Simultaneous Mutation Detection in 90 Retinal Disease Genes in Multiple Patients Using a Custom-designed 300-kb Retinal Resequencing Chip
Simultaneous Mutation Detection in 90 Retinal Disease Genes in Multiple Patients Using a Custom-designed 300-kb Retinal Resequencing Chip
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DOI：
10.1016/j.ophtha.2010.04.022
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发表时间：
2011-01-01
期刊：
影响因子：
13.7
通讯作者：
Florijn, Ralph J.
中科院分区：
文献类型：
--
作者：
Booij, Judith C.;Bakker, Arne;Florijn, Ralph J.
目的：为了开发一种高通量，具有成本效益的诊断策略，用于识别已知的和新的突变，在90视网膜疾病genes.Design：循证研究参与者：60例患者与各种视网膜疾病，包括Leber的先天性黑蒙，眼白化病，弹性假黄瘤，视网膜色素变性，和Stargardt的diseases.Methods：我们设计了一个定制的300 kb重测序芯片。根据Affytechnology的建议进行聚合酶链反应（PCR）扩增、DNA片段化和芯片杂交。使用Sequence pilot模块seq-C突变检测软件（2009）分析杂交信号。桑格序列technology.Main结果措施：致病序列的变化。结果：我们开发了一个视网膜重测序芯片，涵盖了所有外显子的90视网膜疾病基因。我们开发并测试了1445个扩增子的多重引物组，这些扩增子代表了芯片上包含的基因。我们验证了我们的方法，筛选87外显子从25个视网膜疾病基因含有87个已知的序列变化，以前确定在我们的患者组中使用桑格测序。成功杂交的扩增子的检出率为98%至100%。在已知的单核苷酸变化中，99%可以在芯片上检测到。正如预期的那样，删除不能被reliably detected.Conclusions：我们设计了一个自定义的重测序芯片，可以检测已知的和新的序列变化90视网膜疾病基因使用一个新的高通量的策略，具有高灵敏度和特异性的十分之一的成本，传统的直接测序。所开发的扩增策略允许汇集具有非重叠表型的多个患者，使得能够以快速且具有成本效益的方式同时分析许多患者。
Purpose: To develop a high-throughput, cost-effective diagnostic strategy for the identification of known and new mutations in 90 retinal disease genes.Design: Evidence-based study.Participants: Sixty patients with a variety of retinal disorders, including Leber's congenital amaurosis, ocular albinism, pseudoxanthoma elasticum, retinitis pigmentosa, and Stargardt's disease.Methods: We designed a custom 300-kb resequencing chip. Polymerase chain reaction (PCR) amplification, DNA fragmentation, and chip hybridization were performed according to Affymetrix recommendations. Hybridization signals were analyzed using Sequence pilot module seq-C mutation detection software (2009). This resequencing approach was validated by Sanger sequence technology.Main Outcome Measures: Disease-causing sequence changes.Results: We developed a retinal resequencing chip that covers all exons of 90 retinal disease genes. We developed and tested multiplex primer sets for 1445 amplicons representing the genes included on the chip. We validated our approach by screening 87 exons from 25 retinal disease genes containing 87 known sequence changes previously identified in our patient group using Sanger sequencing. Call rates for successfully hybridized amplicons were 98% to 100%. Of the known single nucleotide changes, 99% could be detected on the chip. As expected, deletions could not be detected reliably.Conclusions: We designed a custom resequencing chip that can detect known and new sequence changes in 90 retinal disease genes using a new high-throughput strategy with a high sensitivity and specificity for one tenth of the cost of conventional direct sequencing. The developed amplification strategy allows for the pooling of multiple patients with non-overlapping phenotypes, enabling many patients to be analyzed simultaneously in a fast and cost-effective manner.