PROS1 Research Articles

Influence of single nucleotide polymorphisms on thrombin generation in factor V Leiden heterozygotes

Carriership of the factor V (FV) Leiden mutation increases the risk of venous thromboembolism (VTE) ~4-fold, but the individual risk of each FV Leiden carrier depends on several co-inherited risk and protective factors. Under the hypothesis that thrombin generation might serve as an intermediate phenotype to identify genetic modulators of VTE risk, we enrolled 188 FV Leiden heterozygotes (11 with VTE) and determined the following parameters: thrombin generation in the absence and presence of activated protein C (APC); plasma levels of prothrombin, factor X, antithrombin, protein S and tissue factor pathway inhibitor; and the genotypes of 24 SNPs located in the genes encoding these coagulation factors and inhibitors. Multiple regression analysis was subsequently applied to identify the (genetic) determinants of thrombin generation. The endogenous thrombin potential (ETP) showed a striking inter-individual variability among different FV Leiden carriers and, especially when measured in the presence of APC, correlated with VTE risk. Several SNPs in the F2 (rs1799963, rs3136516), F10 (rs693335), SERPINC1 (rs2227589), PROS1 (Heerlen polymorphism) and TFPI (rs5940) genes significantly affected the ETP-APC and/or the ETP+APC in FV Leiden carriers. Most of these SNPs have shown an association with VTE risk in conventional epidemiological studies, suggesting that the genetic dissection of thrombin generation leads to the detection of clinically relevant SNPs. In conclusion, we have identified several SNPs that modulate thrombin generation in FV Leiden heterozygotes. These SNPs may help explain the large variability in VTE risk observed among different FV Leiden carriers.

Molecular basis of protein S deficiency in China

Protein S (ProS) is a physiological inhibitor of coagulation with an important function in the down-regulation of thrombin generation. ProS deficiency is a major risk factor for venous thrombosis. This study enrolled 40 ProS-deficient probands to investigate the molecular basis of hereditary ProS deficiency in Chinese patients. A mutation analysis was performed by resequencing the PROS1 gene. Large deletions were identified by multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 20 different mutations, including 15 novel mutations, were identified in 21 of the 40 index probands. Small mutations were detected in 18 (45.0%) probands, and large deletions were found in 3 (7.5%) probands, leaving 19 (47.5%) patients without causative variants. To evaluate the functional consequences of 2 novel missense variants, ex vivo thrombin-generation assays, bioinformatics tools, and in vitro expression studies were employed. The p.Asn365Lys ProS variant was found to have moderately impaired secretion and reduced activated protein C cofactor activity. In contrast, the p.Pro410His mutant appeared to have severely impaired secretion but full anticoagulant activity. This study is the largest investigation of ProS deficiency in China and the first investigation of the influence of Type I ProS missense mutations on the global level of coagulation function. The p.K196E mutation, which is common in the neighboring Japanese population, was not found in our Chinese population, and null mutations were common in our Chinese population but not common in Japan. Further genetic analysis is warranted to understand the causes of ProS deficiency in patients without a genetic explanation.

Ru substitution effect on the peak effect in superconducting PrOs4Sb12

Ac susceptibility (χac) measurements on Pr(Os1−xRux)4Sb12 single crystals for x = 0.05 and 0.1 were performed by mutual inductance method. A peak structure caused by anomalously enhanced flux pinning force, which is so-called the peak effect, appears in the H dependence of χac. The peak structure shifts to lower fields as T increases and disappears in T > 1 K in both samples, while in PrOs4Sb12 it is observable up to near Tc. This fact indicates that the Ru substitution suppresses the peak effect. We demonstrate that the observed T dependent behavior of the peak structure can be explained roughly by the synchronization model although there remains deviation from the model curve, suggesting some modification may be needed in the model to be applied to Pr(Os1−xRux)4Sb12.

Quality in Molecular Biology Testing for Inherited Thrombophilia Disorders

As the understanding of the genetic basis of the inherited thrombophilias has increased over recent years, their routine diagnostic genetic analysis has also matured. This review considers methods used to test for the factor V (F5) Leiden mutation and prothrombin 20210A (F2 c.*97G>A) allele, and analysis of the SERPINC1, PROC, and PROS1 genes in cases of antithrombin, protein C (PC), and protein S (PS) deficiency, respectively. Issues relating to quality are explored, highlighting where analytical and sample handling errors may occur. Detection of the factor V Leiden mutation and the prothrombin c.*97G>A allele are best performed using real-time polymerase chain reaction analysis as this relatively simple technique allows their discrimination from rare variants of neighboring nucleotides; not possible using the more time-consuming restriction digestion assays. With the advent of low-cost and high-throughput sequence analysis, direct sequencing has become the first-line method to provide a definitive diagnosis of inherited, rather than acquired, deficiencies. Large cohort studies have shown that antithrombin and PC mutations are identified in between 61 and 87% of patients, whereas the detection rate in PS deficiency is substantially lower in around 40% of patients. Large gene deletions make up between 7 and 10% of PS and antithrombin mutations and only 1% of PC mutations, but it is suggested that dosage analysis techniques such as multiplex ligation-dependent probe amplification should be used for all three genes as part of routine analysis to ensure mutations are not missed. Best practice guidelines are available from EuroGentest covering a wide variety of the issues raised in this review and all laboratories should participate in appropriate external quality assurance schemes to ensure they continue to offer high quality service.

Inherited protein S deficiency due to a novel nonsense mutation in the PROS1 gene in the patient with recurrent vascular access thrombosis: A case report

Vascular access thrombosis is one of the major causes of morbidity in patients maintained on chronic hemodialysis. Thrombophilia has been recognized as a risk factor of vascular access thrombosis. The authors report a case of inherited protein S deficiency associated with vascular access thrombotic events. DNA sequence analysis of the PROS1 gene identified a novel heterozygous nonsense mutation in exon 10 by transition of AAG (lysine) to TAG (stop codon) at codon 473 (c.1417A>T, p.K473X). Results from the study suggest that the inherited protein S deficiency due to a PROS1 gene mutation may cause vascular access thrombosis in hemodialysis patients.

Genotype and Laboratory and Clinical Phenotypes of Protein S Deficiency

The diagnosis of thrombophilia caused by protein S deficiency remains difficult. From 2005 to 2010, we documented 135 patients with suspected hereditary protein S deficiency for whom mutational analysis of the PROS1 gene had been performed by direct double-stranded sequencing of the amplified 15 exons including splice sites. Multiplex ligation-dependent probe amplification was performed on 12 of 15 exons in cases with no mutation found but a large deletion in the PROS1 gene was suspected. Mutations were identified in 49 patients, 9 by familial screening. Altogether, 17 new and 11 previously described mutations of PROS1 were identified among the 49 patients. After the exclusion of acquired protein S deficiency due to pregnancy or hormonal contraceptives, there remained only 1 case with protein S activity levels less than 40% that could not be explained by sequence variations or deletions in the examined regions of the PROS1 gene. After the exclusion of conditions associated with acquired protein S deficiency, persistently low protein S activity levels are highly indicative of a genetic alteration in PROS1. We observed a clear correlation between the laboratory phenotype and the type of mutation.

Small and large PROS1 deletions but no other types of rearrangements detected in patients with protein S deficiency

Protein S deficiency is a dominantly inherited disorder that results from mutations in the PROS1 gene. Previous sequencing of the gene failed to detect mutations in eight out of 18 investigated Swedish families, whereas segregation analyses detected large deletions in three out of the eight families. The present study investigates more thoroughly for the presence of deletions but also for other types of rearrangements. FISH analysis confirmed the existence of the three previously identified large deletions, but failed to identify any other type of rearrangement among the eight analysed families. MLPA analysis of the PROS1 gene revealed two smaller deletions covering two and four exons, respectively. Thus, deletions could be found in five out of eight families where no point mutations could be found despite sequencing of the gene. Twelve additional, not previously analysed, families were subsequently analysed using MLPA. The analysis identified two smaller deletions (3 and 4 exons). Including all PS-deficient families, i.e. also the 10 families where sequencing found a causative point mutation, deletions were identified in seven out of 30 PS-deficient families. A strategy of sequencing followed by MLPA analysis in mutation-negative families identified the causative mutation in 15 out of 18 of Swedish PS-deficient families. Most deletions were different as determined by their sizes, locations and flanking haplotypes. FISH (8 families) and MLPA analysis (20 families) failed to identify other types of rearrangements.

Deficiencies of antithrombin, protein C and protein S – Practical experience in genetic analysis of a large patient cohort

Deficiencies of natural anticoagulant proteins including antithrombin (AT), protein C (PC) and protein S (PS) are important causes of inherited thrombophilia. This study aimed to report on the practical experience gained in performing genetic analyses of a large cohort of patients with AT, PC and PS deficiencies and to relate this knowledge to clinical application. We genotyped a large cohort of 709 unrelated patients with AT (231), PC (234) and PS (244) deficiencies referred to us by physicians throughout Germany. Mutations were detected by direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. Contrastingly, for PC and PS deficiencies, the MDR dropped significantly and mildly lowered to subnormal values. At PS activities >55% for PS no mutations were detected. Mutation profiles of all three genes were similar with the highest prevalence for missense mutations (63-78%), followed by nonsense (7-11%), splice-site mutations (7-13%), small deletions (1-8%), small insertions/duplications (1-4%) and large deletions (3-6%). In conclusion, genetic testing is a useful diagnostic tool for diagnosing thrombophilia. Based on our data, genetic analysis for patients with AT deficiency is indicated for all subnormal activities. In contrast, genotyping is not advisable for PC activities >70% and for PS activities >55%.

Insight in Protein S Deficiency From Mouse Models

Abstract 529Protein S (ProS) is an important negative regulator of blood coagulation. Its physiological importance is evident in purpura fulminans and other life-threatening thrombotic disorders typical of ProS deficient patients. Our previous characterization of ProS deficiency in mouse models has shown similarities with the human phenotypes: heterozygous ProS-deficient mice (Pros+/−) had increased thrombotic risk whereas homozygous deficiency in ProS (Pros−/−) was incompatible with life (Blood 2009; 114:2307-2314). In tissues, ProS exerts cellular functions by binding to and activating tyrosine kinase receptors of the Tyro3 family (TAM) on the cell surface.To extend the analysis of coagulation defects beyond the Pros−/− phenotype and add new insights into the sites of synthesis ProS and its action, we generated mice with inactivated ProS in hepatocytes (Proslox/loxAlbCre+) as well as in endothelial and hematopoietic cells (Proslox/loxTie2Cre+). Both models resulted in significant reduction of circulating ProS levels and in a remarkable increased thrombotic risk in vivo. In a model of tissue factor (TF)-induced venous thromboembolism (VTE), only 17% of Proslox/loxAlbCre+ mice (n=12) and only 13% of Proslox/loxTie2Cre+ mice (n=14) survived, compared with 86% of Proslox/lox mice (n=14; P<0.001).To mimic a severe acquired ProS deficiency, ProS gene was inactivated at the adult stage using the polyI:C-inducible Mx1-Cre system (Proslox/loxMx1Cre+). Ten days after polyI:C treatment, Proslox/loxMx1Cre+ mice developed disseminated intravascular coagulation with extensive lung and liver thrombosis.It is worth noting that no skin lesions compatible with purpura fulminans were observed in any of the above-described models of partial ProS deficiency. In order to shed light on the pathogenesis of purpura fulminans, we exposed the different ProS-deficient mice to warfarin (0.2 mg/day). We observed that Pros+/−, Proslox/loxAlbCre+ and Proslox/loxTie2Cre+ mice developed retiform purpura (characterized by erythematous and necrotic lesions of the genital region and extremities) and died after 3 to 5 days after the first warfarin administration.In human, ProS is also synthesized by megakaryocytes and hence stored at high concentrations in circulating platelets (pProS). The role of pProS has been investigated by generating megakaryocyte ProS-deficient model using the PF4 promoter as Cre driver (Proslox/loxPf4Cre+). In the TF-induced VTE model, Proslox/loxPf4Cre+ (n=15) mice showed a significant increased risk of thrombosis compared to Proslox/lox controls (n=14; survival rate 47% and 86%, respectively; P<0.05). Furthermore, preliminary results suggest survival to be associated with higher circulating ProS levels. In order to evaluate the potential role of pProS in thrombus formation, we investigated the thrombotic response to intravenous injection of collagen-epinephrine in vivo and platelet function in vitro. Both in vivo and in vitro experiments showed similar results between Proslox/loxPf4Cre+ and Proslox/lox, indicating that platelet reactivity was not influenced by the absence of pProS. These data suggest that pProS is delivered at the site of thrombosis to inhibit thrombin generation.We further investigated the ability of ProS to function as a ligand of TAM receptors, by using homozygous and heterozygous deficient mice for both the TAM ligands ProS and Gas6. Gas6−/−Pros−/− mice died in utero and showed comparable dramatic bleeding and thrombotic phenotype as described for Pros−/− embryos.In conclusion, like complete ProS deficiency, double deficiency in ProS and Gas6 was lethal, whereas partial ProS deficiency was not. Mice partially deficient in ProS displayed a prothrombotic phenotype, including those with only deficiency in pProS. Purpura fulminans did not occur spontaneously in mice with partial Pros deficiency but developed upon warfarin administration.Thus, the use of different mice models of ProS deficiency can be instrumental in the study of its highly variable thrombotic phenotype and in the investigation of additional roles of ProS in inflammation and autoimmunity through TAM signaling. Disclosures:No relevant conflicts of interest to declare.

Free and Total Protein S Antigen Levels on the Risk of Venous Thrombosis: Results From the MEGA Study

Abstract 538 Context:Conflicting data have been reported on the risk for venous thrombosis in individuals with low total protein S levels (i.e. type I protein S deficiency) and low free protein S levels with normal total protein S levels (i.e. type III protein S deficiency). This may be due to small numbers, wrong cut off level or inclusion of individuals with mild transitory decrements in protein S levels. Most studies that showed that type I and type III protein S deficiency were related with an increased risk for venous thrombosis, have been performed in thrombophilic families, suggesting that these deficiencies are inherited. As the prevalence of inherited type I or type III protein S deficiency is not known, the relevance of these findings within normal populations remain to be established. Objectives:To assess the risk of first venous thrombosis in persons with low levels of free protein S or total protein S in a large population-based case–control study. Design:MEGA study, 4956 consecutive patients aged 18 to 70 years with a first episode of venous thrombosis were included. Age- and sex-matched controls were partners of patients (n=3297) or individuals recruited by random digit dialing (n=3000). DNA was obtained by standard methods and was available for 4485 patients and 4889 control subjects. Citrated plasma was available for 2471 patients and 2940 controls. Molecular basis for protein S deficiency was investigated by analysis of copy number variation of PROS1 and sequencing of individuals with the lowest levels of protein S in attempt to explain the different findings in risk estimates between families and population studies. Statistical analysis:Odds ratios were adjusted for age and sex (matching factors) for levels of free/total protein S and their 95% confidence levels (95% CIs) with the use of logistic regression. The 2.5th-97.5th percentile of both total and free protein S in control subjects that did not use vitamin K antagonists (VKA) at time of blood draw were considered as the reference range. Individuals that used VKA at time of blood draw were excluded when calculating relative risk estimates. Furthermore, a preplanned sensitivity analysis was performed where we excluded estrogen users and pregnant women at time of blood draw. Results:Individuals with low free protein S levels or low total protein S levels (<2.5th percentile) were not at increased risk of venous thrombosis as compared to individuals with protein S levels in the 2.5th-97.5th percentile; odds ratio 0.82 (95% CI, 0.56–1.21) and 0.90 (95% CI, 0.62–1.31) respectively. Excluding all women who used estrogens or were pregnant/puerperic at time of venous thrombosis or at time of blood sampling increased the odds ratios slightly to 1.55 (95% CI, 0.84–2.88) for individuals with low free protein S levels and to 1.28 (95% CI, 0.70–2.35) in individuals with low total protein S levels. We subsequently compared decreasing cut off values of free and total protein S levels on the risk of venous thrombosis as compared to the same reference group. Although numbers became small, it appeared that a free protein S cut off level of < 0.20th or < 0.10th percentile could identify individuals at high risk of venous thrombosis (odds ratios 2.01; 95% CI, 0.57–7.15, and 5.44; 95% CI, 0.61–48.78, respectively). Even extremely low (<0.10th percentile) total protein S levels were not associated with venous thrombosis. Only one patient had a copy number variation of PROS1 in 2270 consecutive samples tested. Currently, we are sequencing the PROS1 gene in all individuals with protein S levels <1st percentile of which results will be available before the ASH conference of 2011. Conclusion:Low free protein S and low total protein S levels could not identify individuals at risk for venous thrombosis in a population based study. Although extremely low free protein S levels were associated with an increased risk for venous thrombosis, numbers were too small to support testing on free protein S in an unselected group of venous thrombosis patients. Disclosures:No relevant conflicts of interest to declare.

Protein S deficiency with a PROS1 gene mutation in a patient presenting with mesenteric venous thrombosis following total colectomy

Protein S deficiency (PSD) is an inherited or acquired disorder associated with an increased risk of thrombosis. We describe the unusual case of a previously healthy 45-year-old man with PSD. The patient was admitted for work-up of hematochezia and was diagnosed with colorectal cancer. Preoperative evaluation was unremarkable, and he subsequently underwent an uneventful laparoscopic total colectomy. The next day, he complained of chest tightness, and a computed tomography enterography revealed acute thrombosis throughout the entire superior mesenteric vein. Further laboratory evaluation revealed decreased free protein S levels. Polymerase chain reaction and direct sequencing of the PROS1 gene revealed a heterozygous transition (NM_000313.3 c.1907A > G), resulting in an amino acid change (p.Tyr636Cys). Although several case reports have described mesenteric venous thrombosis (MVT) in individuals with PSD, most have just reported decreases in the activity or concentration of protein S. We confirmed a nucleotide alteration of PROS1 associated with PSD. This patient, who had hereditary thrombophilia, may have developed MVT after transient exposure to abdominal surgery. We may need to implement more comprehensive coagulation testing prior to surgery considering the prevalence and possible risk of thromboembolic event.

The induction of epigenetic regulation of PROS1 gene in lung fibroblasts by gold nanoparticles and implications for potential lung injury

Advances in nanotechnology have given rise to the rapid development of novel applications in biomedicine. However, our understanding in the risks and health safety of nanomaterials is still not complete and various investigations are ongoing. Here, we show that gold nanoparticles (AuNPs) significantly altered the expression of 19 genes in human fetal lung fibroblasts (using the Affymetrix Human Gene 1.0 ST Array). Among the differentially expressed genes, up-regulation of microRNA-155 (miR-155) was observed concomitant with down-regulation of the PROS1 gene. Silencing of miR-155 established PROS1 as its possible target gene. DNA methylation profiling analysis of the PROS1 gene revealed no changes in the methylation status of this gene in AuNP-treated fibroblasts. At the ultrastructural level, chromatin condensation and reorganization was observed in the nucleus of fibroblasts exposed to AuNPs. The findings provide further insights into the molecular mechanisms underlying toxicity of AuNPs and their impact on epigenetic processes.

Mesenteric Venous Thrombosis in a Child With Type 2 Protein S Deficiency

A 5-year-old girl presented with abdominal pain and bloody stools 2 weeks after suffering from influenza A infection. Enhanced computed tomographic scan showed widespread splanchnic venous thrombosis and small intestine necrosis. She recovered after the necrotic bowel was resected. The patient continues to receive anticoagulant therapy. Thrombophilia screening after the complete resolution consistently showed mildly decreased protein S (PS) activity with normal PS antigen levels. Sequence analysis detected a heterozygous K196E mutation in the PROS1 gene. Type 2 PS deficiency was diagnosed. This is the first report of mesenteric vein thrombosis in a child with a type 2 PS deficiency.

Investigation on gene mutation from hereditary protein S deficiency pedigree

Objective To identify the clinical phenotypic diagnosis and gene mutation detection of two kindreds with PS deficiency. MethodsPS: A was measured by chromogenic substrate method;TPS:Ag, FPS: Ag levels were measured by ELISA method; PS gene(PROS1 gene)was detected by amplifying 15 exons and flanking intron sequences from the propositus with PCR method. PCR products were purified and directly sequenced. Results For propositus 1,PS: A was 48.6% ,TPS: Ag was 136 mg/L, FPS : Ag was 41 mg/L, PROSI gene exon 2 was in c. Heterozygous base substitutions was detected in C121T locus, which led to Arg-1Cys (R-1C) heterozygous roissense mutation encoded in PS proteins. For propositus 2, PS: A was 29.2%, TPS: Ag was 83 mg/L, FPS: Ag was 26 mg/L, PROSI gene exon 14 was in c. Heterozygous base substitutions was identified in CI687T locus, in which Gln.522Stop heterozygous nonsense mutation was encoded in PS proteins. Conclusions c. C121T is a novel mutation locus detected in PROS1 gene. This heterozygous mutation could lead to type Ⅱ PS hereditary deficiency, while c. C1687T heterozygous mutation could bring about type Ⅰ PS hereditary deficiency. Key words: Protein S deficiency; Pedigree; Blood proteins; Mutation

Intracerebral mass bleeding in a term neonate: Manifestation of hereditary Protein S deficiency with a new mutation in the PROS1 Gene

Intracerebral mass bleeding in a term neonate: Manifestation of hereditary Protein S deficiency with a new mutation in the PROS1 Gene

Down-regulation of PROS1 Gene Expression by 17β-Estradiol via Estrogen Receptor α (ERα)-Sp1 Interaction Recruiting Receptor-interacting Protein 140 and the Corepressor-HDAC3 Complex

Pregnant women show a low level of protein S (PS) in plasma, which is known to be a risk for deep venous thrombosis. 17Beta-estradiol (E(2)), an estrogen that increases in concentration in the late stages of pregnancy, regulates the expression of various genes via the estrogen receptor (ER). Here, we investigated the molecular mechanisms behind the reduction in PS levels caused by E(2) in HepG2-ERalpha cells, which stably express ERalpha, and also the genomic ER signaling pathway, which modulates the ligand-dependent repression of the PSalpha gene (PROS1). We observed that E(2) repressed the production of mRNA and antigen of PS. A luciferase reporter assay revealed that E(2) down-regulated PROS1 promoter activity and that this E(2)-dependent repression disappeared upon the deletion or mutation of two adjacent GC-rich motifs in the promoter. An electrophoretic mobility shift assay and DNA pulldown assay revealed that the GC-rich motifs were associated with Sp1, Sp3, and ERalpha. In a chromatin immunoprecipitation assay, we found ERalpha-Sp protein-promoter interaction involved in the E(2)-dependent repression of PROS1 transcription. Furthermore, we demonstrated that E(2) treatment recruited RIP140 and the NCoR-SMRT-HDAC3 complex to the PROS1 promoter, which hypoacetylated chromatin. Taken together, this suggested that E(2) might repress PROS1 transcription depending upon ERalpha-Sp1 recruiting transcriptional repressors in HepG2-ERalpha cells and, consequently, that high levels of E(2) leading to reduced levels of plasma PS would be a risk for deep venous thrombosis in pregnant women.

Crossing of the crystalline-electric-field and rattling-phonon excitation energies in the filled skutterudite superconductor Pr(Os1−xRux)4Sb12

By specific heat measurements on Pr(Os1−xRux)4Sb12, we have studied the x-dependences of the 4f-electron crystalline-electric-field first excitation energy and the rattling energy of the Pr ion. The former increases monotonically from ~ 10 K to ~ 85 K, while the latter of ~ 45 K does not change much resulting in a crossing of the two energy levels at x ≊ 0.6. The reported minimum in the superconducting transition temperature Tc at x ≊ 0.6 could be associated with the level crossing.

Molecular Basis for A Thrombophilic Patient's Combined Deficiencies in Proteins C and S.

Abstract 4198 Background/objectivesProteins C and S (PC and PS) are vitamin K-dependent plasma proteins with anticoagulant properties. Protein S functions as a non-enzymatic cofactor for activated protein C (APC). APC proteolytically degrades coagulation factors Va and VIIIa, thereby diminishing the activities of the prothrombinase and tenase complexes, respectively. The human PC gene (PROC) is located on the long arm of chromosome 2 (2q13-q14) and contains 9 exons which code for 461 amino acid residues. The human PS (PROS1) gene resides on chromosome 3 (3p11.1-q11.2) and contains 15 exons coding for 636 amino acid residues. Hereditary PS and PC deficiencies are both autosomal dominant disorders in which patients have diminished functional levels of the respective protein (usually ∼50% relative to normal controls). Clinically, this results in increased propensity toward thromboembolic disease, also known as thrombophilia. In this study, we describe a unique thrombophilic patient who has combined deficiencies in both proteins C and S. The objective of the study was to elucidate the precise genetic defect(s) causing these deficiencies. MethodsFollowing purification of the patient's DNA from peripheral blood leukocytes, PCR amplifications were performed using oligonucleotide primers flanking all exons of the PROS1 and PROC genes. In addition, the 400bp region upstream of the first exon of PROS1 (corresponding to the promoter region) was also amplified by PCR. The PCR products were purified and their DNA sequences determined in both forward and reverse directions using the dye-terminator method. The resultant nucleotide sequences were compared with the PROS1 and PROC reference sequences [web address]. ResultsThe patient was found to be heterozygous for a novel missense PROC gene mutation in exon 8, which codes in part for the proteolytic domain of protein C. The resulting ValàGly substitution of residue 221 is in close proximity to the catalytic triad, which could abrogate its enzymatic activity. Although no mutations were present in any of the patient's PROS1 exons, there was a novel heterozygous CàG nucleotide substitution in the PROS1 promoter region. This substitution is present within an Sp1 transcription factor binding site that is highly conserved among mammals. Therefore, this mutation could significantly diminish expression of the otherwise normal PROS1 gene, leading to the observed decreased protein S level. Both mutations are unreported in the literature and are not listed in the PROC and PROS1 mutation databases. ConclusionsWe have successfully identified two novel genetic defects leading to deficiencies of the anticoagulant proteins C and S in a patient with thrombophilia. Further studies are underway to confirm the suggested biochemical effects of the mutation on protein C and protein S gene expression. Disclosures:No relevant conflicts of interest to declare.

Copy Number Variation of PROS1 and Protein S Deficiency Are Rare in a Population-Based Case-Control Study On Venous Thrombosis (MEGA study).

Abstract 3166Poster Board III-106Protein S (PS) deficiency increases the risk of venous thrombosis (VT) in family studies, but it has been difficult to estimate the risk in population-based studies. Moreover, the genetic basis of PS deficiency is only poorly understood as in about 50% of PS deficient families no mutations are found by exon-targeted sequencing of the PS gene PROS1. Further, the laboratory diagnosis of PS deficiency is complicated by physiological variation due to age and sex, as well as interference by pregnancy, use of hormones and medication and vitamin K deficiency. We recently showed with the multiplex ligation-dependent probe amplification (MLPA) methodology, that whole or partial deletion of PROS1 explained PS deficiency in around 40% of point mutation-negative French PS deficient families. Here, we analyzed PS deficiency in the MEGA (Multiple Environmental and Genetic Assessment) study to verify whether gross gene deletions or duplications are also common in unselected individuals with venous thrombosis. The MEGA study included 5000 patients with a first episode of deep VT or pulmonary embolism and 5000 controls. Questionnaires and DNA samples were taken for every participant and blood was collected in around half of patients and controls. Total PS (tPS) was measured in plasma by ELISA. For analysis, vitamin K users were excluded yielding 2198 patients and 2904 controls. The mean tPS (±SD) was 102.6 U/dL (±19.1) in patients and 100.8 U/dL (±19.7) in controls. OR and 95% confidence interval were computed to estimate the relative risk of thrombosis. PS was not a risk factor for thrombosis in the MEGA study when deciles were used to calculate OR neither when lowest 2.5% or 1% PS levels was examined (data not shown). We then randomly analyzed DNA samples from 2270 participants out of 10000 individuals (1395 patients and 875 controls) by MLPA to look for copy number variations (CNVs). In only one individual an abnormal MLPA pattern was found and the test was repeated confirming results. This individual, a female patient of 66 years who was not using estrogens, was heterozygous for a deletion of the complete PROS1 gene. Her tPS was 64.3 U/dL, which was below the 2.5th percentile (tPS = 65.3 U/dL) of the total MEGA control group. To check if the frequency of CNVs in PROS1 was indeed low in the MEGA, we zoomed in on PS deficiency by selecting DNA from individuals with low tPS (cuttof: mean tPS -2SD of the total MEGA control group), not excluding vitamin K users. This selection yielded 404 individuals, out of which 198 had been previously analyzed in the first round. MLPA was performed for the remaining 206 individuals and no new CNV was found in PROS1. In conclusion, PS deficiency was not a risk factor for VT in this population-based study and deletions or duplications in PROS1 were not common. This is in contrast with previous data from family-based studies but in accordance with the fact that the genetic basis of PS deficiency and its risk for VT outside of a family setting was also not evident in other studies. DisclosuresNo relevant conflicts of interest to declare.

Compound heterozygous mutations in the PROS1 gene responsible for quantitative and qualitative protein S deficiency

Protein S (PS) is a vitamin K-dependent plasma glycoprotein, which plays an important role in the blood anticoagulant system. The PS gene (PROS1) spans some 80 kb of genomic DNA, composed of 15 exons and 14 introns, and is closely linked to a highly homologous pseudogene (PROS2), whose sequence is 96% identical to that of PROS1 [1]. Congenital PS deficiency caused by PROS1 mutations is associated with an increased risk of thromboembolic disease. In this study, we investigated the molecular basis of a patient with both quantitative and qualitative PS deficiency. A 69-year-old man had a medical examination in our hospital because of progressive bilateral leg pain and swelling. He was diagnosed with deep vein thrombosis (DVT) based on the venographs of the legs. Laboratory tests showed normal values of platelet count, prothrombin time, activated partial thromboplastin time, ATIII activity and protein C activity. Test results for lupus anticoagulant and anti-cardiolipin b2GPI antibody were negative. Total PS antigen, free-PS antigen and PS activity levels were 49.0% (normal range 65‐135%), 26.7% (60‐150%) and 10% (60‐150%), respectively. From these findings, he was diagnosed as having PS deficiency.