快速检测儿童期起病的激素耐药性肾病综合征的单基因病因。
Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome.
机构信息
Division of Nephrology, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts;
Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, Michigan;
出版信息
Clin J Am Soc Nephrol. 2014 Jun 6;9(6):1109-16. doi: 10.2215/CJN.09010813. Epub 2014 Apr 17.
BACKGROUND AND OBJECTIVES
In steroid-resistant nephrotic syndrome (SRNS), >21 single-gene causes are known. However, mutation analysis of all known SRNS genes is time and cost intensive. This report describes a new high-throughput method of mutation analysis using a PCR-based microfluidic technology that allows rapid simultaneous mutation analysis of 21 single-gene causes of SRNS in a large number of individuals.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: This study screened individuals with SRNS; samples were submitted for mutation analysis from international sources between 1996 and 2012. For proof of principle, a pilot cohort of 48 individuals who harbored known mutations in known SRNS genes was evaluated. After improvements to the method, 48 individuals with an unknown cause of SRNS were then examined in a subsequent diagnostic study. The analysis included 16 recessive SRNS genes and 5 dominant SRNS genes. A 10-fold primer multiplexing was applied, allowing PCR-based amplification of 474 amplicons in 21 genes for 48 DNA samples simultaneously. Forty-eight individuals were indexed in a barcode PCR, and high-throughput sequencing was performed. All disease-causing variants were confirmed via Sanger sequencing.
RESULTS
The pilot study identified the genetic cause of disease in 42 of 48 (87.5%) of the affected individuals. The diagnostic study detected the genetic cause of disease in 16 of 48 (33%) of the affected individuals with a previously unknown cause of SRNS. Seven novel disease-causing mutations in PLCE1 (n=5), NPHS1 (n=1), and LAMB2 (n=1) were identified in <3 weeks. Use of this method could reduce costs to 1/29th of the cost of Sanger sequencing.
CONCLUSION
This highly parallel approach allows rapid (<3 weeks) mutation analysis of 21 genes known to cause SRNS at a greatly reduced cost (1/29th) compared with traditional mutation analysis techniques. It detects mutations in about 33% of childhood-onset SRNS cases.
背景与目的
在类固醇耐药性肾病综合征（SRNS）中，已知有>21 个单基因病因。然而，对所有已知的 SRNS 基因进行突变分析既费时又费钱。本报告描述了一种新的高通量突变分析方法，该方法使用基于 PCR 的微流控技术，可在大量个体中快速同时分析 21 个导致 SRNS 的单基因病因。
设计、地点、参与者和测量方法：本研究筛查了患有 SRNS 的个体；样本于 1996 年至 2012 年期间从国际来源提交进行突变分析。作为原理验证，评估了一个包含 48 名已知存在已知 SRNS 基因突变的个体的试点队列。在方法改进后，随后对 48 名患有未知原因 SRNS 的个体进行了诊断性研究。该分析包括 16 个隐性 SRNS 基因和 5 个显性 SRNS 基因。应用了 10 倍引物多重化，允许同时对 21 个基因的 474 个扩增子进行基于 PCR 的扩增，共对 48 个 DNA 样本进行扩增。48 个个体在条形码 PCR 中进行索引，然后进行高通量测序。所有致病变异均通过 Sanger 测序进行确认。
结果
该试点研究在 48 名受影响个体中的 42 名（87.5%）中确定了疾病的遗传原因。在诊断性研究中，在 48 名先前未知病因的 SRNS 个体中，有 16 名（33%）检测到疾病的遗传原因。在不到 3 周的时间内，在 PLCE1（n=5）、NPHS1（n=1）和 LAMB2（n=1）中发现了 7 个新的致病突变。与传统的突变分析技术相比，该方法的使用可以将成本降低到 1/29。
结论
这种高度并行的方法可在不到 3 周的时间内快速（<3 周）分析已知导致 SRNS 的 21 个基因的突变，成本降低了 1/29（与传统突变分析技术相比）。它可检测到约 33%的儿童发病 SRNS 病例的突变。
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