Periodontitis is a common complex inflammatory disease of the oral cavity and it is characterized by inflammation of gingival tissue and alveolar bone loss. Recently, a genome-wide association study (GWAS) and two GWAS-meta-analyses identified two associated regions at the inhibitory immune receptor gene SIGLEC5 to increase the risk for periodontitis. The GWAS also identified two associated regions at the long non-coding RNA CTD-2353F22.1. Additionally, a gene-gene interaction study identified gene-gene interaction between risk variants at SIGLEC5 and at CTD-2353F22.1. The aims of the current thesis were the identification of the putative causal variants underlying these associations, characterization of their molecular biological effects and validation of SIGLEC5 and CTD-2353F22.1 as the target genes and to identify genes and genetic pathways regulated by CTD-2353F22.1. In addition, this thesis aims to identify the target genes of a miRNA that was differentially expressed in periodontitis patients compared to healthy controls. Employed methods included SNP mapping to DNA elements with predictive features of regulatory functions, screening for transcription factor (TF) binding sites, antibody electrophoretic mobility shift assays (EMSAs), luciferase reporter gene assays, CRISPR/dCas9 gene activation and RNA-sequencing to identify genetic interaction partners of CTD-2353F22.1. Genome-wide mRNA expression was quantified (Clarion Array) to identify the target gene of hsa-miR-374b-5p in primary gingival fibroblasts (pGF) and gene specific regulation was validated by qRT-PCR and Luciferase activity. In summary, EMSA in B lymphocytes showed that TF MAF bZIP (MAFB) binds at the common G-allele of rs4284742, whereas the minor A-allele reduced TF binding by 69%, corresponding to 9-fold reduction of luciferase reporter gene activity by the A-allele (p = 0.01). EMSA in peripheral blood mononuclear cells revealed that E-26 transformation-specific TF related gene (ERG) binds at rs11084095, with almost complete loss of binding at the minor A-allele. Allele-specific reporter genes showed enhancer function of the DNA-sequence at rs11084095, which was abrogated in the background of the A-allele. For the associations at CTD-2353F22.1, allele-specific binding of the TF PR Domain Zinc Finger Protein 14 (PRDM14) at rs1122900 was confirmed using antibody EMSA. Binding was reduced by 76% at the rare C-allele. Luciferase reporter gene assays revealed an allele-specific enhancer effect with a 5.5-fold increase of expression in the background of the common A-allele (p = 0.0003). Using CRISPR-dCas9, the enhancer at rs4284742 showed strong activation of SIGLEC5 expression, validating this gene as the target gene of the association. The enhancer at rs11084095 and rs1122900 demonstrated moderately activated SIGLEC5 and CTD-2353F22.1 expression. RNA sequencing of CTD-2353F22.1 CRISPRa cells indicated significant upregulation of the gene sets “angiogenesis” and “TNFalpha signaling via NFκB”. UHMK1 was the top one downregulated gene in pGFs (p = 2.5x10-04, fold change (FC) = 1.8) in genome-wide expression analysis after hsa-miR-374b-5p overexpression. Using reporter gene assays with the cloned 3’UTR of UHMK1, the downregulation of mRNA level (p = 0.02; FC = 1.5) and reduction of luciferase protein activity (p = 0.013, FC = -1.3) was validated. In conclusion, rs4284742, rs11084095 and rs1122900 were identified as putative causal variants for the genome-wide significant associations with periodontitis that impair MAFB, ERG and PRDM14 binding, respectively and SIGLEC5 and CTD-2353F22.1 were validated as the target genes of the associations. hsa-miR-374b-5p was shown to regulate UHMK1, which has a role in osteoclast differentiation. Data generated from this project can help to improve the understanding of the underlying disease biology, to point to a regulatory genetic pathway and hopefully leading to new treatment options.
