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Carcinogenesis Advance Access originally published online on April 10, 2006
Carcinogenesis 2006 27(9):1867-1875; doi:10.1093/carcin/bgl036
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Polymorphisms in genes involved in GH1 release and their association with breast cancer risk

Kerstin Wagner1,*, Kari Hemminki1,2, Ewa Grzybowska3, Rüdiger Klaes4, Barbara Burwinkel1, Peter Bugert5, Rita K. Schmutzler6,7, Barbara Wappenschmidt6,7, Dorota Butkiewicz3, Jolanta Pamula3, Wioletta Pekala3 and Asta Försti1,2

1 Division of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ) Heidelberg, Germany
2 Department of Biosciences at Novum, Karolinska Institute Huddinge, Sweden
3 Department of Molecular Biology, Centre of Oncology, Maria Sklodowska-Curie Institute Gliwice, Poland
4 Institute of Human Genetics, University of Heidelberg Heidelberg, Germany
5 Institute of Transfusion Medicine and Immunology, Red Cross Blood Service of Baden-Württemberg-Hessia, University of Heidelberg, Faculty of Clinical Medicine Mannheim, Germany
6 Division of Molecular Gynaeco-Oncology, Department of Gynaecology and Obstetrics Clinical Center University of Cologne, Germany
7 Center of Molecular Medicine Cologne (CMMC), University Hospital of Cologne Germany

*To whom correspondence should be addressed at: Kerstin Wagner, Division of Molecular Genetic Epidemiology C050, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany; Tel: +49 6221 421811; Fax: +49 6221 421810; Email: K.Wagner{at}dkfz.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 REFERENCES
 
The regulation of growth hormone 1 (GH1) and insulin-like-growth factor-1 (IGF-1) release is under the influence of three pituitary hormones [growth hormone releasing hormone (GHRH), ghrelin (GHRL) and somatostatin (SST)], which act in an autocrine/paracrine fashion in the breast. By binding to their respective receptors, they control cell proliferation, differentiation and apoptosis in a GH1/IGF-1-dependent manner. We investigated single nucleotide polymorphisms (SNPs) in the GHRH, GHRHR, GHRL, GHSR, SST and SSTR2 gene regions in a Polish and a German cohort of 798 breast cancer cases and 1011 controls. Our study revealed an association of a novel TC repeat polymorphism in the SST promoter with a decreased breast cancer risk in the Polish study population [odds ratio (OR), 0.65; 95% confidence interval (CI), 0.44–0.96]. The closely linked SNP IVS1 A+46G showed the same trend. For both polymorphisms the association was stronger in women above the age of 50 (OR, 0.33; 95% CI, 0.14–0.76 and OR, 0.39; 95% CI, 0.18–0.87, respectively). The protective effect of these polymorphisms was confirmed in a haplotype analysis among women above 50 years of age and carrying the two variant alleles (OR, 0.37; 95% CI, 0.17–0.80). In the independent German population, we observed slightly decreased ORs among women above the age of 50 years. In the SSTR2 gene, carriers of the promoter 21/21 TG repeat genotype were at a decreased breast cancer risk (OR, 0.62; 95% CI, 0.41–0.94) compared to carriers of the other genotypes in the Polish population. Furthermore, we identified a protective effect of the GHRHR C-261T SNP in both populations (joint analysis CT+TT versus CC: OR, 0.80; 95% CI, 0.65–0.99). This effect was carried by a haplotype containing the protective allele. Thus, our study concludes a possible protective influence of distinct polymorphisms in genes involved in GH1 release on breast cancer risk.

Abbreviations: GH1, growth hormone 1; GHRH, growth hormone releasing hormone; GHRL, ghrelin; GHSR, growth hormone secretagogues receptor; LD, Linkage disequilibrium; SNP, single nucleotide polymorphism; SST, somatostatin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 REFERENCES
 
The development of the normal breast and the initiation and progression of breast cancer are under the influence of endocrine hormonal growth factors that, when produced locally, can act in an autocrine/paracrine fashion (1). During the last decade, research has revealed an important role of the growth hormone 1 (GH1)/insulin-like-growth factor-1 (IGF-1)-axis on cell proliferation, differentiation and apoptosis and further on breast cancer development. We have shown earlier that polymorphisms in the genes along this axis contribute to breast cancer risk (2–4). GH1 release is stimulated by growth hormone releasing hormone (GHRH) and ghrelin (GHRL), whereas it is inhibited by somatostatin (SST) as demonstrated in Figure 1 (5). Although these hormones are known to be produced in the hypothalamus and the stomach, a production in local tissues, resulting in an autocrine/paracrine mode of action, has been ascertained (6). The hormones mediate their actions through the corresponding receptors, and expression of both the ligands and the receptors have been found in normal and malignant tissues (1,7–9). It has been shown that elevated serum levels of GH1 and IGF-1 increase the risk of breast cancer (10–12) and transgenic mice overexpressing GH1 develop mammary adenocarcinomas (13). On the other hand, GHRH antagonists strongly restrain the serum levels of GH1 and IGF-1 as well as IGF-1 mRNA expression (14). Additionally, SST analogues not only block hormone hypersecretion but also cause variable degrees of tumour shrinkage (15). Lit/lit mice with a mutation in the GHRHR have dramatically reduced GH1 and IGF-1 levels, leading to a reduced growth of mammary tumour transplants (16).


Figure 1
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  		Fig. 1 Schematic diagram of the regulation of GH1 release. The production and secretion of GH1 from the pituitary is stimulated by hypothalamic GHRH and GHRL, and inhibited by SST. All these hormones act by binding to their corresponding receptors. GH1 enters the circulation and binds to its receptor in the liver, activating the expression and release of IGF-1 and its binding proteins, IGFBPs. In the circulation, IGF-1 is bound to IGFBPs, which regulate the interaction between IGF-1 and its receptor. This interaction starts a signalling cascade resulting in increased cellular growth and reduced apoptosis. Several components of the GH1/IGF-1 axis are produced locally in the breast, indicating a possible autocrine/paracrine function.

 
The contribution of genetic factors in the regulation of average daily GH1 secretion is about 27% (17). Thus, polymorphisms in the genes regulating GH1 release may affect the risk of breast cancer. So far, only a few studies have addressed the question about the genetic variation in these genes and cancer susceptibility (18–20). Even less is known about the effect of these polymorphisms on the gene or protein function. The only study on the functional influence of the polymorphisms has been performed by Torrisani et al. (20). This group has shown that the G allele of the SSTR2 A-167G variant is associated with a 60–70% decreased promoter activity.

In this study, we screened the GHRH, GHRHR, GHRL, GHSR, SST and SSTR2 gene regions for published polymorphisms. We chose 12 SNPs and 3 dinucleotide repeats for further analyses in a Polish breast cancer cohort in order to find a possible association with breast cancer risk. The polymorphisms were selected according to a proven or potential functional effect. The positive findings were confirmed in an independent German population.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 REFERENCES
 
Subjects
The analyses were performed on genomic DNA with 405 Polish familial and early age breast cancer cases (mean age 46 years, range 22–81) and 460 regionally and ethnically matched female controls (mean age 42 years, range 16–79). The inclusion criteria for the cases were (i) at least two first-degree relatives with breast and/or ovarian cancer regardless of age, (ii) breast cancer diagnosed before the age of 35 without family history, (iii) bilateral breast cancer regardless of the family history, (iv) breast and ovarian cancer diagnosed in one patient regardless of the family history and (v) breast cancer diagnosed before 50 years of age regardless of family history (21). The subjects corresponding to criteria (i)–(iv), 220 cases, were collected during the years 1997–2002 by the Chemotherapy Clinics and the Genetic Counselling Service (Gliwice, Poland) and the subjects corresponding to criteria (v), 185 cases, were collected during the time period from December 2002 to March 2004 by the Surgery Clinics (Gliwice, Poland). All cases were unrelated. They were tested for four founder mutations in BRCA1 and two in BRCA2 and were found to be negative. These mutations account for more than 90% of the BRCA1/2 mutations in the Polish population (22). The controls were recruited to earlier studies. They were healthy women, who did not have a family history of breast cancer.

An independent population consisting of 393 German familial breast cancer cases (mean age 45 years, range 21–80) and 551 regionally and ethnically matched female controls (mean age 51 years, range 26–68) was used to confirm the positive findings in the Polish population. This population was collected during the years 1996–2005 through the Institute of Human Genetics, University of Heidelberg (Germany) and the Department of Gynaecology and Obstetrics, Cologne (Germany). They were collected according to the criteria used by the German Consortium for Hereditary Breast and Ovarian Cancer as follows: (A1) families with two or more breast cancer cases including at least two cases with onset before the age of 50; (A2) families with at least one male breast cancer case; (B) families with at least one breast cancer and one ovarian cancer case; (C) families with at least two breast cancer cases comprising one case diagnosed before the age of 50; (D) families with at least two breast cancer cases diagnosed after the age of 50. All the cases were unrelated. The entire coding regions of the BRCA1 and BRCA2 genes were screened and cases carrying deleterious BRCA1/2 mutations were excluded from the study (23). The control group included healthy, unrelated female blood donors who were recruited in 2004 and 2005 by the Institute of Transfusion Medicine and Immunology (Mannheim, Germany) and share their ethnic background with the breast cancer case patients (Caucasian populations). According to the German guidelines for blood donation, all blood donors were examined by a standard questionnaire and consented to the use of their samples for research purposes. Although blood donors may be somewhat healthier than controls drawn from the general population, their use has been recommended (24).

Over 90% of the patients and the controls approved the participation to the study and provided a blood sample. From the rest, no blood/DNA sample was available. The study was approved by the ethical committee of the University of Heidelberg.

Polymorphisms selection
Our general procedure for polymorphisms identification was the screening of the entire gene region of the ligands owing to their rather small gene size. For the receptors, we restricted the screening to exons in which polymorphisms have been published (NCBI SNP database) and their flanking intronic regions. Furthermore, we sequenced the receptors' promoter and 3'-UTR. The selection of polymorphisms for investigation was primarily dependent on a proven or potential functional effect of the variant. Thus, non-synonymous single nucleotide polymorphisms (SNPs) were the first choice, especially if the substitution changed the polarity of the amino acid. A potential impact of the amino acid substitution on protein function was evaluated using the PolyPhen prediction tool (http://tux.embl-heidelberg.de/ramensky/polyphen.cgi). Single base changes in promoter sequences may alter the regulation of gene expression and contribute to tumourigenesis, particularly if the variant affects a transcription factor binding site, and were therefore included to our analyses. Putative transcription factor binding sites were identified using the TESS (http://www.cbil.upenn.edu/tess/) and PROMO (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3) databases. Polymorphisms in the 3'-UTR were included due to their potential to influence gene expression. Dinucleotide repeats in the SST and SSTR2 genes have been reported in the Human Genome Database (http://www.gdb.org/) and they were included into our analyses owing to their potential influence on DNA and RNA stability (25). Since SSTR2 is the most common SSTR in breast cancer, we restricted our analyses to this receptor subtype.

SNP screening by sequencing
Sequencing was used to screen the selected gene regions in 23 breast cancer samples, to analyse the polymorphisms Arg51Gln and Leu72Met in the GHRL gene and to confirm about 5% of the genotypes of randomly chosen samples analysed by TaqMan assays.

Amplification was performed with 5 ng genomic DNA in a 10 µl reaction volume using 1x PCR buffer, 1.5 mM MgCl2, 0.11 µM dNTP Mixture (Invitrogen, Paisley, UK), 0.15 µM of each primer (Thermo Electron Corporation, Ulm, Germany) and 0.3 U Platinum Taq Polymerase (Invitrogen). The PCR was carried out in a GeneAmp 9700 PCR system (Applied Biosystems, Foster City, CA) using 94°C for 2 min, followed by 3 cycles of 94°C for 1 min, annealing temperature T1 for 1 min and 72°C for 1 min and 32 cycles of 94°C for 30 s, annealing temperature T2 for 30 s and 72°C for 30 s. The final extension was for 6 min at 72°C. The primer sequences and corresponding annealing temperatures for the polymorphisms analysed further in a larger cohort are listed in Table I. The remaining primer sequences for screening the gene regions are available on request from the corresponding author. For the amplification of GHRHR and the GHRL C-1062T fragments, we included 5% DMSO (Sigma, Munich, Germany) in the PCR reaction and used a Touch-Down PCR with annealing temperatures ranging from 70 to 62°C.


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  		Table I Genes and polymorphisms selected for our study

 
The PCR product was cleaned-up using 0.75 µl of ExoSapIT (USB Amersham, Uppsala, Sweden) for 40 min at 37°C followed by 15 min at 85°C. The sequencing reaction was carried out using the Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) as described earlier (2). The original data were analysed by Sequencing Analysis 3.7 software (Applied Biosystems) for base calling. The obtained sequences were aligned using DNASTAR Lasergene 5.0 software (DNASTAR Inc., Madison, USA).

TaqMan allelic discrimination
Most of the SNP analyses were performed by the allelic discrimination method. TaqMan assays were ordered from Applied Biosystems as Assay-on-Demand when possible and the Assay IDs are listed in Table I. For the SST IVS1A+46G SNP, an Assay-by-Design was used (Applied Biosystems). The reaction was performed in 5 µl using 5 ng of genomic DNA, 1x TaqMan Universal Master-Mix-2x (Applied Biosystems) and 0.6x Assay-Mix (20x) per reaction. PCR was performed at 50°C for 2 min, 95°C for 10 min and 35–45 cycles at 92°C for 15 s and 60°C for 1 min. PCR was performed in a GeneAmp PCR System 9700 thermocycler and the number of cycles was dependent on the genotype clustering. The samples were read and analysed in an ABI Prism 7900HT sequence detection system using SDS 1.2 software (Applied Biosystems).

Fragment analysis
For the analyses of the microsatellite repeats we used a fluorescent fragment analysis. The PCR amplification was performed as described above using the fluorescently labelled primers and annealing temperatures shown in Table I. PCR products were visualized on ethidium bromide stained agarose gels (Invitrogen) and 1 µl of the 1 : 10 diluted PCR product was added to 10 µl HIDI-Formamide/GeneScan ROX350 standard size marker (mixed according to the manufacturers instruction, Applied Biosystems). The mixture was denatured for 5 min at 95°C, loaded onto the ABI PRISM 3100 Genetic Analyser and analysed by the GeneMapper software version 3.0 (Applied Biosystems).

Statistical analysis
The observed genotype frequencies in the breast cancer cases and controls were tested for Hardy–Weinberg equilibrium (HWE) and the difference between the observed and expected frequencies was tested for significance using the {chi}2-test. Statistical significance for the differences in the genotype and haplotype frequencies between the breast cancer cases and the controls was determined by the {chi}2-test. The joint analysis was carried out using Mantel–Haenszel adjustment. Whenever the expected number of cases was smaller than five, Fisher's exact test was used. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for associations between genotypes and breast cancer. The calculations were carried out using the HWE test tool offered by the Institute of Human Genetics, TU Munich, (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) and Epi Info 2000 software. Haplotype effects were estimated by logistic regression analysis using the Statistical Analysis System software (version 9.1.3, SAS Institute, Cary, NY, USA). The haplotype analyses were carried out using the SNPHAP program created by David Clayton, which can be downloaded from the internet (www.gene.cimr.cam.ac.uk/clayton). Linkage disequilibrium (LD) between the SNPs was evaluated using the Haploview program (http://www.broad.mit.edu/mpg/haploview/documentation.php). Because menopausal status of the women has been shown to affect the levels of IGF-1 and IGFBP3 proteins (11,12), we adjusted the results according to the age of diagnosis, less than and equal to or greater than 50 years.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 REFERENCES
 
Selection of polymorphisms
In order to select polymorphisms for the case–control study, we screened the GHRH, GHRHR, GHRL, GHSR, SST and SSTR2 gene regions in a small sample set of 23 breast cancer cases by sequencing.

In the GHRH gene, we only identified three rare SNPs, one in the coding region, one in intron 3 and one in the 3'-UTR. Since the Leu75Phe SNP was the only one in the coding region and the PolyPhen tool predicted it as possibly damaging, we chose this SNP for further analyses. For the GHRHR gene, we confirmed three common SNPs in the promoter, out of which two were in 100% LD (C-771T and C-235T), and three SNPs in introns 1, 7 and 10. Additionally, we found 6 not reported SNPs in introns 1, 2, 5, 9, and 12. The SNPs leading to an amino acid change were too rare to be investigated further or did not exist. For further analyses, we chose the promoter SNPs C-261T and C-235T owing to their location around an SP1 transcription factor binding site.

For GHRL, we confirmed six tightly linked SNPs in the promoter, eight in intron 3 and one in the 3'-UTR. Additionally, we confirmed the Leu72Met and Gln90Leu SNPs in the coding region but the Arg51Gln SNP was not detected in our small sample set. The Arg51Gln residue corresponds to the last amino acid of the mature GHRL product and an amino acid exchange thus may disturb the conversion of pregroghrelin, the primary translated product of GHRL, to the mature peptide by endoproteases (26). Leu72Met is outside the region of the mature GHRL; its function remains unclear. PolyPhen prediction identified them as benign amino acid substitutions. Since these SNPs were in the same sequencing fragment, we decided to study them in our cohort. Additionally, we analysed the promoter SNP G-1062C and the 3'-UTR SNP T+673C in our larger cohort. In the GHSR gene, we confirmed the synonymous variants Gly57Gly and Arg159Arg, which were investigated further, and five SNPs in the intronic sequences.

In the SST gene, we identified a so far unreported TC repeat in the promoter region 818 bp upstream of the translation start site. We confirmed the existence of seven SNPs. Three SNPs out of four in intron 1 were in 100% LD as well as two SNPs in the 3'-UTR. Additionally, we found a novel but rare Ala11Val SNP. We choose the TC promoter repeat, the SNPs IVS1 A+46G and 3'-UTR A+22G for further analyses owing to their potential impact on gene regulation. Furthermore, a TG repeat in intron 1 was investigated. In the SSTR2 gene, we found 3 SNPs in the promoter. Even though they were not tightly linked, we continued only with the A-167G variant due to its proven functional impact (20). In addition, a TG repeat in the promoter near regulatory elements was studied.

GHRHR C-261T is associated with a decreased breast cancer risk
We investigated the GHRH Leu75Phe variant as well as the GHRHR C-261T and C-235T SNPs in the Polish breast cancer cohort. The observed allele frequencies were concordant with the ones reported for Caucasians in the NBCI SNP database. All genotype distributions followed HWE. We did not observe an effect for the GHRH Leu75Phe or the GHRHR C-235T SNPs on breast cancer risk (Table II). For the GHRHR C-261T variant, we observed an equally high protective effect for the heterozygous and homozygous T allele carriers (CT+TT versus CC: OR, 0.73; 95% CI, 0.54–0.99; P = 0.04) (Table II). In order to confirm this finding, we included an independent German population to our study. In this population, the T allele frequency was also lower among the cases than the controls, but the difference was not statistically significant. In a joint analysis of the two populations, we detected a significantly decreased OR among the T allele carriers (OR, 0.80; 95% CI, 0.65–0.99; P = 0.04). Both the women below and above the age of 50 years had similar ORs (data not shown).


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  		Table II Genotype, allele and haplotype distributions of the SNPs in the GHRH and GHRHR genes

 
Logistic regression analysis showed no overall haplotype effect in the GHRHR gene (P = 0.15). However, one of the two haplotypes containing the –261 T allele, TC, was associated with a decreased breast cancer risk (OR, 0.68; 95% CI, 0.48–0.96; P = 0.02, Table II).

GHRL haplotypes are associated with breast cancer risk
We investigated four SNPs in GHRL (G-1062C, Arg51Gln, Leu72Met and 3'-UTR T+673C) and two SNPs in GHSR (Gly57Gly and Arg159Arg) in the Polish cohort. All observed allele frequencies agreed with the ones published among Caucasians (NCBI SNP database). In the GHRL gene, although we did not detect the Arg51Gln SNP in our initial screening, the variant allele was present in 7% of the Polish cases and 5% of the Polish controls. We did not find any differences in the allele or genotype distributions for any of the four studied SNPs between the cases and the controls (Table III). In the haplotype analysis, we found a small haplotype effect using logistic regression analysis (P = 0.07). A statistically significant protective effect was observed for the rare haplotypes GGAC and GGAT (Table III) while the protective effect of the CGCT haplotype was of borderline significance (P = 0.06). For the GHSR gene, the controls for the Gly57Gly variant did not follow HWE (P = 0.004) which lead to a significantly increased breast cancer risk (OR, 2.54; 95% CI, 1.44–4.47; P = 0.001). We excluded genotyping errors by double-checking the genotypes by sequencing 20% of the samples and found no genotype discordances with our first experiments, pointing to the reliability of the genotyping results. Since all genotype distributions of the remaining 14 polymorphisms followed HWE, study design errors can be excluded. Furthermore, the Arg159Arg SNP in the same gene showed a non-significant trend for a decreased OR with the increasing number of the variant alleles (Table III). Adjustment for age did not change the results. Because the studied SNPs did not affect the risk of breast cancer in the Polish cohort, we did not study them in the German cohort.


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  		Table III Genotype, allele and haplotype distributions of the SNPs in the GHRL and GHSR gene in the Polish population

 
SST polymorphisms are associated with a decreased breast cancer risk
We identified a novel TC repeat 818 bp upstream of the translation start site in the putative SST promoter. Either 7 or 10 TC repeats were present. We investigated this polymorphism in the Polish cohort and observed a significantly decreased risk of breast cancer among the 10 TC repeat carriers (OR, 0.65; 95% CI, 0.44–0.96; P = 0.02). This effect was stronger in women above the age of 50 (OR, 0.33; 95% CI, 0.14–0.76; P = 0.004, Table IV). Similarly, the SNP in intron 1 (IVS1 A+46G), that was found to be in 94% LD with the TC repeat, showed a decreased OR in all women and a decreased breast cancer risk in women above the age of 50 (OR, 0.39; 95% CI, 0.18–0.87; P = 0.01, Table IV). We also investigated these SNPs in the German cohort and observed a slightly decreased OR among women above the age of 50 and no effect among all women. In a joint analysis, we observed a protective effect for women above the age of 50 with a borderline significance. Haplotype analyses with these two polymorphisms did not show any overall effect in Polish women (P = 0.11). However, the haplotype effect was significant in women above the age of 50 (P = 0.03) owing to the protective effect of the 10G haplotype (OR, 0.37; 95% CI, 0.17–0.80; P = 0.005, Table IV). This haplotype contained the protective alleles of the TC repeat and the IVS1 A+46G SNP. In the German population and in the joint analysis, no haplotype effect was observed. The 3'-UTR A+22G SNP was not associated with the risk of breast cancer. We examined also the influence of a TG repeat in intron 1 of the SST gene. The repeat length varied from 19 to 28 TG repeats and the allele distribution was similar to the one reported in the GDB database. Twenty-four repeats was the most common allele length and 47% of the cases and 43% of the controls were homozygous for the 24 repeat allele. No significant differences in the genotype distribution between the cases and the controls were observed (overall P-value 0.39, Figure 2A). Adjustment for age did not change the results.


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  		Table IV Genotype, allele and haplotype frequencies in the SST and SSTR2 genes

 

Figure 2
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  		Fig. 2 Genotype distribution of the TG repeat in (A) the SST IVS1 and (B) the SSTR2 promoter. Asterisk indicates a significant difference between the cases and the controls compared to all other genotypes.

 
In the SSTR2 gene, a TG repeat located 4114 bp upstream of the translation start site and immediately downstream of the non-coding exon 1 was investigated. The number of TG repeats varied between 15 and 26 and the allele distribution was according to the GDB database. Alleles with 20 and 21 TG repeats were the commonest alleles (34 and 37%, respectively). Even though there was no overall genotype effect, we observed a significantly lower frequency of the 21/21 genotype carriers among the cases than among the controls compared to all other genotype carriers (OR, 0.62; 95% CI, 0.41–0.94; P = 0.02) (Figure 2B). The SNP with a proven functional effect, A-167G (20), in the SSTR2 promoter revealed no effect on breast cancer risk (Table IV).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
REFERENCES
 
The regulation of the synthesis and secretion of GH1 is multifactorial, but the predominant regulators are the hypothalamic hormones GHRH and GHRL, which stimulate GH1 release, and SST, which exerts inhibitory influences (1). In return, GH1, and also IGF-1, can act to inhibit GH1 secretion directly and indirectly by suppressing GHRH release and stimulating SST production (27). Local production in the target tissues, such as the breast, has been revealed for all these hormones, facilitating their action in an autocrine/paracrine manner.

We investigated the effect of a total of 12 SNPs and 3 dinucleotide repeats in the genes encoding GHRH, GHRL, SST and their receptors on the risk of breast cancer. We included familial and early onset cases to our study because it has been shown earlier that selection of cases based on the family history of the same disease increases the power to detect low-penetrance variants (28,29). With the present sample size, we had a power of 90% to detect an OR of 0.50 with a variant genotype frequency of 0.20 in the control population. In the analyses of the subpopulations consisting of women above and under the age of 50, the power was lower. As another issue, a case–control association study with multiple SNPs or haplotypes in multiple genes represents a statistical multiple comparisons problem. Even though there are several methods for handling multiple comparisons in molecular epidemiology studies, no standard approach has been universally adopted (30). In addition, it is unclear how the biological relevance of a SNP should be taken into account. We did not correct for multiple comparisons, but whenever a polymorphism was associated with the risk of breast cancer in the Polish cohort, we confirmed the finding in an independent German cohort. Additionally, we also studied whether the effect of a SNP was carried by a haplotype, which would increase the reliability of the results.

The polymorphisms were selected based on a proven or potential functional effect. One SNP in the GHRHR gene was associated with a decreased breast cancer risk. In the GHRL gene, three rare haplotypes showed a protective effect. Finally, two tightly linked polymorphisms in the SST gene and the dinucleotide repeat genotype 21/21 in the SSTR2 gene protected against breast cancer. The remaining polymorphisms were not associated with the risk of breast cancer.

From the SNPs analysed in the GHRH and GHRHR genes, only the C-261T SNP in the GHRHR gene was associated with the risk of breast cancer. The –261T allele consistently correlated with a decreased OR and the effect was traced to a haplotype carrying the T allele. The C-261T SNP lies within a fragment that is necessary for the positive regulation of GHRHR transcription and expression (31). It is located next to a SP1 transcription factor binding site which may influence transcription owing to a conformational change.

In the GHRL gene, individual SNPs did not show any effect on the risk of breast cancer but we observed a protective effect for three rare haplotypes. From the SNPs involved in the haplotypes, G-1062C is a tagging SNP for the promoter (32,33) and the Arg51Gln SNP changes the last amino acid of the mature peptide, which may lead to an altered protein cleavage. The Arg51Gln and Leu72Met SNPs have been associated with obesity and altered hormone levels (26,34), although further studies have not confirmed these associations (19,35–37). The 3'-UTR T+673C SNP may affect mRNA stability and expression. Although a general haplotype effect was seen and the haplotype analysis revealed that the three rare haplotypes CGCT (5%), GGAC (1%) and GGAT (1%) were more frequent in the controls than the cases, no consistency in the presence of variant alleles was observed. None of the GHSR SNPs showed an association with the risk of breast cancer.

In the SST gene, we found a novel TC repeat in the SST promoter that contained either 7 or 10 TC repeats. This polymorphism was in 94% LD in the Polish cohort and in 97% LD in the German cohort with a SNP in intron 1 (IVS1A+46G) that may also influence gene regulation. Recently, the IVS1+46G allele has been associated with an increased breast cancer risk in a large study consisting of 807 breast cancer cases and 1588 matched controls nested within the multicentric European Prospective Investigation into Cancer and Nutrition (EPIC) study (18). In our study, both the 10 TC repeat and the IVS1+46G alleles and their joint haplotype were associated with a decreased breast cancer risk in the Polish women, especially among those above 50 years of age. However, we could not confirm the effect in the independent German population, even though the ORs were marginally decreased among women over the age of 50 years. All our 798 breast cancer cases were women having either a family history of breast cancer or an early onset of breast cancer, which would have been expected to increase the power to detect low-penetrance susceptibility alleles. As we see the effect of the polymorphisms only in the Polish cohort, a regional or a nationality influence may be the reason for these opposite findings. As another alternative, the observed associations may be due to chance, and the polymorphisms may not have any significant effect on the risk of breast cancer. Unfortunately, no characterization of the SST promoter has been reported so far. However, our search in the transcription factor binding site databases revealed the existence of c-Myb, IRF-1, NF-1 and SP1 binding sites immediately flanking the repeat region, thus adding to the likelihood of a functional effect. No effect was observed for the TG repeat in intron 1 and the 3'-UTR A+22G SNP.

In the SSTR2 gene, the first exon is non-coding and the whole region up to the translation start site often is referred to as the promoter sequence (38). We investigated a TG repeat polymorphism 4114 bp upstream of the translation start site, immediately downstream of the non-coding exon 1, and identified the 21/21 genotype as a protective factor for breast cancer. This polymorphism resides in the promoter region that has been identified to contain regulatory elements, including an estrogen response element (38). A study on sporadic breast cancer did not find any effect of the SSTR2 repeat polymorphism on breast cancer risk, but the study was small with 109 cases and only the allele distribution was measured (39). The proximal promoter, 200 bp upstream of the translation start site, contains an initiator element necessary for the enhance-like activity. It harbours the A-167G SNP (numbering relative to the translation start site). This SNP is associated with decreased promoter activity, but it was not associated with pancreatic cancer in a study consisting of 22 cases and 50 controls (20). Another study found no differences in serum levels of GH1 and IGF-1 for –167G allele carriers and this genotype was not associated with acromegaly (40). A recent study identified a marginally increased breast cancer risk, but only in heterozygotes for this SNP (18). We did not observe any effect of this SNP on breast cancer risk.

In summary, we investigated the effect of polymorphisms in the GHRH, GHRHR, GHRL,GHSR, SST and SSTR2 genes on the risk of breast cancer. Our results suggest that the polymorphisms in GHRHR, GHRL, SST and SSTR2 may contribute to a decreased breast cancer risk which would be consistent with a decreased activity of the IGF-1 pathway. However, except for the GHRHR C-261T SNP, no other SNP was associated with the risk of breast cancer in both the Polish and the German cohorts. Further studies are needed to investigate if and how the polymorphisms affect the related gene and the protein functions, and further what, if any, are their effects on the function of the GH1/IGF-1 axis and on the diseases linked to this pathway.


   Acknowledgments
 
The German breast cancer samples were collected within a project funded by the Deutsche Krebshilfe, supported by the Center of Molecular Medicine Cologne (CMMC) and coordinated by Prof. Rita K. Schmutzler. This study was supported by the grants from State Committee for Scientific Research (PBZ-KBN-090 P05/02 to E.G.) and a grant from EU (LSHC-CT-2004-503465 to E.G. and K.H.).

Conflict of Interest Statement: None declared.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received January 12, 2006; revised March 23, 2006; accepted March 31, 2006.


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L. Dossus, J. D. McKay, F. Canzian, S. Wilkening, S. Rinaldi, C. Biessy, A. Olsen, A. Tjonneland, M. U. Jakobsen, K. Overvad, et al.
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