Detection of BRCA1 Gene Mutation in the Breast Cancer Probands by Non-Radioactive PCR-SSCP Method
P. Mehdipour PhD,* M. Atri MD,* F. Pour-Farzad MSc**
*Unit of Cytogenetics/Oncogenetics, Department of Human Genetics, School of Public Health and Public Health Research, Tehran University of Medical Sciences, **Department of Surgery, Cancer Institute, School of Medicine, Tehran University of Medical Sciences
Background-A
worldwide high incidence of breast cancer (BC) has
promoted essential investigational efforts in the fields
of clinics and genetics. A positive family history of BC
is the strongest epidemiologic risk factor which could
provide early detection and preventive measures.
Objective-To establish an
informative bank allowing rapid detection, prevention and
positive counseling and management strategies for
BC-prone pedigrees.
Methods-DNA samples were
extracted from peripheral blood of 12 primary BC patients
and the Polymerase Chain Reaction (PCR) and
non-radioactive Single Strand Conformation Polymorphism
(SSCP) were performed. The electrophoretic gels were
stained with silver nitrate and the variant bands were
assumed a result of mutation for BRCA1-gene, including
exon 5,11A, and 11B.
Results-The
family history of 4 and 2 BC-cases was found in 42%
(5/12) and 58% (7/12) of pedigrees respectively, of those
an affected BC-praband revealed to carry a mutation in
exon 11B.
Conclusion- A genetics-clinical
strategy as a powerful diagnostic tool to prevent breast
cancer in patients and their relatives with high risk
pedigrees.
Introduction
Breast cancer (BC) is considered the most common cancer among women and the cumulative risk by the age of 85 years is 1 in 8 and 1 in 12 in the United States of America and Britain respectively.1
A positive family history of BC is the strongest epidemiologic risk factor.2,3
The background of genetic epidemiology in Iran indicates that 43% (43/100) of BC-probands have the family history of BC in their pedigrees4,5 and the autosomal dominant susceptibility genes contribute to 5%-10% of all breast cancer cases,6,7 BRCA1-gene is involved in the inherited predisposition to BC and accounts for approximately 45% of inherited BC8,9 and this gene has been mapped to chromosome 17q21.10 The general population who may carry a predisposing BRCA1-mutation is estimated to be 1 in every 200 women.6,11
The BRCA1-gene consists of 24 exons and encodes a 7.8-kb transcript composed of 22 coding exons which span > 80 kb of genomic DNA (gDNA).8
The purpose of this initial investigation was to detect BRCA1- mutation (s), including exons 5, 11A, and 11B in 12 BC - probands with the positive family history of BC - in their pedigrees.
Materials and Methods
Polymerase chain reaction (PCR) and non-radioactive single strand conformation polymorphism (SSCP) were performed according to the standard techniques.12 Extraction was carried out on the peripheral blood of BC - patients with application of phenol-chloroform13 and PCR was performed with 50 vl volumes, containing 50 ng GDNA; 1´ PCR buffer (Boehringer Manheim: BM); 200m m dNTP (Gibco BRL); 50 pmol each primer (Table 1); and 1.25 u Taq DNA polymerase (BM). Amplification condition included 35 cycles of denaturation at 94° C for 45S; annealing was at the optimal temperature of each primer pair for 30S; with extension at 72° C for 30S.
The template of PCR was lymphocytes-GDNA, from BRCA1-BC-patients with a positive family history of BC. Amplified DNA was prepared of 1:1 in formamide buffer (98% formamide, 10 Mm EDTA, Ph 8; 0.05% Bromophenol blue, 0.05% xylene cyanol), and was denatured at 95° C for 5 min, then was rapidly cooled to 4° C. Then single strand conformation polymorphism (SSCP) was carried out according to the standard protocol as follows.14
- Five m L of each sample was loaded to a SSCP-gel, containing 0.5´ MDE (FMC, Bioproducts), 0.6´ TBE, 320 m L of 10% ammonium per sulfate, and 30 m l TEMED.
- Gel was run at 70V for 16h in 0.6´ TBE at room temperature. Then the gel was stained with silver nitrate, and the variant bands were assumed to be the result of mutation.
We detected SSCP-variant band of exon 11B-BRCA1-gene in one sample (No. 211) of BC-probands, who had a BC family history in her second degree relatives, i.e., the patient's aunt (Fig. 2).
Results
The germ line mutation of BRCA1-gene, including exons 5, 11A, and 11B was investigated by SSCP, in the peripheral blood-gDNAs from 12 probands, affected with primary - unilateral breast cancer, with at least one family history of BC in a second degree relative.
Distribution of BC patients, including age of onset (38-70 years), histo-pathologic classification, breast side, and the status of BRCA1-gene, is given in Table 2.
As far as the genetic epidemiology concerned, 42% (5/12), and 58% (7/12) of pedigrees had the family history of 4 and 2 BC cases, respectively (Fig. 1). The history of BC was found to be in the second and third degree relative of BC-probands, including aunt, uncle and their offsprings.
There was no consanguinity in parental and grand-parental sides of BC probands.
The purpose of the present initial investigation was to detect BRCA1-mutation (s), including exons 5, 11A and 11B in 12 BC-probands with the positive family history of BC in their pedigrees.
Discussion
Use of the appropriate information to reduce the risk of breast cancer (BC), especially in cancer - prone families remains a paradigm of long-term studies. Fifteen to 20% of BC pedigrees have family history of BC, and although a positive family history is the strongest risk factor in the field of preventive medicine, it is also important to initiate the mutational detection of the predisposing gene, ie, BRCA1 for the BC high risk families in Iran.
An estimated 5% to 10% of all BC - cases are thought to be due to BRCA1 and BRCA2 mutations which account for 80% of hereditary BC and for 1/2 BC only families. The present data are the results of an initiating step for such purposes in Iran. The BRCA1-gene with an autosomal dominant mode of inheritance, consists of 24, including 22 coding exons, and 5, 592 nucleotides. However, the isolation of the BRCA1-gene offers an opportunity for high risk members of hereditary BC and the early detection of BRCA1-gene could lead to positive management strategies.
Twelve BC probands in our series had the family history of BC in second, third and fourth degree relatives. Fifty percent (6/12) included a probands aunt, including 33% (2/6) with an additional affected proband cousin as well; and 50% (6/12) only the proband's cousins and grand cousins. These figures demonstrated that the family history of breast cancer in second and further-removed relatives, including third degrees, is also important and should be considered as the focusing point in high risk families.
Among our 12 BC probands, a 38-year-old female affected with ductal carcinoma, whose aunt was also affected with BC, demonstrated a variant band in exon 11B which was also reconfirmed twice by PCR-SSCP analysis (Fig. 1). The exon 11B with 309bp is a part of exon 11 of BRCA1-gene, in which 60% of BRCA1- mutations occur.
However, the present data, as an initial investigation, is currently being performed on other exons and we hope that we will be able to draw the mutational pattern of BRCA1-gene in Iranian BC patients, in the near future. This could lead to preventive strategies, under effective genetic counselling, for breast cancer - prone families in Iran.
Acknowledgement
We thank International Agency for Research on cancer, especially to Dr. R. Wilmotte, for providing us MDE-electrophoresis gel and the primer for E. 11A and 11B. We are also grateful to S. Hosseini and S. Kheradmand-kia for their assistance.
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