Balkan Journal of Medical Genetics

ASSOCIATION OF E-SELECTIN S128R POLYMORPHISM WITH HEREDITARY BREAST CARCINOMA SUSCEPTIBILITY IN TURKISH PATIENTS WITHOUT BRCA1/2 GERMLINE MUTATIONS
Yararbas K, Atalay PB
*Corresponding Author: Kanay Yararbas, M.D., Assistant Professor, Department of Medical Genetics, Acibadem Mehmet Ali Aydinlar University Faculty of Medicine; İçerenköy Mahallesi, Kayisdagi Caddesi, No: 32, 34742, Istanbul, Turkey. Tel: +90-216-500-4785. Fax: +90-216-500-5076. E-mail: kanay.yararbas@ acibadem.edu.tr
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MATERIALS AND METHODS

Subjects. The present study included 360 genetically unrelated females between 40-50 years of age who were referred to a regional reference laboratory between 2013 and 2016 for genetic counseling and testing. Of these females, 90 were diagnosed with breast carcinoma, clinically resembling the hereditary type according to the National Comprehensive Cancer Network (NCCN) guidelines of genetic/familial high-risk assessment for breast and ovarian cancers [24]. These patients were otherwise healthy. All patients were screened for BRCA1/2 mutations by next generation sequencing (NGS). Briefly, targeted amplification of all coding exons of BRCA1 and BRCA2 was performed using the BRCA MASTR Dx kit from Multiplicom, Agilent Technologies (Santa Clara, CA, USA), as described by the manufacturer and the amplicon pool was sequenced on the Illumina MiSeq secuencing platform. Data analysis was performed with SEQ powered by Genomize (https:// seq.genomize.com). Patients in whom normal results were obtained with no pathogenic variants were included. The control group consisted of 270 females who did not belong to an at-risk population with higher BRCA mutation carrier frequencies, such as Ashkenazi Jewish descent, who had no previous cancer diagnosis and no family history of cancer, or cardiovascular diseases hypothesized to be related with increased SELE polymorphism frequencies. The present study was approved by the Clinical Research Ethics Committee of Maltepe University, Istanbul, Turkey, and written informed consent was obtained from all participants. Histopathological data obtained from patient records revealed that all patients had invasive ductal carcinoma. Genotyping. For genotyping analysis, DNA was extracted from ethylenediaminetetraacetic acid (EDTA)- anticoagulated peripheral blood samples of all consenting subjects using the Qiagen DNA Blood Mini Kit and a QiaCube robotic device (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. All patients and controls were examined for the S128R (A/C) SNP of the E-selectin gene [19] by real-time (RT) polymerase chain reaction (PCR) analysis using TIB Molbiol LightSNiP Genotyping Assay (TIB Molbiol GmbH, Berlin, Germany). The reaction master mix used in the study was commercially obtained from TIB Molbiol GmbH. The RT-PCR reactions were performed using 50 ng genomic template DNA. The Qiagility robotic instrument (Qiagen GmbH) was used to prepare the reagent mix. The RT-PCR cycling conditions used for the S128R polymorphism were as follows: 10 min. of initial denaturation at 95 °C, 45 cycling reactions of 10 seconds at 95 °C, 10 seconds at 60 °C, 15 seconds at 72 °C, melting curves at 95 °C, 40 °C, 75 °C, and cooling to 40 °C. Repeatability of the reactions was checked for internal quality control by repeating the procedure using randomly chosen samples. Melting peaks were obtained at 59 °C for the A allele and at 64 °C for the C allele. Statistical Analyses. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) Statistics for Windows version 21.0 (IBM Corporation, Armonk, NY, USA). Data were expressed as frequencies and percentages. Genotype frequencies in the patient and control groups were compared using the ζ2 test. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to examine the effect of the S128R polymorphism on breast cancer susceptibility in non carriers of the BRCA1/2 mutations. A p value of <0.05 was considered statistically significant.

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