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Clinical Diagnosis of BRCA 1/2 Mutations

Updated on January 12, 2017
PDB Rendering of BRCA1 Gene
PDB Rendering of BRCA1 Gene | Source


Due to the nature of mutations in this gene it is important for individuals to be assessed and identified as a carrier of the mutation. Those who feel that they may be susceptible to this form of cancer and carry BRCA1 mutations will meet one of the following criteria (Palma, Ristori, Ricevuto, Giannini, & Gulino, 2006):

1- A personal early onset of breast cancer or breast and ovarian cancer at any age and or bilateral or multifocal disease. Where early onset is any period under the age of 50 years;

2- A family history of breast cancer (or ovarian cancer) which is linked to autosomal dominant pattern of inheritance;

3- A personal or family history of male breast cancer.

Certain mutations within these genes (founder mutations) are found among certain women of ethnic descent such as Ashkenazi-Jewish. BRCA mutations are also found in certain populations such as in some families in the Netherlands and Iceland (Petrucelli, Daly, Culver, & Feldman, 1998). If a person is found to be eligible for testing they are recommended to undertake genetic counselling first.

Genetic Counselling

Genetic counselling is a process which is designed to deal with the human problems that are associated with the occurrence or risk of occurrence of a genetic disorder in a family. Genetic counselling should be carried out by a healthcare professional such as a nurse educator. Mutational analysis is expensive and laborious and therefore not always carried out on an individual. If the patient meets the above criteria it can sometimes be assumed that they are a carrier of a mutation and they undergo regular screening to limit cancer development. Mutational analysis is commonly performed on tumours removed from patients who develop cancer and meet the above criteria to see if they are carriers for the BRCA1 mutations. If so, this may affect further treatment, management and further surgery in an attempt to reduce the risk of recurrence of metastasis. However those who have not developed cancer are also tested in the hope that they may reduce the risk and enter an early screening program (Rosenthal & Jacobs, 2006).

Types of mutation analysis are dependent on the family history as certain mutations are evident in certain ethnic groups. Ashkenazi-Jewish descent have 3 known mutations within the BRCA1/2 genes (185delAg (BRCA1), 5385insC (BRCA1), and 6174delT (BRCA2)) (Petrucelli, Daly, Culver, & Feldman, 1998). Therefore individuals from families with known mutations or those from certain ethnic groups may be tested specifically for those mutations. Those who have no linkage to these groups are suitable for direct DNA sequencing. Guidelines recommend that analysis start by testing a relative with known breast or ovarian cancer and identifying if they have specific mutations so that results can be compared for the same mutation and screening effort reduced.

DNA Screening

BRCA1 DNA screening is limited as some mutations in the gene are not obvious and clearly known. The BRCA1 gene is characterised by highly heterogeneous patterns of mutations which are found scattered throughout the whole coding sequences and flanking each exon in the intronic sequences (Gaffney, et al., 1998). The Breast Cancer Information Core (BIC) reported in 2004 that within the BRCA1 gene there were 1539 distinct nucleotide variations (Palma, Ristori, Ricevuto, Giannini, & Gulino, 2006). More than 50% of these were reported only once, i.e. belonging to only one family. This makes it difficult to sequence DNA for mutations, when there are a large number of possible mutations to identify. Beside the polymorphisms the majority of the genetic alterations found in the BRCA1 gene are frame shifts, nonsense and splice site mutations. These are responsible for the synthesis of truncated or incomplete protein products and represent pathogenetic mutations. Due to the lack of specific mutation areas this means that the entire coding regions have to be scanned thoroughly, which for BRCA1 is approximately 5500 nucleotides (Boulton, 2006). This is organised into small exons of around 100bp, with exception of two large exons. Direct sequencing would assure the highest sensitivity when assessing the DNA for mutations though it is very expensive and time consuming. There have been attempts to develop more cost effective and less time consuming methods and a large number of DNA scanning methods have been developed. In routine laboratory use there is a combination of methods used from the following: Single Strand Conformation Polymorphism Analysis (SSCA), Hetroduplex Analysis (HAD), Denaturing Gradient Gel Electrophoresis (DGGE), Chemical Cleavage Mismatch (CCM), Protein Truncation Test (PTT) and Denaturing High Performance Liquid Chromatography (DHPLC). All of these techniques have advantages and disadvantages and cannot guarantee to detect all of the cancer predisposing mutations in the genes. Table 1 outlines some of the advantages and disadvantages of the above techniques.

Principles for mutations detection
Adhered electrophoretic mobility of single stranded DNA (non-denaturing gels)
Rapid and easy to carry out
Low detection rate (70-80%), Scans short fragments
Altered electrophoretic mobility of heteroduplex (non-denaturing gels)
Rapid and easy to carry out. Detects insertion/deletion mutations in large fragments
Low detection rate (80%), Poor sensitivity for point mutations
Adhered electrophoretic mobility of heteroduplex based on their melting behaviour (denaturing gradient gels)
Rapid and easy after the initial laborious planning
Low detection rate unless well established conditions (95%), Effort required to set up the technique
Detection of hertoduplex through chemical cleavage at the site of DNA mismatch
Good sensitivity (›95%), Scans large fragments, Provides an approximate location of DNA alterations
Time consuming and labour intensive, Hazardous chemicals are required
Detection of pre-terminal in vitro synthesized protein products
Good sensitivity in identifying pathogenic mutations in large fragments (98%), Provides their approximate location
Only detects sequence alterations responsible of truncated proteins, Unsuitable to analyse small exons using genomic DNA as template
Detection of heteroduplex through their chromatographic elution profile
High sensitivity (≤ 100%), Extremely rapid and easy after initial setting up, Provides an almost precise location of alterations
Efforts required to establish the technique, Initial investment in equipment (but low cost for single analysis)
Direct sequencing of DNA fragments
Best sensitivity (100%), Defines the exactly location and the nature of alterations
Still labour intensive despite automated steps, Expensive if few mutations are expected
Table 1 - Taken from Palma et al (2006) outlining the advantages and disadvantages of techniques used for screening the BRCA1 gene. The further down the table the growing rate of sensitivity

Some mutations of the BRCA1 gene are also large genomic rearrangements including deletions or duplications of entire exons. Homologous recombination is responsible for the formations of large rearrangements mediated by the high frequency of Alu repeats. All methods which are based on polymerase chain reaction (PCR) such as direct sequencing do not detect these large rearrangements. The detection of these requires the use of alternate approaches including southern blotting, long –range PCR, fluorescent in situ hybridization (FISH) carried out on combed DNA (Palma, Ristori, Ricevuto, Giannini, & Gulino, 2006).

In most Western genetic counselling services testing is offered if there is a strong suspicion of possible hereditary breast cancer (discussed above). The BRCA1 gene is 7.8kb in length and is spread over 100kb of genomic DNA making genomic sequencing laborious, and there has to be strong recommendations before testing is undertaken (Petrucelli, Daly, Culver, & Feldman, 1998).

Usually screening occurs by subdividing the gene into smaller fragments and each fragment is screened independently. The gene is screened over 24 exons with exons 2-12 and 12-24 working well, with each of these being around 230 to 450 base pairs in length. Exon 11 however is over 3000 base pairs in length. To resolve this, a series of exon based overlapping primer sets are used which divide this exon into 300 base pair fragments for analysis (Petrucelli, Daly, Culver, & Feldman, 1998). Tissue may be taken at surgery and snap froze quenching the tissue in a life like state or may be taken from paraffin embedded tissues and subjected to testing. The DNA may be removed from the tissues by various methods such as the proteinase K method or the sonication technique for fresh and paraffin tissue respectively. The DNA is then amplified using PCR employing specific primer sets that yield 230 to 450 base pair products. Exon 11 is subjected to 20 overlapping primer sets as mentioned previously. The DNA sequences are separated using electrophoresis and developed giving banding on gels which can be analysed and interpreted.

If a patient is a descendent from certain ethnic origins such as the Ashkenzi -Jewish descent they can be tested using targeted mutational analysis which screens for the specific detection of prevalent mutations. If they are positive for these mutations then the patient may enter a screening program or preventative treatments. If the patient does not test positive for these targeted mutations then this does not rule out that they may be carrying a mutation in the gene and therefore enter DNA sequencing techniques. Those that do not have any comparative mutation and fit the likelihood of carrying germline mutations are also subjected to DNA sequencing technique. This technique analyses the full BRCA1 gene and identifies any mutations which may be present. The lack of any mutation detected does not rule out a germline mutation. They may carry another mutation within another cancer causing gene such as p53. The individual and the family may undergo further genetic testing but also must be warned that they (due to the previous criteria) are at a higher risk of developing cancer and must be screened regularly. An inconclusive result may also occur if the result is a novel variation of the BRCA1 gene. This may be a single change in a nucleotide sequence (missense mutation) which may or may not alter protein structure and function. To further develop this result the laboratory may request samples from family members especially those affected so that results may be compared. This analysis could result in the detection of this mutation to be a pathogenic mutation or a polymorphism resulting in no clinical significance.

The possibility of carrying BRCA1/2 mutations can be calculated using mathematical analysis using two mathematical models Myriad II and BRCApro. The Myriad model uses logistical regression and is applied to various characteristics in family history. These characteristics are absence or presence of breast cancer/ovarian cancer in one or more relatives below the age of 50 and disease status of the proband. This model only applies to women below the age of 50 with early onset breast cancer and at least one relative (first or second degree) with early onset breast cancer or ovarian cancer (Petrucelli, Daly, Culver, & Feldman, 1998). BRCApro utilizes a mendalian approach to autosomal dominant pattern of inheritance, taking into account a person’s family history of breast and ovarian cancer and also taking into account, the disease status (healthy or affected), the affected age of the individual along with the age of all affected second and first degree relatives at diagnosis (Petrucelli, Daly, Culver, & Feldman, 1998). The mendalian characteristic inputs such as gene penetrance and prevalence may be subject to wide variation in different populations and birth cohorts. There are tendencies of this model to provide underestimations of incidence however, but this model has been validated as an accurate counselling tool for determining the probability of carrying BRCA1/2 mutations. The figure below outlines the method of which a person may be clinical managed if they are found to be at risk for the mutation (Palma, Ristori, Ricevuto, Giannini, & Gulino, 2006).

Model of clinical management of an at-risk individual, their identification, the genetic-oncologic counselling and the genetic testing execution
Model of clinical management of an at-risk individual, their identification, the genetic-oncologic counselling and the genetic testing execution | Source


Boulton, S. (2006). Cellular Functions of the BRCA Tumour Supressor Proteins. Biochemical Society Transactions 34 , 633-645

Gaffney, D. K., Brohet, R. M., Lewis, C. M., Holden, J. A., Buys, S. S., Neuhausen, S. L., et al. (1998). Response to Radiation Therapy and Prognosis in Breast Cancer Patients with BRCA1 and BRCA2 Mutations. Radiotherapy and Oncology 47 , 129-136.

Palma, M., Ristori, E., Ricevuto, E., Giannini, G., & Gulino, A. (2006). BRCA1 and BRCA2: The Genetic Testing and the Current Management Options For Mutation Carriers. Critical Reviews In Oncology Hematology 57 , 1-23.

Petrucelli, N., Daly, M., Culver, J., & Feldman, G. (1998). BRCA1 and BRCA2 Hereditary Breast/Ovarian Cancer. In U. o. Washington, Gene Reviews (p. National Center for Biotechnology Information . Accessed First June 2009

Rosenthal, A., & Jacobs, I. (2006). Familial Ovarian Cancer Screening. Best Practice and Research Clincal Obstetrics and Gynaecology 20 , 321-338.


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