TET2, ASXL1, IDH1, and IDH2 Single Nucleotide Polymorphisms in Turkish Patients with Chronic Myeloproliferative Neoplasms

We aimed to determine the genotype distribution, allele frequency, and prognostic impact of IDH1/2, TET2, and ASXL1 single nucleotide polymorphisms (SNPs) in myeloproliferative neoplasms (MPNs). TET2 (rs763480), ASXL1 (rs2208131), and IDH1 (rs11554137) variant homozygous genotype frequencies were found at rates of 1.5%, 9.2%, and 2.3%, respectively. No IDH2 SNP was identified. IDH1 and TET2 frequencies were 5% in essential thrombocythemia (ET) and 1.7% in ET and 5% in primary myelofibrosis (PMF), respectively. ASXL1 frequencies were 8.3%-10% in MPN subgroups. The TET2 mutant allele T and ASXL1 mutant allele G had the highest frequencies with 0.272 in the PMF and 0.322 in the polycythemia vera (PV) group, respectively. There was no impact of the SNPs on prognosis. IDH1 frequency in MPNs was found similar to the literature. ASXL1 frequencies were similar between ET, PV, and PMF patients. The ASXL1 and TET2 allele frequencies of the Turkish population are similar to those of the European population. The role of SNPs in MPNs might be further evaluated in larger multicenter studies.

ASXL1 encodes a member of the chromatin-binding proteins and is involved in epigenetic regulation of gene expression [7]. ASXL1 mutations are rare in ET and PV (<7%) but are frequent  [3,7]. Worsened survival was reported in PMF patients with mutated ASXL1 [8].
TET proteins are enzymes that can convert 5-methylcytosine to 5-hydroxymethylcytosine [9]. TET2 mutation frequency was 16% in PV, 5% in ET, and 17% in PMF. Previous research did not identify an impact of TET2 mutations on survival or leukemic transformation [10].
Although the prognostic impact of these mutations has been investigated in some studies, there is still limited information available [6,8,10]. In this study, we focused only on single nucleotide polymorphisms (SNPs) in the ASXL1, TET2, and IDH1/2 genes. We aimed to determine the genotype distribution, allele frequency, and prognostic impact of selected SNPs in MPNs.

Materials and Methods
The study included 130 MPN patients. The diagnosis of MPNs was performed based on World Health Organization and International Working Group criteria [11]. The Ege University Local Ethics Committee approved the study (13-5.1/8, 15.07.2013). Patient data were collected from the patient files.
SNP analysis was performed on DNA derived from peripheral blood samples that were collected into tubes containing EDTA between February 2008 and September 2009 and were stored at -80 °C until the DNA extraction. DNA extraction was performed using the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche) according to the manufacturer's instructions. After DNA isolation, the purity and concentration of DNA was measured using a NanoDrop spectrophotometer (Thermo Scientific, USA) at 260 nm and 280 nm.
All SNP analysis was carried out with ready-to-use TaqMan assays from Life Technologies (USA): [rs763480-(A/T conversion)], and ASXL1 [rs2208131-(A/G conversion). Realtime polymerase chain reaction (PCR) reactions were performed according to the manufacturer's instructions using the ABI 7500 Fast Real-Time PCR instrument (Applied Biosystems, USA). The real-time PCR cycling conditions were: 95 °C for 10 min for enzyme activation, 40 cycles of 95 °C for 15 s (denaturation), and 60 °C for 1 min (annealing/extension). All assays were evaluated and genotyped using SDS software.

Statistical Analysis
Hardy-Weinberg equilibrium was used for each SNP. All p-values were two sided and p≤0.05 indicated statistical significance. Categorical and continuous variables were compared with chisquare statistics and the Mann-Whitney U test, respectively. Survival analysis was performed by the Kaplan-Meier method, taking the interval from the date of diagnosis to death or last contact. The log-rank test was used to compare the survival data.

Results
The demographic features of patients at the time of diagnosis are shown in Table 1. IDH2 (rs121913503) and IDH2 (rs267606870) SNPs were not detected in any of the patient groups; all of the cases were genotyped as wild-type homozygous (GG and CC, respectively, for the rs numbers). Two (1.5%) of all, 12 (9.2%) of all, and 3 (2.3%) of all patients were variant homozygous for TET2 (rs763480), ASXL1 (rs2208131), and IDH1 (rs11554137) SNPs, respectively ( Table 2).
The TET2 mutant allele T frequency was 0.218 in the PV, 0.20 in the ET, and 0.272 in the PMF group. The ASXL1 mutant allele G frequency was 0.322 in the PV, 0.308 in the ET, and 0.25 in the PMF group. The IDH1 mutant allele T frequency was 0.073 in the PV, 0.108 in the ET, and 0.045 in the PMF group.
The median follow-up time was 8 years (range: 1-25). The estimated 10-year survival rate was 71% for ET, 87.3% for PV, and 71% for PMF patients (Figure 1). We did not find any significant differences between the diagnostic subgroups and the 10-year survival rates. At the time of the analysis, 5 and 10 patients had leukemic transformation and fibrotic transformation, respectively. There were no significant differences between the sexes, diagnoses, JAK2 mutation status (positive/negative), thrombosis status at diagnosis, survival rates, leukemic and fibrotic transformation, and genotyping results of ASXL1, TET2, and IDH1 (Table 3).
In our series, TET2 (rs763480) frequency was higher in PMF patients.
ASXL1 mutation frequency was 10% in MPN patients. These mutations are rare in ET and PV (<7%) but frequent in PMF (19%-40%) [3,7]. In JAK2-and MPL-negative MPN patients, TET2 and ASXL1 mutation frequencies were 8% [14]. ASXL1 mutation frequency was 24.7% in PMF and 8.4% in ET patients [15]. ASXL1 mutation frequency was 12%-13% in PMF patients [16]. The frequency of the ASXL1 (rs2208131) SNP was 9.2% and this was similar between ET, PV, and PMF patients. We did not find any previous study that evaluated ASXL1 (rs2208131) and TET2 (rs763480) SNPs in MPNs. There was no relationship between TET2, IDH1, and ASXL1 SNPs and clinical and laboratory factors in our study. An impact of TET2 mutation on survival and leukemic transformation was not shown [10]. In normal karyotype acute myeloid leukemia, the IDH1 (rs11554137) SNP was an adverse prognostic factor [17]. In PMF, there was a significantly negative impact of IDH mutations on survival [6]. ASXL1 mutations were identified in patients with PMF or post-ET/PV-myelofibrosis and associated with poor survival [8,9,15]. Whether the TET2, IDH1, and ASXL1 SNPs possibly confer any prognostic impact in MPN patients requires further evaluation.
The TET2 mutant allele T and ASXL1 mutant allele G had the highest frequencies at 0.272 in the PMF and 0.322 in the PV group, respectively. The ASXL1 and TET2 mutational statuses in the Turkish population are similar to those of the European population according to HapMap CEU data reporting 0.322 G mutant allele and 0.372 T mutant allele frequencies, respectively (hapmap.ncbi.nlm.nih.gov/).
One of the limitations of this study is the small sample size. Since we did not evaluate MPL and CALR mutations, we could not assess whether there was a relationship between CALR and MPL mutations and these SNPs. Since we had no control group, we could not perform a comparison for these SNPs between patients and controls.

Conclusion
IDH1 frequency in MPNs was found to be similar to the rate reported in the literature. ASXL1 frequencies were similar between ET, PV, and PMF patients. We did not find an impact of the SNPs on survival, JAK2 status, or leukemic and fibrotic transformation. These findings suggest that IDH1 is a rare SNP in MPNs. The role of SNPs in MPNs might be further evaluated in larger multicenter studies.

Authorship Contributions
Surgical