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The Janus kinase 2 (JAK2) is a critical component of diverse signal-transduction pathways. These molecules, with tyrosine kinase activity, play pivotal roles in cellular survival, proliferation, differentiation, and apoptosis, as well as cytokine signaling in both normal and neoplastic cells [1, 2]. The substitution of valine with phenylalanine at amino acid position 617 (V617F) in the JAK2 protein leads to the activation of the JAK/STAT signaling pathway. Genetic mutations in genes encoding JAK2 are partially responsible for tumorigenesis. Currently, the presence of the JAK2 V617F mutation is well-established in chronic myeloproliferative neoplastic disorders (MPNs) [2].
The mutation's frequency ranges from 65% to 97% in polycythemia vera (PV), approximately 23% to 57% in essential thrombocythemia (ET), and 35% to 57% in myelofibrosis with myeloid metaplasia [3-7]. Therefore, the reliable and sensitive detection of these mutations is essential. Notably, one of the major WHO criteria for diagnosing PV is the detection of JAK2 mutations [8].
Several molecular assays for detecting the JAK2 V617F mutation, such as PCR-RFLP and ARMS PCR, have been previously described.
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High-resolution melting (HRM) is a rapid and cost-effective methodology for mutation scanning and genotyping. It has demonstrated high sensitivity and specificity through the chelation of double-stranded DNA (dsDNA) dye. The HRM assay is a suitable and specific closed-tube technique for mutation scanning, making it more convenient than other scanning methodologies [2]. The primary objective of our study was to assess the effectiveness of HRM analysis in identifying the JAK2 V617F missense mutation. To achieve this, we compared HRM analysis with the amplification refractory mutation system (ARMS), Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assays, commercial kits, and considered the clinical background of the patients.
Subsequently, we reported the frequency of the JAK2 V617F missense mutation in Iranian patients diagnosed with PV, ET, and PMF.
Peripheral blood samples were collected from a total of 15 patients diagnosed with Myeloproliferative Disorders (MPD) and 72 control subjects. The control group consisted of 27 patients with chronic diseases such as hypertension, 32 patients with lung disorders including Asthma and Chronic Obstructive Pulmonary Disease (COPD), 8 patients with infectious diseases such as tuberculosis, and 5 healthy individuals without any signs or symptoms of diseases. Demographic data of the subjects are summarized in Table 1. The DNA extraction from leukocyte blood was performed using the Yekta Tajhiz kit following the manufacturer's instructions. This study was approved by the National Institute of Tuberculosis and Lung Diseases (NRITLD) at Shaheed Beheshti University of Iran.
| Minimum | Maximum | Mean | N (%) | |
|---|---|---|---|---|
| Age (y) | 17 | 84 | 53.38 ± 17.43 | |
| Male/female gender | 63/24 | 72.4% / 27.6% | ||
| Hemoglobin | 8 | 24 | 15.64 ± 4.07 | |
| Platelet count | 32,000 | 1,588,000 | 418,045.9 ± 364,649.4 | |
| Hematocrit | 23.40 | 76.30 | 47.58 ± 11.74 | |
| Mean Corpuscular Hemoglobin Concentration (MCHC) | 12.70 | 38.80 | 32.51 ± 3.33 | |
| Red Blood Cells | 2.81 | 9.63 | 5.61 ± 1.47 | |
| Red Blood Cell Distribution Width (RDW) | 12.00 | 33.80 | 16.88 ± 4.29 | |
| White Blood Cells | 4,300 | 74,980 | 13,157.01 ± 11,605.58 |
PCR reactions were conducted in a final volume of 20 µl. The reaction mixture included 1× buffer containing Taq DNA polymerase, nucleotides, and the SYBR green dye, 2.5 mM of each forward (F) primer (5'-AAGCTTTCTCACAAGCATTTGGTTT-3') and reverse (R) primer (5'-AGAAAGGCATTAGAAAGCCTGTAGTT-3'), as well as MgCl2. The amplification targeted a 155 bp region of the JAK2 gene, likely harboring the V617F mutation. The PCR program consisted of an initial denaturation-activation step at 95°C for 15 minutes, followed by a 45-cycle program (denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and elongation at 72°C for 10 seconds, with fluorescence acquisition at 533 nm at the end of the elongation step).
The melting program included a rising melting ramp of 0.1 degree per step, ranging from 65°C to 95°C. This program continuously recorded fluorescence readings per °C. HRM PCR reactions were performed and analyzed using the Rotor Gene real-time analyzer 6000™ (Corbett Life Sciences, Mortlake, Australia). Both negative and positive controls were used as reference curves to generate the difference plot.
To validate the findings obtained through HRM analysis, sequencing analysis was performed on 5 positive samples. The sequencing was conducted using the ABI Prism 310 Genetic Analyzer from Applied Biosystems, and the resulting sequences were analyzed using BioEdit v7.0.5.
The ARMS PCR was carried out using tetra primers as previously described [9]. The primer sequences used were as follows:
In addition, a multiplex PCR was performed with three primers, as described by Baxter [7]. The amplifications were carried out for 30 cycles using 2X master mix (PARS Tous co, IRI), which included 1X buffer, 0.2 µM of each dNTP, 1.5 µM MgCl2, and 1 U Taq DNA polymerase. The reaction mixture also contained 25 ng of genomic DNA. Standard amplification conditions were followed, with an annealing temperature of 58°C. Notably, the final concentrations of the outer primers and the mutant/wild-type-specific inner primers were 1 µmol/l and 0.5 µmol/l, respectively. The resulting products were separated on 2% agarose gels and visualized after staining with a safe stain.
In order to detect the JAK2 V617F mutation through restriction digestion, a portion of the JAK2 region encompassing this mutation (420 base pairs) was amplified using the previously mentioned forward and reverse outer primers (G49-JAK2-FO and G50-JAK2-RO). The resulting fragment was then subjected to digestion with the BsaXI restriction enzyme from BioLabs™, following the manufacturer's instructions. The digested products were visualized on 2% agarose gels stained with a safe stain. The undigested fragment indicated the presence of the JAK2 V617F missense mutation, as it lost the restriction enzyme site. Conversely, wild-type cases were digested and produced two distinct bands (200 and 220bp). The presence of three bands indicated heterozygosity, which may be due to a low allele burden of the mutation alongside a wild-type allele or incomplete endonuclease activity.
In this study, the HRM assay successfully differentiated homozygous (T/T) mutants from wild-type DNA when compared to the melting profile of a wild-type control (G/G). Among the 16 Myeloproliferative Neoplasms (MPNs) patients studied, 13 (81.25%) tested positive for the presence of the JAK2 V617F mutation using the HRM assay. The mutation prevalence varied among different subtypes of MPNs, with rates of 71.4% in Polycythemia Vera (PV) (5 out of 7 patients), 80% in Essential Thrombocythemia (ET) (4 out of 5), and 100% in myelofibrosis (3 out of 3). The background disease of one patient could not be determined, although she was suspected to have MPN disorders, the WHO criteria were not confirmed in this patient (See Table 2).
| Disorders | PCR-RFLP | PCR-ARMS | PCR-HRM | Commercial kit |
|---|---|---|---|---|
| Polycythemia Vera (PV) | 7 | 7 (100%) | 5 (71.4%) | 5 (71.4%) |
| Primary Myelofibrosis (PMF) | 3 | 2 (66.7%) | 2 (66.7%) | 3 (100%) |
| Essential Thrombocythemia | 5 | 4 (80%) | 4 (80%) | 4 (80%) |
| Clinically Suspected MPNs | 1 | 1 (100%) | 1 (100%) | 0 (0%) |
| Total | 16 | 14 (87.5%) | 12 (75%) | 13 (81.25%) |
The study revealed that the sensitivity of HRM analysis in detecting the JAK2 V617F mutation in patients with MPNs and those suspected of having MPNs was 86.6%, compared to 93.3% for PCR-RFLP and 80% for PCR-ARMS assays. The sensitivity of commercial kits, including ipsogen JAK2 RGQ PCR Kit CE from Qiagen (Germany) and JAK2 RG version 2 from Novin gene (Iran), was lower than that of homemade assays, at 66.7% (See Table 3).
| PCR-HRM | PCR-RFLP | ARMS-PCR | Commercial Real-time kit | |
|---|---|---|---|---|
| Sensitivity | 86.6% | 93.3% | 80% | 66.7% |
| Specificity | 100% | 80.5% | 100% | 100% |
| Positive Predictive Value | 100% | 50% | 100% | 100% |
| Negative Predictive Value | 97.3% | 98.3% | 96% | 90.9% |
| General Efficacy | 97.7% | 82.7% | 96.5% | 92.3% |
The objective of this study was to evaluate the efficacy of various PCR-based assays for detecting the JAK2 V617F mutation. The results are crucial for selecting the most suitable method for the benefit of both patients and laboratories involved. Several techniques, including allele-specific polymerase chain reaction (AS-PCR), genomic DNA-PCR-sequencing, PCR-amplification refractory mutation system, PCR-restriction analysis, and DNA-melting curve analysis, have been developed over the past decades for detecting the JAK2 V617F missense mutation. Each method has its own set of advantages and disadvantages.
For instance, while DNA sequencing has the power to detect specific mutations, it suffers from low sensitivity and high cost. PCR-ARMS, known for its high sensitivity (0.01%–5%), requires post-PCR manipulation. PCR-RFLP, on the other hand, can produce false-positive results due to incomplete digestion and is a time-consuming technique. Evaluating these methods should consider important parameters such as sensitivity, specificity, and cost. Unfortunately, there are very few studies evaluating these assays for detecting the JAK2 V617F mutation in Iranian MPNs patients.
In our study, we assessed four different screening methods using a total of 16 confirmed MPNs patients among 87 individuals with various disease symptoms. These assays were compared with the PCR-RFLP method, which is widely used as a reference assay for detecting the JAK2 V617F mutation. Statistical analysis demonstrated good concordance among all assays. However, the melting curve assay proved to be less reliable at lower levels of JAK2 V617F mutation allele (See Table 2). It's possible that the mutant allele burden in PMF patients who tested positive with PCR-HRM was lower than the detection limit of the mutation in a wild-type background using PCR-RFLP. PCR-HRM failed to detect the mutation in only 2 out of 7 PV patients compared to PCR-RFLP. Interestingly, both samples in PCR-RFLP were heterozygote and were also not detected by direct sequencing. This suggests that the results obtained by the PCR-RFLP assay might have been false positives due to incomplete digestion. Therefore, our study indicates that PCR-ARMS and PCR-HRM methods can accurately detect the JAK2 V617F mutation in PV patients.
We used two PCR-ARMS methods, including multiplex PCR and Allele-Specific PCR, and found similar results. The multiplex assay produced an additional smaller fragment, while the allele-specific method used two specific primers to detect wild-type and mutant alleles. Both methods required post-amplification manipulation, which could lead to cross-contamination, a significant disadvantage. Due to PCR-HRM's advantages, such as ease of use, speed, and high sensitivity compared to other molecular assays, we believe PCR-HRM may be a suitable tool for screening JAK2 V617F mutation in clinically suspected patients.
Our evaluation revealed that the two commercial kits for detecting the JAK2 V617F missense mutation were less sensitive methods. The mutation detection limit seemed to be subjective and may depend on the mutant allele's burden level. Unfortunately, the manufacturer did not specify the allele burden limit. It's possible that the commercial kits had a higher threshold, perhaps more than 20% mutant DNA in the background of the wild-type allele, which is higher than the HRM-PCR assay.
In conclusion, our study compared the results of four PCR-based assays, including PCR-RFLP, for detecting the JAK2 V617F mutation in blood samples from clinically confirmed and suspected patients with MPNs disorders. Our findings demonstrate that the PCR-HRM protocol is a quick, easy, and effective method for screening the JAK2 V617F mutation in patients with MPNs disorders. However, for result confirmation, it should be complemented with PCR-RFLP, which serves as the gold standard method for detecting the JAK2 V617F mutation.
Comparing JAK2 V617F Mutation Detection Methods in MPN Patients. (2024, Jan 23). Retrieved from https://studymoose.com/document/comparing-jak2-v617f-mutation-detection-methods-in-mpn-patients
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