纤维蛋白原Bβ启动子区多态性与特发型下肢深静脉血栓的相关性研究
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摘要
目的探寻纤维蛋白原Bβ (FGB)启动子区可能存在的单核苷酸多态性(SNPs)位点(已知或未知的)并由此构筑Logistic回归模型和单倍型模型,从而在遗传背景上揭示FGB启动子区SNPs对特发型下肢深静脉血栓形成(IDVT)易感性、血流动力学和凝血功能(包括血浆纤维蛋白原水平等)的影响。
方法本课题分为两个部分：
第一部分,特发型下肢深静脉血栓形成临床相关因素研究。为了尽量排除混杂因素的干扰,并着重考察遗传因素对DVT发病的影响,病例组入选标准为我院门诊和住院部经彩色多普勒确诊的特发型下肢深静脉血栓患者(Idiopathic deep venous thrombosis, IDVT)。所有患者均为首次发病且未经过任何治疗(包括手术、抗凝和溶栓等)。病例组和对照组各随机选择120名,来自于云南省各县市,均为汉族。两组年龄及性别无明显统计学差异；无甲状腺和肝、肾功能衰退及代谢性疾患等。使用彩色多普勒收集上述人群的血流动力学参数(包括IDVT解剖分型、肢体受累范围和股、膪静脉流速峰值、内径等)；采集上述人群的血细胞分析、血生化指标和凝血功能指标(主要有红细胞沉降率、凝血酶原时间、纤维蛋白原、凝血酶时间和活化部分凝血活酶时间等)。
同时,收集受试者的残余血标本,置于1.5ml EP管,置于-80℃冰箱中冻存备用。
第二部分,纤维蛋白原B β启动子区基因多态性与特发型下肢深静脉血栓形成的相关性研究。采用长段基因测序的方法探寻FGB基因整个启动子区(1560bp)所有可能存在的SNPs并分析它们与IDVT易感性、血流动力学和凝血功能(包括红细胞沉降率、凝血酶原时间、纤维蛋白原、凝血酶时间和活化部分凝血活酶时间等)的关系。同时,为了更好的理解聚合酶链反应·限制性长度多态性(PCR·RFLP)这一传统方法对SNPs研究的贡献；也为了制造DVT相关基因芯片找到便捷、可行的RFLP瑚南在对所有样本成功测序后随机选取40例样本的PCR产物进行PCR·RFLP检测(每组各随机选取20例)。检测结果与测序结果完全相符。
主要方法有：
1、各SNPs位点Hardy-Weinberg遗传平衡度检验；
2、各SNPs位点连锁不平衡分析(Linkage Disequilibrium tests, LD);
3、分析IDVT组和对照组各SNPs的基因型频率和等位基因频率差异；
4、IDVT多因素Logistic回归分析；
5、构筑FGB启动子区SNPs单倍型模型；
6、分析FGB启动子区SNPs对IDVT血流动力学的影响；
7、分析FGB启动子区SNPs对凝血功能的影响(包括血浆纤维蛋白原水平等)。
结果实验结果显示：
第一部分
1、IDVT组和对照组性别、年龄构成比(包括总体比较和各年龄阶段分层比较)均无明显统计学意义(均P>0.05)。
2、分析血流动力学参数后发现：
(1)本组IDVT患者在收治住院时以亚急性期和慢性期最为多见,所占比例达55%,也因此丧失了最佳治疗时机；
(2)解剖分型则以周围型最多见(51.7%),其次是混合型(35%),中央型比例最小(13.3%)；肢体累积范围以左下肢为主(63.3%),其次是右下肢(27.5%)和双下肢(9.2%)；
(3) IDVT组股、胭静脉流速较慢,低于对照组(均氏0.05)；IDVT组股、胭静脉内径增宽,高于对照组(均P<0.05)。
3、分析凝血功能指标后发现：IDVT组红细胞沉降率(ESR)、纤维蛋白原(Fg)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)高于对照组：而凝血酶时间(TT)低于对照组;
4、IDVT组-对照组大部分血常规、血生化指标无统计学差异(均P>0.05)；白细胞计数、血小板计数、白蛋白含量、谷草转氨酶、肌酐含量在2组间虽有统计学差异(均P<0.05),但都在正常生理范围,且这种差异多与DVT疾病本身有关。
第二部分
1、经长段基因测序后证实：(1)在FGB启动子区存在6种SNPs,即β-148C/T(rs1800787), β-249C/T (rs1800788), β-455G/A(rs1800790), β-854G/A(rs1800791), β-993C/T (rs2227389), β-1420G/A(rs1800789):(2)在240例样本中均没有发现β-854AA纯合子基因型；(3)在对照组中-887位点即发现一例疑似CC突变的基因型(-887AA→CC,正常为从野生纯合子基因型)。
2、除了对照组中1个SNP(β-249C/T,χ2=8.661103)外,对照组其余5个SNPs和实验组所有SNPs均达到Hardy-Weinberg遗传平衡。
3、β-993C/T与β-455G/A(r2=0.699)、β-993C/T与β-148C/T(r2=0.509). β-455G/A与β-148C/T(r2=0.556)之间观察到较强的两两连锁不平衡关系。
4、β-1420G/A、β-455G/A, β-249C/T, β-148C/T的基因型频率和等位基因频率在下肢IDVT组和对照组之间存在显著统计学差异(均P<0.05)；β-993C/T和8-854G/A的基因型频率和等位基因频率在IDVT组和对照组之间无统计学差异(P>0.05)。
5、通过多因素Logistic回归分析,我们发现：
(1)纤维蛋白原(Fg)含量越高的人,患下肢IDVT的危险性就越高。纤维蛋白原每增加1个单位,IDVT的发病风险相应增加4.579倍;
(2)β-1420A、β-148T等位基因是IDVT的危险因素且具有统计学意义(P<0.05),上述等位基因携带者IDVT的发病风险分别为非携带者的3.445、5.375倍;
(3)β-455A等位基因是IDVT的保护因素,其发病率是非携带者的的0.088倍,即β-455A等位基因的存在可使IDVT的发病风险降低91.2%。
6、在两组人群中共发现26种单倍型(病例组中21种,对照组中18种)。考虑到单倍型构筑的实际意义,我们在EH程序中将病例-对照组中频率都小于3%的单倍型予以排外,得到8种单倍型。H1.H3.H4.H5.H6和H7共6种单倍型分布频率在病例组和对照组之间存在显著统计学差异(P<0.05)： H3、H6单倍型在IDVT组出现频率较高(0R分别为32.085和1.896),属于IDVT易感单倍型；H1、H4、H5和H7单倍型在对照组出现频率较高(0R分别为0.025、0.119、0.644和0.383),属于保护单倍型；其余单倍型(H2和H8)在两组之间无显著统计学差异(P>0.05),属于无义单倍型。
7、上述6个SNPs与IDVT的解剖类型、肢体累积范围以及血流动力学(包括股、胭静脉内径和流速)之间均未发现统计学关联。
8、FGB启动子区SNPs与凝血功能的关系：
(1)在正常对照组,只有ESR、Fg在β-993C/T两种基因型之间存在统计学差异(均P<0.05);
(2)在IDVT组,所有凝血指标在所有SNPs相应基因型之间均无统计学差异(均P>0.05)。
结论
1、本组受试人群种族、地域以及发病因素选择针对性强,无论在年龄、性别甚至遗传学(Hardy-Weinberg遗传平衡)方面均具有良好的均衡性,适合作基因和遗传学研究。
2、在IDVT的整个自然病程中,随着血栓反复发作,血液高凝和低凝状态可能会交替出现,呈现动态平衡,有时甚至难以界定。
3、SNPs研究方面,将受试人群临床特征(除了IDVT易感性外,还包括解剖类型、凝血功能和血流动力学等参数)与后续基因类型研究结果相结合加以联合考察。从这个意义上讲,该项目的针对性和研究深度都将超越以往为数不多的同类型研究。
4、基因测序技术完全可以满足SNPs研究的绝大部分需求。对于大规模、长序列,特别是多个目标均在同一DNA区域的SNPs研究,基因测序技术比限制性长度多态性方法(PCR-RFLP)更为准确、直观、快捷,利于“由点到线”进行研究以及发现未知SNPs和基因突变位点(本实验在-887位点即发现一例疑似CC突变的基因型)。
5、纤维蛋白原(Fg)每增加1个单位,IDVT的发病风险相应增加4.579倍。因此,Fg很可能是IDVT的独立危险因素；
6、无论是单因素或多因素Logistic回归分析,都提示：
(1)β-1420G/A、β-455G/A,β-148C/T可能直接影响IDVT的易感性—β-1420A、β-455G、β-148T等位基因是IDVT的危险因素,而β-1420G、β-455A、β-148C等位基因是IDVT的保护因素；
(2)β-993C/T可能通过提高血浆Fg基础水平及与β-455G/A、β-148C/T的连锁不平衡方式对IDVT易感性产生间接影响；
(3) β-854G/A多态性可能与云南地区汉族人群IDVT易感性无关；
(4)因对照组β-249C/T未达到Hardy-Weinberg遗传平衡,其对IDVT易感性的影响值得商榷。
7、H3、H6属于IDVT易感单倍型；H1、H4、H5和H7属于保护单倍型； H2和H8属于无义单倍型；
8、在上述6个SNPs与IDVT解剖类型、肢体累积范围以及血流动力学(包括股、胭静脉内径和流速)之间均没有发现统计学关联,但不能据此否定FGB启动子区SNPs对IDVT血流动力学产生的影响；
9、在IDVT状态下,所有凝血指标在6个SNPs相应基因型之间均无统计学差异,但不能据此否定FGB启动子区SNPs对IDVT凝血功能、特别是Fg的影响。
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 gene
2Linkage Disequilibrium analyses (LD) for above-mentioned SNPs
3Analyze the statistical difference of genotype frequencies and allele frequencies between IDVT group and control group by Chi square test
4Multiple Logistic regression analysis of IDVT
5Establish haplotypes model for SNPs determined in the promoter region of β-fibrinogen gene
6Analyze the influence to IDVT haemodynamics caused by above-mentioned SNPs
7Analyze the influence to IDVT thrombophilia (mainly plasma fibrinogen level, e.t.c) caused by above-mentioned SNPs
Results:
Part I
1Neither 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 II
1After 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 (-887AA→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.
方法本课题分为两个部分：
第一部分,特发型下肢深静脉血栓形成临床相关因素研究。为了尽量排除混杂因素的干扰,并着重考察遗传因素对DVT发病的影响,病例组入选标准为我院门诊和住院部经彩色多普勒确诊的特发型下肢深静脉血栓患者(Idiopathic deep venous thrombosis, IDVT)。所有患者均为首次发病且未经过任何治疗(包括手术、抗凝和溶栓等)。病例组和对照组各随机选择120名,来自于云南省各县市,均为汉族。两组年龄及性别无明显统计学差异；无甲状腺和肝、肾功能衰退及代谢性疾患等。使用彩色多普勒收集上述人群的血流动力学参数(包括IDVT解剖分型、肢体受累范围和股、膪静脉流速峰值、内径等)；采集上述人群的血细胞分析、血生化指标和凝血功能指标(主要有红细胞沉降率、凝血酶原时间、纤维蛋白原、凝血酶时间和活化部分凝血活酶时间等)。
同时,收集受试者的残余血标本,置于1.5ml EP管,置于-80℃冰箱中冻存备用。
第二部分,纤维蛋白原B β启动子区基因多态性与特发型下肢深静脉血栓形成的相关性研究。采用长段基因测序的方法探寻FGB基因整个启动子区(1560bp)所有可能存在的SNPs并分析它们与IDVT易感性、血流动力学和凝血功能(包括红细胞沉降率、凝血酶原时间、纤维蛋白原、凝血酶时间和活化部分凝血活酶时间等)的关系。同时,为了更好的理解聚合酶链反应·限制性长度多态性(PCR·RFLP)这一传统方法对SNPs研究的贡献；也为了制造DVT相关基因芯片找到便捷、可行的RFLP瑚南在对所有样本成功测序后随机选取40例样本的PCR产物进行PCR·RFLP检测(每组各随机选取20例)。检测结果与测序结果完全相符。
主要方法有：
1、各SNPs位点Hardy-Weinberg遗传平衡度检验；
2、各SNPs位点连锁不平衡分析(Linkage Disequilibrium tests, LD);
3、分析IDVT组和对照组各SNPs的基因型频率和等位基因频率差异；
4、IDVT多因素Logistic回归分析；
5、构筑FGB启动子区SNPs单倍型模型；
6、分析FGB启动子区SNPs对IDVT血流动力学的影响；
7、分析FGB启动子区SNPs对凝血功能的影响(包括血浆纤维蛋白原水平等)。
结果实验结果显示：
第一部分
1、IDVT组和对照组性别、年龄构成比(包括总体比较和各年龄阶段分层比较)均无明显统计学意义(均P>0.05)。
2、分析血流动力学参数后发现：
(1)本组IDVT患者在收治住院时以亚急性期和慢性期最为多见,所占比例达55%,也因此丧失了最佳治疗时机；
(2)解剖分型则以周围型最多见(51.7%),其次是混合型(35%),中央型比例最小(13.3%)；肢体累积范围以左下肢为主(63.3%),其次是右下肢(27.5%)和双下肢(9.2%)；
(3) IDVT组股、胭静脉流速较慢,低于对照组(均氏0.05)；IDVT组股、胭静脉内径增宽,高于对照组(均P<0.05)。
3、分析凝血功能指标后发现：IDVT组红细胞沉降率(ESR)、纤维蛋白原(Fg)、凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)高于对照组：而凝血酶时间(TT)低于对照组;
4、IDVT组-对照组大部分血常规、血生化指标无统计学差异(均P>0.05)；白细胞计数、血小板计数、白蛋白含量、谷草转氨酶、肌酐含量在2组间虽有统计学差异(均P<0.05),但都在正常生理范围,且这种差异多与DVT疾病本身有关。
第二部分
1、经长段基因测序后证实：(1)在FGB启动子区存在6种SNPs,即β-148C/T(rs1800787), β-249C/T (rs1800788), β-455G/A(rs1800790), β-854G/A(rs1800791), β-993C/T (rs2227389), β-1420G/A(rs1800789):(2)在240例样本中均没有发现β-854AA纯合子基因型；(3)在对照组中-887位点即发现一例疑似CC突变的基因型(-887AA→CC,正常为从野生纯合子基因型)。
2、除了对照组中1个SNP(β-249C/T,χ2=8.661103)外,对照组其余5个SNPs和实验组所有SNPs均达到Hardy-Weinberg遗传平衡。
3、β-993C/T与β-455G/A(r2=0.699)、β-993C/T与β-148C/T(r2=0.509). β-455G/A与β-148C/T(r2=0.556)之间观察到较强的两两连锁不平衡关系。
4、β-1420G/A、β-455G/A, β-249C/T, β-148C/T的基因型频率和等位基因频率在下肢IDVT组和对照组之间存在显著统计学差异(均P<0.05)；β-993C/T和8-854G/A的基因型频率和等位基因频率在IDVT组和对照组之间无统计学差异(P>0.05)。
5、通过多因素Logistic回归分析,我们发现：
(1)纤维蛋白原(Fg)含量越高的人,患下肢IDVT的危险性就越高。纤维蛋白原每增加1个单位,IDVT的发病风险相应增加4.579倍;
(2)β-1420A、β-148T等位基因是IDVT的危险因素且具有统计学意义(P<0.05),上述等位基因携带者IDVT的发病风险分别为非携带者的3.445、5.375倍;
(3)β-455A等位基因是IDVT的保护因素,其发病率是非携带者的的0.088倍,即β-455A等位基因的存在可使IDVT的发病风险降低91.2%。
6、在两组人群中共发现26种单倍型(病例组中21种,对照组中18种)。考虑到单倍型构筑的实际意义,我们在EH程序中将病例-对照组中频率都小于3%的单倍型予以排外,得到8种单倍型。H1.H3.H4.H5.H6和H7共6种单倍型分布频率在病例组和对照组之间存在显著统计学差异(P<0.05)： H3、H6单倍型在IDVT组出现频率较高(0R分别为32.085和1.896),属于IDVT易感单倍型；H1、H4、H5和H7单倍型在对照组出现频率较高(0R分别为0.025、0.119、0.644和0.383),属于保护单倍型；其余单倍型(H2和H8)在两组之间无显著统计学差异(P>0.05),属于无义单倍型。
7、上述6个SNPs与IDVT的解剖类型、肢体累积范围以及血流动力学(包括股、胭静脉内径和流速)之间均未发现统计学关联。
8、FGB启动子区SNPs与凝血功能的关系：
(1)在正常对照组,只有ESR、Fg在β-993C/T两种基因型之间存在统计学差异(均P<0.05);
(2)在IDVT组,所有凝血指标在所有SNPs相应基因型之间均无统计学差异(均P>0.05)。
结论
1、本组受试人群种族、地域以及发病因素选择针对性强,无论在年龄、性别甚至遗传学(Hardy-Weinberg遗传平衡)方面均具有良好的均衡性,适合作基因和遗传学研究。
2、在IDVT的整个自然病程中,随着血栓反复发作,血液高凝和低凝状态可能会交替出现,呈现动态平衡,有时甚至难以界定。
3、SNPs研究方面,将受试人群临床特征(除了IDVT易感性外,还包括解剖类型、凝血功能和血流动力学等参数)与后续基因类型研究结果相结合加以联合考察。从这个意义上讲,该项目的针对性和研究深度都将超越以往为数不多的同类型研究。
4、基因测序技术完全可以满足SNPs研究的绝大部分需求。对于大规模、长序列,特别是多个目标均在同一DNA区域的SNPs研究,基因测序技术比限制性长度多态性方法(PCR-RFLP)更为准确、直观、快捷,利于“由点到线”进行研究以及发现未知SNPs和基因突变位点(本实验在-887位点即发现一例疑似CC突变的基因型)。
5、纤维蛋白原(Fg)每增加1个单位,IDVT的发病风险相应增加4.579倍。因此,Fg很可能是IDVT的独立危险因素；
6、无论是单因素或多因素Logistic回归分析,都提示：
(1)β-1420G/A、β-455G/A,β-148C/T可能直接影响IDVT的易感性—β-1420A、β-455G、β-148T等位基因是IDVT的危险因素,而β-1420G、β-455A、β-148C等位基因是IDVT的保护因素；
(2)β-993C/T可能通过提高血浆Fg基础水平及与β-455G/A、β-148C/T的连锁不平衡方式对IDVT易感性产生间接影响；
(3) β-854G/A多态性可能与云南地区汉族人群IDVT易感性无关；
(4)因对照组β-249C/T未达到Hardy-Weinberg遗传平衡,其对IDVT易感性的影响值得商榷。
7、H3、H6属于IDVT易感单倍型；H1、H4、H5和H7属于保护单倍型； H2和H8属于无义单倍型；
8、在上述6个SNPs与IDVT解剖类型、肢体累积范围以及血流动力学(包括股、胭静脉内径和流速)之间均没有发现统计学关联,但不能据此否定FGB启动子区SNPs对IDVT血流动力学产生的影响；
9、在IDVT状态下,所有凝血指标在6个SNPs相应基因型之间均无统计学差异,但不能据此否定FGB启动子区SNPs对IDVT凝血功能、特别是Fg的影响。
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 gene
2Linkage Disequilibrium analyses (LD) for above-mentioned SNPs
3Analyze the statistical difference of genotype frequencies and allele frequencies between IDVT group and control group by Chi square test
4Multiple Logistic regression analysis of IDVT
5Establish haplotypes model for SNPs determined in the promoter region of β-fibrinogen gene
6Analyze the influence to IDVT haemodynamics caused by above-mentioned SNPs
7Analyze the influence to IDVT thrombophilia (mainly plasma fibrinogen level, e.t.c) caused by above-mentioned SNPs
Results:
Part I
1Neither 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 II
1After 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 (-887AA→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.
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