Genetic Studies of Human Craniofacial Diseases

人类颅面疾病的遗传学研究

• 批准号: 7593384
• 负责人: THOMAS C HART
• 金额: $ 186.11万
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 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/7593384
• 关键词: Affect; Allogenic; Amelogenesis Imperfecta; Area; Bone Density; Bone Diseases; Cathepsin C; Cell Count; Cell Cycle; Cell Cycle Progression; Cell membrane; Cells; Cementoblast; Chromosomes, Human, Pair 8; Clinical; Collagen; Complex; Condition; Critical Care; Cultured Cells; Data; Defect; Deletion Mutation; Dental; Dental Papilla; Dental caries; Dentin; Dentin Dysplasia; Dentin Formation; Dentinogenesis Imperfecta; Development; Diagnostic; Disease; Ectopic Expression; Elevation; Environmental air flow; Enzyme-Linked Immunosorbent Assay; Enzymes; Epithelial; Epithelial Cells; Evaluation; Family; Family Study; Fibroblasts; Fostering; Gene Mutation; Gene Proteins; Genes; Genetic; Genetic Heterogeneity; Gingiva; Gingival Fibromatosis; Gingival Overgrowth; Goals; Growth Factor; Hematopoietic Stem Cell Transplantation; Hereditary Disease; Hospitals; Host Defense; Human; Image; Immune; Immune response; Immunohistochemistry; In Vitro; Individual; Infection; Inflammation; Inflammatory; Inherited; Intensive Care; Interleukin-1 beta; Intervention; Laboratories; Lactoferrin; Leukocytes; Lipopolysaccharides; Localized; Location; Macrophage Inflammatory Proteins; Mass Spectrum Analysis; Mediating; Mesenchymal; Microbe; Modeling; Molecular Biology; Molecular Profiling; Mouth Diseases; Mutation; Numbers; Odontoblasts; Oral; Osteoclasts; Patients; Periodontitis; Phenotype; Phosphorylation; Play; Process; Proteins; Proteome; Proteomics; Rate; Recovery; Recruitment Activity; Reporting; Research Personnel; Risk; Role; Saliva; Salivary; Salivary Proteins; Sampling; Secretory Immunoglobulin A; Serine Protease; Signal Transduction; Site; Skin; Son; Staging; Stimulus; Syndrome; Thick; Tissues; Tooth Diseases; Tooth structure; Transcriptional Activation; Transgenic Mice; Tumor Suppressor Proteins; Up-Regulation; Work; antileukoprotease; base; beta-2 Microglobulin; bone; bone cell; craniofacial; cyclin C; density; design; gel electrophoresis; gene environment interaction; graft vs host disease; human SLPI protein; human disease; insight; interest; leukocyte activation; microbial; mutant; neutrophil; oral bacteria; reconstitution; retinoblastoma tumor suppressor; taurodontism; transcription factor; tricho-dento-osseous syndrome; two-dimensional

The working paradigm of the section is that there is a genetic basis to human disease and that understanding the genetic basis of disease will foster development of better diagnostic and treatment strategies. To this end we are studying a number of Mendelian diseases to identify the underlying gene defect and to understand how the product(s)of this/these gene mutation(s)result in abnormal development or disease. Specific disease conditions studied include: Dentinogenesis Imperfecta (DI): Dentin, the most abundant tissue in teeth is produced by odontoblasts which differentiate from mesenchymal cells in the dental papilla. Genetic mutations of the DSSP gene are responsible for dentinogenesis imperfecta and dentin dysplasia. We have identified mutations in the DSSP in individuals affected with DI and DD. Certain DSSP mutations are the result of defective transport of DSSP gene products. These findings have provided a basis to evaluate strategies to remove defective protein from the odontoblasts, and evaluation of intervention strategies. Amelogenesis Imperfecta (AI): We continue to study families segregating the amelogenesis imperfecta phenotype. Autosomal dominant and recessive families are being evaluated to identify the chromosomal locations of genetic mutations responsible for various forms of AI. Studies of dominant forms of the disease indicate a gene of major effect for AI on chromosome 8 in some families, while other families show no evidence for linkage to known AI genes, indicating additional genetic heterogeneity for the condition. Tricho-dento-osseous syndrome (TDO): Previously we determined that a DLX3 truncation mutation is responsible for TDO in humans. One feature of the condition is increased bone thickness and density. Our goal is to understand how this mutation results in these favorable osseous findings. Results of in vitro studies indicate that mutant Dlx3 may accelerate differentiation of osteoprogenitor cells. To evaluate this observation, we generated transgenic (TG) mice that carry the same deletion mutation. Transgenic mice have phenotypic features including taurodontism as well as enhanced bone density and thickness. These TG mice show defects in odontoblasts and cementoblasts resulting in reduced dentin formation. Bone density and thickness are markedly enhanced in TG mice. Hereditary gingival fibromatosis (HGF): We have previously identified a mutation of the son of sevenless 1 (SOS1) gene HGF. Fibroblasts with the mutation have higher proliferation rates, resulting in increased cell numbers and collagen. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in HGF gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans. Papillon Lefvre syndrome (PLS): Mutations of the cathepsin C gene (CTSC)and resultant inactivity of cathepsin C enzyme are responsible PLS and several allelic conditions. Inactivity of cathepsin C has been demonstrated to result in the inability to activate neutrophil serine proteases (NSP). We have previously related inactivity of CTSC and NSP with dysregulation of MIP-1alpha, and suggested this may be important in the inability to regulate leukocyte at sites of inflammation in PLS, possibly contributing to tissue destruction. Because elevated levels of the macrophage inflammatory protein-1alpha (MIP-1alpha) are reported in inflammatory bone diseases including periodontitis, we evaluated the ability of interleukin-1beta (IL-1beta) and bacterial lipopolysaccharides (LPSs) to modulate MIP-1alpha expression in epithelial cells, fibroblasts, and polymorphonuclear leukocytes (PMNs). We found that MIP-1alpha expression in PMNs and gingival epithelial cells was induced by IL-1beta and LPS, but neither induced MIP-1alpha expression in gingival fibroblasts or osteoblastic cells. MIP-1alpha was highly expressed in the basal epithelial layer of inflamed gingiva but not in healthy gingiva. We also found that MIP-1alpha induced osteoclast formation. These findings suggest that MIP-1alpha expression by gingival epithelial cells may be important in initiating inflammation by facilitating accumulation and activation of leukocytes. The ability of MIP-1alpha to facilitate formation of multinuclear bone cells indicates a possible role in periodontitis-associated bone destruction. These findings indicate MIP-1alpha may play an important role in early and later stages of inflammatory-related periodontitis. We believe these findings have clinical implications for Mendelian and complex forms of periodontitis. To study gene-environment interactions that impact on oral diseases we have been evaluating the utility of incorporating microbial expression profiles and proteomic data into models that can be use to predict disease or to distinguish between diseased individuals and controls. We have assessed changes in the oral flora in intubated critical care patients, in an effort to identify changes in the oral flora that are associated with ventilation in hospital intensive care patients. Findings indicate observable changes in the oral flora, and characterization of the salivary proteome is currently underway. Studies of the salivary proteome led us to investigate changes in salivary proteome following allogeneic hematopoietic stem cell transplantation. As saliva contains many components of adaptive and innate immune response crucial for local host defenses, changes in salivary constituents could reflect systemic processes such as immune reconstitution and development of graft versus host disease(GVHD) that occur posttransplant. Serially collected saliva samples from patients undergoing allo-HCT were evaluated. Changes in salivary proteome were initially examined by SELDI-TOF mass spectrometry. Individual protein changes were identified by 2-dimensional differential in-gel electrophoresis (2D-DIGE) with subsequent MS/MS sequencing and ELISA. Significant increases and decreases in multiple salivary proteins that lasted at least 2 months posttransplant were detected by SELDI-TOF mass spectrometry. Lactoferrin and secretory leukocyte protease inhibitor demonstrated elevations 1 month post-HCT that persisted at least 6 months. Secretory IgA (sIgA) levels were decreased 1 month posttransplant, with recovery at approximately 6 months. Levels of salivary beta(2)-microglobulin were elevated at 6 months and correlated with sIgA levels. We conclude that Allo-HCT is associated with long-term changes in several salivary proteins important for innate immune responses. These results support further studies on the association of salivary proteins with posttransplant complications including infections and GVHD. We have also profiled the salivary proteome from individuals with infectious dental diseases, including caries, to determine if profiling the salivary proteome can add discriminatory power to models to identify individuals with or at risk for developing caries. This work is building on our previous observation that quantitative changes in groups of oral microbes can be related to the presence or absence of caries in groups of individuals.

该部分的工作范式是人类疾病有遗传基础，了解疾病的遗传基础将促进更好的诊断和治疗策略的发展。为此，我们正在研究多种孟德尔疾病，以识别潜在的基因缺陷，并了解该/这些基因突变的产物如何导致异常发育或疾病。研究的具体疾病包括： 牙本质发育不全 (DI)：牙本质是牙齿中最丰富的组织，由成牙本质细胞产生，成牙本质细胞与牙乳头中的间充质细胞分化。 DSSP 基因的基因突变是导致牙本质发育不全和牙本质发育不良的原因。我们已经在 DI 和 DD 患者中发现了 DSSP 突变。某些 DSSP 突变是 DSSP 基因产物运输缺陷的结果。这些发现为评估从成牙本质细胞中去除缺陷蛋白的策略以及评估干预策略提供了基础。 釉质形成不完美 (AI)：我们继续研究分离釉质形成不完美表型的家族。正在评估常染色体显性和隐性家族，以确定导致各种形式人工智能的基因突变的染色体位置。对这种疾病的显性形式的研究表明，在一些家族的 8 号染色体上有一个对 AI 有主要影响的基因，而其他家族则没有证据表明与已知的 AI 基因有联系，这表明该疾病存在额外的遗传异质性。 毛发齿骨综合征 (TDO)：之前我们确定 DLX3 截短突变是导致人类 TDO 的原因。该病的特征之一是骨质厚度和密度增加。我们的目标是了解这种突变如何导致这些有利的骨质发现。体外研究结果表明突变的Dlx3可能加速骨祖细胞的分化。为了评估这一观察结果，我们培育了携带相同缺失突变的转基因（TG）小鼠。转基因小鼠具有牛牙症以及骨密度和厚度增强等表型特征。这些 TG 小鼠表现出成牙本质细胞和成牙骨质细胞的缺陷，导致牙本质形成减少。 TG 小鼠的骨密度和厚度显着增强。 遗传性牙龈纤维瘤病（HGF）：我们之前已经发现了七少之子1（SOS1）基因HGF的突变。具有突变的成纤维细胞具有更高的增殖率，导致细胞数量和胶原蛋白增加。利用野生型和突变型 SOS1 构建体的异位表达，我们发现截短的 SOS1 可以定位于质膜，无需生长因子刺激，导致 Ras/MAPK 信号持续激活。此外，我们观察到 HGF 牙龈成纤维细胞中 ERK 信号强度和持续时间的增加，这与视网膜母细胞瘤肿瘤抑制蛋白的磷酸化和细胞周期调节因子（包括细胞周期蛋白 C、D 和 E 以及 E2F/DP 转录因子）的上调有关。这些因子促进细胞周期进展，它们的上调可能是观察到的牙龈成纤维细胞增殖增加的基础。这些发现阐明了人类 SOS1 基因突变介导的牙龈过度生长的机制。 Papillon Lefvre 综合征 (PLS)：组织蛋白酶 C 基因 (CTSC) 突变和由此导致的组织蛋白酶 C 酶失活是造成 PLS 和多种等位基因病症的原因。组织蛋白酶 C 的失活已被证明会导致无法激活中性粒细胞丝氨酸蛋白酶 (NSP)。我们之前已将 CTSC 和 NSP 的不活动与 MIP-1α 的失调联系起来，并表明这可能对于无法调节 PLS 炎症部位的白细胞很重要，可能导致组织破坏。由于在包括牙周炎在内的炎症性骨病中巨噬细胞炎症蛋白 1α (MIP-1α) 水平升高，因此我们评估了白细胞介素 1β (IL-1β) 和细菌脂多糖 (LPS) 调节上皮细胞、成纤维细胞和多形核白细胞中 MIP-1α 表达的能力 （PMN）。我们发现中性粒细胞和牙龈上皮细胞中的MIP-1α表达是由IL-1β和LPS诱导的，但两者都不诱导牙龈成纤维细胞或成骨细胞中的MIP-1α表达。 MIP-1α 在发炎牙龈的基底上皮层中高表达，但在健康牙龈中不表达。我们还发现 MIP-1α 诱导破骨细胞形成。这些发现表明，牙龈上皮细胞的 MIP-1α 表达可能通过促进白细胞的积累和激活而在引发炎症方面发挥重要作用。 MIP-1α 促进多核骨细胞形成的能力表明其在牙周炎相关骨破坏中可能发挥作用。这些发现表明 MIP-1α 可能在炎症相关牙周炎的早期和晚期发挥重要作用。我们相信这些发现对孟德尔牙周炎和复杂形式的牙周炎具有临床意义。 为了研究影响口腔疾病的基因-环境相互作用，我们一直在评估将微生物表达谱和蛋白质组数据纳入可用于预测疾病或区分患病个体和对照的模型中的效用。我们评估了插管重症监护患者口腔菌群的变化，试图找出与医院重症监护患者通气相关的口腔菌群变化。研究结果表明口腔菌群发生了可观察到的变化，唾液蛋白质组的表征目前正在进行中。对唾液蛋白质组的研究使我们研究了同种异体造血干细胞移植后唾液蛋白质组的变化。由于唾液含有许多对局部宿主防御至关重要的适应性和先天免疫反应的成分，唾液成分的变化可以反映移植后发生的免疫重建和移植物抗宿主病（GVHD）等全身过程。对接受异基因 HCT 的患者连续收集的唾液样本进行了评估。唾液蛋白质组的变化最初通过 SELDI-TOF 质谱法进行检查。通过二维微分凝胶内电泳 (2D-DIGE) 以及随后的 MS/MS 测序和 ELISA 来鉴定单个蛋白质变化。 SELDI-TOF 质谱检测到移植后至少持续 2 个月的多种唾液蛋白的显着增加和减少。 HCT 后 1 个月乳铁蛋白和分泌性白细胞蛋白酶抑制剂表现出升高，并持续至少 6 个月。移植后 1 个月分泌型 IgA (sIgA) 水平下降，约 6 个月后恢复。唾液 β(2)-微球蛋白水平在 6 个月时升高，并与 sIgA 水平相关。我们得出的结论是，Allo-HCT 与几种对先天免疫反应很重要的唾液蛋白的长期变化有关。这些结果支持进一步研究唾液蛋白与移植后并发症（包括感染和 GVHD）之间的关系。我们还对患有传染性牙科疾病（包括龋齿）的个体的唾液蛋白质组进行了分析，以确定唾液蛋白质组分析是否可以为模型增加辨别力，以识别患有龋齿或有患龋齿风险的个体。这项工作建立在我们之前的观察基础上，即口腔微生物群体的数量变化可能与个体群体中是否存在龋齿有关。