Overexpression mRNA of HMGA2 and WT1 Genes in Whole Blood Samples As Potential Diagnostic Markers in MPN
Authors: Stolyar Marina , Gorbenko Aleksey , Mikhalev Mikhail , Olkhovik Tatiana , Khazieva Anna , Belevcova Nataliya , Tabakova Kseniya , Vasiliev Evgeniy , Olkhovskiy Igor 
Published: October 2019

Abstract
Background
High-mobility group AT-hook protein 2 (HMGA2) gene codes for a non-histone protein which expressed during embryogenesis and early hematopoiesis but suppressed in differentiated cells. Overexpression of HMGA2 as well as transcription factor Wilms' tumor 1 (WT1) gene has been found in a variety of human cancers, but only a few reports has included myeloproliferative neoplasms (MPNs) (Guglielmelli P. et al., 2007; Chen C-C. et al., 2017; Tasdemir S. et al, 2015). Furthermore, there were no any data about co-expression of mRNA both genes in blood in patients with MPNs.
Aims: evaluate of the diagnostic potential of the WT1 and HMGA2 mRNA level in MPN patients.
Methods
In our retrospective study was analysed 115 archived frozen peripheral blood samples with RNAse inhibitor from 62 MPN patients: 29 with polycythemia vera (PV), 16 with essential thrombocythemia (ET), and 17 with myelofibrosis (MF) were included. Samples of 28 healthy volunteers were tested as control group. Total RNA was isolated using "Ribo-zol-D" (Aplisens) and then were transcribed using "Reverta-L" (Aplisens). Synthesized cDNA was analysed by real-time PCR to detect of WT1 and HMGA2 expression levels on the CFX96 (Bio-Rad) housekeeping kits using. The results were calculated utilizing the delta Ct method in the software package of "R". The threshold cycles (Ct) WT1 and HMGA2 genes and reference gene ABL determined using Cy0 method. Results are presented as median and range (25% and 75%). Comparisons between the patient and control group were made using the Mann-Whitney U test. Statistical analysis was performed using Statistica 12.0 software.
Results
The level of HMGA2 mRNA in five samples of venous blood in PV patients and 12 samples in MF patients was higher than in healthy subjects (Figure 1). Expression of WT1 mRNA (≥ 0.01%) was observed only in samples among patients with MF (in 19 of 28). In 12 blood samples of patients with MF, transcripts of both genes were detected together (Table 1). The increased level of HMGA2 mRNA expression was associated with a higher level of erythrocytosis in patients with PV (p<0.05). The co-expression of HMGA2 mRNA and WT1 mRNA in patients with MF was associated with a low level of white and red blood cell (p<0.05). Interestingly that in MF patients the most pronounced thrombocytopenia was observed if only WT1 mRNA was detected. In all cases of repeated testing of patients, the growth of HMGA2 or WT1 mRNA level was accompanied by decline of the cellular blood composition. The level of HMGA2 mRNA and WT1 mRNA in our study did not depend on the driver mutations presence or their allele burden. It should be noted that 4 out of 12 patients with MF died within 5 months after the first detection of WT1 mRNA and one patient died more after 15 months. At that time only one MF patient without WT1 or HGMA2 transcripts died during of 20 months' observation.
Summary and Conclusions
Expression of the WT1 and HMGA2 genes is involved in the pathogenesis and progression of the disease and can be used as diagnostic markers. The question of the independent prognostic significance of these transcripts in blood samples for various phenotypes of MPN is promising for further study.

Disclosures
No relevant conflicts of interest to declare.