Clinical Research of Oral Connective Tissue Program

项目A.探讨特发性牙根吸收的遗传易感性及免疫病理机制。特别是，描述IRF 8突变在多发性特发性牙根吸收易感性增加中的作用。 在我们对Bsp基因敲除小鼠的研究中，我们发现了一种特发性牙根吸收表型。基于这一发现，唾液样本获得自个体与特发性牙根吸收（和适当的控制）的全外显子组测序，因为以前的病例报告的家族模式表明遗传易感性的疾病。在获得底特律大学慈善牙科学院和美国国立卫生研究院（NIH）的IRB批准后，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）中收集牙科/病史、X线片、唾液样本和拔牙。经检查，先证者和受影响的儿子和女儿表现出严重的根吸收的多个牙齿，但没有其他重要的病史。脱落牙齿的显微CT显示了一种独特的严重的颈部牙根吸收模式，与蛀牙不同。使用唾液进行的全外显子组测序鉴定了与再吸收表型共分离的23个候选基因中的SNP，包括干扰素调节因子8（IRF 8）中的新的常染色体显性错义突变。在另一项由我们的合作者Steve Holland博士使用血液样本进行的独立研究中，发现了IFR 8中的一种新突变。这两种Kinetamine都表现出慢性牙周炎和/或多发性特发性牙根吸收，在IRF 8的高度保守的N-末端（T96 M，Holland）和C-末端（G388 S，Neely）基序中有两个新的常染色体显性杂合突变。IRF 8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（NFATc 1）活性介导破骨细胞生成。在IRF 8中鉴定的氨基酸变化（G388 S）定位于高度保守的C-末端基序，导致改变的丝氨酸磷酸化基序和磷酸丝氨酸结合结构域，并且预测引起3D蛋白质折叠的大位移。这些数据表明，G388 S突变会损害IRF 8与其他转录因子（包括NFATc 1）的异源二聚体化，从而产生过度活跃的破骨细胞，靶向牙周组织。与这些预测一致，我们注意到，与WT小鼠相比，Irf 8-/-小鼠在牙周组织中表现出破骨细胞活性增加，牙周膜（PDL）间隙变宽，牙槽骨丢失加速。 正在进行中：与Ozato和Holland博士合作，我们正在表征Irf 8 KO小鼠和IRF 8突变患者的牙周表型。WT和人IRF 8突变体构建体将在Irf 8 KO巨噬细胞中表达（已启动），以使用ChIP/ChIPseq和RNAseq绘制全基因组IRF 8突变体结合位点图谱并分析差异调节基因。在初步研究中，我们报道了与IRF 8 WT相比，用突变体人IRF 8 G388 S载体转导到Irf 8-/-巨噬细胞中的破骨细胞数量增加了3倍。其他正在进行的试验包括免疫共沉淀和电泳迁移率变化试验（EMSA），破骨细胞分化和功能试验，以及Irf 8 KO小鼠表型的表征。 项目B。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个体的DOC复合物组织/细胞的改变。此外，我们已经看到ENPP 1（PPi调节因子）突变的患者，突变导致婴儿全身性动脉钙化（GACI），以确定是否存在牙齿表型。初步观察表明牙齿萌出延迟。 C项目Chediak-Higashi综合征（CHS）：CHS是一种罕见的常染色体隐性疾病，由编码溶酶体运输调节因子的基因突变引起，具有免疫缺陷，导致对影响几种组织（包括口腔组织）的感染的易感性增加。在我们与巴西坎皮纳斯州立大学的Nociti和Kantovitz博士的持续合作中，对7名CHS诊断个体（4名非典型和3名经典，年龄8-21岁）在NIH诊所进行了关于其口腔状况的全面检查，并从非典型和经典CHS患者和对照个体中获得牙龈成纤维细胞（GF）。临床资料显示，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，非典型患者比经典患者出血更多，但文献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时以及在用E. coli LPS和F.具核提取物来自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对LPS的反应发生改变，表明对牙齿生物膜的促炎反应更强烈，并且非典型CHS GF中较高水平的TLR-4在对革兰氏阴性菌的过度活跃牙周反应中发挥关键作用（正在审查的手稿，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).