Molecular analysis of MEHMO syndrome mutations in translation factor eIF2

MEHMO 综合征翻译因子 eIF2 突变的分子分析

• 批准号: 10554445
• 负责人: Sara Kathryn Young-Baird
• 金额: $ 24.7万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-12-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10554445
• 关键词: Address; Affect; Automobile Driving; Award; Biological Assay; Biology; CRISPR/Cas technology; Candidate Disease Gene; Cell Line; Characteristics; Development; Diagnosis; Disease; Electrophysiology (science); Epilepsy; Exhibits; Gene Expression; Generations; Genes; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Immunohistochemistry; Impairment; Informatics; Initiator Codon; Knowledge; Link; MEHMO syndrome; Measurement; Mental deficiency; Messenger RNA; Microcephaly; Molecular; Molecular Analysis; Mutation; Neurons; Obesity; Outcome; Pathogenesis; Patients; Peptide Initiation Factors; Phenotype; Play; Positioning Attribute; Prokaryotic Initiation Factor-2; Protein Biosynthesis; Quantitative Reverse Transcriptase PCR; Research; Role; Site; Stains; Symptoms; Syndrome; Techniques; Translation Initiation; Translational Regulation; Translations; Validation; Western Blotting; White Matter Disease; X-linked intellectual disability; acronyms; biological adaptation to stress; career; disease-causing mutation; early childhood; effective therapy; excitatory neuron; experience; experimental study; genome editing; genome-wide; homologous recombination; human disease; improved; induced pluripotent stem cell; mRNA Translation; member; multidisciplinary; mutant; polysome profiling; postsynaptic; presynaptic; protein function; ribosome profiling; therapeutic development; translation factor; treatment strategy; white matter

PROJECT SUMMARY/ABSTRACT Multiple cases of MEHMO syndrome, an X-linked intellectual disability syndrome caused by mutations in EIF2S3 that encodes a core member of the cellular protein synthesis machinery, have recently been identified. However, the causal links between mis-regulated protein synthesis and human disease remain poorly understood. EIF2S3 encodes the γ subunit of translation initiation factor 2 (eIF2γ) that, along with the eIF2 α and β subunits, GTP, and the initiator Met-tRNAiMet, plays a critical role in selection of the translation start site. Thus, patients with MEHMO syndrome are likely to experience changes in protein synthesis on both a global and gene specific level. The acronym MEHMO denotes the symptoms of this syndrome: Mental deficiency, Epilepsy, Hypogenitalism, Microcephaly, and Obesity. How changes in gene expression incurred through mutations in eIF2γ result in the presentation of MEHMO syndrome remains unknown. Thus, there is a critical need to determine the underlying molecular mechanism governing the phenotype and symptoms of MEHMO patients. The long-term goal of these studies is to identify key factors and gene expression changes that underlie MEHMO syndrome in order to develop improved strategies for treating patients. The overall objective in this application is to determine how mis-regulation of protein synthesis contributes to the symptoms of MEHMO syndrome. In Aim 1 ribosome profiling, an informatics-based, genome- wide approach, and additional validation techniques will be used to identify mRNAs subject to changes in start codon selection and translational efficiency in MEHMO-patient derived iPSCs expressing mutant eIF2γ or isogenic iPSCs expressing wild-type eIF2g. In Aim 2 wild-type and eIF2γ mutant iPSCs will be differentiated into neurons that will be utilized in multidisciplinary analyses, including electrophysiological readings, immunohistochemistry, and ribosome profiling, geared toward understanding changes in neuronal biology and function caused by the eIF2γ mutation. In Aim 3 CRISPR Cas9 genome editing will be used to generate isogenic iPSCs with Vanishing White Matter disease-causing eIF2B mutations. Generation of eIF2B mutant iPSCs from the isogenic wild-type control iPSCs utilized in Aim 1 will enable direct comparisons of the protein synthesis changes caused by mutations in eIF2γ and its guanine-nucleotide exchange factor eIF2B. These studies will enhance our understanding of how translation is mis- regulated by alterations in eIF2 function, and ultimately provide critical knowledge for the development of targeted strategies for the treatment of patients with MEHMO syndrome.

项目摘要/摘要 多发性MEHMO综合征，一种X连锁的智力残疾综合征 由编码细胞蛋白质合成核心成员的EIF2S3突变引起 机械，最近已经被发现。然而，监管不当之间的因果联系 蛋白质合成和人类疾病仍然知之甚少。EIF2S3编码γ亚基 翻译起始因子2(eif2γ)，与eif2α和β亚基、gtp和 启动子Met-tRNAiMet在翻译起始点的选择中起着关键作用。因此，患者 患有MEHMO综合征的患者可能在全球范围内经历蛋白质合成的变化 和基因特异性水平。首字母缩写MEHMO表示这种综合征的症状：精神 缺乏症、癫痫、先天性发育不良、小头畸形和肥胖。基因是如何变化的 EIF-2γ突变导致甲氧合酶综合征的表现 仍然不为人知。因此，迫切需要确定潜在的分子 MEHMO患者的表型和症状的控制机制。的长期目标是 这些研究是为了确定MEHMO背后的关键因素和基因表达变化 为了制定更好的治疗患者的策略，我们将继续开展这项工作。年的总体目标 这个应用是为了确定蛋白质合成的错误调节如何导致 MEHMO综合征的症状。在AIM 1核糖体图谱中，一种基于信息学的基因组- 广泛的方法，并将使用其他验证技术来识别符合以下条件的mRNAs MEHMO患者来源的IPSCs起始密码子选择和翻译效率的变化 表达突变型eIF2γ或表达野生型eIF2g的等基因IPSCs。在AIM 2中野生型和 Eif2γ突变的ipscs将被分化为神经元，将用于多学科 分析，包括电生理读数、免疫组织化学和核糖体 简写，旨在了解由神经生物学和功能引起的变化 EIF2γ突变。在AIM 3中，将使用CRISPR Cas9基因组编辑来产生等基因的IPSCs 白质病引起的eIF2B突变正在消失。EIF2B突变体的产生 来自AIM 1中使用的同基因野生型对照IPSCs的IPSCs将能够进行直接比较 EIF2γ及其鸟苷核苷酸突变引起的蛋白质合成变化 交换系数eIF2B。这些研究将加深我们对翻译是如何错位的理解。 受eIF2功能变化的调节，并最终为 制定治疗MEHMO综合征患者的有针对性的策略。