Balkan Journal of Medical Genetics

HIGH-RESOLUTION HLA-DRB1 ALLELE FREQUENCIES IN A ROMANIAN COHORT OF STEM CELL DONORS
Caragea ÌÀ, Ursu IR, Visan DL, Maruntelu I, Iordache P, Constantinescu A, Tizu M, Tălăngescu A, Constantinescu I
*Corresponding Author: Lect., MD, PhD Radu-Ioan Ursu, Carol Davila University of Medicine and Pharmacy, Department of Medical Genetics, Bucharest, Romania, Str. Batistei 12, Bucharest, Romania; tel.: 0040736167020; e-mail: dr.radu.ursu@gmail.com
page: 43

MATERIAL AND METHODS

The study for HLA typing included 405 healthy vol- untary donors (Romanians/Europeans, 61% male, age 43.3 ± 7.7 years) who were registered in the National Registry of Voluntary Hematopoietic Stem Cell Donors (RNDVCSH). This study was conducted at the Fundeni Clinical Institute in the Medical Analysis Laboratory 2, and it in- volved healthy donors who voluntarily registered for stem cell donation in the RNDVCSH between 2020 and 2021. In accordance with the Declaration of Helsinki, writ- ten consent from willing donors was requested for the processing of personal data and evidence. This study was reviewed and approved by the Ethics Committee of the Fundeni Clinical Institute (no. Ten Points: 7916/10.02. 2021). Each donor’s medical file contained medical data that the project’s research team extracted, processed, and statistically examined. We included willing donors be- tween the ages of 20 and 50 in the study who had no underlying health issues. We looked through each donor’s personal medical record to see if their medical history was in compliance with the national protocol. After they donated blood, we also looked at their viral status and biochemical parameters. Peripheral blood collected in vacutainers containing the anticoagulant EDTA (ethylene-diamino-tetra-acetic acid) provided the DNA used in this investigation. DNA was extracted from blood using the manual technique. DNA was extracted using the QIAmp DNA Blood Mini ® extraction kit (QIAGEN, Hilden, Germany). The purifi- cation of total DNA (genomic, mitochondrial) from bone marrow, cell cultures, leukocyte concentrate, and whole blood was made possible by this rapid and easy technique based on silicon dioxide membranes. Each blood sample was thoroughly vortexed, mixed with protease and lysis buffer, and heated in a thermo- block for 10 minutes at 56 degrees Celsius to facilitate rapid lysis. The DNA was still free in the lysate after the cell membranes broke down, so we added 80% alcohol to make it precipitate. The lysate was placed into tubes with silicon membranes, to which DNA adheres, because the two materials had different electrical charges. The DNA was purified by multiple washings and separated from the silicon membrane following the addition of the elution buffer, which neutralizes the electrical charges. Prior to usage, the DNA was divided into tubes and stored at -18 °C. An IMPLEN nanophotometer was used to measure the concentration and purity of the DNA using an A260nm/A280nm ratio between 1.7 and 1.9, certifying solution purity and a DNA concentration at >20 ng/µL. The Mia Fora NGS MFlex kit from Immucor was used to genotype the HLA class II alleles (HLA-DRB1, DRB3/4/5) at a 6-digit resolution. Using Next-Genera- tion Sequencing techniques, HLA genotyping was carried out using the MIA FORA NGS MFlex HLA kit (MIA FORATM NGS MFlex) from Immucor. The three main components of this process are long-range PCR, library construction, and sequencing and data analysis. In the long-range PCR step, the most pertinent HLA genes were amplified. After fragmented probes are used to build librar- ies, adenine nucleotides are added to the ends of each frag- ment to enhance the ligation of the unique index adapters. Each fragment is barcoded to make identification easier during sequencing. To ensure adequate cluster generation, a final amplification of the size-selected library was then required. By using the Pippin Prep system, DNA fragments containing 500–900 base pairs were selected. Before the final library preparation, the concentration was measured using a Qubit ® fluorometer (Thermo Fisher Scientific) and adjusted according to the protocol. Using Illumina reagents, the NGS sequencing library was prepared and then loaded into an Illumina MiniSeq sequencer. Making use of the MIA FORA NGS FLEX pro- gram (Sirona Genomics, Inc. Sirona Genomics and IMGT databases, two reference databases, were used to interpret data after sequencing was completed. To confirm the allele frequency distribution among the analyzed population, the Hardy-Weinberg equilibrium was used.

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