孤独症相关16p13.11拷贝数变异的功能分析及相应斑马鱼模型的建立和研究

Autism spectrum disorders (ASDs) are common, heritable neurodevelopmental disease characterized by communication deficits, impairments of reciprocal social interaction, repetitive and restricted patterns of behavior and interests. It is widely accepted that autism is a multi-gene related multifactorial disorders. Combining with copy number variation (CNV) microarray technologies, numerous genome-wide linkage studies, genome-wide association studies (GWAS) and case studies have been performed to identify the underlying susceptibility genes for autism. Up to now, these studies have provided evidence for susceptibility loci or CNVs on several different chromosomal regions, including 11p12, 15q2, 16p13.11, and so on, which affirmed that ASD is a complex disease regulated by multiple genes. .16p13.11 is a genomic hotspot region for studying on neuropsychiatric disorders. Several genome-wide studies have Identified CNVs exhibiting association to ASD and/or schizophrenia, epilepsy and others, which reflected that genetic architecture of neuropsychiatric disorders are interacted each others. In spite of continuous accumulation of evidence from association studies, function analysis of CNVs is necessary for identifying autism susceptibility genes..Benefited from his abundant experience in gene-brain-behavior-drug study in zebrafish, applicant presents this proposal to build up transgenic zebrafish to perform systematic functional analysis. The objective is to identify autism susceptibility genes from 16p13.11 CNVs and build up animal model for relevant mechanism study, susceptibility study and drug sensitivity study..First, Takifugu rubripes genomic sequences insertion based, or shRNA expression in vivo based transgenic zebrafish will be build up to simulate duplication CNV or deletion CNV separately. Size of CNV is more than hundreds kilo bases normally, multiple genes inside CNV. In transgenic zebrafish, abnormal genes expression will be observed, influences on zebrafish development, morphology, behavior, drug sensitivity will be studied. Therefore the association of autism and CNVs will be functional validated. Second, in order to identify autism susceptibility genes from validated CNVs, similar functional analysis will be carried out at single-gene level. After that, in order to explore for mechanisms, more studies will be carried out at level of molecular and cell..Results of these studies will provide evidences or clues to uncover function of 16p13.11 CNV and autism susceptibility genes; will build up and validate animal models for relevant gene or drug study; will provide solution to functional analyze genomic fragment with size more than hundreds or thousands kilo bases economically, efficiently and rapidly.

孤独症作为神经发育相关多基因遗传病，随拷贝数变异(CNV)的发现及疾病关联研究的进展，在16p13.11发现了多个孤独症相关CNVs。它们可能含孤独症易感基因，目前已拥有关联性研究证据，尚缺乏功能性研究证据。申请者基于长期斑马鱼神经系统发育、基因、行为及药物研究经验，拟以斑马鱼为模式动物对这些CNV进行系统功能分析。首先构建模拟CNV的转基因斑马鱼，通过研究CNV多基因异常表达对斑马鱼发育形态、行为、药物敏感性的影响，分析验证它们与孤独症的功能联系；然后深入CNV，在单基因水平进行功能分析，鉴别孤独症易感基因，并在分子和细胞水平探索其机制。此研究不仅是16p13.11 CNVs与孤独症关联研究的趋势发展，可鉴别分析新的孤独症易感基因及功能，为孤独症个体化诊疗提供新靶点；而且在CNV和单基因水平分别构建孤独症相关斑马鱼模型，为进一步功能机制研究、孤独症易感性和药物敏感性研究了提供有力工具。

本研究利用模式生物斑马鱼对16p13.11片段内孤独症相关多个基因进行了功能分析。研究中我们利用了最新的CRISPR/Cas9技术分别实现了Rrn3、Nomo、C16orf45、Ntan等多个基因的敲除，大部已获得纯合子，可以进行深入的功能分析。行为分析显示，这些基因与个体行为和社交行为密切相关。由此实现了建立了适于研究孤独症等神经精神系统疾病的基因缺陷型斑马鱼模型。同时，本研究也应用了MK-801、育亨宾、乙琥胺等多种小分子化合物，急性或慢性处理斑马鱼胚胎，利用了形态学观察、HPLC、定量PCR、行为学分析等技术分析鉴定了它们对斑马鱼的整体影响，尤其是反应神经精神状态的行为学影响，从而建立了适于研究孤独症等神经精神系统疾病的药物诱导斑马鱼模型。这两种模型的建立和分析，为进一步研究建立了深厚的基础。对于16p13.11中单个基因的功能研究，随着对基因敲除斑马鱼的不断深入分析，可为孤独症等疾病机制的研究提供更多的线索。