Elucidating exonic splice mutations and structural mutations in the cone opsin gene array on Xq28 underlying Blue Cone Monochromatism

蓝锥单色症（Blue Cone Monochromacy，简称蓝锥单色症）是一种罕见的遗传性疾病，由于同时缺乏对红色和绿色敏感的视锥细胞感光功能，从婴儿早期开始表现为低视力、色盲、畏光和频繁的眼球震颤。视锥细胞色素变性是一种X连锁隐性遗传病，由染色体Xq28上的视锥细胞视蛋白基因阵列中的突变引起，该基因阵列包括分别编码红色和绿色敏感视锥细胞色素的脱辅基蛋白的OPN1LW和OPN1MW基因。有三个主要类别的突变的基础上，其中两个，即外显子3剪接突变和结构突变，将在这个项目中进行了前所未有的详细研究。外显子3剪接缺陷是由常见外显子序列变异体的某些组合引起的，迄今为止，这些组合中只有少数被研究过。我们将采用并行多重剪接试验来检测哺乳动物细胞培养模型中所有256种可能的变体组合的剪接能力，从而检测致病性。结果将与外显子3序列中剪接因子结合位点的丢失或获得的生物信息学预测相关，高分相关性将通过细胞培养物中候选剪接因子的选择性敲低进行实验验证。迄今为止，在视锥细胞视蛋白基因阵列中只有大约十几个结构突变（例如缺失、倒位）被精确定位。在这个项目中，一个大的预先筛选的cardiac患者队列的可用性，使实验断点映射在一个更大的和经济的规模。我们期望将精确定位的结构突变的数量增加3倍或更多。这将使我们能够解决一些未解决的问题，如锥视蛋白基因阵列结构突变的分子机制，功能重要序列的定义，以及解释该基因位点结构突变频率高的热点的存在。此外，它将使我们能够验证假设，即视锥细胞视蛋白基因阵列中的大拷贝数易于发生引起疾病的结构突变。该项目的结果将更好地理解潜在的遗传缺陷的分子机制，但预计将具有实际应用，例如开发用于女性携带者检测的简单检测方法，并可能为开发治疗方法提供途径。外显子3剪接缺陷。

Blue Cone Monochromacy (BCM) is a rare genetic disorder which is characterized by low vision, colour blindness, photophobia and frequently nystagmus from earliest infancy due to the simultaneous lack of red and green sensitive cone photoreceptor function. BCM is inherited as an X-linked recessive condition and caused by mutations in the cone opsin gene array on chromosome Xq28 which includes the OPN1LW and OPN1MW genes encoding the apo-protein of the red and green sensitive cone photopigments, respectively. There a three main categories of mutations underlying BCM, two of which, namely exon 3 splicing mutations and structural mutations, will be studied in this project in unprecedented detail. Exon 3 splicing defects are caused by certain combinations of common exonic sequence variants and only few of these combinations have been studied so far. We will adapt and employ a parallelized multiplex splicing assay to test the splicing competence, and thus the pathogenicity, of all 256 possible variant combinations in the mammalian cell culture model. The results will be correlated with bioinformatic predictions on the loss or gain of splicing factor binding sites in exon 3 sequences and high-score correlations will be experimentally validated through selective knockdown of candidate splicing factors in cell culture. Only about a dozen of structural mutations (e.g. deletions, inversions) in the cone opsin gene array have been precisely mapped so far. The availability of a large cohort of pre-screened BCM patients in this project enables experimental breakpoint mapping at a much larger and economical scale. We expect to increase the number of precisely mapped structural mutations by a factor of 3 or more. This will allow us to address a number of unsolved questions such as the molecular mechanism(s) underlying structural mutation at the cone opsin gene array, the definition of functionally important sequences, and the presence of hotspots explaining the high frequency of structural mutations at this gene locus. Moreover it will enable us to test the hypothesis that a large copy number in the cone opsin gene array predisposes to the occurrence of disease causing structural mutations.The results of this project will provide a better understanding of the molecular mechanisms underlying BCM but are expected to have practical application such as the development of simple assays for female carrier detection and may provide a path for the development of therapies for exon 3 splicing defects in BCM patients.