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

Background: Mutations 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.

Methods: One 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.

Conclusion: The 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.

Keywords: Children; High resolution melt; Mutation; Steroid resistant; WT1.

Fig. 1

HRM data for exon 8 and 9 of WT1 gene (standardization assay). The melt plots show five wild type control samples, which have been previously sequenced exons 8 and 9 . For each exon the Derivative plot (dF/dT) (a, b), Normalized melt curve (c, d) and Fluorescence difference plots (Δ Threshold/Temperature) (e, f) is presented

Fig. 2

POLAND Melt profile prediction (Blake and Delcourt algorithm) for WT1- Exon 8 and 9. The differentiated hypochromicity at 260 and 280 nm (dA/dT) vs. temperature plot is depicted. Theta is the calculated fraction of base pairs remaining in helical state. Both exon 8 and 9 were predicted to melt completely at a single temperature and was in concordance with the experimental melt profiles

Fig. 3

HRM Validation assay showing derivative melt plot, normalized melt curve and difference plots for exon 8 and 9 of the WT1 gene. In WT1 exon 8, the wildtype (blue) and the blinded patient sample (maroon) grouped together indicating that they have the same genotype. The mutant samples c.1079G > A; p.C360Y (orange) and c.1119C > A; p.H373Q (green) were distinguished from the wildtype sample (Fig. 3 a, c and e). Similary in WT1exon 9, the wildtype (green) and the blinded patient sample (maroon) grouped together and the mutant samples c.1180C > T; p.R394W (red), c.1190A > C; p.H397P (pink) and c.A1200C > T; p.H401Y (black) were easily distinguished from the wild type (Fig. 3 b, d and f)