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（L）和中（M）波长锥细胞色素基因产生变异，引起异常的前 信使RNA（mRNA）剪接，并导致视力丧失与一组不同的临床表型。95%的 视锥细胞是L或M视锥细胞，并且除了在非常低的光水平下当视杆细胞活跃时，所有视觉都基于视锥细胞。 L和M视锥细胞在视觉引导反馈机制中起着关键作用， 眼生长（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视锥细胞色素，这些基因是常见眼部疾病的重要危险因素， 现代人类L和M（LM）锥状色素基因，命名为OPN 1 LW和OPN 1 MW， 分别在外显子2、3和4中表现出高的单倍型多样性。已知最多的是两种变体， 称为LIAVA和LVAVA，与感光器功能障碍和严重视力相关 损伤它们存在于一系列临床诊断的患者中，包括高度近视，蓝 视锥单色性和视锥营养不良。我们和其他人最近表明， 与这些变体相关的核苷酸多态性（SNP）导致异常的前mRNA剪接。我们 初步数据显示，外显子3多态性的不同组合改变了全长与 外显子3跳跃的mRNA，产生剪接缺陷严重程度的变异性，这将是非常有用的， 阐明了控制该外显子剪接的基本机制。初步数据使用 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单倍型 与低/非跳跃相比，跳跃与平均-1.3屈光度的屈光不正相关 变体。不同突变的病理生理学是复杂的，因为叠加在 由剪接缺陷产生的视蛋白质的低于正常量，是以下因素的某些组合的影响： 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： 目的1：研究L和M视蛋白基因外显子2、3和4的SNPs组合在视网膜病变中的作用。 拼接方式 1.1完全计数转录异构体，并测量它们的相对丰度。 1.2研究和定量外显子3单倍型对视锥细胞剪接的影响 特别是那些作为青少年近视发病风险等位基因的单倍型。 目的2：从生物化学和分子生物学的角度探讨外显子3跳读的机制 目的3：评估外显子特异性U1 snRNAs拯救外显子3跳跃表型的潜力。

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.