Comparison of high-resolution melting analysis with denaturing high-performance liquid chromatography for mutation scanning in the ABCA4 gene.
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.
Aguirre-Lamban
Jana
J
Genetics Department, Fundacion Jimenez Diaz, Madrid, Spain. jaguirre@fjd.es
Riveiro-Alvarez
Rosa
R
Garcia-Hoyos
Maria
M
Cantalapiedra
Diego
D
Avila-Fernandez
Almudena
A
Villaverde-Montero
Cristina
C
Trujillo-Tiebas
Maria Jose
MJ
Ramos
Carmen
C
Ayuso
Carmen
C
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
C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters