II型胶原基因（COL2A1）突变导致单纯眼部异常表型的机制研究

Mutations in the COL2A1 gene could lead to several diseases involving multi systems. For oclular system, early-onset high myopia is the most common feature and the frequent occurrence of retinal detachement will cause serious ocular feature even blindness. COL2A1 precursor mRNA undergoes alternative splicing and results in two transcripts, which are type IIA transcript including exon2 and type IIB transcripts excluding exon 2. The IIA transcript is present in the vitreous of the eye, which is the only tissue containing procollagen IIA in adults. It has been reported that mutations in exon 2 of COL2A1 could cause a predominantly ocular abonrmalities by specially affecting the IIA transcript, which is only present in vitreous in adults. However, several mutations at regions outside of exon 2 could also cause ocular-only phenotypes and the molecular mechanism of these mutations has yet to be studied. In our previous study, nine mutations in COL2A1 were identified in 3% of families with early-onset high myopia, which also cause ocular-only abnormalities but located at different regions of COL2A1 outside of exon 2. It was proposed that these mutations maybe also affect the tissue-specific alternative splice of COL2A1. The current project will study the effect of the mutations on two alternative splice of COL2A1 by constructing different expression vector and transfecting three human cell strains (human chondrosarcoma cells, human chondrocytes, and human fetal scleral fibroblasts cells). We expect to find the possible mechanism through which these mutations cause ocular-only phenotypes. The results will provide basis for prevention of blindness in the future.

COL2A1基因（MIM120140）突变可引起累及全身多个系统异常的表现，其中早发高度近视是最常见的眼部表型，其结局往往因难治性视网膜脱离而严重影响视功能，甚至致盲。COL2A1基因有两种转录剪接方式，根据是否包含第二个外显子分为IIA型转录体和IIB型转录体，其中包含第二个外显子的IIA型转录体在成人仅有玻璃体中存在。既往有研究发现部分COL2A1基因突变导致了单纯的眼部异常，这些突变绝大多数位于第二个外显子附近，通过仅仅影响在成人玻璃体中唯一表达的IIA型转录体，而只引起单纯眼部异常。但是还有个别位于其它区域，也同样导致单纯眼部异常的突变，其致病机制尚未得到深入研究。我们的既往研究在3%的早发高度近视家系中发现9个不同的COL2A1基因突变，同样导致单纯眼部异常，却均分布于第二个外显子以外的区域，我们推测这些突变可能在转录水平对两种组织特异性转录体产生了不同的影响。本项目拟对这9个突变进行研究，通过构建不同突变的表达载体并转染三种不同的细胞系（人骨肉瘤细胞、人软骨细胞、人胚胎眼巩膜成纤维细胞），分析其对基因转录剪接的影响，寻找可能的致病机制，为未来制定防盲策略提供依据。

II型胶原基因（COL2A1）突变可引起累及全身多个系统异常的表现，部分突变导致仅有眼部表型而无眼外全身系统的异常。早发高度近视是COL2A1基因突变最常见表型，结局合并因合并难治性视网膜脱离而致盲。我们既往研究发现COL2A1基因是导致早发高度近视最常见的致病基因，约占3.4%的早发高度近视家系，这些病人初诊均未表现出明显的全身系统的异常，研究这些突变仅导致眼部异常表型的机制，将为高度近视的发病机制以及未来的防盲策略提供依据。本研究在一个常染色体显性遗传的早发型高度近视大家系中，全外显子组测序分析未发现可能的致病突变，进一步通过高度近视候选基因位点连锁分析和突变筛查，发现COL2A1基因深部内含子的突变，体外构建minigene证实该突变可导致COL2A1基因第二外显子的剪接异常。该家系三代不同年龄区域的8个病人，均未发现眼外全身系统的异常表现，提示影响COL2A1基因第二外显子的突变，可引起眼部特异表达转录本的转录剪接异常，导致仅累及眼部异常的表型。此外，增加部分内容，基于单中心来源的11836例眼遗传病患者的外显子组测序数据，系统分析了COL2A1基因的突变频谱和表型谱，结果发现，少部分COL2A1基因突变导致Stickler综合征，大部分导致无明显全身异常表现的早发高度近视，且发现COL2A1基因突变频谱较既往文献报道突变频谱有差异，本研究结果为早发高度近视病人的防盲以及伴发其他系统异常表型的风险预测提供参考依据。