title: Comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning in the ABCA4 gene.
abstract: OBJECTIVE
  Mutations in the ABCA4 gene have been associated with autosomal recessive Stargardt disease (STGD), a few cases of autosomal recessive cone-rod dystrophy (arCRD), and autosomal recessive retinitis pigmentosa (arRP). The purpose of this study was to compare high-resolution melting (HRM) analysis with denaturing high-performance liquid chromatography (dHPLC), to evaluate the efficiency of the different screening methodologies.
  
  
  METHODS
  Thirty-eight STGD, 15 arCRD, and 5 arRP unrelated Spanish patients who had been analyzed with the ABCR microarray were evaluated. The results were confirmed by direct sequencing. In patients with either no or only one mutant allele, ABCA4 was further analyzed by HRM and dHPLC. Haplotype analysis was also performed.
  
  
  RESULTS
  In a previous microarray analysis, 37 ABCA4 variants (37/116; 31.9%) were found. dHPLC and HRM scanning identified 18 different genotypes in 20 samples. Of the samples studied, 19/20 were identified correctly by HRM and 16/20 by dHPLC. One homozygous mutation was not detected by dHPLC; however, the p.Cys2137Tyr homozygote was distinguished from the wild-type by HRM technique. In the same way, one novel change in exon 5 (p.Arg187His) was found only by means of the HRM technique. In addition, dHPLC identified the mutation p.Trp1724Cys in one sample; however, HRM detected the mutation in two samples.
  
  
  CONCLUSIONS
  ABCA4 should be analyzed by an optimal screening technique, to perform further characterization of pathologic alleles. The results seemed to show that HRM had better sensitivity and specificity than did dHPLC, with the advantage that some homozygous sequence alterations were identifiable.
authors: Genetics Department, Fundacion Jimenez Diaz, Madrid, Spain. jaguirre@fjd.es
authors: Jana
authors: J
authors: Aguirre-Lamban
authors: Rosa
authors: R
authors: Riveiro-Alvarez
authors: Maria
authors: M
authors: Garcia-Hoyos
authors: Diego
authors: D
authors: Cantalapiedra
authors: Almudena
authors: A
authors: Avila-Fernandez
authors: Cristina
authors: C
authors: Villaverde-Montero
authors: Maria Jose
authors: MJ
authors: Trujillo-Tiebas
authors: Carmen
authors: C
authors: Ramos
authors: Carmen
authors: C
authors: Ayuso
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D002851:Chromatography, High Pressure Liquid; D004252:DNA Mutational Analysis; D005838:Genotype; D008268:Macular Degeneration / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis; D011489:Protein Denaturation; D012162:Retinal Degeneration / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000235:genetics*; D012680:Sensitivity and Specificity; D044366:Transition Temperature
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters