MOLECULAR ANALYSIS OF CONGENITAL ANOPHTHALMIA

先天性无眼症的分子分析

基本信息

批准号：
6635704
负责人：
Thomas M. Glaser
金额：
$ 25.65万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-05-01 至 2005-04-30
项目状态：
已结题

项目摘要

Clinical anophthalmia and microphthalmia (absent or small eyes) are significant human birth defects, affecting 0.3 and 1 2 per 10,000 children, respectively (Kallen et al. 1996), and are important causes of congenital blindness. These malformations are thought to arise from an inherent failure in optic vesicle growth or lens induction (DukeElder 1964). In spite of remarkably deep evolutionary conservation among patterning genes and pathways in the optic cup, no molecular mechanism has been found for this disorder. The majority of cases are sporadic, although some pedigrees show simple Mendelian inheritance (Kohn et al. 1988). PAX6 gene mutations have been identified in one case of anophthalmia (Glaser et al. 1594). Teratogenic etiologies, polygenic factors or geneenvironment interactions have also been suggested. However, the small size of affected families, and the possibility of heterogeneity and nongenetic factors make it difficult to perform conventional linkage studies. Likewise, the regression of eyes in numerous fossorial (subteranean) and troglobific (cavedwelling) taxa is a fascinating macroevolutionary problem, which is difficult to approach experimentally. Eye regression in vertebrates may involve relatively few genes and may occur rapidly, even within a single species (Behrens et al. 1997). A mouse model, the eyeless ZRDCT inbred strain, was first described many years ago (Chase and Chase, 19423. It is phenotypically and genetically similar to human clinical anophthalmia. Affected mice lack eyes and optic tracts, have attenuated brain visual centers and primary circadian rhythm disturbances. Although F1 mice are normal, heterozygous embryos are more sensitive to teratogeninduced eye defects than other laboratory mice (Beck, 1963). On the basis of breeding experiments, Chase (1944) proposed that the eyeless trait is oligogenic, involving a synthetic interaction between a single major recessive factor (eyl ) and one or a few modifier loci (ey2). In preliminary studies, we discovered that eyl is a point mutation in the Rx/rax rednal homeobox gene. Rx/rax is conserved among bilateral metazoans; is specifically expressed in the anterior neural fold, hypothalamus, optic cup and neurorefina, beginning at day E7.5 of mouse development; and has the potential to promote neural and pigmented retina formation when ectopically expressed in frog anterior blastomeres (Mathers et al. 1997, Furukawa et al. 1997). However, the molecular role, downstream targets and interacting proteins of this major patterning gene for eye development are unknown. The mutation, a M1OL codon substitution, is hypomorphic in comparison to a null (knockout) Rx/rax allele, which causes severe CNS defects and is lethal (Mathers et al. 1997). The M1OL mutation may thus alter translational initiation or disrupt a previously unknown functional domain at the arninoterminus. Homozygosity for the M1OL allele is necessary but not sufficient to produce the eyeless phenotype The ey2 modifier gene(s) are likely to encode proteins that interact directly with Rx/rax or function in a common pathway for optic cup morphogenesis. Mapping of ey2 is thus conceptually similar to enhancer screens routinely performed in Drosophila. In this proposal, we aim to: (1) characterize the MlOL mutation in detail; (2) find target genes and interacting proteins for Rx/rax using ZR about mice and in vitro assays (3) map, clone and characterize ey2 loci in the mouse genome; (4) compare Rx coding and regulatory sequences from the blind mole rat Spalax erhenbergi to Rx genes of mice, hamsters and other sighted Muridae; (5) collect and screen human anophthalmia cases for Rx mutations; and ( about) test the role of Rx mutahons in two other model organisms, the F33 anophthalmic rat and the eyeless (e/e) axolotl Ambystoma mexicanum. These studies should provide valuable new informahon regarding Rx/rax function, opUc cup formahon and retina1 histogenesis, ontogeny of the hypothalamic circadian clock, the efiology of human clinical anophthalmia and rnicrophthalmia, polygenic developmental disorders and geneenvironrnent interachons, and the evolubon of eyelessness in nature.

临床无眼症和小眼症（眼睛缺失或小）很严重人类出生缺陷，分别影响每 10,000 名儿童 0.3 名和 1 2 名（Kallen 等人，1996），并且是先天性失明的重要原因。这些畸形被认为是由视神经泡固有的缺陷引起的生长或晶状体诱导（DukeElder 1964）。尽管非常深视杯中的图案基因和通路之间的进化保守性，尚未发现这种疾病的分子机制。大多数情况是零星的，尽管一些谱系显示出简单的孟德尔遗传（Kohn 等人。 1988）。 PAX6 基因突变已在 1 例病例中被发现无眼症（Glaser 等人，1594）。致畸病因、多基因因素或还提出了基因环境相互作用。不过，尺寸较小受影响家庭的数量，以及异质性和非遗传性的可能性因素使得进行传统的连锁研究变得困难。同样地，许多化石（地下）和穴居动物的眼睛退化（穴居）类群是一个令人着迷的宏观进化问题，很难通过实验接近。脊椎动物的眼睛退化可能涉及基因相对较少，即使在单个物种内也可能快速发生（Behrens 等人，1997）。小鼠模型，即无眼 ZRDCT 近交系，许多年前首次被描述（Chase 和 Chase，19423。它的表型是并且在遗传上与人类临床无眼症相似。受影响的老鼠没有眼睛和视束，减弱了大脑视觉中心和初级昼夜节律节律紊乱。虽然F1小鼠是正常的，但杂合胚胎更多与其他实验室小鼠相比，对致畸引起的眼部缺陷敏感（Beck， 1963）。 Chase (1944) 在育种实验的基础上提出无眼特征是寡基因的，涉及单个个体之间的合成相互作用主要隐性因子 (eyl ) 和一个或几个修饰基因座 (ey2)。在初步研究，我们发现eyl是Rx/rax的点突变红纳尔同源盒基因。 Rx/rax 在双侧后生动物中是保守的；是特异表达于前神经皱襞、下丘脑、视杯和神经视网膜，从小鼠发育的 E7.5 天开始；并且有潜力当异位表达时促进神经和色素性视网膜形成青蛙前卵裂球（Mathers et al. 1997, Furukawa et al. 1997）。然而，该主要分子的分子作用、下游靶标和相互作用蛋白眼睛发育的模式基因尚不清楚。突变，M1OL 密码子与空（敲除）Rx/rax 等位基因相比，取代是低等位的，它会导致严重的中枢神经系统缺陷并且是致命的（Mathers et al. 1997）。 M1OL 因此，突变可能会改变翻译起始或破坏先前的翻译过程。氨基末端的未知功能域。 M1OL 的纯合性等位基因是必要的，但不足以产生无眼表型 ey2 修饰基因可能编码直接与 Rx/rax 或在视杯形态发生的共同途径中发挥作用。映射因此，ey2 在概念上类似于通常在果蝇。在本提案中，我们的目标是：（1）表征 MlOL 突变细节; (2)使用ZR找到Rx/rax的靶基因和相互作用蛋白关于小鼠和体外测定 (3) 绘制、克隆和表征 ey2 位点小鼠基因组； (4) 盲法比较 Rx 编码和调控序列鼹鼠鼹鼠（Spalax erhenbergi）对小鼠、仓鼠和其他有视力的动物的 Rx 基因鼠科； (5) 收集并筛查人类无眼症病例的 Rx 突变；和（关于）测试 Rx 突变体在另外两种模式生物 F33 中的作用无眼大鼠和无眼 (e/e) 蝾螈 Ambystoma mexicanum。这些研究应提供有关 Rx/rax 功能、opUc 的有价值的新信息杯福马洪和视网膜1组织发生，下丘脑昼夜节律的个体发育时钟，人类临床无眼症和小眼症的病因学，多基因发育障碍和基因环境相互作用，以及自然界无眼的进化。