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

Abstract

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.

Keywords: focal segmental glomerulosclerosis; genetic renal disease; human genetics; molecular genetics; nephrotic syndrome.

Figure 1.

Minimum coverage of targeted amplicons. The y axis indicates the number of sequencing reads (coverage). The x axis indicates each amplicon from 1 to 474, sorted by coverage in descending order. Pilot study coverage data are shown by blue triangles, and diagnostic study coverage data are shown by red triangles. Note that in the pilot study, 409 of 474 amplicons (82.3%) had a minimum coverage depth (horizontal black line) >20-fold (blue triangles). In the diagnostic study, 462 of 474 amplicons (97.4%) had a minimum coverage depth >20-fold (red triangles).

Figure 2.

Relative proportion of solved cases in 48 families with steroid-resistant nephrotic syndrome (SRNS) in the diagnostic study. In the diagnostic study, 48 individuals from 48 different families with an unknown cause of SRNS were screened for variants in 21 genes known to cause SRNS. In 16 of 48 individuals, the underlying molecular cause was identified. Numbers in parentheses following the gene symbols represent the number of patients with mutations in each respective gene.