Next-generation sequencing (NGS) 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 complex ethnic admixture characteristic for East Croatian population, we aimed to investigate class-I (HLA-A, -B, -C) and class-II (HLA-DRB1, -DQA1, -DQB1) HLA diversity at the highest, 4-field resolution level in 120 healthy, unrelated, blood donor volunteers. Genomic DNA was extracted and HLA genotypes of class I and DQA1 genes were defined in full-length, -DQB1 from intron 1 to 3' UTR, and -DRB1 from intron 1 to intron 4 (Illumina MiSeq platform, Omixon Twin algorithms, IMGT/HLA release 3.30.0_5). Linkage disequilibrium statistics, Hardy-Weinberg departures, and haplotype frequencies were inferred by exact tests and iterative Expectation-Maximization algorithm using PyPop 0.7.0 and Arlequin v3.5.2.2 software. Our data provide first description of 4-field allele and haplotype frequencies in Croatian population, revealing 192 class-I and class-II alleles and extended haplotypic combinations not apparent from the existing 2-field HLA reports from Croatia. This established reference database complements current knowledge of HLA diversity and should prove useful in future population studies, transplantation settings, and disease-associated HLA screening.

Department of Laboratory Diagnostics and Clinical Transfusion Medicine Clinical Institute of Transfusion Medicine Osijek University Hospital J Huttlera 4 HR 31000 Osijek Croatia

Department of Medical Chemistry Biochemistry and Clinical Chemistry Faculty of Medicine University of Osijek J Huttlera 4 HR 31000 Osijek Croatia

Department of Nuclear Medicine and Oncology Faculty of Medicine University of Osijek J Huttlera 4 HR 31000 Osijek Croatia

Department of Pathological Physiology Faculty of Medicine and Dentistry Palacký University Hnevotinska 3 775 15 Olomouc Czech Republic

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Rendečić Miočević, D. Iliri i Antički Svijet. (The Illyrians and the Ancient World., ed. Mimica, I) Književni krug: Split, Croatia, 1–920 (1989).

Mujadžević D. The other Ottoman serhat in Europe: Ottoman territorial expansion in Bosnia and Croatia in 16th century. Ankara Univ. J. Center South East Eur. Studies. 2012;1:99–111.

Pavičić, S. Slavonija u svojem naselnom razvitku od trinaestog stoljeća do danas. (Slavonia In Its Settlement Development From The Thirteenth Century To The Present., ed. V, Radauš) Zbornik radova I. znanstvenog sabora Slavonije i Baranje, Jugoslavenska akademija znanosti i umjetnosti, 191–236 (1970).

Sanchez-Mazas A, Buhler S, Nunes JM. A new HLA map of Europe: Regional genetic variation and its implication for peopling history, disease-association studies and tissue transplantation. Hum. Hered. 2013;76:162–177. doi: 10.1159/000360855. PubMed DOI

Choo SY. The HLA system: genetics, immunology, clinical testing, and clinical implications. Yonsei. Med. J. 2007;48:11–23. doi: 10.3349/ymj.2007.48.1.11. PubMed DOI PMC

Robinson J, et al. The IPD and IPD-IMGT/HLA Database: allele variant databases. Nucleic Acids Res. 2015;43:D423–431. doi: 10.1093/nar/gku1161. PubMed DOI PMC

Robinson J, Malik A, Parham P, Bodmer JG, Marsh SGE. IMGT/HLA - a sequence database for the human major histocompatibility complex. Tissue Antigens. 2000;55:280–28. doi: 10.1034/j.1399-0039.2000.550314.x. PubMed DOI

Trowsdale J, Knight JC. Major Histocompatibility Complex Genomics and Human Disease. Annu Rev. Genomics Hum. Genet. 2013;14:301–323. doi: 10.1146/annurev-genom-091212-153455. PubMed DOI PMC

Gonzalez-Galarza FF, et al. Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations. Nucleic Acid Res. 2015;28:D784–D788. doi: 10.1093/nar/gku1166. PubMed DOI PMC

Sanchez-Mazas A, et al. Common and well-documented HLA alleles over all of Europe and within European sub-regions: A catalogue from the European Federation for Immunogenetics. HLA. 2017;89:104–113. doi: 10.1111/tan.12956. PubMed DOI

Pröll J, et al. Sequence capture and next generation resequencing of the MHC region highlights potential transplantation determinants in HLA identical haematopoietic stem cell transplantation. DNA Res. 2011;18:201–210. doi: 10.1093/dnares/dsr008. PubMed DOI PMC

Zhao LP, et al. Next-generation sequencing reveals that HLA-DRB3, -DRB4, and -DRB5 may be associated with islet autoantibodies and risk for childhood type 1 diabetes. Diabetes. 2016;65:710–718. doi: 10.2337/db15-1115. PubMed DOI PMC

Carapito R, Radosavljevic M, Bahram S. Next-generation sequencing of the HLA locus: methods and impacts on HLA typing, population genetics and disease association studies. Hum. Immunol. 2016;77:1016–1023. doi: 10.1016/j.humimm.2016.04.002. PubMed DOI

Kishore A, Petrek M. Next-generation sequencing based HLA typing: deciphering immunogenetic aspects of sarcoidosis. Front Genet. 2018;9:503. doi: 10.3389/fgene.2018.00503. PubMed DOI PMC

Sanchez-Mazas A, Meyer D. The relevance of HLA sequencing in population genetics studies. J. Immunol. Res. 2014;2014:971818. doi: 10.1155/2014/971818. PubMed DOI PMC

Grubic Z, Zunec R, Cecuk-Jelicic E, Kerhin-Brkljacic, Kastelan A. Polymorphism of HLA-A, -B, -DRB1, -DQA1, and –DQB1 haplotypes in a Croatian population. Eur. J. Immunogenet. 2000;27:47–51. doi: 10.1046/j.1365-2370.2000.00193.x. PubMed DOI

Grubic Z, et al. HLA-A, HLA-B and HLA-DRB1 allele and haplotype diversity among volunteer bone marrow donors from Croatia. Int. J. Immunogenet. 2014;41:211–221. doi: 10.1111/iji.12117. PubMed DOI

Grubic Z, Maskalan M, Svilicic D, Stingl Jankovic K, Zunec R. Determination of HLA-A, -B, and -DRB1 allele and haplotype frequencies in the croatian population based on a family study. Arch. Immunol. Ther. Exp. (Warsz). 2016;64(Suppl 1):83–88. doi: 10.1007/s00005-016-0445-3. PubMed DOI

Grubic Z, et al. The effect of HLA allele and haplotype polymorphisms on donor matching in hematopoietic stem cell transplantation – Croatian experience. Hum. Immunol. 2016;77:1120–1127. doi: 10.1016/j.humimm.2016.10.004. PubMed DOI

Pingel J, et al. High-resolution HLA haplotype frequencies of stem cell donors in Germany with foreign parentage: how can they be used to improve unrelated donor searches? Hum. Immunol. 2013;74:330–340. doi: 10.1016/j.humimm.2012.10.029. PubMed DOI

Martinović I, et al. Application of HLA class II polymorphism analysis to the study of the population structure of the island Krk, Croatia. Hum Biol. 1997;69:819–829. PubMed

Grubić Z, et al. HLA class II gene and haplotype diversity in the population of the island of Hvar, Croatia. Coll. Antropol. 1998;22:157–168. PubMed

Crnić-Martinović M, et al. HLA class I and class II polymorphism in the population of Rijeka, Croatia. Coll. Antropol. 2002;26:69–75. PubMed

Crnić-Martinović M, et al. HLA class II polymorphism in autochthonous population of Gorski kotar, Croatia. Coll. Antropol. 2007;31:853–858. PubMed

Grubic Z, Burek Kamenaric M, Maskalan M, Stingl Jankovic K, Zunec R. Nonfrequent but well-documented, rare and very rare HLA alleles observed in the Croatian population. Tissue Antigens. 2014;84:560–564. doi: 10.1111/tan.12476. PubMed DOI

Burek Kamenaric M, et al. HLA-DPB1 matching in unrelated hematopoietic stem cell transplantation program contributes to a higher incidence of disease relapse. Hum. Immunol. 2017;78:665–671. doi: 10.1016/j.humimm.2017.08.008. PubMed DOI

Rendine S, et al. Estimation of human leukocyte antigen class I and class II high-resolution allele and haplotype frequencies in the Italian population and comparison with other European populations. Hum. Immunol. 2012;73:399–404. doi: 10.1016/j.humimm.2012.01.005. PubMed DOI

Burek Kamenaric M, Maskalan M, Drabbels J, Golubic Cepulic B, Grubic Z. Identification of the novel HLA-B*18:37:02 allele in a Croatian individual. HLA. 2018;91:299–300. doi: 10.1111/tan.13238. PubMed DOI

Matevosyan L, et al. HLA-A, HLA-B, and HLA-DRB1 allele distribution in a large Armenian population sample. Tissue Antigens. 2011;78:21–30. doi: 10.1111/j.1399-0039.2011.01668.x. PubMed DOI

Schmidt AH, et al. Estimation of high-resolution HLA-A, -B, -C, -DRB1 allele and haplotype frequencies based on 8862 German stem cell donors and implications for strategic donor registry planning. Hum. Immunol. 2009;70:895–902. doi: 10.1016/j.humimm.2009.08.006. PubMed DOI

Meyer, D. et al. 13th IHWS anthropology/human genetic diversity joint report. Chapter 4. Single locus polymorphism of classical HLA genes. (ed. Hansen J. A.) Immunobiology of the human MHC, International Histocompatibility Working Group Press, 1–52 (2006).

Amirzargar A, et al. Kurds HLA Genes: Its Implications in Transplantation and Pharmacogenomics. Open Med. J. 2015;2:43–47. doi: 10.2174/1874220301401010043. DOI

Hajjej A, et al. HLA class I and class II polymorphism in a population from south-eastern Tunisia (Gabes Area) Int. J. Immunogenet. 2011;38:191–199. doi: 10.1111/j.1744-313X.2011.01003.x. PubMed DOI

Ivanova M, et al. HLA polymorphism in Bulgarians defined by high-resolution typing methods in comparison with other populations. Tissue Antigens. 2002;60:496–504. doi: 10.1034/j.1399-0039.2002.600605.x. PubMed DOI

Nowak J, et al. Allele and extended haplotype polymorphism of HLA-A, -C, -B, -DRB1 and -DQB1 loci in Polish population and genetic affinities to other populations. Tissue Antigens. 2008;71:193–205. doi: 10.1111/j.1399-0039.2007.00991.x. PubMed DOI

Davey S, Ord J, Navarrete C, Brown C. HLA-A, -B and -C allele and haplotype frequencies defined by next generation sequencing in a population of 519 English blood donors. Hum. Immunol. 2017;78:397–398. doi: 10.1016/j.humimm.2017.04.001. PubMed DOI

Montero-Martín G, et al. High-resolution characterization of allelic and haplotypic HLA frequency distribution in a Spanish population using high-throughput next-generation sequencing. Hum. Immunol. 2019;80:429–436. doi: 10.1016/j.humimm.2019.02.005. PubMed DOI PMC

Vidan-Jeras B, et al. Resolution of HLA-B*44:02:01G, -DRB1*14:01:01G and-DQB1*03:01:01G reveals a high allelic variability among 12 European populations. Tissue Antigens. 2014;84:459–464. doi: 10.1111/tan.12422. PubMed DOI

Arnaiz-Villena A, et al. HLA genes in Macedonians and the sub-Saharan origin of the Greeks. Tissue Antigens. 2001;57:118–127. doi: 10.1034/j.1399-0039.2001.057002118.x. PubMed DOI

Petlichkovski A, et al. High-resolution typing of HLA-DRB1 locus in the Macedonian population. Tissue Antigens. 2004;64:486–491. doi: 10.1111/j.1399-0039.2004.00273.x. PubMed DOI

Papassavas EC, et al. MHC class I and class II phenotype, gene, and haplotype frequencies in Greeks using molecular typing data. Hum Immunol. 2000;61:615–623. doi: 10.1016/S0198-8859(00)00115-4. PubMed DOI

Zajacova M, Kotrbova-Kozak A, Cerna M. HLA-DRB1, -DQA1 and –DQB1 genotyping of 180 Czech individuals from the Czech Republik pop 3. Hum Immunol. 2016;77:365–366. doi: 10.1016/j.humimm.2016.02.003. PubMed DOI

Kokaraki G, et al. Major histocompatibility complex class II (DRB1*, DQA1*, and DQB1*) and DRB1*04 subtypes’ associations of Hashimoto’s thyroiditis in a Greek population. Tissue Antigens. 2009;73:199–205. doi: 10.1111/j.1399-0039.2008.01182.x. PubMed DOI

Reveille JD, et al. HLA-class II alleles and C4 null genes in Greeks with systemic lupus erythematosus. Tissue Antigens. 1995;46:417–421. doi: 10.1111/j.1399-0039.1995.tb03140.x. PubMed DOI

Wu Z, et al. Molecular analysis of HLA-DQ and -DP genes in caucasoid patients with Hashimoto’s thyroiditis. Tissue Antigens. 1994;43:116–119. doi: 10.1111/j.1399-0039.1994.tb02310.x. PubMed DOI

Kirijas M, Genadieva Stavrik S, Senev A, Efinska Mladenovska O, Petlichkovski A. HLA-A, -B, -C and -DRB1 allele and haplotype frequencies in the Macedonian population based on a family study. Hum. Immunol. 2018;79:145–153. doi: 10.1016/j.humimm.2017.12.003. PubMed DOI

Stingl Jankovic K, et al. The study of HLA-B*44~C haplotype polymorphism in the Croatian population. Mol. Exp. Biol. Med. 2017;1:42–46.

Sulcebe G, et al. HLA allele and haplotype frequencies in the Albanian population and their relationship with the other European populations. Int. J. Immunogenet. 2009;36:337–343. doi: 10.1111/j.1744-313X.2009.00868.x. PubMed DOI

Excoffier L, Laval G, Schneider S. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol. Bioinform. Online. 2007;23:47–50. PubMed PMC

Guo SW, Thompson EA. Performing the exact test of Hardy-Weinberg proportion for multiple alleles. Biometrics. 1992;48:361–372. doi: 10.2307/2532296. PubMed DOI

Excoffier L, Slatkin M. Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population. Mol. Biol. Evol. 1995;12:921–927. PubMed

Lewontin RC. The interaction of selection and linkage. I. General considerations; heterotic models. Genetics. 1964;49:49–67. PubMed PMC

Hedrick PW. Gametic disequilibrium measures: proceed with caution. Genetics. 1987;117:331–341. PubMed PMC

Cramer, H. Mathematical Models Of Statistics. Princeton University Press, Princeton, NJ, p. 575 (1946).

Lancaster AK, Single RM, Solberg OD, Nelson MP, Thomson G. PyPop update—a software pipeline for large-scale multilocus population genomics. Tissue Antigens. 2007;69:192–197. doi: 10.1111/j.1399-0039.2006.00769.x. PubMed DOI PMC

Adapted from Regije Hrvatske, Wikipedia. Retrieved December 12, 2019, from https://bs.wikipedia.org/wiki/Regije_Hrvatske#/media/Datoteka:Croatia_Macroregions.svg, Available for reuse under the Creative Commons Attribution-Share Alike 3.0 (CC BY-SA 3.0) Unported license (https://creativecommons.org/licenses/by-sa/3.0/), work by Tomobe03 using file: Croatia location map.svg by NordNordWest, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=19320911 (2012).

Bhatla, N. Exon-Intron Graphic Maker. Available at, http://wormweb.org/exonintron (2012).

R Core Team R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, http://www.R-project.org (2013).