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

蓝视锥单色性（Blue Cone Monochromacy， BCM）是一种罕见的遗传性疾病，其特征是由于同时缺乏红色和绿色敏感的视锥光感受器功能，从婴儿期开始就表现为视力低下、色盲、畏光和频繁的眼球震颤。BCM是一种遗传的x连锁隐性疾病，由染色体Xq28上的视锥蛋白基因阵列突变引起，其中包括分别编码红色和绿色敏感视锥光色素载蛋白的OPN1LW和OPN1MW基因。BCM主要有三种类型的突变，其中两种，即外显子3剪接突变和结构突变，将在本项目中进行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的某些组合引起的，迄今为止对这些组合的研究很少。我们将采用并行多重剪接实验来测试哺乳动物细胞培养模型中所有256种可能的变异组合的剪接能力，从而测试其致病性。这些结果将与外显子3序列剪接因子结合位点的损失或增加的生物信息学预测相关联，高分相关性将通过在细胞培养中选择性敲除候选剪接因子得到实验验证。到目前为止，锥视蛋白基因阵列中只有大约12个结构突变（例如缺失，倒置）被精确地定位。在这个项目中，大量预先筛选的BCM患者的可用性使实验断点映射能够在更大和更经济的规模上进行。我们期望将精确映射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，如锥体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高频率的热点的存在。此外，这将使我们能够检验一个假设，即锥体视蛋白基因阵列的大量拷贝数易导致引起结构突变的疾病的发生。该项目的研究结果将有助于更好地理解BCM的分子机制，但预计将有实际应用，如开发女性携带者检测的简单检测方法，并可能为BCM患者外显子3剪接缺陷的治疗方法的开发提供途径。

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.