Pereyra Silvana, Velazquez Tatiana, Bertoni Bernardo, Sapiro Rossana
Departament of Histology and Embryology, School of Medicine, University of the Republic, Gral, Flores 2125, Montevideo, Uruguay.
BMC Res Notes. 2012 Jan 26;5:69. doi: 10.1186/1756-0500-5-69.
Complex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs) from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM) analysis is a new method in which polymerase chain reaction (PCR) and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB) is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB.
Here, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism.
We were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB.
Several gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing their association to PTB. It can easily be used for simultaneously analyze any set of SNPs, either as the first choice for new association studies or as a complement to large-scale genotyping analysis. Given that inflammatory pathway is in the base of several diseases, it is potentially useful to analyze a broad range of disorders.
癌症、糖尿病、肥胖症或精神分裂症等复杂性状源于遗传和环境因素之间的复杂相互作用。复杂疾病往往在家族中聚集，但没有明确的遗传模式。全基因组关联研究专注于检测数十个或数百个导致复杂疾病的个体标记。为了测试候选基因中的单核苷酸多态性（SNP）子集是否与特定个体或人群中感兴趣的疾病相关，需要新技术。高分辨率熔解（HRM）分析是一种新方法，其中聚合酶链反应（PCR）和突变扫描在封闭管中同时进行，使该过程快速、廉价且简便。早产（PTB）被认为是一种复杂疾病，其中遗传和环境因素相互作用，导致新生儿在妊娠37周前分娩。炎症在妊娠和早产中起重要作用已被广泛认可。
在此，我们使用实时PCR随后进行HRM分析，以同时鉴定早产中涉及炎症途径的几种基因变异。在病例对照研究中分析了TLR4、IL6、IL1β和IL12RB基因的SNP。结果通过测序或PCR随后进行限制性片段长度多态性分析进行确认。
我们能够以与其他方法相似的准确性同时识别四个基因的变异。为了获得不重叠的熔解温度，该策略的关键步骤是引物设计。每个SNP的基因型频率与先前在相似人群中描述的频率一致。所研究的SNP均与早产无关。
通过一种新的灵活、快速且廉价的方法筛选了与同一炎症途径相关的几种基因变异，以分析它们与早产的关联。它可以轻松用于同时分析任何一组SNP，既可以作为新关联研究的首选方法，也可以作为大规模基因分型分析的补充。鉴于炎症途径是多种疾病的基础，分析广泛的疾病可能具有潜在的用途。
