Polymorphism 10034C>T Is Located in a Functional Cleavage Stimulation Factor (CstF) Binding Site of the Fibrinogen Gamma Gene and Influences the γ′/γA mRNA Ratio.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 543-543 ◽  
Author(s):  
Shirley Uitte de Willige ◽  
Inge M. Rietveld ◽  
Marieke C.H. de Visser ◽  
Hans L. Vos ◽  
Rogier M. Bertina
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Binding Site ◽  
Point Mutations ◽  
Fold Increase ◽  
Nucleotide Polymorphisms ◽  
Functional Variation ◽  
Increased Risk ◽  
Cleavage Stimulation Factor

Abstract Previously we found that haplotype 2 of the fibrinogen gamma gene (FGG-H2) is associated with an increased risk of deep venous thrombosis and with reduced fibrinogen γ′ levels, an alternatively spliced form of FGG, and reduced fibrinogen γ′/total fibrinogen ratios (Blood2005; 106(13): 4176–83). Two of the FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T [rs2066864] and 10034C>T [rs2066865], are located in the region containing the two polyadenylation (pA) signals used in FGG pre-mRNA processing, namely pA1 and pA2, resulting in transcripts coding for γ′-chains and γA-chains, respectively. SNP 10034C>T is located in a Cleavage stimulation Factor (CstF) binding site at nucleotides 10030–10047 of FGG. CstF is a multi-subunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. We hypothesized that 10034T, by strengthening this CstF binding site, favors use of pA2 (synthesis of γA-chains) at the expense of pA1-use (synthesis of γ′-chains). We transiently transfected FGG mini-gene constructs containing exon 9, intron 9 (containing pA1), exon 10 and the 3′ UTR (containing pA2) in liver-derived HepG2 cells and used quantitative real-time PCR to measure the mean relative pA-signal usage (pA1/pA2 ratio) of the different constructs. To study the effects of SNPs 9615C>T and 10034C>T on the relative use of pA1 and pA2, we made constructs carrying the different alleles of these SNPs. Compared to reference construct CC (9615C, 10034C; FGG-H1) of which the pA1/pA2 ratio was set at 100%, the ratio of construct TT (9615T, 10034T; FGG-H2) was 1.4-fold decreased (71.5%, p=0.015). To investigate whether the SNPs individually or collectively caused the reduction in the pA1/pA2 ratio, exchange constructs CT (9615C, 10034T) and TC (9615T, 10034C) were also tested. The ratio of construct CT was almost 1.2-fold decreased (85.3%, p=0.001), while the ratio of construct TC did not differ significantly from the reference construct (101.6%, p=0.890). These results indicate that in our in vitro system SNP 10034C>T, and not SNP 9615C>T, is the major contributor to the reduction of the pA1/pA2 ratio. We further investigated the functionality and importance of the CstF binding site, containing 10034C>T, with a construct in which nucleotides 10013–10056 containing the complete CstF binding site were deleted. Compared to the reference construct, in which the CstF site was still present, the pA1/pA2 ratio of the CstF deletion construct was 2.2-fold increased (222.3%, p<0.001), indicating that this site is involved in the regulation of pA2-usage. The functionality of the CstF site was further confirmed using several mutant constructs in which the CstF consensus was weakened or strengthened by introducing one or two point mutations. Weakening the CstF site resulted in a significant 1.5-fold increase of the pA1/pA2 ratio, while strengthening the site resulted in a significant 1.6-fold decrease of the ratio. From these results we conclude that 10034C>T is located in a functional CstF binding site and is involved in regulating the usage of the pA2-signal of FGG. We propose that the 10034C>T change is the functional variation in the FGG-H2 haplotype and is responsible for the reduction in the fibrinogen γ′/total fibrinogen ratio and the increased risk of deep venous thrombosis.

Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen γ′ levels

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4176-4183 ◽  
Author(s):  
Shirley Uitte de Willige ◽  
Marieke C. H. de Visser ◽  
Jeanine J. Houwing-Duistermaat ◽  
Frits R. Rosendaal ◽  
Hans L. Vos ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Case Control Study ◽  
Population Based ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Thrombosis Risk ◽  
Increased Risk ◽  
Risk For Thrombosis ◽  
Control Study

We investigated the association between haplotypes of fibrinogen alpha (FGA), beta (FGB), and gamma (FGG), total fibrinogen levels, fibrinogen γ′ (γA/γ′ plus γ′/γ′) levels, and risk for deep venous thrombosis. In a population-based case-control study, the Leiden Thrombophilia Study, we typed 15 haplotype-tagging single nucleotide polymorphisms (htSNPs) in this gene cluster. None of these haplotypes was associated with total fibrinogen levels. In each gene, one haplotype increased the thrombosis risk approximately 2-fold. After adjustment for linkage disequilibrium between the genes, only FGG-H2 homozygosity remained associated with risk (odds ratio [OR], 2.4; 95% confidence interval [95% CI], 1.5-3.9). FGG-H2 was also associated with reduced fibrinogen γ′ levels and reduced ratios of fibrinogen γ′ to total fibrinogen. Multivariate analysis showed that reduced fibrinogen γ′ levels and elevated total fibrinogen levels were both associated with an increased risk for thrombosis, even after adjustment for FGG-H2. A reduced fibrinogen γ′ to total fibrinogen ratio (less than 0.69) also increased the risk (OR, 2.4; 95% CI, 1.7-3.5). We propose that FGG-H2 influences thrombosis risk through htSNP 10034C/T [rs2066865] by strengthening the consensus of a CstF site and thus favoring the formation of γA chain above that of γ′ chain. Fibrinogen γ′ contains a unique high-affinity, nonsubstrate binding site for thrombin, which seems critical for the expression of the antithrombin activity that develops during fibrin formation (antithrombin 1).

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

scholarly journals Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

2014 ◽  
Vol 70 (a1) ◽  
pp. C1793-C1793
Author(s):  
Paul Rowland ◽  
Onkar SINGH ◽  
Leila Ross ◽  
Francisco Gamo ◽  
Maria Lafuente-Monasterio ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Crystal Structures ◽  
Point Mutations ◽  
Crystal Form ◽  
Drug Binding ◽  
Binding Modes ◽  
Resistance Mutations ◽  
Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

scholarly journals Mutations in Plasmodium falciparumCytochrome b That Are Associated with Atovaquone Resistance Are Located at a Putative Drug-Binding Site

2000 ◽  
Vol 44 (8) ◽  
pp. 2100-2108 ◽  
Author(s):  
Michael Korsinczky ◽  
Nanhua Chen ◽  
Barbara Kotecka ◽  
Allan Saul ◽  
Karl Rieckmann ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Point Mutations ◽  
Drug Binding ◽  
Single Amino Acid ◽  
Malaria Parasites ◽  
Drug Binding Site ◽  
Sole Agent ◽  
Amino Acid Mutations

ABSTRACT Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYTbc 1) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYTb.

Analysis of the Genetic Expression of Inflammatory Mediators From Patients with Spontaneous Deep Venous Thrombosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5251-5251
Author(s):  
Aline Barnabé ◽  
Fernanda Dutra Santiago-Bassora ◽  
Andrey dos Santos ◽  
Marcelo Falsarella Carazzolle ◽  
Gonçalo AG Pereira ◽  
...  
Keyword(s):  
Gene Expression ◽  
Major Histocompatibility Complex ◽  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Major Histocompatibility Complex Class ◽  
Inflammatory Mediators ◽  
Fold Increase ◽  
Class Ii ◽  
Complex Class ◽  
Histocompatibility Complex

Abstract Abstract 5251 Deep venous thrombosis (DVT) is a multifactorial disease and in about 30% of patients no risk factor can be identified. Association of inflammation and hemostasis is thought to play a significant role in the pathogenesis of DVT. We hypothesized that distinctive patterns of expression of inflammatory mediators in mononuclear cells from patients with previous DVT could be relevant to the pathogenesis of DVT. cDNA microarray technology (CodeLink Bioarrays) was chosen to study the gene expression profile of inflammatory mediators in DVT patients with clinical characteristics of a high penetrance of any putative hereditary risk, and their healthy siblings. These microarrays were hybridized with synthesized probes from pooled samples collected from patients and control siblings. Patients were divided into two clinical groups: (1) patients with one spontaneous DVT (n=2), and (2) patients with recurrent spontaneous DVT episodes (n=1). Preliminary analysis showed that 2% of approximately 55,000 transcripts contained in the array had significant (higher than 2-fold) differences in expression in spontaneous and recurrent spontaneous DVT relative to their asymptomatic siblings. In group 1, 738 genes were upregulated and 504 genes were downregulated. In group 2, 1229 genes were upregulated and 87 genes were downregulated. Genes with different expression compared to controls were associated to immune and inflammatory response, motility, cell adhesion, cell-cell signaling, defense response, signal transduction and apoptosis. Genes that presented the highest differences compared to controls were interleukin 8 (43-fold increase), chemokine (C-X-C motif) ligand 2 (11-fold increase), major histocompatibility complex, class II, DR beta 5 (8-fold increase), major histocompatibility complex, class II, DQ alpha 1 (29-fold decrease), caspase recruitment domain family, member 15 (7-fold increase), carboxypeptidase A5 (11-fold decrease), cathepsin G (8-fold decrease), and azurocidin 1 (3-fold decrease). A comprehensive list of these genes was generated. Evaluation of the significance of these distinctive gene expression profiles between patients with spontaneous and recurrent DVT compared to their asymptomatic siblings could reveal novel insights into the pathophysiology of the first DVT episode and of DVT recurrence, as well as new biomarkers or therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

Molecular Analysis Of Patients With Deep Venous Thrombosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4799-4799
Author(s):  
Soudabeh Hosseeini ◽  
Ebrahim Kalantari ◽  
Akbar Dorgalaleh ◽  
Arash Rozei ◽  
Marzieh Jafari ◽  
...  
Keyword(s):  
Risk Factors ◽  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Mthfr C677t ◽  
Control Group ◽  
Mthfr A1298c ◽  
Increased Risk ◽  
Fv Leiden ◽  
The Impact ◽  
Pai 1

Background Deep venous thrombosis (DVT) refers to the formation of a thrombus within a deep vein that frequently occurs after surgical procedures, trauma, in the presence of cancer and immobilization conditions. DVT is a major health problem that causes high rate of morbidity and mortality in the general population. Hyper coagulation states such as antithrombin-III, protein-C and protein-S deficiencies, contribute to formation of DVT. Congenital and acquired gene mutations are other risk factors that stimulate formation of thrombus. Our aims in this study was to molecularly analyze the patients with DVT and assess the impact of common mutations of MTHFR (C677T) (A1298C), PAI-1, Prothrombin 20210 and FV Leiden mutation on occurrence of deep venous thrombosis. Methods This long-term study was conducted from June 2009 to July 2013 on 221 patients with deep venues thrombosis. Two hundred and twenty-one age and sex matched individuals were also chosen as control group. The diagnosis of venous thromboembolic disease was based on patient’s history, clinical findings and D-dimer test. Finally deep venous thrombosis was confirmed with Doppler ultrasonography. In addition, all participants were asked to complete a standardized questionnaire on acquired risk factors for venous thrombosis. After confirmation of DVT, both groups were assessed molecularly for five mutations including, MTHFR C677T, MTHFR A1298C, PAI-1 4G/5G, Prothrombin 20210 and FV Leiden. The relationship between these mutations and the risk of DVT was calculated using logistic regression and expressed as an OR with a 95% confidence interval (CI). Results The mean age of patients and control group were 38±0.8 and 3.7± 0.7 years. Our results revealed that the MTHFR C677T (OR 2.9, 95% 95% CI 1.1 to 7.5) and MTHFR A1298C in heterozygote manner (OR 4.3, 95% CI 1.4 to 13.7) were each associated with an increased risk of DVT. The OR associated with being a carrier of the PAI-1 4G/5G genotype was 2.9 (95% CI 1.14 to 7.5). There was a 4-fold increased risk of DVT associated with Prothrombin 20210 mutation in heterozygote manner (OR 4.3, 95% CI 1.4 to 13.7). For patients who were heterozygous for FV Leiden mutation OR DVT was 2.6 (95% CI 1.3 to 5.0). Conclusion Our findings suggest that genetic risk factors have a contributory role on occurrence of DVT. Disclosures: No relevant conflicts of interest to declare.

The association between the 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor-1 gene and deep venous thrombosis in young people

2005 ◽  
Vol 20 (1) ◽  
pp. 48-52 ◽  
Author(s):  
A Mansilha ◽  
F Araújo ◽  
M Severo ◽  
S M Sampaio ◽  
T Toledo ◽  
...  
Keyword(s):  
Young People ◽  
Venous Thrombosis ◽  
Deep Venous Thrombosis ◽  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Control Group ◽  
Plasminogen Activator Inhibitor 1 ◽  
Increased Risk ◽  
Activator Inhibitor ◽  
Pai 1

Objective: To evaluate the association between the 4G/5G polymorphism in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene and deep venous thrombosis (DVT) in young people. Methods: Prevalence of the 4G/5G polymorphism was investigated using DNA analysis in a population of 81 consecutive and unrelated patients with an objectively documented first episode of DVT under 40 years old and in a control group of 88 healthy subjects. Results: The frequency of genotypes among patients was 0.27 4G/4G, 0.49 4G/5G and 0.23 5G/5G, corresponding to a frequency of 0.52 for the 4G allele. In the control group the results were, respectively, 0.24, 0.44 and 0.32, corresponding to a frequency of 0.46 for the 4G allele. The odds ratio (OR) for homozygous 4G genotype was 1.5 (95% confidence interval: 0.7–3.6), which was not statistically significant ( P = 0.51). Conclusion: In this study, the 4G/5G polymorphism in the promoter of the PAI-1 gene, including the homozygous 4G genotype, was not associated with a significantly increased risk of DVT in young people.

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