A Mouse Knock-In Model for ENGRAILED 2 Autism Susceptibility

描述(申请人提供)：自闭症谱系障碍(ASD)是一种常见的和衰弱的神经发育障碍。虽然有强有力的证据表明基因对ASD有贡献，但分离特定的致病基因缺陷一直很困难。我们之前的研究主要集中在同源框转录因子Engraile2(EN2)上。我们已经在三个不同的数据集中证明了两个内含子EN2 SNP，rs1861972和rs1861973的一致关联。这些发现决定了en2可能是ASD的易感基因。LD作图和测序证实关联的A-C单倍型rs1861972-rs1816973为候选疾病等位基因。功能研究表明，EN2内含子发挥转录抑制作用，A-C单倍型与非关联G-T单倍型相比，导致较弱的抑制因子。EMSA已经确定DNA结合蛋白与这两个SNP的相关等位基因特异地相互作用，为观察到的功能差异提供了一种机制。这些研究已经确定A-C单倍型是自闭症的第一个常见风险因素。这项建议的目标是通过重组酶介导的基因组置换(RMGR)来建立EN2风险等位基因的小鼠模型。RMGR比标准的靶向方法更受欢迎，因为小鼠基因组的大片段(&gt；100kb)可以被同源的人类区域取代。这减少了人们对人类基因适当调控的担忧。我们的计划是使用RMGR将~72kb的小鼠EN2基因座替换为人类的同步区，并为相关的A-C和非相关的G-T单倍型产生敲入线。一个IRES：红色荧光蛋白(DsRed-E5/pTIMER)也将被用来修饰EN2基因座，以便识别风险等位基因对水平和空间/时间表达的微妙影响。然后，将对敲入基因进行检查，以确定EN2风险等位基因具有功能的发育细胞类型和年龄。对EN2基因敲除的研究揭示了与ASD相关的解剖、发育和神经化学表型。同样的分析将对敲入基因重复，作为确定受风险等位基因影响的细胞通路的第一步。这些信息将对开发更好的ASD诊断、治疗和预防措施至关重要。

DESCRIPTION (provided by applicant): Autism spectrum disorder (ASD) is a common and debilitating neurodevelopmental disorder. Although there is strong evidence for a genetic contribution to ASD, the isolation of specific causative genetic defects has been difficult. Our previous research has focused on the homeobox transcription factor ENGRAILED 2 (EN2). We have demonstrated consistent association for two intronic EN2 SNPs, rs1861972 and rs1861973, in three separate datasets. These findings determined that EN2 is a likely ASD susceptibility gene. LD mapping and re-sequencing identified the associated A-C rs1861972-rs1816973 haplotype as a candidate disease allele. Functional studies have now demonstrated that the EN2 intron acts a transcriptional repressor and that the A-C haplotype results in a weaker repressor compared to the non-associated G-T haplotype. EMSAs have determined that DNA binding proteins specifically interact with the associated alleles for both SNPs, providing a mechanism for the observed functional difference. These studies have identified the A-C haplotype as the first common risk factor for autism. The goal of this proposal is to generate a mouse model for the EN2 risk allele by using recombinase- mediated genomic replacement (RMGR). RMGR is preferred over standard targeting approaches because large segments of the mouse genome (>100kb) can be replaced with the syntenic human region. This reduces concerns about the proper regulation of the human gene. Our plan is to use RMGR to replace ~72kb of the mouse En2 locus with the syntenic human region and to generate knock-in lines for both the associated A-C and the non-associated G-T haplotypes. An IRES:red fluorescent protein (DsRed-E5/pTIMER) will also be used to modify the EN2 locus so that subtle effects of the risk allele on levels and spatial/temporal expression can be identified. The knock-ins will then be examined to determine the developmental cell types and ages in which the EN2 risk allele is functional. Studies with the En2 knock-out have uncovered anatomical, developmental and neurochemical phenotypes relevant to ASD. The same analysis will be repeated for the knock-ins as a first step in determining the cellular pathways affected by the risk allele. This information will be critical in the development of better diagnoses, treatments and preventions for ASD.