BRCA 1 ( 185 delAG ) Mutation among Egyptian Breast Cancer Female Patients

This study was conducted to estimate the frequency of BRCA1 (185delAG) mutation among Egyptian female patients with breast cancer. Forty selected female patients with breast cancer, 80 of their female relatives and 10 healthy females as a control group were included in this study. Result: The age of onset of breast cancer was below 40 years in 25 (62.5%) patients and above 40 years in 15 (37.5%) patients. There were significant differences among the patients regarding the age at menarche before 13 years (p=0.011, p<0.05), onset of breast cancer (p=0.000, p<0.001), parity (p=0.000, p<0.001), first delivery before 30 years of age (p=0.04, p<0.05), breast feeding (p=0.002, p<0.05), and positive family history (p=0.000, p<0.001). The frequency of BRCA1 (185delAG) mutation was found among 10% of the patients’ group. Eight percent of patients with early onset below 40 years and 13.5% of patients with onset after 40 years were heterozygotes for the mutation. Three percent of patients with unilateral breast cancer, 40% of patients with bilateral breast cancer and 50% of patients with breast ovarian cancer were carrying the mutation. Our results indicated that breast ovarian cancer and bilateral breast cancer patients were likely to have BRCA1 (185delAG) mutation than in unilateral breast cancer.


INTRODUCTION
Breast cancer (BC) is the most common malignancy in women, accounting for 31% of all female cancers, and responsible for 15% of cancer deaths in women. (1)One million females world wide are diagnosed with BC every year.Treatment of advanced BC is futile and disfiguring, making early detection has a high priority in medical management of the disease. (2)e risk factors of BC include genetic, environmental, and hormonal.Genetic risk factors contribute to about 5%-10% of all Bull High Inst Public Health Vol.38 No. 2 [2008]   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)ny genes have been found to increase susceptibility to cancer and are also associated with familial breast cancer.

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)CA1 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 BRAC1 gene and predispose to development of cancer.(8) The  a-DNA isolation and purification from peripheral blood using standard method. (9)Polymerase chain reaction (PCR) amplification: according to Lahad et   al., (1977). (10)The sequence of the primers used were as follows: d-Agarose gel electrophoresis: was carried out on a 3% agarose gel and visualized with ethidium bromide. (11)ull High Inst Public Health Vol.38 No. 2 [2008]   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 85°C 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 1 X TBE buffer.
Detection was carried out using silver stain and ethidium bromide.

3-Statistical analysis: Statistical
differences between patients and controls, also among patients were determined with the Fisher's Exact Test.

RESULTS
This study included 40 female patients with BC or BOC, 80 of their close female relatives and 10 healthy adult females as a control group.

Characteristics of cases
The age of menarche was below 13 years in 28 patients (70%) and over 13

Classification of cases
The female patients were classified into (Table 2)

Molecular study
DNA was amplified using intronic specific primers spanning exon 2 (262bp).
Similar amplified PCR products for all samples subjected to the present study were detected (Fig. 1).
The SSCP analysis revealed 4 patients (10%) carring 185 del AG mutation in BRCA1 gene.(Fig. 2 and 3).No mutation was detected in the patient's relatives or in control groups.
The main findings of the four patients with BRCA1 mutation are summarized in (Table 3).

DISCUSSION
Multiple menstrual and reproductive events; menarche, pregnancy, breast feeding; may alter BC risk.In the present study there was a significant increase in the patients with menarche before 13 years of age (P<0.05) and the mean age of menarche of patients was 13.2 years and menopause at 49 years.These results were similar to those reported by Knudson et al (14) who found that both early age at menarche (before 13 years) and late age at menopause (more than 45 years) were found to be associated with increased risk of BC especially in susceptible women.
This may be related to a higher life time exposure to the hormones estrogen and progesterone.
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>0.05).(17) However, the married women were significantly higher in the patients group (P<0.05).
Single and nulliparous women were reported to have an increased risk of BC, about 1.4 times the risk of parous women.
(18) The finding of the present study was contrary to that as parous women were significantly higher in the patients group (P<0.05).
These parous women may be exposed to a strong risk factors that lead to the BC inspite 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)oreover, 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 Bull High Inst Public Health Vol.38 No.2 [2008]   patients and controls, moreover, the parous patients who gave birth to their first child before 30 years of age were significantly higher (P<0.05)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 >0.05) between patients and controls was detected but the patients who had experienced breast feeding were significantly higher (p< 0.05) among patients group.
Our results indicated that parity, age at first delivery, and breast feeding had no effect on the BC risk.
In the preset study, the frequency of BRCA1 (185delAG) mutation was 10% among the patients.This is similar to that reported by Santrosa et al., (24) who found the mutation in 10% of Italian females with familial BC, but lower than that reported by Lahad et al., (19%) (10) in Ashkenazi Jewish.
However our result was higher than that reported by Peelen et al., (1.2%). (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%).
Other studies reported frequencies of 1.9% (26) and 2% (27) in patients above 40 years of age .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 studies reported a mutation frequency of 20% (32) to 100% (33) in BOC.In the present study, although unequal number of patients with unilateral, bilateral BC and BOC, are present, 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 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.
(Result ) Our finding is higher than that reported by Friedman et al., (36) (7.4%) and

2 -
Molecular studies to detect BRCA1 (185 del AG) mutation as follows: 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 95°C for 5 minutes followed by 33 cycles at 94°C for 1 minute, 58°C for 1 minute and 72°C for 1 minute, 1 cycle at 72°C for 10 minutes.

Figure ( 3 ):
Figure (3): Single strand conformational polymorphism (SSCP) analysis of PCR product for BRCA1 exon2 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.