Sophie Gandrille

Mutations in Promoter Region of Thrombomodulin and Venous Thromboembolic Disease

The present study was designed to analyze the thrombomodulin proximal promoter region spanning nucleotides -293 to -12 to search for polymorphisms that could modify thrombomodulin gene expression in patients with venous thromboembolic disease. The study population comprised 205 patients and 394 healthy subjects of similar age and sex distribution. No polymorphisms and only 1 point mutation (G-33A) were found. The G-33A mutation was present at the heterozygous state in 2 patients and in 1 control. Being more frequent in the patients (0.97%) than in the controls (0.25%), the G-33A mutation might be a risk factor for venous thrombosis. To investigate the effect of this mutation on the thrombomodulin promoter activity, the proximal promoter region of the gene (bearing or not bearing the G-33A mutation) was inserted into a promotorless expression vector, upstream of the firefly luciferase gene, and transiently transfected into EA.hy926 endothelial cells. Under the conditions of the assay, the G-33A mutation mildly decreased the promoter activity. This study confirms that abnormalities of the thrombomodulin proximal promoter are not frequent in patients with venous thromboembolism.

Complex association of protein C gene promoter polymorphism with circulating protein C levels and thrombotic risk.

The allele and haplotype frequency of the -1654 C/T and -1641 A/G protein C (PC) gene promoter polymorphisms was determined and analyzed according to circulating PC concentrations in 394 healthy subjects aged 20 to 60 years. The CG haplotype was associated with a lower PC concentration in both homozygous and heterozygous subjects compared with noncarriers. The TA allele had the reverse effect, but only in homozygotes. The distribution of the CG and TA alleles was significantly different in 242 patients, aged 17 to 60 years, with venous thromboembolism. The CG allele increased the risk of thrombosis, with an OR of 1.39 (95% confidence interval (CI), 1.04 to 1.87). The TA allele was protective in subjects aged <45 years, with an OR of 0.68 (95% CI, 0.44 to 1.04). TA was also significantly associated with older age at the first thrombosis. This study confirms the link between the PC gene promoter and circulating PC levels, and suggests a complex effect on the risk of thrombosis.

Molecular basis for protein S hereditary deficiency: Genetic defects observed in 118 patients with type I and type IIa deficiencies

Circulating protein S (PS) is partly bound to C4b-binding protein, and only free PS can act as a cofactor for protein C (PC), a natural anticoagulant. Two types of PS deficiencies are commonly observed in patients with unexplained thrombosis, and they are characterized by having both a low total PS level and a low free PS level (type I) or by having only a low free PS level (type IIa). To elucidate the genetic mechanisms responsible for these two plasma phenotypes, we screened 118 symptomatic patients with type I or type IIa PS deficiency for a PS gene coding sequence variation. A total of 34 mutations, 17 of which were novel, were identified in 65 propositi (70% in type I and 44% in type IIa). In type I deficiency, 29 different mutations were distributed throughout the coding sequence. In type IIa deficiency, five different missense mutations were clustered in exons XII and XIII, with a Ser 460 to Pro mutation accounting for most cases (82%). This points to a role of the domain encoded by exons XII and XIII in the distribution between bound and free PS. The Ser 460 to Pro mutation was associated with the factor V Arg 506 to Gin mutation or a PC gene mutation in about half the patients, suggesting a cooperative effect on clinical expression.

Gross deletions/duplications in PROS1 are relatively common in point mutation-negative hereditary protein S deficiency

Hereditary protein S (PS) deficiency is an autosomal disorder caused by mutations in the PS gene (PROS1). Conventional PCR-based mutation detection identifies PROS1 point mutations in approximately 50% of the cases. To verify if gross copy number variations (CNVs) are often present in point mutation-negative hereditary PS deficiency we used multiplex ligation-dependent probe amplification (MLPA) as a detection tool in samples from individuals with a high probability of having true PS deficiency. To this end, DNA samples from nine PS deficient probands with family members (seven type I and two type III) and nine isolated probands (three type I and six type III), in whom PROS1 mutations were not found by DNA sequencing, were evaluated. An independent quantitative PCR (qPCR) was performed to confirm the findings of the MLPA assay. Family members were also tested when DNA was available. Gross abnormalities of PROS1 were found in six out of eighteen probands. In three probands complete deletion of the gene was detected. Two probands had a partial deletion involving different parts of the gene (one from exon 4 through 9 and another from exon 9 through 11). One family showed a duplication of part of PROS1. qPCR analysis was in accordance with these results. In conclusion, this study substantiates that gross gene abnormalities in PROS1 are relatively common in hereditary PS deficient patients and that MLPA is a useful tool for direct screening of CNVs in PROS1 point mutation-negative individuals.

Identification of functional polymorphisms of the thromboxane A2 receptor gene in healthy volunteers

Thromboxane A2 receptor (TP) is an important actor in vascular physiology and plays a crucial role in the platelet activation process. Genetic polymorphisms of the gene coding for the TP have been described, but their impacts on platelet function tests are unknown. The aim of this study was to investigate the relationship between genetic polymorphisms of the coding sequence of the TP gene and platelet function tests. We investigated 100 healthy volunteers twice, one week apart by performing platelet aggregation and secretion tests. We sequenced the coding region of the TP gene and confronted the genetic variants with the phenotypic results. We identified five single nucleotide polymorphisms (SNP); one of them, T1712C, replaces Leu by Pro at position 133 of the isoform beta of the TP. Homozygosity for the minor allele of the C795T, C924T or the G1686A SNP was associated with a decreased expression of CD62P when platelets were stimulated with the TP agonist U46619. As C795T and C924T have been linked to clinical disorders in which TxA2 plays a key role, the possible role of the G1686A and T1712C SNP should also be examined in selected diseases.

Lack of sequence variations in the C4b-BP β-chain in patients with type III protein S deficiency bearing the Ser 460 to Pro mutation : Description of two new intragenic isomorphisms in the C4b-BP β-chain gene (C4BPB)

Type III protein S (PS) deficiency, characterized by low levels of free PS and normal total PS levels, is often associated with the Ser 460 to Pro substitution. However, some patients bearing this mutation have normal PS levels, suggesting that another gene defect may account for this phenotype. We postulated that this defect was located in the C4b‐BP β‐chain gene (C4BPB) and searched for a mutation in the coding regions of this gene in 35 propositi with type III PS deficiency and bearing the Ser 460 to Pro mutation. No mutations explaining the phenotype of type III PS deficiency were identified. We did, however, find two frequent nucleotide changes, one being located in the donor splice site of intron d and the second in the codon corresponding to Asn 137. We used these two polymorphisms to establish C4BPB gene haplotype in five informative type III PS‐deficient families and exclude a role of the C4BPB gene in this phenotype of three of them. Finally, increased C4b‐BP β‐chain levels were not responsible for the phenotype of type III PS deficiency as the C4BPB haplotype did not correlate with C4b‐BP β‐chain levels.

Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant.

Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations.

Implication of protein S thrombin-sensitive region with membrane binding via conformational changes in the gamma-carboxyglutamic acid-rich domain.

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the gamma-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the gamma-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.

Influence of three potential genetic risk factors for thrombosis in 43 families carrying the factor V Arg 506 to Gln mutation

The factor V (FV) Arg 506 to Gln mutation is the most common abnormality observed in familial thrombophilia. Many studies have shown that its clinical expression differs among families and among carriers. Some thrombotic patients carry an additional genetic risk factor such as protein C, protein S or antithrombin deficiency. We sought to identify other genetic risk factors potentially favouring expression of the thrombotic phenotype in 370 members of 43 families with the FV Arg 506 to Gln mutation. We analysed three candidate polymorphisms in genes involved in the PC anticoagulant pathway, consisting of two polymorphic sites in the 5′ non‐transcribed region of the PC gene, −1654 C/T and −1641 A/G, with three known combinations (TA, CA and CG) that influence the protein C plasma level; one polymorphic site (4070 A/G) in exon 13 of the FV gene, which influences the plasma factor V concentration, and one polymorphic site (677 C/T) in the methylenetetrahydrofolate reductase gene, which is often associated with moderate hyperhomocysteinaemia. The distribution of these different polymorphisms was similar in patients with a history of thrombosis and those who remained asymptomatic, ruling out the possibility that each of these polymorphisms alone can play a role in the onset of thrombosis in carriers of the FV Arg 506 to Gln mutation.

Cleaved Protein S (PS), Total PS, Free PS, and Activated Protein C Cofactor Activity as Risk Factors for Venous Thromboembolism

BACKGROUND Although hereditary protein S (PS) deficiency is clearly associated with venous thromboembolism (VTE), the importance of low PS concentrations as a risk factor for VTE in other patients is still a matter of debate. To clarify this issue, we designed a case-control study to evaluate the role of different molecular forms of plasma PS. METHODS We quantified plasma cleaved, total, and free PS and activated protein C (APC) cofactor activity in 87 VTE patients and 174 controls matched for age, sex, and hormonal treatment. Free PS was measured by ELISA or by enzyme-linked ligand sorbent assay (ELSA). Cleaved and total PS were measured by ELISA. RESULTS In controls, the mean (SD) concentration of circulating cleaved PS was 39 (14) nmol/L, corresponding to 10% (3.5%) of total PS. Concentrations of cleaved PS and total PS were not significantly different in patients with VTE compared with controls. However, in our population, low free PS measured by ELISA or ELSA, as well as APC cofactor activity values were significantly associated with VTE with odds ratios (95% confidence intervals) of 2.9 (1.3-6.3), 2.5 (1.1-5.6), and 2.9 (1.3-6.4), respectively, in multivariate analyses. CONCLUSION Phenotypic low PS detected by APC cofactor activity assay or by an assay specific for free PS should be considered a risk factor for VTE.