Prevalence of the most frequent BRCA1 mutations in Polish population.

The purpose of our study was to establish the frequency and distribution of the four most common BRCA1 mutations in Polish general population and in a series of breast cancer patients. Analysis of the population frequency of 5382insC (c.5266dupC), 300T >G (p.181T >G), 185delAG (c.68_69delAG) and 3819del5 (c.3700_3704del5) mutations of the BRCA1 gene were performed on a group of respectively 16,849, 13,462, 12,485 and 3923 anonymous samples collected at birth in seven Polish provinces. The patient group consisted of 1845 consecutive female breast cancer cases. The most frequent BRCA1 mutation in the general population was 5382insC found in 29 out of 16,849 samples (0.17%). 300T >G and 3819del5 mutations were found in respectively 11 of 13,462 (0.08%) and four of 3923 (0.1%) samples. The population prevalence for combined Polish founder 5382insC and 300T >G mutations was 0.25% (1/400). The frequencies of 5382insC and 300T >G carriers among consecutive breast cancer cases were, respectively, 1.9% (35/1845) and 1.2% (18/1486). Comparing these data with the population frequency, we calculated the relative risk of breast cancer for 5382insC mutation at OR = 17 and for 300T >G mutation at OR = 26. Our results, based on large population studies, show high frequencies of founder 5382insC and 300T >G BRCA1 mutations in Polish general population. Carriage of one of these mutations is connected with a very high relative risk of breast cancer.

A functional promoter polymorphism in the TERT gene does not affect inherited susceptibility to breast cancer.

Telomere dysfunction is a key mechanism in cancer development. The human telomerase reverse transcriptase (TERT) is the rate-limiting catalytic subunit of the telomerase enzyme, which is necessary for the maintenance of telomere DNA length, chromosomal stability, and cellular immortality. In our attempt to identify functional polymorphisms in the TERT gene and their effect on breast cancer risk, we sequenced the promoter of the gene and identified three single nucleotide polymorphisms (SNPs) with a frequency of at least 10%. One of these SNPs, rs2853669 (-244 T > C), has been shown to affect telomerase activity and telomere length. Recently, this SNP has been suggested to affect familial breast cancer risk. In our case-control study using two large breast cancer sample series, including one with 841 cases with inherited susceptibility to breast cancer, we did not find any association with familial or sporadic breast cancer risk. This well-powered study excludes an effect of the functional -244 T > C SNP and two other correlated SNPs on breast cancer risk.

High-throughput genotyping of a common deletion polymorphism disrupting the TRY6 gene and its association with breast cancer risk.

BACKGROUND: Copy number polymorphisms caused by genomic rearrangements like deletions, make a significant contribution to the genomic differences between two individuals and may add to disease predisposition. Therefore, genotyping of such deletion polymorphisms in case-control studies could give important insights into risk associations. RESULTS: We mapped the breakpoints and developed a fluorescent fragment analysis for a deletion disrupting the TRY6 gene to exemplify a quick and cheap genotyping approach for such structural variants. We showed that the deletion is larger than predicted and encompasses also the pseudogene TRY5. We performed a case-control study to test an association of the TRY6 deletion polymorphism with breast cancer using a single nucleotide polymorphism which is in 100% linkage disequilibrium with the deletion. We did not observe an effect of the deletion on breast cancer risk (OR 1.05, 95% CI 0.71-1.56). CONCLUSION: Although we did not observe an association between the TRY6 deletion polymorphism and breast cancer risk, the identification and investigation of further deletions using the present approach may help to elucidate their effect on disease susceptibility.

Polymorphisms in the KDR and POSTN genes: association with breast cancer susceptibility and prognosis.

Angiogenesis is an important step in the development of cancer. Vascular endothelial growth factor is a major regulator of breast cancer angiogenesis, the effects of which are transmitted through the kinase domain receptor (KDR). Up-regulation of KDR by periostin (POSTN) induces angiogenesis. We screened the KDR and the POSTN genes for published single nucleotide polymorphisms (SNPs) and chose two SNPs in each gene for further analyses. We carried out a case-control study consisting of 412 familial and 912 unselected breast cancer cases together with ethnically and geographically selected controls. Genotype, haplotype and genotype combination analyses were carried out to evaluate their effect on susceptibility to and prognosis of breast cancer. A haplotype in the POSTN gene was associated with an increased risk even after correction for multiple comparisons. Nominal associations between the SNPs and prognostic indicators were also observed. Tumors of the KDR 472His allele carriers were less often progesterone receptor negative according to both genotype and haplotype analyses (OR 0.61, 95%CI 0.40-0.92 and OR 0.60, 95%CI 0.40-0.91, respectively). The POSTN -33G allele carriers had more often high grade and estrogen receptor negative tumors (OR 1.75, 95%CI 1.02-3.01 and OR 1.70, 95%CI 1.04-2.78, respectively). The overall and cancer specific survival after 15 years of follow-up was more than 75%, and it did not depend on the genotype. Although a major effect of the SNPs in the KDR and the POSTN genes on breast cancer susceptibility and prognosis was excluded, the effect of the POSTN C-33G SNP on prognosis needs further characterization.

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

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.

Polymorphisms in the growth hormone receptor: a case-control study in breast cancer.

The human growth hormone receptor (GHR) mediates the effects of growth hormone (GH1), starting a signalling cascade that is involved in the regulation of proliferation, differentiation and apoptosis. Recently, an isoform of the GHR gene lacking exon 3 (GHRd3) was associated with accelerated responsiveness to growth hormone. In this study, we investigated the association of the GHRd3 polymorphism with breast cancer risk and performed a haplotype analysis with 3 additional single nucleotide polymorphisms (SNPs) (Gly186Gly, Cys440Phe and Ile544Leu) in the GHR coding region in a Polish cohort. We did not observe any effect of the 4 polymorphisms on breast cancer risk. Neither did the 3 most common haplotypes influence breast cancer risk. However, a rare haplotype (dGGC), containing the GHRd3 allele, was associated with a decreased breast cancer risk (OR 0.30, 95% CI 0.11-0.80).

Functional Impact of Sequence Alterations Found in BRCA1 Promoter/5'UTR Region in Breast/Ovarian Cancer Families from Upper Silesia, Poland.

The 5' region of BRCA1 contains multiple regulatory sequences flanking the two alternative promoters alpha and beta and two alternative, non-coding exons, 1a and 1b. Aberrations within the 5' region BRCA1 (encompassing two alternative promoters alpha and beta and exons 1a and 1b) may be associated with an increased risk of breast and ovarian cancer. In this study we screened 150 patients for polymorphism and mutations in this region of BRCA1. All probands came from familial breast and/or ovarian cancer that had been found to be mutation-negative in a previous search for founder mutations in BRCA1 (185delAG, C61G, 4153delA, 5382insC) or BRCA2 (6174delT, 9631delC). In our study we found several sequence alterations within the non-coding region of BRCA1 by using direct DNA sequencing and allele-specific PCR amplification. Three families with a polymorphic deletion in BRCA1 exon 1b (2223delAAAAA, Acc. U37574) were found. Moreover, two linked nucleotide substitutions (2642A>T, 2743T>C, Acc. U37574) in BRCA1 intron 1 were detected in 16 patients. In order to assess the functional significance of these two sequence variants, we constructed a reporter vector encoding firefly luciferase under the transcriptional and translational control of wild type and altered BRCA1 promoter region. The reporter assay was performed using a lung cancer cell line (NCI-H1299) and a breast cancer cell line (MCF7). We have demonstrated that the analysed sequence variants have no functional significance in our experimental system. However, we have found that the BRCA1 promoter has lower relative activity in the breast cancer cell line compared with the lung cancer cell line. Based on the results of our functional experiments we conclude that the polymorphic deletion 2223delAAAAA and two linked substitutions 2642A>T and 2743T>C do not significantly alter BRCA1 expression and are probably not disease-causing mutations.

Gene Expression Profiling in Hereditary, BRCA1-linked Breast Cancer: Preliminary Report.

Global analysis of gene expression by DNA microarrays is nowadays a widely used tool, especially relevant for cancer research. It helps the understanding of complex biology of cancer tissue, allows identification of novel molecular markers, reveals previously unknown molecular subtypes of cancer that differ by clinical features like drug susceptibility or general prognosis. Our aim was to compare gene expression profiles in breast cancer that develop against a background of inherited predisposing mutations versus sporadic breast cancer. In this preliminary study we analysed seven hereditary, BRCA1 mutation-linked breast cancer tissues and seven sporadic cases that were carefully matched by histopathology and ER status. Additionally, we analysed 6 samples of normal breast tissue. We found that while the difference in gene expression profiles between tumour tissue and normal breast can be easily recognized by unsupervised algorithms, the difference between those two types of tumours is more discrete. However, by supervised methods of data analysis, we were able to select a set of genes that may differentiate between hereditary and sporadic tumours. The most significant difference concerns genes that code for proteins engaged in regulation of transcription, cellular metabolism, signalling, proliferation and cell death. Microarray results for chosen genes (TOB1, SEPHS2) were validated by real-time RT-PCR.

c-MYC Asn11Ser is associated with increased risk for familial breast cancer.

c-MYC is a multifaceted protein that regulates cell proliferation, differentiation and apoptosis. Its crucial role in diverse cancers has been demonstrated in several studies. Here, we analysed the influence of the rare c-MYC Asn11Ser polymorphism on familial breast cancer risk by performing a case-control study with a Polish (cases n = 349; controls n = 441) and a German (cases n = 356; controls n = 655) study population. All cases have been tested negative for mutations in the BRCA1 and BRCA2 genes. A joint analysis of the Polish and the German study population revealed a 54% increased risk for breast cancer associated with the heterozygous Asn11Ser variant (OR = 1.54, 95% CI 1.05-2.26, p = 0.028). The breast cancer risk associated with this genotype increases above the age of 50 years (OR = 2.24, 95% CI 1.20-4.21, p = 0.012). The wild-type amino acid Asn of this polymorphism is located in the N-terminal MYC transactivation domain and is highly conserved not only among most diverse species but also in the N-MYC homologue. Due to the pivotal role of c-MYC in diverse tumours, this variant might affect the genetic susceptibility of other cancers as well.

Polymorphisms in the IGF-1 and IGFBP 3 promoter and the risk of breast cancer.

Binding of IGF-1 to the type I IGF receptor starts a signalling cascade that plays an important role in regulating cell proliferation, differentiation and apoptosis. The interaction between the IGF-1 and its receptor is mainly regulated by a binding protein, IGFBP 3. We studied a CA repeat polymorphism 969 bp upstream of the transcription start site in the IGF-1 gene and an A-202 C polymorphism in the IGFBP 3 gene and tested their association with breast cancer risk using four case-control series with a total of 787 cases and 900 controls. We did not find any association between the breast cancer risk and the IGF-1 repeat length (19 versus non-19) or the IGFBP 3 A-202 C polymorphism in the postmenopausal breast cancer series or in women diagnosed for breast cancer under the age of 50. In the familial breast cancer series we observed a non-significantly increased odds-ratio (OR) in homozygotes for the non-19 alleles of the IGF-1 gene (OR 1.51, 95% CI 0.96-2.39, p=0.07). Similarly, in the familial breast cancer series we detected an increased frequency of the IGFBP 3 -202 C allele carriers (OR 1.50, 95% CI 1.05--2.14, p=0.03). The association was stronger in individuals homozygous for these alleles (OR 3.76, 95% CI 1.44-v-9.81, p=0.006). Thus, the polymorphisms in the IGF-1 and IGFBP 3 genes associated with an increased risk of breast cancer in familial cases carrying the variant alleles.

Vascular endothelial growth factor polymorphisms in relation to breast cancer development and prognosis.

PURPOSE: Angiogenesis is a necessary step in tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is a major mediator of breast cancer angiogenesis. Therefore, we investigated the association of polymorphisms in the VEGF gene with breast cancer risk and prognostic characteristics of the tumors in a large case-control study. EXPERIMENTAL DESIGN: We examined three polymorphisms in the VEGF gene (-2578C/A, -1154G/A, and +936C/T) in 571 familial breast cancer cases from Poland and Germany and -2578C/A, -634G/C, and +936C/T polymorphisms in 974 unselected breast cancer cases from Sweden together with ethnically and geographically selected controls. RESULTS: None of the polymorphisms or any haplotype was significantly associated with either familial or unselected breast cancers. Our study suggests that the +936C/T polymorphism is unlikely to be associated with breast cancer. We also analyzed the unselected cases for genotypes or haplotypes that associated with tumor characteristics. The -634CC genotype and the -2578/-634 CC haplotype were significantly associated with high tumor aggressiveness (large tumor size and high histologic grade, P < 0.01) and the -2578AA genotype and the -2578/-634 AG haplotype with low histologic grade tumors (P = 0.04). The genotypes and haplotypes were not related with other tumor characteristics such as regional or distant metastasis, stage at diagnosis, or estrogen or progesterone receptor status. CONCLUSIONS: Although none of the polymorphisms studied in the VEGF gene was found to influence susceptibility to breast cancer significantly, some of the VEGF genotypes and haplotypes may influence tumor growth through an altered expression of VEGF and tumor angiogenesis.

Association of NCOA3 polymorphisms with breast cancer risk.

The nuclear receptor coactivator 3 (NCOA3, also known as AIB1) is a coactivator of nuclear receptors like the estrogen receptor. NCOA3 is overexpressed in approximately 60% of primary human breast tumors, and high levels of NCOA3 expression are associated with tamoxifen resistance and worse survival rate. In contrast, NCOA3 deficiency suppresses v-Ha-ras-induced breast cancer initiation and progression in mice. Here, we analyzed the influence of NCOA3 coding single nucleotide polymorphisms on breast cancer risk by performing a case-control study using a German and a Polish study population and identified an association between NCOA3 polymorphisms and breast cancer. A joint analysis of the German and the Polish study population revealed a significant protective effect for the 1758G>C (Q586H) and 2880A>G (T960T) variants. In addition, haplotype analysis showed a protective effect of the 1758C-2880A and 1758G-2880G haplotypes (odds ratio 0.79; 95% confidence interval, 0.67-0.93; P = 0.004). Because of the impact of NCOA3 in antiestrogen therapy resistance, these polymorphisms might also influence therapy outcome in breast cancer.

Polymorphisms and haplotype structures in genes for transforming growth factor beta1 and its receptors in familial and unselected breast cancers.

Alterations in TGF-beta signaling appear to be associated with an altered risk of developing cancer, including breast cancer. We carried out a case-control study on 8 polymorphisms, including 5 in the TGF-beta1 gene (G-800A, C-509T, Leu10-->Pro, Arg25-->Pro and Thr263-->Ile), a polyalanine polymorphism (9A-->6A) in the TGF-betaRI gene and 2 (G-875A and A-364G) in the TGF-betaRII gene, using samples from 2 different populations, Polish familial and Finnish unselected breast cancer cases, together with ethnically and geographically matched controls. Additionally, familial breast cancer cases with respective controls from Sweden and Germany were studied in the Leu10-->Pro polymorphism, making the total number of familial cases 659. Allele, genotype and haplotype analysis on the TGF-beta1 gene as well as an analysis of the combinations of genotypes of the TGF-beta1 and its receptor genes in each individual were performed. Population differences in the allele and genotype distributions were found from 5 of the polymorphisms and 3 common haplotypes from the TGF-beta1 gene between the Finnish and other populations. However, no statistically significant difference between the breast cancer and healthy control groups was found for any of the 8 polymorphisms nor did the haplotype or genotype combination analysis reach statistical significance. Thus, none of the studied polymorphisms from the TGF-beta1 and its receptor genes was found to influence significantly susceptibility to breast cancer. The possible contribution of 6A/6A homozygosity in the TGF-betaRI gene to breast cancer needs to be confirmed in an independent study.

The insulin-like growth factor-1 pathway mediator genes: SHC1 Met300Val shows a protective effect in breast cancer.

The insulin-like growth factor 1 (IGF-1) pathway plays an important role in regulating cell proliferation, differentiation and apoptosis. IRS1, IRS2 and SHC1 are the key mediators for the downstream pathway processes. Genetic variation within these genes may lead to altered signalling. We screened IRS1, IRS2 and SHC1 for published coding region polymorphisms and choose five of them, IRS1 Ala804Ala and Gly972Arg, IRS2 Cys816Cys and Gly1057Asp and SHC1 Met300Val, for further analysis. We studied the association of the polymorphisms with breast cancer risk using a case-control design with Polish familial breast cancer cases and respective controls. For the polymorphisms in IRS1 and IRS2 no differences in the allele, genotype or haplotype distributions could be detected between the case and control subjects. Carriers of the variant allele of the SHC1 polymorphism were at decreased risk of breast cancer (OR 0.54, 95% CI 0.32-0.90, P = 0.016). A non-significant trend for a protective effect of the SHC1 Val300 allele was also seen in an independent population consisting of German familial breast cancer cases and matched controls. The joint analysis after Mantel-Haenzsel adjustment of the two populations gave an OR of 0.62 (95% CI 0.41-0.93, P = 0.02) for the SHC1 Val300 carriers. A stronger effect was detected in women diagnosed below the age of 50 (OR 0.54, 95% CI 0.32-0.89, P = 0.01). A genotype combination analysis of the non-synonymous polymorphisms in the IRS1, IRS2 and SHC1 genes did not show any effect on breast cancer risk.

Single nucleotide polymorphisms in breast cancer.

A limited number of genes have been identified that explain heritable risks of breast cancer (BC). We searched for low-penetrant genes in an association study using two populations: 223 Finnish unselected patients and 172 Polish familial cases, both with locally collected healthy controls. Candidate genes included DNA repair genes, methylenetetrahydrofolate reductase (MTHFR) and cyclin D1 genes. The frequencies for single nucleotide polymorphisms (SNPs) were measured in the following genes: NBS1, XPC, XPD, XRCC1, XRCC3, MTHFR, and cyclin D1. Odds ratios (ORs) were calculated to the wild-type genotype. The positive findings in the Finnish series were repeated in the Polish series. Significant findings among Finns were associations to XPC exon 15, XPD exon 10 and XRCC3 exon 7, the latter of borderline significance. None of these results could be repeated in the Polish series. The XPC result among Finns was probably an artifact of the control group deviating from the Hardy-Weinberg Equilibrium (HWE). The attempt to repeat the result for the XPD polymorphism among Poles was probably not valid because the control group deviated from the HWE. We conclude that within statistical power of the present study, none of the tested polymorphisms associated with BC, with the probable exception of XPD.