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

这项研究的长期目标是了解人类混合的机制 长波长(L)和中波长(M)锥体光调色基因产生变异，导致异常的前 信使RNA(MRNA)剪接，并导致视力丧失与一套不同的临床表型。我们95%的员工 视锥是L视锥或M视锥，除了当视杆被激活时在非常低的光线下，所有的视觉都建立在视锥的基础上。 L视锥细胞和M视锥细胞在负责控制的视觉引导反馈机制中起着关键作用 眼球生长(正视化)。视力的每一个方面，包括高敏锐度和色觉，都取决于L 和M CONE感光色素和这些基因是困扰常见眼病的重要危险因素 现代人。L和M(Lm)锥体光色基因，分别命名为OPN1LW和OPN1MW， 分别在外显子2、3和4上表现出高度的单倍型多样性。最已知的是两个变体， 与光感受器功能障碍和严重视力相关的Liava和LVAVA 减损。它们在有一系列临床诊断的患者中发现，包括高度近视、蓝色 视锥细胞单色和视锥细胞营养不良。我们和其他人最近已经证明，单一的组合 与这些变异相关的核苷酸多态(SNPs)会导致异常的前mRNA剪接。我们的 初步数据显示，外显子3多态的不同组合将全长比率转移到 外显子3跳过了mRNA，产生了剪接缺陷严重程度的可变性，这将在 阐明控制该外显子剪接的基本机制。获取的初步数据 基于细胞培养的剪接分析也表明，产生中等水平外显子3的外显子3的单倍型 与低/非跳过相比，跳过与平均-1.3屈光度相关 变种。不同突变的病理生理学是复杂的，因为叠加在 剪接缺陷产生的视蛋白含量低于正常值，这是一些组合的影响 氨基酸对蛋白质功能的影响。为了实现我们的目标，我们提出： 目的1：探讨L和M-视蛋白基因外显子2、3和4的SNPs组合在骨质疏松症发病中的作用。 拼接方式 1.1充分计数转录本异构体，并测量其相对丰度。 1.2研究和定量外显子3单倍型对视锥细胞感光细胞剪接的影响 尤其是那些单倍型，这些单倍型是青少年发作性近视的危险等位基因。 目的2：用生化和分子生物学方法研究外显子3跳跃的机制 目的：评价外显子特异的U1单链RNA挽救外显子3跳跃表型的可能性。

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.