Direct dissection of genomic features determining transcription factor binding, and systematic characterization of long noncoding RNAs, which are associated with an increased risk of aggressive periodontitis

牙周炎（PD）是世界上最常见的炎症性疾病之一，遗传率为50%。它影响人群，严重形式的流行率为11%。口腔炎症会影响牙周组织的大片区域，从而对免疫系统造成严重的压力。目前还缺乏精确的遗传分子生物学机制的详细知识，这些机制驱动PD发病过程中的各个步骤。为了确定PD的主要易感基因，我们进行了一项与侵袭性牙周炎（AgP）的大型全基因组关联研究（GWAS）， AgP是PD最严重和早发的疾病表型。通过整合我们输入的GWAS数据和强连锁不平衡的变异，以及最具暗示性的GWAS导联多态性与ENCODE （DNA元件百科全书）联盟关于细胞类型特异性基因组特征的数据，我们确定了推测的致病变异（pcv），这些变异高度暗示在基因表达中具有调节作用。为了确定它们的因果关系和功能，我们制定了以下研究目标。(1)用EMSA（电泳迁移位移法）分析指定PCVs的效应等位基因对蛋白质- dna结合的功能影响。(2)为了证明PCVs对基因表达的直接影响，我们将在永生化的牙龈和淋巴母细胞系中设计染色体整合报告细胞试验。(3)在鉴定的转录因子过表达后，我们将通过定量表达谱和蛋白印迹验证预测的pcv靶基因。此外，我们将通过CRISPR-dCas9激活和RACE-PCR （dna末端快速扩增）在血液和牙龈原代细胞中表征三种最具提示性的agp相关长链非编码rna (lncRNAs)，然后进行全转录组mRNA测序。lncRNA调控的基因将通过蛋白印迹法进行验证。生成的数据将把AgP相关的lncRNA聚合到病原学相关的基因网络中，并将极大地有助于识别和表征关键的pcv及其对驱动AgP疾病风险的信号通路的影响。

Periodontitis (PD) is among the most common inflammatory diseases worldwide with a heritability of 50%. It affects human populations at prevalence rates of 11% for the severe forms. The oral inflammation can affect large areas of the periodontal tissues and can thus pose a substantial stress for the immune system. Detailed knowledge of the precise genetic molecular biological mechanisms that drive the individual steps in the pathogenesis of PD is currently missing. To identify major susceptibility genes of PD, we performed a large genome-wide association study (GWAS) with aggressive periodontitis (AgP), the most severe and early-onset disease phenotype of PD. By integrating our imputed GWAS data together with variants in strong linkage disequilibrium to the most suggestive GWAS lead-polymorphisms with data of the ENCODE (Encyclopedia of DNA Elements) consortium on cell type specific genomic features, we identified putative causative variants (PCVs) that are highly suggestive to have a regulatory role in gene expression. To establish their causality and function, we formulated the following aims of research. (1) Dissection of the functional impacts of the effect alleles of the nominated PCVs on protein-DNA binding by EMSA (electrophoretic mobility shift assays). (2) To provide evidence for the direct effects of the PCVs on gene expression, we will design chromosomally integrated reporter assays in immortalized gingival and lymphoblast cell lines. (3) Following overexpression of the identified transcription factors, we will validate the predicted target genes of the PCVs by quantitative expression profiling and protein blotting. In addition, we will characterize the three most suggestive AgP-associated long noncoding RNAs (lncRNAs) by CRISPR-dCas9 activation and RACE-PCR (rapid amplification of cDNA-ends) in primary cells of blood and the gingiva, followed by whole transcriptome mRNA sequencing. The genes that are regulated by the lncRNA will be validated by protein blotting. The generated data will aggregate the AgP-associated lncRNA into etiological relevant gene networks and will substantially contribute to identify and characterize key PCVs and their effects on signaling pathways that drive the disease risk of AgP.