KIR AND HLA HAPLOTYPE ANALYSIS IN A FAMILY LACKING THE KIR 2DL1-2DP1 GENES
Vojvodić S, Ademović-Sazdanić D
*Corresponding Author: Associate Professor Svetlana Vojvodić, Institute for Blood Transfusion of Vojvodina, Tissue Typing Compartment, Medical Faculty of the University of Novi Sad, Hajduk Veljkova 9a, 21000 Novi Sad, Serbia; Tel: +381-21-4877-963, Fax: +381-21-4877-978; E-mail: svetlana.vojvodic021@gmail.com
page: 55

MATERIAL AND METHODS

Patient History. An 8-year old female patient (M.M.), suffering from acute myelogenous leukemia (AML), her parents [father (M.A.) 37 years old, mother (M.Z.) 32 years old], two siblings (M.M. and M.Z.), aged 14 and 10, maternal grandparents: grandfather (M.M., 67 years old), grandmother (M.M., 56 years old), and paternal grandmother: (M.T., 63 years old), were reported. The paternal grandfather (M.F.), who passed away 2 years ago, was not included in the case study, therefore, we were unable to show both his KIR and HLA genotypes, but only the one inherited by his son, the patient’s father (M.A.). Family History. There were no known disorders from which direct blood relatives of the patient suffered, with the exception of hypertension, which both of the patient’s grandmothers suffer. There are the data on consanguinity among family members: great-grandmother of the patient’s father (M.A.) and grandfather of the patient’s mother (M.Z.), were siblings. Therefore, the mother and the father of the patient are third-degree cousins. DNA Extraction and Human Leukocyte Antigen Genotyping. DNA extraction was performed by a silica-based extraction method, using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) or the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Inc., Vilnius, Lithuania) from 200 μL of buffy-coat obtained from 3 mL of blood collected in EDTAcontaining vacutainers. The DNA was rehydrated with pure water and the DNA concentration and purity was determined by Eppendorf BioPhotometer (Eppendorf AG, Hamburg, Germany). The HLA and KIR genotyping was performed by a reverse sequence-specific oligonucleotide (SSO) probe method coupled with the multiplexed microspherebased suspension array platform xMAP technology designed for use with the Luminex® system to identify HLA and KIR alleles encoded by sample DNA using Lifecodes KIR/HLA SSO Typing kits, Gen-Probe Transplant Diagnostics, Inc. (Stamford, CT, USA). The target DNA was first amplified by polymerase chain reaction (PCR) with biotinylated primers specifically designed for each HLA and KIR locus. The PCR product was denatured and hybridized to complementary oligonucleotide probes immobilized on fluorescently coded microsphere beads. At the same time, the biotinylated PCR product was labeled with phycoerythrin-conjugated streptavidin to allow it to be detected by the Luminex® system. The HLA and KIR alleles were assigned by analysis of the reaction (hybridization) pattern of the target sample using the Quicktype for Lifecodes software. Verification of HLA and KIR genotyping was based on a PCR-SSP (sequence-specific primers) technique using HLA-Ready Gene and KIR-Ready Gene kits (Inno-Train Diagnostik GmbH, Kronberg, Germany). The cycling program involved the initial denaturation at 96 °C for 2 min. followed by 10 cycles of denaturation at 96 °C for 15 seconds, annealing at 65 °C for 60 seconds, and followed by 20 cycles of denaturation at 96 °C for 15 seconds, annealing at 61 °C for 50 seconds, and elongation at 72 °C for 30 seconds. Electrophoresis using 2.0% agarose gel was done. After PCR, created bands in each well were interpreted based on standard protocol available in the kit, using Helmberg SCORE™ software. The genotype of each individual was typed specifically.



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