Rapid detection of monogenic causes of childhood-onset steroid-resistant nephrotic syndrome.

Background and objectives In steroid-resistant nephrotic syndrome (SRNS), .21 single-gene causes are known. However,mutationanalysisofallknownSRNSgenesistimeandcostintensive.Thisreportdescribesanewhighthroughput 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 10fold 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.