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.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
authors.forename: Annes
authors.initials: A
authors.lastname: Siji
authors.affiliation: Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
authors.forename: Varsha Chhotusing
authors.initials: VC
authors.lastname: Pardeshi
authors.affiliation: Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
authors.forename: Shilpa
authors.initials: S
authors.lastname: Ravindran
authors.affiliation: Division of Molecular Medicine, St. John's Research Institute, Bangalore, India.
authors.forename: Ambily
authors.initials: A
authors.lastname: Vasudevan
authors.affiliation: Division of Molecular Medicine, St. John's Research Institute, Bangalore, India. anil.vasudevan@sjri.res.in.|Department of Pediatric Nephrology, St. John's Medical College Hospital, Bangalore, 560034, India. anil.vasudevan@sjri.res.in.
authors.forename: Anil
authors.initials: A
authors.lastname: Vasudevan
mesh_terms: D002648:Child; D002675:Child, Preschool; D004351:Drug Resistance; D005091:Exons; D005260:Female; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D006801:Humans; D007223:Infant; D008297:Male; D009154:Mutation; D009404:Nephrotic Syndrome / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards; D013256:Steroids / Q000627:therapeutic use; D025721:WT1 Proteins / Q000235:genetics*
chemical_list: D013256:Steroids; D025721:WT1 Proteins; C583751:WT1 protein, human
keywords: Children; High resolution melt; Mutation; Steroid resistant; WT1