The Genetics of Uveal Coloboma

葡萄膜缺损的遗传学

基本信息

批准号：
10930511
负责人：
Brian Brooks
金额：
$ 167.05万
依托单位：
NATIONAL EYE INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/10930511
关键词：
ATOH7 gene Adult Affect Alleles Amacrine Cells Anatomy Anophthalmos Biological Assay Blood CLIA certified sequencing CRISPR screen CRISPR/Cas technology Candidate Disease Gene Cell Count Cells Child Chromosome 13 Clinical Clinical Data Coloboma Congenital Abnormality DNA Sequence Rearrangement Data Defect Development Developmental Gene Developmental Process Dorsal Down-Regulation Embryo Embryology Enrollment Exhibits Eye Eye Development Eye diseases FAT gene Failure Family Family history of Family member First Pregnancy Trimester Funding Gene Expression Gene Expression Profiling Genes Genetic Genetic Counseling Genomics Genotype Goals Habits Health Heterozygote Histology Human Hyperplasia Hypopigmentation Individual Inherited Iris Junk DNA Knock-out Knockout Mice Knowledge Laboratory Study Life Style Lung Mediating Melanins Methods Microphthalmos Molecular Molecular Diagnostic Testing Molecular Profiling Mothers Mus Mutation Neural Retina Nuclear Optic Nerve Optics Overlapping Genes Parents Participant Pathway interactions Patient Care Patients Penetrance Perinatal mortality demographics Phenotype Pigments Pregnancy Prevention Proteins Protocols documentation Publishing Questionnaires Reflex action Registries Reporting Repression Research Retina Role Signal Pathway Signal Transduction Signaling Protein Site Structure of retinal pigment epithelium Surface Syndrome Telephone Test Result Testing Texas Time Transcript Transgenes Transgenic Organisms United States National Institutes of Health Up-Regulation Variant Vascular Endothelial Growth Factors Work Zebrafish Zinc Fingers clinical care cohort differential expression dosage exome experimental study ganglion cell genetic disorder diagnosis genetic epidemiology genetic signature genetic testing genome editing genome sequencing glycoprotein NMB inhibitor insulinoma associated 1 integration site loss of function malformation mouse model mutant ophthalmic examination optic stalk population based protein expression recruit rib bone structure screening transcription factor transcriptome sequencing whole genome

项目摘要

1. Clinical and Genomic Studies Recruitment of coloboma patients in the Genetics of Uveal Coloboma protocol (13-EI-0049) and the Whole Exome and Whole Genome Sequencing for Genotyping of Inherited and Congenital Eye Conditions protocol (14-EI-0064) continues. As of August 2023, we had enrolled approximately ___ participants (___ affected) from ___ families. Whenever possible, all affected individuals and their first degree family members have undergone a complete ophthalmic examination and blood draw for genetics. Affected individuals have undergone a battery of systemic testing looking for potential phenotypic associations. They have CLIA-certified sequencing of known developmental eye disease genes on an exome background followed by reflex full genome sequencing for negative reports. The protocol has been modified recently to include individuals with microphthalmia and anophthalmia--two phenotypes on the same continuum of developmental abnormalities. This change leverages our recently-funded U01 research effort with Drs. Philip Lupo and Laura Mitchell to perform population-based genetic epidemiology for microphthalmia/anophthalmia/coloboma phenotypes (MAC) as part of the Texas Birth Defects Registry. Recruitment numbers have included patients that have been ascertained through the MAGIC protocol. Our protocol "Potential Environmental Causes of Uveal Coloboma" (000366-EI) explores maternal factors and exposures during the first trimester of pregnancy as potential causes of uveal coloboma to correlate exposure data to clinical data from affected children. We have begun administering a questionnaire over the phone about parents' health, lifestyle and habits before and during pregnancy. The questionnaire is adapted from the National Birth Defects Prevention Study (NBDPS) Mother Questionnaire. Furthermore, the study will use existing data from NBDPS and NIH studies, including family data such as eye exam, genetic test results, and family history of coloboma. Enrollment has commenced. 2. Laboratory Studies A. The RICO mouse model of coloboma The RICO (Retinal & Iris COloboma) mouse arose from the random insertion of a transgene (NSE-VEGF) on chromosome 13 in the C57BL/6 background. Both homozygous and heterozygous mutants developed coloboma. . Long-read sequencing detected approximately fifteen copies of the transgene at the insertion site, an inversion, one duplications and a deletion in a gene desert on chromosome 13. We detect hVEGF in the developing eye. An open question is whether coloboma is caused by this abnormal expression of VEGF and/or disruption of gene expression caused by the genomic rearrangement. Creation of two additional transgenic lines with NSE-VEGF did not result in coloboma, although copy numbers were considerably lower; as such, a dosage-dependent effect cannot be ruled out. We are pursuing RNA-Seq experiments to identify changes in gene expression, particularly those surrounding the integration site. B. Coloboma candidate gene studies (Zfp703/Nlz1 and Zfp503/Nlz2) Using developmental gene profiling, we previously identified two zinc-finger motif-containing genes, Zfp703/Nlz1 and Zfp503/Nlz2, that are important in regulating optic fissure closure in zebrafish. Nlz2 KO mice were perinatally lethal and exhibited coloboma. Protein expression studies in mouse eye indicated that Nlz2 is expressed transiently during development in the retinal pigment epithelium (RPE) at the time around optic fissure closure, and in developing and adult amacrine and ganglion cells. Zfp503 knockout embryos display coloboma and hypopigmentation of the presumptive RPE (pRPE) at 100% penetrance. Around the time of OF closure, the pRPE becomes hyperplastic in a ventral-to-dorsal fashion and expresses VSX2 immunostaining, indication of a more neural-retina-like phenotype. We have characterized viable Zfp503+/- mice, showing congenital optic nerve excavation by OCT and histology. We completed the assessment of developmentally regulated ocular transcription factors, revealing down-regulation of MITF and OTX2 and up-regulation and/or anatomically expanded expression of PAX6, PAX2, VSX2 proteins, and Vax1 and Vax2 transcripts, particularly in the ventral, proximal pRPE, accompanied by an expansion of cell number. Zfp503-/- mice were never observed in live born litters, likely because of hypoplastic lung, and/or rib cage abnormalities. Gene expression profiling by RNA-Seq revealed significant downregulation of melanin pigment-related genes(e.g., Tyr, Dct, Slc45A2, Slc24a5, Gpnmb, Pmel, Gpr132, Mlana, Mlph) and RPE signature genes (<9.5x10-15, hypergeometric testing), consistent with a defect in RPE differentiation. A number of differentially expressed genes overlapped with those we previously identified by molecular profiling of OF closure (p<3.2x10-9, hypergeometric testing), including Strmn4, Insm1, Itgb8, Dcc, Tox3, Atoh7, Ascl1, Dkk3, Myb, Hes5, Fgf15, and Onecut1. Sequencing of a cohort of patients with uveal coloboma did not reveal convincing loss-of-function alleles. These data were published during the reporting period in IOVS. C. CRISPR screening for genes associated with optic fissure closure Using CRISPR/Cas9-mediated genome editing as a screening method, we generated KO zebrafish lines to investigate the roles of candidate genes from developmental gene profiling--combining data from our experiments as well as those from similar studies published since. We are pursuing validating several candidates that have coloboma using standard markers and rescue experiments. D. Downstream effectors of FAT1 Our collaborators and we have reported that biallelic mutations in FAT1 result in a syndromic form of uveal coloboma. We have since sought to identify potential downstream effectors of FAT1 in ocular development. Recent work has focused on the role of the Hippo signaling pathway and the RERE pathway. We have shown that the zebrafish rerea mutant (babyface) robustly recapitulates optic fissure closure defects resulting from loss of RERE function, as observed in humans with NEDBEH syndrome. Mutants exhibit expansion of proximal retinal optic stalk and reduced expression of some ventral retinal fate genes due to deregulated protein signaling. Using zebrafish and cell-based assays, we determined that NEDBEH-associated human RERE variants function as hypomorphs in their ability to repress shh signaling; some exhibit abnormal nuclear localization. Inhibiting shh signaling by the protein inhibitor HPI-1 rescues coloboma, confirming our observation that coloboma in rerea mutants is indeed due to deregulation of shh signaling. We have also confirmed that

1. 临床和基因组研究葡萄膜缺损遗传学方案 (13-EI-0049) 和遗传性和先天性眼部疾病基因分型的全外显子组和全基因组测序方案 (14-EI-0064) 继续招募缺损患者。截至 2023 年 8 月，我们已招募了来自 ___ 个家庭的大约 ___ 名参与者（___ 受影响）。只要有可能，所有受影响的个人及其一级家庭成员都接受了完整的眼科检查和抽血进行遗传学检查。受影响的个体已经接受了一系列系统测试，寻找潜在的表型关联。他们在外显子组背景上对已知的发育性眼病基因进行了 CLIA 认证的测序，然后针对阴性报告进行反射全基因组测序。该方案最近经过修改，纳入了患有小眼症和无眼症的个体——这两种表型属于发育异常的同一连续体。这一变化利用了我们最近资助的 U01 与 Drs. 的研究工作。菲利普·卢波 (Philip Lupo) 和劳拉·米切尔 (Laura Mitchell) 对小眼症/无眼症/缺损表型 (MAC) 进行基于人群的遗传流行病学研究，作为德克萨斯州出生缺陷登记的一部分。招募人数包括通过 MAGIC 方案确定的患者。我们的方案“葡萄膜缺损的潜在环境原因”(000366-EI) 探讨了妊娠前三个月的母体因素和暴露作为葡萄膜缺损的潜在原因，将暴露数据与受影响儿童的临床数据相关联。我们已经开始通过电话进行问卷调查，了解父母怀孕前和怀孕期间的健康、生活方式和习惯。该问卷改编自国家出生缺陷预防研究 (NBDPS) 母亲问卷。此外，该研究将使用 NBDPS 和 NIH 研究的现有数据，包括眼科检查、基因检测结果和缺损家族史等家庭数据。报名已开始。 2. 实验室研究 A. RICO 缺损小鼠模型 RICO（视网膜和虹膜 COloboma）小鼠源自 C57BL/6 背景下 13 号染色体上随机插入的转基因 (NSE-VEGF)。纯合子和杂合子突变体都会出现缺损。。长读长测序在插入位点检测到大约 15 个转基因拷贝、一个倒位、一个重复以及 13 号染色体上基因荒漠中的一个缺失。我们在发育中的眼睛中检测到 hVEGF。一个悬而未决的问题是缺损是否是由 VEGF 的异常表达和/或基因组重排引起的基因表达破坏引起的。用 NSE-VEGF 创建另外两个转基因系并没有导致缺损，尽管拷贝数显着降低；因此，不能排除剂量依赖性效应。我们正在进行 RNA 测序实验来识别基因表达的变化，特别是整合位点周围的变化。 B. 缺损候选基因研究（Zfp703/Nlz1 和 Zfp503/Nlz2）通过发育基因分析，我们之前鉴定了两个含有锌指基序的基因，Zfp703/Nlz1 和 Zfp503/Nlz2，它们对于调节斑马鱼视裂闭合很重要。 Nlz2 KO 小鼠围产期致死并表现出缺损。小鼠眼中的蛋白质表达研究表明，Nlz2 在视裂闭合前后的视网膜色素上皮 (RPE) 发育过程中以及在发育中和成年的无长突细胞和神经节细胞中短暂表达。 Zfp503 敲除胚胎在 100% 外显率下显示出缺损和假定 RPE (pRPE) 色素沉着不足。在 OF 闭合时，pRPE 以从腹侧到背侧的方式增生，并表达 VSX2 免疫染色，表明更类似于神经视网膜的表型。我们对活的 Zfp503+/- 小鼠进行了表征，通过 OCT 和组织学显示先天性视神经凹陷。我们完成了对发育调节眼部转录因子的评估，揭示了 MITF 和 OTX2 的下调以及 PAX6、PAX2、VSX2 蛋白以及 Vax1 和 Vax2 转录本的上调和/或解剖学扩展表达，特别是在腹侧、近端 pRPE 中，伴随着细胞数量的增加。在活产仔猪中从未观察到 Zfp503-/- 小鼠，可能是由于肺发育不良和/或胸腔异常。 RNA-Seq 基因表达谱显示黑色素相关基因（例如 Tyr、Dct、Slc45A2、Slc24a5、Gpnmb、Pmel、Gpr132、Mlana、Mlph）和 RPE 特征基因（<9.5x10-15，超几何测试）显着下调），与 RPE 分化的缺陷一致。许多差异表达基因与我们之前通过 OF 闭合的分子分析鉴定的基因重叠（p<3.2x10-9，超几何测试），包括 Strmn4、Insm1、Itgb8、Dcc、Tox3、Atoh7、Ascl1、Dkk3、Myb、Hes5 、Fgf15 和 Onecut1。对葡萄膜缺损患者队列的测序未发现令人信服的功能丧失等位基因。这些数据已在报告期内发布在 IOVS 上。 C. CRISPR 筛选与视裂闭合相关的基因使用 CRISPR/Cas9 介导的基因组编辑作为筛选方法，我们生成了 KO 斑马鱼品系，以研究发育基因分析中候选基因的作用——结合我们的实验数据以及此后发表的类似研究的数据。我们正在利用标准标记和救援实验来验证几名患有缺损的候选人。 D. FAT1 的下游效应器我们的合作者和我们已经报道，FAT1 的双等位基因突变会导致葡萄膜缺损综合征。此后，我们一直致力于寻找 FAT1 在眼部发育中的潜在下游效应子。最近的工作重点是 Hippo 信号通路和 RERE 通路的作用。我们已经证明，斑马鱼 rerea 突变体（babyface）能够稳健地再现因 RERE 功能丧失而导致的视裂闭合缺陷，正如在患有 NEDBEH 综合征的人类中观察到的那样。由于蛋白质信号传导失调，突变体表现出近端视网膜视柄的扩张和一些腹侧视网膜命运基因的表达减少。使用斑马鱼和基于细胞的检测，我们确定 NEDBEH 相关的人类 RERE 变体在抑制 shh 信号传导的能力方面发挥着亚型的作用；有些表现出异常的核定位。通过蛋白质抑制剂 HPI-1 抑制 shh 信号传导可以挽救缺损，这证实了我们的观察结果，即 rerea 突变体中的缺损确实是由于 shh 信号传导失调所致。我们还确认了

项目成果

期刊论文数量（21）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ocular and Systemic Findings in Adults with Uveal Coloboma.

患有葡萄膜缺损的成人的眼部和全身检查结果。

DOI：
发表时间：
2020
期刊：
影响因子：
13.7
作者：
Daich Varela, Malena;Huryn, Laryssa A;Hufnagel, Robert B;Zein, Wadih M;Blain, Delphine;Brooks, Brian P
通讯作者：
Brooks, Brian P

De novo frameshift mutation in YAP1 associated with bilateral uveal coloboma and microphthalmia.

YAP1 的从头移码突变与双侧葡萄膜缺损和小眼症相关。

DOI：
发表时间：
2022
期刊：
影响因子：
1.2
作者：
DeYoung, Charles;Guan, Bin;Ullah, Ehsan;Blain, Delphine;Hufnagel, Robert B;Brooks, Brian P
通讯作者：
Brooks, Brian P

Zebrafish model of RERE syndrome recapitulates key ophthalmic defects that are rescued by small molecule inhibitor of shh signaling.

RERE 综合征的斑马鱼模型概括了通过 shh 信号小分子抑制剂可挽救的关键眼部缺陷。

DOI：
发表时间：
2023-04
期刊：
影响因子：
0
作者：
George, Aman;Lee, Jerry;Liu, James;Kim, Suzie;Brooks, Brian P
通讯作者：
Brooks, Brian P

Detailed analysis of chick optic fissure closure reveals Netrin-1 as an essential mediator of epithelial fusion.

对鸡视神经裂闭合的详细分析表明 Netrin-1 是上皮融合的重要介质。

DOI：
发表时间：
2019
期刊：
影响因子：
7.7
作者：
Hardy, Holly;Prendergast, James Gd;Patel, Aara;Dutta, Sunit;Trejo;Kroeger, Hannah;Yung, Andrea R;Goodrich, Lisa V;Brooks, Brian;Sowden, Jane C;Rainger, Joe
通讯作者：
Rainger, Joe

eyeGENE(R): a novel approach to combine clinical testing and researching genetic ocular disease.

eyeGENE(R)：一种将临床测试和研究遗传性眼病相结合的新方法。