These authors contributed equally to this work.
JAK2 V617F mutation and 46/1 haplotype in Chinese Budd-Chiari syndrome patients
Article first published online: 19 DEC 2013
© 2013 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd
Journal of Gastroenterology and Hepatology
Volume 29, Issue 1, pages 208–214, January 2014
How to Cite
Wang, H., Sun, G., Zhang, P., Zhang, J., Gui, E., Zu, M., Jia, E., Xu, H., Xu, L., Zhang, J. and Lu, Z. (2014), JAK2 V617F mutation and 46/1 haplotype in Chinese Budd-Chiari syndrome patients. Journal of Gastroenterology and Hepatology, 29: 208–214. doi: 10.1111/jgh.12379
Potential conflicts of interest: The authors declare no conflicts of interest.
- Issue published online: 19 DEC 2013
- Article first published online: 19 DEC 2013
- Accepted manuscript online: 23 AUG 2013 06:05AM EST
- Manuscript Accepted: 9 AUG 2013
- National Natural Science Foundation of China. Grant Number: 81172604
- Budd-Chiari syndrome;
Background and Aim
The presence of JAK2V617F was reported to be associated with JAK2 46/1 haplotype, which was considered as an independent risk factor for Budd-Chiari syndrome (BCS) in Western countries. However, little is known in China. Therefore, the aim of this study was to determine whether the 46/1 haplotype is associated with such patients.
Patients with primary BCS and controls were consecutively admitted in our study from October 2009 to December 2012. The subjects were detected for the JAK2V617F mutation by allele-specific polymerase chain reaction (AS-PCR) and the JAK2 46/1 haplotype by real-time PCR.
The prevalence of JAK2V617F mutation was 2.37% (7/295) in BCS patients, and 46/1 haplotype was overrepresented in JAK2V617F-positive BCS patients compared with controls (P < 0.01). The risk for the JAK2V617F-positive BCS with CC genotype was elevated compared with subjects presented TT genotype (OR = 13.4, 95%CI = 2.01–89.5) and non-CC genotype (OR = 15.0, 95%CI = 2.45–91.7).
Our study showed that the presence of 46/1 haplotype increased the risk of JAK2V617F-positive BCS in China. In addition, low prevalence of JAK2V617F mutation in BCS patients suggested that myeloproliferative neoplasms (MPNs) should not be an etiological factor of BCS in China.
Budd-Chiari syndrome (BCS) is defined as hepatic venous outflow obstruction at any level from the small hepatic veins to the junction of the inferior vena cava (IVC) and the right atrium, regardless of the cause of obstruction.[1-3] BCS is divided into primary when related to a primarily venous disease and secondary when resulting from compression or invasion by a lesion originating outside the veins. BCS is a rare disorder with an incidence of around 1 to 2 per million inhabitants in the Western world, while a relatively higher prevalence in Asia countries such as China, Japan, and India. In China, Henan, Shandong, Jiangsu, and Anhui provinces in Yellow River valley are considered as areas with high prevalence.[6, 7]
The etiology of BCS has been attributed to a variety of genetic and environmental factors while myeloproliferative neoplasms (MPNs) is considered to be the leading cause of BCS. A somatic mutation identified in 40–59% of patients with BCS,[8-10] and 80% of MPNs is JAK2V617F mutation providing a robust diagnostic tool. The prevalence of JAK2V617F mutation in Chinese BCS and the exact role in the pathogenic mechanism is unclear.
Further somatic mutations have been reported in JAK2V617F-negative MPNs and BCS patients, including a cluster of different mutations in exon 12 of JAK2, MPLW515L/K mutation, Factor V Leiden (FVL) mutation, and prothrombin G20210A mutation.[12-15]
Recently, a series of studies showed that the presence of JAK2V617F is associated with an inherited JAK2 46/1 haplotype in MPNs and BCS.[16-19] Simultaneously, other studies suggested this haplotype also frequently occurs in JAK2V617F-negative BCS patients.[20, 21] These findings all implied that JAK2 46/1 haplotype might represent as a susceptible locus for the development of BCS.
To investigate whether JAK2 46/1 haplotype is associated with BCS patients in China, we conducted a case-control study in Chinese individuals.
Subjects and definitions
We performed the study in the hospital affiliated to Xuzhou Medical Collage, China. Two-hundred and ninety-five Chinese BCS patients aged 10–79 years were involved in this study. BCS was diagnosed by Doppler ultrasound, computed tomography(CT), magnetic resonance imaging (MRI) and angiography according to the previously published criteria. Outflow obstruction caused by hepatic veno-occlusive disease and cardiac disorders were excluded. Three hundred fifty-seven volunteers aged 15–82 from the same demographic area were recruited as controls. Healthy controls who had history of thrombosis, hypertension, diabetes, circulatory system disease, metabolic disease history were excluded. This study was approved by the Ethics Committee of the hospital affiliated to Xuzhou Medical College. Written informed consents were obtained from all individuals.
Blood sampling and genotyping
Whole blood samples were collected from the participants and then stored at −80°C. DNA was extracted using the UltraPureTM genomic DNA purification kit (SBS, Shanghai, China). The allele-specific polymerase chain reaction (AS-PCR) was performed to detect the following mutations: JAK2V617F mutation, JAK2 exon12 mutation (K539L, H538QK539L, E543-D544del, N542-E543del, F537-K539delinsl, 547insl+I540-F47dup8, I540-N542delinS), MPLW515L/K mutation, FV Leiden mutation, and prothrombin G20210A mutation. Rs12343867(C/T) was used as a tag single nucleotide polymorphism (SNP) to examine the JAK2 46/1 haplotype. Genomic DNA was amplified using the Applied Biosystems 7900HT Fast Real-Time PCR System (Foster City, CA, USA) according to the manufacturer's instructions. The Assay ID for rs12343867 of the genotyping assays from Applied Biosystems was C__31941689_10. Amplifications were performed in a 384-well format, and post-PCR analysis performed with SDS 2.4 automated software. Approximately, 10% of samples were randomly selected for repeat assays, and results were in agreement with the results of the initial assays.
Baseline data were extracted from the medical records, including demographic characteristics, clinical presentations, laboratory tests, and Child-Pugh score before the study began. Furthermore, all participants filled in a personal history questionnaire, which addressed smoking habits, alcohol ingestion, and women taking contraceptives.
Genotypes were tested for Hardy–Weinberg equilibrium among all participants using a two-sided χ2 test. Continuous variables were summarized as the median values (interquartile range, 25–75 percentiles) and compared using the Mann–Whitney U test. Categorical variables were expressed as frequencies and compared using χ2 tests or the Fisher exact test. The association between JAK2 46/1 haplotype and risk of BCS was estimated by odds ratios (OR) and their 95% confidence intervals (95% CI) using logistic regression. P value of two-tailed and statistical significance was set at P < 0.05. All statistical calculations were performed using the SPSS 16.0 software (Chicago, IL, USA).
General characteristics of the study population
A total of 295 patients and 357 controls were enrolled into the present study. The demographic, clinical, and laboratory characteristics of these individuals were described in Table 1. The median age of patients at diagnosis was 44.0 years while 41.0 years in controls. Compared with controls, BCS patients had significantly higher levels of alpha fetoprotein, aspartate aminotransferase, alanine aminotransferase, total bilirubin, and direct bilirubin while lower levels of high density lipoprotein, low density lipoprotein, white blood cell count, red blood cell count, hemoglobin, platelets count, and a higher prevalence of smoking. Additionally, the patients group had a higher proportion of the poverty (P < 0.01) and manual workers (P < 0.01). However, the alcohol consumption and oral contraceptives use were similar between the two groups.
|Variables||BCS (n = 295)||Controls (n = 357)||P|
|Age, (years)||44.0 (36.0–54.0)||41.0 (31.0–48.0)||0.06|
|Poverty (%)†||117 (51.8)||2 (0.6)||< 0.01|
|Manual workers (%)||179 (68.3)||9 (20.0)||< 0.01|
|Oral contraceptives (Y/N)||12/122||16/149||0.83**|
|AFP, ng/mL||3.7 (2.5–5.9)||2.6 (1.9–3.4)||< 0.01|
|CA125, U/mL||33.1 (15.4–184.7)||10.8 (1.5–21.6)||0.05|
|AST, U/L||30.0 (23.0–42.0)||25.0 (22.0–29.0)||< 0.01|
|ALT, U/L||21.0 (16.0–29.0)||16.0 (11.0–24.8)||< 0.01|
|ALB, g/L||37.8 (32.5–42.1)||44.7 (43.3–46.0)||< 0.01|
|TBIL, μmol/L||30.9 (20.6–48.9)||14.9 (11.7–18.5)||< 0.01|
|DBIL, μmol/L||11.6 (7.4–18.9)||4.6 (3.6–5.6)||< 0.01|
|HDL, mmol/L||1.4 (1.1–1.7)||1.5 (1.3–1.8)||< 0.01|
|LDL, mmol/L||1.8 (1.4–2.2)||2.8 (2.3–3.3)||< 0.01|
|WBC count, 109/L||3.8 (2.8–5.4)||5.9 (5.0–6.8)||< 0.01|
|RBC count, 1012/L||4.2 (1.4–2.2)||4.6 (4.25–5.01)||< 0.01|
|Hb, g/L||121.0 (103.0–137.0)||138 (126–150)||< 0.01|
|PLT count, 109/L||98.0 (73.0–174.0)||187.5 (159.0–221.0)||< 0.01|
Differences between the patients with and without the JAK2V617F mutation
JAK2V617F mutation was detected in 2.37% (7/295) of these patients. The differences between patients with and without the JAK2V617F mutation were described in Table 2. Compared with those without the JAK2V617F mutation, the patients with the mutation had higher levels of direct bilirubin (P = 0.03), prothrombin time (P = 0.01), and international normalized ratio (P = 0.01). The clinical symptoms and signs were similar between two groups.
|n = 7||n = 288|
|Age, (years)||39 (20–64)||44 (36–54)||0.58|
|AFP, ng/mL||3.74 (1.88–5.69)||3.71 (2.45–5.88)||0.86|
|CA125, U/mL||93.1 (21.83–200.53)||30.3 (15.3–183.5)||0.60|
|AST, U/L||45 (31–59)||29 (23–41)||0.05|
|ALT, U/L||32 (18–38)||21 (15.25–29)||0.07|
|ALB, g/L||32.1 (30.23–39.78)||37.9 (32.8–42.1)||0.20|
|TBIL, μmol/L||52.8 (31.5–84.25)||30.65 (20.38–48.05)||0.08|
|DBIL, μmol/L||22.5 (15.6–38.05)||11.4 (7.3–18.35)||0.03|
|WBC count, 109/L||4.53 (2.06–19.18)||3.77 (2.79–5.38)||0.27|
|RBC count, 1012/L||4.52 (3.08–6.59)||4.16 (3.69–4.65)||0.31|
|Hb, g/L||124 (87–167)||121 (103–136.5)||0.64|
|PLT count, 109/L||134 (99–268)||97 (71.25–146)||0.07|
|Abdominal pain (%)||7 (100)||19 (7.2)||1.00|
|Abdominal wall varices (%)||1 (14.3)||159 (57.4)||0.06|
|Disease duration, month (%)||12 (0.9–60)||60 (10.8–120)||0.07|
|Lower back varices (%)||1 (14.3)||78 (28.4)||0.69|
|Edema of lower limbs (%)||4 (57.1)||157 (56.3)||1.00|
|Varices of lower limbs (%)||1 (14.3)||62 (22.9)||0.94|
|Pigmentation of lower limbs (%)||2 (28.6)||124 (45.1)||0.63|
|Ulcer of lower limbs (%)||0 (0)||49 (18.1)||0.46|
|Fatigue (%)||7 (100)||201 (74.7)||0.13|
|Portal hypertension (%)||0 (0)||49 (18.1)||0.14|
|Hepatomegaly (%)||1 (14.3)||85 (32)||1.00|
|Cirrhosis (%)||1 (14.3)||62 (23.4)||0.91|
|Splenomegaly (%)||4 (57.1)||181 (67.8)||0.85|
|Ascites (%)||4 (57.1)||146 (54.9)||1.00|
|Child-Pugh class: A/B/C||5/2/0||66/87/38||0.07|
|PT, sec||17.8 (16.2–23.7)||14.9 (13.9–16.5)||0.01|
|INR||1.49 (1.35–2.12)||1.22 (1.13–1.38)||0.01|
Association between the JAK2 46/1 haplotype and patients with Budd-Chiari syndrome
The SNP rs12343867 genotype distributions of the study population and the OR for BCS were presented in Table 3. DNA samples for this study were available for 295 BCS patients and 332 healthy controls. Of these, 274 BCS patients (93%) and 310 controls (93%) were successfully genotyped. The number of BCS patients with CC, CT, and TT genotypes were 16, 83, and 175, respectively, while the controls were 10, 99, and 201. Genotype distribution of our study population was in Hardy–Weinberg equilibrium. In the overall group of BCS, there was no significant difference in frequency of JAK2 46/1 haplotype (that is the rs12343867 C-allele frequency) compared with the controls (21% vs 19%; P = 0.56). However, when stratified for the presence of the JAK2V617F mutation, 46/1 haplotype was presented more frequently in the patients of JAK2V617F-positive mutation than in controls (42% vs 19%, P < 0.01). Meanwhile, the risk for the JAK2V617F-positive BCS in subjects with the CC genotype was elevated compared with subjects with the common TT genotype (OR = 13.4, 95% CI = 2.01–81.5). When combined TT with CT, we could also find a significantly increased risk of JAK2V617F-positive BCS associated with CC (OR = 15.0, 95%CI = 2.45–91.7). No difference in JAK2 46/1 haplotype frequency was observed in the JAK2V617F-negative individuals with BCS compared with controls (21% vs 19%; P = 0.72).
|Study population||n||Rs12343867 genotype||C allele frequency||P||Odds ratio (95% CI)|
|CC||CT||TT||TT versus CT||TT versus CC||TT versus (CC + CT)||(TT + CT) vs CC|
|Overall||274||16||83||175||0.21||0.72||0.96 (0.67–1.36)||1.68 (0.76–3.71)||1.03 (0.73–1.44)||1.86 (0.83–4.17)|
|JAK2V617F-positive||6||2||1||3||0.42||< 0.01||0.67 (0.07–6.56)||13.4 (2.01–89.50)||1.82 (0.36–9.17)||15.0 (2.45–91.71)|
|JAK2V617F-negative||268||14||82||172||0.21||0.72||0.96 (0.68–1.38)||1.50 (0.66–3.38)||1.02 (0.72–1.43)||1.65 (0.72–3.79)|
Characteristics associated with JAK2 46/1 haplotype in patients with Budd-Chiari syndrome
We examined the association between JAK2 46/1 and demographic/clinical features of the BCS patients (Table 4). The results showed that no difference was observed.
|Variables||rs12343867 genotype (BCS)||P1||P2||P3|
|Age, years||47 (30–56)||43 (37–53)||44 (35–56)||0.94||1.00||0.75|
|AFP, ng/mL||3.49 (2.52–6.56)||3.68 (2.52–5.75)||4.14 (3.04–7.79)||0.62||0.35||0.67|
|CA125, U/mL||18.3 (6.95–160.1)||37.2 (14.9–196.9)||28.2 (21.9–53.8)||0.99||0.86||0.97|
|AST, U/L||30.0 (23.3–42.8)||29.0 (21.0–41.0)||35.0 (23.0–41.0)||0.80||0.78||0.73|
|ALT, U/L||21.0 (16.0–29.0)||20.0 (14.0–32.0)||22.0 (18.0–33.0)||0.51||0.50||0.62|
|ALB, g/L||37.4 (29.4–44.4)||38.5 (32.5–42.5)||40.6 (36.2–43.7)||0.35||0.15||0.35|
|TBIL, μmol/L||32.6 (25.0–62.7)||28.1 (19.0–42.0)||37.9 (29.7–55.0)||0.16||0.16||0.70|
|DBIL, μmol/L||15.8 (11.0–24.3)||10.1 (7.0–15.0)||14.3 (8.7–22.5)||0.25||0.28||0.58|
|HDL, mmol/L||1.41 (1.07–1.59)||1.54 (1.15–1.83)||1.58 (1.34–1.82)||0.52||0.34||0.29|
|LDL, mmol/L||1.77 (1.41–2.19)||1.55 (1.18–2.20)||1.92 (1.70–2.43)||0.12||0.51||0.15|
|WBC count, 109/L||4.38 (3.24–6.58)||3.68 (2.81–5.38)||3.43 (2.52–3.89)||0.62||0.36||0.44|
|RBC count, 1012/L||4.37 (3.92–4.67)||4.09 (3.59–4.52)||4.52 (3.72–4.75)||0.30||0.52||0.35|
|Hb, g/L||132 (113–138)||115 (103–132)||132 (106–142)||0.39||0.43||0.57|
|PLT count, 109/L||104 (76–148)||91 (61.8–145.3)||93 (76–118)||0.68||0.76||0.39|
|PT, sec||15.9 (14.8–19.1)||14.6 (13.5–16.4)||14.9 (14.5–18.4)||0.10||0.20||0.36|
|INR||1.33 (1.21–1.64)||1.20 (1.10–1.35)||1.22 (1.16–1.55)||0.32||0.33||0.57|
Other genetic mutations detected in BCS patients
The results of gene mutation screening were shown in Table 5. In the gene of JAK2 exon12, both K539L and H538QK539L were found five in 295 patients, no other mutations were found. Neither MPLW515L/K mutation nor FVL mutation was found in any of 295 patients tested as well as prothrombin G20210A mutation.
|Positive JAK2V617F mutation, n/n (%)||7/295 (2.37)|
|Positive JAK2 exon12 mutation, n/n (%)|
|Positive MPLW515L/K mutation, n/n (%)||0/295 (0)|
|Positive FV Leiden mutation, n/n (%)||0/295 (0)|
|Positive prothrombin G20210A mutation, n/n (%)||0/295 (0)|
|Positive JAK2 46/1 haplotype, n/n (%)|
|Homozygous rs12343867 CC genotype||16/274 (5.84)|
|Heterozygous rs12343867 CT genotype||83/274 (30.29)|
The present study is the first to evaluate the prevalence of the JAK2V617F mutation and 46/1 haplotype in such a relatively large cohort of BCS patients in China.
Previous studies reported JAK2V617F mutation was detected in a larger number of sporadic BCS patients,[22-24] and the prevalence of mutation was different. In 2006, Patel et al. and Primignani M et al. identified a high prevalence, 58.5% and 40%, respectively. Then, Regina et al. found that JAK2V617F was specifically associated with idiopathic splanchnic vein thrombosis, with a prevalence of 18.2% in BCS patients. In India, several studies[25, 26] also conducted to detect such mutation which ranged from 8.8% to 40%. Compared with previous studies, our study showed a low prevalence in Chinese BCS patients, which was significantly lower than 37% reported in a recent meta-analysis. The contradictory results could be explained by the known different incidences of MPNs in BCS. Our result was consistent with another study conducted in China (4.3%), which indicated that MPNs could be an uncommon risk factor of BCS in China. In the year of 2007, JAK2V617F mutation was detected in a large Chinese hospital population by Xu et al. The 37 samples from a total of 3935 were found to be positive cases whose red cell counts, white blood, and platelet counts were all within the normal range. This data suggested that the JAK2V617F mutation was apparently much more common than MPNs in Chinese, which confirmed our conclusion from another point. Furthermore, higher levels of prothrombin time and international normalized ratio were closely associated with JAK2V617F mutation in Chinese BCS patients which was different from previous reports with elevated peripheral blood cell counts.[16, 22] Given low prevalence of JAK2V617F mutation, further study needs to confirm these findings.
Additionally, Andrikovics H reported that JAK2V617F-associated disease was highly associated with a specific haplotype named JAK2 46/1 haplotype which was a 280 kb-long region on chromosome 9p including the entire JAK2, INSL6, and INSL4 genes. In our study, we found that the JAK2 46/1 haplotype frequency was similar between BCS and controls. It is noteworthy that only one previous study examined the role of 46/1 haplotype in BCS on larger number of patients, which showed the 46/1 haplotype presented more frequently in patients. In this study, JAK2V617F positive patients accounted for 32% in overall BCS while 2.37% in our study; could this be the reason leading to different prevalence of 46/1 haplotype? But to date, it is not clear why JAK2V617F mutation is associated with a particular inherited haplotype, and two hypotheses have been suggested,[18, 30, 31] the hypermutability hypothesis and fertile ground hypothesis. The first hypothesized that 46/1 may be more easily to acquire V617F mutation than other haplotypes for its genetic instability. The second hypothesis suggested that V617F may appear on all haplotypes with same rate, but 46/1 may carry specific properties that either give a selective advantage to the V617F-positive clone or gain proliferative advantage in some way. Nevertheless, our result showed that the risk of BCS occurrence significantly elevated in JAK2V617F-positive patients in homozygous carriers of 46/1 compared with noncarriers. That meant that the presence of 46/1 haplotype increased the risk of occurrence of JAK2V617F-positive BCS in an allele-dependent manner. This result was in agreement with the previous study which showed an association between 46/1 haplotype and the risk of developing BCS with JAK2V617F mutation. Additionally, the current data demonstrated that no difference was found between patients with different rs12343867 genotypes, which implied JAK2 46/1 haplotype seem not to be associated with distinct clinical and laboratory characteristics of BCS in China. Combined with the above two hypotheses, a possible explanation for the higher incidence of rs12343867 CC genotype in patients with JAK2V617F mutation is that the presence of CC genotype is not sufficient in itself for the disease but appears to be in linkage with JAK2V617F or other unidentified variations. Clearly, this explanation deserves further studies.
Interestingly, the JAK2 46/1 haplotype was a risk factor for MPNs in China,[32, 33] which were in line with previous reports conducted in Western countries.[17, 18, 21] According to researches, MPNs were only accounted for 4.1–5.0% in Chinese BCS patients.[34, 35] Taken with low prevalence of JAK2V617F mutation together, MPNs seemed not to be the etiological factor for Chinese BCS patients.
Reviewed the researches about BCS in western countries, underlying inherited or acquired thrombotic risk factors were reported including MPNs, protein C deficiency, protein S deficiency, antithrombinIII deficiency, FVL mutation, prothrombin G20210A mutation, JAK2 exon12 mutation, MPLW515L/K mutation, paroxysmal nocturnal hemoglobinuria (PNH), antiphospholipid syndrome (APS), Behcet's disease.[36-39] However, in China, literatures indicated that FVL mutation, prothrombin G20210A mutation, and PNH were rarely found in BCS patients.[34, 39-41] Our research also showed a low prevalence of JAK2 exon12 mutation, while the other gene mutation showed negative. In addition, oral contraceptive use has been shown to increase the risk of BCS with odds ratios about 2.5 as compared to nonusers, which was not demonstrated in our population. Combined with the results of our study, we could infer that the etiological distribution of BCS is geographically and ethnically different. A case-control study conducted in Nepal showed that IVC obstruction was associated with a very poor standard of living. Our survey also displayed that majority of BCS patients presented with IVC obstruction (157/282, data not shown) and were engaged in manual work with family financial difficulties.
In conclusion, our study suggested that the presence of 46/1 haplotype increased the risk of occurrence of JAK2V617F-positive BCS in China. In addition, BCS patients had a very low prevalence of the JAK2V617F mutation, which revealed that MPNs might not be the etiological factor of Chinese patients. However, further studies addressing these issues are required, and these results prompt us to explore the actual etiology of BCS in China.
This study was supported by the National Natural Science Foundation of China (grant number 81172604).
- 6Surgical treatment of 1360 cases of Budd-Chiari syndrome: 20-year experience. Hepatobiliary Pancreat. Dis. Int. 2004; 3: 391–1394., , et al.
- 17The JAK2 46/1 haplotype confers susceptibility to essential thrombocythemia regardless of JAK2V617F mutational status—clinical correlates in a study of 226 consecutive patients. Leukemia 2009; 24: 110–114., , et al.
- 26Prevalence of JAK2V617F mutation in intra-abdominal venous thrombosis. Trop. Gastroenterol. 2011; 32: 279–284., , , , , .