Mutation Analysis Of Selected Lymphoid Immune Disorders

该项目是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。目前正在利用荧光探针对基因组DNA进行Sanger测序，评估涉及共同γ链（X-SCID）和Fas （ALPS）基因的突变。这些研究继续在这两种疾病中确定了许多新的突变，这些数据已被输入NIH NHGRI网站，以支持这两种疾病。随后纳入了针对编码CD40L和NEMO基因的高IgM综合征患者的突变分析，然后对涉及机会性细胞内生物的复发性感染的宿主防御缺陷的免疫缺陷相关突变进行测序，包括编码干扰素γ受体1和2，IL-12P40和IL-12受体β 1基因的基因。最后，新增加的基因包括AIRE、ARTEMIS、BTK、FOXP3、ICOS、IL-7Ralpha、JAK3、mu重链、SAP、WASp。这项初步工作现在得到了NextGen测序的补充，该测序使用Ion Torrent PGM平台，专注于扩大与原发性免疫缺陷相关的基因评估数量，目前专注于172个已知或可能的原发性免疫缺陷基因，以筛选提交给NIH的患者和疑似原发性免疫缺陷疾病的临床研究方案。这种基因突变筛选方法依赖于已在内部验证的乳液PCR平台（Haloplex系统）。这与选择合适的文库一起，已经使用去鉴定的患者gDNA样本进行了验证，这些样本具有先前定义的与原发性免疫缺陷疾病相关的突变。到目前为止，我们已经确定了至少一些与以前未表征的原发性免疫缺陷相关的新基因，这些基因已被Sanger测序和功能测试证实。我们将继续评估更多的样品，并将使用相同的原理转向更强大的平台，但可能允许全外显子组测序以及靶向基因测序。我们正在准备发表我们在筛选大量患者样本中使用靶向基因方法（使用Ion Torrent和Haloplex技术）的总体经验。迄今为止，我们的经验表明，这是一种成本有效的方法，用于筛选有明确证据表明宿主防御缺陷的转诊患者，在特定情况下，使用标准Sanger方法进行重测序可能是不必要的。

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.