Page 69 - GTM-3-2

Global Translational Medicine Genes and blood cells in Ph-negative MPNs

(PCR) method by setting up a multiplex reaction. Sets 4.01]), PV (1.45 [0.85 – 2.12]), and PMF (1.79 [1.10 – 2.52])
of reagents were used to detect polymorphisms in the compared to the control group (0.96 [0.79 – 1.16]) (P <
human genome through the single nucleotide method. 0.0001, P = 0.012, and P < 0.0001, respectively). Similarly,
Allele1 (wild type) was labeled with the fluorophore the PLT/WBC ratio was significantly elevated in patients
HEX, while Allele2 (mutated) was determined using with ET (83.8 [71.7 – 124.1]) and PV (43.7 [34.0 – 69.3])
the FAM channel. Amplification was performed on the compared to the control group (36.6 [28.3 – 45.2]), with
CFX96 Real-Time System (Bio-Rad, USA). The results P < 0.0001 and P = 0.031, respectively (Figure 1).
TM
were analyzed using CFX Maestro Software (Bio-
TM
Rad, USA). The amplification reaction was performed 3.2. JAK2V617F mutation and its association with
according to the following protocol: one cycle of the blood cell count and ICIs
temperature 80°C for 2 min; one cycle of temperature The JAK2V617F mutation was detected in 44 (78.6%)
94°C for 3 min; 40 cycles of temperature 94°C for 15 s; patients: 13 (59.1%) with ET, 18 (94.7%) with PV (P = 0.011
and 40 cycles of temperature 64°C for 40 s (reading compared to the ET group), and 13 (86.7%) with PMF.
phase). No significant differences in ICIs were observed between
subgroups of patients with and without the JAK2V617F
2.3. Distribution of genotypes of the polymorphic mutation. However, patients with ET who had this
locus 4G/5G of the PAI-1 gene
mutation showed significantly higher blood cell counts
The distribution of genotypes and alleles of the compared to those without the mutation: PLT counts
polymorphic locus −675 4G/5G ins/del of the PAI-1 gene were 918 (837 – 1025) ×10 /L vs. 732 (583 –837) ×10 /L
9
9
was determined through molecular genetic analysis of the (P = 0.021), RBC counts were 4.96 (4.44 –5.35) ×10 /L
12
DNA sequence using the PCR method in high-resolution vs. 4.19 (4.05 –4.54) ×10 /L (P = 0.015), and WBC counts
12
melting mode. This was accomplished using the GenETics were 11.5 (7.1 – 15.1) ×10 /L vs. 6.3 (5.9 – 7.5) ×10 /L
9
9
Haemostatic Disease (PAI-1/ITGB3) Kit from Adaltis (P = 0.012). This finding suggests that in patients with ET
(Italy) on the CFX96TMReal-Time System (Bio-Rad, and the JAK2V617F mutation, the increase in blood cell
USA). The results were analyzed using the CFXMaestro counts is likely due to excessive production resulting from
TM
Software and RealBest Diagnostics programs. the activation of the JAK-STAT pathway in hematopoietic
stem cells and progenitor cells. 23
2.4. Blood cell count and ICIs
Hematopoiesis features were assessed through automated 3.3. The PAI-1 4G/5G genotypes and their
counting of RBCs, PLTs, and WBCs. ICIs were calculated, associations with the JAK2V617F mutation
including the PLT/WBC ratio and C×PLT/RBC. The In patients with Ph-negative MPNs, the PAI-1 4G/4G,
conventional coefficient, C=20, was used to estimate 4G/5G, and 5G/5G genotypes were detected in
the synchronization of reproduction of the PLT and 11 (19.6%), 29 (51.8%), and 16 (28.6%) cases, respectively.
RBC populations, considering that the PLT population The distribution rate was 4G/5G > 5G/5G > 4G/4G.
typically recovers 20 times faster than the RBC population. According to molecular genetic analysis conducted by
Therefore, approximately the same amount of PLTs and Makukh et al., the distribution of these genotypes in
24
RBCs is produced per conventional unit of time. DNA samples of 225 healthy residents of the Western
2.5. Statistical analysis Ukrainian region was 33.33% for 4G/4G, 45.33% for
4G/5G, and 21.33% for 5G/5G, with the distribution
Statistical processing of the obtained data was performed rate being 4G/5G > 4G/4G > 5G/5G. The frequency of
using the Statistica for Windows software package (Statsoft, the 4G allele was 45.5% in the examined MPN patients
USA). Quantitative parameters were compared using the and 56.0% in the general population. No significant
Mann–Whitney U-test and were presented as median differences in the distribution of PAI-1 gene genotypes
(interquartile range). The Fisher’s test and the χ test were and alleles were found between the general population
2
used to compare qualitative parameters. sample and our study.
3. Results In patients with ET, the detection rates of the genotypes
4G/4G, 4G/5G, and 5G/5G were in the following
3.1. Blood cell count and ICIs order: 5G/5G > 4G/5G > 4G/4G (40.9%, 36.4%, and
Before the initiation of treatment, thrombocytopoiesis was 22.7%, respectively). In patients with PV, the order was
predominant over erythropoiesis and leukopoiesis in all 4G/5G > 5G/5G > 4G/4G (63.15%, 21.05%, and 15.8%,
patients with Ph-negative MPNs. The C×PLT/RBC index respectively). For patients with PMF, the order was 4G/5G
was significantly higher in patients with ET (3.70 [2.84 – > 5G/5G = 4G/4G (60.0%, 20.0%, and 20.0%, respectively).

Volume 3 Issue 2 (2024) 5 doi: 10.36922/gtm.2559