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*47)), also called type 2 mutation. These two genetic variants represent 80% of all CALR genetic variants. The other ones have been classified as type 1–like or type 2–like using algorithms based on the preservation of an α helix close to wild type CALR⁴. Here, we present one of the highly sensitive and rapid methods for CALR genetic variant detection, the high resolution melting analysis method (HRM). This method enables the rapid detection of type 1 and type 2 genetic variants, which represent the majority of CALR mutations⁵. HRM was introduced in combination with real time »polymerase chain reaction« (qPCR) in 1997 as a tool to detect the mutation in factor V Leiden⁶. In comparison to Sanger sequencing that represents the golden standard technique, HRM is a more sensitive and less specific method⁵. The HRM method is a good screening method that enables a rapid analysis of a large number of samples with a great cost-benefit⁵. It is a simple PCR method performed in the presence of a fluorescent dye and does not require specific skills. Another benefit is that the procedure itself does not damage or destroy the analyzed sample that allows us to reuse the sample for electrophoresis or Sanger sequencing after the HRM procedure⁷. The only disadvantage is that it is sometimes difficult to interpret the results. Additionally, HRM does not detect the exact mutation in patients with non-type 1 or type 2 mutations⁸. In these patients, Sanger sequencing should be performed (Figure 1).

HRM is based on the amplification of the specific DNA region in the presence of saturating DNA fluorescent dye, which is incorporated in double-stranded DNA (dsDNA). The fluorescent dye emits light when incorporated in the dsDNA. After a progressive increase in temperature, dsDNA breaks down into single stranded DNA, which can be detected on the melting curve as a sudden decrease in fluorescence intensity. The shape of the melting curve depends on the DNA sequence that is used to detect the mutation. Melting curves of samples are compared to melting curves of known mutations or wild type CALR. Distinct melting curves represent a different mutation that is non type 1 or type 2⁹.

The algorithm for the somatic genetic variant detection in the CALR gene by HRM, agarose gel electrophoresis and sequencing method (Figure 1) was used and validated in the retrospective study published before¹⁰.

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 manufacturer’s instruction¹¹ (see Table of Materials). Prepare a 20 ng/µL DNA solution using 10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0 (see Table of Materials).
   1. In addition to DNA samples, prepare at least three DNA controls: two positive controls with the NM_004343.3 (CALR): c.1099_1150del52, p.(Leu367Thrfs*46) (52 bp deletion or type 1 mutation), and NM_004343.3 (CALR): c.1154_1155insTTGTC, p.(Lys385Asnfs*47) (5 bp insertion or type 2 mutation) genetic variant, as well as wild-type DNA and negative control as a non-template (NTC) control.
      NOTE: Before performing the HRM setup, confirm that the qPCR instrument is calibrated for HRM experiments.
3. Prepare qPCR HRM Master Mix according to manufacturer’s protocol (see Table of Materials) as follows: 10 µL of 2x qPCR Master Mix with Dye, 0.2 µL of 10 µM Forward Primer, 0.2 µL of 10 µM Reverse Primer, 8.6 µL of sterile, RNase and DNase free H₂O. Use the primers from the Table of Materials. Run three replicates for each DNA sample and control.
   1. Prepare the qPCR HRM Master Mix according to the number of samples being processed. Include excess volume of at least 10% in the calculations to provide excess volume for the loss that occurs during reagent transfers.
4. Mix the reaction contents by gently tapping and inverting the tube and vortexing for 2-3 s. Collect all the scattered droplets from the wall of the tube to the bottom by a brief spin.
5. Prepare a reaction plate appropriate for the instrument and HRM experiment (see Table of Materials). Transfer 19 µL of the qPCR HRM Master Mix to the appropriate wells of the 96-well optical reaction plate.
6. Pipet 1 µL of the negative controls, positive controls, and samples into the appropriate wells of the optical reaction plate. For the NTC, transfer 1 μL of sterile, RNase and DNase free H₂O used for preparing the qPCR HRM Master Mix instead of DNA.
7. Seal the reaction plate with the optical adhesive film. Do it firmly to prevent evaporation during the run. Check that the optical adhesive film is plane across all the wells in the reaction plate to ensure correct fluorescence detection.
8. Spin the reaction plate at 780 x g at room temperature (RT) for 1 minute. Check that the liquid is at the bottom of the wells in the reaction plate. Proceed to run the assay.
   NOTE: The protocol can be paused here. Store the plate at 4 °C for no more than 24 h. Allow the plate stored at 4 °C to warm to RT, and then spin the plate briefly before running it.
9. According to the manufacturer’s instruction prepare and start the run to amplify and melt the DNA and to generate HRM fluorescence data in the qPCR instrument. In the instrument system software, assign the controls and samples to the appropriate wells.
10. For the CALR genetic variant detection, change the default instrument amplification protocol and amplify the DNA using the following thermal cycling protocol: 95 °C for 10 minutes, followed by 50 cycles of 95 °C for 10 s and 62.5 °C for 60 s.
11. Perform the melt curve/dissociation (HRM) stage immediately after qPCR according to the manufacturer’s instructions¹² as follows: 95 °C for 10 s, 60 °C for 1 min, and then a ramp rate of 0.025 °C/s until 95 °C. Hold the temperature at 95 °C for 15 s and at 60 °C for 15 s.
12. The run ends automatically. First, review and verify the amplification plot (Figure 2).
13. In the experiment menu of the instrument system software, select the amplification plot option. If no data are displayed, click the green button Analyze.
    1. In the amplification plot tab, from the plot type drop-down menu, select the plot that displays the amplification data as the raw fluorescence readings normalized to the fluorescence from the passive reference (ΔRn) as a function of cycle number (ΔRn vs Cycle). In the plot color drop-down menu, select Sample.
14. From the graph type drop-down menu, select the linear amplification graph type. Show the baseline start and end cycle on the linear amplification graph by selecting the baseline start option. Verify that the baseline is set correctly. The end cycle should be set a few cycles before the cycle number where significant fluorescent signal is detected. The baseline is usually set from 3 to 15 cycles (Figure 2).
15. From the graph type drop-down menu, select the log10 amplification graph type. Show the threshold line on the graph by selecting the threshold option. Adjust the threshold accordingly if not set correctly. The correctly set threshold means that the threshold line crosses the exponential phase of the qPCR curves (Figure 2).
16. Verify that normal amplification curves are in all sample and positive control wells. Verify that there is no amplification in the NTC wells before cycle 40. A normal amplification plot shows an exponential increase in fluorescence that exceeds the threshold between cycles 15 and 35 (Figure 2). Exclude the sample wells from the analysis if there is no amplification in the well position.
    NOTE: If the amplification plot looks abnormal or the NTC well indicates the amplification, identify and resolve the problem according to the manufacturer’s troubleshooting guide.
17. Exclude the outlier with the quantification cycle (Cq) value that differs from replicates by more than 2 in the instrument system software¹². The outliers may produce erroneous HRM results.
18. In the derivative melt curves, review the pre- and post-melt regions/temperature lines. The pre- and post-melt regions should be within a flat area where there are no large peaks or slopes in the fluorescent levels (Figure 3). If needed adjust it accordingly. Set up the start and stop of the pre- and post-melt temperature lines approximately 0.5 °C apart from each other (Figure 3). Restart the analysis if the parameters are adjusted by clicking on the Analyze button.
19. In the difference plot tab, in the plot settings tab, choose one of the wild-type control (homozygote) replicates as the reference DNA and restart the analysis by clicking on the Analyze button.
20. In the aligned melt curves tab, confirm that all positive controls have the correct genotype and NTC failed to amplify (Figure 4). From the well table, select the wells containing a positive control to highlight the corresponding melt curve in the analysis plots. Confirm that the color of the line corresponds to the correct genotype. Repeat steps for the wells containing the other positive controls and NTC.
21. In the aligned melt curves tab, carefully review the plot displays for the unknown samples and compare them to the plot displays for controls (Figure 4). From the well table, select the wells containing the unknown sample replicates, check the color of the melt curve and align them with the controls in an ordered sequence by selecting the wells containing positive controls one by one. Repeat the process for all the unknown samples.
    NOTE: The unknown sample contains the variant of one of the controls if its melting curve is tightly aligned to it. Different variant groups (different colors) could be displayed indicating that the unknown sample consists of an unknown variant, not corresponding to the controls (Figure 8). A low level of the somatic genetic variant can be present in the patient's sample. This could influence the interpretation of the HRM result, particularly at the detection limit of the assay (Figure 9). In these cases, the color or even the shape of the line could closely resemble that of the wild-type genotype.
    1. When the result is inconclusive, combine the HRM results with the results of the agarose gel electrophoresis and sequencing methods. Retest the sample or request and retest a new sample if needed.
    2. Carefully review the data set for replicate curves and check that the alignment of each replicate within the group is tight. Exclude the replicate if it does not align tightly with the other samples in the group (outlier). Retest the sample if more outliers are present and the results are inconclusive. Be aware that the quantity and quality of the DNA sample influence the HRM results.
22. Analyze the result for the unknown sample in the Difference plot tab. Repeat the procedure described in the step 1.21 and verify that the results obtained for the unknown sample are the same.
23. Then, run the qPCR HRM products on the agarose gel.
    NOTE: The protocol can be paused here. Store the plate at 4 °C for no more than 24 hours or at -20 °C for a longer period time but no more than 7 days. Allow the plate stored at 4 °C to warm to RT, and then spin the plate briefly before running it. Allow the plate stored at - 20 °C to thaw and warm to RT, and then spin the plate briefly before running it.

2. Agarose gel electrophoresis

1. Run qPCR HRM products on a 4% agarose pre-cast gel containing a fluorescent nucleic acid stain using the appropriate gel electrophoresis system (see Table of Materials, Figure 5). Run only one positive, negative, NTC and sample qPCR HRM replicate.
   NOTE: Gloves should always be worn when handling gels. Any other gel electrophoresis system can fulfill this purpose if the equivalent agarose percentage, fluorescent nucleic acid stain and well format are chosen.
2. Run the gel electrophoresis system according to the manufacturer’s protocol (see Table of Materials). Remove the pre-cast gel in the cassette from the package, remove the comb and insert it into the apparatus according to the manufacturer's instructions (see Table of Materials).
   NOTE: Load the gel within 15 minutes of opening the package.
3. Dilute a 10 μL sample to 20 μL with sterile, RNase and DNase free H₂O. Load each well with 20 μL of diluted sample.
4. Dilute 3 μL of DNA size standard solution to 20 μL with sterile, RNase and DNase free H₂O (see Table of Materials). Load the M well with 20 µL of diluted DNA size standard solution (Figure 6).
5. Fill any empty wells with 20 µL of sterile, RNase and DNase free H₂O.
6. Immediately select the program according to the percentage of the gel being run and set the run time on the apparatus to 10 minutes.
7. Start the electrophoresis within 1 min of loading sample by pressing the GO button. The electrophoresis time can be extended if insufficient resolution is obtained.
   NOTE: Do not exceed the maximum recommended agarose electrophoresis running time according to manufacturer's instructions.
8. When the electrophoresis is completed, visualize DNA in the gel using a blue light or UV transillumination. Visualizing, analyzing, and storing the electrophoresis images are mostly done by a gel imager with photo-documentation system (Figure 5).
9. Analyze and interpret the visualized qPCR HRM products gel pattern by comparing the results of the unknown sample to positive controls (Figure 7 and Figure 8).
10. If the unknown sample HRM and gel electrophoresis results indicates that an unknown genetic variant is present, sequence the qPCR HRM product using primers described in Table of Materials according to the protocol described¹³.
    CAUTION: The agarose gels containing a fluorescent nucleic acid stain must be properly disposed of per institution regulations.

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