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D. Manasses, A. G. Robson, G. E. Holder, B. Scott, S. S. Bhattacharya, A. T. Moore, A. R. Webster; Analysis of the ABCA4 Gene in 200 Unrelated Probands With Macular Dystrophy Using Multiplex Ligation-Dependent Probe Assay (MLPA) and DNA Microarrays. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1664.
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© ARVO (1962-2015); The Authors (2016-present)
Mutation of the ABCA4 gene is associated with autosomal recessive macular and cone-rod dystrophy. However, sensitivity of mutation detection using standard methods is low. The purpose of this study is to survey 200 well-characterised, unrelated individuals with suspected ABCA4-retinopathy for mutations in the gene, using a microarray to detect known intragenic variants, as well as MLPA to detect gene rearrangements.
Clinical investigation was undertaken prospectively on a cohort of 200 affected, unrelated individuals attending Moorfields Eye Hospital, fitting the following criteria: retinal disease confined to, or more severely affecting the retinal macula, segregation compatible with autosomal recessive inheritance, absence of a history or signs suggesting an acquired aetiology. Clinical data included visual acuity, colour fundus imaging, fundal autofluorescence imaging (FAF) and electroretinography. 484 DNA variants were assayed using the Apex array (AsperBiotech) and MLPA probes used to seek gene rearrangements were designed in collaboration with MRC-Holland.
The mean age of the cohort was 37 years (range of 7 to 76 years). Of 150 patients screened for mutations using the Apex DNA microarray, 115 (77%) had one or more disease-associated variants identified. Of these 56 individuals had one, 51 had two and 8 >2 such variants. In 24 (16%) at least one variant was a likely null mutation. The most common single variants were c.863g->a (11% of patients) and IVS39-10 t->g (also 11% of patients). Analysis of common polymorphisms suggested the presence of at least one common disease associated haplotype . The genomic region of ABCA4 was scanned using 47 probes to cover 50 coding exons, together with 24 control probes, in two MLPA reactions. Of 11 patients so far analysed completely, a reproducible reduction in copy number of exon 44 in one patient, suggests a heterozygous deletion.
Known variants are present on at most 58% of disease alleles. A common haplotype suggests an, as yet unknown, DNA variant associated with disease. MLPA shows some promise in finding mutations in ABCA4 missed by other methods. MLPA analysis of the remaining cohort is ongoing.
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