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Elena Buena-Atienza, Britta Baumann, Nicole Weisschuh, Klaus W Ruether, Susanne Kohl, Bernd Wissinger, ; OPN1LW/MW exon 3 haplotypes associated with Blue Cone Monochromacy disrupt exonic regulatory elements of splicing and can arise by gene conversion. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2886.
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© ARVO (1962-2015); The Authors (2016-present)
Cone Dysfunction syndromes such as Blue Cone Monochromacy (BCM) and Bornholm Eye Disease (BED) are caused by defects in the OPN1LW/MW (LW/MW) gene cluster. Subjects carrying a structurally intact opsin gene cluster and no known pathogenic point mutations were investigated for a novel third category of opsin mutations characterized by rare combinations of common single nucleotide polymorphisms (SNP) in exon 3 of LW/MW. These rare haplotypes commonly termed by the amino acid combination they encode, such as LIAVA, may cause misplicing of the respective opsin gene.
16 affected subjects with a clinical diagnose of BCM or BCM-like were recruited and genotyped. Sequencing of LW/MW exon 3 revealed novel haplotypes. The effect of various exon 3 haplotypes on transcript processing was tested applying minigene constructs heterologously expressed in HEK293 cells. Relative quantification of transcripts was performed by fragment analysis of FAM-labeled RT-PCR products. Genotyping of microsatellite markers and allelic cloning were performed to analyze the segregation and integrity of LW/MW in a family with evidence for gene conversion (GC).
In this cohort, 4 out of 13 different haplotypes analyzed in the minigene assay, LIAVA, LVAVA, MIAVA and a second MIAVA with one synonymous variant, resulted in a strong abundance of aberrantly spliced transcripts (90-100%). Further 4 haplotypes resulted in 40-80% of aberrantly spliced transcripts. Correlation of haplotype sequences with the minigene assay outcome suggests that the penultimate SNP in the haplotype, encoding for p.I178V is a strong exonic splicing silencer. In a case in which the phenotype switches from macular dystrophy with deuteranopia in the grandfather to BCM in the grandson, we found that intrachromosomal GC in the male germline introduced a deleterious LIAVA haplotype into LW.
We found a variety of rare LW/MW exon 3 haplotypes associated with BCM or BCM-like phenotype and demonstrated fully or partially penetrant splicing defects caused by these variants. The 8 SNPs comprised in these haplotypes overlap with several exonic splicing silencer, enhancer and regulator signals involved in splicing regulation of LW/MW, and result in different outcomes when disrupted. Furthermore, we provide direct evidence that LIAVA haplotype in LW can be originated by intrachromosomal GC.
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