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. 确定导致特发性牙根吸收的遗传易感性和免疫病理学机制。具体来说，描述 IRF8 突变在增加对多种特发性牙根吸收的易感性中的作用 在我们对 Bsp KO 小鼠的研究中，我们发现了特发性牙根吸收表型。基于这一发现，从患有特发性牙根吸收的个体（和适当的对照）中获取唾液样本进行全外显子组测序，因为之前的家族模式病例报告表明对该疾病具有遗传易感性。经底特律大学梅西牙科学院和美国国立卫生研究院 (NIH) 批准后，IRB 从表现出特发性牙根吸收的亲属（3 名受影响成员和 3 名未受影响成员）收集了牙科/病史、X 射线、唾液样本和拔除的牙齿。经过检查，先证者和受影响的儿子和女儿表现出多颗牙齿的严重牙根吸收，但没有其他重要的病史。脱落牙齿的显微 CT 显示了与蛀牙不同的严重颈根吸收的独特模式。使用唾液进行的全外显子组测序鉴定了 23 个与吸收表型共分离的候选基因中的 S​​NP，其中包括干扰素调节因子 8 (IRF8) 中的一种新型常染色体显性错义突变。在我们的合作者 Steve Holland 博士使用血液样本进行的另一项独立研究中，发现了 IFR8 的新突变。两个亲属均表现出慢性牙周炎和/或多发特发性牙根吸收，在 IRF8 高度保守的 N 端（T96M，Holland）和 C 端（G388S，Neely）基序中存在两个新的常染色体显性杂合突变。 IRF8 主要在免疫细胞中表达，是炎症和骨代谢的关键调节因子，其抑制通过增强活化 T 细胞核因子 c1 (NFATc1) 活性来介导破骨细胞生成。 IRF8 中已鉴定的氨基酸变化 (G388S) 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丝氨酸结合域发生改变，预计会导致 3D 蛋白质折叠发生巨大变化。这些数据表明，G388S 突变会损害 IRF8 与包括 NFATc1 在内的其他转录因子的异二聚化，从而产生针对牙周组织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-/- 小鼠表现出牙周破骨细胞活性增加、牙周韧带 (PDL) 间隙扩大和牙槽骨丢失加速。 正在进行：与博士合作。 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（已启动），以绘制全基因组 IRF8 突变体结合位点，并使用 ChIP/ChIPseq 和 RNAseq 分析差异调节基因。在初步研究中，我们报道，与 IRF8WT 相比，将突变型人 IRF8G388S 载体转导到 Irf8-/- 巨噬细胞中，破骨细胞数量增加了 3 倍。其他正在进行的测定包括免疫共沉淀和电泳迁移率变化测定 (EMSA)、破骨细胞分化和功能测定以及 Irf8 KO 小鼠表型的表征。 项目 B. 矿化障碍：我们与 NIDCR 临床研究人员和其他 IC 临床医生合作，一直在检查患有矿化组织代谢障碍的个体 DOC 复合体组织/细胞的变化。此外，我们还观察到 ENPP1（一种 PPi 调节因子）发生突变的患者，这些突变会导致婴儿期广泛动脉钙化 (GACI)，以确定是否存在牙齿表型。初步观察表明牙齿萌出延迟。 项目 C. Chediak-Higashi 综合征 (CHS)：CHS 是一种罕见的常染色体隐性遗传疾病，由编码溶酶体运输调节因子的基因突变引起，患有免疫缺陷，导致对影响多个组织（包括口腔组织）的感染的易感性增加。在我们与博士的持续合作中。巴西坎皮纳斯州立大学的 Nociti 和 Kantovitz 等人在 NIH 诊所对 7 名中枢性低通气综合症诊断者（4 名非典型型和 3 名典型型，年龄 8-21 岁）的口腔状况进行了全面检查，并从非典型和典型中枢性低通气综合症患者和对照个体中获取牙龈成纤维细胞 (GF)。临床数据表明，与健康志愿者的牙周组织相比，牙周组织的出血和探诊深度增加，非典型患者与典型患者相比，牙周组织的出血更多，但文献中报道的严重牙周病并不明显。迄今为止进行的体外研究揭示了非典型细胞与对照细胞在基线以及用大肠杆菌 LPS 和具核梭菌提取物攻击时的不同特征。来自 qPCR 和多重蛋白质分析的数据表明，非典型患者的牙龈成纤维细胞对 LPS 的反应发生了改变，表明对牙齿生物膜有更强烈的促炎反应，并且非典型 CHS GF 中较高水平的 TLR-4 在牙周对革兰氏阴性菌的过度活跃反应中发挥着关键作用（手稿正在审查中，2016）。