Clinical Research of Oral Connective Tissue Program

口腔结缔组织项目临床研究

• 批准号: 9359804
• 负责人: Martha Somerman
• 金额: $ 17.58万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9359804
• 关键词: Affect; Age; Alveolar Bone Loss; Amino Acids; Binding; Binding Sites; Biological Assay; Blood specimen; Brazil; C-terminal; CHS protein; Candidate Disease Gene; Case Study; Cells; Cervical; Clinic; Clinical; Clinical Data; Clinical Research; Co-Immunoprecipitations; Collaborations; Connective Tissue; Data; Daughter; Dental; Dental caries; Development; Diagnosis; Disease; Electrophoretic Mobility Shift Assay; Escherichia coli; Exhibits; Extramural Activities; Fibroblasts; Fusobacterium nucleatum; Gene Proteins; Genes; Genetic Predisposition to Disease; Gingiva; Goals; Gram-Negative Bacteria; Hemorrhage; Heterodimerization; Human; IFN consensus sequence binding protein; Immune; In Vitro; Individual; Infection; Inflammation; Inflammatory Response; Institutional Review Boards; Knockout Mice; Literature; Maintenance; Manuscripts; Maps; Mediating; Medical History; Metabolic Diseases; Microbial Biofilms; Missense Mutation; Molecular Analysis; Mus; Mutation; N-terminal; National Institute of Dental and Craniofacial Research; Natural History; Natural regeneration; Oral; Osteoclasts; Pathology; Patients; Pattern; Periodontal Diseases; Periodontal Ligament; Periodontitis; Phenotype; Phosphorylation; Phosphoserine Motif; Play; Predisposition; Proteins; Protocols documentation; Reporting; Repression; Research; Research Personnel; Role; Root Resorption; Saliva; Salivary; Sampling; School Dentistry; Serine; Son; Tissues; Tooth eruption; Tooth root structure; Tooth structure; United States National Institutes of Health; Universities; Work; base; bone metabolism; calcification; chediak-higashi syndrome; craniofacial complex; disease-causing mutation; exome sequencing; genome-wide; healthy volunteer; infancy; kindred; macrophage; member; microCT; mineralization; mutant; novel; nuclear factors of activated T-cells; oral status; oral tissue; osteoclastogenesis; proband; programs; protein folding; repaired; response; transcription factor; transcriptome sequencing; vector

Project A. Determine the genetic susceptibility and immunopathological mechanisms contributing to idiopathic tooth root resorption. Specifically, delineate the role of IRF8 mutations in increased susceptibility to multiple idiopathic root resorption During our research of the Bsp KO mice, we identified an idiopathic tooth root resorption phenotype. Based on this finding, salivary samples were obtained from individuals with idiopathic root resorption (and appropriate controls) for whole exome sequencing because previous case reports of familial pattern suggested a genetic susceptibility to the disease. Following IRB approval from the University of Detroit Mercy School of Dentistry and the National Institutes of Health (NIH), dental/medical histories, x-rays, saliva samples, and extracted teeth were collected from a kindred (3 affected and 3 unaffected members) exhibiting idiopathic root resorption. On examination, the proband and the affected son and daughter exhibited severe root resorption of multiple teeth, but had no other significant medical history. Micro-CT of exfoliated teeth revealed a unique pattern of severe cervical root resorption distinct from tooth decay. Whole exome sequencing performed using saliva identified SNPs in twenty-three candidate genes that co-segregated with the resorption phenotype, including a novel autosomal dominant missense mutation in Interferon Regulatory Factor 8 (IRF8). In another independent study using blood samples by our collaborator, Dr. Steve Holland, a novel mutation in IFR8 was identified. Both Kindreds exhibited chronic periodontitis and/or multiple idiopathic root resorption, with two novel autosomal dominant heterozygous mutations in the highly conserved N-terminal (T96M, Holland)) and C-terminal (G388S, Neely) motifs of IRF8. Primarily expressed in immune cells, IRF8 is a key regulator of inflammation and bone metabolism, and its repression mediates osteoclastogenesis by enhancing nuclear factor of activated T cells c1 (NFATc1) activity. The identified amino acid change (G388S) in IRF8 was localized to a highly conserved C-terminal motif, leading to altered serine phosphorylation motifs and phosphoserine binding domains, and is predicted to cause a large shift in 3D protein folding. These data suggest that the G388S mutation would impair IRF8 heterodimerization with other transcription factors including NFATc1, thereby producing overactive osteoclasts that target the periodontia. Consistent with these predictions, we noted that compared to WT mice, Irf8-/- mice exhibited increased osteoclast activity in the periodontia, widened periodontal ligament (PDL) space, and accelerated alveolar bone loss. Ongoing: In collaboration with Drs. Ozato and Holland, we are characterizing the periodontal phenotype in Irf8 KO mice and patients with IRF8 mutations. WT and human IRF8 mutant constructs will be expressed (already initiated) in Irf8 KO macrophages to map genome-wide IRF8 mutant binding sites and profile differentially regulated genes using ChIP/ChIPseq and RNAseq. In preliminary studies, we reported a 3-fold increase in osteoclast numbers with mutant human IRF8G388S vector transduced into Irf8-/- macrophages compared to IRF8WT. Other ongoing assays include co-immunoprecipitation and Electrophoretic Mobility Shift Assays (EMSA), osteoclast differentiation and function assays, and characterization of Irf8 KO mouse phenotype. Project B. Disorders of mineralization: In collaboration with NIDCR clinical researchers and other IC clinicians, we have been examining individuals with mineralized tissue metabolism disorders for alterations in tissues/cells of the DOC complex. In addition, we have been seeing patients with mutations in ENPP1, a PPi regulator, with mutations causing generalized arterial calcification of infancy (GACI) to determine if there is a dental phenotype. Initial observations indicated delayed tooth eruption. Project C. Chediak-Higashi Syndrome (CHS): Individuals with CHS, a rare autosomal recessive disease caused by mutations in the gene encoding lysosomal trafficking regulator, are immunodeficient, resulting in increased susceptibility to infections impacting several tissues, including oral tissues. In our ongoing collaboration with Drs. Nociti and Kantovitz from the State University of Campinas Brazil, seven CHS-diagnosed individuals (four atypical and three classic, ages 8-21) were comprehensively examined regarding their oral status in the NIH clinic, and gingival fibroblasts (GF) were obtained from atypical and classic CHS patients and control individuals. Clinical data indicated increased bleeding and probing depths of periodontal tissues vs periodontal tissues from healthy volunteers and more bleeding for atypical vs classic patients, yet the severe periodontal disease reported in the literature was not apparent. In vitro studies performed to date, reveal a different profile for atypical vs control cells at baseline and also when challenged with E. coli LPS and F. nucleatum extract. Data from qPCR and multiplex protein analyses suggest that gingival fibroblasts from atypical patients exhibit an altered response to LPS suggestive of a more robust pro-inflammatory response to dental biofilm, and that higher levels of TLR-4 in atypical CHS GF play a key role in a hyperactive periodontal response to gram-negative bacteria (manuscript under review,2016).

项目A.确定有助于特发性牙根吸收的遗传敏感性和免疫病理学机制。具体而言，描述了IRF8突变在增加对多种特发根吸收的敏感性中的作用 在对BSP KO小鼠的研究期间，我们确定了特发性牙根吸收表型。基于这一发现，从具有特发根吸收（和适当的对照）的个体中获得唾液样本，用于整个外显子组测序，因为先前的家族模式病例报告表明对该疾病具有遗传敏感性。在底特律慈悲大学牙科学院和美国国立卫生研究院（NIH）的IRB批准之后，牙科/医学历史，X射线，唾液样本和拔牙是从一名（3个受影响和3个受影响的成员）中收集的，表现出特发性根部吸收。经过检查，探险和受影响的儿子和女儿表现出严重的牙齿吸收多种牙齿，但没有其他重要的病史。去角质牙齿的微分离表现出了与牙齿衰减不同的严重宫颈根吸收的独特模式。使用唾液进行的整个外显子组测序在23个候选基因中鉴定出与吸收表型共隔离的SNP，包括干扰素调节因子8（IRF8）中新型的常染色体显性错义突变（IRF8）。在另一项独立研究中，我们的合作者史蒂夫·霍兰德（Steve Holland）博士使用了血液样本，这是IFR8中的一种新型突变。两种幼虫均表现出慢性牙周炎和/或多种特发性根部吸收，在高度保守的N末端（T96M，荷兰）中，有两个新型的常染色体显性杂合突变）和IRF8的C-末端（G388S，neely）。 IRF8主要在免疫细胞中表达，是炎症和骨骼代谢的关键调节剂，其抑制作用通过增强活化T细胞C1（NFATC1）活性的核因子来介导整骨质发生。 IRF8中鉴定出的氨基酸变化（G388）定位于高度保守的C末端基序，导致丝氨酸磷酸化基序和磷酸碱结合结构域改变，并预计会导致3D蛋白折叠的大变化。这些数据表明，G388S突变会与其他转录因子（包括NFATC1）损害IRF8异二聚化，从而产生针对周期的过度活跃的破骨细胞。与这些预测一致，我们注意到，与WT小鼠相比，IRF8 - / - 小鼠在牙周上表现出增加的破骨细胞活性，牙周韧带扩大（PDL）空间，并加速了肺泡骨损失。 正在进行：与Drs合作。 Ozato和Holland，我们正在表征IRF8 KO小鼠和IRF8突变患者的牙周表型。 WT和人IRF8突变体构建体将在IRF8 KO巨噬细胞中表达（已经启动），以绘制全基因组IRF8突变体结合位点，并使用CHIP/CHIPSEQ和RNASEQ对基因进行差异调节的基因。在初步研究中，我们报道了与IRF8WT相比，将突变的人IRF8G388S载体载体的破骨细胞数增加了3倍。其他正在进行的测定包括共免疫沉淀和电泳迁移率转移测定法（EMSA），破骨细胞分化和功能分析以及IRF8 KO小鼠表型的表征。 B.矿化疾病：与NIDCR临床研究人员和其他IC临床医生合作，我们一直在检查患有矿化组织代谢疾病的个体，以改变DOC复合物的组织/细胞。此外，我们一直在看到PPI调节剂ENPP1中有突变的患者，其突变导致了婴儿期的广义动脉钙化（GACI），以确定是否存在牙型表型。初始观察结果表明牙齿喷发延迟。 C. Chediak-Higashi综合征（CHS）项目：CHS的个体是由编码溶酶体运输调节剂的基因突变引起的一种罕见的常染色体隐性疾病，是免疫缺陷的，导致对包括口腔组织在内的多个组织的感染的敏感性增加。在我们与Drs的持续合作中。全面检查了来自巴西州立大学巴西州立大学的Nociti和Kantovitz，七个CHS诊断的个体（四名非典型和三个经典，8-21岁），从NIH诊所进行口头状态以及牙龈成纤维成纤维细胞（GF）进行了全面检查，并从非典型和经典的CHS患者和经典的CHS患者和对照组中获得。临床数据表明，牙周组织与健康志愿者的牙周组织的出血和探测深度增加，对于非典型与经典患者的出血更多，但文献中报道的严重牙周疾病并不显而易见。迄今为止进行的体外研究揭示了基线时非典型与对照细胞的不同特征，当时也揭示了大肠杆菌LPS和F. nucleatum提取物的挑战。来自QPCR和多重蛋白分析的数据表明，非典型患者的牙龈成纤维细胞表现出对LP的反应的改变，暗示了对牙齿生物膜的更强大的促炎反应，并且在非典型CHS中，TLR-4的较高水平在非典型CHS中，在非典型元素反应中对Gram-nerusia（Man）（Man）（Man）（MAN）的关键作用（均在高度元素反应中）。