MOLECULAR CHARACTERIZATION OF IRANIAN PATIENTS WITH INHERITED COAGULATION FACTOR VII DEFICIENCY
Shahbazi S, Mahdian R, Karimi K, Mashayekhi A
*Corresponding Author: Dr. Shirin Shahbazi, Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Al-e-Ahmad and Chamran Cross, POB 14115-111Tehran, Iran. Tel: +98-21-82-884-556. Fax: +98-21- 82-884-555. E-mail: sh.shahbazi@modares.ac.ir
page: 8

MATERIALS AND METHODS

Patients and Sample Collection. This study was approved by the ethics committee of the Iran National Science Foundation (INSF). Eight FVII deficiency patients participated in this study after we obtained written informed consent. They were selected from eight unrelated families living in different parts of Tehran Province, Iran. The patients were referred to the Clinic of Hematology and Oncology, Imam Khomeini Hospital in Tehran, Iran. All of them were evaluated by a hematologist for clinical manifestation and tested for bleeding abnormalities and coagulation factor levels. For DNA and RNA extraction, 5 mL of peripheral blood was collected from each patient in EDTA-containing vacutainers. Each sample was divided into two aliquots, one to be used for peripheral blood mononuclear cells (PBMCs) and RNA isolation and the other for DNA extraction. In order to separate PBMCs, the blood samples were centrifuged at 150 g for 20 min. and the plasma content (upper phase) was removed. The cells in the lower phase were resuspended (1:2 V/V) with phosphate-buffered saline (PBS), overlaid on Ficoll-Plaque™ (Sigma-Aldrich Co. Ltd., Gillingham, Dorset, UK) and centrifuged at 400 g for 30 min. at room temperature. The PBMC layer were collected and stored at –80 °C until used for RNA isolation. DNA Extraction and Mutation Detection. Genomic DNA was extracted from the whole blood samples using a salting-out method. The concentration and purity of the isolated DNA were determined by a spectrophotometer (Nanophotometer™; Implen GmbH, München, Germany). High quality DNA (A260/280≥1.8) was selected and kept at –20 °C. The primers were designed to amplify all F7 gene exons and their flanking intronic sequences using Oligo explorer (Gene Link Inc., Hawthorne, NY, USA) software (V 1.2) and verified for specificity using the BLAST website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). The sequence of the primers and their polymerase chain reaction (PCR) product size are described in Table 1. Polymerase chain reactions included 0.4 μM of each primer and 50 ng of genomic DNA in 2×PCR master mix (Ampliqon; Pishgam Biotech, Tehran, Iran ) containing 0.2 units/μL Ampliqon Taq DNA polymerase and 2.5mM MgCl2. The PCR amplification involved an initial denaturation step for 5 min. at 94 °C and 30 cycles of 30 seconds at 94 °C, primer annealing temperature for 30 seconds and 72 °C for 1 min., followed by a final extension step at 72 °C for 5 min. Amplified fragments were sequenced and the data were analyzed using Chromas software (http:// technelysium.com.au/ wp/chromas/). All sequence changes were confirmed on both strands. RNA Extraction and Reverse Transcription-Polymerase Chain Reaction (RT-PCR). RNA was extracted from PBMC pellets using NucleoSpin® RNA Blood kit (Macherey-Nagel; Bahar-Tashkhis, Tehran, Iran) as recommended by the manufacturer. The concentration and purity of the purified RNA were determined by spectrophotometry and gel electrophoresis. High quality RNA (A260/280≥1.8) was selected and kept at –80 °C until used for cDNA synthesis. Up to 1 μg RNA was converted to cDNA using RevertAid First Strand cDNA synthesis kit (Thermo Scientific; NedayeFan Company, Tehran, Iran) according to the manufacturer’s instructions. To verify the integrity of the cDNA, a RT-PCR experiment was performed using GAPDH gene specific primers. The purified cDNA was subjected to amplification and sequencing. The primers were designed by Oligo explorer software (V 1.2) and specified using the BLAST website as described in Table 2. Reverse transcription-polymerase chain reactions were performed in a final volume of 25 μL containing 2 μL of cDNA, 1 μL of each primer in 2×PCR master mix. The RT-PCR amplification program started with a single denaturation step at 94 °C for 5 min. and followed by 30 cycles of 30 seconds at 94 °C, primer annealing temperature for 30 seconds and 72 °C for 1 min., followed by a final extension step at 72 °C for 5 min. Amplified fragments were analyzed by DNA sequencing to determine the proportion of mutation-containing transcript. Relative allele-specific mRNA quantitation by DNA sequencing was carried out as described previously [14,15].



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