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.

Novel multiplex bead-based assay with LNA-modified probes for detection of MPL exon 10 mutations.

MPL exon 10 mutations were the second class of mutations shown to be associated with the pathogenesis of some Philadelphia chromosome - negative myeloproliferative neoplasms (MPNs). Recently, their identification gained wide recognition in the diagnostic work-up for suspected cases of JAK2 V617F negative MPNs. Various molecular approaches have been applied, yet universally accepted method is still lacking. We aimed at development and validation of a novel bead-based liquid assay using Locked nucleic acids (LNA)-modified oligonucleotide probes for multiplexed detection of the following MPL mutations: W515L/K/A/R. Testing on both artificial plasmid constructs and on clinical samples revealed that the method was comparable in terms of specificity to direct sequencing and had a much higher sensitivity of 1% mutant alleles. This method could be successfully implemented in the diagnostic work-up for MPNs. Furthermore, this system allows further multiplexing for single-tube identification of different mutations associated with MPNs.

Cytokine gene polymorphism in kidney transplantation--impact of TGF-beta 1, TNF-alpha and IL-6 on graft outcome.

Chronic allograft nephropathy (CAN) is one of the main causes of graft loss in renal transplantation. Polymorphisms with functional significance in the promoter and coding regions of cytokine genes have been suggested as a possible factor for graft rejection. The aim of this study was to investigate the impact of cytokine gene polymorphism of pro and anti-inflammatory cytokines on development of CAN in a group of renal transplant patients and donors. Eight single nucleotide polymorphisms (SNPs) including TNFA (-308), TGFB1 (cdns10, 25), IL-10 (-1082, -819, -592), IL-6 (-174) and IFNG (+874) were analyzed in 56 patients with stable graft function (SGF), 10 with CAN and 28 kidney donors by PCR-SSP method. CAN was significantly associated with the recipient TGFB1 cod10 T/T and combination of cods10, 25 T/T G/G genotypes (high producer), (p<0.05). Influence of patient's TNFA genotype correlated with high level of gene expression on the development of CAN was further demonstrated when the patients were stratified according to the HLA mismatches (HLA-DRB MMs). Additionally donor TNFA-308 G/A (high) and IL-6-174 CC (low) genotypes were increased in cases with CAN. No statistically significant differences in distribution of IL-10, IL-6 and IFNG genotypes between recipients with SGF and CAN were found. In conclusion our data suggest that the high producer genotype of profibrogenetic TGF-beta1, pro-inflammatory TNF-alpha and genetically determined low production of immunoregulatory IL-6 cytokine might be risk factors for CAN development.

Association of cytokine gene polymorphisms with malignant melanoma in Caucasian population.

It has been hypothesized that polymorphisms expected to result in functional changes in cytokine genes may influence susceptibility to cancer, including malignant melanoma (MM). Here, we have screened 24 potentially functional polymorphisms in five cytokine genes by PCR-SBT and PCR-SSP methods in 122 MM cell lines derived from Caucasian patients. The polymorphic positions studied were: TNFA -1031, -863, -857, -851, -574, -376, -308, -238, +488; TGFB1 -988, -800, -509, +869, +915, +652, +673, +713, +788; IL10 -1082, -819, -592; IL6 -174; IFNG -333, +874. The frequencies of cytokine genotypes of melanoma tumours were compared with those published for healthy Caucasians. It was found that TNFA -238 GA, TGFB1 -509 CT, -800 GG, IFNG +874 AT, IL6 -174 GG, IL10 -1082 GA genotypes were significantly decreased, while TNFA -238 AA, -857 CC, TGFB1 -509 TT, IFNG +874 AA, IL6 -174 CC, IL10 -1082 AA, -819 TT, -592 AA genotypes were significantly increased, in MM. This suggests that genotypes provisionally associated with low expression of pro-inflammatory and immunomodulatory TNF-alpha, IFN-gamma and IL-6 and anti-inflammatory IL-10 and TGF-beta1 could be involved in the mechanisms of cancer progression and escape from immunosurveillance.