High-resolution characterization of KIR genes polymorphism in healthy subjects from the Bulgarian population-A pilot study.

Although killer-cell immunoglobulin-like receptor (KIR) gene content has been widely studied in health and disease, with the advancement of next-generation sequencing (NGS) technology the high-resolution characterization of this complex gene region has become achievable. KIR allele-level diversity has lately been described across human populations. The present study aimed to analyze for the first time the allele-level polymorphism of nine KIR genes in 155 healthy, unrelated individuals from the Bulgarian population by applying NGS. The highest degree of polymorphism was detected for the KIR3DL3 gene with 40 observed alleles at five-digit resolution in total, 22 of which were common. On the other hand, the KIR3DS1 gene was found to have the lowest degree of polymorphism among the studied KIR genes with one common allele: KIR3DS1*01301 (31.6%). To better understand KIR allelic associations and patterns in Bulgarians, we have estimated the pairwise linkage disequilibrium (LD) for the 10 KIR loci, where KIR2DL3*00501 allele was found in strong LD with KIR2DL1*00101 (D' = 1.00, R2 = 0.742). This is the first study investigating KIR polymorphism at the allele level in a population from the South-East European region. Considering the effect of the populationally shaped KIR allelic polymorphism on NK cell function, this data could lead to a better understanding of the genetic heterogeneity of this region and can be carried into clinical practice by improvement of the strategies taken for NK-mediated diseases.

Elevated IgE Levels-An Allergy or an Underlying Inborn Error of Immunity in Children with Recurrent Infections?

Elevated immunoglobulin E (IgE) is a hallmark of allergic diseases. However, high IgE levels also occur in a number of other infectious and noninfectious diseases. In most cases, elevated IgE levels indicate allergy, eczema, or chronic skin infection. Very high IgE levels are not uncommon in patients with active eczema but more often indicate monogenic atopic disorder or inborn errors of immunity with an atopic phenotype. We conducted a retrospective study of 385 children with suspected immune deficiency referred to the clinic over a 9-year period. Measurement of IgE, IgG, IgA, IgM, and IgG subclasses in blood samples revealed that nearly one-third of the patients had elevated serum IgE levels. Most of the cases with elevated IgE were children with underlying atopy-mainly atopic dermatitis and, to a lesser extent, bronchial asthma-whereas 40.12% (37 children) had no atopy at all. In the most severe cases (with extremely elevated IgE or severe dermatitis), we confirmed genetic mutations for underlying immunodeficiency. Our results indicate that allergic phenotype should not be underestimated and that children with more severe allergic disease should be evaluated for an underlying inborn error of immunity. If inborn error of immunity (IEI) is suspected, a comprehensive immunologic evaluation is required. Genetic testing helps identify the specific genetic abnormality, which provides important insight into the immunopathogenesis of the disease and accurate determination of optimal therapy.

HLA class I chain-related MICA and MICB genes polymorphism in healthy individuals from the Bulgarian population.

Although human leukocyte antigen (HLA) gene polymorphism has been investigated in many populations around the world, the data on MHC class I chain-related (MIC) genes are still limited. The present study is aimed to analyze the allelic polymorphism of MICA and MICB genes and haplotype associations with HLA-B locus in 132 healthy, unrelated individuals from the Bulgarian population by next generation sequencing (NGS). A total of 36 MICA and 16 MICB alleles were observed with the highest frequency detected for MICA*008:01 (17.1%) and MICB*005:02 (32.4%). Further, two and three-loci haplotype frequencies and pairwise linkage disequilibrium were estimated. Highly significant global linkage disequilibrium was found between either HLA-B and MICA and MICB genes. This is the first study on MICA and MICB allelic polymorphism, linkage disequilibrium, and haplotype polymorphism in the Bulgarian population. These results will allow for better characterization of the genetic heterogeneity of the Bulgarian population and could contribute to further analyses on MICA and MICB clinical significance.

Probable HLA-mediated immunoediting of JAK2 V617F-driven oncogenesis.

Human leukocyte antigen class I (HLA-I) genotype has been found to influence cancer development through the presentation of mutational neoepitopes. However, our understanding of its effect on the development of myeloproliferative neoplasms (MPNs) remains limited. We aimed to elucidate the putative protective role of HLA-I alleles in the development of JAK2 V617F-driven MPNs using a population genetics approach. The variability of the HLA-I genotype had no effect on the presence of JAK2 V617F mutation. However, three alleles were found to be inversely correlated with the presence of JAK2 V617F mutation: HLA-A*02:01 (p = 0.036), HLA-B*35:01 (p = 0.017), and HLA-C*15:02 (p = 0.033). The HLA-B*35:01 allele was predicted to bind to a 9-mer peptide derived from JAK2 V617F mutant protein. Gene expression analysis revealed a lower expression of HLA-A and -B in MPN CD34+ cells compared with normal CD34+ cells, which was modulated by ruxolitinib and interferon-α treatment. In summary, we provide robust evidence that specific HLA-I molecules restrict JAK2 V617F-driven oncogenesis. JAK2 V617F+ stem cells evade immune surveillance through downregulation of the HLA-I expression. Therefore, the presence of specific HLA-I alleles might be a predictive marker for response to certain immunotherapies upregulating HLA-I expression. Finally, our findings have implications in the development of mutational neoepitope-based vaccines in MPNs.

17th IHIW component "Immunogenetics of Ageing" - New NGS data.

The 'Immunogenetics of Aging' project is a component introduced in the 14th International HLA and Immunogenetics Workshop (IHIW) and developed further within subsequent workshops. The aim was to determine the relevance of immunogenetic markers, focusing on HLA, cytokine genes, and some innate immunity genes, for successful aging and an increased capacity to reach the extreme limits of life-span. Within the 17th IHIW we applied Next Generation Sequencing methods to refine further HLA associations at allele level in longevity, and to extend our knowledge to additional loci such as HLA-DQA1, HLA-DPB1 and HLA-DPA1. Analysis of relatively small number of healthy elderly and young controls from four populations showed that some HLA class I and class II alleles were significantly positively associated with healthy aging. Additionally we observed statistically significant differences in HLA allele distribution when the analysis was performed separately in elderly females and males compared to sex-matched young controls. Haplotypes, probably associated with better control of viral and malignant diseases were increased in the elderly sample. These preliminary NGS data could confirm our hypotheses that survival and longevity might be associated with selection of HLA alleles and haplotypes conferring disease resistance or susceptibility. Therefore HLA alleles and haplotypes could be informative immunogenetic markers for successful ageing.

Evaluation of lymphocyte subpopulations in cord blood of Bulgarian newborns.

BACKGROUND: Umbilical cord blood (UCB) is a clinically useful source of hematopoietic stem and progenitor cells for treatment of a wide variety of malignant and non-malignant disorders. An important way to completing infor- mation on the quality and composition of units for transplantation is more extensive immunophenotyping of UCB. Moreover, phenotyping of lymphocyte subpopulations is essential for the diagnosis and follow-up of children with immunodeficiencies and other immune disorders and therefore, establishment of age-matched reference values of lymphocyte subsets is a necessity for each population. The aim of this study was to determine the normal range of T and B lymphocytes, and NK cells as well as the CD4 and CD8 subpopulations of T cells in cord blood collected from healthy term infants. METHODS: The relative and absolute number distributions (median, 5th and 95th percentile) of lymphocyte subsets in cord blood samples from 72 healthy newborns were examined by multi-colour flow cytometry with a view to obtaining reference values for Bulgarian neonates at birth. RESULTS: Mean percentages of lymphocyte subpopulations were: CD3 (62.27 ± 9.64), CD19 (17.47 ± 5.46), CD3- CD16/CD56+ (17.27 ± 8.4). Our results show the prevalence of helper-inducer CD3+CD4+ (44.88-8.21) compared to the suppressor-cytotoxic CD3+CD8+ (16.65 ± 4.54) T-cell subpopulation, which determines the positive CD4/CD8 ratio (2.86 ± 0.82; 1.4-4.8). Also, the absolute numbers of studied populations varied widely due to differences of the absolute number of lymphocytes in the samples. CONCLUSIONS: This study on distribution of lymphocyte subpopulations in UCB helps to enhance our knowledge about cell phenotypes in cord blood and improve characterization of products for cellular therapy, as well as contributes to the correct interpretation of laboratory results for infants with possible immune disorders. Our data can be used as normal intervals for lymphocyte subsets in Bulgarian neonates.