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 settingReportar como inadecuado




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 - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Medical Genetics

, 18:3

Clinical-Molecular Genetics and Cytogenetics

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.

ResultsWT1 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.

KeywordsChildren WT1 Steroid resistant High resolution melt Mutation AbbreviationsCKDChronic kidney disease

DDSDenys-Drash syndrome

DHPLCDenaturing high performance liquid chromatography

DMSDiffuse mesangial sclerosis

ESRDEnd-stage renal disease

FMPAFluorescent multiplexed-PCR analysis

FSFrasier syndrome

HRMHigh resolution melt

NSNephrotic syndrome

PCRPolymerase chain reaction

SNPSingle nucleotide polymorphism

SRNSSteroid resistant nephrotic syndrome

TmMelting temperature

WT1Wilm’s tumor 1

Electronic supplementary materialThe online version of this article doi:10.1186-s12881-016-0362-7 contains supplementary material, which is available to authorized users.

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Autor: Annes Siji - Varsha Chhotusing Pardeshi - Shilpa Ravindran - Ambily Vasudevan - Anil Vasudevan

Fuente: https://link.springer.com/



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