The Genetics of Uveal Coloboma

葡萄膜缺损的遗传学

• 批准号: 10706111
• 负责人: Brian Brooks
• 金额: $ 311.91万
• 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/10706111
• 关键词: ATOH7 gene; Adult; Affect; Alleles; Amacrine Cells; Anatomy; Anophthalmos; Architecture; Backcrossings; Blood; Brain; C57BL/6 Mouse; CLIA certified sequencing; COVID-19; COVID-19 pandemic; CRISPR interference; CRISPR screen; CRISPR/Cas technology; Candidate Disease Gene; Cell Count; Chick; Child; Chromosome 13; Clinical; Clinical Data; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Coloboma; Congenital Abnormality; DNA Sequence Rearrangement; Data; Defect; Development; Developmental Process; Dorsal; Down-Regulation; Embryo; Embryology; Enrollment; Epithelial; Exhibits; Eye; Eye Development; Eye diseases; Failure; Family; Family history of; Family member; First Pregnancy Trimester; Fishes; Fissural; Funding; Future; Gene Expression Profiling; Gene Mutation; Gene Targeting; Generations; Genes; Genetic; Genetic Counseling; Genomics; Genotype; Goals; Habits; Health; Histology; Human; Human Resources; Hyperplasia; Hypopigmentation; Image; Individual; Inherited; Institutional Review Boards; Investigation; Iris; Junk DNA; Knock-out; Knockout Mice; Knowledge; Laboratories; Laboratory Study; Lead; Life Style; Light; Lung; MLPH gene; Manuscripts; Mediating; Medical Genetics; Melanins; Methods; Microphthalmos; Molecular; Molecular Diagnostic Testing; Molecular Profiling; Mothers; Mus; Mutation; Neural Retina; Optic Nerve; Optics; Overlapping Genes; Parents; Participant; Pathology; Patient Care; Patients; Penetrance; Perinatal mortality demographics; Phenotype; Pigments; Pregnancy; Prevention; Proteins; Proteomics; Protocols documentation; Questionnaires; Reflex action; Registries; Regulation; Reporting; Research; Retina; Role; Severities; Site; Structure of retinal pigment epithelium; Surface; Techniques; Telephone; Test Result; Testing; Texas; Time; Transcript; Transgenes; Transgenic Mice; United States National Institutes of Health; Up-Regulation; Variant; Vascular Endothelial Growth Factors; Work; Workplace; Zebrafish; Zinc Fingers; axon guidance; base; cell motility; clinical care; cohort; differential expression; exome; exome sequencing; ganglion cell; genetic disorder diagnosis; genetic epidemiology; genetic signature; genetic testing; genome editing; genome sequencing; glycoprotein NMB; imaging system; knock-down; loss of function; mRNA Expression; malformation; mouse model; mutant; ophthalmic examination; overexpression; population based; protein expression; receptor; recruit; rib bone structure; screening; transcription factor; transcriptome sequencing; transcriptomics; 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 after the slow down imposed by the Covid-19 pandemic. As of February 2022, we had enrolled 245 participants (85 affected) from 79 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 followed by reflex full exome 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. We received IRB approval for a new protocol "Potential Environmental Causes of Uveal Coloboma" (000366-EI). The aim is to explore maternal factors and exposures during the first trimester of pregnancy as potential causes of uveal coloboma and to correlate exposure data to clinical data from affected children. This study focuses on the mothers of children with coloboma. They will be administered a questionnaire over the phone about their 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. 2. Laboratory Studies Work in the laboratory has suffered a major slowing down because of Covid-19 and the limitations in personnel allowed to the physical workplace. 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 and the homozygous transgene insertion was lethal. The genomic organization at the insertion site included approximately 30 copies of the transgene, an inversion, three duplications and a deletion in a gene desert. We are now using long-read sequencing to better evaluate this genomic architecture. We detected transient VEGF mRNA and protein expression during eye and brain development. Upon outcrossing of the RICO mouse from the C57BL/6 to the 129/SvJ background, the homozygous mutant survived to adulthood and the severity of the coloboma phenotype persisted, albeit with reduced penetrance in the heterozygous mice. Since the phenotype was maintained across a different background, it remained to be tested whether it was a direct consequence of VEGF overexpression or of genomic rearrangements at the site of transgene insertion. To this end, we generated additional transgenic mouse lines (NSE-VEGF) using the same construct as in the RICO mouse. Three lines showed integration at different chromosomal sites with various degrees of VEGF transcript expression. Mice backcrossed to the C57BL/6 background have been examined and do not have coloboma. However, the total number of copies of the NSE-VEGF transgene incorporated are low compared to parent RICO line, making interpretation of a negative result difficult. B. Coloboma candidate gene studies (Zfp703/Nlz1 and Zfp503/Nlz2) We identified two zinc-finger motif-containing genes, Zfp703/Nlz1 and Zfp503/Nlz2, that are important in regulating optic fissure closure in zebrafish (Brown et al., Proc Natl Acad Sci U S A. 2009). Both Nlz1 knockout (KO) and 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. Since the time of the last BSC, 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)37, 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, 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 Brown et al., Proc Natl Acad Sci U S A. 2009, in the ontogenesis of coloboma. Out of 84 genes targeted with CRISPR, 17 displayed coloboma in the F1 progenies with underlying compound heterozygous/homozygous variants, although numbers were small in many cases and will need further confirmation. Of these, we have studied netrin1 (ntn1)encoding a protein associated with axon guidance regulation of cell migration and epithelial plasticitymost extensively, showing that mutation/knockdown consistently produced a coloboma phenotype; the gene coding for a Ntn1 receptor, Dcc, was also identified in ourscreen. Notably, ntn1 was similarly identified in an RNA-seq-based screen in chick OF closure and our data were incorporated into a collaborative manuscript. The CRISPR screen has been hampered by several factors: 1) systemic pathology caused by some induced gene mutations and 2) loss of mutant alleles with outcrossing of mutation-carrying F0 fish to wild-type (WT) fish; leading us to readdress this project using a CRISPRi strategy. Because the numbers of fish with coloboma in the F1 generation were low, we did not pursue transcriptomics or proteomics at present, but feel that such techniques will be useful in our future studies. In light of recent progress by others with live imaging of OF closure, we have temporarily de-emphasized this line of research*. We have engaged Dr. Kristen Kwan, who has developed an elegant live imaging system to look at early eye morphogenesis and OF closure, and we intend to forge collaborations around the investigation of specific mutants.

1。临床和基因组研究 在卵子古罗巴马方案（13-EI-0049）的遗传学中募集了古罗巴马患者，以及在遗传和先天性眼睛条件方案（14-EI-0064）基因分型的整个外显子组和整个基因组测序中，在COVID-19 PANDECOGY施加的慢速后继续进行。截至2022年2月，我们已经招募了来自79个家庭的245名参与者（受影响的85名参与者）。 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。 他们对已知发育眼病基因的CLIA认证测序进行了反射外显子组测序，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。 我们获得了IRB的批准，该方案是“紫veal山地山的潜在环境原因”（000366-EI）。目的是探索妊娠前期三个月的产妇因素和暴露，作为紫veal山山地的潜在原因，并将暴露数据与受影响儿童的临床数据相关联。这项研究的重点是儿童造成儿童的母亲。他们将通过电话向问卷调查，以了解他们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。 2。实验室研究 由于19号，实验室的工作遭受了重大减慢，并且人员限制到了身体工作场所。 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均出现山骨，纯合转基因插入都是致命的。插入部位的基因组组织包括大约30份转基因副本，倒置，三个重复和基因沙漠中的删除。现在，我们正在使用长阅读测序来更好地评估这种基因组体系结构。 我们在眼睛和脑发育过程中检测到瞬时VEGF mRNA和蛋白质表达。 从C57BL/6到129/SVJ背景的RICO小鼠过头后，纯合突变体存活到成年，而古罗映表型的严重程度持续存在，尽管在杂合小鼠中的渗透率降低。由于表型在不同的背景上保持不变，因此仍有待测试是在转基因插入部位的VEGF过表达或基因组重排的直接结果。为此，我们使用与RICO鼠标相同的结构生成了其他转基因鼠标线（NSE-VEGF）。三条线在不同程度的VEGF转录本表达的不同染色体位点显示了整合。已经检查了回到C57BL/6背景的小鼠已经进行了检查，并且没有造成山状的小鼠。 但是，与母体RICO线相比，NSE-VEGF转基因掺入的副本总数很低，因此难以解释负面结果。 B. Coloboma候选基因研究（ZFP703/NLZ1和ZFP503/NLZ2） 我们确定了两个含锌指基序的基因ZFP703/NLZ1和ZFP503/NLZ2，这些基因在调节斑马鱼中的视觉裂隙闭合非常重要（Brown等，Proc Natl Acad Acad Sci u s A.2009）。 NLZ1基因敲除（KO）和NLZ2 KO小鼠均致命致死，并表现出coloboma。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。 自上次BSC的时间以来，我们已经表征了可行的ZFP503 +/-小鼠，显示了OCT和组织学通过OCT和组织学进行了先天性视神经挖掘。 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。 RNA-SEQ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 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， 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，研究了Brown等人，Proc Natl Acad Sci u S A. 2009年在Coloboma的成生中的候选基因的作用。在以CRISPR为目标的84个基因中，有17个在F1后代中显示出具有基本化合物杂合/纯合型变体的Coloboma，尽管在许多情况下数量很小，并且需要进一步确认。 其中，我们研究了Netrin1（NTN1），该蛋白质与轴突指导调节有关细胞迁移和上皮可塑性最广泛的蛋白质，表明突变/敲低始终产生了造型的表型。在我们的屏幕上也发现了编码NTN1受体DCC的基因。 值得注意的是，NTN1在闭合小鸡的基于RNA-Seq的屏幕上类似地鉴定出来，我们的数据被纳入了协作手稿中。 CRISPR筛查受到了几个因素的阻碍：1）由于某些诱导的基因突变引起的系统病理和2）突变等位基因的丧失，突变携带F0 FISH到野生型（WT）鱼的脱落；带领我们使用CRISPRI策略来读取该项目。 由于F1一代中具有山糖果的鱼类数量很少，因此我们目前没有追求转录组学或蛋白质组学，而是认为这种技术将在我们未来的研究中有用。 鉴于其他人的最新进展以及封闭的实时成像，我们暂时取消了这一研究*。 我们聘请了克里斯汀·夸（Kristen Kwan）博士，他开发了一种优雅的现场成像系统来研究早期的形态发生和闭合，我们打算围绕调查特定突变体的调查进行合作。