The risk factors of BC include genetic, environmental and hormonal. Genetic risk factors contribute to about 5%-10% of all cases, 90%-95% of them result from somatic mutation and about 5%-10% are inherited as the result of germ line mutation in autosomal dominant BC susceptibility genes[3,4].

Many genes have been found to increase susceptibility to cancer and are also associated with familial breast cancer. These genes include Breast Cancer Anti-estrogen resistance-1 (BRCA1), Breast Cancer Anti-estrogen resistance- 2 (BRCA2), Ataxia Telangiectasia mutant gene (ATM), Phosphate and Tensin homology (PTEN) and Tumor Protein (P53). Several other less frequently predisposing genes are also involved but to lesser extent[3,4].

BRCA1 gene (chr 17q21) is a tumor suppressor gene that encodes tumor suppressor protein which acts as a negative regulator for tumor growth. It accounts for 45% of inherited BC and 90% of inherited breast-ovarian cancer (BOC) in highly affected families[5]. Different types of mutation have been found in the BRCA1 gene and predispose to development of cancer. The most common of them is the frame shift mutation at position 185 in exon 2, involving deletion of adenine and guanine (185 del AG mutation)[6-8]. The identification and study of this mutation could facilitate early diagnosis and proper counseling for BC. Therefore, the present study aimed to identify the 185 del AG mutation (exon 2) of BRCA1 gene in Egyptian females with early BC and BOC and their female relatives. This will allow early diagnosis and proper genetic counseling for susceptible cases in the families.

Female patients participated in the study were selected according to having one of the following:

1- Early onset breast cancer (below 40 years).
2- Bilateral breast cancer.
3- Breast-ovarian cancer.
4- Positive family history of breast cancer.

The patients and control group were subjected to the following:

1- Detailed history:
a- Personal history including age, age of menarche, marriage, age of first full term pregnancy, lactation and menopause.
b- Family history of breast cancer or any other cancer (for patients and relatives).

2- Molecular studies to detect BRCA1 (185 del AG) mutation as follows:

a- DNA isolation and purification from peripheral blood using standard method[9]
b- Polymerase chain reaction (PCR) amplification: according to Lahad et al.[10] The sequence of the primers used was as follows:

Forward: 5'-GAAGTTGTCATTTTATAAACCTTT-3'
Reverse: 5'-TGTCTTTTCTTCCCTAGTATGT-3'

The PCR mix was performed in a final volume of 25 µL containing 100 ng (3µl) DNA, 10 pmole (3µl) of each primer, 12.5 µl of the ready made PCR master mix (Promega Chemical Co.) and 3.5 µl of nuclease free water was added to adjust the volume to 25 µl.

c- PCR conditions as follows: 1 cycle at 95ºC for 5 minutes followed by 33 cycles at 94ºC for 1 minute, 58ºC for 1 minute and 72ºC for 1 minute, 1 cycle at 72ºC for 10 minutes.

d- Agarose gel electrophoresis: was carried out on a 3% agarose gel and visualized with ethidium bromide[11]

e- Single Strand Conformational Polymorphism (SSCP) Analysis[12,13]: Ten µl of PCR product was diluted 1:1 in formamide buffer (95% formamide, 20 mM EDTA pH 8.0, 0.05 xylene cyanol and 0.05% bromophenol blue), kept at 85ºC for 5 minutes and then cooled quickly in ice. The samples were loaded onto a non-denaturating polyacrylamide and run at room temperature for 4 hours using constant voltage (150V) and 1x TBE buffer. Detection was carried out using silver stain and ethidium bromide.

Statistical analysis
Statistical differences between patients and controls were determined with the Fisher’s Exact Test.

Characteristics of cases
The age of menarche was below 13 years in 28 patients (70%) and over 13 years in 12 patients (30%) with a mean age of 13.20±1.3 years. The age of menopause was more than 45 years in 8 patients (20%) with a mean age of 49.10±3.4 years. The remaining 32 patients (80%) did not experience menopause as they developed cancer at young age. The majority of the patients, 37 cases (92.5%), were married. Nullipara constituted 6 patients (15%). Among 34 patients (85%); 23 (67.6%) had their 1^st child below 30 years of age while 11 (32.4%) were over 30 years. Breast feeding was practiced in 30 patients (75%).

Table 1 shows the characteristics of the patients and the controls. There was no significant differences between patients and controls in age at menarche (p=0.138), marital status (p=1.00), parity (p=0.653), age at first delivery (p=0.402) and breast feeding (p=0.707).

Table 1: Characteristics of patients with breast cancer and controls

Classification of cases
The female patients were classified into 2 groups according to the age of onset of the disease. The first group included 25 (62.5%) patients with early onset BC before 40 years. Among this group 2 patients (8%) were with bilateral BC, 22 patients (88%) with unilateral BC and one patient (4%) with BOC. The second group included 15 patients (37.5%) with late onset BC above 40 years of age. Three of them (20%) were with bilateral BC, 11 patients (73.3%) with unilateral BC and one patient (6.7%) with BOC. Positive family history with BC was detected in 2 patients (8%) in the first group and in 13 patients (86.6%) in the second group. Positive family history was significantly higher in the second group patients (p<0.001) (Table 2).

Table 2: Distribution of patients according to age of onset of the disease

Molecular study
DNA was amplified using intronic specific primers spanning exon 2 (262 bp). Similar amplified PCR products for all samples subjected to the present study was detected (Figure 1).

Fig 1: Agarose gel (3%) electrophoresis of PCR products for BRCA1 exon 2 gene (lanes 2-8). Lane 1 represents 50 bp ladder molecular weight marker

The SSCP analysis revealed 4 patients (10%) carried 185 del AG mutation in BRCA1 gene (Figures 2&3). No mutation was detected in the patient’s relatives or in control groups.

Fig 2: Single strand conformational polymorphism (SSCP) analysis of PCR products for BRCA1 exon 2 gene (using ethidium bromide stain). Lanes (1,7) show band variation: upper bands represent the single stranded DNA; the lower bands represent the reannealed double strand DNA. Lanes (10,11) control PCR product (negative for the mutation). Lanes (2,3,4,5,6,8,9) represent normal PCR products for patients without the mutation

Fig 3: Single strand conformational polymorphism (SSCP) analysis of PCR product for BRCA1 exon 2 gene (using silver staining). Lanes (1,2) show band variation: upper bands represent the single stranded DNA and lower bands represent the reannealed double stranded DNA. Lane 5 control PCR product (negative for the mutation). Lanes (3,4) represent PCR products for patients without the mutation

The main findings of the four patients with BRCA1 mutation are summarized in (Table 3).

Table 3: Characteristics of the patients with BRCA1 (185 del AG) mutation

No association between BC and marital status of the patients was detected in the present study as there was no significant difference between patients and controls (p=1.000). This is consistent with other studies that confirmed the absence of significant association between BC and marital status which may indicate that marital status alone without parity has no role in hormone level change in the body[15-17]. However, the married women were significantly higher in the patients group (p<0.001).

Single and nulliparous women were reported to have an increased risk of BC, about 1.4 times the risk of parous women. This low risk of BC in parous women might result from the protective effect of age at first pregnancy and child birth[16-18]. The finding of the present study was contrary to that as parous women were significantly higher in the patients group (p<0.001). These parous women may be exposed to strong risk factors that lead to the BC in spite of the protective effect of parity.

Late age at first child birth increases the life time incidence of BC[19,20]. It was reported that being older than 30 years at first delivery is a risk factor for BC[21]. The highest risk was in those who have a first child after the age of 35 years, they appear to be at even a higher risk than that of nulliparous women[22]. Moreover, epidemiological studies have consistently shown that women who undergo an early first full term pregnancy have a significantly reduced life time risk of BC, this association is independent of parity, i.e. number of live birth[19,20]. In the present study there was no significant difference in age at first delivery between parous patients and controls, moreover the parous patients who gave birth to their first child before 30 years of age were significantly higher (p=0.040) than those who gave birth after 30 years of age.

It has been suggested that breast feeding may protect against BC and increasing years of nursing experience may decrease the BC risk[23]. In the present study no significant difference (p=0.707) between patients and controls was detected but the patients who had experienced breast feeding were significantly higher (p=0.002) among patients group.

Our results indicated that parity, age at first delivery and breast feeding had no effect on the BC risk.

In the present study, the frequency of BRCA1 (185delAG) mutation was 10% in the patients. This is similar to that reported by Santarosa et al.[24] who found the mutation in 10% of Italian females with familial BC, but lower than 19% reported by Lahad et al.[10] in Ashkenazi Jewish. However our result was higher than 1.2% reported by Peelen et al.[25]. This variation in the frequency may be attributed to the ethnic differences.

The age is an important risk factor for breast cancer as the risk increases steadily with age, so occurrence of breast cancer in young age group gives strong implication for the presence of inherited genetic predisposition for breast cancer[10]. In the present study, two patients with 185delAG mutation were below 40 years (2/25; 8%). Several studies found frequencies of 3.5%, 6.7%, 9% and 20% in the patients below 40 years of age[26-29]. In the present study the other 2 patients carrying the mutation were above 40 years (2/15; 13.5%) at the age of onset of the disease. Other studies reported frequencies of 1.9% and 2% in patients above 40 years of age[26,27]. Although the differences between the results of many studies conducted in different populations, most of these studies concluded that BRCA1 185delAG mutation is a strong candidate for early onset breast cancer than in late onset breast cancer. This was inconsistent with our results as the frequency of the mutation was higher in the old age group. This may be attributed to a possible impact of gene-environment interaction which delays the onset of the BC in the old age group.

The occurrence of multilocular cancer is usually associated with inherited mutation of one of cancer predisposing genes, so occurrence of bilateral BC or BOV is suggestive for the presence of dominantly inherited mutation of one of the breast cancer susceptibility genes[30]. In the present study 2 out of 5 (40%) patients with bilateral breast cancer, 1 out of 2 (50%) patients with BOC and 1 out of 33 (3.0%) patients with unilateral breast cancer were found to have the mutation. Steinmann et al.[31] found that the frequency of the mutation was not different between the unilateral and bilateral BC patients and explained the development of bilateral BC to familial aggregation of additional susceptibility factors modifying the penetrance of BRCA1 mutation. Other studies reported a mutation frequency of 20% to 100% in BOC[32,33]. In the present study, although unequal number of patients with unilateral, bilateral BC and BOC, our results indicate that BRCA1 (185delAG) frequency is higher in bilateral BC and BOC than in unilateral BC Egyptian female patients.

A positive family history of breast cancer usually reflects genetic susceptibility and it can be considered as one of the strongest risk factors for the disease[34,35]. In the present study, 2 out of 15 (13.5%) patients with positive family history of BC had BRCA1 gene mutation while 2 out of 25 (8%) patients with negative family history had the mutation. Our finding is higher than 7.4% reported by Friedman et al.[36] and 5.8% reported by Guran et al.[37] in patients with positive family history of BC. However it was lower than the 21% reported by Kumer et al.[38] and 40% by Schubert et al.[39]. Our results indicated that a considerable proportion of the familial risk of BC is attributable to genes other than BRCA1 (185delAG) mutation.

In conclusion, our results indicated that BRCA1 (185delAG) mutation has a role in breast cancer but a considerable proportion of the early breast cancer and familial breast cancer may be due to genes other than BRCA1 (185delAG) mutation. Also, bilateral breast cancer and breast ovarian cancer patients were likely to have the mutation than unilateral breast cancer patients.

1) Alexandria Cancer Registry annual report, 2003 Alexandria, Egypt, Alexandria Cancer Registry, Medical Research Institute, Alexandria University, 2003.

2) Frolov A, Prowse AH, Vanderveer L, et al. DNA arraybased method for detection of large rearrangements in the BRCA1 gene. Genes Chromosomes Cancer 2002;35:232-41.

3) Gad S, Caux MV, Pages BS, et al. Significant contribution of large BRCA1 gene rearrangements in 120 French breast and ovarian cancer families. Oncogene 2002;21:6841-7.

4) Lux MP, Fasching PA, Beckmann MW. Hereditary breast cancer and ovarian cancer: review and future perspectives. J Mol Med 2006;84:16-28.

5) Barois M, Bieche I, Mazoyer S, et al. Real time PCR based gene dosage assay for detecting BRCA1 rearrangements in breast ovarian cancer families. Clin Genet 2004;65:131-6.

6) Farshid G, Balleine RL, Cummings M, et al. Kathleen Cuningham Consortium for Research into Familial Breast Cancer (KConFab). Morphology of breast cancer as a triage of patients for BRCA1 genetic testing. Am J Surg Pathol 2006;30:1357-66.

7) Martin AM, Blackwood MA, Antin OD, et al. Germline mutations in BRCA1 and BRCA2 in breastovarian families from a breast cancer risk elevation clinic. J Clin Oncol 2001;19:2247-53.

8) Bonatti F, Pepe C, Tancredi M, et al. RNA-based analysis of BRCA1 and BRCA2 gene alterations. Cancer Genet Cytogenet 2006;170:93-101.

9) Bellus GA, Hefferon TW, Ortiz de Luna RI, et al. Achondroplasia is defined by recurrent G380R mutations of FGFR3. Am J Hum Genet 1994;56:368-73

10) Lahad EL, Catane R, Eisenberg S, et al. Founder BRCA1 and BRCA2 mutations in Ashkenazi Jewish: Frequency and differential penetrance in ovarian cancer and in breast/ovarian cancer families. Am J Hum Genet 1997;60:1059-67.

11) Sambrook J, Fritch EF, Maniatis T, editors. In: Molecular Cloning, A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, USA, 1989.

12) Hayashi K. Laboratory protocols for mutation detection. Oxford University Press 1996;14-22.

13) Yap EPH, Mc Gee J. Non isotopic SSCP and competitive PCR for DNA quantification: P53 in breast cancer cells. Nucl Acids Res 1992;20:145.

14) Knudson AG. Two genetic hits (more or less) to cancer. Nat Rev Cancer 2001;1:157-62.

15) Pearson PL, Van der Luijt RB. The genetic analysis of cancer. J Intern Med 1998;243:413-7.

16) MacMahon B, Purde M, Cramer D, et al. Association of breast cancer risk with age at first and subsequent births: in a population of the Estonian republic. J Nat Cancer Inst 1982;69:1035-8.

17) Melbye M, Wohlfahrt J, Olsen JH, et al. Induced abortion and the risk of breast cancer. New Engl J Med 1997;336:81-5.

18) Antoniou AC, Shenton A, Maher ER, et al. Parity and breast cancer risk among BRCA1 and BRCA2 mutation carriers. Breast Cancer Res 2006;8:72.

19) Wang Q-S, Ross PK, Yu MC, et al. A case-control study of breast cancer in Tianjin, China. Cancer Epidemiol Biomark Prev 1992;1:435-9.

20) Rosner B, Coldiz GA. Nurses’ Health Study: logincidence mathematical model of breast cancer incidence. J Natl Inst 1996;88:359-64.

21) Bouchardy C, LE MG, Hill C. Risk factors for breast cancer according to age at diagnosis in a French casecontrol study. J Clin Epidiology 1990;43:267-74.

22) Rilke F, Di Palma S. Pathology. In: Textbook of breast cancer. A clinical guide to therapy. Bonadonna G, Hortobagyi GN, Gianni AM, editors. 1st ed. Martin Dunitz Ltd 1998;2-3.

23) Newcomb P, Storer B, Longnecker M, et al. Lactation and a reduced risk premenopausal breast cancer. New Engle J Med 1994;330:81-7.

24) Santarosa M, Dolcetti R, Magri MD, et al. BRCA1 and BRCA2 gene: role in hereditary breast and ovarian cancer in Italy. Int J Cancer 1999;83:5-9.

25) Peelen T, Van Vliet M, Peter-Bosch A, et al. A high proportion of noval mutations in BRCA1 with stronger founder effects among Dutch and Belgian hereditary breast and ovarian cancer families. Am J Hum Genet 1997;60:1041-9.

26) Peto J, Collins N, Barfoot R, et al. Prevalence of BRCA1 and BRCA2 Gene Mutations in patients with Early-Onset Breast Cancer. J Natl Cancer Inst 1999;91:943-9.

27) Abeliovich D, Kaduri L, Lerer I, et al. The founder mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women. Am J Hum Genet 1997;60:505-14

28) Pholreich P, Stribrne J, Kleibl Z, et al. Mutations of the BRCA1 gene in hereditary breast and ovarian cancer in the Czech Republic. Med Princ Parct 2003;12:23-9

29) Roa BB, Friedman L, Kuezli G, et al. Screening for 185delAG in the Ashkenazim. Am J Hum Genet 1997;60:1085-98.

30) Perera FP. Environment and cancer: who are susceptible? Science 1997;278:1068-73.

31) Steinmann D, Bremer M, Rades D, et al. Mutations of the BRCA1 and BRCA2 genes in patients with bilateral breast cancer. Br J Cancer 2001;85:850-8.

32) Phelan CM, Kwan E, Jack E, et al. A low frequency of non-founder BRCA1 mutations in Ashkenazi Jewish breast-ovarian cancer families. Hum Mutat 2002;20:352-7.

33) Hodgson SV, Heap E, Cameron J, et al. Risk factors for detecting germline BRCA1 and BRCA2 founder mutations in Ashkenazi Jewish women with breast or ovarian cancer. J Med Genet 1999;36:369-73.

34) Offit K. Clinical Cancer Genetics: Risk Counseling and Management. New York: Wiley-Liss, 1997.

35) Margolin S, Lindblom A. Familial breast cancer, underlying genes and clinical implications: a review. Crit Rev Oncog 2006;12:75-113.

36) Friedman LS, Szabo CI, Ostermeyer EA, et al. Novel inherited mutations and variable expressivity of BRCA1 alleles, including the founder mutation 185delAG in Ashkenazi Jewish families. Am J Hum Genet 1995;57:1284-97.

37) Guran S, Ozet A, Dede M, et al. Hereditary breast cancer syndromes in a Turkish population. Results of molecular germline analysis. Cancer Genet Cytogenet 2005;160:164-8.

38) Kumer BV, Lakhotia S, Ankathil R, et al. Germline BRCA1 mutation analysis in Indian breast/ovarian cancer families. Cancer Biol Ther 2002;1:22-3.

39) Schubert EL, Mefford HC, Dann JL, et al. BRAC1 and BRAC2 mutations in Ashkenazi Jewish families with breast and ovarian cancer. Genet Test 1997;1:41-6.