Mutation Analysis Of Selected Lymphoid Immune Disorders

该项目代表了为更好地描述和了解免疫缺陷而进行的一系列合作研究的继续。涉及共同伽马链(X-SCID)和Fas(Alps)基因的突变正在使用带有荧光探针的基因组DNA的桑格测序进行评估。这些研究继续在这两种疾病中发现一些新的突变，这些数据已经输入NIH NHGRI网站，支持这两种疾病。随后对高IgM综合征患者进行了针对CD40L和NEMO编码基因的突变分析，然后对免疫缺陷相关突变进行了测序，这些突变集中在宿主防御缺陷和反复感染涉及机会性细胞内生物，包括编码干扰素伽马受体1和2的基因、IL-12P40和IL-12受体β1基因。最后，新增加了新的基因，包括编码：AIRE、Artemis、BTK、FOXP3、ICOS、IL-7Rpha、JAK3、MU重链、SAP、黄蜂的基因。这项初步工作现在得到了使用Ion Torrent PGM平台的NextGen测序的补充，该平台专注于扩大与初级免疫缺陷相关的评估基因的数量，目前重点是172个已知或可能的初级免疫缺陷基因，以筛查转诊至NIH的患者，以及关于疑似初级免疫缺陷疾病的临床研究方案。这种基因突变筛查的方法依赖于已经在室内得到验证的乳胶聚合酶链式反应平台(Halopex系统)。这种选择适当文库的方法已经使用已识别的患者gDNA样本进行了验证，这些样本具有先前定义的与原发免疫缺陷疾病有关的突变。到目前为止，我们已经确定了至少一些与先前未描述的初级免疫缺陷相关的新基因，这些基因已经通过Sanger测序和功能测试得到证实。我们继续评估更多的样本，并将使用相同的原则转向更强大的平台，但可能允许整个外显子组测序以及有针对性的基因测序。我们正在准备出版我们在筛查大量患者样本中使用靶向基因方法(使用Ion Torrent和Halopex技术)的总体经验。到目前为止，我们的经验表明，对于有明确证据表明宿主防御存在缺陷的转诊患者来说，这是一种成本效益高的方法，在特定情况下使用标准Sanger方法进行重新测序可能没有必要。

This project represents is a continuation of series of collaborative studies performed to better characterize and understand immune deficiency. Mutations involving the genes for the common gamma chain (X-SCID) and Fas (ALPS) are being evaluated using Sanger sequencing of genomic DNA with fluorescent probes. These studies have continued to identify a number of new mutations in both diseases and these data have been entered into the NIH NHGRI web site supporting each of these two disorders. This was followed by the inclusion of mutation analysis of patients with hyper IgM syndrome directed at the genes encoding CD40L and NEMO followed by sequencing for immune deficiency associated mutations focused on host defense defects with recurrent infections involving opportunisitc intracellular organisms including genes encoding the interferon gamma receptor 1 and 2, the IL-12P40 and IL-12 receptor beta 1 genes. Finally, new additional genes have been added to the repertoire including genes encoding: AIRE, ARTEMIS, BTK, FOXP3, ICOS, IL-7Ralpha, JAK3, mu heavy chain,SAP, WASp. This initial work is now complemented by NextGen sequencing using the Ion Torrent PGM platform focused on expanding number of genes evaluated associated with primary immunodeficiencies that currently is focused on 172 known or possible primary immunodeficiency genes to screen patients refered to the NIH and on clinical research protocols with suspected primary immunodeficiency disorders. THis approach to gene mutation screeningd depends on emulsion PCR platform (Haloplex system) that has been validated in house. This togetehr with the choice of the appropriate library has been validated using deidentified patient gDNA samples with previously defined mutations linked to primary immunodeficiency disorders. To date we have identified at least a number of new genes associated with a previously uncharacterized primary immunodeficiency that have been confirmed by Sanger sequencing and functional testing. We continue to evaluate additional samples and will be moving to a more robust platform using the same principle but potentially allowing whole exome sequencing as well as targeted gene sequencing. We are in the process of preparing for publication our overall experience with the targeted gene approach (using the Ion Torrent and Haloplex technology) in screening a substantial number of patient samples. Our expereince to date suggests that this is a cost effective approach for screening referred patients with clear evidence of defects in host defense and that resequencing using the standard Sanger method under defined circumstances smay not be necessary.