DSPP Function, Pathophysiology, and Genetic Diagnosis

遗传性牙本质缺陷（HDD）影响8，000人中的1人。大多数HDD的遗传原因与以下因素有关： 牙本质蛋白I型胶原和牙本质涎磷蛋白（DSPP）功能障碍。所有DSPP 迄今报道的突变显示出显性遗传模式。这是因为DSPP突变 通过显性负性或功能机制的获得来表现其表型，而不是通过单倍型， 不足如在Dspp杂合子小鼠中，将DSPP的正常量减少一半， 牙本质畸形Dspp-/-敲除小鼠由于缺乏DSPP而表现出严重的表型，而不是由Dspp-/-敲除小鼠引起的。 导致人类HDD的常染色体显性病理机制。这一区别很重要。 在治疗上，Dspp-/-缺失时的HDD可以通过恢复DSPP表达来逆转，而Dspp-/-缺失时的HDD可以通过恢复DSPP表达来逆转。 由DSPP突变引起的人类HDD不能以这种方式恢复，因为条件不是由于 DSPP蛋白的缺乏，而是由于成牙本质细胞中异常DSPP的病理作用。 这项提案“DSPP功能，病理生理学和遗传诊断”旨在改善我们的下- DSPP衍生蛋白在正常牙本质形成过程中的作用， Dspp-1移码突变，以及3）开发用于HDD基因检测的实用方法， 确定致病突变并建立明确的诊断。提出了三个具体目标： SA1：确定DSPP衍生蛋白在初始牙本质矿物形成和聚结过程中的作用 通过表征Dspp +/+、Dspp-1fs/-1fs、Dspp-2fs/-2fs和Dspp-/-小鼠的早期牙本质矿化。 SA 2：在体内定位DSPP-1移码蛋白，以确定其在何处积聚并导致 成牙本质细胞病理学 SA3：通过建立有效的基因检测来改善HDD的诊断和管理 算法（确定给定个体中HDD的确切遗传原因的动作序列）。 策略：我们假设DSPP有助于启动牙本质钙球的矿化， 促进它们的生长和聚结成连续的矿物层。通过描述和比较 使用聚焦离子束扫描在Dspp +/+、Dspp-/-和Dspp-2fs/-2fs小鼠中的早期牙本质矿化 电子显微镜（FIB-SEM），我们可以确定是否牙本质涎蛋白（DSP）或牙本质磷蛋白 (DPP)促进牙质的起始和/或聚结。我们假设DSPP-1移码 突变可能通过ER应激引起成牙本质细胞病变。我们用以下方法来检验这一假设： dspp-1移码基因敲入小鼠与人类疾病非常相似。成牙本质细胞病理学通过以下评估： FIB-扫描电镜和透射电镜免疫金双标记法用于体内突变蛋白和细胞器标记。到 为了提高HDD的诊断和管理水平，我们将遗传检测算法应用于HDD的招募 家族，以优化其在识别潜在致病突变方面的可重复性和效率。

Hereditary Dentin Defects (HDD) affect 1 in 8,000 people. The genetic causes of most HDD correlate with the dysfunction of dentin proteins: type I collagen and dentin sialophosphoprotein (DSPP). All DSPP mutations reported to date show a dominant pattern of inheritance. This is because DSPP mutations manifest their phenotype through a dominant negative or gain of function mechanism—not by haplo- insufficiency. Reducing the normal amount of DSPP by half, as in Dspp heterozygous mice, does not cause dentin malformations. Dspp-/- null mice show a severe phenotype due to the absence of DSPP—not by the autosomal dominant pathological mechanism that causes HDD in humans. This distinction is important. Therapeutically, HDD in the absence of Dspp-/- could be reversed by restoring DSPP expression, whereas human HDD caused by DSPP mutations could not be restored in this way because the condition is not due to a lack of DSPP protein, but rather, is due to the pathological effects of aberrant DSPP in odontoblasts. This proposal “DSPP Function, Pathophysiology, and Genetic Diagnosis” seeks to improve our under- standings of 1) DSPP-derived proteins during normal dentinogenesis, 2) the pathological mechanism of Dspp -1 frameshift mutations, and 3) to develop a practical approach for HDD genetic testing to specifically identify the causative mutation and establish a definitive diagnosis. Three Specific Aims are proposed: SA1: Determine the role of DSPP-derived proteins during initial dentin mineral formation and coalescence by characterizing early dentin mineralization in Dspp+/+, Dspp-1fs/-1fs, Dspp-2fs/-2fs and Dspp-/- mice. SA2: Localize the DSPP -1 frameshift protein in vivo to determine where it accumulates and causes odontoblast cell pathology. SA3: Improve the diagnosis and management of HDDs by establishing an efficient genetic testing algorithm (sequence of actions that identifies the exact genetic cause of HDD in a given individual). Strategy: We hypothesize that DSPP helps initiate the mineralization of dentin calcospherites and promotes their growth and coalescence into a continuous mineral layer. By characterizing and comparing early dentin mineralization in Dspp+/+, Dspp-/-, and Dspp-2fs/-2fs mice using Focus Ion Beam Scanning Electron Microscopy (FIB-SEM), we can determine if dentin sialoprotein (DSP) or dentin phosphoprotein (DPP) is promoting the initiation and/or coalescence of dentin. We hypothesize that DSPP -1 frameshift mutations cause odontoblast cell pathology, possibly through ER stress. We test this hypothesis using Dspp -1 frameshift knockin mice that closely mimic human disease. Odontoblast pathology is assessed by FIB-SEM and TEM double immunogold labeling for the mutant protein and organelle markers in vivo. To improve the diagnosis and management of HDD, we apply a genetic testing algorithm to recruited HDD families to optimize its reproducibility and efficiency in identifying the underlying disease-causing mutations.