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Jana Zernant, Carmen Ayuso, Francesca Simonelli, Mette Bertelsen, Thomas Rosenberg, Michael Gorin, Bo Yuan, Peter L Nagy, Rando Allikmets; Analysis of the entire ABCA4 genomic locus in Stargardt disease patients in search for missing mutations. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3253. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Stargardt disease (STGD) is inherited as an autosomal recessive trait. Complete sequencing of the causal ABCA4 gene coding sequences in STGD patients finds the expected two disease-associated alleles in 70-80% of patients and only one mutation in 15-20% of patients. This study was designed to find the missing ABCA4 mutations by a combination of next-generation sequencing and in silico analyses.
The entire140kb ABCA4 genomic locus was sequenced in 114 STGD patients with one known ABCA4 exonic mutation by either RainDance microdroplet-PCR target enrichment or Illumina Truseq Custom Amplicon methods. The resulting variants were filtered to exclude false positives and those frequent in the general population by comparing to in-house data and to 1000 Genomes and Exome Sequencing Projects. The resulting rare variants were verified via Sanger sequencing, followed by segregation analyses in families and screening of additional cohorts of STGD patients and controls. All remaining variants were analyzed by in silico methods for evolutionary conservation and pathogenicity.
Locus sequencing revealed ~200 intronic variants per sample. Filtering of these data resulted in 142 candidates for new mutations. Two variants were detected in 4 samples, two in 3 samples, and 21 variants in 2 samples, the remaining 117 new variants were detected only once. Multimodal analysis suggested 10 new likely pathogenic intronic ABCA4 variants, some of which were specific to (isolated) ethnic groups. Some confirmed pathogenic variants included c.4539+1729G>T, c.570+1798A>G, c.2161-8G>A, and c.859-9T>C. The latter is specific for Asian Indian population. Many variants, including some which were suggested recently (Braun et al., Hum Mol Genet 22:5136-45, 2013), were excluded since they were conserved in non-human primates, were frequent in African populations and, therefore, represented ancestral, and not disease-associated, variants.
The sequence variability in the ABCA4 locus is vast and the non-coding sequences do not harbor frequent mutations in STGD patients of European-American descent. Since photoreceptor-specific ABCA4 mRNA is not accessible for analysis in patients, defining of disease-associated alleles in the ABCA4 locus requires exceptionally well characterized large cohorts and a tedious analysis by a combination of various approaches.
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