Dataset Information

High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

ABSTRACT: BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. METHODS: We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. RESULTS: We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. CONCLUSION: HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

SUBMITTER: Bataille S

PROVIDER: S-EPMC3206831 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

Bataille Stanislas S Berland Yvon Y Fontes Michel M Burtey Stéphane S

BMC nephrology 20111018

<h4>Background</h4>Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious.<h4>Methods</h4>We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate m ...[more]

PMID: 22008521

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Project description:AimThis study has three broad aims: to (a) develop genus-specific primers for High Resolution Melt analysis (HRM) of members of Cyclopia Vent., (b) test the haplotype discrimination of HRM compared to Sanger sequencing, and (c) provide an example of using HRM to detect novel haplotype variation in wild C. subternata Vogel. populations.LocationThe Cape Floristic Region (CFR), located along the southern Cape of South Africa.MethodsPolymorphic loci were detected through a screening process of sequencing 12 non-coding chloroplast DNA segments across 14 Cyclopia species. Twelve genus-specific primer combinations were designed around variable cpDNA loci, four of which failed to amplify under PCR; the eight remaining were applied to test the specificity, sensitivity and accuracy of HRM. The three top performing HRM Primer combinations were then applied to detect novel haplotypes in wild C. subternata populations, and phylogeographic patterns of C. subternata were explored.ResultsWe present a framework for applying HRM to non-model systems. HRM accuracy varied across the PCR products screened using the genus-specific primers developed, ranging between 56 and 100%. The nucleotide variation failing to produce distinct melt curves is discussed. The top three performing regions, having 100% specificity (i.e. different haplotypes were never grouped into the same cluster, no false negatives), were able to detect novel haplotypes in wild C. subternata populations with high accuracy (96%). Sensitivity below 100% (i.e. a single haplotype being clustered into multiple unique groups during HRM curve analysis, false positives) was resolved through sequence confirmation of each cluster resulting in a final accuracy of 100%. Phylogeographic analyses revealed that wild C. subternata populations tend to exhibit phylogeographic structuring across mountain ranges (accounting for 73.8% of genetic variation base on an AMOVA), and genetic differentiation between populations increases with distance (p < 0.05 for IBD analyses).ConclusionsAfter screening for regions with high HRM clustering specificity-akin to the screening process associated with most PCR based markers-the technology was found to be a high throughput tool for detecting genetic variation in non-model plants.

Dataset Information

High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

Publications

High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

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