Research Article Polymorphisms rs12998 and rs5780218 in...

Research ArticlePolymorphisms rs12998 and rs5780218 in KiSS1 SuppressorMetastasis Gene in Mexican Patients with Breast Cancer

Edhit Guadalupe Cruz Quevedo1 Gabriela Monserrat Mimendi Aguilar12

Luis Anselmo Juaacuterez Aguilar12 Susan Andrea Gutierrez Rubio3

Silvia Esperanza Flores Martiacutenez1 Ingrid Patricia Daacutevalos Rodriacuteguez4

Joseacute Saacutenchez Corona1 Martha Isabel Torres Moraacuten5

Roberto Carlos Rosales Goacutemez1 and Mariacutea Cristina Moraacuten Moguel1

1Division de Medicina Molecular Centro de Investigacion Biomedica de Occidente Instituto Mexicano del Seguro SocialSierra Mojada No 800 Colonia Independencia 44340 Guadalajara JAL Mexico2Centro Universitario de Ciencias de la Salud Universidad de Guadalajara Sierra Mojada No 950 Colonia Independencia44340 Guadalajara JAL Mexico3Laboratorio de Inmunologıa Departamento de Fisiologıa Centro Universitario de Ciencias de la Salud Universidad de GuadalajaraSierra Mojada No 950 Colonia Independencia 44340 Guadalajara JAL Mexico4Division de Genetica Centro de Investigacion Biomedica de Occidente Instituto Mexicano del Seguro Social Sierra MojadaNo 800 Colonia Independencia 44340 Guadalajara JAL Mexico5IMAREFI Centro Universitario de Ciencias Biologicas y Agropecuarias Universidad de GuadalajaraCamino Ing Ramon Padilla Sanchez No 2100 Nextipac 44600 Zapopan JAL Mexico

Correspondence should be addressed to Marıa Cristina Moran Moguel cmmoguelyahoocommx

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

Academic Editor Natacha Turck

Copyright copy 2015 Edhit Guadalupe Cruz Quevedo et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Aims KiSS1 is ametastasis suppressor gene associated with inhibition of cellular chemotaxis and invasion attenuating themetastasisin melanoma and breast cancer cell lines Along the KiSS-1 gene at least 294 SNPs have been described however the associationof these polymorphisms as genetic markers for metastasis in breast cancer studies has not been investigated Here we describetwo simple PCR-RFLPs protocols to identify the rs5780218 (9DelT) and the rs12998 (E20K) KiSS1 polymorphisms and the allelicgenotypic and haplotypic frequencies inMexican general population (GP) and patients with benign breast disease (BBD) or breastcancer (BC) Results The rs5780218 polymorphism was individually associated with breast cancer (119875 = 00332) and the rs12998polymorphism shows statistically significant differences when GP versus case (BC and BBD) groups were compared (119875 lt 00001)The H1 Haplotype (G-) occurred more frequently in BC group (04256) whereas H2 haplotype (GT) was the most prevalentin BBD group (04674) Conclusions Our data indicated that the rs5780218 polymorphism individually confers susceptibility fordevelopment of breast cancer in Mexican population and a possible role as a genetic marker in breast cancer metastasis for H1haplotype (Wtvariant) in KiSS1 gene must be analyzed in other populations

1 Introduction

Breast cancer is the most common malignancy amongMexican mestizo women accounting for 212 of all femalecancers [1] Breast cancer metastasis is a leading cause ofdeath in cancer patients rather than as a primary tumorgrowth

KiSS1 is a metastasis suppressor gene located on chro-mosome 1q32 It is 6151 base pairs in length and has fourexons The first exon is not translated while exons 2 and 3 arecoding exons KiSS-1 can inhibit chemotaxis and invasion byattenuating the metastasis of breast cancer and melanomas[2] Several studies suggest a possible role for KiSS1 inregulating events after cell-matrix adhesion and cytoskeleton

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 365845 6 pageshttpdxdoiorg1011552015365845

2 Disease Markers

Table 1 Diagnosis and age distribution for the studied BC and BBD groups

Breast cancer group diagnosis 119899 Age 119899

Adenocarcinoma 2 lt30 2Ductal adenocarcinoma 4 30ndash39 10Invasive ductal adenocarcinoma 47 40ndash49 20Invasive lobular carcinoma 5 50ndash59 12Lobular adenocarcinoma 1 60ndash69 11Lobular and ductal adenocarcinoma 1 gt70 4Residual adenocarcinoma 3 Not available 6Adenocarcinoma 2Ductal adenocarcinoma 4BBD group diagnosis 119899 Age 119899

Cyst 1 lt30 6Fibroadenoma 10 30ndash39 12Fibrocystic mastopathy 14 40ndash49 10Fibrosis 2 50ndash59 7Hyperplasia 2 60ndash69 1Lipoma 2 gt70 4Negative for malignance 9

reorganization [2 3] KiSS1 expression reduces the metastaticpotential by 95 but does not suppress tumorigenicity Itregulates adhesion molecules such as E-cadherin and dimin-ishesMMP9 expression throughNF-120581B binding inhibition tothe promoter [4ndash6] The encoded protein shows a 54-aminoacid COOH-terminal extreme which participates as a ligandin G protein-coupled receptors in humans [7ndash9] Along thegene KiSS-1 at least 294 SNPs have been described of which42 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 allelicgenotypic and haplotypic frequencies in Mexican generalpopulation (GP) and patients with benign breast disease(BBD) or breast cancer (BC)

The variants in KiSS1 gene analyzed in this study wereselected on the basis of theoretical functional relevance orbiological implications The 9 del T (rs5780218) polymor-phism of KiSS1 gene is located at the minus146 position of the51015840UTR of the mRNA transcript and corresponds to the dele-tion of an A (adenine) The E20K (rs12998) polymorphismis a GrarrA transition at position 212 of the cDNA whichdetermines the change of Glu-Lys at residue 20 of the protein[10]

So far it is unknownwhether these changes are associatedwith some alteration in the protein function or whether theyconfer susceptibility to develop metastasis in breast cancerpatients

2 Material and Methods

21 Study Population We used information from 225 con-secutive breast biopsies and 45 mastectomies from thehistopathology files of the Pathological Anatomy Unit atHospital de Especialidades from Centro Medico Nacional

from Instituto Mexicano del Seguro Social The selectioncriteria for all patients were unrelated persons age above 18years only female of the same ethnicity (Mexican mestizos)for two previous generations no family history of breastcancer and neither radiotherapy or chemotherapy treatmentprevious to biopsy Exclusion criteria included duplicatedsamples inability to access patients clinical chart insufficientamounts of sample or bad quality DNA after extractionOnly 130265 samples met the criteria for inclusion in thisstudy The tissue was histologically typed based on standardcriteria [11] Slides from all cases were reviewed to select spe-cific paraffin-embedded tissue blocks containing primarilyaffected tissue from which genomic DNA was extracted andanalyzed previously for other polymorphisms [12]

One hundred and thirty genomic DNA samples wererecovered from paraffin-embedded breast tissue (cases) forthe present study numbered and then processed for genotypeanalysis by another coauthor who has no knowledge of thehistopathological diagnosis After genotyping was completecases were distributed in two groups according to pathologi-cal assessment into a breast cancer (BC) group and a benignbreast disease (BBD) group (Table 1) Data from 90 genomicDNA extracted from blood samples from general populationindividuals were also included for comparison (GP group)this group comprisesmale and female nonrelated and healthyadults (Hardy-Weinberg Equilibrium) and an age range of 18ndash65 years All individuals belong to the sameMexicanmestizosethnic group

22 Genetic Analysis DNA was extracted from paraffin-embedded tissue and from blood samples using conventionalmethods [13 14] and stored at minus20∘C Genotypes for bothrs12998 and rs5780218 polymorphisms in KiSS1 gene weredetermined by PCR-RFLPs protocols implemented by ourgroup Table 2 shows the primer sequences and restriction

Disease Markers 3

Table 2 Primer sequences PCR conditions and restriction enzymes (RE) used for genotyping rs12998 and rs5780218 polymorphisms inKiSS1 gene AT annealing temperature

Polymorphism Primers sequence AT (∘C) Amplicon (bp) RE

rs 12998 AG F 51015840-ACT TgC TCA CAT TCC ACA gg-31015840R 51015840-gCA TCT CTC TgC TCT TgC AC-31015840 65 238 Nla IV

rs 5780218 mdashA F 51015840-CCT TTg CCT gCC Tgg ATg CA-31015840R 51015840-Tgg gCC TgT gCT Tgg AgA Cg-31015840 65 294 Sml I

Table 3 Genotype frequencies for KiSS1 suppressor metastasis gene rs12998 and rs5780218 polymorphisms in three groups and statisticalanalysis between groups BC breast cancer BCD breast cancer disease GP general population

Genotype frequency Statistical analysisGroups (119899) BC (78) BBD (52) GP (90) BC versus

BBD BC versus GP BBD versusGP

BC + BBDversus GPGG GA AA GG GA AA GG GA AA

rs12998 043 047 008 048 046 005 091 008 0 1205942 = 05660P= 07688

1205942 = 4492

P = 000001205942 = 336018P = 00000

1205942 = 488025P = 00000

AA Amdash mdash AA Amdash mdash AA Amdash mdash

rs5780218 026 039 033 032 044 017 038 044 016 1205942 = 40800P= 01368

1205942 = 67695P = 00332

1205942 = 05724119875= 07751

1205942 = 39609119875= 01415

enzymes used PCR was done in the final volume of 10 120583Ladding MgCl

2(35mM) dNTPs (2mM) primers forward

(01 120583M) reverse (01 120583M)DNA (10 ng) PCR conditionswereperformed with an initial denaturation of 4min at 94∘Cfollowed by 30 cycles (94∘C 45 sec 65∘C 45 sec 72∘C 1min)and a final extention of 10min at 72∘C

After PCR-RFLPs the 238 bp fragment (rs12998) andthe 294 bp fragment (rs5780218) show two bands each of158 bp and 80 bp and 224 bp and 70 bp respectively In thepresence of variant alleles the restriction sites are lost in bothpolymorphisms (Figures 1(a) and 1(b))

23 Statistical Analysis To infer the haplotypes for the groupswe used the Haplotype Reconstruction Program V = 06Hardy-Weinberg equilibrium was tested on the observedgenotypes All groups were compared using 1198832 and Fisherrsquosexact test when needed a 119875 value of le005 was consideredsignificantThe strength of any given SNP-cancer associationwas measured by odds ratio (OR) and its corresponding 95confidence intervals (CI)

Allelic and genotypic distributions were determined bygene counting and are presented as simple frequencies Con-tingency tables and Chi-squared tests were used to comparethe allele and genotype frequencies and haplotype associationamong the BC patients and BBD or healthy individuals aswell as to test for the presence ofHardy-Weinberg equilibriumamong the genotypes

3 Results

31 Polymorphisms A total of 220 DNA samples were ana-lyzed 78 were from patients that were classified within theBC group whilst 52 were of the BBD group and 90 sampleswere from the GP group (general population individuals)

Table 3 shows the allelic and genotypic frequencies forrs12998 and rs5780218 polymorphisms in KiSS1 gene in the

three study groups There is no difference between BC andBBD groups for both polymorphisms however when GPwas compared versus BC or BBD groups there are differenceswhich could be due to the type of tissue (blood versusparaffin-embedded tissue) from which DNA was extracted(Table 3)

32 Haplotypes The haplotype construction used the wildtype (Wt) or the variant for each allele at rs12998 andrs5780218 polymorphisms Based on segregation analysis andhomozygosity the haplotype phase could be established in 156chromosomes from BC 104 chromosomes from BBD and180 chromosomes from GP groups The four possible haplo-types (rs12998 allele 1 rs5780218 allele 2) are H1 Wtvariant(G-) H2 WtWt (GA) H3 variantvariant (A-) and H4variantWt (AA) All of them were identified in BC andBBD groups while only three haplotypes were found in theGP group The haplotype frequencies are shown in Table 4The comparison between BBD and GP group showed nodifferences for neither haplotype H1 (WtVariant) nor H2(WtWt) However the comparative analysis of haplotypesbetween BC and BBD groups showed significant differences(119875 = 00001 OR = 332) as well as the BC or BBD groupsversus GP (119875 = 0 OR = 3)

4 Discussion

InKiSS1 gene at least 294 SNPs have been identified [10] nonepreviously associated with breast cancer The rs12998 andrs5780218 SNPs have not been reported previously in relatingto cancer studies In this association study individuals froma Mexican general population and patients with benignbreast disease or breast cancer were consideredThe genotypeand allele frequencies of both polymorphisms were similarin the studied groups (119875 gt 005) suggesting that thesepolymorphisms individually do not confer susceptibility to

4 Disease Markers

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 fromMexican general population (GP) Wt wild type Haplotypes H1ndashH4

Group (119899chromosomes) Haplotype rs12998rs5780218 Statistical analysisH1

WtvariantH2

WtWtH3

VariantvariantH4

VariantWtBC versus

BBDBC versus

GPBBD versus

GPBCM versusBC NM

BC (156) 04256 02474 01064 022051205942 = 219537119875 = 00002

(OR = 332)

1205942 = 706430119875 = 00000(OR = 3)

1205942 = 05724119875 = 07801

1205942 = 105813119875 = 00130

BCM (32) 05101 01773 00836 02288BC NM (26) 01431 04337 02799 01431

BBD (104) 02441 04673 01789 01095GP (180) 03388 06166 00444 0

1 2 3 4 5 6

238bp

158bp

80bp

250

125

75

(bp)

(a)

1 2 3 4 5

294bp224 bp

70bp

300

200

100

50

(bp)

(b)

Figure 1 Genotyping of KiSS1 gene rs12998 (a) and rs5780218 (b) polymorphisms PCR and hydrolysed products digested with restrictionenzymes Nla IV or Sml I separated on 8 polyacrylamide gels with silver nitrate staining The molecular weight markers used were a 25 bpladder and a 50 bp ladder respectively

the development of breast cancer in our population Further-more information contained in the clinical history of somepatients from BC group allowed us to identify breast cancerpatients with or without metastasis (16 and 13 resp) Whenthe allele and genotype frequencies were analyzed againfor both polymorphisms no significant differences betweengroups were observed (119875 gt 05 data not shown) The smallsample size has limitation whereby in this study we can onlydescribe these findings without a clear conclusion

We found differences by comparing the results of rs12998polymorphism between the case group (BC and BBD) andthe general population group (119875 = 00000) and between BCand GP groups for rs5780218 polymorphism (119875 = 00332)However it is necessary to consider that the biological sampletype from which genomic DNA was obtained was differentin each group paraffin-embedded tissue and peripheralblood So in general population individuals we identifiedconstitutive genotypes of both polymorphisms while thegenotypes in the case group (BC and BBD) were identifiedin specific tissue (breast) with or without cancer Loss of

heterozygosity (LOH) is the loss of one allele at a specificlocus caused by a deletionmutation or loss of a chromosomefrom a chromosome pair resulting in abnormal hemizygosity[15] It is detected when heterozygous markers for a locusappear monomorphic because one of the alleles was deletedMoreover in several studies in which polymorphisms incancer related genes (oncogenes or tumor suppressor genes)have been analyzed LOH is reported when a discordancebetween germinal and tumoral genotypes is observed (DNAobtained from peripheral blood and tumor tissue) [15]Paraffin tissue blocks analyzed in this study were obtained inthe 2002ndash2005 period [11] therefore we did not have accessto peripheral blood samples from these patients and we didnot identify the constitutive genotypes in order to establishthe possible loss of heterozygosity in rs12998 or rs5780218polymorphisms that could explain the differences observedbetween genotypes by type of biological sample

The haplotype distribution between BC BBD and GPgroups shows that the H1 haplotype frequency is significantlyhigher in BC (04256) and particularly among patients with

Disease Markers 5

breast cancer and metastasis (05101) than in BC NM BBDor GP groups (Table 3) therefore H1 haplotype might beconsidered as a possible risk haplotype for breast cancer andmetastasis in our population which had not been describedbefore However it is necessary to consider the main limita-tions of this study as the small sample size However evenwith this limitation our study is the first analysis of theusefulness of these polymorphisms as potential genetic riskmarkers for breast cancer and it was possible to establishthe allele genotype and haplotype frequencies in Mexicanpopulation Moreover it was also limiting to not know thegermline genotypes in the study subjects since we only knowthe genotype present in the tissue from which DNA wasobtained and therefore it is not possible to analyze the lossof heterozygosity in these polymorphic markers Finally theinformation in the clinical charts or medical records waslimited or poor in several cases of selected patients

Moreover we do not know the biological significanceof these polymorphisms in cancer cells and there is noinformation about other gene variants described in KiSS1Kisspeptin encoded by KiSS1 gene was first considered as atumormetastasis suppressor however it plays amajor role inregulating the hypothalamic-pituitary-gonadal axis so manystudies have analyzed polymorphisms or haplotypes in KiSS1gene particularly in central precocious puberty Huijbregts etal show that polymorphisms in a G-rich sequence located inthe 31015840UTR upstream of the transcription end site of KiSS1gene influence its expression level [16 17] Likewise in poly-morphisms and gene expression studies it has been shownthat KiSS1 low mRNA expressions correlate with venousinvasion advanced clinical stage occurrence of metastasisand recurrence in patients with different types of cancer[18 19] In breast cancer Kiss-1GPR54 system estrogen-related gene expression profiles regulation and transcriptisoforms of KiSS1 gene have been described [20ndash24] and Xieet al showed significant relationship between lymph nodeinvolvement and absence of Kiss-1 expression in early breastcarcinoma patients (119875 = 0001) [25] however there is noinformation about whether the presence of a polymorphismcould be involved in the differential KiSS1 gene expressionTherefore it will be necessary to confirm in future studieswith a larger sample size if H1 haplotype described here is apossible risk haplotype for cancer andor metastasis in breastcancer to analyze their association with metastasis in othercancer types and analyze mRNA expression levels in theircorrelation with the presence of polymorphisms to knowtheir possible biological significance

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was partially supported by the Fondo de Inves-tigacion en Salud Instituto Mexicano del Seguro Social

(FISIMSSPROTC2007064) and Fondo Sectorial CienciaBasica CONACyT 2007 Reg 082292

References

[1] J Ferlay H R Shin F Bray D Forman C Mathers andD M Parkin ldquoGLOBOCAN 2008 v20rdquo Cancer Incidenceand Mortality Worldwide IARC Cancer Base No 10 Inter-national Agency for Research on Cancer Lyon France 2010httpglobocaniarcfr

[2] J H Lee M E Miele D J Hicks et al ldquoKiSS-1 a novel humanmalignant melanoma metastasis-suppressor generdquo Journal ofthe National Cancer Institute vol 88 no 23 pp 1731ndash1737 1996

[3] J-M Navenot N Fujii and S C Peiper ldquoActivation of Rhoand Rho-associated kinase by GPR54 and KiSS1 metastasissuppressor gene product induces changes of cell morphologyand contributes to apoptosisrdquoMolecular Pharmacology vol 75no 6 pp 1300ndash1306 2009

[4] J-H Lee and D RWelch ldquoSuppression of metastasis in humanbreast carcinomaMDA-MB-435 cells after transfection with themetastasis suppressor gene KiSS-1rdquoCancer Research vol 57 no12 pp 2384ndash2387 1997

[5] C Yan HWang andDD Boyd ldquoKiSS-1 represses 92-kDa typeIV collagenase expression by down-regulating NF-120581B bindingto the promoter as a consequence of I120581B120572-induced blockof p65p50 nuclear translocationrdquo The Journal of BiologicalChemistry vol 276 no 2 pp 1164ndash1172 2001

[6] L Kostadima G Pentheroudakis andN Pavlidis ldquoThemissingkiss of life transcriptional activity of the metastasis suppressorgene KiSS1 in early breast cancerrdquo Anticancer Research vol 27no 4 pp 2499ndash2504 2007

[7] M Kotani M Detheux A Vandenbogaerde et al ldquoThe metas-tasis suppressor gene KiSS-1 encodes kisspeptinas the naturalligands of the orphan G protein coupled receptor GPR54rdquo TheJournal of Biological Chemistry vol 276 no 37 pp 34631ndash346362001

[8] T Ohtaki Y Shintani S Honda et al ldquoMetastasis suppressorgene KiSS-1 encodes peptide ligand of a G-protein-coupledreceptorrdquo Nature vol 411 no 6837 pp 613ndash617 2001

[9] A I Muir L Chamberlain N A Elshourbagy et al ldquoAXOR12a novel human G protein-coupled receptor activated by thepeptide KiSS-1rdquo Journal of Biological Chemistry vol 276 no 31pp 28969ndash28975 2001

[10] ldquoA database of human single nucleotide polymorphismsrdquo 2014httpwwwncbinlmnihgovSNP

[11] F A Tavassoli and P Devilee World Health OrganizationInternational Agency for Research on Cancer Pathology andGenetics of Tumours of the Breast and Female Genital OrgansIARC Press Lyon France 2003

[12] A P Mendizabal-Ruiz J Morales X G Martinez et al ldquoRASpolymorphisms in cancerous and benign breast tissuerdquo Journalof the Renin-Angiotensin-Aldosterone System vol 12 no 2 pp85ndash92 2011

[13] S Gustincich G Manfioletti G Del Sal C Schneider andP Carninci ldquoA fast method for high-quality genomic DNAextraction fromwhole human bloodrdquo BioTechniques vol 11 no3 pp 298ndash302 1991

[14] D K Wright and M M Manos ldquoSample preparation fromparaffin-embedded tissuesrdquo in PCR Protocols A Guide toMethods and Applications M A Innis D H Gelfand J JSninsky and T JWhite Eds pp 153ndash158 Academic Press NewYork NY USA 1990

6 Disease Markers

[15] J E Krebs E S Goldstein and S T Kilpatrick Lewinrsquos GenesXI Jones amp Bartlett Learning 11th edition 2014

[16] L Huijbregts C Roze G Bonafe et al ldquoDNA polymorphismsof the KiSS1 31015840 Untranslated region interfere with the folding ofa G-rich sequence into G-quadruplexrdquo Molecular and CellularEndocrinology vol 351 no 2 pp 239ndash248 2012

[17] X Luan Y Zhou W Wang et al ldquoAssociation study of thepolymorphisms in the KISS1 gene with central precociouspuberty in Chinese girlsrdquo European Journal of Endocrinologyvol 157 no 1 pp 113ndash118 2007

[18] L Kostadima G Pentheroudakis andN Pavlidis ldquoThemissingkiss of life transcriptional activity of the metastasis suppressorgene KiSS1 in early breast cancerrdquo Anticancer Research vol 27no 4 B pp 2499ndash2504 2007

[19] M T Ruiz A L S Galbiatti E C Pavarino J V Maniglia andE M Goloni-Bertollo ldquoQ36R polymorphism of KiSS-1 genein Brazilian head and neck cancer patientsrdquo Molecular BiologyReports vol 39 no 5 pp 6029ndash6034 2012

[20] D Cvetkovic A V Babwah and M Bhattacharya ldquoKisspeptinKISSIR system in breast cancerrdquo Journal of Cancer vol 4 no 8pp 653ndash661 2013

[21] K Jarzabek L KozŁowski R Milewski and S WoŁczynskildquoKiSS1GPR54 and estrogen-related gene expression profiles inprimary breast cancerrdquo Oncology Letters vol 3 no 4 pp 930ndash934 2012

[22] D C Mitchell M Abdelrahim J Weng et al ldquoRegulation ofKiSS-1 metastasis suppressor gene expression in breast cancercells by direct interaction of transcription factors activatorprotein-2120572 and specificity protein-1rdquo The Journal of BiologicalChemistry vol 281 no 1 pp 51ndash58 2006

[23] S Mooez F A Malik M A Kayani R Rashid A Zahid andA Khan ldquoExpressional alterations and transcript isoforms ofmetastasis suppressor genes (KAI1 amp KiSS19 in breast cancerpatientsrdquo Asian Pacific Journal of Cancer Prevention vol 12 pp2785ndash2791 2011

[24] E PapaoiconomouM Lymperi C Petraki et al ldquoKiss-1GPR54protein expression in breast cancerrdquo Anticancer Research vol34 no 3 pp 1401ndash1407 2014

[25] F Xie H Yang S Wang et al ldquoA logistic regression modelfor predicting axillary lymph node metastases in early breastcarcinoma patientsrdquo Sensors vol 12 no 7 pp 9936ndash9950 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

Table 1 Diagnosis and age distribution for the studied BC and BBD groups

Breast cancer group diagnosis 119899 Age 119899

Adenocarcinoma 2 lt30 2Ductal adenocarcinoma 4 30ndash39 10Invasive ductal adenocarcinoma 47 40ndash49 20Invasive lobular carcinoma 5 50ndash59 12Lobular adenocarcinoma 1 60ndash69 11Lobular and ductal adenocarcinoma 1 gt70 4Residual adenocarcinoma 3 Not available 6Adenocarcinoma 2Ductal adenocarcinoma 4BBD group diagnosis 119899 Age 119899

Cyst 1 lt30 6Fibroadenoma 10 30ndash39 12Fibrocystic mastopathy 14 40ndash49 10Fibrosis 2 50ndash59 7Hyperplasia 2 60ndash69 1Lipoma 2 gt70 4Negative for malignance 9

reorganization [2 3] KiSS1 expression reduces the metastaticpotential by 95 but does not suppress tumorigenicity Itregulates adhesion molecules such as E-cadherin and dimin-ishesMMP9 expression throughNF-120581B binding inhibition tothe promoter [4ndash6] The encoded protein shows a 54-aminoacid COOH-terminal extreme which participates as a ligandin G protein-coupled receptors in humans [7ndash9] Along thegene KiSS-1 at least 294 SNPs have been described of which42 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 allelicgenotypic and haplotypic frequencies in Mexican generalpopulation (GP) and patients with benign breast disease(BBD) or breast cancer (BC)

The variants in KiSS1 gene analyzed in this study wereselected on the basis of theoretical functional relevance orbiological implications The 9 del T (rs5780218) polymor-phism of KiSS1 gene is located at the minus146 position of the51015840UTR of the mRNA transcript and corresponds to the dele-tion of an A (adenine) The E20K (rs12998) polymorphismis a GrarrA transition at position 212 of the cDNA whichdetermines the change of Glu-Lys at residue 20 of the protein[10]

So far it is unknownwhether these changes are associatedwith some alteration in the protein function or whether theyconfer susceptibility to develop metastasis in breast cancerpatients

2 Material and Methods

21 Study Population We used information from 225 con-secutive breast biopsies and 45 mastectomies from thehistopathology files of the Pathological Anatomy Unit atHospital de Especialidades from Centro Medico Nacional

from Instituto Mexicano del Seguro Social The selectioncriteria for all patients were unrelated persons age above 18years only female of the same ethnicity (Mexican mestizos)for two previous generations no family history of breastcancer and neither radiotherapy or chemotherapy treatmentprevious to biopsy Exclusion criteria included duplicatedsamples inability to access patients clinical chart insufficientamounts of sample or bad quality DNA after extractionOnly 130265 samples met the criteria for inclusion in thisstudy The tissue was histologically typed based on standardcriteria [11] Slides from all cases were reviewed to select spe-cific paraffin-embedded tissue blocks containing primarilyaffected tissue from which genomic DNA was extracted andanalyzed previously for other polymorphisms [12]

One hundred and thirty genomic DNA samples wererecovered from paraffin-embedded breast tissue (cases) forthe present study numbered and then processed for genotypeanalysis by another coauthor who has no knowledge of thehistopathological diagnosis After genotyping was completecases were distributed in two groups according to pathologi-cal assessment into a breast cancer (BC) group and a benignbreast disease (BBD) group (Table 1) Data from 90 genomicDNA extracted from blood samples from general populationindividuals were also included for comparison (GP group)this group comprisesmale and female nonrelated and healthyadults (Hardy-Weinberg Equilibrium) and an age range of 18ndash65 years All individuals belong to the sameMexicanmestizosethnic group

22 Genetic Analysis DNA was extracted from paraffin-embedded tissue and from blood samples using conventionalmethods [13 14] and stored at minus20∘C Genotypes for bothrs12998 and rs5780218 polymorphisms in KiSS1 gene weredetermined by PCR-RFLPs protocols implemented by ourgroup Table 2 shows the primer sequences and restriction

Disease Markers 3









rs12998 043 047 008 048 046 005 091 008 0 1205942 = 05660P= 07688

1205942 = 4492

P = 000001205942 = 336018P = 00000

1205942 = 488025P = 00000


rs5780218 026 039 033 032 044 017 038 044 016 1205942 = 40800P= 01368

1205942 = 67695P = 00332

1205942 = 05724119875= 07751

1205942 = 39609119875= 01415







3 Results





4 Discussion


4 Disease Markers



WtvariantH2

WtWtH3

VariantvariantH4

VariantWtBC versus

BBDBC versus

GPBBD versus

GPBCM versusBC NM

BC (156) 04256 02474 01064 022051205942 = 219537119875 = 00002

(OR = 332)

1205942 = 706430119875 = 00000(OR = 3)

1205942 = 05724119875 = 07801

1205942 = 105813119875 = 00130

BCM (32) 05101 01773 00836 02288BC NM (26) 01431 04337 02799 01431

BBD (104) 02441 04673 01789 01095GP (180) 03388 06166 00444 0

1 2 3 4 5 6

238bp

158bp

80bp

250

125

75

(bp)

(a)

1 2 3 4 5

294bp224 bp

70bp

300

200

100

50

(bp)

(b)






Disease Markers 5





Acknowledgments



References















6 Disease Markers

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 3









rs12998 043 047 008 048 046 005 091 008 0 1205942 = 05660P= 07688

1205942 = 4492

P = 000001205942 = 336018P = 00000

1205942 = 488025P = 00000


rs5780218 026 039 033 032 044 017 038 044 016 1205942 = 40800P= 01368

1205942 = 67695P = 00332

1205942 = 05724119875= 07751

1205942 = 39609119875= 01415







3 Results





4 Discussion


4 Disease Markers



WtvariantH2

WtWtH3

VariantvariantH4

VariantWtBC versus

BBDBC versus

GPBBD versus

GPBCM versusBC NM

BC (156) 04256 02474 01064 022051205942 = 219537119875 = 00002

(OR = 332)

1205942 = 706430119875 = 00000(OR = 3)

1205942 = 05724119875 = 07801

1205942 = 105813119875 = 00130

BCM (32) 05101 01773 00836 02288BC NM (26) 01431 04337 02799 01431

BBD (104) 02441 04673 01789 01095GP (180) 03388 06166 00444 0

1 2 3 4 5 6

238bp

158bp

80bp

250

125

75

(bp)

(a)

1 2 3 4 5

294bp224 bp

70bp

300

200

100

50

(bp)

(b)






Disease Markers 5





Acknowledgments



References















6 Disease Markers

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Disease Markers



WtvariantH2

WtWtH3

VariantvariantH4

VariantWtBC versus

BBDBC versus

GPBBD versus

GPBCM versusBC NM

BC (156) 04256 02474 01064 022051205942 = 219537119875 = 00002

(OR = 332)

1205942 = 706430119875 = 00000(OR = 3)

1205942 = 05724119875 = 07801

1205942 = 105813119875 = 00130

BCM (32) 05101 01773 00836 02288BC NM (26) 01431 04337 02799 01431

BBD (104) 02441 04673 01789 01095GP (180) 03388 06166 00444 0

1 2 3 4 5 6

238bp

158bp

80bp

250

125

75

(bp)

(a)

1 2 3 4 5

294bp224 bp

70bp

300

200

100

50

(bp)

(b)






Disease Markers 5





Acknowledgments



References















6 Disease Markers

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 5





Acknowledgments



References















6 Disease Markers

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Disease Markers

















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Polymorphisms rs12998 and rs5780218 in...

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