Laboratory of Molecular Biology, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy.
Clin Chem Lab Med. 2012 Jan 13;50(6):1031-4. doi: 10.1515/cclm-2011-0859.
Age-related macular degeneration (AMD) is a complex disorder causing irreversible central vision loss. Complement Factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) are now widely accepted as important AMD susceptibility genes. In particular, two specific variants, CFH p.Y402H and ARMS2 p.A69S, have been reported as strongly AMD associated. In order to perform the genetic screening of these single nucleotide polymorphisms (SNPs), we describe a high resolution melting analysis (HRM) as a rapid closed tube mutation scanning assay.
To validate HRM genotyping, 94 DNA samples from AMD patients (previously genotyped by sequence analysis) were analyzed. PCR amplification and melting curve analysis were performed in the LightCycler 480 Real-Time PCR System. In order to evaluate the accuracy of the HRM assay, we performed a blinded study of 20 unknown independent samples.
We correctly genotyped all samples. In fact, all samples corresponded to the previous genotype assignments.
Early identification of individuals with genetic risk variants CFH p.Y402H and ARMS2 p.A69S is clinically important for the definition of AMD status. High-resolution DNA melting is homogenous, accurate and rapid method for CFH and ARMS2 genotyping.
年龄相关性黄斑变性（AMD）是一种复杂的疾病，会导致不可逆转的中心视力丧失。补体因子 H（CFH）和年龄相关性黄斑变性易感性 2 基因（ARMS2）现已被广泛认为是重要的 AMD 易感基因。特别是两个特定的变体，CFH p.Y402H 和 ARMS2 p.A69S，被报道与 AMD 密切相关。为了对这些单核苷酸多态性（SNP）进行遗传筛查，我们描述了一种高分辨率熔解分析（HRM）作为一种快速的闭管突变扫描检测方法。
为了验证 HRM 基因分型，我们分析了 94 份来自 AMD 患者的 DNA 样本（先前通过序列分析进行了基因分型）。PCR 扩增和熔解曲线分析在 LightCycler 480 实时 PCR 系统中进行。为了评估 HRM 检测的准确性，我们对 20 份未知的独立样本进行了盲法研究。
我们正确地对所有样本进行了基因分型。事实上，所有样本都与之前的基因型分配相对应。
早期识别具有 CFH p.Y402H 和 ARMS2 p.A69S 遗传风险变异的个体对于确定 AMD 状态具有重要的临床意义。高分辨率 DNA 熔解是一种同质、准确、快速的 CFH 和 ARMS2 基因分型方法。
