广州开角型青光眼家系致病基因定位与功能初步研究

目的 鉴定常染色体显性遗传家族性开角型青光眼家系（GZ.1）致病基因并探讨其致病机制。方法 应用全基因组扫描、连锁分析、核苷酸直接测序法筛查GZ.1家系的致病性基因突变,进一步利用体外基因转染技术,观察突变型基因在小梁细胞中的表达、分布及对细胞凋亡的作用。结果 GZ.1家系所有患者均存在myocilin基因第三外显子Pro370Leu杂合突变（16／16）,而家系正常人中均未检测到该突变（0／8）。小梁细胞体外转染野生型和突变型myocilin基因后,经免疫印迹法检测证实,培养上清液中突变型myocilin蛋白的细胞外分泌较野生型明显减少;荧光免疫组化检测结果证实野生型myocilin蛋白在小梁细胞内呈点状、弥漫分布;而Pro370Leu突变型myocilin蛋白以积聚体形式存在于胞质内;经流式细胞仪检测突变型转染组凋亡细胞百分比较野生型转染组及对照组轻度升高。结论 突变型（Pro370Leu）myocilin基因为GZ.1家系的致病基因,突变型（Pro370Leu）myocilin蛋白在小梁细胞内以积聚体形式存在,可能是由于蛋白质错误折叠所致,并可能诱导小梁细胞凋亡。

A preliminary study on gene mutation and its function in a large family (GZ.1 pedigree) with open-angle glaucoma

Objective To determine the causative mutation of myocilin gene and to investigate its pathogenic function in a large Chinese pedigree (GZ.1) with familial open-angle glaucoma. Methods Genome-wide scanning was performed and the Lod scores were calculated. Candidate gene was amplified and screened for mutations using direct sequencing. To elucidate its expression, distribution and cytotoxicity of mutant myocilin, human trabecular cells (HTM) cells were transfected with pcDNA-wild-type and mutant myocilin vectors using liposomes. Results Mutation analysis of the myocilin gene showed a C-to-T transition at the 1,109th nucleotide in exon 3 resulting in a change of amino acid from proline to leucine (Pro370Leu). This mutation cosegregated with all affected individuals (16/16) and never presented in unaffected individuals (0/8). In transfected HTM cells, the mutant myocilin protein was not correctly processed in ER and accumulated as aggresome-like structures in the cytoplasma instead of being secreted. In addition, the expression of mutant protein also led to apoptosis of trabecular cells and the occurrence of.Conclusion The mutation of Pro370Leu in myocilin gene could cause the accumulation of misfolding myocilin protein in HTM cells, which might lead to glaucoma in GZ.1 pedigree.