The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population

In the current study conducted at the Tokyo Medical University Hospital, 138 constitutive Japanese MPN patients aged 30-87 years with known JAK2 V617F status were included: 33 patients with JAK2 V617F-positive PV, 57 patients with JAK2 V617F-positive ET, 39 patients with JAK2 V617F-negative ET, and 9 patients with PMF. The patients experienced no familial MPNs. We revised their classification at diagnosis according to the latest World Health Organization classification of MPNs. As controls, 107 healthy volunteers aged 24-86 years from the same demographic area in Japan were used. The JAK2 V617F mutation detection system used was reported elsewhere [14], and the JAK2 V617F mutational status was categorized according to the allele burden of mutated T allele. This study was approved by the institutional review board of Tokyo Medical University (no. 975). Written informed consent according to the Declaration of Helsinki was obtained from all patients prior to collection of the specimens.

Genomic DNA was obtained from whole blood using an automated system (Qiagen). To identify novel SNPs in the JAK2 locus in this Japanese population, primer sets for the amplification of JAK2 were designed according to GenBank AL161450 (Figure 1A; Additional file 1A). PCR conditions were 94°C for 30 s, 58°C for 30 s, and 72°C for 3 min for 36 cycles using High Fidelity^PLUS PCR System dNTPack (Roche Diagnostics, Mannheim, Germany). The PCR products were purified by a High Pure PCR Product Purification Kit (Roche Molecular Biochemical Diagnostics, Indianapolis, IN, USA) and sequenced using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with an Applied Biosystems 3130 Genetic Analyzer. The obtained sequences were compared with the JAK2 sequence.

To determine whether minor alleles of JAK2 SNPs favor the cis acquisition of JAK2 V617F, we performed allele-specific analysis of six SNPs in patients with PV. The sequence JAK2 nt51936-nt55084 (3158 bp) was amplified using forward primer int12-F and reverse primer V617F-R or exon14-R. The primer set for the amplification of JAK2 nt55038-nt55636 (598 bp) was forward primer V617F-F or exon14-F and reverse primer int14-R. Both primers V617F-R and V617F-F could amplify the T allele of JAK2 V617F (Figure 1B; Additional file 1A). The PCR products were purified and sequenced as described above.

Allele-specific PCR was performed with a common forward primer and two allele-specific reverse primers (Additional file 1B) using High Fidelity^PLUS PCR System dNTPack (Roche Diagnostics) and SYBR Green I (Lonza, Rockland, ME, USA). The PCR conditions were 94°C for 30 s, 58°C for 30 s, and 72°C for 3 min for 40 cycles using an iCycler iQ Real-time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). To avoid nonspecific PCR products, melting analysis was performed by denaturing at 95°C for 1 min and cooling to 55°C for 1 min followed by heating at the rate of 0.5°C/10 s from 55 to 95°C.

GraphPad Prism 5.0 software (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. Associations with risk of MPNs were estimated by odds ratios (ORs) and their 95% confidence intervals (95% CIs) using logistic regression. A Mann-Whitney U-test was used to determine the statistical significance of differences between the control and test groups. P-values less than 0.05 were considered to indicate statistically significant differences. We also performed multivariate analysis using College analysis software (version 4.5, Fukuyama Heisei University, Fukuyama, Japan) to exclude possible false correlation between genotype and clinical manifestations.

We first screened for 24 SNPs of the JAK2 locus around exons 12 to 14 in 28 Japanese patients with JAK2 V617F-positive PV from whom we obtained sufficient DNA for this analysis (Figure 1A). Among them, minor allele frequencies in six SNPs (rs10974944, rs12686652, rs12335546, rs4495487, rs1028730, and rs12343867) were significantly higher in PV patients than in healthy volunteers (Table 1). There were no significant differences in age or sex between the PV population and healthy controls (data not shown). Minor allele frequency was estimated by total cases that had at least one minor allele (heterozygous) or two minor alleles (homozygous). The JAK2 SNP rs4495487, which has not been reported previously in Caucasian populations, showed the highest OR (13.8, 95% CI: 3.79-50.21) among the six SNPs.

To determine whether minor alleles of JAK2 SNPs favor the cis acquisition of JAK2 V617F, we next sequenced six SNPs using allele-specific primers (Additional file 1A). In accordance with a previous report [8], in the genotype with minor alleles in all six SNPs, the T allele was more frequently observed in JAK2 V617F than the G allele in normal controls; the OR was 7.74 (95% CI: 2.32-25.75) (Additional file 2).

We genotyped 138 MPN patients with known JAK2 mutational status and 107 controls for JAK2 SNP rs449587 in addition to two SNPs that are known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Overall, 95 MPN patients (68.8%) were JAK2 V617F positive. When analyzing each MPN entity separately, we found that 33 PV patients (100%), 57 ET patients (63.3%), and 5 PMF patients (55.5%) were JAK2 V617F positive. The distribution of JAK2 SNPs (rs10974944, rs4495487, and rs12343867) is listed in Table 2. Allelic variation of three JAK2 SNPs was strongly associated with JAK2-positive MPN (all patients with PV, 57 patients with ET, and 5 patients with PMF) and much less strongly associated with JAK2-negative MPN (39 patients with ET and 4 patients with PMF). It is notable that allelic variation of the JAK2 SNP rs4495487 showed the highest OR in each population. The JAK2 SNP variation in 9 PMF patients is listed in Additional file 3. Because allelic variation of rs4495487 showed the highest OR in each patient population, we arbitrarily named genetic variation as the "GCC genotype," which has at least one minor allele in each of the three SNPs (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867); patients having the 46/1 haplotype and C allele of rs4495487. The GCC genotype is strongly associated with JAK2 V617F-positive MPNs (OR: 3.07; 95% CI: 1.73-5.46) and modestly associated with JAK2 V617F-negative MPN (OR: 2.26, 95% CI: 1.01-4.7) compared to normal controls (Table 3). The occurrence of the GCC genotype, however, did not depend on JAK2 V671F allele burden (Table 4).

We compared the clinical and hematological features of PV and ET patients with or without the GCC genotype. We subdivided ET patients into four groups: JAK2 V617F-negative ET patients with or without the GCC genotype and JAK2 V617F-positive ET patients with or without the GCC genotype (Table 5). None of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis (p = 0.0446), and splenomegaly was more frequently seen in this subset of ET patients (p = 0.0448), indicating that JAK2 V617F-negative ET without genetic variation shows a distinct clinical feature. White blood cell counts were significantly elevated in patients with JAK2 V617F-positive ET, regardless of GCC genotype status (p = 0.0399) (Figure 2A). Hemoglobin levels were significantly elevated in patients with both JAK2 V617F and the GCC genotype compared to those with the GCC genotype but lacking JAK2 V617F (p = 0.002) (Figure 2B). There was no significant difference in platelet counts among the four groups. These results indicate that the proliferative nature of MPNs, such as increased white blood cell and hemoglobin counts, may be linked to JAK2 V617F, regardless of JAK2 genetic variations.

Because all the PV patients in the current study were JAK2 V617F positive, we compared the clinical and hematological features between PV patients with or without the GCC genotype (Table 6). Although there were no significant differences in age, sex, clinical manifestations, or survival between the two groups, platelet count was significantly elevated in PV patients without the GCC genotype (p = 0.015) (Figure 2). These findings suggest that germline genetic variation also affects PV patients.