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BMC Medical Genetics

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Open Access 01-12-2017 | Technical advance

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

Authors: Annes Siji, Varsha Chhotusing Pardeshi, Shilpa Ravindran, Ambily Vasudevan, Anil Vasudevan

Published in: BMC Medical Genetics | Issue 1/2017

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.

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Title: 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
Authors: Annes Siji
Varsha Chhotusing Pardeshi
Shilpa Ravindran
Ambily Vasudevan
Anil Vasudevan
Publication date: 01-12-2017
Publisher: BioMed Central
Published in: BMC Medical Genetics / Issue 1/2017
Electronic ISSN: 1471-2350
DOI: https://doi.org/10.1186/s12881-016-0362-7

Keynote webinar | Spotlight on medication adherence

Springer Medicine

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

Methods

Results

Conclusion

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

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