Role of a dual splicing and amino acid code in myopia, cone dysfunction and cone dystrophy associated with L/M opsin interchange mutations

双剪接和氨基酸密码在与 L/M 视蛋白互换突变相关的近视、视锥细胞功能障碍和视锥细胞营养不良中的作用

• 批准号: 9893919
• 负责人: MAUREEN E NEITZ
• 金额: $ 44.13万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893919
• 关键词: Adolescent; Alleles; Amino Acids; Area; Benign; Binding; Biochemical; Biological Assay; Blindness; Catalogs; Cell Culture Techniques; Code; Color Visions; Complex; Cone; Cone dystrophy; Data; Defect; Disease; Exclusion; Exhibits; Exons; Eye; Eye diseases; Feedback; Frequencies; Functional disorder; Genes; Genetic Polymorphism; Genetic Recombination; Genetic Variation; Goals; Growth; Haplotypes; Human; Individual; Lead; Length; Light; Link; MW opsin; Measures; Messenger RNA; Modernization; Molecular Biology; Mutation; Myopia; Nucleotides; Opsin; Outcome; Pathology; Patients; Phenotype; Photoreceptors; Plague; Play; Population; Positioning Attribute; Protein Isoforms; Proteins; RNA Splicing; RNA, Messenger, Splicing; Red-Green Color Blindness; Refractive Errors; Research; Retina; Retinal Cone; Retinal Degeneration; Risk; Risk Factors; Rod; Role; Severities; Single Nucleotide Polymorphism; Spliced Genes; Technology; Therapeutic; Time; Transcript; U1 small nuclear RNA; Uncertainty; Variant; Vision; Vision Disorders; Visual impairment; Visual system structure; Work; base; blue cone monochromacy; clinical Diagnosis; clinical phenotype; emmetropization; exon skipping; family genetics; genetic variant; genome editing; insight; protein function; risk variant; visual feedback

The long-term objective of this research is to understand the mechanism by which intermixing of the human long- (L) and middle- (M) wavelength cone photopigment genes give rise to variants that cause aberrant pre- messenger RNA (mRNA) splicing, and lead to vision loss with a diverse set of clinical phenotypes. 95% of our cones are L or M cones, and except at very low light levels when rods are active, all vision is based on cones. The L and M cones play a critical role in the visually guided feedback mechanism responsible for controlling eye growth (emmetropization). Every aspect of seeing, including high acuity and color vision, depends on the L and M cone photopigments and these genes are important risk factors in common eye disorders that plague modern humans. The L and M (LM) cone photopigment genes, designated OPN1LW and OPN1MW, respectively, exhibit high haplotype diversity in exons 2, 3 and 4. The most is known about two variants, designated LIAVA and LVAVA that are associated with photoreceptor dysfunction and severe vision impairment. They are found in patients with a range of clinical diagnoses including high grade myopia, blue cone monochromacy and cone dystrophy. We and others have recently shown that combinations of single nucleotide polymorphisms (SNPs) associated with these variants cause aberrant pre-mRNA splicing. Our preliminary data show that different combinations of the exon 3 polymorphisms shift the ratio of full length to exon 3 skipped mRNA, producing variability in the severity of the splicing defect that will be extremely useful in elucidating the fundamental mechanisms controlling splicing of this exon. Preliminary data obtained using a cell culture-based splicing assay also suggests that haplotypes of exon 3 that yield moderate levels of exon 3 skipping are associated with an average -1.3 diopters of refractive error compared to low/non-skipping variants. The pathophysiology of the different mutations is complex because superimposed on the effects of subnormal amounts of opsin protein produced by the splicing defects, are the effects of some combinations of the amino acids on protein function. To achieve our goal we propose: Aim 1: To investigate the role of combinations of SNPs in exons 2, 3 and 4 of the L and M opsin genes in splicing by 1.1 fully enumerating the transcript isoforms, and measuring their relative abundances. 1.2 investigating and quantitating the effects of exon 3 haplotypes on splicing in cone photoreceptors and particularly those haplotypes that are risk alleles for juvenile onset myopia. Aim 2: To investigate the mechanism of exon 3 skipping using biochemical and molecular biology approaches Aim 3: To evaluate the potential for exon specific U1 snRNAs to rescue the exon 3 skipping phenotype.

这项研究的长期目标是了解人类混合的机制 长（L）和中（M）波长锥体感光色素基因产生变异，导致异常的前- 信使 RNA (mRNA) 剪接，并导致具有多种临床表型的视力丧失。我们的 95% 视锥细胞是 L 或 M 视锥细胞，除了视杆细胞处于活动状态的极低光照水平外，所有视觉都基于视锥细胞。 L 和 M 视锥细胞在负责控制的视觉引导反馈机制中发挥着关键作用 眼睛生长（正视化）。视觉的各个方面，包括高敏锐度和色觉，都取决于 L 和 M 视锥细胞感光色素，这些基因是困扰常见眼部疾病的重要危险因素 现代人类。 L 和 M (LM) 视锥感光色素基因，指定为 OPN1LW 和 OPN1MW， 分别在外显子 2、3 和 4 中表现出高单倍型多样性。最了解的是两个变体， 指定为 LIAVA 和 LVAVA 与光感受器功能障碍和严重视力相关 损害。它们存在于患有一系列临床诊断的患者中，包括高度近视、蓝色 视锥细胞单色性和视锥细胞营养不良。我们和其他人最近表明，单一的组合 与这些变异相关的核苷酸多态性 (SNP) 会导致异常的前 mRNA 剪接。我们的 初步数据表明，外显子 3 多态性的不同组合会改变全长与 外显子 3 跳过 mRNA，产生剪接缺陷严重程度的变异性，这在 阐明控制该外显子剪接的基本机制。使用获得的初步数据 基于细胞培养的剪接测定还表明，外显子 3 的单倍型产生中等水平的外显子 3 与低/非跳视相比，跳视与平均 -1.3 屈光度的屈光不正相关 变体。不同突变的病理生理学很复杂，因为叠加了 剪接缺陷产生的视蛋白数量低于正常水平，是某些组合的影响 氨基酸对蛋白质功能的影响。为了实现我们的目标，我们建议： 目标 1：研究 L 和 M 视蛋白基因外显子 2、3 和 4 中 SNP 组合的作用 拼接通过 1.1 全面计数转录本亚型，并测量它们的相对丰度。 1.2 研究并定量外显子3单倍型对视锥细胞剪接的影响 特别是那些作为青少年发病近视风险等位基因的单倍型。 目标 2：利用生化和分子生物学方法研究外显子 3 跳跃的机制 目标 3：评估外显子特异性 U1 snRNA 拯救外显子 3 跳跃表型的潜力。