The Genetics of Uveal Coloboma

葡萄膜缺损的遗传学

• 批准号: 8737645
• 负责人: Brian Brooks
• 金额: $ 165.71万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8737645
• 关键词: A Mouse; Adult; Affect; Alanine; American; Biological Models; C57BL/6 Mouse; Candidate Disease Gene; Cell Adhesion Molecules; Cell Death; Cell division; Chest; Chromosome Deletion; Chromosomes; Chromosomes, Human, Pair 13; Clinical; Clinical Research; Coloboma; Cornea; Cystic kidney; Data; Defect; Development; Developmental Process; Diagnosis; Echocardiography; Embryo; Embryology; Embryonic Development; Embryonic Eye; Eye; Eye Development; Failure; Family; Family member; Fertility; First Degree Relative; Fishes; Folic Acid; Gene Expression; Gene Family; Genes; Genetic; Genetic Counseling; Goals; Homozygote; Human; Journals; Junk DNA; Kidney; Knock-out; Knockout Mice; Knowledge; Laboratory Study; Lasers; Lead; Manuscripts; Measurement; Messenger RNA; Methods; Microforms; Missense Mutation; Modeling; Molecular; Molecular Diagnostic Testing; Mus; Mutation; Ophthalmology; Optic Nerve; Optics; Paper; Parents; Participant; Patients; Pattern; Phenotype; Physical Examination; Pregnancy; Process; Publications; Publishing; Recruitment Activity; Research; Site; Surface; Syndrome; System; Testing; Threonine; Time; Transgenes; Ultrasonography; Vascular Endothelial Growth Factors; Work; Zebrafish; Zinc Fingers; aldehyde dehydrogenases; cardiogenesis; chromosome 5q loss; clinical care; developmental genetics; exome; face bone structure; genetic pedigree; homologous recombination; interest; interstitial; loss of function; lumbar vertebra bone structure; lymphoblastoid cell line; malformation; member; mouse model; mutant mouse model; novel; optic cup; pressure; proband; transmission process

I. Clinical Studies of Uveal Coloboma Since initiating this research, I have recruited and examined over 100 famlies where at least one member is affected by uveal coloboma. All probands and their first degree relatives (when available) have complete ophthalmic exams. General physical examinations and targeted systemic testing (e.g., renal ultrasounds, echocardiograms) were performed on probands, as needed. Lymphoblastoid cell lines were established on all participants for candidate gene analysis. Preliminary analysis of these patients has resulted in four important clinical observations. First, patients with coloboma often have thickened corneas as part of their developmental defect. This affects the measurement of their eye pressures. Second, I have identified a new syndrome in which missing thoracic and/or lumbar vertebrae co-segregates with coloboma. This was seen in two, unrelated autosomal dominant pedigrees. We have recently published this finding as part of a broader paper on systemic findings in patients with uveal coloboma. We are currently pursuing a whole-exome strategy to try to find the gene responsible. Third, I have determined the yield of systemic testing on patients with apparent isolated coloboma, resulting in the publication in the American Journal of Ophthalmology mentioned above.. Fourth, I have defined microforms of coloboma that aid in genetic counseling of families. II. Laboratory Studies of Uveal Coloboma A. Mouse Models of Coloboma. 1. A novel Pax2 mutant mouse model of coloboma. My lab identified and characterized a mouse model of autosomal dominant congenital optic nerve excavation caused by a missense mutation predicted to change a highly-conserved threonine to alanine in the paired domain of the Pax2 gene. Pax2 is dynamically expressed at the closing edges of the optic fissure and homozygous mutation results in uveal coloboma. Details of our characterization of this model and the Pax2 mutation have been published in PLoS Genetics. More recently, we have used this model system to look at the effects of Pax2 loss-of-function on other genes of interest, such as Nlz1 and Fat1/Fat4. 2. The RICO Mouse Model of Coloboma The RICO mouse arose from the random insertion of a transgene (NSE-VEGF) on chromosome 13 of C57BL/6 mice. Although the transgene is not expressed in the adult or embryonic mouse, its insertion led to a classic coloboma phenotype. The region of insertion did not suggest any previously-identified coloboma genes. This phenotype is dominant and does not significantly affect the viability or fertility of heteorygous mice. We have recently determined that homozygous mice are rarely viable postnatally. Tiling arrays of chromosome 13 show no evidence of major deletion. Pax2 expression is not affected in RICO embryos, indicating that the responsible gene is either independent or downstream of Pax2. We pursued a high-throughput sequencing approach to identify the transgene insertion and have identified both ends of a junctional fragment. This region of mouse chromosome 13 is a gene desert, but is well-conserved across species, suggesting that it contains an important regulatory motif. We are currently using FISH with several BAC clones in the region of the insertion--which appears to have caused an inversion in the process. B. Identification of coloboma candidate genes by molecular characterization of gene expression during optic fissure closure. 1. Zfp503 and Zfp703 Our previous work, published in PNAS, identified two zinc-finger motif-containing genes, Zfp703 and Zfp503 to be important in regulating optic fissure closure in zebrafish. We have created knockout mice for both Zfp703 and Zfp503 and documented germline transmission. We have demonstrated that homozygous knockout of Zfp503 is lethal in late embryonic development. The reason for non-viability is currently being pursued. We have also demonstrated that homozygous embryos develop coloboma, confirming our results from zebrafish work in a mammalian system. In addition, we have characterized the insertion site in the Zfp703 mouse, confirming its homologous recombination. We have determined that homozygotes are not viable postnatally, although the reason is currently unclear. We are currently examining the eye phenotype of these mice. In addition, we have made a more careful study of the zfp703 zebrafish morphant phenotype. We have identified that morphant fish have several important phenotypes such as cystic kidneys and abnormalities in heart development. As such, this model likely represents a syndromic form of coloboma. We have extended our mutation screen to include patients with syndromic forms of coloboma in hopes of identifying causative mutations. Current results are ambiguous are being confirmed using independent methods. In addition, we have identified that zfp703 morphants show a reduced rate of cell division in the optic cup, suggesting that the mechanism of coloboma is non-apposition of the edges of the fissure. 2. FAT protocadherins Another gene family that was suggested by our laser-capture screen was the FAT protocadherins. As previously described, we have found that Fat1 and Fat4 are the members of this family that are most highly-expressed during embryonic eye development and that homozygous knockout of Fat1--but not Fat4--results in coloboma. We have shown that the coloboma in Fat1-/- mice is not the result of a global patterning defect and that the eyes of these embryos are approximately normal size until the time of optic fissure closure. The rate of cell division in the developing optic cup is mildly elevated compared to wild-type and there is no obvious change in the rate of cell death. Real-time PCR of embryos on a panel of genes has revealed RPE-specific changes in several important cell adhesion molecules, suggesting that that mechanism of non-closure is directly related to non-adhesion. We are currently organizing our data for publication. 3. 5q deletion and coloboma We have identified a family with an interstitial deletion on chromosome 5q and coloboma. High throughput sequencing of the proband and his parents did not reveal any mutations in known coloboma genes, providing additional support that the chromosomal deletion and the phenotype were related. Previous work identified the aldehyde-dehydrogenase 7, which is highly expressed at the edges of the closing optic fissure in zebrafish, as a potential candidate. Morpholino studies show a coloboma, facial bone malformations and fin shortening phenotype with ALDH7 knockdown, similar to the patient phenotype. Unlike Fat1, knockdown of aldh7a1 results in reduced cell division in the optic cup. The mechanism of coloboma is therefore most likely a failure of apposition of the edges of the fissure at the right time in development. This effect can be partially rescued with nlz1 mRNA and with folic acid, suggesting that these factors are functionally downstream of aldh7a1. We are currently preparing a manuscript to publish these results.

I.紫val骨的临床研究 自从启动这项研究以来，我已经招募并检查了100多个家族，其中至少一名成员受紫外成coloboma的影响。 所有概率及其一级亲戚（如果有的话）都有完整的眼科考试。 根据需要，对一般的身体检查和靶向的全身测试（例如肾功能超声，超声检查）进行了根据需要进行的。 在所有参与者上建立了淋巴细胞细胞系以进行候选基因分析。 对这些患者的初步分析导致了四个重要的临床观察。 首先，作为其发育缺陷的一部分，造成骨的患者经常会变厚角膜。这会影响他们的眼压的测量。其次，我确定了一种新综合征，其中缺少胸腔和/或腰椎与coloboma共隔离。 这是在两个无关的常染色体显性谱系中看到的。 我们最近发表了这一发现，这是有关紫veal骨患者系统性发现的广泛论文的一部分。 我们目前正在采取全面策略来寻找负责的基因。 第三，我已经确定了对明显孤立的古罗氏菌患者进行全身测试的产率，从而在上面提到的《美国眼科杂志》上发表了。第四，我确定了有助于家庭遗传咨询的古罗巴马的微型细菌。 ii。 卵巢coloboma的实验室研究 A.成山的小鼠模型。 1。一种新型的Pax2突变小鼠模型。 我的实验室确定并表征了由常染色体显性前神经神经发掘的小鼠模型，该模型是由预测将高度保存的苏氨酸更改为PAX2基因配对域中的丙氨酸的错义突变引起的。 PAX2在视裂裂片的闭合边缘和纯合突变的闭合边缘产生了动态表达。 我们对该模型和PAX2突变的表征的细节已在PLOS遗传学中发表。 最近，我们使用此模型系统来研究PAX2功能丧失对其他感兴趣基因的影响，例如NLZ1和FAT1/FAT4。 2。哥洛巴马的RICO小鼠模型 RICO小鼠源于在C57BL/6小鼠的13染色体上的转基因（NSE-VEGF）随机插入。 尽管转基因未在成年或胚胎小鼠中表达，但其插入导致经典的古罗映表型。 插入区域并未提示任何先前鉴定的coloboma基因。 该表型是主导的，不会显着影响霍特里果小鼠的生存能力或生育能力。 我们最近确定纯合小鼠在产后很少可行。 13染色体的平铺阵列没有大量缺失的证据。 PAX2表达在RICO胚胎中不受影响，表明负责的基因是PAX2的独立或下游。我们采用了一种高通量测序方法来识别转基因插入，并确定了连接片段的两端。小鼠13染色体的区域是一个基因沙漠，但在各种物种之间保存良好，表明它包含一个重要的调节基序。 我们目前正在插入区域中使用带有几个BAC克隆的鱼类 - 这似乎在此过程中引起了反转。 B.通过在视觉裂缝闭合过程中基因表达的分子表征来鉴定coloboma候选基因。 1。ZFP503和ZFP703 我们以前发表在PNA中的工作确定了两个含锌指基序的基因ZFP703和ZFP503对于调节斑马鱼的光学裂隙闭合很重要。 我们为ZFP703和ZFP503创建了淘汰小鼠，并记录了种​​系变速器。 我们已经证明，ZFP503的纯合敲除在胚胎后期发育中是致命的。 目前正在追求不可验证的原因。 我们还证明，纯合胚胎发展了卵巢瘤，这证实了我们在哺乳动物系统中斑马鱼的结果。 此外，我们还表征了ZFP703小鼠中的插入位点，证实了其同源重组。 我们已经确定纯合子在产后不可行，尽管目前尚不清楚原因。 我们目前正在研究这些小鼠的眼睛表型。 此外，我们对ZFP703斑马鱼形态表型进行了更仔细的研究。 我们已经确定形态鱼具有几种重要的表型，例如囊性肾脏和心脏发育中的异常。 因此，该模型可能代表了coloboma的综合征形式。 我们已经扩展了突变筛查，以包括具有综合征形式的coloboma的患者，以期鉴定致病突变。 目前的结果是使用独立方法确认的。 此外，我们已经确定ZFP703形态在视觉杯中显示出细胞分裂的速度降低，这表明coloboma的机理是裂缝边缘的不贴合。 2。脂肪原钙蛋白 我们的激光捕获筛选建议的另一个基因家族是脂肪原钙蛋白。 如前所述，我们发现FAT1和FAT4是该家族的成员，在胚胎眼发发育过程中表达最高，并且纯合子敲除fat1-但不是FAT4-在古罗巴马中的肿块。 我们已经表明，FAT1 - / - 小鼠中的coloboma并不是全局模式缺陷的结果，并且这些胚胎的眼睛大小近似正常，直到闭合效率闭合为止。 与野生型相比，发育中的视频杯中的细胞分裂速率有些升高，并且细胞死亡率没有明显变化。 在一个基因面板上的胚胎的实时PCR揭示了几个重要的细胞粘附分子的RPE特异性变化，这表明非闭合的机制与非粘附直接相关。 我们目前正在组织我们的数据进行发布。 3。5q缺失和coloboma 我们已经确定了一个在5q和coloboma染色体上具有间质缺失的家庭。概率和他的父母的高吞吐量测序没有揭示已知的coloboma基因中的任何突变，从而提供了其他支持，即染色体缺失和表型是相关的。先前的工作确定了醛脱氢酶7，该醛在斑马鱼的关闭视神经裂片的边缘高度表达为潜在的候选者。 Morpholino研究表明，与患者表型相似，具有ALDH7敲低的山地骨，面部骨畸形和鳍片缩短表型。与FAT1不同，ALDH7A1的敲低导致光学杯中的细胞分裂减少。 因此，山地瘤的机制很可能是在正确发育中的裂痕边缘的分配失败。 该作用可以用NLZ1 mRNA和叶酸部分挽救，这表明这些因素在功能上是Aldh7a1的下游。 我们目前正在准备一份手稿以发布这些结果。