High‐Resolution Melt as a Screening Method in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Issue 4 (22nd March 2014)

• Record Type: Journal Article
• Authors: Virzì, Grazia Maria; Bruson, Alice; Corradi, Valentina; Gastaldon, Fiorella; de Cal, Massimo; Donà, Marta; Cruz, Dinna N.; Clementi, Maurizio; Ronco, Claudio
• Is Part Of: Journal of clinical laboratory analysis. Volume 28:Issue 4(2014:Jul.)
• Page Start: 328
• Page End: 334
• Publication Date: 2014-03-22
• Subjects: Diagnosis, Laboratory -- Periodicals; Medical laboratory technology -- Periodicals; 616
• DOI: 10.1002/jcla.21689
• Languages: English
• ISSNs: 0887-8013

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="jcla21689-sec-0010" sec-type="section"> <title>Background</title> <p>Autosomal dominant polycystic kidney disease (ADPKD) is an inherited condition caused by <italic>PKD1</italic> and <italic>PKD2</italic> mutations. Complete analysis of both genes is typically required in each patient. In this study, we explored the utility of High‐Resolution Melt (HRM) as a tool for mutation analysis of the <italic>PKD2</italic> gene in ADPKD families.</p> </sec> <sec id="jcla21689-sec-0020" sec-type="section"> <title>Methods</title> <p>HRM is a mismatch‐detection method based on the difference of fluorescence absorbance behavior during the melting of the DNA double strand to denatured single strands in a mutant sample as compared to a reference control. Our families were previously screened by linkage analysis. Subsequently, HRM was used to characterize <italic>PKD2</italic>‐linked families. Amplicons that produced an overlapping profile sample versus wild‐type control were not further evaluated, while those amplicons with profile deviated from the control were consequently sequenced.</p> </sec> <sec id="jcla21689-sec-0030" sec-type="section"> <title>Results</title> <p>We analyzed 16 <italic>PKD2‐</italic>linked families by HRM analysis. We observed ten different variations: six single‐nucleotide polymorphisms and four mutations. The mutations detected by HRM and confirmed by sequencing were as follows: 1158T>A, 2159delA, 2224C>T, and 2533C>T. In particular, the same haplotype block and nonsense mutation 2533C>T was found in 8 of 16 families, so we suggested the presence of a founder effect in our province.</p> </sec> <sec id="jcla21689-sec-0040" sec-type="section"> <title>Conclusions</title> <p>We have developed a strategy for rapid mutation analysis of the <italic>PKD2</italic> gene in ADPKD families, which utilizes an HRM‐based prescreening followed by direct sequencing of amplicons with abnormal profiles. This is a simple and good technique for <italic>PKD2</italic> genotyping and may significantly reduce the time and cost for diagnosis in ADPKD.</p> </sec> </abstract>