A TET2 rs3733609 C/T genotype is associated with predisposition to the myeloproliferative neoplasms harboring JAK2(V617F) and confers a proliferative potential on erythroid lineages

Abstract

Common germline single-nucleotide polymorphisms (SNPs) at JAK2 locus have been associated with Myeloproliferative neoplasms (MPN). And, the germline sequence variant rs2736100 C in TERT is related to risk of MPN, suggesting a complex association between SNPs and the pathogenesis of MPN. Our previous study (unpublished data) showed that there was a high frequency distribution in rs3733609 C/T genotype at Ten-Eleven Translocation 2 (TET2) locus in one Chinese familial primary myelofibrosis. In the present study, we evaluate the role and clinical significance of rs3733609 C/T genotype in JAK2V617F-positive sporadic MPN (n = 181). TET2 rs3733609 C/T genotype had a higher incidence (13.81%; 25/181) in JAK2V617F-positive sporadic MPN patients than that in normal controls (n = 236) (6.35%; 15/236), which was predisposing to MPN (odds ratio(OR) = 2.361; P = 0.01). MPN patients with rs3733609 C/T genotype had increased leukocyte and platelets counts, elevated hemoglobin concentration in comparison with T/T genotype. Thrombotic events were more common in MPN patients with rs3733609 C/T than those with T/T genotype (P < 0.01). We confirmed that rs3733609 C/T genotype downregulated TET2 mRNA transcription, and the mechanism may be involved in a disruption of the interaction between CCAAT/enhancer binding protein alpha (C/EBPA) and TET2 rs3733609 C/T locus.TET2 rs3733609 C/T genotype stimulated the erythroid hematopoiesis in MPN patients. Altogether, we found a novel hereditary susceptible factor-TET2 rs3733609 C/T variant for the development of MPN, suggesting the variant may be partially responsible for the pathogenesis and accumulation of MPN.

Keywords: TET2; clinical significance; mechanisms; myeloproliferative neoplasms; rs3733609.

Figure 1. A representative DNA sequence of TET2 rs3733609 genotype (Left: T/T genotype; Right: C/T genotype)

Figure 2. The mRNA expression levels for TET2 gene in bone marrow mononuclear cells between C/T and T/T genotype MPN patients

Compared with T/T genotype, TET2 mRNA levels were markedly decreased by 71.2% in rs3733609 C/T genotype group (P = 0.006).

Figure 3. C/EBPA binds to the exon 9 of TET2

(A) A schematic diagram for the positions of putative C/EBPA protein binding sites in the exon 9 of TET2. Arrows indicate the regions for PCR primers amplification. (B) CHIP assay was performed on human bone marrow mononuclear cells (BMMNCs) using C/EBPA antibody or mouse IgG and the immunoprecipitated chromatin DNA was subjected to PCR using primers to amplify the region of biding sites for C/EBPA. (C) Precipitated chromatin DNA were subjected to qPCR to amplify this region, and the results showed that the region of +86 to +98 in TET2 exon 9 was enriched 15.7 fold in the C/EBPA chromatin immunoprecipitates in patients with rs3733609 T/T genotype compared with C/T genotype group (P < 0.001).

Figure 4. Luciferase activities assay

(A) A schematic diagram of the reporter constructs containing the sequences of TET2 exon 9. The mutant construct containing identified mutation site (position +96) in the TET2 exon 9 is shown. (B) Results are shown as fold change of luciferase activity corresponding to pGL3-promoter vector, the wild vector displayed the stronger luciferase activity than the pGL3-promoter-vector (P = 0.014); the luciferase activity of mutant vector was dramatically reduced to 0.38 fold in transfected HEL cells than that of the wild-type vector (P = 0.009). Values are means ± SD of triplet data from 3 different experiments.

Figure 5. Photomicrographs of BFU-E colonies in vitro (Left: magnification 40×; Right: magnification 100×)

(A) The BFU-E colonies derived from the bone marrow mononuclear cells with C/T genotype. (B) The BFU-E colonies derived from the bone marrow mononuclear cells with T/T genotype. (C) Colonies were stained by benzidine staining. Blue-black colonies were BFU-E colonies. As shown in Figure A and B, the colony numbers and volume from C/T genotype patients were more and larger than that of T/T genotype.