Screening of WT1 mutations in exon 8 and 9 in children with steroid resistant nephrotic syndrome from a single centre and establishment of a rapid screening assay using high-resolution melting analysis in a clinical setting

Abstract

BackgroundMutations in Wilm’s tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only mutation positive samples identified by HRM will be further processed for sequencing it will help in reducing the sequencing burden and speed up the screening process.MethodsOne hundred SRNS children were screened for WT1 mutations in Exon 8 and 9 using Sanger sequencing. HRM assay was standardized and validated by performing analysis for exon 8 and 9 on 3 healthy control and 5 abnormal variants created by site directed mutagenesis and verified by sequencing. To further test the clinical applicability of the assay, we screened additional 91 samples for HRM testing and performed a blinded assessment.Results WT1 mutations were not observed in the cohort of children with SRNS. The results of HRM analysis were concordant with the sequencing results.ConclusionThe WT1 gene mutations were not observed in the SRNS cohort indicating it has a low prevalence. We propose applying this simple, rapid and cost effective assay using HRM technique as the first step for screening the WT1 gene hot spot region in a clinical setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0362-7) contains supplementary material, which is available to authorized users.

Targeted gene panel for genetic testing of south Indian children with steroid resistant nephrotic syndrome

“…However, reports from India including from our center showed that the prevalence of NPHS2 mutations is much lower in Indian population when compared with Europe and North American population [4% vs. 10.5–28%)] [ 7 – 12 ]. Kumar et al, reported low prevalence of WT1 mutation in south Indian population, whereas we did not detect any mutation in WT1 gene in 100 SRNS children [ 13 , 14 ]. These data suggest that a traditional genetic testing using an algorithmic approach based on age of onset of NS to prioritize the genes to be sequenced by Sanger may not be useful [ 15 , 16 ].…”

“…Socio demographic information, clinical and treatment details were recorded in case record forms. All these children were previously analyzed by Sanger sequencing for all the exons of NPHS2 and exon 8 and 9 of WT1 genes [ 7 , 14 ].We also included three subjects with pathogenic mutations in NPHS2 reported previously to determine the sensitivity of the targeted-NGS method [ 7 ]. Three healthy individuals were included to check sequencing efficiency.…”

The Inflammatory Process Modulates the Expression and Localization of WT1 in Podocytes Leading to Kidney Damage

“…Mutation of WT1 is commonly reported in children with steroid-resistant nephrotic syndrome, a rare disease that affect 10-15% of patients with nephrotic syndrome (12,13). About 80% of all children with sporadic nephrotic syndrome respond to steroid treatment, however, no mutations were found in WT1 in a large cohort study (14).…”