Background. Polycythemia vera (PV) is characterized by increased proliferation and accumulation of erythroid and mature myeloid cells and megakaryocyte in the bone marrow and peripheral blood. The JAK2V617F mutation is present in most PV patients. Deubiquitinase (DUB) genes, including TNFAIP3 (A20), CYLD and Cezanne, function as negative regulators of inflammatory reaction through nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling.
Objectives. To determine single nucleotide polymorphisms (SNPs) profiling and gene expression of the DUB genes as well as the immunophenotype of PV cells.
Material and methods. Seventy-seven patients with PV and 55 healthy individuals with well-characterized clinical profiles were enrolled. Gene expression profile was determined using quantitative real-time polymerase chain reaction (qRT-PCR), the immunophenotype with flow cytometry, secretion of cytokines using enzyme-linked immunosorbent assay (ELISA), and gene polymorphisms using direct DNA sequencing.
Results. Inactivation of A20, CYLD and Cezanne, and increases in interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels, as well as the enhanced number of CD25+CD4 T, Th1 and regulatory T cells were observed in PV patients. The genetic analysis of the CYLD gene identified 11 SNPs, in which a novel W736G nsSNP in exon 15 and a SNP c.2483+6 T>G in intron 15 were observed in PV cases with the frequencies of 18.2% and 5.2%, respectively. The W736G non-synonymous SNP (nsSNP) was found to be most likely to exert deleterious effect and the intronic SNP c.2483+6 T>G was identified as aberrant splicing. Sequencing of Cezanne gene identified 7 SNPs in intron 10 and PV carriers of the SNPs had at least 2 SNPs in this gene. Importantly, PV carriers of the W736G nsSNP had multiple SNPs in CYLD, but not in A20 or Cezanne gene.
Conclusions. Two identified SNPs, including the W736G nsSNP and the SNP c.2483+6 T>G, in CYLD gene might be associated with a risk of PV disease, in which the deleterious effect of the W736G nsSNP in CYLD gene could contribute to the pathogenesis of PV.
Key words: JAK2, CYLD, A20, Cezanne, polycythemia vera
Polycythemia vera (PV) is one of the classic Philadelphia chromosome/BCR-ABL1 negative myeloproliferative neoplasms (MPNs) characterized by increased proliferation and accumulation of erythroid and mature myeloid cells and megakaryocyte in the bone marrow and peripheral blood, with subsequent increases in hematocrit and hemoglobin, and overproduction of red blood cells.1, 2 The most serious complication of PV are the symptoms of thrombosis and cardiovascular disease (CVD), which are major causes of the morbidity and mortality in these patients. The V617F activating mutation in exon 12 of the tyrosine kinase JAK2 gene frequently occurs in PV patients3 and constitutively induces persistent activation of Janus kinase and signal transducer and activator of transcription (JAK-STAT) pathway in hematopoietic cell precursors to promote multiple cellular processes, including survival, differentiation and proliferation.4 In addition to the common JAK2V617F mutation, the MPLW515L/K and calreticulin (CALR) exon 9 indel mutations within the JAK/STAT signaling pathway were also shown to increase the risk of the disease.5
Deubiquitinases (DUB) genes, which include tumor necrosis factor alpha (TNF-α)-induced protein 3 (TNFAIP3, A20), tumor suppressor cylindromatosis (CYLD) and Cezanne, play important roles in deubiquitinating target proteins by cleaving their polyubiquitin chains to suppress activation of downstream signaling pathways. Lack of A20 or CYLD in mouse immune cells results in constitutive activity of several pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcriptions (STATs).6, 7 In humans, inactivation of A20 leads to the progression of lymphomas by inducing the proliferation of lymphoma cells.8, 9 Mutations in exon 3 of A20 gene cause the risk of malignant T-cell acute lymphoblastic leukemia (T-ALL)10 and chronic lymphocytic leukemia (CLL).11 Genetic aberration and reduced expression of CYLD gene are related to solid cancers and lymphoblastic leukemia.12, 13, 14, 15 The presence of CYLD is known to promote cell death in ALL and CLL cells.16, 17 Patients having a 9 nucleotide deletion in exon 7 and a single nucleotide substitution in exon 10 of CYLD gene are at risk of B-cell acute lymphoblastic leukemia (B-ALL).12 The CYLD expression is inhibited by miR-19 in T-ALL, while miR-19 is highly expressed in leukemia and several tumor cell lines.18 Similar to A20 and CYLD, Cezanne inhibits NF-κB signaling by deconjugating K63-polyubiquitin chain,19 and Cezanne inactivation is associated with the progression and poor prognosis in hepatocellular carcinoma.20 In contrast, Cezanne enhances tumor progression in lung squamous carcinoma and adenocarcinoma.21
Several investigations on immune features indicated that A20 and CYLD participate in suppressing inflammatory reaction and accumulation of leukocytes in systemic organs,6, 7 which is associated with risk for the development of cancers.22 The A20-deficient mice develop severe inflammation and cachexia by recruitment of activated lymphocytes, granulocytes and macrophages into liver and spleen.23 The CYLD-knockout mice exhibit abnormalities in the activation and development of T cells and B cells.24, 25 In PV patients, regulatory T (Treg) cells are expanded to suppress function of immune effector cells, therefore contributing to the pathogenesis of PV.26
In this study, single nucleotide polymorphisms (SNPs) profiling and gene expression of A20, CYLD and Cezanne genes in 77 patients with PV and 55 healthy individuals using direct DNA sequencing and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to determine disease-associated SNPs in the DUB genes. Besides, immunophenotypic property of PV cells was also assessed with flow cytometry.
Materials and methods
Patients and control subjects
Total peripheral blood samples from 77 untreated PV patients with a median age of 58.7 (19–81) years and 55 normal healthy volunteers used as controls were taken at the 103 Hospital, Military Medical University, Hanoi, Vietnam. The diagnosis of PV was based on the 2016 WHO criteria.2 The presence of JAKV617F mutation in PV patients was determined according to Baxter et al.27 No individuals in the control population took any medication or suffered from any known acute or chronic disease. All patients and volunteers gave a written consent to participate in the study. All care and experimental procedures were performed according to the Vietnamese law on the welfare of patients and were approved by the Ethical Committee of Institute of Genome Research, Vietnam Academy of Science and Technology, Hanoi, Vietnam. All experimental protocols on human subjects were in accordance with 1975 Declaration of Helsinki, as revised in 2008.
Isolation of PV cells and PBMCs
The PV cells from PV patients and peripheral blood mononuclear cells (PBMCs) from healthy donors were collected by venipuncture and transferred to sterile tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant and isolated using density gradient centrifugation (Ficoll-Paque Plus; GE Healthcare Life Sciences, Chicago, USA). The cells were next counted in a Neubauer chamber, washed with phosphate-buffered saline (PBS) and analyzed for further experiments.
Sera were isolated from the blood samples of PV patients and healthy subjects and stored at −20°C until being used for enzyme-linked immunosorbent assay (ELISA). The TNF-α, interleukin 6 (IL-6) and IL-1β concentrations were determined using ELISA kits (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s protocol.
RNA extraction and RT-PCR
Total mRNA was isolated using the QIAshredder and RNeasy Mini Kit from Qiagen (Hilden, Germany), according to the manufacturer’s instructions. For cDNA first strand synthesis, 1 µg of total RNA in 12.5 µL of diethyl pyrocarbonate (DEPC)-H2O was mixed with 1 µL of oligo-dT primer (500 µg/mL; Invitrogen, Waltham, USA) and heated for 2 min at 70°C. To determine transcript levels of A20, Cezanne, CYLD, OTUB-1, OTUB-2, and GAPDH, qRT-PCR with the LightCycler System (Roche, Basel, Switzerland) was applied. The following primers were used: A20 primers: 5’-TCCTCAGGCTTTGTATTTGA-3’ (forward) and 5’-TGTGTATCGGTGCATGGTTTT-3’ (reverse); OTUB-1 primers: 5’-ACAGAAGATCAAGGACCTCCA-3’ (forward) and 5’-CAACTCCTTGCTGTCATCCA-3’ (reverse); OTUB-2 primers: 5’-CTCACGTCGGCCTTCATCA-3’ (forward) and 5’-GCCATGGGCTCTACTTCGT-3’ (reverse); Cezanne primers: 5’-ACAATGTCCGATTGGCCAGT-3’ (forward) and 5’-ACAGTGGGATCCACTTCACATTC-3’ (reverse); CYLD primers: 5’-TGCCTTCCAACTCTCGTCTTG-3’ (forward) and 5’-AATCCGCTCTTCCCAGTAGG-3’ (reverse), and GAPDH primers: 5’-GGAGCGAGATCCCTCCAAA-3’ (forward) and 5’-GGCTGTTGTCATACTTCTCAT-3’ (reverse). The qRT-PCR reactions were performed in a final volume of 20 µL containing 2 µL of cDNA, 2.4 µL of MgCl2 (3 µM), 1 µL of primer mix (0.5 µM of both primers), 2 µL of cDNA SYBR Green I Master mix (Roche), and 12.6 µL of DEPC-treated water. The target DNA was amplified during 40 cycles of 95°C for 10 s, 62°C for 10 s and 72°C for 16 s, each with a temperature transition rate of 20°C/s, a secondary target temperature of 50°C and a step size of 0.5°C. Melting curve analysis was performed at 95°C (0 s), 60°C (10 s) and 95°C (0 s) to determine the melting temperature of primer dimers and the specific qRT-PCR products. The ratio between the respective gene and corresponding GAPDH was calculated per sample according to the ∆∆ cycle threshold method.28
DNA sequencing of JAK2
and the DUB genes
Genomic DNA was isolated from peripheral blood samples using a DNeasy blood and tissue kit (Qiagen). To determine the polymorphisms of the JAK2, A20, CYLD, and Cezanne genes, qRT-PCR and DNA sequencing (3500 Genetic Analyzers; Thermo Fisher Scientific) were performed as previously described.29 The GenBank accession numbers NM_004972.4, NM_00137874.1, NM_001270508.2 and NM_020205.4 were used for DNA sequence analysis of JAK2, CYLD, A20 and Cezanne, respectively, by using primers: JAK2-F: 5’-TCCTCAGAACGTTGATGGCAG-3’
and JAK2-R: 5’-ATTGCTTTCCTTTTTCACAAGAT-3’;
CYLD-F: 5’-TAAGGTCTTGTGCCTGAGCA-3’; CYLD-R: 5’-TTCTTTGGCAGCAGAAATCC-3’; A20-F: 5’-TGAGCTAATGATGTAAAATCTTGTG-3’ and A20-R: 5’-AGGAGGCCTCTGCTGTAGTC-3’; Cezanne-F: 5’-GCCTCCTGCATCAACTTCCT-3’ and Cezanne-R: 5’-TCAGAGGACAGTGGGATCCA-3. The amplification product lengths of JAK2, CYLD, A20, and Cezanne were 453 bp, 546 bp, 731 bp, and 600 bp, respectively. All obtained PCR fragments were purified with a GeneJET PCR purification kit (Thermo Fisher Scientific). The PCR products were sequenced on both strands with the same primers as the ones used for the PCR.
Immunostaining and flow cytometry
Immunophenotypic features of PV cells and PBMCs were determined with flow cytometry (FACSAria Fusion; Becton Dickinson Biosciences, Franklin Lakes, USA), as previously described.30 Cells (2 × 106) were incubated in 100 µL of flow cytometry staining (FACS) buffer (PBS plus 0.1% function control sequence (FCS)) containing fluorochrome-coupled antibodies to CD45, CD3, CD4, CD8α, CD11b, CD25, CD44, and FoxP3 (all from eBioscience; Thermo Fisher Scientific) at a concentration of 10 µg/mL. For intracellular cytokine staining of IL-4, IL-17 and interferon gamma (IFN-γ), cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich, St. Louis, USA) and ionomycin (500 ng/mL; Sigma-Aldrich) for 3 h, followed by addition of brefeldin A (10 μg/mL; Sigma-Aldrich) for another 4 h. The cells were then stained with anti-human IL-4, IL-17 and IFN-γ antibodies (Thermo Fisher Scientific). After incubating with the antibodies for 60 min at 4°C, the cells were washed twice and resuspended in FACS buffer for flow cytometry analysis.
Data related to the human JAK2, A20, CYLD, and Cezanne genes were collected from National Center for Biotechnology Information (NCBI; https://www.ncbi.nlm.nih.gov/). The information for SNP ID of these genes was retrieved from the NCBI SNP database (https://www.ncbi.nlm.nih.gov/snp/). BioEdit 7.2 software (https://bioedit.software.informer.com/7.2/) was used for the initial analysis of the sequences.
To analyze the functional consequence of deleterious SNPs of the DUB genes, the PolyPhen2 program (http://genetics.bwh.harvard.edu/pph2/index.shtml) was used. The PolyPhen-2 score varies from 0.0 (tolerated) to 1.0 (deleterious), in which the SNPs were designated “probably damaging”, “potentially damaging”, “benign”, or “unknown”. In addition, the possible impact of intronic SNPs on slicing was predicted using SD-Score (https://www.med.nagoya-u.ac.jp/neurogenetics/SD_Score/sd_score.html)31 or MaxEntScan (http://hollywood.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html)32 predictor programs.
The IBM SPSS v. 20 software (IBM Corp., Armonk, USA) was used for statistical analysis. The χ2 test was used to test whether allele distribution of each SNP follows the Hardy–Weinberg equilibrium (HWE). To examine the genotype association of control and PV groups, the Fisher’s exact test was used for SNPs with expected sample sizes less than 20 and χ2 test for those with larger expected sample sizes. Differences between control and PV groups or SNP relevance were tested for significance using Mann–Whitney U test or Kruskal–Wallis test (for many comparisons). All of the statistical tests were two-sided and represented the number of independent experiments. In all statistical analyses, the significance was determined at the level of p < 0.05.
Analysis of gene expression and immunophenotypic profiles in PV patients
For expression of the DUB genes, we observed that mRNA levels of A20, CYLD and Cezanne were significantly downregulated in PV cells as compared to the control group; however, no difference in transcript expression of the other DUB genes, including OTUB-1 and OTUB-2, between patient and control groups was detected (Figure 1A). The inactivated expression of A20, CYLD and Cezanne is predicted to be caused by their genetic alterations in PV.
Inactivation of the DUBs may lead to constitutive activation and recruitment of immune cells into systemic organs6, 7; therefore, changes in the number and activation of myeloid (CD11b+) and CD4 T cells present in PV cells were examined. In our results, CD45+ cells considered as leukocytes were gated in all experiments. Flow cytometry analysis showed that the percentage of CD11b+ myeloid and CD3+ T cells were increased in blood circulation of PV patients (Figure 1B,E). However, the activation of CD11b+ myeloid cells in PV cases was found to be similar to that in healthy individuals, as the number of CD11b+CD86+ and CD11b+CD40+ expressing cells remained unaltered in these patients (Figure 1B,C). Unlike CD11b+ myeloid cells, the activation of CD3+CD4+ T cells was partially enhanced, as the number of CD3+CD4+CD25+ (CD25+CD4 T cells), but not CD3+CD4+CD44+ (CD44+CD4 T cells) was significantly increased in PV cases (Figure 1D,E).
Next, analysis of CD4 T cells subsets showed that the CD3+CD4+CD25+ FoxP3+ (Treg) and CD3+CD4+IFN-γ+ (Th1) expressing cells were recruited into the circulation, whereas the number of CD3+CD4+IL-4+ (Th2) and CD3+CD4+IL-17+ (Th17) expressing cells was unaltered in PV cases (Figure 1F,G). In agreement with a previous study, Treg cells are recruited to circulatory system to facilitate the development of PV,26 suggesting that the inactivated expression of A20, CYLD and Cezanne genes in PV patients could be involved in the recruitment of CD25+ CD4 T, T regulatory and Th1 cells into blood.
The A20 and CYLD are also known as inhibitors of inflammatory reaction7, 33; thus, cytokine production in sera of PV patients was examined. Similar to a recent study,34 we also observed that levels of IL-6 and TNF-α in PV patients were found to be higher than in control individuals; however, these patients showed no change in the serum level of IL-1β (Figure 1H).
Mutational analysis of JAK2
and DUB genes in PV patients
Firstly, the p.V617F (c.1849 G>T) mutation of JAK2 gene is well known to be pathogenic, has been identified in about 90% PV cases3 and is used as an important diagnostic marker of PV. In this study, the results indicated that 51 out of 77 (66.23%) untreated PV cases were positive with the JAK2V617F (Figure 2A and Table 1, Table 2).
Sequencing of the CYLD gene identified 6 nucleotide changes in exon 15 (Figure 2B), in which 5 out of the 6 SNPs, including p.A705P (c.2355 G>C), p.Q731H (c.2435 G>C), p.E735K (c.2445 G>A), p.W736G (c.2448 T>G), and p.E747K (c.2481 G>A), were non-synonymous SNPs (nsSNPs), causing changes in the amino acid residues, and the remaining SNP p.E723E (c.2411 G>A) was silent. Five intronic nucleotide changes, including 2 SNPs c.2351-118delA and c.2351-31 T>G in intron 14 and 3 SNPs c.2483+6 T>G, c. 2483+39 T>G and c. 2483+53 G>A in intron 15, were found (Figure 2C). The genotype distribution of the observed SNPs, except for the 2 SNPs p.E747K and c.2483+53 G>A in CYLD gene, were in agreement with HWE. Importantly, we noted that the minor allele frequency (MAF) of the W736G nsSNP was significantly higher in PV group compared to control group (p = 0.022), and the difference in the MAFs for the 10 remaining SNPs between the 2 groups was not observed (Table 1).
For determination of susceptibility to PV by evaluating the deleterious effect of the nsSNPs in CYLD gene, the results indicated that among the 5 nsSNPs, only the W736G nsSNP was predicted to be probably damaging by PolyPhen-2 with a score of 0.9456 (score range: 0–1; sensitivity: 0.8; specificity: 0.95) (Figure 2D). Accordingly, the W736G nsSNP might be one of the most deleterious nsSNPs in CYLD gene. Moreover, TG genotype of the W736G nsSNP showed higher frequency in PV patients (18.2%) compared to healthy individuals (1.81%; p = 0.018, Table 2), while the 10 other SNPs in CYLD gene were not significantly associated with PV phenotype. The evidence revealed the association of the W736G nsSNP in susceptibility to the progression of PV.
For the analysis of the possible impact of the 5 intronic SNPs in CYLD gene on splicing, only the SNP c. 2483+6 T>G was predicted as an aberrant splicing according to the SD-Score prediction program (Figure 2E).31 Besides, the MaxEntScan32 splicing prediction through the score analysis of MaxENT (Maximum Entropy Model), MDD (Maximum Dependence Decomposition Model), MM (First-order Markov Model), and WMM (Weight Matrix Model) indicated that the mutant scores of the SNP c.2483+6 T>G were lower than of the wild-type scores (Figure 2F), suggesting that the SNP c.2483+6 T>G may be a splice-donor-site mutation.
Further analysis of an alignment of CYLD protein using the PolyPhen-2 software showed that the p.W736 residue is a highly conserved site among different species, including humans (Homo sapiens), rat (Rattus norvegicus), gray short-tailed opossum (Monodelphis domestica), Tasmanian devil (Sarcophilus harrisii), duckbill platypus (Ornithorhynchus anatinus), little brown bat (Myotis lucifugus), chicken (Gallus gallus), wild turkey (Meleagris gallopavo), western clawed frog (Xenopus tropicalis), nile tilapia (Oreochromis niloticus), zebrafish (Danio rerio), three-spined stickleback (Gasterosteus aculeatus), african elephant (Loxodonta africana), and spotted green pufferfish (Tetraodon nigroviridis) (Figure 2G).
Next, the sequencing of A20 gene identified 3 nucleotide changes, including rs776591390 G>T, rs141376366 G>A and rs745670694 G>A, in exon 7 (Figure 3). The genotype distributions of the 3 SNPs in this gene were in accordance with HWE (p > 0.05) (Table 1). The MAF for the SNP rs776591390 was slightly higher, whereas the MAFs for the 2 remaining SNPs were lower in PV patients than in the control group. Among the 3 SNPs, the missense SNP rs776591390 of A20 gene was identified in 1 out of 77 PV patients (1.81%) and the 2 remaining SNPs rs141376366 and rs745670694 were found only in control individuals with the carrier frequencies of 1.81% and 3.63%, respectively (Table 2).
Finally, the sequencing of Cezanne gene identified 7 nucleotide changes in intron 10, 3 out of the 7 intronic SNPs (c.1584-287 C>G, c.1584-167 G>T and c.1584-122 G>T) were unidentified SNPs and the 4 remaining intronic SNPs (rs1168285629 A>G, rs1394369937 A>G, rs1158787149 C>G, and rs1553772411 C>G) are reported in NCBI SNP database (Figure 4). The genotype distributions of the 7 SNPs in Cezanne gene were in accordance with HWE (p > 0.05) (Table 1). The MAFs of the 7 intronic SNPs were slightly higher in the PV group than in healthy individuals. Among these SNPs, 5 out the 7 intronic SNPs (c.1584-287 C>G, rs1394369937 A>G, c.1584-167 G>T, rs1158787149 C>G and rs1553772411 C>G) appeared in PV patients, but not in the control group, with the carrier frequencies of 7.79%, 5.19%, 2.6%, 5.19%, and 3.89%, respectively (Table 2). Importantly, 7 out of 77 (9.09%) PV patients carried at least 2 SNPs in Cezanne gene.
Association between the different SNPs in JAK2 and the DUB genes in PV patients
For determination of the correlation among the SNPs in JAK2 and the DUB genes, we observed that 10 out of 14 (71.4%) PV cases carrying the W736G nsSNP and 3 out of 3 (100%) carriers of the intronic SNP c. 2483+6 T>G in CYLD gene were positive for the JAK2V617F mutation (data not shown), whereas, the JAK2V617F-positive rate of PV carriers of the other SNPs in the DUB genes were similar to healthy individuals (data not shown). The evidence indicated that there was a significant positive correlation between PV carriers of the W736G nsSNP or/and the intronic SNP c.2483+6 T>G and PV patients with JAK2V617F mutation.
Interestingly, out of the 14 patients carrying the W736G nsSNP in CYLD gene, 13 (92.8%) cases were found to carry the SNP p.E747K; 11 (78.6%) cases had both the SNP p.Q731H G>C and the SNP c.2483+53 G>A; 9 (64.3%) cases were found to carry the SNP p.E735K; and 3 (21.4%) carriers of the SNP c.2483+6 T>G and 5 (35.7%) cases infected with the SNP c.2483+39 T>G were observed (Figure 5). According to the Kruskal–Wallis test results, significant relevance was observed among the SNPs in CYLD gene (χ2=7.364, p = 0.007), suggesting that PV carriers of the W736G nsSNP had multiple SNPs in CYLD gene. Additionally, we observed that PV carriers of the W736G nsSNP and the intronic SNP c.2483+6 T>G in CYLD gene did not have SNP in A20 or Cezanne gene (Figure 5).
In this study, inactivated expression of A20, Cezanne and CYLD in PV patients was revealed for the first time. Among the DUB genes, A20 and CYLD but not Cezanne are known as inhibitors of immune reaction through JAK/STAT signaling, whose activation results in the development and pathogenesis of leukemia and lymphoma, including PV.4, 5 A recent study reported that the aberrant expression and mutations in CYLD gene are associated with the susceptibility to leukemia and lymphoma.12, 15 Among the 11 SNPs examined in CYLD genes, W736G nsSNP was found to be most likely to exert deleterious effect – it was observed using the PolyPhen-2 prediction tool and the intronic SNP c.2483+6 T>G was identified as an aberrant splicing by the SD-Score or MaxEntScan predictor program. Importantly, the 2 aberrant SNPs were not found in all samples from 90 patients with chronic myeloid leukemia (CML), 32 patients with acute myeloid leukemia (AML), 16 patients with acute lymphoblastic leukemia (ALL), and 21 patients with chronic lymphocytic leukemia (CLL) (unpublished data), suggesting that the two SNPs could be associated with significant risk of PV, but not leukemia.
Investigations on genetic alteration of A20 gene indicated that carriers of SNPs in the A20 gene at exons 5, 6 and 7 are at high risk for autoinflammatory disease and lymphocytic leukemia.10, 11, 35, 36 In this study, the SNP rs776591390 in A20 gene was detected in PV patients with the frequency of 1.29%, pointing out that A20 polymorphisms associated with disease susceptibility are different.
Unlike the impact of A20 and CYLD, the involvement of SNPs in Cezanne gene with the possible risk of leukemia is not fully documented, although Cezanne expression is linked to poor prognosis in hepatocellular carcinoma.20 In this study, we also revealed for the first time that changes of 7 nucleotides at intron 10 in Cezanne gene were found in PV patients and in 7 out of 77 (9.09%) PV patients who carried at least the 2 SNPs in this gene. Similar to the 2 aberrant SNPs in CYLD gene, the 7 intronic SNPs in Cezanne gene were not carried by AML and CML patients (unpublished data). In addition, the MAFs of the 7 intronic SNPs had slightly higher frequencies in PV groups than in control groups, indicating that carriers of SNPs in Cezanne gene could tend to be at risk of the progression of PV.
In addition to the determination of DNA sequences of the DUB genes, the presence of the JAK2V617F mutation in PV patients was also conducted to determine the correlation between SNPs in the JAK2 and the DUB genes. The JAK2V617F mutation was detected in 66.67% of PV patients in this study, whereas other study indicated the presence of this mutation is about 90% of PV cases.3 The frequency of JAK2V617F allele burden is known to time-dependently increase in PV cases.37 A recent study indicated that the absence of the JAK2V617F mutation in PV patients does not affect treatment effectiveness with JAK/STAT pathway inhibitors,38 suggesting that the constitutive activation of the JAK-STAT pathway could be caused by not only JAK2V617F mutation. Besides the known mutations involved in the pathogenesis of PV, including JAK2, MPL and CALR, we additionally revealed that PV patients carrying the W736G nsSNP had multiple SNPs in the CYLD, but not in A20 or Cezanne gene, suggesting that the effect of the W736G nsSNP in CYLD gene might be one of potential treatment targets for PV. The CYLD is known to suppress inflammatory reaction through the activation of several pathways, including JAK/STAT7; therefore, further studies would investigate the effects of the W736G nsSNP in regulating function of PV cells and underlying mechanisms.
In the analysis of involvement in SNPs and immunophenotype in PV patients, the JAK2V617F mutation is reported not to affect the function of T cells3; therefore, the recruitment of CD25+CD4T, Th1 and Treg cells into the circulatory system in PV cases would be related to the activation of the 2 aberrant SNPs in CYLD gene. Importantly, the enhanced activation of Th1 cells in PV cells was also revealed for the first time in our study. As part of the immune response, Treg cells play an important role in suppressing tumor-specific immunity.30 The aberrant expression of CD25 is associated with poor prognosis in AML40 and CML.41 A previous study indicated that factors related to thrombosis in PV include increased hematocrit, thrombocytosis, platelet activation, and leukocyte activation,42 suggesting the regulatory effects of the 2 aberrant SNPs in CYLD gene on the development of thrombosis in PV. In addition, we did not find any significant association of the SNPs in A20 and CYLD and Cezanne genes with increased levels of IL-6 and TNF-α in PV patients.
There are some limitations to the current study. First, the sample size was not sufficient to verify a potential association between the SNPs in the DUB genes and risk of PV disease in the Vietnamese population. Second, further functional research is necessary for investigating the impacts of the W736G nsSNP and/or intronic SNP c.2483+6 T>G in CYLD gene on PV cell activation for the development of PV treatment. Finally, we only examined number and activation of myeloid (CD11b+) and CD4 T cells present in PV cells, while other cell types, such as macrophages, inflammatory monocytes and B cells, might be also related to the activation of the 2 aberrant SNPs in CYLD gene.
The deleterious effect of the W736G nsSNP in CYLD gene could contribute to the pathogenesis of PV and be a good candidate for further study on its role in regulating functional activation of PV cells.