苗族巴德-毕氏综合征家系相关致病基因鉴定及其与纤毛功能初步研究

Bardet-Biedl Syndrome (BBS) is a rare autosomal recessive ciliopathy with a wide spectrum of common clinical features including obesity, retinitis pigmentosa, polydactyly, mental retardation, hypogonadism, and renal abnormalities. Sixteen genes independently causing BBS have been identified recently (BBS1 through BBS16).Though mutations of these genes are sufficient to cause BBS in 70-75%, the molecular etiology of 20-25% BBS are still unknown.We established immortalized lymphoblastoid cell lines of a Miao core pedigree with Bardet-Biedl syndrome on previous research. Screened reported BBS genes on BBS patients and found no overlapped mutations on the reported BBS genotypes. Thus, to identify of the gene(s) involved in the Miao BBS families and the related molecular mechanisms, we design to 1) By using exome sequencing and bioinformation techniques, to screen and identify the causative gene(s) of these Miao BBS ;2）By using immunofluorescence technique and co-immunoprecipitation, to detect the relationship between the new BBS gene(s) and the BBSome; 3) By using RNA interference technique to silence the new BBS gene, and then to detect the expression of key proteins involved in Leptin receptor pathway such as POMC,α-MSH and MC4R We hope by the research, we could identify the novel causative BBS genotype（s） and learn the relationship between new BBS gene(s) and ciliopathy, which will give new clues of BBS etiology.

Bardet-Biedl综合症是纤毛疾病，虽然为罕见高度基因异质性遗传病，但其疾病表型及基因型与肥胖、高血压及视网膜退化等常见纤毛疾病谱有较大重叠。迄今发现的致病基因只能解释70－75％的患者。本研究前期已建立一个苗族BBS核心家系永生化细胞系，对3例BBS患者未检测到与现有报导相重叠的致病基因型，故本次拟1）通过外显子测序及生物信息学分析，鉴定导致该家系表型的基因及基因型；2）通过免疫荧光及免疫共沉淀技术，观测鉴定基因与纤毛结构蛋白BBS体是否有相互作用关系；3）通过RNA干扰技术沉默鉴定基因表达，同时采用RT－PCR及Western Blot，观测该基因对纤毛参与的痩素受体信号通路关键基因POMC、α-MSH及MC4R表达是否有影响；预期获得苗族BBS家系致病基因型，并在细胞水平初步了解该基因与纤毛功能的关系，为该病致病机理提供新解释

Bardet-Biedl综合症是纤毛疾病，致病基因具高度异质性，其疾病表型及基因型与肥胖、高血压及视网膜退化等常见纤毛疾病谱有较大重叠。本研究前期建立一个苗族BBS核心家系永生化细胞系，对3例BBS患者进行了外周血基因表达谱分析，本次实验1）利用Agilent SureSelect 试剂盒及HiSeq 2000 (Illumina）测序平台对2例患者及1例对照开展全外显子测序，根据隐性遗传模式，筛查出368个已知SNP，对其中MAF小于等于0.2的82个SNP位点进行SNP分型验证；同时，通过功能突变基因分析，筛选出28个候选基因进行进一步Sanger测序验证。经985例表型正常对照验证，仅BBS7基因5号外显子上的c.389_ 390delAC缺失突变符合家系常染色体隐性遗传模式，改突变导致读框移码，产生仅133个氨基酸的截短多肽，为该家系致病突变；2）构建BBS7、BBS1、Kif2a高表达及敲低细胞模型。发现Annexin A1 、Kif2a、Kif3a基因受BBS1基因的调控，Kif2a基因表达与BBS7、Kif3a基因表达呈正相关。BBS1基因上调有减缓293T细胞的增殖速率及降低293T细胞迁移速率的趋势，BBS7基因上调对细胞增殖有升高趋势；3）通过Crisprl/Cas9技术敲低BBS7，经qPCR及Western Blot验证，瘦素通路关键基因POMC、MC1R及MC4RmRNA及蛋白表达与BBS7表达改变与无统计学相关；4）在BBS7敲低细胞模型上进行了泛素化修饰的探索，发现BBS7敲低组较正常对照有明显赖氨酸泛素化修饰水平差异，具体位点正在进行质谱分析。本研究首次在中国BBS家系中报道了新的BBS7缺失突变位点，丰富了BBS致病基因谱。同时，本研究中发现的BBS7缺失导致的赖氨酸泛素化修饰的差异，为BBS致病机理提供了新的方向和治疗策略的可能。

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