ABCB5基因错义突变SNP协同DCC基因遗传结构变异在家族性腺瘤样息肉病发病机制中的研究

Familial adenomatous polyposis (FAP) is an autosomal dominant colorectal cancer with poor prognosis. FAP has been linked to germline mutations in the APC gene, however, about 30% patients with FAP have no APC mutation. We previously have conducted whole genome sequencing in an APC mutation negative FAP family with 7 individuals, and the linkage analysis indicated that a missense mutation in ABCB5 gene (ABCB5-mSNP) and a structural variant in DCC gene (DCC-SV) are the candidates inducing the development of tumor. Further analysis of these mutations by bioinformatics suggest that ABCB5-mSNP act as a gain of function mutation and activate cMYC gene through ABCB5-cMYC axis, meanwhile, DCC-SV disrupts the functional domain in DCC and leads the corresponding apoptotic signal pathway to be inactivated. Therefore, we assume that the ABCB5-mSNP and DCC-SV may be the pathogenic changes predisposing to FAP. In order to study the relationship between ABCB5-mSNP, DCC-SV and FAP, the above genetic variations have been confirmed at the genomic level first, and then ABCB5-mSNP and DCC-SV mutations are designed to knock in normal intestinal epithelial cells and tumor cells by CRISPR-CAS9 method and then the tumor cells are inoculated into mice to investigate the function and phenotype. Biochemical and transcriptomic methods are designed to analyze the signaling network of the ABCB5-mSNP and DCC-SV mutations.

家族性腺瘤样息肉病(FAP)是一类预后极差的遗传性大肠癌,FAP多由APC基因突变导致，但约30%的FAP病因不明。申请人前期在一个病因不明的FAP家系中新发现两种致病性突变，包括ABCB5基因错义突变SNP（ABCB5-mSNP）和DCC基因遗传结构变异（DCC-SV），初步分析提示：ABCB5-mSNP活化ABCB5-cMYC轴激活肠癌相关cMYC基因；DCC-SV破坏抑癌基因DCC结构域导致下游凋亡信号通路失活。我们提出科学假说,ABCB5-mSNP突变在早期引起细胞异常增殖，随后DCC-SV突变使DCC抑癌功能丧失导致FAP。在基因组水平对上述遗传变异进行验证后,我们拟利用CRISPR/CAS9等技术将ABCB5-mSNP及DCC-SV导入正常或肠癌细胞系,研究上述变异在细胞异常增殖、凋亡等协同效应,通过裸鼠成瘤实验研究生理功能,利用生化和转录组学等方法解析上述变异的分子调控机理。

APC基因胚系突变引起Wnt/β-catenin信号通路活化是导致FAP主要病因，但是，按照现有常规筛查手段，仍有许多FAP患者病因未能明确，国内外将此类患者归为APC(-)/FAP，而探索并研究APC(-)/FAP患者的病因及相关机制是本研究领域的热点。.为探索云南地区FAP相关致病基因及其发病机制，项目组对1个研究筛选认定为APC(-)/FAP家系进行WGS测序并进行家系连锁分析。结果我们新发现一个ABCB5基因的错义突变即ABCB5810m可使ABCB5蛋白过表达并激活ABCB5-c-MYC轴。此外，我们还在改家系中发现了抑癌基因DCC的一种遗传结构变异即DCC△E2-3，此种遗传结构变异使得DCC基因出现基因断裂，断裂点后半部的所有结构域功能丧失无法与其配体Netrin-1结合，导致DCC基因相关的细胞凋亡通路失活。.为研究ABCB5基因错义突变与DCC遗传结构变异这两种突变在肠癌发生及发展中的作用，我们利用CRISPR/CAS9技术在正常上皮细胞BEAS-2B和大肠癌SW480细胞中分别将DCC基因敲除及ABCB5810m定点突变，通过基因筛选获取稳定表达DCC基因敲除的SW480细胞及BEAS-2B 细胞，细胞水平实验提示，突变型BEAS-2B细胞出现肿瘤样恶性增殖并具备侵袭性，动物实验提示，突变型BEAS-2B细胞接种小鼠成瘤。.在人肠癌细胞SW480中将DCC基因进行敲除后，其出现典型的干细胞生长特性，在对突变型SW480进行干细胞标记物的检测后发现，突变型SW480细胞的干细胞标记物中LGR5表型较野生型明显升高，而CD29表型明显降低。在将突变型SW480细胞接种小鼠后，我们同样发现，具备干性特征的突变型SW480细胞成瘤速度较野生型SW480细胞更快，而转录组学分析结果提示，EPAS1，NDRG1、CADM1、SLC2A3、PRSS56、CST1、IL17RD、VAT1L、ATP9A及TENT5C等10种基因在转录组水平发生改变。.通过本研究，我们证实，DCC基因遗传结构变异及ABCB5错义突变与肠癌发生发展密切相关，DCC基因遗传结构变异及ABCB5的基因错义突变同样可以作为FAP的基因筛查的靶标。

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