Figueredo Lago Juan Emilio, Armas Cayarga Anny, González González Yaimé Josefina, Collazo Mesa Teresa
Department of Molecular Biology, Immunoassay Center (CIE) Cubanacan, Playa, Havana, Cuba.
National Center of Medical Genetics, Havana, Cuba.
BMC Med Genet. 2017 May 25;18(1):58. doi: 10.1186/s12881-017-0420-9.
Mutation scanning methods in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene may not distinguish between a Cystic Fibrosis (CF) causing mutation and a benign variant. We have developed a simple and fast method for scanning 14 selected CF-causing mutations which have high frequency in Latin America.
In a group of 35 samples coming from CF patients previously characterized and using two allele-specific real-time multiplex PCRs targeting wild-type and mutant alleles respectively, we detect the presence of mutations by analyzing the Ct variation. Twenty-five samples without mutations considered non-carrier samples, were also included in this study. High Resolution Melting Analysis (HRMA) was performed to confirm the result of the scanning method and in most cases allowed the genotype determination.
The results validate this method for CF diagnosis. A least one CFTR gene mutation was detected in the samples of CF patients, as predicted by their ΔCt values. The ΔCt value also indicated the zygosity of the sample according to the distribution of CFTR gene mutations. In most cases, HRMA allowed the identification of the mutation(s), thereby confirming the efficiency of this scanning strategy.
This strategy simplifies the detection of CF, reducing the analysis of 14 CF-causing mutations to two parallel reactions and making the procedure compatible with the analysis of a large number of samples. As the method is fast, inexpensive and highly reliable, it is advisable for scanning CFTR gene mutations in newborns, patients with a clinical suspicion of CF as well as in the preconception carrier screening.
囊性纤维化跨膜传导调节因子（CFTR）基因突变扫描方法可能无法区分导致囊性纤维化（CF）的突变和良性变异。我们开发了一种简单快速的方法，用于扫描在拉丁美洲具有高频率的14种选定的导致CF的突变。
在一组来自先前已鉴定的CF患者的35个样本中，分别使用针对野生型和突变型等位基因的两个等位基因特异性实时多重PCR，通过分析Ct变化来检测突变的存在。本研究还纳入了25个无突变的样本，这些样本被视为非携带者样本。进行高分辨率熔解分析（HRMA）以确认扫描方法的结果，并且在大多数情况下可以确定基因型。
结果验证了该方法用于CF诊断的有效性。正如通过其ΔCt值所预测的那样，在CF患者的样本中检测到至少一种CFTR基因突变。根据CFTR基因突变的分布，ΔCt值还表明了样本的纯合性。在大多数情况下，HRMA能够识别出突变，从而证实了这种扫描策略的有效性。
该策略简化了CF的检测，将对14种导致CF的突变的分析简化为两个平行反应，并使该程序与大量样本的分析兼容。由于该方法快速、廉价且高度可靠，因此建议用于新生儿、临床怀疑患有CF的患者以及孕前携带者筛查中的CFTR基因突变扫描。
