 mutation can show earlier onset of breast cancer and have more severe disease than single heterozygous BRCA mutation carriers. By contrast, Lavie et al, 2012 (PMID 20924075), who found a prevalence of 1,85% of double heterozygotes for one BRCA1 and one BRCA2 disease-causing mutation in a large cohort of Jewish individuals, stated that, besides a slightly younger age at onset, those patients are not actually suffering from a more severe disease than those ones with just one single mutation. Of note, Lavie et al highlighted that, in their cohort, no patients with two mutations in BRCA1 were identified, speculating that such an occurrence might not be compatible with life. Indeed, Stoppa-Lyonnet et al, 1996 (PMID 8755940) had previously identified a family with two BRCA1 mutation, but, also in this case, no family members carrying both mutations at once were identified. Very interestingly, Stoppa-Lyonnet et al observed how linkage studies may fail if a second pathogenic allele is not suspected in a family. An additional report of double heterozygous patients is provided by Leegte et al 2012 (PMID 15744030): also in this case the authors concluded that double heterozygosity does not seem to correlate with a more severe phenotype (they observed that the clinical spectrum of BRCA double heterozygosity is ranging from unilateral breast cancer at age 26 to cancer-free survival at age 70). Cortesi L et al 2003 (PMID 12673274) reported a family with one BRCA1 and one BRCA2 mutation. However, in this family, no patients with double heterozygosity are reported: all affected patients were bearing either the mutation in BRCA1 or the mutation in BRCA2. Also in the family of Ashkenazy Jewish ancestry reported by Liede A et al patients were carrying one single mutation, although three different pathogenic alleles (two in BRCA1 and one in BRCA2) were segregating in the family. Notably, if it’s true that no homozygous mutations in BRCA1 have been reported to date (being presumably not compatible with life), homozygous or compound heterozygous mutations in BRCA2 can cause Fanconi anemia. Interestingly, a 13 years old boy affected with medulloblastoma was identified to carry a homozygous missense BRCA2 mutation. The father of this patient was heterozygous for the mutation (Bayrakli F et al 2011, PMID 22044372).
To summarize, the following instances must be considered about multiple mutations in BRCA1 and BRCA2:
- Segregation of one BRCA1 and one BRCA2 mutation in one family: family members may carry one of the two or both mutations (double heterozygosity). It is still controversial whether double heterozygosity may influence the severity of the phenotype, even if instances of earlier onset have been reported.
- Segregation of two BRCA1 mutations in one family: patients can have just one of the two mutations, as compound heterozygosis or homozygosis for two BRCA1 mutations is thought to be not compatible with life.
- BRCA2 homozygous mutations: homozygous or compound heterozygous BRCA2 mutations are actually causing Fanconi anemia. An adolescent with medulloblastoma and a homozygous BRCA1 missense mutation was reported.
- Segregation of three BRCA gene mutations: one family was reported with two BRCA1 and one BRCA2 mutation. The mutations were segregating separately in different family members.
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