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Characterization of the novel HLA-DRB1*04:311 and HLA-DRB1*11:277 alleles in two Greek individuals of Cretan origin.

Characterization of the novel HLA-C*15:228 and HLA-C*04:434 alleles in two Greek individuals of Cretan origin.

Characterization of HLA-DQB1*03:439 allele in a Greek individual of Cretan origin.

Background aims The high genetic diversity of HLA across populations significantly confines the effectiveness of a donor or umbilical cord blood search for allogeneic hematopoietic stem cell transplantation (HSCT). This study aims to probe the HLA immunogenetic profile of the population of Crete, a Greek region with specific geographic and historical characteristics, and to investigate potential patterns in HLA distribution following comparison with the Deutsche Knochenmarkspenderdatei (DKMS) donor registry. It also aims to highlight the importance of regional public cord blood banks (PCBBs) in fulfilling HSCT needs, especially in countries with significant genetic diversity. Methods A cohort of 1835 samples representative of the Cretan population was typed for HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1, HLA-DPB1) loci by high-resolution second field next-generation sequencing. Data were compared with the respective HLA profiles of 12 DKMS populations (n = 20 032). Advanced statistical and bioinformatics methods were employed to assess specific intra- and inter-population genetic indexes associated with the regional and geographic distribution of HLA alleles and haplotypes. Results A considerable HLA allelic and haplotypic diversity was identified among the Cretan samples and between the latter and the pooled DKMS cohort. Even though the HLA allele and haplotype frequency distribution was similar to regions of close geographic proximity to Crete, a clinal distribution pattern from the northern to southern regions was identified. Significant differences were also observed between Crete and the Greek population of DKMS. Conclusions This study provides an in-depth characterization of the HLA immunogenetic profile in Crete and reveals the importance of demographic history in HLA heterogeneity and donor selection. The novel HLA allele and haplotype frequency comparative data between the Cretan and other European populations signify the importance of regional PCBBs in prioritizing HLA diversity to efficiently promote the HSCT program at the national level and beyond.

Characterization of the HLA-B*51:232:02 allele in a Greek individual of Cretan origin.

MicroRNA signature of THP1 cells revealed a 5.9-fold decreased expression of miR-4520a following siRNA-mediated knockdown of MEFV gene that encodes pyrin [1].

Background MicroRNA signature of THP1 cells revealed a 5.9 fold decreased expression of miR-4520a following siRNA-mediated knockdown of MEFV gene that encodes pyrin (1). Objectives We herein sought to validate the expression levels of miR-4520a in monocytes isolated from peripheral blood mononuclear cells (PBMCs) of FMF patients. Methods Dual luciferase assay and t-test analyses were used to determine the predicted target of miR-4520a, Rheb, after cloning its 3'UTR region containing miR-4520a recognition element into PGL3-prom vector. The expression levels of pyrin, miR-4520a and its putative target Rheb were validated in monocytes from FMF patients (n=10) and compared with healthy controls (n=8). Patients were off colchicine for two days (attack-free period) and monocytes were isolated from PBMCs. Total RNA together with the respective miRNA enriched fractions were isolated from monocytes and used for mRNA and miR-4520a quantitation by real-time quantitative PCR. Expression levels of mRNAs and miRNA were determined with the 2-ΔΔCt method after normalizing to 18S RNA and RNU6B housekeeping genes, respectively. Protein levels of pyrin and Rheb were detected by western blotting. Results The relative expression levels of miR-4520a were variable among FMF patients and not significantly different between patients and controls. Stratification of patients group by genotype revealed an intriguing difference in miR-4520a relative expression. Specifically, miR-4520a expression was lower in carriers of M694V variant (combined group of homo- and heterozygotes), whereas the rest of FMF genotypes had higher levels (p

Background Mutations in MEFV gene encoding pyrin [1] account for Familial Mediterranean fever FMF, a recessively inherited disease but the high number of heterozygote patients is puzzling and requires additional investigation [2,3]. Objectives Elucidation of the pathophysiology of the disease, through the identification of novel genes and miRNAs that interact with or regulate the MEFV gene. Methods Silencing of MEFV gene by AccellsiRNAs was performed in THP-1 cell line that expresses endogenous pyrin. Global changes in mRNA and microRNA expression were assessed by GeneChip HuGene-1.0-ST-v1 and GeneChipmiRNA 3.0 Array, respectively. Putative target genes of the differentially regulated miRNAs were identified by using appropriate algorithms (TargetScan, PicTar). Functional classifications and biological processes of differentially expressed mRNAs and miRNA target genes were assigned according to Gene Ontology (GO) and the updated Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. mRNA and miRNA target genetic or protein interactions were constructed using Cytoscape 3.0.2 (Plugins GeneMania and BioGrid). Results We achieved 62% knockdown of the MEFV mRNA as measured by Taqman PCR, following siRNA-mediated knockdown of the respective gene.Global transcriptome analysis revealed 89 differentially expressed genes and 25 deregulated miRNAs involved in inflammatory-mediated responses, cell cycle, cytoskeleton/microtubule dynamics, intracellular trafficking and survival. Specifically, miR-4520a and miR-19b demonstrate altered expression levels during TLR-mediated signaling, which corroborate the role of pyrin in the regulation of inflammatory responses. Interestingly, the aforementioned deregulated miRNAs are also shown to play a significant role in intracellular membrane trafficking and subsequently autophagy through regulation of small RabGTPases. Bioinformatic analyses predicted high cooperativity within the significantly deregulated miRNAs, with the clustering of 8 (out of 11) members of the miR-378/422a superfamily being the most pertinent example. Conclusions These findings reiterate the involvement of pyrin in innate immune responses and suggest a potential role of this protein in autophagy, probably through regulation of membrane trafficking RabGTPases. Validation of this data in monocytes from FMF patients is in progress. References The International FMF Consortium (1997). Cell 90:797–807. Booty M.G., Chae J.J., et al (2009). Ann. Rheum. Dis. 60:1851-1861. Touitou I, Picot MC, Domingo C, et al. (2001). Arthritis Rheum. 44:163–169. Acknowledgements Mir-Farzin Mashreghi, Helen Latsoudis and Joachim Gruen contributed equally. Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.2470