Table of Contents Author Guidelines Submit a Manuscript
Disease Markers
Volume 2015, Article ID 365845, 6 pages
http://dx.doi.org/10.1155/2015/365845
Research Article

Polymorphisms rs12998 and rs5780218 in KiSS1 Suppressor Metastasis Gene in Mexican Patients with Breast Cancer

1División de Medicina Molecular, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Sierra Mojada, No. 800, Colonia Independencia, 44340 Guadalajara, JAL, Mexico
2Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada, No. 950, Colonia Independencia, 44340 Guadalajara, JAL, Mexico
3Laboratorio de Inmunología, Departamento de Fisiología, Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Sierra Mojada, No. 950, Colonia Independencia, 44340 Guadalajara, JAL, Mexico
4División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social, Sierra Mojada, No. 800, Colonia Independencia, 44340 Guadalajara, JAL, Mexico
5IMAREFI, Centro Universitario de Ciencias Biológicas y Agropecuarias, Universidad de Guadalajara, Camino Ing. Ramón Padilla Sánchez, No. 2100, Nextipac, 44600 Zapopan, JAL, Mexico

Received 10 July 2014; Revised 26 January 2015; Accepted 4 February 2015

Academic Editor: Natacha Turck

Copyright © 2015 Edhit Guadalupe Cruz Quevedo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Aims. KiSS1 is a metastasis suppressor gene associated with inhibition of cellular chemotaxis and invasion attenuating the metastasis in melanoma and breast cancer cell lines. Along the KiSS-1 gene at least 294 SNPs have been described; however the association of these polymorphisms as genetic markers for metastasis in breast cancer studies has not been investigated. Here we describe two simple PCR-RFLPs protocols to identify the rs5780218 (9DelT) and the rs12998 (E20K) KiSS1 polymorphisms and the allelic, genotypic, and haplotypic frequencies in Mexican general population (GP) and patients with benign breast disease (BBD) or breast cancer (BC). Results. The rs5780218 polymorphism was individually associated with breast cancer and the rs12998 polymorphism shows statistically significant differences when GP versus case (BC and BBD) groups were compared . The H1 Haplotype (G/-) occurred more frequently in BC group (0.4256) whereas H2 haplotype (G/T) was the most prevalent in BBD group (0.4674). Conclusions. Our data indicated that the rs5780218 polymorphism individually confers susceptibility for development of breast cancer in Mexican population and a possible role as a genetic marker in breast cancer metastasis for H1 haplotype (Wt/variant) in KiSS1 gene must be analyzed in other populations.

1. Introduction

Breast cancer is the most common malignancy among Mexican mestizo women, accounting for 21.2% of all female cancers [1]. Breast cancer metastasis is a leading cause of death in cancer patients rather than as a primary tumor growth.

KiSS1 is a metastasis suppressor gene located on chromosome 1q32. It is 6151 base pairs in length and has four exons. The first exon is not translated while exons 2 and 3 are coding exons. KiSS-1 can inhibit chemotaxis and invasion by attenuating the metastasis of breast cancer and melanomas [2]. Several studies suggest a possible role for KiSS1 in regulating events after cell-matrix adhesion and cytoskeleton reorganization [2, 3]. KiSS1 expression reduces the metastatic potential by 95% but does not suppress tumorigenicity. It regulates adhesion molecules such as E-cadherin and diminishes MMP9 expression through NF-κB binding inhibition to the promoter [46]. The encoded protein shows a 54-amino acid COOH-terminal extreme which participates as a ligand in G protein-coupled receptors in humans [79]. Along the gene KiSS-1 at least 294 SNPs have been described of which 42 correspond to mutations located in untranslated regions (UTR), 30 in exonic and the rest in intronic regions [10].

The aim of this study is to identify the rs5780218 (9DelT) and the rs12998 (E20K) KiSS1 polymorphisms and the allelic, genotypic, and haplotypic frequencies in Mexican general population (GP) and patients with benign breast disease (BBD) or breast cancer (BC).

The variants in KiSS1 gene analyzed in this study were selected on the basis of theoretical functional relevance or biological implications. The 9 del T (rs5780218) polymorphism of KiSS1 gene is located at the −146 position of the 5′UTR of the mRNA transcript and corresponds to the deletion of an A (adenine). The E20K (rs12998) polymorphism is a GA transition at position 212 of the cDNA which determines the change of Glu-Lys at residue 20 of the protein [10].

So far it is unknown whether these changes are associated with some alteration in the protein function or whether they confer susceptibility to develop metastasis in breast cancer patients.

2. Material and Methods

2.1. Study Population

We used information from 225 consecutive breast biopsies and 45 mastectomies from the histopathology files of the Pathological Anatomy Unit at Hospital de Especialidades from Centro Médico Nacional from Instituto Mexicano del Seguro Social. The selection criteria for all patients were unrelated persons, age above 18 years, only female, of the same ethnicity (Mexican mestizos) for two previous generations, no family history of breast cancer, and neither radiotherapy or chemotherapy treatment previous to biopsy. Exclusion criteria included duplicated samples, inability to access patients clinical chart, insufficient amounts of sample, or bad quality DNA after extraction. Only 130/265 samples met the criteria for inclusion in this study. The tissue was histologically typed based on standard criteria [11]. Slides from all cases were reviewed to select specific paraffin-embedded tissue blocks containing primarily affected tissue from which genomic DNA was extracted and analyzed previously for other polymorphisms [12].

One hundred and thirty genomic DNA samples were recovered from paraffin-embedded breast tissue (cases) for the present study, numbered and then processed for genotype analysis by another coauthor who has no knowledge of the histopathological diagnosis. After genotyping was complete, cases were distributed in two groups according to pathological assessment into a breast cancer (BC) group and a benign breast disease (BBD) group (Table 1). Data from 90 genomic DNA extracted from blood samples from general population individuals were also included for comparison (GP group); this group comprises male and female nonrelated and healthy adults (Hardy-Weinberg Equilibrium) and an age range of 18–65 years. All individuals belong to the same Mexican mestizos ethnic group.

Table 1: Diagnosis and age distribution for the studied BC and BBD groups.
2.2. Genetic Analysis

DNA was extracted from paraffin-embedded tissue and from blood samples using conventional methods [13, 14] and stored at −20°C. Genotypes for both rs12998 and rs5780218 polymorphisms in KiSS1 gene were determined by PCR-RFLPs protocols implemented by our group. Table 2 shows the primer sequences and restriction enzymes used. PCR was done in the final volume of 10 μL, adding MgCl2 (3.5 mM), dNTPs (2 mM), primers: forward (0.1 μM), reverse (0.1 μM), DNA (10 ng). PCR conditions were performed with an initial denaturation of 4 min at 94°C followed by 30 cycles (94°C 45 sec; 65°C 45 sec; 72°C 1 min) and a final extention of 10 min at 72°C.

Table 2: Primer sequences, PCR conditions, and restriction enzymes (RE) used for genotyping rs12998 and rs5780218 polymorphisms in KiSS1 gene. AT: annealing temperature.

After PCR-RFLPs the 238 bp fragment (rs12998) and the 294 bp fragment (rs5780218) show two bands each of 158 bp and 80 bp and 224 bp and 70 bp, respectively. In the presence of variant alleles the restriction sites are lost in both polymorphisms (Figures 1(a) and 1(b)).

Figure 1: Genotyping of KiSS1 gene rs12998 (a) and rs5780218 (b) polymorphisms. PCR and hydrolysed products digested with restriction enzymes Nla IV or Sml I separated on 8% polyacrylamide gels with silver nitrate staining. The molecular weight markers used were a 25 bp ladder and a 50 bp ladder, respectively.
2.3. Statistical Analysis

To infer the haplotypes for the groups we used the Haplotype Reconstruction Program . Hardy-Weinberg equilibrium was tested on the observed genotypes. All groups were compared using and Fisher’s exact test when needed; a value of ≤0.05 was considered significant. The strength of any given SNP-cancer association was measured by odds ratio (OR) and its corresponding 95% confidence intervals (CI).

Allelic and genotypic distributions were determined by gene counting and are presented as simple frequencies. Contingency tables and Chi-squared tests were used to compare the allele and genotype frequencies and haplotype association among the BC patients and BBD or healthy individuals as well as to test for the presence of Hardy-Weinberg equilibrium among the genotypes.

3. Results

3.1. Polymorphisms

A total of 220 DNA samples were analyzed: 78 were from patients that were classified within the BC group whilst 52 were of the BBD group and 90 samples were from the GP group (general population individuals).

Table 3 shows the allelic and genotypic frequencies for rs12998 and rs5780218 polymorphisms in KiSS1 gene in the three study groups. There is no difference between BC and BBD groups for both polymorphisms; however, when GP was compared versus BC or BBD groups there are differences which could be due to the type of tissue (blood versus paraffin-embedded tissue) from which DNA was extracted (Table 3).

Table 3: Genotype frequencies for KiSS1 suppressor metastasis gene rs12998 and rs5780218 polymorphisms in three groups and statistical analysis between groups. BC: breast cancer; BCD: breast cancer disease; GP: general population.
3.2. Haplotypes

The haplotype construction used the wild type (Wt) or the variant for each allele at rs12998 and rs5780218 polymorphisms. Based on segregation analysis and homozygosity the haplotype phase could be established in 156 chromosomes from BC, 104 chromosomes from BBD, and 180 chromosomes from GP groups. The four possible haplotypes (rs12998: allele 1, rs5780218: allele 2) are H1: Wt/variant (G/-); H2: Wt/Wt (G/A); H3: variant/variant (A/-); and H4 variant/Wt (A/A). All of them were identified in BC and BBD groups, while only three haplotypes were found in the GP group. The haplotype frequencies are shown in Table 4. The comparison between BBD and GP group showed no differences for neither haplotype H1 (Wt/Variant) nor H2 (Wt/Wt). However, the comparative analysis of haplotypes between BC and BBD groups showed significant differences (, OR = 3.32) as well as the BC or BBD groups versus GP (, OR = 3).

Table 4: Haplotype distribution in patients with breast cancer (BC), breast cancer with metastasis (BC M), breast cancer without metastasis (BC NM), benign breast disease (BBD), and individuals from Mexican general population (GP). Wt: wild type. Haplotypes: H1–H4.

4. Discussion

In KiSS1 gene at least 294 SNPs have been identified [10], none previously associated with breast cancer. The rs12998 and rs5780218 SNPs have not been reported previously in relating to cancer studies. In this association study, individuals from a Mexican general population and patients with benign breast disease or breast cancer were considered. The genotype and allele frequencies of both polymorphisms were similar in the studied groups (), suggesting that these polymorphisms individually do not confer susceptibility to the development of breast cancer in our population. Furthermore, information contained in the clinical history of some patients from BC group allowed us to identify breast cancer patients with or without metastasis (16 and 13, resp.). When the allele and genotype frequencies were analyzed again for both polymorphisms no significant differences between groups were observed (; data not shown). The small sample size has limitation whereby in this study we can only describe these findings without a clear conclusion.

We found differences by comparing the results of rs12998 polymorphism between the case group (BC and BBD) and the general population group () and between BC and GP groups for rs5780218 polymorphism (). However, it is necessary to consider that the biological sample type from which genomic DNA was obtained was different in each group: paraffin-embedded tissue and peripheral blood. So in general population individuals we identified constitutive genotypes of both polymorphisms, while the genotypes in the case group (BC and BBD) were identified in specific tissue (breast) with or without cancer. Loss of heterozygosity (LOH) is the loss of one allele at a specific locus, caused by a deletion mutation, or loss of a chromosome from a chromosome pair, resulting in abnormal hemizygosity [15]. It is detected when heterozygous markers for a locus appear monomorphic because one of the alleles was deleted. Moreover, in several studies in which polymorphisms in cancer related genes (oncogenes or tumor suppressor genes) have been analyzed, LOH is reported when a discordance between germinal and tumoral genotypes is observed (DNA obtained from peripheral blood and tumor tissue) [15]. Paraffin tissue blocks analyzed in this study were obtained in the 2002–2005 period [11]; therefore we did not have access to peripheral blood samples from these patients and we did not identify the constitutive genotypes in order to establish the possible loss of heterozygosity in rs12998 or rs5780218 polymorphisms that could explain the differences observed between genotypes by type of biological sample.

The haplotype distribution between BC, BBD, and GP groups shows that the H1 haplotype frequency is significantly higher in BC (0.4256) and particularly among patients with breast cancer and metastasis (0.5101), than in BC NM, BBD, or GP groups (Table 3); therefore H1 haplotype might be considered as a possible risk haplotype for breast cancer and metastasis in our population, which had not been described before. However it is necessary to consider the main limitations of this study, as the small sample size. However, even with this limitation, our study is the first analysis of the usefulness of these polymorphisms as potential genetic risk markers for breast cancer, and it was possible to establish the allele, genotype and haplotype frequencies in Mexican population. Moreover it was also limiting to not know the germline genotypes in the study subjects, since we only know the genotype present in the tissue from which DNA was obtained and therefore it is not possible to analyze the loss of heterozygosity in these polymorphic markers. Finally, the information in the clinical charts or medical records was limited or poor in several cases of selected patients.

Moreover, we do not know the biological significance of these polymorphisms in cancer cells, and there is no information about other gene variants described in KiSS1. Kisspeptin, encoded by KiSS1 gene was first considered as a tumor metastasis suppressor; however, it plays a major role in regulating the hypothalamic-pituitary-gonadal axis, so many studies have analyzed polymorphisms or haplotypes in KiSS1 gene particularly in central precocious puberty. Huijbregts et al. show that polymorphisms in a G-rich sequence located in the 3′UTR, upstream of the transcription end site of KiSS1 gene, influence its expression level [16, 17]. Likewise, in polymorphisms and gene expression studies it has been shown that KiSS1 low mRNA expressions correlate with venous invasion, advanced clinical stage, occurrence of metastasis, and recurrence in patients with different types of cancer [18, 19]. In breast cancer, Kiss-1/GPR54 system, estrogen-related gene expression profiles, regulation, and transcript isoforms of KiSS1 gene have been described [2024] and Xie et al. showed significant relationship between lymph node involvement and absence of Kiss-1 expression in early breast carcinoma patients () [25]; however, there is no information about whether the presence of a polymorphism could be involved in the differential KiSS1 gene expression. Therefore, it will be necessary to confirm in future studies with a larger sample size, if H1 haplotype described here is a possible risk haplotype for cancer and/or metastasis in breast cancer, to analyze their association with metastasis in other cancer types and analyze mRNA expression levels in their correlation with the presence of polymorphisms to know their possible biological significance.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

This work was partially supported by the Fondo de Investigación en Salud, Instituto Mexicano del Seguro Social (FIS/IMSS/PROT/C2007/064), and Fondo Sectorial Ciencia Básica, CONACyT 2007 Reg. 082292.

References

  1. J. Ferlay, H. R. Shin, F. Bray, D. Forman, C. Mathers, and D. M. Parkin, “GLOBOCAN 2008 v2.0,” Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 10, International Agency for Research on Cancer, Lyon, France, 2010, http://globocan.iarc.fr. View at Google Scholar
  2. J. H. Lee, M. E. Miele, D. J. Hicks et al., “KiSS-1, a novel human malignant melanoma metastasis-suppressor gene,” Journal of the National Cancer Institute, vol. 88, no. 23, pp. 1731–1737, 1996. View at Publisher · View at Google Scholar · View at Scopus
  3. J.-M. Navenot, N. Fujii, and S. C. Peiper, “Activation of Rho and Rho-associated kinase by GPR54 and KiSS1 metastasis suppressor gene product induces changes of cell morphology and contributes to apoptosis,” Molecular Pharmacology, vol. 75, no. 6, pp. 1300–1306, 2009. View at Publisher · View at Google Scholar · View at Scopus
  4. J.-H. Lee and D. R. Welch, “Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1,” Cancer Research, vol. 57, no. 12, pp. 2384–2387, 1997. View at Google Scholar · View at Scopus
  5. C. Yan, H. Wang, and D. D. Boyd, “KiSS-1 represses 92-kDa type IV collagenase expression by down-regulating NF-κB binding to the promoter as a consequence of IκBα-induced block of p65/p50 nuclear translocation,” The Journal of Biological Chemistry, vol. 276, no. 2, pp. 1164–1172, 2001. View at Publisher · View at Google Scholar · View at Scopus
  6. L. Kostadima, G. Pentheroudakis, and N. Pavlidis, “The missing kiss of life: transcriptional activity of the metastasis suppressor gene KiSS1 in early breast cancer,” Anticancer Research, vol. 27, no. 4, pp. 2499–2504, 2007. View at Google Scholar · View at Scopus
  7. M. Kotani, M. Detheux, A. Vandenbogaerde et al., “The metastasis suppressor gene KiSS-1 encodes kisspeptinas, the natural ligands of the orphan G protein coupled receptor GPR54,” The Journal of Biological Chemistry, vol. 276, no. 37, pp. 34631–34636, 2001. View at Publisher · View at Google Scholar · View at Scopus
  8. T. Ohtaki, Y. Shintani, S. Honda et al., “Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor,” Nature, vol. 411, no. 6837, pp. 613–617, 2001. View at Publisher · View at Google Scholar · View at Scopus
  9. A. I. Muir, L. Chamberlain, N. A. Elshourbagy et al., “AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1,” Journal of Biological Chemistry, vol. 276, no. 31, pp. 28969–28975, 2001. View at Publisher · View at Google Scholar · View at Scopus
  10. “A database of human single nucleotide polymorphisms,” 2014, http://www.ncbi.nlm.nih.gov/SNP/.
  11. F. A. Tavassoli and P. Devilee, World Health Organization, International Agency for Research on Cancer. Pathology and Genetics of Tumours of the Breast and Female Genital Organs, IARC Press, Lyon, France, 2003.
  12. A. P. Mendizábal-Ruiz, J. Morales, X. G. Martinez et al., “RAS polymorphisms in cancerous and benign breast tissue,” Journal of the Renin-Angiotensin-Aldosterone System, vol. 12, no. 2, pp. 85–92, 2011. View at Publisher · View at Google Scholar · View at Scopus
  13. S. Gustincich, G. Manfioletti, G. Del Sal, C. Schneider, and P. Carninci, “A fast method for high-quality genomic DNA extraction from whole human blood,” BioTechniques, vol. 11, no. 3, pp. 298–302, 1991. View at Google Scholar · View at Scopus
  14. D. K. Wright and M. M. Manos, “Sample preparation from paraffin-embedded tissues,” in PCR Protocols: A Guide to Methods and Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, Eds., pp. 153–158, Academic Press, New York, NY, USA, 1990. View at Google Scholar
  15. J. E. Krebs, E. S. Goldstein, and S. T. Kilpatrick, Lewin's Genes XI, Jones & Bartlett Learning, 11th edition, 2014.
  16. L. Huijbregts, C. Roze, G. Bonafe et al., “DNA polymorphisms of the KiSS1 3′ Untranslated region interfere with the folding of a G-rich sequence into G-quadruplex,” Molecular and Cellular Endocrinology, vol. 351, no. 2, pp. 239–248, 2012. View at Publisher · View at Google Scholar · View at Scopus
  17. X. Luan, Y. Zhou, W. Wang et al., “Association study of the polymorphisms in the KISS1 gene with central precocious puberty in Chinese girls,” European Journal of Endocrinology, vol. 157, no. 1, pp. 113–118, 2007. View at Publisher · View at Google Scholar · View at Scopus
  18. L. Kostadima, G. Pentheroudakis, and N. Pavlidis, “The missing kiss of life: transcriptional activity of the metastasis suppressor gene KiSS1 in early breast cancer,” Anticancer Research, vol. 27, no. 4 B, pp. 2499–2504, 2007. View at Google Scholar · View at Scopus
  19. M. T. Ruiz, A. L. S. Galbiatti, É. C. Pavarino, J. V. Maniglia, and E. M. Goloni-Bertollo, “Q36R polymorphism of KiSS-1 gene in Brazilian head and neck cancer patients,” Molecular Biology Reports, vol. 39, no. 5, pp. 6029–6034, 2012. View at Publisher · View at Google Scholar · View at Scopus
  20. D. Cvetković, A. V. Babwah, and M. Bhattacharya, “Kisspeptin/KISSIR system in breast cancer,” Journal of Cancer, vol. 4, no. 8, pp. 653–661, 2013. View at Publisher · View at Google Scholar · View at Scopus
  21. K. Jarzabek, L. KozŁowski, R. Milewski, and S. WoŁczyński, “KiSS1/GPR54 and estrogen-related gene expression profiles in primary breast cancer,” Oncology Letters, vol. 3, no. 4, pp. 930–934, 2012. View at Publisher · View at Google Scholar · View at Scopus
  22. D. C. Mitchell, M. Abdelrahim, J. Weng et al., “Regulation of KiSS-1 metastasis suppressor gene expression in breast cancer cells by direct interaction of transcription factors activator protein-2α and specificity protein-1,” The Journal of Biological Chemistry, vol. 281, no. 1, pp. 51–58, 2006. View at Publisher · View at Google Scholar · View at Scopus
  23. S. Mooez, F. A. Malik, M. A. Kayani, R. Rashid, A. Zahid, and A. Khan, “Expressional alterations and transcript isoforms of metastasis suppressor genes (KAI1 & KiSS19 in breast cancer patients,” Asian Pacific Journal of Cancer Prevention, vol. 12, pp. 2785–2791, 2011. View at Google Scholar
  24. E. Papaoiconomou, M. Lymperi, C. Petraki et al., “Kiss-1/GPR54 protein expression in breast cancer,” Anticancer Research, vol. 34, no. 3, pp. 1401–1407, 2014. View at Google Scholar · View at Scopus
  25. F. Xie, H. Yang, S. Wang et al., “A logistic regression model for predicting axillary lymph node metastases in early breast carcinoma patients,” Sensors, vol. 12, no. 7, pp. 9936–9950, 2012. View at Publisher · View at Google Scholar · View at Scopus