Background/Aims: Mutations in ABCA4 have been associated to autosomal recessive Stargardt disease (STGD), a few cases with autosomal recessive cone-rod dystrophy (arCRD) and autosomal recessive retinitis pigmentosa (arRP). The purpose of this study was three-folded: to molecularly characterise families with no mutations or partially characterised families; to determine the specificity and sensitivity of the genotyping microarray; to evaluate the efficiency of different methodologies.
Methods: We re-evaluated 23 STGD, 5 arCRD and 3 arRP Spanish patients who were previously analysed with the ABCR400 microarray. Results were confirmed by direct sequencing. In patients with either none or only one mutant allele, ABCA4 was further analysed by dHPLC and MLPA. Haplotype analysis was also performed.
Results: In the first analysis performed with the microarray, 27 ABCA4 variants (27/62; 43.5%) were found. By dHPLC scanning, 12 novel mutations were additionally identified. In addition, 2 previously described mutations, one false negative (1/62; 1.6%) and one false positive (1.6%) were detected. MLPA analysis did not reveal additional substitutions. The new strategy yieldean increment of 21% compared to the approach used in the first round.
Conclusion: ABCA4 should be analysed by optimal combination of high through-put screening techniques as microarray, dHPLC and direct sequencing. To the best of our knowledge, this strategy yielded significant mutational spectrum identification in Spanish patients with ABCA4-associated phenotypes. Follow up of patients, presenting an early onset of the disease and severe mutations, seems essential to perform accurate genotype-phenotype correlations and further characterization of pathologic ABCA4 alleles.