Array-Based Sequence Capture and Next-Generation Sequencing for the Identification of Primary Immunodeficiencies
Array-Based Sequence Capture and Next-Generation Sequencing for the Identification of Primary Immunodeficiencies
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DOI：
10.1111/j.1365-3083.2011.02658.x
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发表时间：
2012-03-01
影响因子：
3.7
通讯作者：
Borkhardt, A.
中科院分区：
文献类型：
--
作者：
Ghosh, S.;Krux, F.;Borkhardt, A.
原发性免疫缺陷是一种遗传性疾病，其中免疫途径的组成部分缺失或失调。随着下一代测序的出现，在具有异质遗传背景的条件下测试基因似乎更有希望。我们设计了具有 385K 探针能力的定制微阵列，可捕获 395 个人类基因的外显子，这些基因已知或预测与原发性免疫缺陷和免疫调节相关。使用 GS FLX Titanium 454 平台对富集的目标 DNA 进行测序。所选患者可能患有潜在的免疫缺陷。在一名患有肝脾肿大、反复感染和 IgM 水平升高的患者中，对该患者及其两位未受影响的父母进行的序列分析确定 ATM（毛细血管扩张共济失调突变）是潜在的缺陷。在第二个具有临床 SCID 表型的孩子中，我们在关注 SCID 相关基因后检测到了 ARTEMIS 基因的突变。 454 测序为每位患者产生了 152,000397,000 条高质量读数。 7899% 的目标核苷酸至少被覆盖一次，7682% 至少被覆盖五次。基于阵列的序列捕获扩展了我们以更省力、更省时的方法对大型目标 DNA 区域进行测序的能力。我们的阵列能够发现两名疑似原发性免疫缺陷患者的潜在遗传缺陷。即将到来的全外显子组测序肯定会增加更多有价值的数据，但生物信息分析和变异验证已经提出了重大挑战。
Primary immunodeficiencies are genetic disorders in which components of immunological pathways are either missing or dysregulated. With the advent of next-generation sequencing, testing for genes in conditions with a heterogeneous genetic background seems more promising. We designed a custom microarray with 385K probe capacity to capture exons of 395 human genes, known or predicted to be associated with primary immunodeficiency and immune regulation. Enriched target DNA was sequenced using a GS FLX Titanium 454 platform. The patients selected were likely to have an underlying immunodeficiency. In one patient with hepatosplenomegaly, recurrent infections and an elevated IgM level, sequence analysis of the patient and his two unaffected parents identified ATM (ataxia telangiectasia mutated) as the underlying defect. In a second child with a clinical SCID phenotype, we detected a mutation in the ARTEMIS gene after focusing on SCID-associated genes. 454 sequencing yielded 152,000397,000 high-quality reads per patient. 7899% of the targeted nucleotides were covered at least one time, 7682% at least five times. Array-based sequence capture expands our capacities to sequence large targeted DNA regions in a less laborious and time-consuming approach. Our array was capable to find the underlying genetic defect in two patients with suspected primary immunodeficiency. Upcoming whole-exome sequencing definitely will add more valuable data, but bioinformatical analysis and validation of variants already pose major challenges.