Velissari Aliki, Skalidakis Iosif, Oliveira Samantha C, Koutsandrea Chryssanthi, Kitsos George, Petersen Michael B, Kroupis Christos
Clin Chem Lab Med. 2015 Sep 1;53(10):1521-9. doi: 10.1515/cclm-2014-0920.
Age-related macular degeneration (AMD) is a degenerative ocular disease, which may lead to loss of central vision. In Caucasian populations, a strong correlation has been established with polymorphism Y402H (rs1061170) in the complement factor H gene (CFH). The H131R polymorphism (rs1801274) in the FCGR2A gene has been associated with many inflammatory diseases, but has not been investigated in relation to AMD. The goal of our study was the development of a novel method for Y402H (g.43097C>T) genotyping, the confirmation of its association with AMD in the Greek population and the investigation of the H131R polymorphism in AMD.
DNAs were extracted from blood samples of 120 patients with the severe wet form of AMD and 103 age- and sex-matched controls, all of whom were clinically evaluated. A real-time PCR and melting curve analysis method for Y402H genotyping was developed in the LightCycler platform, after in silico design of appropriate primers and probes. Genotyping for H131R was performed using a real-time PCR method previously described by our group.
The novel genotyping method for Y402H in the CFH gene is fast, reproducible (Efficiency=1.79, reproducibility CVCq=3.33%, Tm C allele 53.36 °C and T allele 61.91 °C, ΔTm=8.55) and accurate as results were confirmed with the gold standard DNA Sequencing method.
The present study confirmed the association between CFH Y402H SNP and wet AMD in the Greek population (OR=1.77, p=0.002). FCGR2A H131R polymorphism was investigated for the first time in this present study for possible correlation with wet AMD and a statistically significant association was detected (OR=1.74, p=0.006), that awaits further confirmation in a larger set of samples.
年龄相关性黄斑变性（AMD）是一种退行性眼病，可能导致中心视力丧失。在白种人群中，已证实其与补体因子H基因（CFH）中的Y402H（rs1061170）多态性密切相关。FCGR2A基因中的H131R多态性（rs1801274）与许多炎症性疾病有关，但尚未针对AMD进行研究。我们研究的目的是开发一种新的Y402H（g.43097C>T）基因分型方法，在希腊人群中证实其与AMD的关联，并研究AMD中的H131R多态性。
从120例严重湿性AMD患者和103例年龄及性别匹配的对照者的血样中提取DNA，所有受试者均经过临床评估。在对合适的引物和探针进行计算机设计后，在LightCycler平台上开发了一种用于Y402H基因分型的实时PCR和熔解曲线分析方法。使用我们小组先前描述的实时PCR方法对H131R进行基因分型。
CFH基因中Y402H的新型基因分型方法快速、可重复（效率=1.79，重复性CVCq=3.33%，C等位基因熔解温度53.36°C，T等位基因熔解温度61.91°C，ΔTm=8.55），并且由于结果通过金标准DNA测序方法得到了证实，所以该方法准确。
本研究证实了希腊人群中CFH Y402H单核苷酸多态性与湿性AMD之间的关联（OR=1.77，p=0.002）。本研究首次对FCGR2A H131R多态性与湿性AMD的可能相关性进行了研究，并检测到具有统计学意义的关联（OR=1.74，p=0.006），这有待在更大样本量中进一步证实。
