ABCB6基因在眼缺损发病机制中的功能研究

Ocular coloboma is a developmental defect of the eye due to abnormal or incomplete closure of optic fissure. This disorder displays genetic and clinical heterogeneity. Using a positional cloning approach, a mutation in the ATP-binding Cassette (ABC) Transporters ABCB6 was identified from a Chinese pedigree with autosomal dominant coloboma. The L811V mutation was identified in seven affected members of the pedigree and was absent in six unaffected members from three generations. Sequence analysis of the ABCB6 exons from 116 sporadic cases of microphthalmia with coloboma (MAC), isolated coloboma and aniridia, an additional mutation (A57T) was identified in three patients with MAC. These two mutations were not present in 200 normal controls. Immunostaining of transiently transfected myc-tagged ABCB6 in retinal epithelial cells (RPE) showed it localizing to the ER and Golgi apparatus of RPE. RT-PCR for ABCB6 mRNA in human cell lines and tissues indicated that ABCB6 is expressed in the retina and RPE. Using zebrafish we show that abcb6 is expressed in the eye and central nervous system. Morpholino knockdown of abcb6 in zebrafish produces a phenotype characteristic of coloboma and replicates the clinical phenotype observed in our index cases. The knockdown phenotype can be complemented with co-injection of the wild type, but not mutant human ABCB6 mRNA, suggesting that the phenotypes observed in zebrafish is due to insufficiency of abcb6 function. Our results demonstrate that ABCB6 mutations cause ocular coloboma. In this study, we will construct ABCB6 transgenic mice and ABCB6 knockout mouse model; To test ABCB6 expression as the optic fissures closeing during early mouse development; To reveal the interaction between ABCB6, PAX6 and SHH; To identify ABCB6 interacting protein; The role of mutated ABCB6 in proliferation and migration of retinal pigment epithelial cell, and intracellular iron ion transporter.

眼缺损是严重的儿童致盲性眼病，是由于胚胎期6-7周时胚裂闭合不全所导致的一种先天性眼部异常。先天性眼缺损常有遗传倾向，多表现为常染色体显性遗传或隐性遗传。ABCB6是一个新发现的眼缺损致病基因。我们通过基因连锁分析和对候选基因进行的广泛的外显子测序，发现了致病基因是ABCB6。L811V是在家系突变筛查得到，在116例散发病例中又发现新突变（A57T）。在斑马鱼模型中，我们通过morpholino敲低ABCB6复制了眼缺损的表型；共注射野生mRNA ABCB可拯救表型而突变的则不行。 在本课题中，我们将构建ABCB6转基因小鼠和ABCB6敲除小鼠模型，观察小鼠发育早期，ABCB6对视裂隙闭合的影响；揭示ABCB6与PAX6，SHH间的关系；找出ABCB6相互作用蛋白；ABCB6突变对视网膜色素上皮细胞增殖、迁移，对细胞内铁离子的转运作用的影响