Balkan Journal of Medical Genetics

HIGH RISK OF GESTATIONAL TROPHOBLASTIC NEOPLASIA DEVELOPMENT IN RECURRENT HYDATIDIFORM MOLES WITH NLRP7 PATHOGENIC VARIATIONS
Kocabey M.1,a, Gulhan I.2, Koc A.1,b, Cankaya T.1, Karatasli V.2, Ileri A.3
*Corresponding Author: MD Mehmet Kocabey, Address: Güzelburc District Kıbrıs Street No: 81, 31175 Antioch/Hatay, e-mail: mehmet_kocabey@hotmail.com
page: 45

MATERIALS AND METHODS

Patient Selection RHM patients who were referred to the Department of Gynecologic Oncology (Tepecik Training and Research Hospital) and to the Center for Genetic Diagnosis (Dokuz Eylul University) between the years 2018 and 2020 were retrospectively reviewed. All 3 cases were included in the study. In addition to the gynecologic work up, Sanger Sequencing of the NLRP7, KHDC3L coding regions and exon-intron boundaries were performed. This study was in conformity with the Declaration of Helsinki on ethical principles for medical research. All individuals provided written informed consent for molecular analysis and also for the publication of this paper. Sample Collection and DNA Extraction Total genomic DNA was extracted from 4 ml peripheral blood from all patients via magnetic bead purification method. MagPurix Blood DNA Extraction Kit (Zinexts, Taiwan) was used with the MagPurix Automated Extraction System (Zinexts, Taiwan) according to the manufacturer’s protocol. DNA quality and concentration measurements were performed by NanoDrop ND1000® Spectrophotometer (Thermo Fisher Scientific, USA). After proper quality (50-100 ng/μl concentration and A260/ A280: 1.8-2.0 purity) of DNA was ensured, the DNA was stored at -20°C until further use. Sanger Sequencing Direct amplification and sequencing of the KHDC3L and NLRP7 genes were performed primarily using the primer sequences shown on Table 1. Exons and splice-site junctions were amplified using a standard PCR procedure that utilized the AmpliTaq Gold™ 360 DNA Polymerase (Applied Biosystems - Thermo Fisher Scientific, USA). Amplifications were performed using Eppendorf 5332 Mastercycler (Eppendorf AG, Germany). PCR products were verified by 2% agarose gel electrophoresis and ethidium bromide staining. Sequencing reactions were performed with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems - Thermo Fisher Scientific, USA), and electrophoresed on an ABI 3130 Capillary Electrophoresis System (Applied Biosystems - Thermo Fisher Scientific, USA). Sequence alignment and evaluation were performed using CLC Genomics Workbench v3.6.5 (Qiagen, Germany). GRCh38.p13 human genome reference in the “Ensembl” database, with a ENST00000370367 transcript of the KHDC3L gene and a ENST00000592784 transcript of the NLRP7 gene as reference. Detected variants were classified according to the current guidelines [8].

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