Multiplex Ligation-Dependent Probe Assay (MLPA) of the ABCA4 Gene in 212 Unrelated Probands With Macular Dystrophy.

Abstract

Purpose: : 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 (microarray analysis of our cohort of 212 patients has identified known variants in a maximum of 55% of potential disease alleles). Although high allelic heterogeneity of the ABCA4 gene is likely to contribute to this, another possibility is that gene rearrangements which include primer binding sites are not identified using traditional techniques. The purpose of this study is to survey 212 well-characterised, unrelated individuals with suspected ABCA4-retinopathy for mutations in the gene, using MLPA to detect gene rearrangements.

Methods: : Clinical investigation was undertaken prospectively on a cohort of 212 affected, unrelated individuals attending Moorfields Eye Hospital, fitting the following criteria: retinal disease confined to, or more severely affecting the 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. MLPA probes designed in collaboration with MRC-Holland were used to seek gene rearrangements.

Results: : The genomic region of ABCA4 was scanned using 48 probes to cover 50 coding exons, together with 25 control probes, in two MLPA reactions. Of 126 successful assays on 63 patients so far analysed, 116 (92%) did not show a reduction in copy number for any of the ABCA4 probes. Of the remaining 10 assays, 3 demonstrated a reduction in probe amplification in an exon where microarray analysis has demonstrated a mutation near the probe hybridization site. In a further 2 assays, the apparent copy number change has been shown by direct sequencing to be due to a novel mutation near the probe hybridization site. In the other 5 assays, the reduction in copy number cannot be explained by a mutation directly localized at the probe hybridization site, and is likely to represent a gene rearrangement.

Conclusions: : MLPA has so far demonstrated that gene rearrangements may occur in 5 of 126 alleles (4.0%). The technique has also identified novel mutations localized to the probe binding site in some samples. MLPA analysis of the remaining cohort is ongoing.

Keywords: retinal degenerations: hereditary • genetics