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Contents lists available at ScienceDirect

Maturitas
journal homepage: www.elsevier.com/locate/maturitas

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Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population
Sadia Javed a , Muhammad Ali a , Sobia Sadia a , Muhammad Asad Aslam a , Ahmed Ijaz Masood b , Rehan Sadiq Shaikh a , Ali H. Sayyed a,
a b

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Institute of Biotechnology, Bahauddin Zakariya University, Multan 60800, Pakistan Department of Radiotherapy & Oncology, Nishtar Medical College, Multan, Pakistan

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a r t i c l e

i n f o

a b s t r a c t
Cancer incidences and mortality rates are rapidly increasing and breast cancer is among the most frequent malignancy experienced in women worldwide. The occurrence of breast cancer could be associated with various social, cultural, environmental, life-style, hormonal and genetic factors. Objective: To establish if PvuII and XbaI polymorphisms of estrogen receptor alpha would make Pakistani women more susceptible to breast cancer. Furthermore, association between breast cancer and various factors was also explored to establish the contributing factors in breast cancer in Pakistani population. Subjects and methods: Two hundred samples, aged 1565 years, consisting of 100 breast cancer patients and 100 control samples were ascertained for this casecontrol study in order to evaluate the factors related to disease incidence. 57 ml of blood sample of each participating women in the study was collected and analyzed for polymorphisms of PvuII and XbaI using PCR-RFLP method. Results: The menopause had strong inuence on incidences of cancer with ca 18-fold increase in risk of breast cancer in women with menopause compared with non-menopaused. Furthermore signicant impact of menopause age (P < 0.0001) was observed on the incidence of cancer, as high rate of cancer incidence was observed in patients with age between 36 and 45 years (P < 0.0001). Similarly, the genotype XbaI had signicant inuence on the incidence of the disease with heterozygous genotype of XbaI was 45% higher than wild type in cancerous cases. The menopausal women having heterozygous and homozygous mutants of PvuII or XbaI genotypes were strongly correlated with breast cancer (P < 0.01). Conclusion: The polymorphism of genes involving estrogen-metabolizing pathway and estrogen receptor pathway may play an important role in the etiology of breast cancer in Pakistani women. 2011 Published by Elsevier Ireland Ltd.

Article history: Received 8 May 2011 Received in revised form 11 May 2011 Accepted 13 May 2011 Available online xxx Keywords: Breast cancer Polymorphism XbaI PvuII Menopause age

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1. Introduction Globally carcinoma of breast is the most widespread malignancy and the leading cause of death in women [1,2]. In Pakistan, it is the most common at early age compared with the western countries (normal age in West is >60 year) as all women irrespective of their racial or ethnic origin are at the risk of developing breast cancer [3]. Family history is one of the major risk factors for breast cancer and ca 30% cases of the disease are heritable [4]. It has been suggested that the genes of the estrogen pathway may inuence breast cancer risk and can be divided into high and low penetrance genes depending upon the relative risk conferred by them to develop cancer [5]. Breast cancer is heterogeneous in its clinical, genetic and biochemical prole and greatly inuenced by hormonal factors. There

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Corresponding author. Tel.: +92 315 623 7604. E-mail address: ali sayyed@hotmail.com (A.H. Sayyed). 0378-5122/$ see front matter 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.maturitas.2011.05.008

are several established risk factors, such as early menarche, late menopause, nulliparity and late age at rst pregnancy are involved in sufcient exposure to circulating estrogens in the mammary gland [6,7]. The estrogen can inuence the growth, differentiation and function of several target tissues at the onset of puberty including the breast, uterus, vagina, ovary, testis, epididymis, and prostate. The biological effects of estrogens are mainly mediated through estrogen receptors (ERs) such as ER- and ER- [8]. However, estrogen can also have carcinogenic affects, which can be executed in two different ways. First, estrogens promote cell proliferation through ER-mediated pathways and a large number of estrogen-induced molecules have been identied which function to promote cell proliferation, growth and invasiveness due to reduced sensitivity to apoptotic stimuli. Secondly estrogen metabolites also have direct genotoxic effect on DNA damage, mutation and cell transformation [9]. Several common polymorphisms of the ER- gene have been reported to be associated with the alterations in receptor expression and function. Among the widely studied variants of this

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Please cite this article in press as: Javed S, et al. Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population. Maturitas (2011), doi:10.1016/j.maturitas.2011.05.008

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Fig. 1. Ethidium bromide stained gel electrophoresis of restriction product of ER- . 1 kb ladder (lane M); PvuII wild type (lanes 7 and 8) and XbaI wild type (lane 17); PvuII and XbaI mutant genotype (lanes 2, 5 and 10, 12, 15, respectively); PvuII and XbaI heterozygous genotype (lanes 1, 3, 4, 6, 9 and 11, 13, 14, 16, respectively).

54 55 56 57 58 59 60 61 62 63 64 65 66 67

gene implicated with breast cancer risk [8] are the PvuII and XbaI polymorphism in intron 1 [10,11] which have been reported to alter gene expression by inuencing transcription. As estrogen is a risk factor of breast cancer therefore it can be assumed that polymorphic forms of estrogen receptor alpha will be associated to susceptibility of breast cancer and this hypothesis has been investigated in various populations representing diverse results [12]. Apropos above, the objective of the present study was to explore whether PvuII and XbaI polymorphisms in estrogen receptor alpha make Pakistani women more susceptible to breast cancer. Furthermore, association between breast cancer and different demographic factors, hormonal and reproductive factors was also investigated to establish the factors contributing in breast cancer prevalence in Pakistani population. 2. Materials and methods 2.1. Sample collection This study was approved by the ethical review committee of Institute of Biotechnology (IBT), Bahauddin Zakariya University (BZU), Multan and after careful discussion a questionnaire was prepared having questions on variables that could be linked to breast cancer risk [12]. The questionnaire included the information on demographic characteristics, personal and family medical history, detailed menstrual, reproductive and lactation history. One hundred breast cancer patients and 100 control samples were randomly selected, between 15 and 65 years of age and were enrolled for the study after getting proper written consents from each participant. The samples were divided into four age groups viz. 1525 years, 2635 years, 3645 years and 4560 years. It was observed in our preliminary studies that females in the age group of 3645 years were at more risk to breast cancer than early age group. In case of environmental factors smoking and exposure to pesticides were also considered. 2.2. DNA extraction and genotyping of the samples Genomic DNA was extracted from the whole blood samples using non-organic method [13] and quantied using standard protocol. The genotypes for PvuII and XbaI polymorphisms in each sample were determined by polymerase chain reaction restriction fragment lengths polymorphism (PCR-RFLP) using forward and reverse primers 5 CTG CCA CCC TAT CTG TAT CTT TTC CTA TTC TCC 3 and 5 TCT TTC TCT GCC ACC CTG GCG TCG ATT ATC TGA 3 . The 50 l of PCR was set up on ABI 9700 Thermal Cycler (Applied Biosystems, USA) using 400 ng of DNA, 1 PCR buffer, 2.5 mM MgCl2 , 0.25 mM dNTPs, 16 pM of each primer and 5 U of Taq DNA polymerase and following PCR prole was used for amplication; initial denaturation at 94 C for 7 min, followed by 40 cycles of 94 C for 45 s, 61 C for 45 s and 72 C for 90 s. The 1015 l PCR product was cleaved using 5 U of PvuII and XbaI restriction endonuclease at 37 C

for overnight. Restricted products were resolved on 1.5% ethidium bromide stained agarose gel. 2.3. Statistical analysis Genotype frequencies were calculated as the number of participants with a particular genotype divided by the total number of participants. The signicance was described as Pearson P value. Associations between various factors or genotypes and breast cancer risk were calculated as odds ratio (ORs) with 95% condence intervals (CIs), and its statistical signicance was determined by 2 test using SPSS v17 software. The two parameters were considered signicant at P < 0.05 or otherwise stated. 3. Results

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In this study, PvuII and XbaI polymorphisms of estrogen receptor alpha were examined by PCR-RFLP to study their relation with breast cancer susceptibility. The primers generated a 1.372 kb fragment, a part of intron 1 and exon 2 of the estrogen receptor alpha gene (Fig. 1) which was analyzed on 1.5% agarose gel stained by ethidium bromide. Both PvuII and XbaI exist in intron 1, therefore same PCR product was used for digestion with these two restriction enzymes. Genotypes for PvuII and XbaI polymorphisms were denoted by PP, Pp, and pp and XX, Xx, and xx, respectively. Uppercase letters represent the absence of restriction site i.e. 1372 bp, lowercase letters indicate the presence of restriction sites. Restriction by PvuII produces fragments of 936 and 436 bp while restriction by XbaI produces fragments of 982 and 390 bp and heterozygous conditions exhibit three bands. Heterozygous Pp genotype exhibited fragments of 1372, 936 and 436 bp and heterozygous Xx exhibited fragments of 1372, 982 and 390 bp (Fig. 1). The menopause had strong inuence on incidences of cancer ( 2 = 20.22; df = 91; P < 0.0001). A strong correlation was observed between menopause and breast cancer with ca 18-fold increase in risk of breast cancer in menopause women compared with nonmenopause (OR 17.5; 95% CI = 4.0376.01; P < 0.001). Since 75% of the tested cancerous cases were from menopausal women while only 25% were the cancerous cases from normal women (Table 1). Furthermore menopause age had signicant impact ( 2 = 16.25; df 168; P < 0.0001) on the incidence of cancer as 83.3% had menopause before the age of 48 years. The cases of cancer were signicantly higher (t = 26.06; df = 167; P < 0.0001) between the age of 3645 years (Table 1). However other factors such as marital status, age at rst pregnancy, total number of pregnancies, breast feeding and age at last breast feeding, smoking and pesticides exposure were not associated with breast cancer (Table 1). The genotype PvuII had no signicant correlation with phenotype (F = 2.48; df = 2, 181; P > 0.05), heterozygous genotype of PvuII was carried in 42.3% of the total individuals surveyed while 35.7% had homozygous mutant genotype while only 21.9% carried wild type genotype. The prevalence of heterozygous was

113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149

Please cite this article in press as: Javed S, et al. Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population. Maturitas (2011), doi:10.1016/j.maturitas.2011.05.008

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S. Javed et al. / Maturitas xxx (2011) xxxxxx 3 Category 1525 2635 3645 4660 6070 Post menopause Pre-menopause 2838 3948 4958 N/A Yes No 1525 2635 No, N/A 2535 3645 4655 5665 N/A Yes No 1525 2635 No, N/A 15 610 1115 No, N/A Yes No 1525 2635 3645 N/A Yes No Yes No Controln = 100 28 (28%) 38 (38%) 17 (17%) 13 (13%) 4 (4%) 16 (16%) 84 (84%) 2 (2%) 7 (7%) 7 (7%) 84 (84%) 91 (91%) 9 (9%) 85 (85%) 6 (6%) 9 (9%) 0 0 0 0 100 (100%) 0 100 73 (73%) 13 (13%) 14 (14%) 62 (62%) 21 (21%) 3 (3%) 14 (14%) 72 (72%) 28 (28%) 20 (20%) 50 (50%) 15 (15%) 15 (15%) 13 (13%) 87 (87%) 29 (29%) 71 (71%) Casen = 100 20 (20%) 48 (48%) 20 (20%) 11 (11%) 1 (1%) 49 (49%) 51 (51%) 6 (6%) 34 (34%) 8 (8%) 52 (52%) 97 (97%) 3 (3%) 89 (89%) 7 (7%) 4 (4.0%) 26 (26%) 43 (43%) 22 (22%) 9 (9%) 0% 16 (16%) 84 (84%) 71 (71%) 16 (16%) 13 (13%) 53 (53%) 32 (32%) 2 (2%) 13 (13%) 82 (82%) 18 (18%) 8 (8%) 52 (52%) 24 (24%) 16 (16%) 4 (4%) 96 (96%) 16 (16%) 84 (84%) P value

Table 1 Selected risk factors of breast cancer for individuals of Southern Punjab. Characteristics

Age

0.319ns

Menopause

0.000***

Menopause age

0.000***

Marriage

0.074ns

Marriage age

0.351ns

Breast cancer age

0.000***

History of BC

0.000***

Age at 1st pregnancy

0.829ns

Total no of pregnancies

0.358
ns

Breast feeding

0.093ns

Breast feeding age

0.063*

Smoking Pesticide exposure

0.022** 0.028**

ns, non-signicant. * Marginally signicant ** Signicant. *** Highly signicant.

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found to be higher than other genotypes; however mutant genotype was 1.32-fold higher in infected individuals than normal (OR = 1.32; 95% CI = 0.247.34). In contrast, wild type genotype was 2-fold (OR = 1.96; 95% CI = 0.616.32) greater in normal than infected individuals (Table 2). However, the genotype XbaI had

signicant inuence on the incidence of the disease (F = 4.44; df 2, 180; P < 0.05). The heterozygous genotype of XbaI was found in 45.2% higher than wild type present in 17.1% and mutant in 37.6% of individuals surveyed. The wild type genotype was 1.7fold greater in normal samples than infected while heterozygous

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Table 2 PvuII and XbaI polymorphisms and breast cancer. Genotypes PP Pp pp XX Xx xx Control 25 (25.3%) 41 (41.4%) 33 (33.3%) 20 (20.2%) 39 (39.4%) 40 (40.4%) Case 18 (18.6%) 42 (43.3%) 37 (38.1%) 12 (12.4%) 47 (48.5%) 38 (39.2%) Total 43 (21.9%) 83 (42.3%) 70 (35.7%) 32 (16.3%) 86 (43.9%) 78 (39.8%) P value 0.507ns

PvuII

XbaI

0.250
ns

ns, non-signicant.

Please cite this article in press as: Javed S, et al. Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population. Maturitas (2011), doi:10.1016/j.maturitas.2011.05.008

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S. Javed et al. / Maturitas xxx (2011) xxxxxx Normal 19 (19.19%) 6 (6.06%) 0 (0.00%) 1 (1.01%) 31 (31.31%) 9 (9.09%) 0 (0.00%) 2 (2.02%) 31 (31.31%) Affected 12 (12.37%) 6 (6.18%) 0 (0.00%) 0 (0.00%) 37 (38.14%) 5 (5.15%) 0 (0.00%) 4 (4.12%) 33 (34.02%) Total 31 (15.82%) 12 (6.12%) 0 (0.00%) 1 (0.51%) 68 (34.69%) 14 (7.14%) 0 (0.00%) 6 (3.06%) 64 (32.65%)

Table 3 Cross-tabulation of PvuII and XbaI combination with breast cancer. Genotype combination PvuII++/XbaI++ PvuII++/XbaI+ PvuII++/XbaI PvuII+/XbaI++ PvuII+/XbaI+ PvuII+/XbaI PvuII/XbaI++ PvuII/XbaI+ PvuII/XbaI

160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187

were 1.2-fold higher in infected individuals compared with normal (Table 2). With the combined analysis of wild type genotypes of both the genes, their prevalence was 1.6-fold higher in normal samples than affected. In contrast the PvuII mutant and heterozygous XbaI was 2-fold higher in affected samples compared with normal samples (Table 3). A strong correlation between PvuII or XbaI polymorphism and menopause with breast cancer was observed (Table 4). All three forms of PvuII (homozygous wild, homozygous mutant and heterozygous genotypes) in combination with menopause were associated with breast cancer susceptibility but menopause women having heterozygous and homozygous mutant genotypes had strong correlation with breast cancer than wild type genotype (P < 0.01). In contrast the wild type genotype of XbaI had no correlation with breast cancer (P = 0.187) but the heterozygous and homozygous mutants of XbaI had signicant correlation with breast cancer in menopausal women (P < 0.01). Cross tabulation of different combinations of PvuII, XbaI and menopause was done with breast cancer. It was observed that menopausal women with the combinations of heterozygous and homozygous mutant allelic forms had correlation with breast cancer. The combinations of PvuII+ and XbaI+ (P < 0.05), PvuII+/XbaI (P < 0.05) and PvuII/XbaI (P < 0.05) had signicant correlation with breast cancer (Table 5). Cross tabulation of different combinations of PvuII, XbaI and pesticide exposure was also done with breast cancer but no signicant results were observed (Table 6). 4. Discussion Estrogen is implicated in the development and progression of numerous diseases such as breast cancer, ovarian cancer, colorectal cancer, prostate cancer, and endometrial cancer, osteoporosis, neurodegenerative diseases, cardiovascular diseases, and obesity.
Table 4 Cross-tabulation of PvuII and XbaI polymorphism and menopause with breast cancer. Genotype Menopause

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However breast cancer and prostate cancer are greatly affected than any other disease. Estrogen induces cellular changes by binding to its receptors ESR- and ESR- and polymorphism exists in these receptors. Among these polymorphisms PvuII and XbaI are most widely studied polymorphisms [9,11,14,15]. In the present studies all three forms of PvuII polymorphism were identical in cases as well as control samples and only mutant seems to contribute to the increased risk of breast cancer. Similarly in XbaI polymorphism mutant and heterozygotes seems to be highly distributed among cases than controls and therefore could contribute in breast cancer. In a hospital based study in Norway, Andersen et al. [16] found that allele frequencies of the PvuII polymorphism did not differ between cases and controls. The frequency of the mutant allele of the XbaI polymorphism among breast cancer patients, however, was 1.4fold of that of controls [8]. Similarly South Korean women carrying mutant genotype of XbaI has 2.5-fold higher risk of breast cancer compared with control but the PvuII polymorphism distribution was identical between the breast cancer patients and the controls [17]. In contrast, a large-scale population-based casecontrol studies conducted in urban Shanghai showed that the pp genotype of PvuII was associated with a relative risk of 1.4-fold when compared with women with PP genotype [8]. The XbaI polymorphism was associated with a non-signicantly elevated risk, however, for Xx and xx genotype, a higher elevated risks was mainly conned to older or postmenopausal women [18]. But in case of Caucasian postmenopausal women, PvuII and XbaI polymorphisms of estrogen receptor alpha do not play a role in breast cancer risk [19]. The differences observed in our study and the previous studies (hospital based versus population based) or Caucasian versus Chinese might be due to differences in experimental design. Along with genotype analysis, various biological and environmental factors were also investigated to determine the inuence of different factors on incidences of breast cancer. The most of the factors analyzed in the present studies include age at rst pregnancy, number of total pregnancies, breast feeding and age at the time of last breast feeding, were found to have no association with breast cancer. The most probable reason for such non-signicant association could be due to socio-economic group from which blood samples were taken. In contrast menopause and occurrence of breast cancer was signicantly correlated when PvuII and XbaI polymorphisms were combined together. The association seems to be stronger among breast cancer cases diagnosed younger than 45 years and was among women without a family history. Age group in which breast cancer was found to be most prevalent was 3645 years of age which shows that in Pakistan women of middle age are at the highest risk of breast cancer development. However no such association was observed between risk of breast cancer in a population-based casecontrol study among younger women (<40 years) carried out in Australia [20].

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Total no. %

Chi-square test (P-value)

Post menopause Control PvuII (TT) PvuII (TC) PvuII (CC) XbaI (AA) XbaI (AG) XbaI (GG) ns, non-signicant. 3 (12%) 7 (17.1%) 6 (18.2%) 4 (20%) 6 (15.4%) 6 (15%) Case 7 (38.9%) 22 (52.4%) 18 (48.6%) 5 (41.7%) 23 (48.9%) 19 (50%)

Pre menopause Control 22 (88%) 34 (82.9%) 27 (81.8%) 16 (80%) 33 (84.6%) 34 (85%) Case 11 (61.1%) 22 (47.6%) 19 (51.4%) 7 (58.3%) 24 (51.1%) 19 (50%) 43 (100%) 83 (100%) 70 (100%) 32 (100%) 86 (100%) 78 (100%) 0.039** 0.001*** 0.007*** 0.187ns 0.001*** 0.001***

Q2 * Marginally signicant.
** ***

Signicant. Highly signicant.

Please cite this article in press as: Javed S, et al. Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population. Maturitas (2011), doi:10.1016/j.maturitas.2011.05.008

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Table 5 Cross-tabulation of combination of PvuII and XbaI and menopause with breast cancer. Gene combination

Normal PvuII++/XbaI++ PvuII++/XbaI+ PvuII++/XbaI PvuII+/XbaI++ PvuII+/XbaI+ PvuII+/XbaI PvuII/XbaI++ PvuII/XbaI+ PvuII/XbaI ns, non-signicant. ** Signicant. *** Highly signicant. 3 (9.7%) 0 (0.00%) 1 (100%) 6 (8.8%) 0 (0.00%) 0 (0.00%) 6 (9.4%)

Affected 5 (16.1%) 2 (16.7%) 0 (0.00%) 18 (26.5%) 4 (28.6%) 3 (50%) 15 (23.4%)

Normal 16 (51.6%) 6 (50%) 0 (0.00%) 25 (36.8%) 9 (64.3%) 2 (33.3%) 25 (39.1%)

Affected 7 (22.6%) 4 (33.3%) 0 (0.00%) 19 (27.9%) 1 (7.1%) 1 (16.7%) 18 (28.1%) 31 (15.8%) 12 (6.12%) 1 (0.5%) 68 (34.69%) 14 (7.14%) 6 (3.06%) 64 (32.65%) 0.206ns 0.455ns

0.021** 0.005*** 0.400ns 0.035**

242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274

The PvuII (CC), PvuII (TC), XbaI (AG) and XbaI (GG) polymorphism though non-signicantly associated with elevated risk but the risk of incident of breast cancer in post menopause association with these genotype is almost 3-fold greater than the control. However, this nding needs to be further conrmed as the sample size in the present studies was not high enough. In the present studies there are some evidences that combination of PvuII+/XbaI+, PvuII+/XbaI and PvuII/XbaI genotypes may jointly increase susceptibility to breast cancer through the pathway of estrogen-metabolizing and estrogen-binding response. Examination of genegene interaction requires bigger sample size therefore very limited studies have inspected the effect of genetic polymorphism of multiple genes on the risk of breast cancer. For example, Huang et al. [21] reported that three estrogenmetabolizing genes had the joint effect of increasing the risk of breast cancer. Similarly Mitrunen et al. [22] reported that the combination of COMT-L allele with the GSTM1 null genotype posed a signicantly increased risk of breast cancer. Those and the present studies indicated that multiple genes may be involved in increasing the risk of breast cancer. In summary, in Pakistani women population, more than 3-fold increased risk of breast cancer was observed to be associated with heterozygote and mutant genotypes of PvuI and XbaI. There was some indication that womens age at diagnosis of breast cancer may modify the association between these polymorphisms and breast cancer risk. The risk of breast cancer was further increased when combination of those susceptible genotypes was present simultaneously. The ndings of the present study suggest that polymorphism of genes involving estrogen-metabolizing pathway and estrogen receptor pathway may play an important role in the etiology of breast cancer as different results for different studies have been reported. However, further studies could clarify the role of the genes in development and progression of different diseases and

may help to identify diagnostic or therapeutic markers. It would allow tailoring the type and duration of hormone therapy based on genetic prole and provides a means to optimize treatment for each patient [11].

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Contributors
Q1

279

Sadia Javed, as the study was part of her MPhil degree therefore whole experiments were carried out by her. She collected the sample from the hospital, conducted the survey by interviewing the patients and normal samples and has written the rst draft of the manuscript. Muhammad Ali, MPhil supervisor of Ms Sadia Javed therefore he designed the project and helped in accomplishing her studies. Sobia Sadia, helped Ms Sadia Javed during genetic analysis of collected samples and also helped in sample collections. Muhammad Asad Aslam, helped Ms Sadia Javed in optimization/standardization of samples to carry out genetic analysis. He also has seen and approved the nal version. Ahmed Ijaz Masood, As he is the head of the radiotherapy unit of Nishtar Hospital, Multan, Pakistan, he helped in Ms Sadia Javed in identication and collection of blood samples of cancer patients and controls. He also has seen and approved the nal version. Rehan Sadiq Shaikh, co-supervisor of Ms Sadia Javed for her MPhil degree, in collaboration with Prof. Ali he designed the project and helped Ms Sadia Javed in collecting blood samples and accomplishing the survey. He also helped Ms Sadia Javed in preliminary analysis of the data and completing earlier version of the thesis and manuscript. Ali H. Sayyed, re-analyzed the whole data set and has rewritten the whole manuscript.

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Table 6 Cross-tabulation of combination of PvuII and XbaI and pesticide exposure with breast cancer. Gene combination Pesticide exposed Not Exposed to pesticides Total Chi-square test (P-value)

Normal PvuII++/XbaI++ PvuII++/XbaI+ PvuII++/XbaI PvuII+/XbaI++ PvuII+/XbaI+ PvuII+/XbaI PvuII/XbaI++ PvuII/XbaI+ PvuII/XbaI ns, non-signicant. 6 (19.4%) 1 (8.3%) 1 (100%) 9 (13.2%) 1 (7.1%) 0 (0.00%) 10 (15.6%)

Affected 5 (16.1%) 1 (8.3%) 0 (0.00%) 5 (7.4%) 1 (7.1%) 0 (0.00%) 4 (6.3%)

Normal 13 (41.9%) 5 (41.7%) 0 (0.00%) 22 (32.4%) 8 (57.1%) 2 (33.3%) 21 (32.8%)

Affected 7 (22.6%) 5 (41.7%) 0 (0.00%) 32 (47.1%) 4 (28.6%) 4 (66.7%) 29 (45.3%) 31 (15.8%) 12 (6.12%) 1 (0.5%) 68 (34.69%) 14 (7.14%) 6 (3.06%) 64 (32.65%) 0.705ns 1.000ns 0.140ns 1.000ns 0.071ns

Please cite this article in press as: Javed S, et al. Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population. Maturitas (2011), doi:10.1016/j.maturitas.2011.05.008

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Competing interest None. Funding The study was the part of MPhil degree and no funding agency contributed in completing this study. Ms Javed was solely responsible for carrying out the study therefore the funding for this study came from Ms Javeds own nancial resources as well as contribution from the department. References
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Please cite this article in press as: Javed S, et al. Combined effect of menopause age and genotype on occurrence of breast cancer risk in Pakistani population. Maturitas (2011), doi:10.1016/j.maturitas.2011.05.008