Molecular genetics and cell biology of a glaucoma gene and the gene product

采用单链构象多态性分析和直接测序法对150余例原发性开角型青光眼患者的myocilin基因进行了突变筛查。没有一个患者有青光眼家族史和myocilin基因突变，但除了我们以前报道的一个家系之外，还有一个家系在myocilin基因的第1109个核苷酸对发生突变。突变为C1109T，导致Pro370Leu氨基酸突变。患者的表型与我们以前报道的几乎相同：患者在20岁之前出现非常高的眼内压，并且患有青少年型开角型青光眼。为了研究肌球蛋白的功能，我们利用培养的牛眼小梁细胞克隆了牛肌球蛋白的cDNA。根据测定的cDNA序列，我们制备了核苷酸探针，用原位杂交技术研究了肌球蛋白在牛眼组织中的分布。采用半定量PCR和实时定量PCR方法，研究类固醇刺激对肌球蛋白mRNA表达的影响。根据已测定的牛肌细胞素cDNA序列，构建了引物对和荧光素探针。结果表明，地塞米松处理诱导myocilin mRNA的表达呈剂量依赖性。

Mutations in myocilin gene were screened in more than 150 primary open-angle glaucoma patients with single strand conformation polymorphism and direct sequencing. There was no patient who had family history of glaucoma and a mutation in myocilin gene but one pedigree in addition to the ones we previously reported had a mutation in myocilin gene at the 1109th nucleotide pair. The mutation was C1109T which resulted in Pro370Leu amino acid mutation. The phenotype of the patients was almost the same as the one we previously reported : the patients developed very high intraocular pressure earlier than 20 years old and had a juvenile onset type open-angle glaucoma. For the purpose of investigating the function of myocilin protein, we cloned bovine myocilin cDNA using the cultured bovine trabecular meshwork cells. According to the determined cDNA sequence, we made nucleotide probes and investigated distribution of myocilin in bovine eye by in situ hybridization. Using semi-quantitive PCR and realtime quantitive PCR, we investigated the change of expression of myocilin mRNA by steroid stimulation. The primer pairs and fluorescein probes were constructed based on the determined bovine myocilin cDNA sequence. As a result, dexamethason treatment induced myocilin mRNA expression dose-dependently.