Targeted next-generation sequencing in steroid-resistant nephrotic syndrome: mutations in multiple glomerular genes may influence disease severity

Abstract

Genetic diagnosis of steroid-resistant nephrotic syndrome (SRNS) using Sanger sequencing is complicated by the high genetic heterogeneity and phenotypic variability of this disease. We aimed to improve the genetic diagnosis of SRNS by simultaneously sequencing 26 glomerular genes using massive parallel sequencing and to study whether mutations in multiple genes increase disease severity. High-throughput mutation analysis was performed in 50 SRNS and/or focal segmental glomerulosclerosis (FSGS) patients, a validation cohort of 25 patients with known pathogenic mutations, and a discovery cohort of 25 uncharacterized patients with probable genetic etiology. In the validation cohort, we identified the 42 previously known pathogenic mutations across NPHS1, NPHS2, WT1, TRPC6, and INF2 genes. In the discovery cohort, disease-causing mutations in SRNS/FSGS genes were found in nine patients. We detected three patients with mutations in an SRNS/FSGS gene and COL4A3. Two of them were familial cases and presented a more severe phenotype than family members with mutation in only one gene. In conclusion, our results show that massive parallel sequencing is feasible and robust for genetic diagnosis of SRNS/FSGS. Our results indicate that patients carrying mutations in an SRNS/FSGS gene and also in COL4A3 gene have increased disease severity.

Figure 1

Pedigrees of two families with mutations in an SRNS/FSGS gene and COL4A3. (a) In family 10, both siblings had compound heterozygous pathogenic mutations in NPHS2 gene and the more severely affected individual (10-1) carried an additional likely pathogenic variant in COL4A3 gene. (b) In family 253, individuals 253-1 to -4 carried a pathogenic mutation in COL4A3 gene demonstrated to produce exon 46 skipping by reverse transcriptase-PCR and Sanger sequencing and predicted to result in a protein lacking 42 amino acids. Patient 253-1 carried an additional variant in INF2 gene inherited from her mother and developed a more aggressive phenotype than the other affected family members. Cr, creatinine; wt, wild type. The arrows indicate probands. Squares denote males, circles denote females. Filled symbols indicate affected status. Quarter solid symbols indicate microhematuria.