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Susanne Kohl, Marc Sturm, Britta Baumann, Bernd Wissinger, Nicole Weisschuh; Deep-intronic variants in CNGB3 are an important cause of CNGB3-related autosomal recessive achromatopsia. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2392.
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Mutations in CNGB3, encoding the alpha-subunit of the cone photoreceptor cyclic nucleotide-gated ion channel are the most common cause of achromatopsia (ACHM) in patients of European descent. Yet a considerable proportion of such ACHM patients have been only shown to carry only a single heterozygous CNGB3 mutation and the second allele remained elusive. Recently, copy number variations have been shown to account for some but not all of the missing alleles.
We sequenced the entire CNGB3 locus in 33 monoallelic CNGB3 cases by next generation sequencing. The library was prepared by targeted enrichment of CNGB3 by long distance PCR and Nextera XT DNA Sample Preparation Kit for indexed paired-end library prep and a 300 cycle sequencing run using the MiniSeq System Mid-Output Kit on the MiniSeq instrument (Illumina). Variant calling and annotation was performed using the in-house bioinformatic pipeline. Intronic variants in CNGB3 were filtered according to their frequency. Those that were found in cis with pathogenic alleles were excluded from further validation. In silico prediction tools were used to further filter for intronic variants predicted to impair splicing. These were analysed by means of a heterologous splicing assay in HEK293 cells.
Two intronic variants in CNGB3, c.1663-1205G>A and variant c.1663-2137C>T were indeed shown to exert a splicing defect by causing insertion of an out-of-frame pseudoexon into the transcript. Subsequent screening of further monoallelic CNGB3 subjects identified in total 18 cases harboring the c.1663-1205G>A variant while the c.1663-2137C>T variant was observed in 2 independent individuals. This makes the c.1663-1205G>A variant the eighth most frequent CNGB3 mutant variant in our cohort of >1000 ACHM patients.
Our study demonstrates that whole gene sequencing is a powerful tool to identify the 'missing' disease allele in patients with autosomal recessive disease, and that deep intronic variants are an important cause of CNGB3-associated ACHM.
This is a 2020 ARVO Annual Meeting abstract.
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