Department of Periodontology, Oral Medicine and Oral Surgery, Institute for Dental and Craniofacial Sciences, Charité-University Medicine Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany.
Department of Biology, Chemistry and Pharmacy, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany.
J Dent Res. 2022 May;101(5):551-558. doi: 10.1177/00220345211049984. Epub 2021 Dec 2.
Periodontitis is a common complex inflammatory disease of the oral cavity. It is characterized by inflammation of gingival tissues and alveolar bone loss. Recently, a genome-wide association study and 2 genome-wide association study meta-analyses found 2 associated regions (haplotype blocks) at the inhibitory immune receptor gene to increase the risk for periodontitis. The aims of the current study were the identification of the putative causal variants underlying these associations, characterization of their molecular biological effects, and validation of as the target gene. We mapped the associated single-nucleotide polymorphisms to DNA elements with predictive features of regulatory functions and screened the associated alleles for transcription factor (TF) binding sites. Antibody electrophoretic mobility shift assays (EMSAs) with allele-specific probes were used to identify TF binding and to quantify allele-specific effects on binding affinities. Luciferase reporter assays were used to quantify the effect directions and allele-specific strength of the associated regulatory elements. We used CRISPR-dCas9 gene activation to validate as a target of the association. EMSA in peripheral blood mononuclear cells showed 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. 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. Using CRISPR-dCas9, we showed that the enhancer at rs4284742 strongly activated expression, validating this gene as the target gene of the association. We conclude that rs11084095 and rs4284742 are putatively causal for the genome-wide significant associations with periodontitis at that impair ERG and MAFB binding, respectively.
牙周炎是一种常见的口腔复杂炎症性疾病。其特征为牙龈组织炎症和牙槽骨丧失。最近，一项全基因组关联研究和两项全基因组关联研究荟萃分析发现，抑制性免疫受体基因 上的两个相关区域（单倍型块）增加了牙周炎的风险。本研究的目的是确定这些关联背后的假定因果变异体，阐明其分子生物学效应，并验证 作为靶基因。我们将相关的单核苷酸多态性映射到具有预测调控功能的 DNA 元件上，并筛选与转录因子（TF）结合位点相关的等位基因。等位基因特异性探针的抗体电泳迁移率变动分析（EMSA）用于鉴定 TF 结合，并量化结合亲和力的等位基因特异性效应。荧光素酶报告基因测定用于量化相关调控元件的效应方向和等位基因特异性强度。我们使用 CRISPR-dCas9 基因激活来验证 作为关联的靶基因。外周血单核细胞中的 EMSA 显示 E-26 转化特异性 TF 相关基因（ERG）与 rs11084095 结合，在 minor A-allele 时几乎完全丧失结合。等位基因特异性报告基因显示 rs11084095 处的 DNA 序列具有增强子功能，在 A-allele 背景下被废除。B 淋巴细胞中的 EMSA 显示 TF MAF bZIP（MAFB）与 rs4284742 的常见 G-allele 结合，而 minor A-allele 使 TF 结合减少 69%，相应地，A-allele 使荧光素酶报告基因活性降低 9 倍。使用 CRISPR-dCas9，我们表明 rs4284742 处的增强子强烈激活 表达，验证了该基因是关联的靶基因。我们得出结论，rs11084095 和 rs4284742 分别是与牙周炎相关的全基因组显著关联的假定因果变异体，它们分别损害了 ERG 和 MAFB 的结合。
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