Br J Ophthalmol 93:614-621 doi:10.1136/bjo.2008.145193
  • Clinical science
    • Original Article

Molecular analysis of the ABCA4 gene for reliable detection of allelic variations in Spanish patients: identification of 21 novel variants

Open Access
  1. J Aguirre-Lamban1,2,
  2. R Riveiro-Alvarez1,2,
  3. S Maia-Lopes3,
  4. D Cantalapiedra1,2,
  5. E Vallespin1,2,
  6. A Avila-Fernandez1,2,
  7. C Villaverde-Montero1,2,
  8. M J Trujillo-Tiebas1,2,
  9. C Ramos1,2,
  10. C Ayuso1,2
  1. 1
    Genetics Department, Fundacion Jimenez Diaz, Madrid, Spain
  2. 2
    Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII, Madrid, Spain
  3. 3
    Visual Neuroscience Laboratory, IBILI, Faculty of Medicine, Coimbra, Portugal
  1. Dr J Aguirre-Lamban, Fundacion Jimenez Diaz, Genetics Department, Reyes Catolicos 2, 28.040 Madrid, Spain; jaguirre{at}
  • Accepted 6 October 2008
  • Published Online First 21 November 2008


Background/aims: Mutations in ABCA4 have been associated with autosomal recessive Stargardt disease (STGD), a few cases with autosomal recessive cone–rod dystrophy (arCRD) and autosomal recessive retinitis pigmentosa (arRP). The purpose of the study was threefold: to molecularly characterise families with no mutations or partially characterised families; to determine the specificity and sensitivity of the genotyping microarray; and to evaluate the efficiency of different methodologies.

Methods: 23 STGD, five arCRD and three arRP Spanish patients who were previously analysed with the ABCR400 microarray were re-evaluated. Results were confirmed by direct sequencing. In patients with either none or only one mutant allele, ABCA4 was further analysed by denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (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, two 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 yielded an increment of 21% compared with the approach used in the first round.

Conclusion: ABCA4 should be analysed by optimal combination of high-throughput screening techniques such 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 characterisation of pathological ABCA4 alleles.


  • Competing interests: None.

  • Funding: Thanks to FIS (Sanitary Research Fund) 06/0027, Fundacion Mutua Madrileña (30171/005), CIBERER (06/07/0036) and EviGenoRet LSHG-CT-2005-512036 for their support. JA-L’s work is supported by FIS from the Instituto de Salud Carlos III (PI06/0027).

  • Ethics approval: Ethics approval was provided by the Ethics Committee of the Hospital (Fundacion Jimenez Diaz).

  • Patient consent: Obtained.

  • This is an open-access article distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.