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Frauke Coppieters, Takuro Fujimaki, Marieke De Bruyne, Annelot Baert, Mineo Kondo, Akira Murakami, Elfride De Baere; Partial NMNAT1 deletions cause Leber Congenital Amaurosis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3285.
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© ARVO (1962-2015); The Authors (2016-present)
In 2012, the NMNAT1 gene was identified as a novel disease gene for Leber Congenital Amaurosis 9 (LCA9), characterized by a recognizable phenotype with severe macular lesions. The mutation spectrum of this gene is quite broad, varying from coding to regulatory mutations (Coppieters et al. personal communication). The starting point of this study was pseudohomozygosity of a known NMNAT1 mutation (c.709C>T, p.Arg237Cys) in exon 5 in the probands of two unrelated Japanese families, assuming hemizygosity for this mutation. Here, we aimed to identify and characterize the putative NMNAT1 deletions, contributing to the LCA phenotype in these families.
Copy number variation (CNV) screening was performed for all NMNAT1 exons using one qPCR amplicon per exon. Subsequently, identified deletions were refined with additional qPCR amplicons located in the breakpoint regions. Finally, the junction product was amplified with long-range PCR using the primers of the last and first non-deleted amplicons, and sequenced using both Sanger and next-generation sequencing (Nextera XT, MiSeq, Illumina).
In the first family (F1), CNV analysis showed a heterozygous deletion of exon 4 and 5. Subsequent qPCR with 7 additional amplicons refined the deletion at both ends to a region of 13.0-18.7 kb. In the second family (F2), the amplicon for exon 4 was deleted, whereas the copy number of the amplicon for exon 5, located downstream of the p.Arg237Cys mutation, was normal. Subsequent refinement at the 5’end delineated the deletion to a region of 1.5-6.7 kb. Long-range PCR revealed a band of 2.4 kb and 2.1 kb in F1 and F2, pointing to two different deletions of approximately 16.6 kb and 4.8 kb, respectively. Sequencing analysis of the junction products is currently ongoing to determine the exact breakpoints and the underlying mechanisms. In both probands, early-onset macular degeneration was observed, which is typical for NMNAT1-associated LCA.
In conclusion, we report the first deletions of NMNAT1, expanding the mutation spectrum of this gene. The complexity of the repeat-rich NMNAT1 genomic region likely contributes to the formation of these deletions. Hence, it can be anticipated that the high number of patients in which only a single heterozygous mutation has been reported so far, can be explained by CNVs or structural variations of NMNAT1, requiring additional strategies apart from resequencing of the NMNAT1 coding region.
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