Greenwald Scott H, Kuchenbecker James A, Rowlan Jessica S, Neitz Jay, Neitz Maureen
Department of Ophthalmology, University of Washington, Seattle, WA, USA.
Transl Vis Sci Technol. 2017 May 10;6(3):2. doi: 10.1167/tvst.6.3.2. eCollection 2017 May.
Human long () and middle () wavelength cone opsin genes are highly variable due to intermixing. Two / cone opsin interchange mutants, designated and , are associated with clinical diagnoses, including red-green color vision deficiency, blue cone monochromacy, cone degeneration, myopia, and Bornholm Eye Disease. Because the protein and splicing codes are carried by the same nucleotides, intermixing and genes can cause disease by affecting protein structure and splicing.
Genetically engineered mice were created to allow investigation of the consequences of altered protein structure alone, and the effects on cone morphology were examined using immunohistochemistry. In humans and mice, cone function was evaluated using the electroretinogram (ERG) under /- or short (S) wavelength cone isolating conditions. Effects of and genes on splicing were evaluated using a minigene assay.
ERGs and histology in mice revealed protein toxicity for the LVAVA but not for the LIAVA opsin. Minigene assays showed that the dominant messenger RNA (mRNA) was aberrantly spliced for both variants; however, the gene produced a small but significant amount of full-length mRNA and subjects had correspondingly reduced ERG amplitudes. In contrast, the subject had no / cone ERG.
Dramatic differences in phenotype can result from seemingly minor differences in genotype through divergent effects on the dual amino acid and splicing codes.
The mechanism by which individual mutations contribute to clinical phenotypes provides valuable information for diagnosis and prognosis of vision disorders associated with / interchange mutations, and it informs strategies for developing therapies.
由于基因混合，人类长波长（L）和中波长（M）视锥蛋白基因具有高度变异性。两种L/M视锥蛋白互换突变体，分别命名为LVAVA和LIAVA，与临床诊断相关，包括红绿色觉缺陷、蓝锥单色色觉、视锥细胞变性、近视和博恩霍尔姆眼病。由于蛋白质编码和剪接编码由相同的核苷酸携带，L和M基因的混合会通过影响蛋白质结构和剪接而导致疾病。
构建基因工程小鼠，以单独研究蛋白质结构改变的后果，并使用免疫组织化学检查对视锥细胞形态的影响。在人类和小鼠中，在L-或短（S）波长视锥细胞隔离条件下，使用视网膜电图（ERG）评估视锥细胞功能。使用小基因检测评估L和M基因对剪接的影响。
小鼠的ERG和组织学显示LVAVA视蛋白具有蛋白质毒性，而LIAVA视蛋白没有。小基因检测表明，两种变体的主要信使核糖核酸（mRNA）均发生异常剪接；然而，M基因产生少量但显著的全长mRNA，且携带M基因的受试者ERG振幅相应降低。相比之下，携带L基因的受试者没有L/M视锥细胞ERG。
基因型看似微小的差异通过对双氨基酸编码和剪接编码的不同影响，可导致表型的显著差异。
个体突变导致临床表型的机制为与L/M互换突变相关的视力障碍的诊断和预后提供了有价值的信息，并为制定治疗策略提供了依据。
