Association Between Idiopathic Deep Venous Thrombosis And Genetic Polymorphisms In The Promoter Region Ofβ-fibrinogen Gene

Objective:To discover the possible single nucleotide polymorphism (SNPs)(known or unknown) in the promoter region of β-fibrinogen gene(FGB) and establish haplotypes and Logistic regression model, with intent to reveal the association between genetic polymorphisms in the promoter region of β-fibrinogen gene and features of idiopathic deep venous thrombosis(IDVT) such as susceptibility, haemodynamics and thrombophilia(including blood plasma fibrinogen concentration, erythrocyte sedimentation rate, prothrombin time, thrombin time, activated partial thromboplastin time) under genetic background.Subjects and Methods:The thesis contains two parts:Part I Correlated research of clinical features for idiopathic deep venous thrombosis In order to exclude confounding factors and emphasize on the influence of genetic factors to deep venous thrombosis, IDVT patients were selected carefully as case group after confirmed with ultrasounography from either out-patient department or in-patient department. Each patient suffered their first episode of IDVT and had not accepted any therapy such as thrombectomy, anti-coagulation and thrombolysis before recruited. Both case group and control group comprised of120cases, respectively. All subjects came from cities or counties of Yunnan province, belonging to Han population. Neither age nor gender differences were observed between this two groups statistically. Thyroid, hepatic, renal disfunctioning and other metabolic diseases were excluded from the subjects. Duplex scanning were used to assemble haemodynamic parameters (anatomical categories, thrombus-involving limbs, femoral/popliteal vein velocity and femoral/popliteal vein diameter). Hemacyte analysis, biochemical indicators and coagulation state of each subject were evaluated routinely and cautiously before the residual blood was collected and reserved into Eppendorf tubes in refrigerator (-80℃).Part II Association between idiopathic deep venous thrombosis and genetic polymorphisms in the promoter region of β-fibrinogen gene Long-fragment gene sequencing technique was adopted to scan the whole promoter region of β-fibrinogen gene (1560bp) intent to detecting the possible SNPs (known or unknown) and analyzing their interaction with IDVT predisposition. Meanwhile, with purpose to achieve a more concrete apprehension of the dedication made by the traditional technique-restriction fragment length polymorphism (RFLP) in SNPs research, and to search for a suitable and swift RFLP method for SNPs detection in the future DVT related gene chip manufacture, we randomly selected40samples of DNA products (20samples for each group) for further RFLP testing after the previous gene sequencing having been accomplished successfully. Completely the outcome of RFLP testing was in accordance with that of gene sequencing. Principal methods are described as bellow:1Hardy-Weinberg equilibrium test for SNPs determined in the promoter region of β-fibrinogen gene2Linkage Disequilibrium analyses (LD) for above-mentioned SNPs3Analyze the statistical difference of genotype frequencies and allele frequencies between IDVT group and control group by Chi square test4Multiple Logistic regression analysis of IDVT5Establish haplotypes model for SNPs determined in the promoter region of β-fibrinogen gene6Analyze the influence to IDVT haemodynamics caused by above-mentioned SNPs7Analyze the influence to IDVT thrombophilia (mainly plasma fibrinogen level, e.t.c) caused by above-mentioned SNPsResults:Part I1Neither age nor gender differences were observed between this two groups statistically(both for population and stratified)(P>0.05).2Haemodynamic analyses:A. According to the course of disease, all IDVT patients were subdivided as acute, sub-acute and chronic phase. Sub-acute and chronic phase embraced the highest percentage (55%), and accordingly lost the optimal opportunity to get cured.B. Anatomical categories division of IDVT cases showed that peripheral type composed of the most proportion(51.7%) followed by mixed type(35%) and then central type(13.3%); the limb susceptible to IDVT was Left lower extremity(63.3%), right lower extremity(27.5%) and both lower extremities(9.2%).C. Femoral and popliteal vein velocity in IDVT group were slower than that of control group(P<0.05) while femoral and popliteal diameter in IDVT group were longer than that of control group(P<0.05).3Thrombophilia analyses:Plasma fibrinogen level, erythrocyte sedimentation rate, prothrombin time and activated partial thromboplastin time in case group exceeded that of control group, respectively(P<0.05) while thrombin time was shorter than that of control group(P<0.05).4The majority of hemacyte analysis and biochemical indicators showed no difference between two groups(P>0.05). Statistical significance was observed in white blood cell count, blood platelet count, albumin level, glutamic-oxaloacetic transaminase (AST) and creatinine level between IDVT group and control group. However, the indicators were still in physiological range, IDVT associated fluctuation was considered as the cause for these slight differences.Part II1After successful gene sequencing,6kinds of SNPs were determined in the promoter region of β-fibrinogen gene, i.e. β-148C/T (rs1800787), β-249C/T (rs1800788), P-455G/A (rs1800790), β-854G/A (rs1800791), p-993C/T (rs2227389) and β-1420G/A (rs1800789). Homozygous for theβ-854G/A polymorphism (P-854AA) was not detected in each sample of240cases from both groups.2Hardy-Weinberg equilibrium was achieved for all SNPs except for P-249C/T (χ2=8.661103) in the control group. 3A strong linkage disequilibrium relationship was confirmed between β-993C/T and β-455G/A (r2=0.699),β-993C/T and β-148C/T (r2=0.509), β-455G/A and β-148C/T (r2=0.556).4Statistical differences of genotype frequencies and allele frequencies between IDVT group and control group were observed in β-1420G/A, β-455G/A,β-249C/T and β-148C/T(P<0.05) polymorphisms. There were no statistical significances in β-993C/T and β-854G/A polymorphisms as far as genotype frequencies and allele frequencies were concerned.5Multiple Logistic regression analysis of IDVT suggested that:A. The risk of IDVT increased with fibrinogen concentration rising. The risk of IDVT was4.579times than before when fibrinogen concentration elevated by1g/L.B. Allele β-1420A and allele β-148T were risk factors of IDVT(P<0.05), the risk of IDVT onset of the carriers of these alleles was3.445and5.375times that the non-carriers took, respectively.C. Allele β-455A was the protective factor of IDVT onset, the incidence was0.088times compared with allele β-455G, meaning that a91.2%cut-down of risk was achieved through the allele alteration.6Twenty-six categories of haplotypes were found among the subjects (21for IDVT group and18for control group). Considering the practical significance of haplotypes-establishing, those with a frequency of less than3%in both case group and control group would be discharged. Ultimately, eight sorts of haplotypes remained. Frequencies of haplotypes-H1, H3, H4, H5, H6and H7between groups were statistically different(P<0.05):Haplotypes-H3and H6presented a higher incidence in IDVT group (OR=32.085,1.896, respectively), belonging to the susceptible type; Haplotypes-H1, H4, H5and H7presented a higher incidence in control group (OR=0.025,0.119,0.644and0.383, respectively), belonging to the protective type. The rest haplotypes(H2and H8) showed no discrepancy(P>0.05), belonging to nonsense type.7None statistical correlation was found between the6sorts of SNPs and anatomical categories, thrombus-involving limbs and haemodynamics (femoral/popliteal vein velocity and diameter).8The influence to coagulation function caused by above-mentioned SNPs:A. In control group, discrepancy was only observed in plasma fibrinogen concentration and erythrocyte sedimentation rate between allele β-993C and allele β-993T (P<0.05). B. In IDVT group, however, no variance appeared in all coagulation indicators between corresponding alleles of all SNPs.Conclusion:1The preponderance of our subjects lay in two facts:A. Strict selection standard for ethnicity, region and DVT associated risk factors (the secondary DVT patients were excluded from the IDVT group meant that a relatively "simple" case group formed in favour of our concentrating on genetic back-ground research).B. A perfect equilibrium was realized in case-control study:not only age and gender but genetic characteristic were concerned and confirmed to be in good balance from the following Hardy-Weinberg equilibrium test. Therefore, the subject was extraordinarily suitable for genetic research.2During the nature course of IDVT, along with DVT relieving and recovering, the coagulation state-hypercoagulable or hypocoagulable condition would change alternatively, fluctuating on a dynamic balance. Sometimes it is very difficult to define clinically.3Our SNPs research was combined with clinical features (susceptibility, haemodynamics and thrombophilia). From this perspective, the direction and depth of our research will exceed most of the few similar DVT-SNPs research.4Gene sequencing technique will perfectly meet the overwhelming majority of SNPs research requirement. For those with large scale, long-fragment and, especially, a great many targets in the same DNA plasm, gene sequencing technique is more accurate, direct and swift, facilitating to detect each base in target gene and discover the possible SNPs (known or unknown) and gene mutation points. By the way, we found one case of probable gene mutation (-887AAâ†'CC) in control group.5The risk of IDVT was4.579times than before when fibrinogen concentration elevated by1g/L. Therefore, fibrinogen is probably the independent risk factor of IDVT onset.6Based on logistic regression analysis, the following conclusions might be drawn:A. SNPs-β-1420G/A, β-455G/A and β-148C/T could directly influence the predisposition of IDVT onset—Allele β-1420A,β-455G and β-148T are risk factors while Allele β-1420G,(3-455A and β-148C are protective factors.B. SNP-β-993C/T may participate in IDVT onset via elevating basic blood plasma fibrinogen concentration or through linkage disequilibrium with SNPs-β-455G/A and β-148C/T.C. SNP-β-854G/A could not influence the susceptibility of IDVT in Han Population of Yunnan province.D. Because SNP-β-249C/T in control group failed to reach Hardy-Weinberg equilibrium, the effect of SNP-β-249C/T to IDVT susceptibility deserves further study.7. Haplotypes H3and H6are susceptible for IDVT, Haplotypes H1, H4, H5and H7are protective while H2and H8belong to nonsense types.8. None statistical correlation was found between the6kinds of SNPs and haemodynamics, however, we can not deny the contribution the SNPs in the promoter region of β-fibrinogen gene made to IDVT haemodynamics.9. No variance appeared in all coagulation indicators between corresponding alleles of all SNPs in IDVT group, but still, we can not draw the conclusion that there was no association between SNPs in the promoter region of β-fibrinogen gene and IDVT thrombophilia.