Laboratório de Genética Molecular Humana, Universidade Federal do Paraná, Curitiba, Brazil.
Department of Neurology, University of California San Francisco, San Francisco, California.
HLA. 2018 Dec;92(6):384-391. doi: 10.1111/tan.13430.
Killer cell immunoglobulin-like receptor (KIR) genes encode cell surface molecules that recognize HLA molecules and modulate the activity of natural killer (NK) cells. KIR genes exhibit presence and absence polymorphism, which generates a variety of gene-content haplotypes in worldwide populations. KIR gene-content variation is implicated in many diseases and is also important for placentation and transplantation. Because of the complexity of KIR polymorphism, variation in this family is still mostly studied at the gene-content level, even with the advent of next-generation sequencing (NGS) methods. Gene-content determination is generally expensive and/or time-consuming. To overcome these difficulties, we developed a method based on multiplex polymerase chain reaction with specific sequence primers (PCR-SSP) followed by melting curve analysis that allows cost-effective, precise and fast generation of results. Our method was 100% concordant with a gel-based method and 99.9% concordant with presence and absence determination by NGS. The limit of detection for accurate typing was 30 ng of DNA (0.42 μM) with 260/230 and 260/280 ratios as low as 0.19 and of 0.44. In addition, we developed a user-friendly Java-based computational application called killerPeak that interprets the raw data generated by Viia7 or QuantStudio 7 quantitative PCR machines and reliably exports the final genotyping results in spreadsheet file format. The combination of a reliable method that requires low amount of DNA with an automated interpretation of results allows scaling the KIR genotyping in large cohorts with reduced turnaround time.
杀伤细胞免疫球蛋白样受体 (KIR) 基因编码细胞表面分子，可识别 HLA 分子并调节自然杀伤 (NK) 细胞的活性。KIR 基因表现出存在和缺失多态性，在全球人群中产生了多种基因含量单倍型。KIR 基因含量的变异与许多疾病有关，对胎盘形成和移植也很重要。由于 KIR 多态性的复杂性，该家族的变异在很大程度上仍主要在基因含量水平上进行研究，即使有了下一代测序 (NGS) 方法也是如此。基因含量的测定通常既昂贵又耗时。为了克服这些困难，我们开发了一种基于多重聚合酶链反应与特定序列引物 (PCR-SSP) 结合熔解曲线分析的方法，该方法允许以具有成本效益、精确和快速的方式生成结果。我们的方法与凝胶方法 100%一致，与 NGS 存在和缺失的测定 99.9%一致。准确分型的检测限为 30ng DNA（0.42 μM），260/230 和 260/280 比值低至 0.19 和 0.44。此外，我们开发了一种名为 killerPeak 的基于 Java 的用户友好型计算应用程序，可解释 Viia7 或 QuantStudio 7 定量 PCR 仪器生成的原始数据，并可靠地以电子表格文件格式导出最终的基因分型结果。这种需要少量 DNA 的可靠方法与结果的自动解释相结合，允许在减少周转时间的情况下，对大型队列进行 KIR 基因分型。
