Genetic Variant Detection in the CALR gene using High Resolution Melting Analysis

Summary

High resolution melting analysis (HRM) is a sensitive and rapid solution for genetic variant detection. It depends on sequence differences that result in heteroduplexes changing the shape of the melting curve. By combing HRM and agarose gel electrophoresis, different types of genetic variants such as indels can be identified.

Abstract

High resolution melting analysis (HRM) is a powerful method for genotyping and genetic variation scanning. Most HRM applications depend on saturating DNA dyes that detect sequence differences, and heteroduplexes that change the shape of the melting curve. Excellent instrument resolution and special data analysis software are needed to identify the small melting curve differences that identify a variant or genotype. Different types of genetic variants with diverse frequencies can be observed in the gene specific for patients with a specific disease, especially cancer and in the CALR gene in patients with Philadelphia chromosome–negative myeloproliferative neoplasms. Single nucleotide changes, insertions and/or deletions (indels) in the gene of interest can be detected by the HRM analysis. The identification of different types of genetic variants is mostly based on the controls used in the qPCR HRM assay. However, as the product length increases, the difference between wild-type and heterozygote curves becomes smaller, and the type of genetic variant is more difficult to determine. Therefore, where indels are the prevalent genetic variant expected in the gene of interest, an additional method such as agarose gel electrophoresis can be used for the clarification of the HRM result. In some instances, an inconclusive result must be re-checked/re-diagnosed by standard Sanger sequencing. In this retrospective study, we applied the method to JAK2 V617F-negative patients with MPN.

Introduction

Somatic genetic variants in the calreticulin gene (CALR) were recognized in 2013 in patients with myeloproliferative neoplasms (MPN) such as essential thrombocythemia and primary myelofibrosis^1,2. Since then, more than 50 genetic variants in the CALR gene have been discovered, inducing a +1 (−1+2) frameshift³. The two most frequent CALR genetic variants are a 52 bp deletion (NM_004343.3 (CALR):c.1099_1150del52, p.(Leu367Thrfs*46)), also called type 1 mutation, and a 5 bp insertion (NM_004343.3 (CALR):c.1154_1155insTTGTC, p.(Lys385Asnfs*....

Protocol

The study was approved by the Committee of Medical Ethics of the Republic of Slovenia. All procedures were in accordance with the Helsinki declaration.

1. Fluorescence-based quantitative real-time PCR (qPCR) and post-qPCR analysis by HRM

1. Resuspend primers listed in the Table of Materials to 100 µM with sterile, RNase and DNase free H₂O (see Table of Materials). Make a 10 µM working concentration primer. Primers used in the protocol were published before².
2. Quantitate granulocytes DNA by the fluorescence staining method following the manufactu....

Results

The successfully amplified DNA region of interest with an exponential increase in fluorescence that exceeds the threshold between cycles 15 and 35 and very narrow values of the cycle of quantification (Cq) in all replicated samples and controls (Figure 2) is a prerequisite for the reliable identification of genetic variants by HRM analysis. This is achieved by using a precise determination of DNA with fluorescence staining and an equal amount of DNA in the qPCR HRM experiment (see step 1.2)........

Discussion

High-resolution melting of DNA is a simple solution for genotyping and genetic variant scanning¹⁴. It depends on sequence differences that result in heteroduplexes that change the shape of the melting curve. Different types of genetic variants with diverse frequencies can be observed in the gene specific for a certain group of patients with cancer^1,2,15,16,

Materials

Name	Company	Catalog Number	Comments
E-Gel EX 4% Agarose	Invitrogen, Thermo Fischer Scientific	G401004
Fuorometer 3.0 QUBIT	Invitrogen, Thermo Fischer Scientific	Q33216
Invitrogen E-Gel iBase and E-Gel Safe Imager Combo Kit	Invitrogen, Thermo Fischer Scientific	G6465EU
MeltDoctor HRM MasterMix 2X	Applied Biosystem, Thermo Fischer Scientific	4415440	Components: AmpliTaqGold 360 DNA Polymerase, MeltDoctor trade HRM dye, dNTP blend including dUTP, Magnesium salts and other buffer components, precisely formulated to obtain optimal HRM results
MicroAmp Fast 96-well Reaction Plate (0.1 mL)	Applied Biosystems, Thermo Fischer Scientific	4346907
MicroAmp Optical adhesive film	Applied Biosystems, Thermo Fischer Scientific	4311971
NuGenius	Syngene	NG-1045	Gel documentation systems
Primer CALRex9 Forward	Eurofins Genomics		Sequence: 5'GGCAAGGCCCTGAGGTGT'3 (High-Purity, Salt-Free)
Primer CALRex9 Reverse	Eurofins Genomics		Sequence: 5'GGCCTCAGTCCAGCCCTG'3 (High-Purity, Salt-Free)
QIAamp DNA Mini Kit	QIAGEN	51306	DNA isolation kit with the buffer for DNA dilution.
Qubit Assay Tubes	Invitrogen, Thermo Fischer Scientific	Q32856
QUBIT dsDNA HS assay	Invitrogen, Thermo Fischer Scientific	Q32854
Trackit 100bp DNA Ladder	Invitrogen, Thermo Fischer Scientific	10488058	Ladder consists of 13 individual fragments with the reference bands at 2000, 1500, and 600 bp.
ViiA7 Real-Time PCR System	Applied Biosystems, Thermo Fischer Scientific	4453534
Water nuclease free	VWR, Life Science	436912C	RNase, DNase and Protease free water