The antibody genetics of multiple sclerosis: comparing next-generation sequencing to sanger sequencing.
The antibody genetics of multiple sclerosis: comparing next-generation sequencing to sanger sequencing.
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DOI：
10.3389/fneur.2014.00166
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发表时间：
2014
影响因子：
3.4
通讯作者：
Monson NL
中科院分区：
文献类型：
--
作者：
Rounds WH;Ligocki AJ;Levin MK;Greenberg BM;Bigwood DW;Eastman EM;Cowell LG;Monson NL
我们之前在脑脊液（CSF）中分离的B细胞抗体基因中发现了一种独特的突变模式，可以识别复发-缓解型多发性硬化症（RRMS）患者和临床分离综合征将转化为RRMS的患者。该抗体基因标记（AGS）是利用单个B细胞的Sanger测序开发的。虽然对患者有潜在的帮助，但Sanger测序并不是一种可以在临床环境中实际应用的检测方法。为了给患者提供AGS评估作为其诊断检查的一部分，我们开发了使用下一代DNA测序（NGS）对csf来源的细胞颗粒生成AGS评分的方案，而无需分离单个细胞。这种方法有可能增加被分析的b细胞群的覆盖率，减少生成AGS评分所需的时间，并可能在未来提高AGS方法作为诊断测试的整体性能。然而，没有研究关注基于ngs的谱库是否能正确反映Sanger测序定义的抗体基因频率和体细胞超突变模式。为了解决这一问题，我们从8名患有多发性硬化症或有患多发性硬化症风险的患者中分离了成对的脑脊液样本。在这里，我们提供的数据表明，这些成对脑脊液样本中基于Sanger和ngs的抗体库的抗体基因频率和体细胞超突变模式相似。此外，来自NGS数据库的AGS评分正确地识别了最初患有或随后转化为RRMS的患者，其精度与Sanger测序方法相似。有必要在更大的患者队列中进一步研究AGS在预测使用ngs衍生抗体库转化为多发性硬化症方面的效用。
We previously identified a distinct mutation pattern in the antibody genes of B cells isolated from cerebrospinal fluid (CSF) that can identify patients who have relapsing-remitting multiple sclerosis (RRMS) and patients with clinically isolated syndromes who will convert to RRMS. This antibody gene signature (AGS) was developed using Sanger sequencing of single B cells. While potentially helpful to patients, Sanger sequencing is not an assay that can be practically deployed in clinical settings. In order to provide AGS evaluations to patients as part of their diagnostic workup, we developed protocols to generate AGS scores using next-generation DNA sequencing (NGS) on CSF-derived cell pellets without the need to isolate single cells. This approach has the potential to increase the coverage of the B-cell population being analyzed, reduce the time needed to generate AGS scores, and may improve the overall performance of the AGS approach as a diagnostic test in the future. However, no investigations have focused on whether NGS-based repertoires will properly reflect antibody gene frequencies and somatic hypermutation patterns defined by Sanger sequencing. To address this issue, we isolated paired CSF samples from eight patients who either had MS or were at risk to develop MS. Here, we present data that antibody gene frequencies and somatic hypermutation patterns are similar in Sanger and NGS-based antibody repertoires from these paired CSF samples. In addition, AGS scores derived from the NGS database correctly identified the patients who initially had or subsequently converted to RRMS, with precision similar to that of the Sanger sequencing approach. Further investigation of the utility of the AGS in predicting conversion to MS using NGS-derived antibody repertoires in a larger cohort of patients is warranted.
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通讯作者：
Monson, Nancy L.
影响因子：
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