DE PEDIATRIC COBRE: MOLECULAR MECHANISMS IN PELIZAEUS MERZBACHER DISEASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。Pelizaeus-Merzbacher病(PMD)是一种X连锁的脑白质营养不良，由编码中枢神经系统髓鞘蛋白的蛋白脂蛋白1基因(PLP1)缺陷引起。大约60%的家庭有包括PLP1基因的基因组重复，15%-20%的家庭有PLP1点突变。对小鼠的研究表明，PLP1剂量的增加可以解释疾病的发病机制。此外，研究表明，非编码区的突变可以改变PLP1的表达水平或选择性剪接形式PLP1和DM20的比例。我们的长期目标是了解PMD表型产生的分子机制，以便开发合理的治疗方法和改进的诊断技术。在第一个目的中，关于PMD复制的位置、大小、结构和序列的结果支持这样的假设，即大多数复制是由一种新的耦合的同源和非同源机制引起的。我们将继续采用细胞遗传学、分子遗传学和电子计算机相结合的方法来完善我们对这一机制的理解。在第二个目标中，通过在ES细胞系中设计不同的复制，并分析分化为少突胶质细胞前后结构对基因表达的影响，验证了复制结构影响PLP1表达的假设。在第三个目的中，通过基因转移和RNA-蛋白质结合分析来检验某些PMD突变对PLP1剪接的调节作用的假设，以研究PLP1/DM20选择性剪接中涉及的顺式作用元件和反式作用因子。我们已经确定，外显子和内含子剪接增强剂以及PLP1和DM20供体剪接位点的相对强度在PLP1选择性剪接中起着重要作用。这些研究的结果具有最大的影响潜力，因为它们可以推广到其他基因组和剪接疾病，这些疾病直到最近才被确认为重要的遗传病类型。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pelizaeus-Merzbacher disease (PMD), an X-linked leukodystrophy, is caused by defects of the proteolipid protein 1 gene (PLP1) that encodes the major CNS myelin protein. Approximately 60% of families have a genomic duplication that includes the PLP1 gene, and 15-20% of families have PLP1 point mutations. Studies in mice have shown that an increased dosage of PLP1 can account for disease pathogenesis. Further, studies have shown that mutations in noncoding regions can alter PLP1 expression levels or the ratio of the alternatively spliced forms PLP1 and DM20. Our long-term objective is to understand the molecular mechanisms involved in generating the PMD phenotype so rational treatments and improved diagnostic techniques can be developed. In the first aim, results on location, size, structure and sequence at the junctions of PMD duplications support the hypothesis that most are caused by a novel coupled homologous and nonhomologous mechanism. We will continue a combined cytogenetic, molecular, and in silico approach to refine our understanding of the mechanism. In the second aim, the hypothesis that the duplication structure affects expression of PLP1 is being tested by engineering different duplications into ES cell lines and analyzing the structural effects on gene expression before and after differentiation into oligodendrocytes. In the third aim, the hypothesis that some PMD mutations dysregulate splicing of PLP1 is being tested by using gene transfer and RNA-protein binding assays to investigate cis-acting elements and trans-acting factors involved in PLP1/DM20 alternative splicing. We have determined that exon and intron splicing enhancers and the relative strength of the PLP1 and DM20 donor splice sites play an important role in PLP1 alternative splicing. The results of these studies have greatest impact potential through their generalizability to other genomic and splicing diseases, which have only recently been recognized as important types of genetic disease.