Childhood-onset hypomyelinating leukodystrophy and the multi-tRNA synthetase complex

项目总结/摘要 儿童发病的低髓鞘化脑白质营养不良（HLD）是一组遗传异质性的神经退行性病变， 以脑髓鞘形成减少为特征的疾病。临床特征包括认知以及 儿童时期出现的运动障碍。没有治愈性的治疗方法。我们的合作者格蕾丝·尹博士 评估了两个兄弟姐妹表现出严重的神经功能缺损和共同的表型，包括全球 发育迟缓和智力残疾，产前发病的生长不足和小头畸形，旋转 眼球震颤、共济失调和进行性步态障碍/痉挛以及脑MRI上的髓鞘形成不足。整个 外显子组测序显示EPRS 1基因中的c.4444C>A突变的纯合性。突变是在 该基因的编码序列（cds）引起氨基酸位置1482处的Pro-to-Thr点突变。EPRS1 基因编码双功能谷氨酰-脯氨酰tRNA合成酶（EPRS 1），该合成酶存在于细胞质多- tRNA合成酶复合物（MSC）。P1482 T突变位于蛋白质的C末端附近， 在催化或反密码子识别结构域之外的Pro合成酶区域。的比活性 重组P1482 T突变体EPRS 1与野生型没有区别。相反，EPRS 1蛋白表达 在受影响同胞的永生化淋巴母细胞系（LCL）中， 对照EPRS 1 mRNA水平在同胞和对照中是相同的，表明转录后调节。 我们的初步研究表明，在受影响的LCL中， 即突变mRNA的核输出减少，随后细胞质翻译减少。我们表明 EPRS 1表达的降低导致其他MSC成分的释放。值得注意的是， 与HLD有因果关系的其他几种MSC成分也诱导MSC成分释放，这表明 一整类HLD的共同病因我们假设，低效率的核出口和翻译， c.4444C> EPRS 1 mRNA减少MSC结合的EPRS 1，引起MSC成分的释放并驱动细胞增殖。 HLD表型。同样，编码其他MSC成分的基因缺陷也有共同的途径， HLD的病因我们将通过追求这些具体目标来检验这一假设：在目标1中，我们将阐明 EPRS 1 P1482 T变异体表达减少的分子机制，重点关注m6 A的作用 甲基化目的2确定HLD相关表型，我们新产生的，基因修饰的， Eprs 1 P1482 T小鼠。在目标3中，我们将研究MSC成分缺乏对MSC完整性的影响， 对髓鞘生成少突胶质细胞的不良后果。CNS特异性转录组学分析将通过 单核RNA测序。这些研究的完成将阐明一个独特的机制， 因此，本发明提供了一种诱导HLD的失调，并且将提供一种独特的HLD小鼠模型，其将允许详细的体内研究。 HLD中细胞缺陷的分析。重要的是，这些结果表明了特异性的，基于RNA的 治疗性干预以挽救变体EPRS 1 mRNA的翻译并挽救神经缺陷。

Project Summary/Abstract Child-onset hypomyelinating leukodystrophy (HLD) is a genetically heterogeneous group of neurodegenerative diseases characterized by reduced cerebral myelin formation. Clinical features include cognitive as well as motor impairment appearing in childhood. There are no curative treatments. Our collaborator, Dr. Grace Yoon, evaluated two siblings presenting with severe neurological deficit and a shared phenotype consisting of global developmental delay and intellectual disability, prenatal onset undergrowth and microcephaly, rotatory nystagmus, ataxia and progressive gait disturbance/spasticity, and hypomyelination on brain MRI. Whole exome sequencing revealed homozygosity of a c.4444C>A mutation in the EPRS1 gene. The mutation is in the coding sequence (cds) of the gene causing a Pro-to-Thr point mutation at aa position 1482. The EPRS1 gene encodes the bifunctional, glutamyl-prolyl tRNA synthetase (EPRS1) that resides in the cytoplasmic multi- tRNA synthetase complex (MSC). The P1482T mutation is located near the C-terminus of the protein in a region of the Pro synthetase outside the catalytic or anti-codon recognition domains. The specific activity of recombinant P1482T mutant EPRS1 is indistinguishable from wild-type. In contrast, EPRS1 protein expression in immortalized lymphoblastoid cell lines (LCL) from affected siblings is about 20% of that in unaffected controls. EPRS1 mRNA levels are identical in siblings and controls indicating post-transcriptional regulation. Our preliminary studies show a dual mechanism determining diminished EPRS1 level in affected LCLs, namely, decreased nuclear export of mutant mRNA, followed by decreased cytoplasmic translation. We show that decreased EPRS1 expression causes release of other MSC constituents. Remarkably, suppression of several other MSC components causally implicated in HLD also induce MSC constituent release, suggesting a common etiology of an entire class of HLDs. We hypothesize that inefficient nuclear export and translation of c.4444C>A EPRS1 mRNA reduces MSC-bound EPRS1, causing release of MSC constituents and driving the HLD phenotype. Likewise, defects in genes encoding other MSC constituents share a common pathway and etiology of HLD. We will test this hypothesis by pursuing these Specific Aims: In Aim 1 we will elucidate molecular mechanisms underlying reduced expression of EPRS1P1482T variant, focusing on the role of m6A methylation. Aim 2 determines the HLD-related phenotype of our newly generated, genetically-modified Eprs1P1482T mice. In Aim 3 we will investigate effect of deficiency of MSC constituents on MSC integrity and adverse consequences in myelinating oligodendrocytes. A CNS-specific transcriptomic analysis will be done by single-nucleus RNA-sequencing. Completion of these studies will elucidate a unique mechanism of gene dysregulation that induces HLD, and will provide a unique mouse model of HLD that will permit detailed in vivo analysis of the cellular defects in HLD. Importantly, these results suggest the possibility of specific, RNA-based therapeutic intervention to rescue translation of the variant EPRS1 mRNA and rescue the neurologic defect.