Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

Abstract

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

RETRACTED ARTICLE: Frequency of TP53 Mutations and its Impact on Drug Sensitivity in Acute Myeloid Leukemia?

“…It is a closed technique so very less chances of contamination and also it is non-destructive so the same sample can be used to analyse further. The development of new fluorescent dye makes this method more attractive and target oriented for mutation scanning [23][24][25].…”

Detection of High-Frequency and Novel DNMT3A Mutations in Acute Myeloid Leukemia by High-Resolution Melting Curve Analysis

“…HRM analysis relies on conformational differences and altered melting curves monitored in real time by the release of fluorescent double-strand DNA binding dye 15 and has been used to assess for mutations in clinically important genes including PIK3CA, KRAS, TP53, IDH1, and IDH2. 16,17 In this study, we used HRM analysis to screen for mutations in exons 8 through 10 and 15 through 23 that encode PWWP (proline-tryptophan-tryptophan-proline), ADD (ATR-DNMT3-DNMT3L) zinc finger, and MTase domains of the DNMT3A. All samples were also assessed by bidirectional Sanger sequencing.…”

A Novel HRM Strategy for Detection of BRCA Mutations on Mexican Population

“…The purpose of our study was to evaluate the efficacy of the HRM method in screening BRCA1/2 mutations in a sample of Mexican population. High Resolution DNA Melting Analysis was first described in 2003 [33] and it has been used to screen many different mutations relevant in cancer, specifically breast cancer related mutations on BRCA genes [33][34][35][36].…”

“…Recently we performed a meta-analysis to look for some of the most probable mutations affecting this particular ethnic group [34]. These results together with a previous bibliographic research focused exclusively on Mexican population, lead us to select the mutations named in this work.…”