title: Molecular analysis of the ABCA4 gene for reliable detection of allelic variations in Spanish patients: identification of 21 novel variants.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Fundacion Jimenez Diaz, Genetics Department, Madrid, Spain. jaguirre@fjd.es
authors.forename: J
authors.initials: J
authors.lastname: Aguirre-Lamban
authors.forename: R
authors.initials: R
authors.lastname: Riveiro-Alvarez
authors.forename: S
authors.initials: S
authors.lastname: Maia-Lopes
authors.forename: D
authors.initials: D
authors.lastname: Cantalapiedra
authors.forename: E
authors.initials: E
authors.lastname: Vallespin
authors.forename: A
authors.initials: A
authors.lastname: Avila-Fernandez
authors.forename: C
authors.initials: C
authors.lastname: Villaverde-Montero
authors.forename: M J
authors.initials: MJ
authors.lastname: Trujillo-Tiebas
authors.forename: C
authors.initials: C
authors.lastname: Ramos
authors.forename: C
authors.initials: C
authors.lastname: Ayuso
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000293:Adolescent; D000328:Adult; D015785:Eye Diseases, Hereditary / Q000235:genetics*; D005260:Female; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics; D008297:Male; D008875:Middle Aged; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010375:Pedigree; D010641:Phenotype; D012162:Retinal Degeneration / Q000235:genetics*; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters