A Mouse Knock-In Model for ENGRAILED 2 Autism Susceptibility

描述（由申请人提供）：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遗传因素对ASD有贡献，但很难分离出特定的致病遗传缺陷。我们以前的研究主要集中在同源异型盒转录因子ENGRAP 2（EN 2）。我们已经在三个独立的数据集中证明了两个内含子EN 2 SNP rs 1861972和rs 1861973的一致关联。这些结果确定EN 2可能是ASD易感基因。LD定位和重新测序鉴定了相关的A-C rs 1861972-rs 1816973单倍型作为候选疾病等位基因。功能研究表明，EN 2内含子作为一个转录阻遏物，A-C单倍型的结果在一个较弱的阻遏物相比，非相关的G-T单倍型。EMSA已经确定DNA结合蛋白与两种SNP的相关等位基因特异性相互作用，为观察到的功能差异提供了机制。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。该提议的目标是通过使用重组酶介导的基因组置换（RMGR）产生EN 2风险等位基因的小鼠模型。RMGR优于标准靶向方法，因为小鼠基因组的大片段（> 100 kb）可以被同线人类区域替换。这减少了对人类基因适当调控的担忧。我们的计划是使用RMGR将小鼠En 2基因座的~ 72 kb替换为同线人类区域，并产生相关A-C和非相关G-T单倍型的敲入系。IRES：红色荧光蛋白（DsRed-E5/pTIMER）也将用于修饰EN 2基因座，以便可以鉴定风险等位基因对水平和空间/时间表达的微妙影响。然后将检查敲入以确定其中EN 2风险等位基因起作用的发育细胞类型和年龄。En 2基因敲除的研究揭示了与ASD相关的解剖学、发育和神经化学表型。将对敲入重复相同的分析，作为确定受风险等位基因影响的细胞途径的第一步。这些信息对于开发更好的ASD诊断，治疗和预防至关重要。

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.