A Novel MALDI-TOF Assay for Rapid KIR Complex Sequencing

描述（由申请人提供）：我们建议开发一种高通量测序方法，用于使用基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱法对杀伤免疫球蛋白样受体（KIR）基因进行基因分型。KIR是在自然杀伤（NK）细胞上表达的几个受体家族之一，自然杀伤（NK）细胞是先天免疫系统的组成部分，专门用于早期防御感染以及肿瘤。KIR与I类人白细胞抗原（HLA）配体相互作用，所述配体在感染和转化的细胞中下调。宿主免疫系统的这些早期应答者的活化最终是来自许多细胞表面受体（包括活化性和抑制性KIR）的信号整合的结果。KIR在个体中存在的基因的数量、类型和组合方面是多样的，并且在核苷酸水平上显示出广泛的多态性。这些高度可变的宿主遗传因素可能会显著影响对入侵病毒的免疫反应的强度和广度。我们实验室和其他实验室的初步数据2-5支持KIR及其人类白细胞抗原（HLA）配体在许多病毒传播疾病的疾病结局中起重要作用的假设。我们建议使用Sequenom公司的MassCLEAVE（TM）系统开发一种创新系统，用于从~30 ng基因组DNA中对KIR基因进行完全测序。将来自扩增靶区域的碱基特异性切割反应数据与从参考序列生成的二氧化硅切割模式进行比较，以重建靶序列。当序列变化导致裂解模式和质谱特征发生变化时，可识别多态性。一个高通量和基于序列的KIR基因分型方法将显着地进一步加深我们对这种基因复合体的多样性的理解，也将显着地有助于未来的分析KIR在疾病相关性研究的统计能力所需的大型队列。

DESCRIPTION (provided by applicant): We propose to develop a high-throughput sequencing methodology for genotyping the Killer Immunoglobulin-like Receptor (KIR) genes using matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry. KIR is 1 of several families of receptors expressed on natural killer (NK) cells, components of the innate immune system specialized for early defense against infection as well as tumors. KIR interact with class I Human Leukocyte Antigen (HLA) ligands which are down regulated in infected and transformed cells. The activation of these early responders of the host immune system is ultimately the result of signal integration from numerous cell surface receptors, including both activating and inhibitory KIR. KIR is diverse in terms of the number, the type, and the combination of genes present in individuals, and display extensive polymorphism at the nucleotide level. These highly variable host genetic factors may significantly affect both the strength and breadth of the immune response to invading viruses. Preliminary data from our lab and others 2-5 support the hypothesis that KIR and their Human Leukocyte Antigen (HLA) ligands play an important role in disease outcome for a number of virally transmitted diseases. We propose to develop an innovative system for complete sequencing of KIR genes from ~30 ng of genomic DMA using the MassCLEAVE(tm) system from Sequenom, Inc. Base-specific cleavage reaction data from amplified target areas are compared to in silica cleavage patterns generated from reference sequences in order to reconstruct the target sequence. Polymorphisms are identified when sequence change leads to changes in cleavage patterns and the resultant characteristics of the mass spectra. A high-throughput and sequence-based method of KIR genotyping would significantly further our understanding of the diversity of this gene complex, and would also contribute significantly to future analysis of KIR in the large cohorts needed for statistical power in disease association studies.