engleski

HLA-A, -B, -C, -DRB1, -DQA1, -DQB1 and -DPB1 allele and haplotype frequencies defined by next generation sequencing in a population of East Croatia blood donors

Next-generation sequencing is increasingly used in transplantation settings, but also as a method of choice for in-depth analysis of population-specific HLA genetic architecture and its linkage to various diseases. With respect to ethnic admixture characteristic for East Croatian population, we aimed to investigate the frequency and linkage between 7 HLA loci in 112 healthy, unrelated, blood donor volunteers (female/male-32/80, 20-61 years, median age 34.5) originating from 4 eastern Croatia counties. Genomic DNA was extracted and HLA genotypes determined using high-resolution Omixon Holotype HLA 96/7 and 24/7 kits on Illumina MiSeq sequencing platform. HLA-A, -B, -C, and -DQA1 loci were sequenced for their entire length, -DQB1 and -DPB1 from intron 1 to 3' UTR, and -DRB1 from intron 1 to intron 4. Collected reads were analysed using Omixon Twin software and IMGT/HLA release 3.29. Allele frequencies and linkage disequilibrium statistics were obtained by direct counting and PyPop 0.7.0 software. Arlequin v3.5.2.2 was used for haplotype frequencies (ML-EM algorithm) and Hardy-Weinberg departures (Guo-Thompson test). In total, 215 alleles were determined at third/fourth field resolution, with 25, 40, 28, 39, 34, 20 and 29 alleles identified for HLA-A, - B, -C, -DRB1, -DQA1, -DQB1 and -DPB1, respectively. The most frequent alleles observed per locus were A*02:01:01G (31.08%), B*51:01:01:01 (8.56%), C*04:01:01:01 (13.96%), DRB1*16:01:01 (13.06%), DQA1*01:02:02 (15.76%), DQB1*05:02:01 (15.76%) and DPB1*04:01:01:01 (31.98%). Of the 189 determined 6-loci haplotypes, five had a frequency of greater than 1%, the most common of which was HLA-A*01:01:01:01-B*08:01:01G- C*07:01:01:01-DRB1*03:01:01:01-DQA1*05:01:01:02- DQB1*02:01:01 (3.6%). In conclusion, a powerful tool for HLA typing was used to establish a reference database that should prove useful in future population and anthropologic studies, and in identifying high-risk, disease-associated HLA variants in East Croatian population.

Next-generation sequencing, HLA, blood donors, East Croatia

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