Anopthalmia Spectrum Disorders

描述（由申请人提供）：无眼症，或没有眼睛，在5000到10000个人中发现1个，是一种毁灭性的先天缺陷，因为由此导致的视力障碍。无眼症与小眼症（小眼睛）和结肠瘤（脉络膜裂隙或视膜裂隙无法闭合）密切相关。我们研究了一位患有严重小眼症的男性患者，他在染色体18q22.1处有一个新的染色体缺失。我们认为18q22.1的一个缺失基因TXNDC10与眼睛发育有关。原位杂交显示Txndc10在发育中的小鼠眼视网膜神经上皮和晶状体上皮中表达。我们对66例无眼症或小眼症患者的TXNDC10进行了重新测序，发现c.260G>A，预测了一个不相关的小眼症和视网膜缺损患者的错义替换p.R39Q。我们利用反义morpholinos靶向斑马鱼Txndc10同源基因Zgc110025，发现斑马鱼变体幼虫存在小眼症和结肠瘤。与Pax2原位杂交显示，与对照组相比，变形体的脉络膜裂隙染色增加，表明Pax2失调参与了结肠瘤的形成。变形视网膜外周抗组蛋白H3染色增加，后期分化细胞类型减少。人类野生型TXNDC10 mRNA与morpholino共注射可挽救morphant表型，而人类TXNDC10/(p。含morpholino的R39Q突变体mRNA则没有。我们的研究结果表明，TXNDC10的单倍不足会干扰眼睛的发育。我们建议从两个方向继续对TXNDC10进行研究。我们假设Txndc10可能是腹侧视网膜发育已知途径的一部分，并希望利用斑马鱼来确定Zgc110025是否与其他参与脉络膜裂隙闭合和腹侧视网膜形成的已知基因如Vax1、Vax2和Shh （Syu）相互作用。我们将采用原位杂交、双重注射反义morpholinos和mRNA拯救实验来实现这一目标。我们的目的是试图揭示Txndc10影响眼睛发育的机制。其次，我们建议建立Txndc10功能缺失的小鼠模型，在基因缺失模型中检测眼睛表型，而不是在仅部分基因功能缺失的动物模型中。我们假设Txndc10功能的缺失会导致小鼠出现小眼症和结肠瘤，我们将使用缺失Txndc10的小鼠来检测缺失Txndc10的动物的眼睛形态，并通过表达阵列来评估Txndc10缺失小鼠和野生型产仔配偶之间的基因表达差异。我们的最终目标是提高对腹侧视网膜形成和脉络膜裂隙闭合所需基因的现有知识，以便为无眼症和相关眼缺陷的人类患者提供改进的遗传咨询和检测。

DESCRIPTION (provided by applicant): Anophthalmia, or absence of the eye, is found in 1 in 5,000 to 10,000 individuals and is a devastating birth defect because of the resulting visual impairment. Anopththalmia is closely related to microphthalmia (small eyes) and coloboma (failure of the choroid fissure or the optic fissure to close). We have studied a male patient with severe microphthalmia who had a novel chromosome deletion at chromosome 18q22.1. We have implicated one of the deleted genes at 18q22.1, TXNDC10, in eye development. In situ hybridization showed that Txndc10 was expressed in the retinal neuroepithelium and lens epithelium in the developing murine eye. We re-sequenced TXNDC10 in 66 patients with anophthalmia or microphthalmia, and found c.260G>A, predicting a missense substitution, p.R39Q, in an unrelated patient with microphthalmia and retinal coloboma. We used antisense morpholinos targeted against the zebrafish Txndc10 orthologue, Zgc110025, to show that the morphant larvae had microphthalmia and coloboma. In-situ hybridization with Pax2 showed increased choroid fissure staining in morphants compared to controls, suggesting that Pax2 dysregulation was involved in the coloboma formation. The morphant retinas had increased anti-histone H3 staining in the peripheral retina and we observed fewer late-differentiating cell types in the morphant retinas. Co-injection of human wild type TXNDC10 mRNA with a morpholino rescued the morphant phenotype, whereas co-injection of human TXNDC10/(p.R39Q) mutant mRNA with morpholino did not. Our results suggest that haploinsufficiency for TXNDC10 perturbs eye development. We propose to continue our research on TXNDC10 in two directions. We hypothesize that Txndc10 may be part of a known pathway for ventral retinal development, and would like to use zebrafish to determine whether Zgc110025 interacts with other known genes that are involved in choroid fissure closure and the formation of the ventral retina, such as Vax1, Vax2 and Shh (Syu). We will use in-situ hybridization, dual injections of antisense morpholinos and mRNA rescue experiments to accomplish this aim. Our purpose is to try to uncover the mechanism whereby Txndc10 affects eye development. Secondly, we propose to make a mouse model of loss of Txndc10 function to examine the eye phenotype in a genetic null model rather than in an animal model with only partial loss of gene function. We hypothesize that loss of Txndc10 function will produce mice with microphthalmia and coloboma, and we will use the resultant knock-out mice to examine eye morphology in a Txndc10 null animal and to perform expression arrays to evaluate for differences in gene expression between Txndc10 null mice and wildtype litter mates. Our ultimate goal is to improve the existing knowledge about the genes that are required for ventral retina formation and choroid fissure closure so that human patients with anophthalmia and related eye defects can be offered improved genetic counseling and testing. PUBLIC HEALTH RELEVANCE: Birth defects affect an estimated 120,000 (1 in 33) babies born in the United States each year, and are the leading case of mortality in the first year of life. Anophthalmia (absent eye), microphthalmia (small eye) and coloboma (failure of the choroid fissure or the optic fissure to close) can result in a significant burden for affected individuals and their families because of the medical and social effects of reduced vision. This application seeks to improve the existing knowledge pertaining to the genetic causes of these eye defects so that patient counseling, management and treatment can be improved.