Genetics Department, Fundacion Jimenez Diaz, Madrid, Spain.
Invest Ophthalmol Vis Sci. 2010 May;51(5):2615-9. doi: 10.1167/iovs.09-4518. Epub 2009 Dec 3.
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.
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.
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.
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.
ABCA4 基因突变与常染色体隐性型 Stargardt 病（STGD）、少数常染色体隐性型 cone-rod 营养不良（arCRD）和常染色体隐性型视网膜色素变性（arRP）相关。本研究旨在比较高分辨率熔解（HRM）分析与变性高效液相色谱（dHPLC），评估不同筛选方法的效率。
对 38 例 STGD、15 例 arCRD 和 5 例 arRP 无关的西班牙患者进行 ABCR 微阵列分析，结果经直接测序确认。在只有一个或没有突变等位基因的患者中，进一步通过 HRM 和 dHPLC 分析 ABCA4。还进行了单体型分析。
在之前的微阵列分析中，发现 37 个 ABCA4 变异（37/116；31.9%）。dHPLC 和 HRM 扫描在 20 个样本中鉴定出 18 种不同的基因型。通过 HRM 鉴定的 20 个样本中，19/20 正确，16/20 通过 dHPLC 正确鉴定。dHPLC 未能检测到一个纯合突变；然而，HRM 技术可将 p.Cys2137Tyr 纯合子与野生型区分开来。同样，仅通过 HRM 技术发现外显子 5 中的一个新变化（p.Arg187His）。此外，dHPLC 在外显子 5 中发现一个 p.Trp1724Cys 突变，但 HRM 在两个样本中检测到该突变。
ABCA4 应通过最佳筛选技术进行分析，以进一步鉴定病理性等位基因。结果似乎表明 HRM 比 dHPLC 具有更好的灵敏度和特异性，具有识别某些纯合序列改变的优势。
