Sex hormone-binding globulin polymorphisms in familial and sporadic breast cancer

Asta Försti1,7, Qianren Jin1, Ewa Grzybowska2, Magnus Söderberg3, Helena Zientek2, Marzena Sieminska2, Jadwiga Rogozinska-Szczepka4, Ewa Chmielik5, Beata Utracka-Hutka6 and Kari Hemminki1

1 Department of Biosciences at Novum, Karolinska Institute, SE-14157 Huddinge, Sweden,
2 Department of Tumor Biology, Centre of Oncology, Maria Sklodowska-Curie Institute, Gliwice, Poland,
3 Department of Pathology, Huddinge Hospital, SE-14186 Huddinge, Sweden,
4 I Clinics of Radiotherapy, Centre of Oncology, Maria Sklodowska-Curie Institute, Gliwice, Poland,
5 Department of Pathology, Centre of Oncology, Maria Sklodowska-Curie Institute, Gliwice, Poland and
6 Clinics of Chemotherapy, Centre of Oncology, Maria Sklodowska-Curie Institute, Gliwice, Poland


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ovarian steroids are one of the strongest risk factors for breast cancer. Sex hormone-binding globulin (SHBG) binds and transports sex steroids in the blood, regulating their bioavailable fraction and access to target cells. It can also inhibit the estradiol-induced proliferation of breast cancer cells through its membrane receptor. Three coding-region polymorphisms, which lead to an amino acid change, have been reported. We studied the influence of these three polymorphisms on breast cancer risk in three different populations: Polish familial breast cancer cases, 27% of them carrying a BRCA1/BRCA2 mutation, Nordic familial and sporadic breast cancer cases. The reported G to A polymorphism in exon 1 was not found in the 423 analyzed samples. Instead, we found a C to T transition causing an arg to cys amino acid change within the same codon in one Polish breast cancer patient and her daughter. Both of them were heterozygotes for the exon 8 G to A polymorphism as well. They were diagnosed for bilateral breast cancer and carried a BRCA1 mutation (5382insC). Analysis of the tumor samples showed that they had lost the wild-type allele both at exons 1 and 8 of the SHBG gene. Analysis of the other Polish samples showed no correlation of the exon 8 polymorphism to breast cancer, bilateral breast cancer, BRCA1/BRCA2 mutations or age at diagnosis. No association of the exon 8 polymorphism with breast cancer in the Nordic familial or sporadic cases was found. The C to T polymorphism located in exon 4 was rare in all the studied populations (overall allele frequency 0.011). However, in each of the study populations there was a trend for a lower variant allele frequency in cancer cases than in controls. Variant allele frequency in all the breast cancer cases was significantly lower than in all the controls ({chi}2 = 5.27, P-value 0.02; odds ratio = 0.23, 95% confidence interval 0.05–0.84).

Abbreviations: CI, confidence interval; OR, odds ratio; RFLP, restriction fragment length polymorphism; SHBG, sex hormone-binding globulin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Family history of breast cancer is a well-established and important risk factor for breast cancer. Recent twin studies suggest that hereditary accounts for some 30% of breast cancer which is about 10 times more than the contribution of the currently identified breast cancer susceptibility genes (1–4). Among the identified hereditary risk factors of breast cancer, BRCA1 and BRCA2 are estimated to account for ~2% and ATM for 1–2% of all cases (5–7). Family pedigree linkage studies were important in the identification of the two high-penetrant breast cancer genes, BRCA1 and BRCA2 (8–12). However, attempts to clone further breast cancer genes by linkage analysis have not been successful, probably because of heterogeneity and low penetrance (3). Statistically, genetic association studies are considered more powerful in identifying low-penetrance alleles than linkage studies within pedigrees (13–15). Association studies have been carried out between breast cancer risks and polymorphisms in steroid and xenobiotic metabolizing genes and BRCA1, yet without convincing evidence on consistent relationships (13).

Breast cancer risk is increased in women, who have relatively high premenopausal level of estradiol or whose exposure to ovarian steroids is long because of early menarche and late menopause (16). The effects are probably mediated through the cellular growth-promoting effects of estradiol (17). The factors that regulate serum levels of estrogen include sex hormone-binding globulin (SHBG). Data from prospective studies have shown that postmenopausal women who develop breast cancer have higher pre-diagnostic serum concentrations of estradiol and lower SHBG levels than postmenopausal women who remain healthy (18–20). SHBG is a plasma carrier of androgens and estradiol and it is involved in the regulation of their bioavailability in the target cells (21). In estrogen-dependent breast cancer, SHBG is also able to inhibit estrogen-induced cell proliferation through its membrane receptor (21).

In this paper we have studied the effects of three reported coding-region polymorphisms of the SHBG gene on breast cancer risk in familial and sporadic breast cancer cases. The polymorphisms were selected according to functional considerations. Each of the selected polymorphisms causes an amino acid change and may lead to a change of function of SHBG. The first polymorphism is located in exon 1 within the signal peptide sequence and may thus influence the transport of SHBG across cellular membranes. The second polymorphism is located in exon 4 within the splice donor site and may thus have an effect on correct splicing. The third polymorphism, located in exon 8, causes an Asp to Asn amino acid substitution and introduces an additional N-glycosylation site. Because the glycidic residues of the wild-type SHBG are critical for the correct binding to the receptor, the interaction between SHBG and its receptor may be modified.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This genotype study was of a case-control type, in which the most important criterion in choosing cases and controls is that they are drawn from the same ethnic population (22). Assuming that the polymorphism does not cause early mortality, there is no need to match cases and controls according to other parameters, like age and sex.

Polish samples
The breast cancer cases from 118 Polish families from the Upper Silesia region were included in the study. The inclusion criteria were: (i) at least two first degree relatives with breast and/or ovarian cancer regardless of age; (ii) breast or ovarian cancer diagnosed below 35 years without family history; (iii) bilateral breast cancer regardless of the family history; or (iv) breast and ovarian cancer diagnosed in one patient regardless of the family history. Fifty-eight cases had unilateral breast cancer, 48 had bilateral breast cancer and 12 had breast and ovarian cancer. Twenty-seven of them were BRCA1 mutation carriers, five were BRCA2 mutation carriers and one had a TP53 mutation. Mutation analyses of the BRCA1, BRCA2 and TP53 genes were carried out using PCR single-strand conformation polymorphism and heteroduplex analysis combined with direct sequencing as described earlier (23). Age at diagnosis of the Polish familial cases varied from 23 to 77 years (mean 47 years). Patients with the BRCA1 (5382insC) mutation were diagnosed at ages 28–66 years (mean 45 years). Paraffin-embedded tumor tissue samples were available for loss of heterozygosity analysis. The 66 control samples were from healthy, non-smoking Silesian women, who were between 32 and 45 years of age (mean 39 years). They were office workers and did not have a family history of breast cancer. Genomic DNA was isolated from the blood lymphocytes as reported earlier (24).

Nordic samples
The 48 Nordic familial breast cancer cases were from 40 families with at least two first degree relatives diagnosed for breast cancer and at least one of them diagnosed for bilateral breast cancer. They were diagnosed for breast cancer at ages 27–77 years (mean 50 years). An additional 223 sporadic breast cancer cases were included to the study. The mean age of diagnosis for them was 63 years, ranging from 50 to 76 years. The control samples were selected from a population of 1066 healthy male blood donors. Information on family history of breast cancer was not available for the controls. However, they represented the same ethnic group and were from the same geographic area as the breast cancer cases. Thus, the allele frequencies in our control group represent the allele frequencies in the general population, which is accurate enough in the case of breast cancer, which affects about one in 10 women during their lifetime in Western countries. Genomic DNA was isolated using standard phenol–chloroform extraction method followed by ethanol precipitation.

DNA isolation from paraffin-embedded tissue samples
DNA was isolated from the paraffin-embedded tissue samples as described earlier with some modifications (25). Briefly, 10 µm paraffin sections on glass slides were deparafinized by xylene and rehydrated through lowering concentrations of ethanol. After microdissection of the tumor and normal tissues, the paraffin chips were suspended in 100 µl of digestion buffer (10 mM Tris–HCl pH 8.3, 1 mM EDTA, 1% Tween 20). Fifty micrograms of proteinase K was added and the mixture was incubated at 55°C over night. Proteinase K was inactivated by heating at 80°C for 10 min. Aliquots of the supernatant were used for PCR amplification.

PCR–RFLP
The primer sequences for the polymorphisms in exons 1, 4 and 8 of the SHBG gene, respectively, were designed based on the published GenBank SHBG gene sequence M31651 (Table IGo) (26). The three fragments, 151, 227 and 208 bp, respectively, were amplified by PCR using 10 ng genomic DNA in a 10 µl reaction volume containing 1xPCR buffer, 2.0 mM (SHBG1F/R and 4F/R) or 1.0 mM (SHBG8F/R) MgCl2, 0.2 mM each dNTP, 0.2 µM each primer and 0.3 U Platinum Taq DNA polymerase (Invitrogen, UK). PCR was programmed as 94°C for 2 min followed by 35 cycles of 94°C for 1 min, 63°C (SHBG1F/R and 4F/R) or 57°C (SHBG8F/R) for 1 min, 72°C for 1 min. The final extension was at 72°C for 7 min. The PCR machine used was a PTC-200 from MJ Research.


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Table I. Primers used for the PCR amplification of the sequences in the SHBG gene containing the analysed polymorphisms.
 
The three polymorphisms analyzed in the SHBG gene result in loss of existing restriction sites. We used those restriction sites to detect genotypes by restriction fragment length polymorphism (RFLP) analysis. The PCR products were digested with the restriction enzymes AciI (cleaves at 5'CCGC-3', New England BioLabs), MspI (cleaves at 5'––3', MB, Fermentas, Lithuania) and HinfI (cleaves at 5'GANTC-3', MBI Fermentas), respectively, and analysed on an 8–10% PAGE gel stained with ethidium bromide. All polymorphisms detected were confirmed by a new PCR–RFLP.

DNA sequencing
DNA sequencing was used to confirm variants in the SHBG gene following PCR–RFLP. PCR for sequencing reactions was run in a 50 µl volume using the same conditions and primers as above. The PCR products were purified using Microspin S-400 HR Columns (Amersham Pharmacia Biotech, Uppsala, Sweden). The sequencing reaction was carried out using ABI Prism® Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Warrington, UK). The sequencing reactions were performed using forward and reverse primers separately under temperature conditions of 96°C for 1 min, 25 cycles of 96°C for 15 s, 55°C for 15 s and 60°C for 4 min. The precipitated sequencing products were resuspended in 5 µl of the loading buffer, denatured at 95°C for 2 min and 2.5 µl were loaded onto ABI 377 sequencing gel. The original data were analyzed by ABI prism DNA sequence analysis software for tracking and base calling. The obtained sequence was compared with the published GenBank sequence M31651 using Align software in DNA Star package.

Statistical analysis
Statistical significance for the differences in allelic frequencies of the studied polymorphisms in the SHBG gene between the breast cancer cases and healthy controls was determined by the {chi}2 test. Odds ratios (OR) for allelic frequencies between breast cancer cases and controls were also determined. All the statistical tests were carried out using Epi Info 2000 software.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Analysis of the SHBG polymorphisms
The three populations were screened for the presence of the three published non-synonymous coding-region polymorphisms in the SHBG gene using PCR–RFLP analysis. The published exon 1 G to A polymorphism at nucleotide 2831 (M31651, GenBank) was not found in the analysed 239 Nordic and 184 Polish samples (Table IIGo). Also, the allele frequency of exon 4 C to T polymorphism at nucleotide 3945 (M31651, GenBank) was very low, varying between 0% in the Nordic familial breast cancer cases and 2.3% in the Polish controls (Table IIGo). There was a trend of lower allele frequency in the breast cancer samples than in the control samples in each of the study populations (Table IIGo), but the difference was not significant in any of the different populations. However, the difference in the variant allele frequency was significant when all the breast cancer cases were compared with all the controls [{chi}2 = 5.27, P = 0.02; OR = 0.23, 95% confidence interval (CI) 0.05–0.84]. Exon 8 G to A polymorphism at nucleotide 5790 (M31651, GenBank) was the most common one (Table IIGo). The variant allele frequency in the Nordic familial and sporadic cancer cases (0.063 and 0.056, respectively) was lower than in the controls (0.078), but the difference was not statistically significant. The five Nordic familial breast cancer cases, which were heterozygous for the exon 8 polymorphism, were all from different families. In the Polish familial breast cancer cases the variant allele frequency was higher than in the controls (0.076 and 0.045, respectively), but the difference was not statistically significant.


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Table II. Frequency of variant alleles for different polymorphisms in the SHBG gene in Polish and Nordic populationsa
 
New mutation in SHBG exon 1
A new C to T mutation at nucleotide 2830 (M31651, GenBank) was found in one Polish case and her daughter (Figure 1Go, left panel). This caused an arg to cys amino acid change at codon 25 within the signal peptide sequence. Both the case and her daughter were heterozygotes for the exon 8 G to A polymorphism as well (Figure 1Go, right panel). Both of them were diagnosed for bilateral breast cancer and they were carriers for a BRCA1 5382insC mutation (23). The restriction enzyme fragment pattern showed that the wild-type allele at exon 1 was lost in all the four tumors of the case and her daughter compared to their blood samples (Figure 1AGo, left panel). One tumor from each of the case and her daughter could be analyzed for allelic loss at exon 8 polymorphism by PCR–RFLP, which showed the loss of the wild-type allele in both tumors (Figure 1AGo, right panel). The weak bands of the wild-type alleles seen in the tumor samples (108 and 43 bp in exon 1 and 64 and 69 bp in exon 8) are due to contamination of the tumor tissues by normal tissues. The loss of the wild-type allele was confirmed by sequencing (Figure 1B–DGo).



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Fig. 1. Loss of the wild-type allele at exons 1 (left panel) and 8 (right panel) polymorphisms in the SHBG gene in one Polish breast cancer case and her daughter. (A) PCR–RFLP analysis of the blood samples of the case (mb) and her daughter (db) and their tumors (mt1, mt2, dt1 and dt2, respectively). One wild-type sample is also shown (wt). (B) Sequence analysis of the blood sample of the case: the arrows point to C to T alteration in exon 1 and G to A polymorphism in exon 8. The tumors of the case (C) and her daughter (D) have lost the wild-type allele C in exon 1 and G in exon 8.

 
Because both the case and her daughter were heterozygous for the exon 8 polymorphism, had lost the wild-type allele in their tumor tissues, were BRCA1 5382insC mutation carriers and were diagnosed for bilateral breast cancer, we studied the possibility that polymorphism was linked to BRCA1 mutations, bilateral breast cancer or age at diagnosis. No association was detected (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Family history is an important predictive factor for breast cancer risk in women unlikely to carry BRCA1 or BRCA2 mutations (3,5,27), implying that other common, low-penetrance variants may be more important predisposing factors for breast cancer. Also, within BRCA1/2 mutation carriers the penetrance estimates vary depending on the studied population (28–31), indicating involvement of other, both genetic and environmental, modifiers. Association studies using large populations are commonly used in attempts to identify low-penetrance alleles (13). Another approach is to evaluate candidate low-penetrance genes as modifiers of high-penetrance genes (32). We combined these two approaches in our study by using three different populations with low to high risks for breast cancer. The Nordic familial breast cancer cases with at least one first degree family member affected by bilateral breast cancer have a relative risk of about three compared with the Nordic sporadic cases (33). For the Polish familial breast cancer cases, 27% of them being BRCA1/2 mutation carriers, the risk is probably even higher.

We studied the influence of the three published non-synonymous coding-region polymorphisms in the SHBG gene on breast cancer risk. The three polymorphisms were chosen because each of them can have an influence on the function of the SHBG protein. The three polymorphisms locate at the signal peptide sequence, at the splice donor site and create an additional N-glycosylation site, respectively, and may thus modify the transport of SHBG across cellular membranes, correct splicing and interaction between SHBG and its receptor, respectively.

The allele frequencies in the breast cancer groups were compared with the allele frequencies in the general population by using ethnically and regionally matched controls. The Polish samples were additionally sex matched. Matching ethnicity rather than age and sex of the cases and controls is more relevant unless the polymorphism is a cause of an early gender-specific mortality (34). The effect on mortality should show deviation from the Hardy–Weinberg equilibrium (HWE). In our study, the genotypes followed the HWE, but because the exon 4 polymorphism was rare only a large deviation would have shown.

The exon 1 polymorphism has been reported to occur in a frequency of 5–15% in European, Asian, African-American and African Pygmy populations (http://www.genome.wi.mit.edu/cvar_snps). However, we did not find any variant alleles in our Nordic and Polish populations. Interestingly, we found another nucleotide change 5' to the published one. The C to T transition caused an amino acid change at the same codon 25 in the signal peptide sequence as the published polymorphism, but instead of replacing arginine by histidine, the new variant caused an arginine to cysteine change. This new C to T alteration was found in one Polish bilateral breast cancer case, who was a BRCA1 5382insC mutation carrier (23). Her daughter was also diagnosed for bilateral breast cancer and carried the same BRCA1 mutation as her mother. She was found to be heterozygous for the C to T alteration as well. All four tumors of these two patients showed a loss of the wild-type allele. Both of them were heterozygous for the exon 8 polymorphism and had lost the wild-type allele in at least one tumor each. The probability that the four tumors would have lost the wild-type allele at exon 1 polymorphism by chance is 1/8. In at least two of the tumors also the wild-type allele at exon 8 polymorphism was lost indicating that these two alterations were linked together. Because no correlation between the exon 8 polymorphism and breast cancer, bilateral breast cancer, BRCA1/2 mutations or age at diagnosis was found in the other Polish samples, it is unlikely that the new alteration or the exon 8 polymorphism modifies the risk for breast cancer in the Polish high-risk families. Loss of the wild-type allele may just be a consequence of genetic instability reported earlier in tumors from BRCA1 mutation carriers (35,36).

No data of the variant allele frequency for the exon 4 polymorphism has been reported (http://www.genome.wi.mit.edu/cvar_snps). In our study populations the variant allele frequency was very low varying from 0% in the Nordic familial breast cancer cases to 2.3% in the Polish controls. There was a trend for a lower variant allele frequency in the breast cancer cases than in the controls, but the difference was significant only when all the breast cancer cases were compared with all the controls. Because of the low frequency of the polymorphism the effect, if any, of this polymorphism on breast cancer risk would be only marginal at the population level.

One study has been carried out on the association of the exon 8 polymorphism and breast cancer in an Italian population (37). The variant allele frequency for the breast cancer cases (10.8%) was significantly higher than in the controls (5.8%), and it was correlated for the ER+/PR+ tumors and tumors diagnosed in patients over 50 years of age. The variant allele frequencies in our study were at the same level as in the Italian study (4.5–7.8%). In the Polish population variant allele frequency was higher in the breast cancer cases than in the controls, while the opposite occurred for the Nordic populations. The variant alleles detected in the Nordic familial breast cancer cases were all in different families. None of the differences were statistically significant. The Polish population was also studied with regard to BRCA1/2 mutations, bilateral breast cancer and age at diagnosis, but no correlation between the polymorphism and these parameters were found.

According to our results none of the published non-synonymous coding-region polymorphisms in the SHBG gene influence the breast cancer risk, either in the high-risk Polish and Nordic families or the Nordic sporadic cases. However, whether the protection of the exon 4 polymorphism is real, a much larger confirmatory study is needed in addition to functional studies.


    Notes
 
7 To whom correspondence should be addressed Email asta.forsti{at}cnt.ki.se Back


    Acknowledgments
 
We thank P.Vaittinen for identification of the Nordic familial breast cancer cases and K.Håkansson and J.Mourad for technical assistance. This work was supported by grant from State Commitee for Scientific Research No. 6P05A 14221 to E.G.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 6, 2002; revised April 22, 2002; accepted April 26, 2002.