乙酰胆碱受体（AChR）α1和δ亚单位基因在AChR抗体介导的重症肌无力发病和表型中的作用

AChR is the major auto-antigen of myasthenia gravis (MG). There is explicit heterogeneity in the phenotypes of AChR antibody-mediated MG (AChR-MG). We previously reported that polymorphism of AChR was strongly associated with MG and AChR antibody levels. In this study, we sequence the genes of alpha 1 and delta subunits of AChR and search for SNP of differential distribution between MG patients and the controls. Then, we select the candidate SNPs which is associated with MG and with its severity by high-throughput phenotyping and association studies among patients with different severity. For the associated SNPs which might influence the transcription levels of AChR, we examine the expression levels of subunits in the muscles from MG patients with different genotypes, and in the transfected muscle-line cells, to analyze the relationship between the basic and the compensational expression of subunits in muscles and the severity of MG; and examine the expression levels of subunits in the thymus and the strength of the autoimmune response against AChR, to analyze the effect of subunit expression levels in the thymus on the immune tolerance and its relationship to the susceptibility and severity of MG. For the associated SNPs which might influence the transcription sequence of mRNA, we examine the mRNA sequence and the strength of autoimmune response against AChR in patients with different genotypes, and examine the pathogenicity of the patients with transfected cells, to analyze the relationship between different gene sequence and its three-dimensional conformation and the susceptibility and severity of MG. This study starts from the individual variation of AChR genes and stops in explanation of heterogeneity in the key phenotype of MG with functional studies in individual levels, and will help to search for the treatment target and biomarkers which might guide individual treatments.

AChR是MG的主要自身抗原,AChR抗体介导的MG(AChR-MG)的临床表型有明确的异质性。我们报道了AChR基因多态性与MG发病及抗体水平高度相关。本研究对AChR的α1和δ亚单位测序和高通量基因分型,与AChR-MG发病及严重程度进行关联分析获得候选SNP。对可能影响mRNA 转录量的关联SNP,从不同基因型患者肌肉及转染细胞内亚单位的表达量来分析是否影响肌肉的抗损害能力,从不同基因型患者胸腺内亚单位表达量及免疫反应强度来分析是否改变自身耐受;对可能影响mRNA转录序列的关联SNP,从不同基因型患者转录的mRN A序列与免疫反应的强度及转染细胞与抗体的结合力,分析基因序列改变引起的抗原构象改变是否导致主要免疫致病力的改变,从而分析基因多态性与MG发病和严重程度的关系。从个体水平发现关联SNP并进行功能验证,阐释AChR-MG的异质性,以寻找潜在治疗靶点或生物标志物。

为了研究乙酰胆碱受体亚单位的基因多态性与重症肌无力表型的关系及其机制，收集患者临床资料和血液样本并检测AChR抗体，并采用采用靶向测序方法对AChR基因及免疫调节基因关联性的进一步分析。通过临床资料的分析，基于国际上常用量表，在对其信度和效度评估的基础上，用Delphi法形成条目池，优选最佳条目建立MG严重程度新量表；明确了相对评分是MG近期疗效的更好指标；在建立干预后状态评估流程的基础上明确了MMS或更好是MG长期疗效的重要指标。完善了AChR抗体检测方法学，确定阳性界值作为分组依据并与严重程度进行相关性分析。以上工作对MG的表型进一步做了优化和标准化，在此基础上精准分析AChR基因多态性影响MG表型的机制，并可用于临床研究。对AChR基因及免疫调节基因关联性的分析进一步证实AChR的α亚单位和γ亚单位的多个SNP与重症肌无力患者的AChR抗体水平和严重程度的有关，而免疫调节基因的多态性与上述两个关键表型无明确关联性。通过双荧光素酶方法对已经发现的与重症肌无力AChR抗体水平和严重程度相关的rs16862847位点的等位基因在肌细胞系中的转录活性改变，提示该位点可能影响AChR亚单位的表达量，从而影响神经肌肉接头传导而影响MG的严重程度。正在进一步通过构建SNP转染细胞系进一步研究启动子和外显子区域的SNP对肌细胞AChR表达量和表达结构的影响是否改变神经肌肉接头电生理活动，是否改变胸腺内AChR表达量而影响免疫耐受和AChR抗体的亲和力，以及表达结构改变是否影响AChR抗体的亲和力，以期对AChR基因多态性是否影响MG的AChR抗体水平和严重程度这两个关键表型做出完整的解释。

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