GENETIC ANALYSES OF THE NF1 GENE IN TURKISH NEUROFIBROMATOSIS TYPE I PATIENTS AND DEFINITION OF THREE NOVEL VARIANTS
Ulusal SD1,*, Gürkan H1, Atlı E1, Özal SA2, Çiftdemir M3, Tozkır H1, Karal Y4, Güçlü H2, Eker D1, Görker I5
*Corresponding Author: Dr. Selma D. Ulusal, Department of Medical Genetics, Trakya University Faculty of Medicine, D100, Edirne, Turkey. Tel: +90-284-235-7642/2330. Fax: +90-284-235-7652. E-mail: selmaulusal@trakya.edu.tr
page: 13

MATERIALS AND METHODS

Patients. Genetic screening results for the NF1 gene of 24 patients including 14 males (mean age 9.64 ± 10.24) and 10 females (mean age 16.30 ± 15.61) living in the Trakya region of Turkey, who were referred to our department because they were suspected of having NF1 were included in this study. Whole Blood Collection and DNA Isolation. Peripheral blood samples were obtained from patients after they signed an informed consent forms for genetic investigation. DNA was isolated from blood samples collected in EDTA vacutainers, by BioRobot EZ1 system from Qiagen GmbH (Hilden, Germany) following the manufacturer’s instructions. DNA quality (OD260/OD280 1.8-2.0) and concentration analysis were determined both with Nano Drop (Thermo Scientific NanoDrop Products, Wilmington, DE, USA) and Qubit 2.0 fluorometer (Invitrogen, Life Technologies, Eugene, OR, USA) prior to downstream applications. Multiplex Ligation-Dependent Probe Amplification. The MLPA analysis was performed according to the manufacturer’s instructions (MRC-Holland, Amsterdam, The Netherlands), using P081.B2 and P082, B2 probe mixes. The MLPA products were identified and quantified by capillary electrophoresis using ABI PRISM® 3130xl (Applied Biosystems®, Invitrogen Life Technologies, Carlsbad, CA, USA). Gene Mapper(Applied Biosystems, Foster City, CA, USA) and Coffalyser software (MRCHolland) were used for the MLPA analysis. Next Generation Sequencing. Amino acid coding regions of the NF1 gene (NM_000267) were amplified using primers designed with the Ion AmpliSeq Designer (Life Technologies). Libraries were amplified with the mix including 4 µL of 5X Ion AmpliSeqTM HiFi mix, 10 µL of 2X Ion AmpliSeq TM primer pool, 10 ng of gDNA per reaction and 4 µL of nuclease-free water. Thermal cycling conditions were 99 °C for 2 min., 99 °C for 15 seconds, 60 °C for 4 min. (19 cycles), with a final hold at 10 °C. Following partial digestion of primer sequences, adapters, and barcodes (Ion Xpress, Thermo Fisher Scientific, Waltham, MA, USA) Life Technologies) ligated to the amplicons as described in Ion AmpliSeqTM library preparation manual. Barcoded libraries were equalized using Ion Library Equalizer Kit (Thermo Fisher Scientific). Enriched, templatepositive ion sphere particles (ISPs) were prepared on an Ion One Touch TM 2 System and the Ion One Touch TM ES Instrument (Thermo Fisher Scientific) using the 200 bp chemistry following the manufacturer’s manual. Sequencing of enriched particles was performed on the PGM (Thermo Fisher Scientific) with 314 chips according to the user guide for the on Ion PGM sequencing 200 kit version 2 (Thermo Fisher Scientific). Raw data were processed and aligned to hg19 human reference genome (Genome Reference Consortium GRCh37) using the Torrent Suite Software version 5 (Thermo Fisher Scientific). A coverage analysis plugin was used for each sample to define total reads at the target bases. A minimum 100X coverage for all bases in the targeted region was accepted for a reliable variant calling. Ion Reporter version 4.0 software was used to annotating variants. Integrated Genomics Viewer (IGV) (http://software.broadinstitute.org/software/ igv/) [15] was used to visual assessment of aligned amplicons. The Human Genome Variation Society (HGVS) (http://www.hgvs.org/mutnomen) recommendations were followed for naming novel variants and checked on the Mutalyzer tool (https://mutalyzer.nl/about) [16]. Sanger Sequencing. Sanger sequencing with inhouse designed primer sets was used for segregation analysis and confirmation of the novel pathogenic variants found in NGS. Polymerase chain reaction (PCR) was performed with intronic primers for indicated exons of the NF1 gene. Sequencing reaction was performed with BigDye Terminator 3.1 Kit (PE AppliedBiosystems, Foster City, CA, USA). Dideoxy-terminated products were analyzed on the ABI-PRISM® 3130 Genetic Analyzer (PE Applied Bio-systems), according to the manufacturer’s instructions. Classification of Novel Variants. The Human Gene Mutation Database (HGMD) [9], LOVD (Leiden Open Variation Database) [12] and ClinVar databases were used to search the known disease causing NF1 variants. Mutation Taster and Exac Database (http://exac.broadinstitute. org) were considered for all novel variants. The final decision about the classification of variants was given according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG) 2015 Guidelines [17].



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