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.