Balkan Journal of Medical Genetics

BRCA 1/BRCA 2 PATHOGENIC/LIKELY PATHOGENIC VARIANT PATIENTS WITH BREAST, OVARIAN, AND OTHER CANCERS
Osman K.1,*, Ahmet K.2, Hilmi T.3, İlker N.O.4, Ercan Ö.5, Devrim Ç.5, Murat S.1, Emre Ç.6, İlhan H.6, Mustafa G.7, Yüksel Ü.7, Bahiddin Y.8, Cihan E.9, Mehmet Ali N. Ş.9, Emrah E.10, Umut D.10, Zeynep O.11, Mehmet Ali K.12, Ali G.2, İvo G.2, Erkan Ö.2, Muhammet B. H.2, Bülent E.2, Selma D.12, Sernaz U.2, Mahmut G.4, Hakan G.12, İrfan Ç.2
*Corresponding Author: Assoc. Prof. Osman Köstek, MD, Marmara University, School of Medicine, Department of Medical Oncology Address: Marmara University, Basıbuyuk Campus, Maltepe, Istanbul, Turkey. Email: osmankostek@hotmail.com, Telephone: +90 554 585 73 90
page: 5

MATERIAL AND METHODS

Study subjects. This retrospective multicenter study includes the results of sequencing analysis of 200 patients (190 women, 10 men) who have been directed to genetic counseling with an indication BRCA1/ BRCA2 test from different regions of Turkey. This study was approved by the local ethics committee. DNA isolation. Genomic DNA was isolated from peripheral blood samples by using the EasyOne DNA isolation system (Qiagen, Hilden, Germany) and isolated DNA samples were assessed spectrophotometrically with NanoDrop (Thermo Fisher Scientific). Samples whose A260/280 values were between 1.8-2.0 were used for Next-Generation Sequencing. Low quality DNA samples were re-extracted from stored blood samples. Next Generation Sequencing (NGS). For NGS, a QIAseq Targeted Amplicon Panel (Qiagen, Hilden, Germany), covering the coding regions of BRCA1 and BRCA2 genes with 20bp intron padding primers was used. Amplicon libraries were prepared according to the instructions of the manufacturer (Qiagen, Hilden, Germany). Pooled libraries were sequenced on the MiSeq System (Illumina, San Diego, CA, USA) following the target enrichment process. Fastq generation was performed on MiseqReporter Software (Illumina, San Diego, CA). Quality control of sequenced amplicons and variant call format (vcf) file generation were performed using QCI analysis (Qiagen, Hilden, Germany) software. Variant analysis was performed using Ingenuity and Clinical Insight Softwares (Qiagen, Hilden, Germany), and all rare and novel variants were visually controlled by using IGV 2.4.8 (www.broadinstitute.com). Segregation analysis of family members were performed using Sanger Sequencing with in-house designed primer sets covering the mutation regions. Data Analysis and Variant Classification. The latest versions of gnomAD [4], dbSNP [5], and ClinVar [6] databases were considered for comparing known variant frequencies. HGMD [7] and literature accessions were also considered. ACMG 2015 [8] guidelines were used for final classification of the variants . All statistical analyses were performed using IBM SPSS ver. 22 (SPSS Inc., Chicago, IL). Data were presented as median (25th-75th interquartile range). Categorical variables were reported as frequencies and group percentages. Progression-free survival was defined as the time from the date of initial diagnosis to disease progression or death due to any cause. The Pearson chi-square test was used to compare the categorical variables of the two groups, and the independent sample t-test or Mann–Whitney U-test was used to compare the continuous variables of the two groups. The Kaplan–Meier method was used for the survival analysis. P < 0.05 was considered statistically significant.

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