Clinical Research of Oral Connective Tissue Program

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机制。具体地说，描述了IRF8突变在增加多发性特发性牙根吸收易感性中的作用 在我们对BSP KO小鼠的研究中，我们发现了一种特发性牙根吸收表型。根据这一发现，唾液样本是从特发性牙根吸收患者(和适当的对照)获得的，用于整个外显子组测序，因为以前的家族性病例报告表明该疾病具有遗传易感性。在底特律大学牙科慈善学院和美国国立卫生研究院(NIH)批准IRB之后，从一个表现出特发性牙根吸收的家庭(3名患者和3名未受影响的成员)收集了牙科/病史、X光、唾液样本和拔除的牙齿。在检查中，先证者和受影响的儿子和女儿表现出多颗牙齿严重的牙根吸收，但没有其他明显的病史。剥脱牙齿的显微CT显示了一种与龋齿不同的严重颈根吸收的独特模式。使用唾液进行的整个外显子组测序发现了23个候选基因中的SNPs，这些基因与吸收表型共分离，包括干扰素调节因子8(IRF8)的一个新的常染色体显性显性错义突变。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的一个新突变。这两个家系均表现为慢性牙周炎和/或多发性特发性牙根吸收，在IRF8高度保守的N端(T96M，Holland)和C端(G388S，Neely)基序中有两个新的常染色体显性显性杂合突变。IRF8主要表达于免疫细胞，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)的活性来介导破骨细胞的形成。IRF8中已发现的氨基酸变化(G388S)定位于一个高度保守的C-末端基序，导致丝氨酸磷酸化基序和磷酸丝氨酸结合结构域的改变，并被预测导致3D蛋白质折叠的大幅移动。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-/-小鼠牙周组织中破骨细胞活性增加，牙周膜(PDL)间隙增宽，牙槽骨丢失加速。 正在进行中：与Ozato博士和Holland博士合作，我们正在研究IRF8 KO小鼠和IRF8突变患者的牙周表型。WT和人类IRF8突变构建体将在IRF8 KO巨噬细胞中表达(已经启动)，以绘制全基因组IRF8突变结合位点图，并使用CHIP/ChIPseq和RNAseq分析差异调控基因。在初步研究中，我们报告了突变的人IRF8G388S载体转导到IRF8-/-巨噬细胞中的破骨细胞数量是IRF8WT的3倍。其他正在进行的检测包括免疫共沉淀和凝胶迁移分析(EMSA)、破骨细胞分化和功能分析以及IRF8 KO小鼠的表型特征。 矿化障碍：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障碍的患者DOC复合体的组织/细胞的变化。此外，我们一直在观察PPI调节因子ENPP1突变的患者，突变会导致婴儿期全身动脉钙化(GACI)，以确定是否存在牙表型。初步观察显示牙齿萌出延迟。 项目C.Chediak-Higashi综合征(CHS)：CHS是一种罕见的常染色体隐性遗传病，由编码溶酶体运输调节因子的基因突变引起，患者存在免疫缺陷，导致对影响几个组织(包括口腔组织)的感染易感性增加。在我们与巴西坎皮纳斯州立大学的Nociti博士和Kantovitz博士的持续合作中，我们对7名CHS确诊患者(4名非典型CHS患者和3名典型CHS患者，年龄8-21岁)在NIH诊所的口腔状况进行了全面检查，并从非典型CHS患者和典型CHS患者和对照组获得了牙龈成纤维细胞(GF)。临床数据显示牙周组织的出血和探查深度较健康志愿者增加，不典型患者较典型患者出血较多，但文献中所报道的严重牙周病并不明显。到目前为止进行的体外研究显示，非典型细胞与对照细胞在基线时以及在大肠杆菌脂多糖和核杆菌提取物攻击时都有不同的特征。来自qPCR和多重蛋白质分析的数据表明，来自非典型患者的牙龈成纤维细胞对脂多糖的反应发生了变化，这表明对牙科生物膜的促炎反应更强烈，而非典型CHS GF中较高水平的TLR-4在牙周对革兰氏阴性细菌的过度活跃反应中发挥了关键作用(手稿正在审查中，2016)。

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).