免疫球蛋白轻链的基因型及其蛋白质结构改变在淀粉样变发病机制中作用

Immunoglobulin light chain amyloidosis (AL) is the most common type of systemic amyloidosis, which affected multiple organs including the kidney, heart, etc, and caused dysfunction of organs progressively with poor prognosis. AL amyloidosis is characterized by deposition of insoluble amyloid fibrils derived from monoclonal immunoglobulin light chains and its fragments secreted by an underlying plasma cell dysplasia. Patients with AL amyloidosis manifested different phenotypes, such as cardiac involvement and dominant vascular deposition of amyloid fibrils, which is supposed to correlate with specific germline gene usage and protein structural alterations of light chains. This project is designed to investigate the germline gene use and its somatic mutations of variable regions of light chains in a large cohort of Chinese patients with different phenotypes of AL amyloidosis. The isolation and extraction of amyloid protein deposited in renal tissue and urine light chains will be performed and measured by liquid chromatography-mass spectrometry. The primary structure of light chain protein including amino acid replacement in patients with different phenotypes will be analyzed, to explore the possible correlation between protein structural alterations of light chains and phenotypes of AL amyloidosis. By in vitro expression of proteins and site-directed mutagenesis technique, the effect of specific structural alterations of light chains on protein stability and in vitro fibril formation will be studied, so as to investigate the role of protein structural alterations of light chains in the pathogenesis of amyloidosis.

免疫球蛋白轻链型（AL）淀粉样变是最常见的系统性淀粉样变，可累及肾脏、心脏等全身多器官，进展性发展导致器官衰竭，预后较差。AL型淀粉样变是由浆细胞分泌的单克隆免疫球蛋白轻链及其片段形成不溶性的纤维结构沉积于组织所致。不同临床表型包括心脏受累及血管沉积为主的淀粉样变病例，可能与轻链蛋白的不同胚系基因型及其基因突变导致蛋白质结构改变有关。本研究拟通过对我国大宗AL型淀粉样变病例的基因分析，探讨与淀粉样变形成相关的轻链胚系基因型及其体细胞突变特点；通过质谱分析尿轻链和肾组织沉积的淀粉样变蛋白的一级结构，探讨与临床表型相关的轻链蛋白分子结构改变包括氨基酸替换等特点。通过定点突变和体外蛋白表达技术，研究携带特定氨基酸改变的轻链在体外形成淀粉样变纤维的变化，探讨轻链蛋白的结构改变在淀粉样变发病机制中作用。

轻链型（AL）淀粉样变是由单克隆免疫球蛋白轻链异常折叠形成不溶性的纤维丝结构，并沉积于全身多系统的组织间隙而导致的一种慢性进展性疾病，肾脏是最常见和早期受累的器官之一，心脏受累提示预后不良。淀粉样变蛋白可沉积于肾脏的各个部位，肾血管壁的沉积程度与肾功能不全及肾外受累的发生率显著正相关。轻链由于基因重排形成不同的胚系基因亚型，或发生体细胞突变导致其分子结构的改变和不稳定性，可能参与淀粉样变的发病机制或与其靶器官选择的倾向性有关。本项目进行了如下的研究：（1）我国AL型淀粉样变的轻链胚系基因亚型与不同临床病理表型的相关性: 选取肾活检病理证实的AL型淀粉样变病例76例，通过激光微切割联合质谱分析的蛋白质组学分析，56/76例（73.7%）获得轻链蛋白的可变区序列及其胚系基因亚型。其中，轻链的λⅢ，λⅥ是最常见的轻链胚系基因亚型。肾脏受累的病例以λⅥ亚型最常见；伴有心脏受累的病例，以λⅢ，λⅥ和κⅠ亚型为常见类型；根据淀粉样变的沉积部位分类，肾小球沉积为主型以λⅡ、λⅥ和κⅣ为常见类型；肾血管沉积为主型以λⅢ、λⅥ为常见类型；肾间质沉积为主型以λⅢ和κⅠ亚型为常见类型。结果提示，轻链的λⅢ，λⅥ和κⅠ基因亚型与心脏受累相关，并可能提示预后不良。（2）致淀粉样变轻链基因及其蛋白质结构的初步分析：通过克隆测序法获得2例淀粉样变和1例骨髓瘤的轻链全长基因序列,2例淀粉样变的轻链基因亚型均为λⅠ（Vλ1-44）；其轻链基因 CDR超突变率（分别为37.0%和38.5%）低于骨髓瘤（42.9%）；但其编码蛋白的疏水性及其二级结构的β折叠比例均较骨髓瘤增加，提示轻链的分子结构改变导致其易于聚集形成纤维丝结构。（3）尿液轻链蛋白的提取及其分子结构分析：通过亲和层析法提取淀粉样变病例的尿液轻链蛋白，质谱分析可获得轻链蛋白约80%的氨基酸序列，还需要通过增加酶切位点，以期获得轻链的全长氨基酸序列。