The Genetics of Uveal Coloboma

葡萄膜缺损的遗传学

• 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
• 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; 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

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 在紫veal卵形遗传学方案（13-EI-0049）中募集了古罗巴马患者以及整个外显子组和整个基因组测序，用于遗传和先天性眼睛条件方案的基因分型（14-EI-0064）。截至2023年8月，我们已经从___家庭中注册了大约___参与者（受影响的___）。 只要有可能，所有受影响的个人及其一级家庭成员都对遗传学进行了完整的眼科检查和抽血。 受影响的个体已经进行了一系列全身测试，以寻求潜在的表型关联。 他们在外显型背景上对已知发育眼病基因的CLIA认证测序进行了反射完整的基因组测序，以进行阴性报告。 该方案最近已被修改，以包括具有微观性心脏病和性质的个体 - 两种表型在相同的发育异常连续性上。 这一变化利用了我们最近资助的U01研究工作的DRS。菲利普·卢波（Philip Lupo）和劳拉·米切尔（Laura Mitchell）作为德克萨斯州出生缺陷注册中心的一部分，对微观恐怖/性疾病/古罗巴马表型（MAC）进行基于人群的遗传流行病学。 招聘人数包括通过魔术方案确定的患者。 我们的方案“紫veal卵形的潜在环境原因”（000366-EI）探讨了妊娠头三个月的母体因素和暴露，这是卵巢古罗巴马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向问卷调查，以了解父母在怀孕之前和期间的健康，生活方式和习惯。调查表是根据《国家先天缺陷预防研究》（NBDPS）的母亲问卷调查的。此外，该研究将使用来自NBDP和NIH研究的现有数据，包括眼科检查，基因测试结果和古罗伯氏菌家族史。入学开始了。 2。实验室研究 A. coloboma的RICO小鼠模型 RICO（视网膜和虹膜coloboma）小鼠来自C57BL/6背景中的转基因（NSE-VEGF）在13染色体上的随机插入。纯合子和杂合突变体都形成了造型。 。长阅读的测序在插入位点检测到了大约15份转基因副本，一个反转，一种重复和在13号染色体上的基因沙漠中的删除。我们在发育中的眼中检测到HVEGF。 一个开放的问题是，coloboma是否是由VEGF的异常表达和/或由基因组重排引起的基因表达的异常引起的。 尽管拷贝数要低得多，但使用NSE-VEGF创建另外两条转基因线并未导致造成山地。因此，不能排除剂量依赖性效果。 我们正在追求RNA-seq实验，以识别基因表达的变化，尤其是围绕整合位点的变化。 B. Coloboma候选基因研究（ZFP703/NLZ1和ZFP503/NLZ2） 使用发育基因分析，我们先前鉴定了两个含锌指基序的基因ZFP703/NLZ1和ZFP503/NLZ2，这些基因对于调节Zebrafish的视觉裂隙闭合非常重要。 NLZ2 KO小鼠是致命的，并表现出哥洛巴马。小鼠眼中的蛋白质表达研究表明，NLZ2在视网膜色素上皮（RPE）的发育过程中瞬时表达，并在视觉裂缝闭合以及发育和成人的无链氨酸和神经节细胞中表达。 ZFP503敲除胚胎以100％的渗透率显示coloboma和推定RPE（PRPE）的不形成。 在闭合时期，PRPE以腹侧到外侧的方式变得更增生，并表达VSX2免疫染色，这表明更神经逆转的表型。 我们已经表征了可行的ZFP503 +/-小鼠，显示了OCT和组织学的先天视神经发掘。 我们完成了对开发调节的眼镜转录因子的评估，揭示了MITF和OTX2的下调以及上调和/或解剖学扩展的PAX6，PAX2，PAX2，VSX2蛋白，VAX1和VAX2蛋白质，VAX1和VAX2转录本，尤其是在腹侧，近端PRPE中，伴随着细胞数量的扩展。 ZFP503 - / - 小鼠从未在活出生的垃圾中观察到，这可能是由于肺动脉症不良和/或肋骨笼子异常。通过RNA-SEQ进行基因表达分析表明，黑色素色素相关基因的显着下调（例如，Tyr，DCT，SLC45A2，SLC24A5，GPNMB，PMEL，PMEL，GPR132，MLANA，MLANA，MLPH）和RPE签名基因（<9.5x10-15-15，超级测试）。 许多差异表达的基因与我们先前通过闭合分子分析（P <3.2x10-9，超几何测试）重叠的基因，包括Strmn4，Insm1，Insm1，ItGB8，DCC，DCC，TOX3，TOX3，ATOH7，ATOH7，ASCL1，ASCL1，ASCL1，ASCL1，DKK3，DKK3，MYB，MYB，MYB，MYB，MYB，MYB，MYB，FGF15和ONECUT1和ONECUT1和ONECUT1和ONECUT1和ONECUT1和ONECUT1和ONECUT1和ONECUT1。一组紫veal骨coloboma患者的测序并未揭示令人信服的功能丧失等位基因。 这些数据在报告期间在IOV中发布。 C. CRISPR筛选与闭合闭合相关的基因 使用CRISPR/CAS9介导的基因组编辑作为筛查方法，我们生成了KO斑马鱼线来研究从发育基因分析中的候选基因的作用 - 从我们的实验以及此后发表的类似研究的数据中综合数据。 我们正在追求验证几个使用标准标记和救援实验的coloboma的候选者。 D. FAT1的下游效应器 我们的合作者，我们已经报道了FAT1中的双重突变导致一种综合征的紫veal骨coloboma。 此后，我们试图确定FAT1在眼部发育中的潜在下游效应因子。 最近的工作集中在河马信号通路和RERE途径的作用上。 我们已经表明，斑马鱼重生突变体（babyface）可靠地概括了由于nedbeh综合征的人类所观察到的，导致RERE功能丧失而导致的视膜闭合缺陷。突变体表现出由于蛋白质信号传导而导致的近端视网膜视觉茎的扩张和一些腹侧视网膜命运基因的表达降低。使用斑马鱼和基于细胞的测定法，我们确定与NEDBEH相关的人类Rere变体在抑制SHH信号传导的能力方面起着降压性的作用。一些表现出异常的核定位。蛋白质抑制剂HPI-1抑制SHH信号传导挽救了造型果实，这证实了我们的观察结果，即重生突变体中的coloboma确实是由于对SHH信号传导的放松调节所致。 我们还确认