Project 2: Impact of H1/H2 haplotypes on cellular disease-associated phenotypes driven by FTD-causing MAPT mutations

项目 2：H1/H2 单倍型对 FTD 引起的 MAPT 突变驱动的细胞疾病相关表型的影响

• 批准号: 10834336
• 负责人: DANIEL H GESCHWIND
• 金额: $ 2.37万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10834336
• 关键词: 17q21; 3-Dimensional; ATAC-seq; Address; Affect; Alleles; Architecture; Astrocytes; Automobile Driving; Binding; Biological Assay; Brain region; CRISPR interference; CRISPR-mediated transcriptional activation; Cell Line; Cell Nucleus; Cells; Chromatin; Chromatin Structure; Chromosome inversion; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA Footprint; Data; Disease; Enhancers; Epigenetic Process; Frontotemporal Dementia; Frontotemporal Lobar Degenerations; Gene Expression; Genes; Genetic; Genetic Recombination; Genetic Risk; Genomics; Glutamates; Haplotypes; High Prevalence; Human; Immunohistochemistry; Inheritance Patterns; Lead; Linkage Disequilibrium; MAPT gene; Maps; Measures; Mediating; Microglia; Modeling; Molecular; Molecular Disease; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nucleic Acid Regulatory Sequences; Pathogenicity; Pathologic; Pathway interactions; Patients; Pattern; Penetrance; Phenotype; Predisposition; Progressive Supranuclear Palsy; Protein Isoforms; Proteomics; Regulator Genes; Regulon; Reporter; Reporting; Risk; Risk Reduction; Severities; Signal Transduction; Splice-Site Mutation; Spliced Genes; Stress; Tauopathies; Testing; Transcript; Transcriptional Regulation; Variant; Work; age related; brain tissue; causal variant; cell type; conditioning; genetic variant; genome editing; genome wide association study; induced pluripotent stem cell; multiple omics; novel therapeutic intervention; promoter; protein expression; rare variant; risk variant; screening; stressor; tau Proteins; tau aggregation; tau mutation; tau-1; transcription factor; transcriptome sequencing; transcriptomics

PROJECT SUMMARY (PROJECT 2) Common and rare variants at the 17q21.31/MAPT locus contribute to Frontotemporal Dementia (FTD-tau) and the Frontotemporal Lobar Degeneration (FTLD) spectrum disorder, Progressive Supranuclear Palsy (PSP). Dominantly acting, rare missense and splice-site mutations in the MAPT gene have been reported to cause both FTD-tau and PSP. Interestingly, it appears as though the majority of reported pathogenic MAPT mutations causing familial PSP and FTD-tau occur on the H1 haplotype background, which is consistent with the increased genetic risk associated with this haplotype in sporadic disease, although P301L, N279K and IVS10+16 have been described on both haplotypes. However, it is unknown whether these haplotypes influence the phenotypic expression of MAPT mutations. In addition to rare variation contributions to disease in this region, the strongest genome-wide association signal for common variation in PSP is in the 17q21.31/MAPT locus. Identification of causal variants in this region has been hampered by the broad patterns of linkage disequilibrium created by the 970 Kb chromosomal inversion, which limits local recombination. Work by our group and by others, including our preliminary data using massively parallel reporter assays (MPRA), indicates that this 17q21.31/MAPT region harbors several different risk loci and hundreds of contributory common causal variants. Furthermore, because >96% of patients with PSP have the H1 haplotype, the effects of these SNPs driving this major association signal likely occur in the context of the H1 haplotype, and not H2. These observations lead to two major hypotheses that drive this project: 1) H1/H2 haplotypes not only influence risk for sporadic FTD-tau/PSP but also modulate expression of the pathogenic phenotypes associated with specific MAPT variants. Therefore, we hypothesize that the changes to chromatin structure and gene expression or splicing that occur in H2/H2 neural cells compared to H1/H1 will ameliorate the pathogenic effects of PSP/FTD MAPT mutations; and 2) That the regulatory effects of common PSP-associated variation in this region will be similarly blunted on an H2 haplotype. We will use a combination of single-cell genomic, and proteomic approaches to characterize the impact of PSP/FTD-associated MAPT mutations in human brain tissue and in iPSC-derived assembloids. We will use CRISPR genome editing to introduce PSP/FTD-associated MAPT mutations into cell lines from Project 1 with either H1/H1 or H2/H2 backgrounds, and will use single-cell multi-OMICs (RNAseq and ATACseq) combined with proteomics and ISOseq to comprehensively assess the impact of the mutations on each background. We will further characterize the impact of MAPT mutations on disease-associated phenotypes on each background using cellular and molecular assays, and will then use CRISPRa/i screens to validate the functional consequences of key haplotype-associated regulatory regions containing candidate common causal variants on disease-related Tau phenotypes on gene expression in both H1 and H2 haplotypes, in order to test the hypothesis that causal disease variants have more activity on the H1 background.

项目概要（项目 2） 17q21.31/MAPT 位点的常见和罕见变异导致额颞叶痴呆 (FTD-tau) 和 额颞叶变性 (FTLD) 谱系障碍、进行性核上性麻痹 (PSP)。 据报道，MAPT 基因中的显性作用、罕见错义和剪接位点突变可导致这两种情况 FTD-tau 和 PSP。有趣的是，似乎大多数报告的致病性 MAPT 突变 导致家族性 PSP 和 FTD-tau 发生在 H1 单倍型背景上，这与增加的 尽管 P301L、N279K 和 IVS10+16 具有与散发性疾病中该单倍型相关的遗传风险 两种单倍型都有描述。然而，尚不清楚这些单倍型是否影响表型 MAPT 突变的表达。除了该地区疾病的罕见变异贡献之外，最强的 PSP 常见变异的全基因组关联信号位于 17q21.31/MAPT 基因座。鉴定 该区域的因果变异受到了连锁不平衡的广泛模式的阻碍 970 Kb 染色体倒转，限制局部重组。我们的团队和其他人（包括我们的团队）的工作 使用大规模并行报告分析 (MPRA) 的初步数据表明，该 17q21.31/MAPT 区域 包含几个不同的风险位点和数百个有贡献的常见因果变异。此外，因为 >96% 的 PSP 患者具有 H1 单倍型，这些 SNP 的作用驱动了这一主要关联信号 可能发生在 H1 单倍型的背景下，而不是 H2。这些观察结果引出了两个主要假设 推动该项目的因素：1) H1/H2 单倍型不仅影响散发性 FTD-tau/PSP 的风险，而且还可以调节 与特定 MAPT 变异相关的致病表型的表达。因此，我们假设 H2/H2 神经细胞中染色质结构和基因表达或剪接发生的变化 与H1/H1相比，将改善PSP/FTD MAPT突变的致病作用；和 2） 该区域常见 PSP 相关变异的调节作用在 H2 单倍型上也会同样减弱。 我们将结合使用单细胞基因组和蛋白质组方法来表征 人脑组织和 iPSC 衍生的组合体中 PSP/FTD 相关的 MAPT 突变。我们将使用 CRISPR 基因组编辑将 PSP/FTD 相关的 MAPT 突变引入项目 1 的细胞系中 H1/H1 或 H2/H2 背景，并将使用单细胞多组学（RNAseq 和 ATACseq）组合 使用蛋白质组学和 ISOseq 来全面评估突变对每个背景的影响。我们 将进一步表征 MAPT 突变对每种背景下疾病相关表型的影响 使用细胞和分子测定，然后使用 CRISPRa/i 筛选来验证功能 包含候选常见因果变异的关键单倍型相关调控区域的后果 疾病相关的 Tau 表型对 H1 和 H2 单倍型基因表达的影响，以测试 假设致病变异在 H1 背景上具有更多活性。