Homeotic hotspot in the human genome for eye and brain disease

人类眼部和脑部疾病基因组中的同源异型热点

• 批准号: 10416324
• 负责人: Thomas M. Glaser
• 金额: $ 39.26万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10416324
• 关键词: 3-Dimensional; 9q21; Affect; Alleles; Anatomy; Anophthalmos; Anterior; Apoptosis; Architecture; Bilateral; Biological Assay; Blindness; Brain; Brain Diseases; CRISPR/Cas technology; Cell Differentiation process; Cells; Cerebellar Ataxia; Cerebellum; Childhood; Choroid Plexus Epithelium; Chromatin; Chromosome Fragile Sites; Chromosomes; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coloboma; Constitutional; Cornea; Cre driver; DNA; DNA Binding; Defect; Development; Disease; Drosophila genus; Ectoderm; Electroporation; Embryonic Development; Enhancers; Epithelial; Evolution; Exhibits; Exons; Eye; Eye diseases; Female; Genetic Transcription; Genome; Gonadal structure; Heterogeneity; Hi-C; Human; Human Genetics; Human Genome; In Vitro; Inheritance Patterns; Iris; Kinetics; Lens Placodes; Life; Light; Link; Meiosis; Methods; Microphthalmos; Modeling; Molecular; Mosaicism; Mus; Mutation; Neural tube; Nucleotides; Optic vesicle; Organ; Pathogenesis; Pathogenicity; Patients; Pattern; Phenotype; Pigments; Pluripotent Stem Cells; Positioning Attribute; Prevalence; Purkinje Cells; Retina; Retinal Photoreceptors; Retinitis Pigmentosa; Rod; Running; Series; Signal Transduction; Site; Specific qualifier value; Specificity; Spinocerebellar Ataxias; Structure of retinal pigment epithelium; Surface; Syndrome; System; Techniques; Testing; Testis; Tissues; Transcriptional Regulation; Transgenes; Visual Fields; X Inactivation; arm; brain malformation; cell type; embryo tissue; gain of function; histogenesis; in utero; induced pluripotent stem cell; male; malformation; mouse model; nerve stem cell; novel; optic cup; photoreceptor degeneration; prenatal; proband; promoter; relating to nervous system; retinal neuron; sex; sex determination; single-cell RNA sequencing; stem cells; transcription factor

PROJECT SUMMARY Classical homeotic mutations (e.g. Drosophila Ubx) disrupt embryonic development, transforming one tissue type into another. We have discovered the molecular basis of four human X-linked disorders affecting the eye or brain – BASR syndrome, foveal dysgenesis, retinitis pigmentosa and spinocerebellar ataxia, which have a homeotic or degenerative basis. Each disorder is caused by insertion of a large autosomal DNA segment at the same Xq27 palindromic site near SOX3, which encodes a potent trans- cription factor homologous to SRY (testis determinant). The Xq insertions are predicted to disrupt chromatin architecture, activating SOX3 ectopically in tissues defined by newly juxtaposed enhancers, and altering cell fate (homeosis) via a gain-of-function (GOF). We propose that SOX3 changes retinal pigment epithelia (RPE) into neuroretina in BASR, reprograms cerebellar Purkinje cells in SCAX5, and triggers photoreceptor degeneration in RP24. Using a novel palinsert PCR assay, we defined the breakpoints and candidate enhancers for each insertion. We also identified >10 further Xq27 disorders affecting the eye, brain or other organs – including unsolved cases with a likely similar mechanism. We will define new Xq27 palindrome insertions and test our hypothesis for disease pathogenesis at chromatin and developmental levels, using [1] patient-derived iPSCs, 3D chromatin interaction assays (Hi-C), in vitro differentiation, serial scRNA-seq profiles; and [2] (homol informative mouse transgenes, including the binary CRISPR/Cas9 Hprt HoP-In ogy promoted integration) GOF system we pioneered – with Sox3HA expression activated in RPE, rods, Purkinje cells, or other tissues via established Cre drivers, in a constitutional (XY) or mosaic (XX) pattern, and in a sustained or Dox-inducible manner.

项目概要 经典的同源突变（例如果蝇 Ubx）会破坏胚胎发育， 将一种组织类型转变为另一种组织类型。我们发现了分子基础 影响眼睛或大脑的四种人类 X 连锁疾病 – BASR 综合征、黄斑中心凹 发育不全、色素性视网膜炎和脊髓小脑性共济失调，具有同源异型或 退化的基础。每种疾病都是由插入大量常染色体 DNA 引起的 SOX3 附近同一 Xq27 回文位点的片段，编码一个有效的反式 与SRY（睾丸决定因子）同源的转录因子。预测 Xq 插入 破坏染色质结构，在新定义的组织中异位激活 SOX3 并置的增强子，并通过功能获得（GOF）改变细胞命运（同源异质）。我们 提出 SOX3 将 BASR 中的视网膜色素上皮 (RPE) 转变为神经视网膜， 在 SCAX5 中重新编程小脑浦肯野细胞，并引发光感受器变性 在RP24中。使用新型 palinsert PCR 检测，我们定义了断点和候选点 每个插入的增强子。我们还发现了超过 10 种进一步影响 Xq27 的疾病 眼睛、大脑或其他器官——包括可能具有类似机制的未解决案例。 我们将定义新的 Xq27 回文插入并测试我们的疾病假设 使用 [1] 患者来源的 iPSC 在染色质和发育水平上研究发病机制， 3D 染色质相互作用测定 (Hi-C)、体外分化、连续 scRNA-seq 谱； 和[2] （霍莫尔 信息丰富的小鼠转基因，包括二元 CRISPR/Cas9 Hprt HoP-In ogy 促进整合）我们首创的 GOF 系统 – 具有 Sox3HA 表达 通过已建立的 Cre 驱动程序在 RPE、视杆细胞、浦肯野细胞或其他组织中激活 组成 (XY) 或镶嵌 (XX) 模式，并且以持续或 Dox 诱导的方式。