Assessing gene expression of coagulation factors and Factor XIII polymorphisms in type 2 diabetes mellitus patients

Abstract

Background: Type 2 diabetes mellitus (T2DM) is characterized by chronic hyperglycaemia, inflammation and coagulopathies due to cytokine activation of IL-1_ and IL-6. The aim of the study was to assess the mRNA levels of coagulation factors and the identification of two FXIII SNPs (Val34Leu and Tyr204Phe) in T2DM patients (n=100) compared to controls (n=100). Methodology: Thromboelastography (TEG) was used to assess the viscoelastic properties of pre-selected clot parameters. Quantitative PCR (qPCR) was used to assess mRNA levels of key coagulation factors (factor XIII-A, factor XII and tissue factor). PCR-Restriction fragment length polymorphism (PCR-RFLP) was used to identify the presence or absence of Val34Leu and Tyr204Phe using specific restriction enzymes. Results: From the pre-selected viscoelastic properties, TEG showed that the R time and TMRTG were prolonged by 17.67 mins (26.15±2.56 min vs. 8.48±0,92 min) and 12.76 mins (24.85±2.41 min vs. 12.09±1.15 min) (p<0.0001) respectively. There was a reduction in MA by 2.55 dyn.cm-2s-1 (3.94±0.61 dyn.cm_2 s_1 vs. 5.73±0.54 dyn.cm_2 s_1, p=0.0074) and TTG by 121 dyn.cm-2 (213.8±26.29 dyn cm_2 vs. 335.5 ± 32.33 dyn cm_2, p= 0.0385) in T2DM patients compared to controls. A 4.38-fold decrease in mRNA levels of FXIII-A in T2DM (delta ct: 10.94±0.44) was observed, compared to controls (delta ct: 13.07±0.48), p=0.0033. The genotype distribution in both T2DM and control individuals conformed to the Hardy-Weinberg Equilibrium (HWE) (p>0.05). An association of the Val34Leu SNP and T2DM was found, (OR) = 1.670, 95% CI=0.97-2.89, p=0.0747) . An even stronger association was found with the Tyr204Phe SNP and T2DM (OR=3.57, 95% CI=2.37-5.45, p<0.0001 ). The levels of FXIII-A mRNA was significantly altered in T2DM (delta ct: 14.46±0.82 vs. 12.03±0.49, p=0.0105) in the presence of the wild type (Tyr/Tyr) compared to the variant (Tyr/Phe and Phe/Phe), respectively. The polymorphism had an effect on the viscoelastic properties as showed by measurements obtained in the TEG variables in T2DM patients, the 34Leu variant increased the MRTG by 1.35 Dyn.cm-2.s-1 (4.92±0.84 Dyn.cm-2s-1 vs. 3.57±0.78 Dyn.cm-2s-1, p=0.0457) and TTG by 120 Dyn.cm-2 (301.3±38.22 Dyn.cm-2 vs. 181.3±31.68 Dyn.cm-2, p=0.0367). The 204Phe variant reduced the R time by 3.79 mins (6.68± 0.73min vs. 10.47± 1.63min, p=0.033), the TMRTG by 13.1 min (14.49±2.59 min vs. Tyr/Tyr 27.52±3.01 min, p=0.0017) and increased the MRTG by 3.95 Dyn.cm-2.s-1 (6.74±1.17 Dyn.cm-2s-1 vs. Tyr/Tyr 2.79±0.47 Dyn.cm-2s-1, p=0.0020). In controls the 34Leu variant had no effect on the viscoelastic properties and the 204Phe variant reduced the R time by 3.88 min (6.59±0.73 min vs. 10.47±1.63 min, p=0.0334). Conclusion: This study has showed that T2DM patients have a hypocoagulable (PPP) clot profile while the formed clot has an increased clot strength. An association of the Val34Leu SNP as well as the Tyr204Phe SNP with T2DM was observed, with an indication that the polymorphic variant, 204Phe, offering a protective effect being more prevalent in controls.