|Year : 2017 | Volume
| Issue : 4 | Page : 1044-1050
Association of janus kinase 2 with primary Budd–Chiari syndrome in Egyptian patients
Hatem M El Sebay1, Manal A Safan1, Ashraf A Daoud1, Safaa I Tayel1, Alaa Nouh2, Shymaa El Shafie1
1 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Menoufia University, Menoufia, Egypt
|Date of Submission||15-Mar-2015|
|Date of Acceptance||12-May-2015|
|Date of Web Publication||04-Apr-2018|
Shymaa El Shafie
Shebein Elkom, Menoufia
Source of Support: None, Conflict of Interest: None
The aim of this study was to evaluate the association of janus kinase 2 (JAK2) V617F mutation with primary Budd–Chiari syndrome (BCS) in Egyptian patients.
BCS is a rare disorder caused by thrombosis of the hepatic veins or the terminal portion of the inferior vena cava. JAK2 V617F mutation is an objective tool for diagnosing BCS patients.
Patients and methods
This study was conducted on 50 patients: 35 patients with primary BCS, and 15 age-matched and sex-matched healthy individuals who were taken as the control group. Patient and control groups were subjected to full history taking, general clinical examination, and laboratory investigations including measurement of complete blood count; liver function tests – serum aspartate aminotransferase and serum alanine aminotransferase (ALT), total and direct bilirubin, serum albumin, prothrombin time, partial thromboplastin time, and international normalized ratio; and renal function tests – serum blood urea and serum creatinine. Genotyping of JAK2 mutation was performed by PCR-RFLP method to diagnose the underlying etiology of BCS.
There was a statistically significant difference between patients and controls regarding hemoglobin, platelets, ALT, aspartate aminotransferase, direct and total bilirubin, albumin, prothrombin time, partial thromboplastin time, international normalized ratio, urea, and creatinine. There was no statistically significant difference between studied patients and control groups regarding JAK2 mutations. Mutant form of JAK2 had significantly increased platelets count and ALT serum levels than wild form, whereas there was no significant difference regarding other laboratory parameters.
JAK2 V617F mutation was not significantly different between patients and controls.
Keywords: Budd–Chiari syndrome, janus kinase 2 V617F, myeloproliferative disorders, PCR-RFLP
|How to cite this article:|
El Sebay HM, Safan MA, Daoud AA, Tayel SI, Nouh A, El Shafie S. Association of janus kinase 2 with primary Budd–Chiari syndrome in Egyptian patients. Menoufia Med J 2017;30:1044-50
|How to cite this URL:|
El Sebay HM, Safan MA, Daoud AA, Tayel SI, Nouh A, El Shafie S. Association of janus kinase 2 with primary Budd–Chiari syndrome in Egyptian patients. Menoufia Med J [serial online] 2017 [cited 2019 Mar 21];30:1044-50. Available from: http://www.mmj.eg.net/text.asp?2017/30/4/1044/229198
| Introduction|| |
Budd–Chiari syndrome (BCS) is a clinical condition caused by hepatic venous outflow obstruction located anywhere from the small hepatic veins (HVs) to the junction of the inferior vena cava (IVC) and the right atrium,.
BCS can be classified into two types: primary, due to venous thrombosis, and secondary, from extrinsic compression by a neighboring mass.
Primary BCS is a rare disorder; its estimated incidence ranges from 0.2 to 0.8 per million per year worldwide. In Egypt, BCS commonly occurs during the third decade of life and is more predominant in women. BCS was present all over Egypt, but it was more prevalent in Delta (42.5%) than Cairo (36.3%) and Upper Egypt (21.25%).
The decreased hepatic perfusion and resultant congestion cause ischemic injury to liver cells that culminates in the development of hepatocyte necrosis, which is followed by progressive fibrosis, nodular regenerative hyperplasia, and ultimately liver cirrhosis. The most common signs and symptoms of BCS are ascites, hepatomegaly, and abdominal pain.
Myeloproliferative disorders (MPDs) account for about 50% of BCS patients; janus kinase 2 (JAK2) mutation (V617F) had been detected in 37–45% of them. JAK2 is a cytoplasmic tyrosine kinase signal transducer. A point mutation at exon 14 substitutes phenylalanine for valine at domain 617 (V617F) and results in a loss of inhibition, uncontrolled signal transduction, and cytokine hypersensitivity of affected cells.
The most common gain-of-function mutation leading to development of MPDs is the JAK2 V617F mutation, which had been found in about 95 and 60% of patients with polycythemia vera (PCV) and essential thrombocythemia (ET) or idiopathic myelofibrosis (MF), respectively.
Screening for theJAK2 V617F mutation is an objective tool for diagnosing MPDs in these patients and is now part of the standard diagnostic workup in splanchnic vein thrombosis,.
The aim of this study is to evaluate the association of JAK2 V617F mutation with primary BCS in Egyptian patients.
| Patients and Methods|| |
This study was conducted on 50 patients: 35 patients with primary BCS and 15 age-matched and sex-matched healthy individuals who were taken as the control group. The patients were selected from BCS Society, Faculty of Medicine, Menoufia University.
They were categorized into two groups: group I included 35 patients with primary BCS. Diagnosis of BCS was based on clinical examination, laboratory investigations, and Doppler-duplex of the HVs and IVC. Group II included 15 age-matched and sex-matched healthy individuals. All cases with secondary BCS were excluded. Written informed consent was obtained from every patient who participated in this study. The protocol of the study was approved by the Ethical Committee of Medical Research of Faculty of Medicine, Menoufia University.
A volume of 10 ml of venous blood was collected from all patients included in this study by venipuncture from the cubital vein, as follows: 4 ml of blood was collected into EDTA tubes for complete blood count and genotyping; 2 ml of blood was collected into citrated tube for prothrombin time (PT) and concentration; and the remaining 4 ml was collected in a plain vacutainer tube, left for 15 min for coagulation, then centrifuged at 3000 rpm for 10 min, and the serum was separated into aliquots for measurement of liver function tests and renal function tests.
Patient and control groups were subjected to full history taking, general clinical examination, and laboratory investigations including measurement of complete blood count: hemoglobin (Hb) level, red blood cell (RBC) count, white blood cell (WBC) count, and platelet (PLT) count using Beckman coulter counter model 750 (Beckman Int, 1320 West Colton Avenue Redlands, CA 92374, USA).
The following tests were performed: liver function tests such as serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT), which were performed using the kinetic UV-optimized method International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) (LTEC kit; Diamond Diagnostics, Houston, USA); total and direct bilirubin and serum albumin, which were tested using the Diamond Diagnostics kit (Diamond Diagnostics inclusion, Houston, USA); PT, which was determined using the Biomed-Liquiplastin Diagnostic kit (Lab Supply company, Heliopolis, Egypt Cairo); and international normalized ratio (INR). Renal function tests such as serum blood urea and serum creatinine were performed using the Diamond Diagnostics kit.
Genotyping of JAK2 mutation (amplicon ∼502 bp) was performed by PCR-RFLP method to diagnose the underlying etiology of BCS. DNA was extracted from whole blood using commercially available spin-column technique kit for DNA extraction (QIAampDNA Blood Mini kit; Qiagen, Germany) (Berlin). A PCR master mix was prepared from 5 μl of 5× buffer green, 2 μl of 25 mmol/l MgCl2(final 2 mmol/l), 0.3 μl of 10 mmol/l dNTPs, 1 μl each forward and reverse primer (10 pmol/μl; Metabiol, Germany), 1 U of Taq polymerase (0.5 μl; Invitrogen) (Waltham, Massachusetts, USA), 5.2 μl of distilled H2O, and 10 μl of template DNA to obtain a final volume of 25 μl. DNA was amplified by thermal cycler (model 2720; Applied Biosystems, Singapore, Singapore) using initial denaturation at 94°C for 3 min followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 59°C for 30 s and extension at 72°C for 30 s, and final extension at 72°C for 5 min. The length of the PCR amplicon (JAK2 V617F) was detected by gel electrophoresis at 502 bp. The PCR products for the JAK2 polymorphism were mixed with 10 U of Bsa XI restriction enzyme (New England Biolabs). The reaction was incubated overnight at 37°C and then 10 μl of the product was loaded into 2% agarose gel containing ethidium bromide for electrophoresis. Wild sequences occur with digestion, producing 246, 226, and 30 bp. The 502-bp PCR remained intact in the presence of the JAK2 V617F mutation (no enzyme digestion).
Results were collected, tabulated, and statistically analyzed by IBM personal computer and statistical package SPSS, version 16. Descriptive statistics were percentage, mean, (SPSS Inc. Released 2007. SPSS for Windows, Version 16.0. Chicago, SPSS Inc.) and SD, and analytic statistics were c2-test, t-test, and Mann–Whitney test. Significant difference was P less than and equal to 0.05.
| Results|| |
BCS patients and healthy control were matched regarding age and sex [Table 1]. The main onset of BCS was the chronic form (71.4%), whereas each of the acute and subacute onset was present in 14.3% of the studied patients. Abdominal enlargement and abdominal pain were the most common clinical symptoms: 77.1% and 51.4%, respectively. The most common clinical signs were ascites and hepatomegaly: 80.0% and 62.9%, respectively [Table 2]. By using duplex ultrasonography, ascites, hepatomegaly, and splenomegaly were found to be present in most of the patients: 74.3, 68.4, and 28.6%, respectively. HV obstruction was the most common pattern of venous obstruction in the studied patients (71.4%). IVC obstruction was found in only 8.6%, whereas combined HVS and IVC obstruction was found in 20% of patients [Table 3]. There was a statistically significant difference between patients and controls regarding Hb, PLT, ALT, AST, DB, TB, albumin, PT, partial thromboplastin time, INR, urea, and creatinine, whereas there was no statistically significant difference between the two groups regarding RBC count and WBC count [Table 4]. There was no statistically significant difference between studied patients and control groups regarding JAK2 mutations [Table 5]. Mutant form of JAK2 had significantly increased PLT count and ALT serum levels than wild form, whereas there was no significant difference regarding other laboratory parameters [Table 6].
|Table 4: Statistical comparison between patient and control groups regarding laboratory data|
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|Table 5: Statistical comparison of janus kinase 2 mutation between patients and controls|
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|Table 6: Statistical comparison between janus kinase 2 mutant and wild-type patients regarding laboratory data|
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| Discussion|| |
MPDs comprise a group of stem cell disorders with defective regulation of myeloid cell proliferation. This results in an overproduction of mature erythrocytes, granulocytes, and megakaryocytes. According to the French–American–British classification, myeloproliferative diseases consist of four diseases: chronic myelogenous leukemia; PCV; ET; and agnogenic myeloid metaplasia, which is also known as MF.
MPDs were found to be the leading causes of BCS in western countries, with a range between 20 and 53%. PCV was found in 10–40% of patients, whereas ET and MF were less prevalent causes.
Ubiquitous tyrosine kinase domains (JAK homolog) JH1 and JH2 play a crucial role in regulation. Normal activation of JAK2 involves cytokine-dependent phosphorylation of JH1 and JH2; the negative regulatory effect of JH2 on JH1 is essential for normal signaling.
Genetic mutation of JAK2 (V617F) has been implicated in MPD, and it results in a loss of inhibition, uncontrolled signal transduction, and cytokine hypersensitivity of affected cells. Other somatic mutations of the JAK2 gene or other genes have been identified in MPD, but they appear to account for only a minor proportion of the cases.
In this study, there was no statistically significant difference between studied groups regarding age and sex. The mean age of BCS patients was 31.57 ± 8.23 years, and there were more women (60%) than men (40%). These results are approximate to the results of Roy, who reported thatBCS affects patients during the third or fourth decades of life, and it may be slightly predominant in women.
These results are also in agreement withthe study of Sakr et al., who found that more women were affected than men (61.7 vs. 38.3%) and mean patient age at the time of first visit was 28.64 ± 8.35 years for men and 28.88 ± 9.08 years for women.
On the other hand, previous results found that male and female patients were equally presented. Mean age of the studied patients at first visit was 25.28 ± 6.08 years for men and 29.24 ± 8.09 years for women.
Most of the patients in the current study were diagnosed during the chronic phase of the disease (71.4%); the rest of the patients were diagnosed during acute (14.3%) or subacute (14.3%) stages.
These results support other studies carried out by Dilawari et al., who studied 177 patients with BCS and reported the following distribution regarding the pattern of clinical presentation: 65% chronic, 28% acute, and 7% subacute.
These results are consistent with those of Hassan et al., who found that most of the patients in their study were diagnosed during the chronic phase of the disease (76%), whereas 10 and 14% of patients were diagnosed during acute or fulminant stages, respectively.
Sakr et al. stated that 75 (79.8%) patients had chronic BCS, 18 (19.1%) had acute or subacute BCS, and one (1.1%) had fulminant BCS.
In the current study, the relevant symptoms in patients with BCS at the time of diagnosis were abdominal enlargement in 77.1%, abdominal pain in 51.4%, and jaundice in 14.3%. The BCS signs in the present study were as follows: ascites (80%), hepatomegaly (62.9%), splenomegaly (25.7%), lower limb edema (14.3%), jaundice (14.3%), and eye disease in 2.9% of the patients.
The clinical manifestations of BCS have been well characterized. Abdominal pain, ascites, hepatosplenomegaly, and portal hypertension are important features, as well as a prominent dilation of subcutaneous veins of the trunk in those patients with long-standing IVC obstruction. However, asymptomatic forms appear to account for about 15% of patients.
In a study conducted by Chun-Min et al. in China, it was reported that ascites was evident in 100%, hepatomegaly in 80.6%, edema of the lower limb in 80.3%, splenomegaly in 74.2%, abdominal pain in 35.5%, jaundice in 6.5%, and hepatic coma in 3.2% of the cases; however, with regard to liver disease, these symptoms of BCS are not specific.
The relevant symptoms in 50 patients with BCS at the time of diagnosis were abdominal enlargement in 84%, abdominal pain in 74%, lower limb swelling in 30%, and history of DVT in 12%, and regarding the signs hepatomegaly was present in 86% and the liver was tender in 20%. Ascites was evident in 84%, splenomegaly in 70%, lower limb edema in 30%, jaundice in 42%, and dilated veins over the trunk and body in 34% of the cases. In addition, bouts of hepatic encephalopathy were recorded in 4% of the patients.
Regarding the pattern of vascular involvement by duplex ultrasonography in the current study, isolated HV obstruction was found in 71.4%, isolated IVC obstruction in 8.6%, and combined HV and IVC obstruction in 20% of patients.
These results agree with a Turkish study conducted by Murad et al., who stated that isolated HV obstruction, isolated IVC obstruction, and combined HV and IVC obstruction were found in 62, 7, and 31% of patients, respectively.
In another pattern of vascular involvement conducted by an Indian study on 97 patients with BCS, the authors reported that isolated HV obstruction or isolated IVC obstruction or combined HV and IVC obstruction were found in 23.94, 18.3, and 57.74% of patients, respectively.
Sakr et al. found isolated HV involvement in 74.5% of patients, isolated IVC involvement in 3.2%, and combined HV and IVC occlusion in 17%.
It is apparent that the patterns of vascular involvement in BCS patients differ from one country to another with evident ethnic variation between the west and east.
In this study, there was a statistically significant difference between patients and controls regarding Hb, PLT, ALT, AST, direct bilirubin, total bilirubin, albumin, PT, partial thromboplastin time, INR, urea, and creatinine. No significant difference was found between the two groups regarding RBC and WBC counts.
There were various theories about thrombocytopenia in chronic liver diseases and BCS. These theories included decreased thrombopoietin levels, splenic sequestration of PLT due to portal hypertension, autoantibody destruction of PLT, and bone marrow suppression due to underlying liver disease. They also explained the decrease of prothrombin concentration and the increase in INR by the decreased production of tissue factor, factor VII, vitamin K-dependent factors, which are synthesized by hepatocytes, and coagulation factors in the common pathway (prothrombin, factors V and X, and fibrinogen). The associated anemia likely results from increased portal pressure generated from the resulting cirrhosis, which leads to a relative hypersplenism,.
Peripheral blood cell counts remain within normal values in most patients with MPD when BCS is present because of hypersplenism, hemodilution, and iron deficiency that affect Hb. Regarding liver function tests, they are altered to various extents according to patients,.
Regarding the biochemical profile of the recruited patients in a study conducted by Chung et al., most of the cases had normal serum albumin, with a mean of 3.26 g/dl in spite of the presence of ascites. This indicated that hepatic congestion and portal hypertension play major roles in the development of ascites.
In the current study, JAK2 V617 F was identified in 11.4% of patients and all were heterozygous. These disorders were confirmed by JAK2 V617F mutation. Recently, clusters of dystrophic megakaryocytes at bone marrow biopsy had been proven to be a specific feature for MPD. JAK2 V617F mutation was not significantly different between patients and controls.
Spivak and Patel et al. found that 40 and 58.5% of patients with BCS have JAK2 mutation, respectively. Colaizzo et al. reported that JAK2 mutation was found in 34.4% of patients of BCS. JAK2 mutation was positive in the Sakr et al. study in 29% of the cases of BCS.
In patients with primary BCS, the JAK2 mutation had been detected in 37–45% of patients.
In BCS, the presence of MPD carried significantly poorer baseline prognostic features that required hepatic decompression procedures earlier but had no impact on 5-year survival. These results suggest that JAK2 V617F testing should replace bone marrow investigations as initial test for MPD in patients with splanchnic vein thrombosis. Underlying MPD is associated with severe forms of BCS, but current therapy appears to cause deleterious effects of MPD on the medium-term outcome.
JAK2 V617F mutation was frequently found in patients with BCS, but there was a huge variation of prevalence among the included studies. In addition, it was more specific to thrombosis in splanchnic areas and strongly associated with the development of MPD.
| Conclusion|| |
JAK2 V617F mutation was not significantly different between patients and control. The discrepancy between results of the present study and results of previous studies could be attributed to the use of different procedures, ethnic variation, and difference in the number of patients involved.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Hong YD, Xin WZ, Gao WJ. China mesojugular shunt for Budd–Chiari syndrome: a case report with long-term follow-up. Ann Vasc surg 2010; 1
Zahn A, Gotthardt D, Weiss K. Budd–Chiari syndrome: long term success via hepatic decompression using transjugular intrahepatic porto-systemic shunt. Gastroenterology 2010; 10
Charles F, Brunicardi FA. Liver and portal venous system. Specific considerations in Schwartz's [Part II; Chapter 31]. In: F. Charles Brunicardi, MD, FACS Los Angeles, California. Principles of surgery
ed. McGraw-Hill; 2010. 465–470.
Wang H, Sun G, Zhang P, Zhang J, Xu H, Zu M, et al.
JAK2 V617F mutation and 46/1 haplotype in Chinese Budd–Chiari syndrome patients. J Gastroenterol Hepatol 2014; 29
Sakr M, Barakat E, Abdelhakam S, Dabbous H, Yousuf S, Eldorry A. Epidemiological aspects of Budd–Chiari in Egyptian patients: a single-center study. World J Gastroenterol 2011; 7
Bayraktar UD, Seren S, Bayraktar Y. Hepatic venous outflow obstruction: three similar syndromes. World J Gastroenterol 2007; 13
Mercier E, Lissalde-Lavigne G, Gris JC. JAK2 V617F mutation in unexplained loss of first pregnancy. N
Engl J Med 2007; 357
Donadini MP, Dentali F, Ageno W. Splanchnic vein thrombosis: new risk factors and management. Thromb Res 2012; 129
Alessandro MV. JAK2 mutation and thrombosis in the myeloproliferative neoplasms. Curr Hemat Malig Rep 2010; 5
Kiladjian JJ, Cervantes F, Leebeek FW, Marzac C, Cassinat B, Chevret S, et al.
The impact of JAK2 and MPL mutations on diagnosis and prognosis of splanchnic vein thrombosis. a report on 241 cases. Blood. 2008; 111
Janssen HL, Leebeek FW. JAK2 mutation: the best diagnostic tool for myeloproliferative disease in splanchnic vein thrombosis? Hepatology 2006; 44
Bergemeyer HU, Horder M, Rej R. International federation of Clinical Chemistry (IFCC) Scientific Committee, Analytical Section: approved recommendation (1985) on IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase (l-alanine: 2-oxoglutarate aminotransferase, EC 18.104.22.168). J Clin Chem Clin Biochem 1986; 24
Young DS. The determination of bilirubin with the photoelectric colorimeter. J Biol Chem 2001; 132
Friedman RB, Anderson RE, Entine SM, Hirshberg SB. effect of disease on clinical laboratory test Clin Chem. 1980;26(4 Suppl):1D-476D.
Hirsh J, Dalen JE, Deykin D, Poller L. Oral anticoagulant, mechanism of action, clinical effectiveness and optimal therapeutic range. Chest 1995; 108
Tobacco A, Meiathini F, Moda E, Tarli P. Simplified enzymatic/colorimetric serum urea nitrogen determination. Clin Chem 1979; 25
Bowers L, Wong E. Kinetic serum creatinine assays. A critical evaluation and review. Clin Chem 1980; 26
Kim J, Rami Y, Haddad F, Ehab A. Myeloproliferative neoplasms: molecular pathophysiology, essential clinical understanding, and treatment strategies. J Clin Oncol 2012; 58
Rasool HJ, Groshek D. Myeloproliferative disease. Blood 2008; 122
Linnemann B, Klaffling C, Schindewolf M, Luxembourg B. JAK2 V617 F
mutation is acquired secondary to the predisposing alteration in familial polycythemia vera. Blood Rev 2011; 107
Kiladjian JJ, Cervantes F, Leebeek FW. Role of JAK2 mutation detection in Budd–Chiari syndrome and portal vein thrombosis associated to MPD. Blood 2006; 106
Roy PK. Budd–Chiari syndrome. Int J Hematol 2006; 77
Hassan M, Sakr M, Dorry A, Abd El-Hakam S. Involvement of inferior vena cava in Egyptian patients with Budd–Chiari syndrome: frequency, relation to etiology, and impact on clinical presentation [Thesis]. Faculty of Medicine, Ain Shams University: Cairo; 2011.
Dilawari JB, Bambery P, Chawla Y, Kaur U, Bhusnurmath SR, Malhotra HS. Hepatic outflow obstruction (Budd–Chiari syndrome). Experience with 177 patients and a review of the literature. Medicine (Baltimore) 1994; 73
Valla DC. Primary Budd–Chiari syndrome. J Hepatol 2009; 50
Chun-Min L, Zhong G, Wang C. Management of Complex Patients with Budd–Chiari syndrome. Ann Vasc Surg 2010; 24
Murad SD, Valla DC, DeGroen PC. Determinants of survival and the effect of portosystemic shunting in patients with Budd–Chiari syndrome. Hepatology 2004; 39
Chandrasekaran S, Velliyappillil J, Kuma A, Joseph G, Venkataraman J. Alternate pathways in hepatic venous outflow obstruction by color Doppler imaging. Ann Gastroenterol 2007; 20
Franca AV, Elias Junior J, Lima BL, Martinelli AL, Carrilho FJ. Diagnosis and treatment of venous thrombosis. Braz J Med Biol Res 2004; 37
Labos C, Dasgupta K. Pancytopenia and atrial fibrillation associated with chronic hepatitis C infection and presumed hepatocellular carcinoma: a case report. J Med Case Reports 2008; 11
Chung RT, Iafrate AJ, Amrein PC. Case records of the Massachusetts general hospital. Case 15-2006, a 46-year-old woman with sudden onset of abdominal distention. N
Engl J Med 2006; 354
Spivak JL. The optimal management of polycythemia vera. Br J Haematol 2002; 116
Patel RK, Lea NC, Heneghan MA, Westwood NB, Milojkovic D, Thanigaikumar M. Prevalence of the activating JAK2 tyrosine kinase mutation V617F in the Budd–Chiari syndrome. Gastroenterology 2006; 130
Colaizzo D, Amitrano L, Tiscia GL, Iannaccone L, Gallone A, Grandone E. Occurrence of the JAK2 V617F mutation in the Budd–Chiari syndrome. Blood Coagul Fibrinolysis 2008; 19
Qi X, Yang Z, Bai M, Shi X, Han G, Fan D. Meta-analysis: the significance of screening for JAK2V617F mutation in Budd–Chiari syndrome and portal venous system thrombosis. Aliment Pharmacol Ther 2011; 33
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]