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C0182: First Observation of De Novo MYH9 Gene Mutation in a Patient with Macrothrombocytopenia
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POSTERS / Thrombosis Research 133S3 (2014) S35–S123
The aim of present study is to evaluate the role of PAI-1 4G/5G polymorphism in intracranial and extracranial hemorrhages in factor XIII deficient patients. Methods: This case control study was conducted on 34 FXIII deficient patients with CNS bleeding episodes and 36 patients as control group with factor XIII deficiency but without any history of CNS bleeding. Initially both groups were evaluated for the most frequent polymorphism of factor XIII (Trp187Arg polymorphism) as reported in a previous study in Sistan and Baluchistan province in order to confirm the molecular defect. Then sample of similar patients were also assessed for PAI-1 4G/5G polymorphism using PCR-RFLP analysis. Eventually the obtained data was analyzed using SPSS software. Results: The results of this study revealed that all patients in our study group were homozygote for Trp187 Arg polymorphism. We also found that an equal numbers of patients (4 individuals) in each case and control group were heterozygote for PAI-1 4G/5G polymorphism and none of them showed homozygote for this polymorphism. All heterozygote patients in our study group had intracranial hemorrhage and those with extracranial hemorrhage showed no PAI-1 4G/5G polymorphism. Conclusions: It seems that PAI-1 4G/5G polymorphism does not have any effect on occurrence of intra- and extracranial hemorrhage in these patients with factor XIII deficiency. C0122 IDENTIFICATION OF 6 MUTATIONS IN THE PROTEIN C GENE (PROC) IN A PANEL OF 83 SPANISH FAMILIES WITH PROTEIN C DEFICIENCY L. Martos1 , E. Bonet1 , P. Medina1 , A. Vaya´ 1 , R. Lecumberri2 , 1 F. Ferrando3 , Y. Mira1 , P. Marco4 , Tomas ´ J. Gonzalez-L ´ opez ´ , 1 1 1 1 J. Hermida , F. Iba´ nez ˜ , R. Montes , A. Estelles ´ , S. Bonanad1 , S. Navarro Rosales1 , F. Espana ˜ 1 . 1 Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Group. La Fe University and Polytechnic Hospital La Fe. Valencia, Spain; 2 Hematology Service, University Clinic of Navarra, Spain; 3 Hemotology Service. La Fe University and Polytechnic Hospital. Valencia, Spain; 4 Thrombosis and Haemostasis Unit. Hematology Service. Universitary General Hospital, Alicante, Spain Background: There are no data on characteristics and molecular basis of the hereditary protein C (PC) deficiency in Spain. The aim of this study was to analyze a panel of probands with PC deficiency from symptomatic Spanish families and identify type II mutations in the PC gene (PROC). Methods: We sequenced the 9 exons (E) and their flanking intron (I) regions of PROC in 83 unrelated probands with venous thromboembolism (VTE) and 174 relatives of 52 probands. Results: In 71 probands (86%), we identified 35 different mutations in heterozygosis associated with the PC deficiency. Six were type II mutations, with reduced functional PC levels and normal antigen levels. (1) Pamplona-1: g.1388G>A mutation located in E3, already reported, which results in the p.Arg(−1)His change. It is located in the propeptide cleavage site, which may prevent the cleavage and/or the correct g-carboxylation of the Gla-domain. (2) Valencia26: g.1390G>A in E3 that induces a p.Ala1Thr change, already reported, with similar effect to the previous one. (3) Valencia-8 and -10, and Alicante-1 and -4: a Glu>Lys change in E3 not reported in vivo, but in vitro studies demonstrated that the mutated protein lost its anticoagulant activity. (4) Valencia-C1: g.3139C>A mutation, which results in the p.His66Asn change, already reported. (5) Valencia-25: a G>A change in E5, not yet reported. Mutations 4 and 5 are located in a epidermal growth factor-like domain involved in the interaction of PC with protein S. (6) Pamplona-1b, Burgos-1: g.7163A>C in E8, resulting in the p.Lys193Gln change not yet reported, but in vitro studies showed that the mutated protein displays a slightly reduced (80%) anticoagulant activity.
Two patients were double heterozygous for p.Arg(−1)His and p.Lys193Gln and other carried only the p.Lys193Gln mutation. According to their PC levels, we inferred that the Arg(−1)His PC has less than 7% of anticoagulant activity, which agrees with the levels seen in other PC mutations located near to this one. Conclusions: These results outline the importance of some PC regions for its anticoagulant function, broadening the knowledge of the PC structure–function relationship. (ISCIII PI12/00027), FEDER and Redes RECAVA (RD06/0014/0004) and RIC (RD12/0042/0029 and 0009), Conseller´ıa de Educacion, ´ Generalitat Valenciana (Prometeo 2011/027), and Instituto de Investigacion ´ Sanitaria La Fe. PM is a Miguel Servet researcher (FIS-CP09/00065). C0169 POLYMORPHISMS OF THE LIPOPROTEIN LIPASE GENE AS GENETIC MARKERS FOR STROKE IN COLOMBIAN POPULATION C. Velasquez Pereira1 , C. Vargas1 . 1 Steenstraat, Meulebeke, Belgium Background: Polymmorphisms of the lipoprotein lipase gene have been extensively studied in different populations finding variations about its association with stroke among societies. Showing that HindIII and PvuII polimorphisms can act as risk markers for the development of stroke by increasin levels of TG and decreasing HDL. However, Ser447X can be protective marker causing an increase of HDL levels and reducing TG levels. Thus, the aim of this study was to analyze if there is an association between the presence of polymorphisms in the LPL gene (HindIII, PvuII and Ser447X) with development of ischemic stroke in Colombian population. Methods: Sample size was 347 stroke patients (clinical diagnosis and x-ray CT) and 347 healthy subjects. PCR-RFLP was the technique to detect Ser447X; HindIII, and PvuII polymorphisms in the LPL gene. Results were analyzed by Statat12 and Arlequin. Results: Allele and genotypic frequencies of the studied polymorphims did not show a statistically significant difference between cases and controls. In the present research was no found any association between any of the LPL gene polymorphism and stroke in the population samples used. Conclusions: LPL gene polymorphism are not genetic markers for the development of stroke in the Colombian sample used. C0182 FIRST OBSERVATION OF DE NOVO MYH9 GENE MUTATION IN A PATIENT WITH MACROTHROMBOCYTOPENIA D. Torun1 , T. Duman1 , N. Akar1 . 1 TOBB-ETU Hospital. Department: Pediatrics. Ankara, Turkey Background: Myosin heavy chain 9 (MYH9)-related platelet disorders belong to the group of inherited macrothrombocytopenias. The MYH9 gene encodes the non-muscle myosin heavy chain IIA (NMMHC-IIA), a cytoskeletal contractile protein. Several mutations in the MYH9 gene lead to premature release of platelets from the bone marrow, macrothrombocytopenia and cytoplasmic inclusion bodies with in leukocytes. In this study, we aimed to analyze mutations in MYH9 gene in a Turkish family with macrotrombocytopenia. Methods: Peripheral blood was collected from a Turkish patient with macrothrombocytopenia and his family members. A written informed consent for genetic analysis was obtained from the patients. DNA was isolated by proteinase K and phenol/chloroform extraction. MYH9 gene was screened by polymerase chain reaction (PCR). Then samples were sequenced using a DNA sequencer (Beckman Coulter, USA). Results: Screening the seven exons of the MYH9 gene from a Turkish patient with macrothrombocytopenia revealed a coexistence of two novel mutations. The first of these was in exon 26, a transition C to A at nucleotide 3756, resulting in Leucine to Methionine at CD 1156 in heterozygote form. The other, c.3762
POSTERS / Thrombosis Research 133S3 (2014) S35–S123
G>A missense mutation in exon 26 resulted in a substitution p.E1182K (E:Glutamat, K:Lysine) in heterozygote form. These were not previously reported (Figure 1). The Leu1156 and the Glu1182 aminoacid residues are highly conserved among species. His mother did not carry a mutation. In order to identify the mutation as a “de novo mutation”, DNA fingerprint paternity test was performed to the family members. Analysis data revealed that father and mother were his biological parents about 99.9% accuracy (Table 1). Conclusions: In this study, de novo mutations were shown for the first time and these mutations are not defined Human Gene Mutation Database (HGMD) previously. It is interesting that these two mutations occurred at the same exon. C0203 VARIABILITY OF THE GP6 GENE IN PATIENTS WITH PLATELET HYPERAGGREGABILITY AND FETAL LOSS J. Sokol1 , J. Stasko1 , I. Skornova1 , M. Skerenova1 , L. Lisa1 , P. Kubisz1 . 1 Department of Hematology and Transfusiology, Jessenius Faculty of Medicine, Martin, Slovakia Background: Sticky platelet syndrome (SPS) is one of the most common thrombophilia associated with otherwise unexplained arterial thrombosis. Moreover according to recent reports (Bick et al., 2000, 2005) the SPS is after antiphospholipid syndrome probably the second most common thrombophilia that causes recurrent spontaneous abortions or fetal loss syndrome. Aim of the study was to evaluate the genetic variability of the GP6 gene in women with SPS type I or II and fetal loss. Methods: 27 women with SPS type I or II, clinically manifested as fetal loss, and 42 healthy women (blood donors) without SPS and with no previous history of fetal loss as well as venous or arterial thrombosis were enrolled. All subjects gave their informed consent with participation in the study. SPS was diagnosed by platelet aggregometry (PACKS-4 aggregometer, Helena Laboratories) according to the method and criteria described by Mammen and Bick. Eight SNPs, i.e. 4 SNPs for each end of GP6 gene were chosen for analysis. For the 3 end: rs4281840, rs12981732, rs10417943, rs1671152 and for the 5’ end: rs1654433, rs1671215, rs10418743, rs8113032. Results: We found a higher occurrence of three SNPs (in SPS group compared to controls (rs1671152: P = 0.018; OR 4.54; CI 1.346– 15.34; rs1654433: P = 0.009; OR 5.11; CI 1.536–17.03; and rs1671215: P = 0.05; OR 2.15; CI 1.009–4.609). The haplotype analysis showed a significantly higher occurrence (p < 0.05) of two haplotypes in SPS women (ACGG, 0.204 vs. 0.048; OR 5.115; CI 1.536–17.032 and CCGT, 0.155 vs. 0.04; OR 4.52; CI 1.211–16.881). Conclusions: Our results, especially the higher occurrence of two GP6 haplotypes in SPS women with fetal loss, can support an idea that variability of the GP6 gene may be associated with the platelet hyperaggregability in these women. Acknowledgement: The work was supported by projects APVV 0222–11, Vega 1/0016/12 and Vega 1/0272/14. C0218 A HIGH PROPHYLACTIC LMWH DOSE SUCCESSFULLY SUPPRESSED HAEMOSTATIC ACTIVITY IN PREGNANT WOMAN WITH A NEW PROTHROMBIN FII G20031T (C.1787G>A) MUTATION M. Kovac1 , Z. Mikovic1 , V. Mandic1 , V. Djordjevic1 , D. Radojkovic1 , S. Lalic-Cosic1 , I. Elezovic1 . 1 Faculty of Medicine, University of Belgrade, Belgrade, Serbia Background: The treatment of pregnant women with severe inherited thrombophilia is a challenge. Since there are no evidence data about anticoagulant therapy during pregnancy in case of a new prothrombin mutation FII G20031T (c.1787G>A) caused antithrombin resistance, we describe the first case of successful pregnancy in a woman heterozygous for this mutation.
S79
Methods: Considering that our patient already had a serious VTE and family present recurrent VTE, in case of her older sister associated with recurrent fetal loss despite the LMWH, a high level of LMWH was introduced from the early pregnancy. In the follow up D-dimer, and anti-Xa level were monitored. In order to determine the full anticoagulant effect of LMWH the thrombin generation test (ETP) was performed from blood samples collected during pregnancy. In the same time the plasma samples from 30 healthy pregnant women were collected to establish D-dimer and ETP to apply as control. Results: Anti-Xa ranged from 0.65–0.80 IU/ml (mean 0.67). D-dimer results showed a slight increase of haemostatic activation and they didn’t differ in comparison to control group related to gestational age. ETP levels showed significant increase in the second trimester and remained stable through pregnancy similarly in both healthy and woman with FII G20031T (c.1787G>A) mutation. The patient was delivered at 37th gestation week of a male baby 49 cm in length and 2550 g in weight (Appgar score 9/9). Conclusions: D-dimer and ETP showed that a high prophylactic LMWH dose successfully suppressed haemostatic activity in case of FII G20031T (c.1787G>A) mutation. C0221 GENOTYPE CYP2C19 EFFECTS ON THERAPEUTIC RESPONSE DURING CLOPIDOGREL TREATMENT IN PATIENTS WITH CAROTID ARTERY STENOSIS D. Backovic1 , M. Kovac1 , B. Calija1 , E. Strugarevic1 , D. Radak1 , L. Rakicevic1 , J. Kusic-Tisma1 , D. Radojkovic1 , S. Ignjatovic1 . 1 Faculty of Medicine, University of Belgrade, Belgrade, Serbia Background: Dual antiplatelet therapy, aspirin and clopidogrel, is a “gold” standard of care prevention of platelet activation and thrombosis. But, clopidogrel faces limitation in its utility due to differences in the ability to metabolize pro-drug. Several studies have showed that different CYP2C19 genotypes affect the pharmacokinetics and pharmacodynamics of clopidogrel. Methods: A prospective observational study included 59 participants with carotid artery stenosis undergoing endarterectomy. All patients, who received clopidogrel (75 mg) for at least 30 days from intervention, were eligible for the study. The mean age at entry was 64.35±11.62 and 55% of the study participants were male. In all of them the influence of CYP2C19 genotype on-clopidogrel platelet reactivity were followed. Platelet aggregation was measured by impedance aggregometry using Multiplate analyzer (Roche Diagnostics, Mannheim, Germany) before endarterectomy (baseline value) and 24h after first dose of clopidogrel. Aggregation was followed-up after 7 days and 30 days of taking the drug. Genotyping of CYP2C19 variant was performed with TaqMan SNP genotyping assay (Appllied Biosystems, Foster City, California). Results: Genotyping results showed that 71% of patients were homozygous for wild-type allele, 27% were heterozygous for the CYP2C19*2 variant allele and 2% were CYP2C19*2 homozygous. Platelet aggregation showed that 25% of patients were clopidogrel non-responders. Among patients carrying CYP2C19 loss-of-function allele (*2) 31% were clopidogrel non-responders, while 23% of noncarriers didn’t have therapeutic response after 30 days of taking the clopidogrel. Patient who was CYP2C19*2 homozygous had no therapeutic response from the beginning of the treatment. Conclusions: Correlation between genotype and phenotype showed that CYP2C19*2 genotype was associated with diminished platelet response to clopidogrel treatment and poorer clinical outcomes.
POSTERS / Thrombosis Research 133S3 (2014) S35–S123
The aim of present study is to evaluate the role of PAI-1 4G/5G polymorphism in intracranial and extracranial hemorrhages in factor XIII deficient patients. Methods: This case control study was conducted on 34 FXIII deficient patients with CNS bleeding episodes and 36 patients as control group with factor XIII deficiency but without any history of CNS bleeding. Initially both groups were evaluated for the most frequent polymorphism of factor XIII (Trp187Arg polymorphism) as reported in a previous study in Sistan and Baluchistan province in order to confirm the molecular defect. Then sample of similar patients were also assessed for PAI-1 4G/5G polymorphism using PCR-RFLP analysis. Eventually the obtained data was analyzed using SPSS software. Results: The results of this study revealed that all patients in our study group were homozygote for Trp187 Arg polymorphism. We also found that an equal numbers of patients (4 individuals) in each case and control group were heterozygote for PAI-1 4G/5G polymorphism and none of them showed homozygote for this polymorphism. All heterozygote patients in our study group had intracranial hemorrhage and those with extracranial hemorrhage showed no PAI-1 4G/5G polymorphism. Conclusions: It seems that PAI-1 4G/5G polymorphism does not have any effect on occurrence of intra- and extracranial hemorrhage in these patients with factor XIII deficiency. C0122 IDENTIFICATION OF 6 MUTATIONS IN THE PROTEIN C GENE (PROC) IN A PANEL OF 83 SPANISH FAMILIES WITH PROTEIN C DEFICIENCY L. Martos1 , E. Bonet1 , P. Medina1 , A. Vaya´ 1 , R. Lecumberri2 , 1 F. Ferrando3 , Y. Mira1 , P. Marco4 , Tomas ´ J. Gonzalez-L ´ opez ´ , 1 1 1 1 J. Hermida , F. Iba´ nez ˜ , R. Montes , A. Estelles ´ , S. Bonanad1 , S. Navarro Rosales1 , F. Espana ˜ 1 . 1 Haemostasis, Thrombosis, Arteriosclerosis and Vascular Biology Group. La Fe University and Polytechnic Hospital La Fe. Valencia, Spain; 2 Hematology Service, University Clinic of Navarra, Spain; 3 Hemotology Service. La Fe University and Polytechnic Hospital. Valencia, Spain; 4 Thrombosis and Haemostasis Unit. Hematology Service. Universitary General Hospital, Alicante, Spain Background: There are no data on characteristics and molecular basis of the hereditary protein C (PC) deficiency in Spain. The aim of this study was to analyze a panel of probands with PC deficiency from symptomatic Spanish families and identify type II mutations in the PC gene (PROC). Methods: We sequenced the 9 exons (E) and their flanking intron (I) regions of PROC in 83 unrelated probands with venous thromboembolism (VTE) and 174 relatives of 52 probands. Results: In 71 probands (86%), we identified 35 different mutations in heterozygosis associated with the PC deficiency. Six were type II mutations, with reduced functional PC levels and normal antigen levels. (1) Pamplona-1: g.1388G>A mutation located in E3, already reported, which results in the p.Arg(−1)His change. It is located in the propeptide cleavage site, which may prevent the cleavage and/or the correct g-carboxylation of the Gla-domain. (2) Valencia26: g.1390G>A in E3 that induces a p.Ala1Thr change, already reported, with similar effect to the previous one. (3) Valencia-8 and -10, and Alicante-1 and -4: a Glu>Lys change in E3 not reported in vivo, but in vitro studies demonstrated that the mutated protein lost its anticoagulant activity. (4) Valencia-C1: g.3139C>A mutation, which results in the p.His66Asn change, already reported. (5) Valencia-25: a G>A change in E5, not yet reported. Mutations 4 and 5 are located in a epidermal growth factor-like domain involved in the interaction of PC with protein S. (6) Pamplona-1b, Burgos-1: g.7163A>C in E8, resulting in the p.Lys193Gln change not yet reported, but in vitro studies showed that the mutated protein displays a slightly reduced (80%) anticoagulant activity.
Two patients were double heterozygous for p.Arg(−1)His and p.Lys193Gln and other carried only the p.Lys193Gln mutation. According to their PC levels, we inferred that the Arg(−1)His PC has less than 7% of anticoagulant activity, which agrees with the levels seen in other PC mutations located near to this one. Conclusions: These results outline the importance of some PC regions for its anticoagulant function, broadening the knowledge of the PC structure–function relationship. (ISCIII PI12/00027), FEDER and Redes RECAVA (RD06/0014/0004) and RIC (RD12/0042/0029 and 0009), Conseller´ıa de Educacion, ´ Generalitat Valenciana (Prometeo 2011/027), and Instituto de Investigacion ´ Sanitaria La Fe. PM is a Miguel Servet researcher (FIS-CP09/00065). C0169 POLYMORPHISMS OF THE LIPOPROTEIN LIPASE GENE AS GENETIC MARKERS FOR STROKE IN COLOMBIAN POPULATION C. Velasquez Pereira1 , C. Vargas1 . 1 Steenstraat, Meulebeke, Belgium Background: Polymmorphisms of the lipoprotein lipase gene have been extensively studied in different populations finding variations about its association with stroke among societies. Showing that HindIII and PvuII polimorphisms can act as risk markers for the development of stroke by increasin levels of TG and decreasing HDL. However, Ser447X can be protective marker causing an increase of HDL levels and reducing TG levels. Thus, the aim of this study was to analyze if there is an association between the presence of polymorphisms in the LPL gene (HindIII, PvuII and Ser447X) with development of ischemic stroke in Colombian population. Methods: Sample size was 347 stroke patients (clinical diagnosis and x-ray CT) and 347 healthy subjects. PCR-RFLP was the technique to detect Ser447X; HindIII, and PvuII polymorphisms in the LPL gene. Results were analyzed by Statat12 and Arlequin. Results: Allele and genotypic frequencies of the studied polymorphims did not show a statistically significant difference between cases and controls. In the present research was no found any association between any of the LPL gene polymorphism and stroke in the population samples used. Conclusions: LPL gene polymorphism are not genetic markers for the development of stroke in the Colombian sample used. C0182 FIRST OBSERVATION OF DE NOVO MYH9 GENE MUTATION IN A PATIENT WITH MACROTHROMBOCYTOPENIA D. Torun1 , T. Duman1 , N. Akar1 . 1 TOBB-ETU Hospital. Department: Pediatrics. Ankara, Turkey Background: Myosin heavy chain 9 (MYH9)-related platelet disorders belong to the group of inherited macrothrombocytopenias. The MYH9 gene encodes the non-muscle myosin heavy chain IIA (NMMHC-IIA), a cytoskeletal contractile protein. Several mutations in the MYH9 gene lead to premature release of platelets from the bone marrow, macrothrombocytopenia and cytoplasmic inclusion bodies with in leukocytes. In this study, we aimed to analyze mutations in MYH9 gene in a Turkish family with macrotrombocytopenia. Methods: Peripheral blood was collected from a Turkish patient with macrothrombocytopenia and his family members. A written informed consent for genetic analysis was obtained from the patients. DNA was isolated by proteinase K and phenol/chloroform extraction. MYH9 gene was screened by polymerase chain reaction (PCR). Then samples were sequenced using a DNA sequencer (Beckman Coulter, USA). Results: Screening the seven exons of the MYH9 gene from a Turkish patient with macrothrombocytopenia revealed a coexistence of two novel mutations. The first of these was in exon 26, a transition C to A at nucleotide 3756, resulting in Leucine to Methionine at CD 1156 in heterozygote form. The other, c.3762
POSTERS / Thrombosis Research 133S3 (2014) S35–S123
G>A missense mutation in exon 26 resulted in a substitution p.E1182K (E:Glutamat, K:Lysine) in heterozygote form. These were not previously reported (Figure 1). The Leu1156 and the Glu1182 aminoacid residues are highly conserved among species. His mother did not carry a mutation. In order to identify the mutation as a “de novo mutation”, DNA fingerprint paternity test was performed to the family members. Analysis data revealed that father and mother were his biological parents about 99.9% accuracy (Table 1). Conclusions: In this study, de novo mutations were shown for the first time and these mutations are not defined Human Gene Mutation Database (HGMD) previously. It is interesting that these two mutations occurred at the same exon. C0203 VARIABILITY OF THE GP6 GENE IN PATIENTS WITH PLATELET HYPERAGGREGABILITY AND FETAL LOSS J. Sokol1 , J. Stasko1 , I. Skornova1 , M. Skerenova1 , L. Lisa1 , P. Kubisz1 . 1 Department of Hematology and Transfusiology, Jessenius Faculty of Medicine, Martin, Slovakia Background: Sticky platelet syndrome (SPS) is one of the most common thrombophilia associated with otherwise unexplained arterial thrombosis. Moreover according to recent reports (Bick et al., 2000, 2005) the SPS is after antiphospholipid syndrome probably the second most common thrombophilia that causes recurrent spontaneous abortions or fetal loss syndrome. Aim of the study was to evaluate the genetic variability of the GP6 gene in women with SPS type I or II and fetal loss. Methods: 27 women with SPS type I or II, clinically manifested as fetal loss, and 42 healthy women (blood donors) without SPS and with no previous history of fetal loss as well as venous or arterial thrombosis were enrolled. All subjects gave their informed consent with participation in the study. SPS was diagnosed by platelet aggregometry (PACKS-4 aggregometer, Helena Laboratories) according to the method and criteria described by Mammen and Bick. Eight SNPs, i.e. 4 SNPs for each end of GP6 gene were chosen for analysis. For the 3 end: rs4281840, rs12981732, rs10417943, rs1671152 and for the 5’ end: rs1654433, rs1671215, rs10418743, rs8113032. Results: We found a higher occurrence of three SNPs (in SPS group compared to controls (rs1671152: P = 0.018; OR 4.54; CI 1.346– 15.34; rs1654433: P = 0.009; OR 5.11; CI 1.536–17.03; and rs1671215: P = 0.05; OR 2.15; CI 1.009–4.609). The haplotype analysis showed a significantly higher occurrence (p < 0.05) of two haplotypes in SPS women (ACGG, 0.204 vs. 0.048; OR 5.115; CI 1.536–17.032 and CCGT, 0.155 vs. 0.04; OR 4.52; CI 1.211–16.881). Conclusions: Our results, especially the higher occurrence of two GP6 haplotypes in SPS women with fetal loss, can support an idea that variability of the GP6 gene may be associated with the platelet hyperaggregability in these women. Acknowledgement: The work was supported by projects APVV 0222–11, Vega 1/0016/12 and Vega 1/0272/14. C0218 A HIGH PROPHYLACTIC LMWH DOSE SUCCESSFULLY SUPPRESSED HAEMOSTATIC ACTIVITY IN PREGNANT WOMAN WITH A NEW PROTHROMBIN FII G20031T (C.1787G>A) MUTATION M. Kovac1 , Z. Mikovic1 , V. Mandic1 , V. Djordjevic1 , D. Radojkovic1 , S. Lalic-Cosic1 , I. Elezovic1 . 1 Faculty of Medicine, University of Belgrade, Belgrade, Serbia Background: The treatment of pregnant women with severe inherited thrombophilia is a challenge. Since there are no evidence data about anticoagulant therapy during pregnancy in case of a new prothrombin mutation FII G20031T (c.1787G>A) caused antithrombin resistance, we describe the first case of successful pregnancy in a woman heterozygous for this mutation.
S79
Methods: Considering that our patient already had a serious VTE and family present recurrent VTE, in case of her older sister associated with recurrent fetal loss despite the LMWH, a high level of LMWH was introduced from the early pregnancy. In the follow up D-dimer, and anti-Xa level were monitored. In order to determine the full anticoagulant effect of LMWH the thrombin generation test (ETP) was performed from blood samples collected during pregnancy. In the same time the plasma samples from 30 healthy pregnant women were collected to establish D-dimer and ETP to apply as control. Results: Anti-Xa ranged from 0.65–0.80 IU/ml (mean 0.67). D-dimer results showed a slight increase of haemostatic activation and they didn’t differ in comparison to control group related to gestational age. ETP levels showed significant increase in the second trimester and remained stable through pregnancy similarly in both healthy and woman with FII G20031T (c.1787G>A) mutation. The patient was delivered at 37th gestation week of a male baby 49 cm in length and 2550 g in weight (Appgar score 9/9). Conclusions: D-dimer and ETP showed that a high prophylactic LMWH dose successfully suppressed haemostatic activity in case of FII G20031T (c.1787G>A) mutation. C0221 GENOTYPE CYP2C19 EFFECTS ON THERAPEUTIC RESPONSE DURING CLOPIDOGREL TREATMENT IN PATIENTS WITH CAROTID ARTERY STENOSIS D. Backovic1 , M. Kovac1 , B. Calija1 , E. Strugarevic1 , D. Radak1 , L. Rakicevic1 , J. Kusic-Tisma1 , D. Radojkovic1 , S. Ignjatovic1 . 1 Faculty of Medicine, University of Belgrade, Belgrade, Serbia Background: Dual antiplatelet therapy, aspirin and clopidogrel, is a “gold” standard of care prevention of platelet activation and thrombosis. But, clopidogrel faces limitation in its utility due to differences in the ability to metabolize pro-drug. Several studies have showed that different CYP2C19 genotypes affect the pharmacokinetics and pharmacodynamics of clopidogrel. Methods: A prospective observational study included 59 participants with carotid artery stenosis undergoing endarterectomy. All patients, who received clopidogrel (75 mg) for at least 30 days from intervention, were eligible for the study. The mean age at entry was 64.35±11.62 and 55% of the study participants were male. In all of them the influence of CYP2C19 genotype on-clopidogrel platelet reactivity were followed. Platelet aggregation was measured by impedance aggregometry using Multiplate analyzer (Roche Diagnostics, Mannheim, Germany) before endarterectomy (baseline value) and 24h after first dose of clopidogrel. Aggregation was followed-up after 7 days and 30 days of taking the drug. Genotyping of CYP2C19 variant was performed with TaqMan SNP genotyping assay (Appllied Biosystems, Foster City, California). Results: Genotyping results showed that 71% of patients were homozygous for wild-type allele, 27% were heterozygous for the CYP2C19*2 variant allele and 2% were CYP2C19*2 homozygous. Platelet aggregation showed that 25% of patients were clopidogrel non-responders. Among patients carrying CYP2C19 loss-of-function allele (*2) 31% were clopidogrel non-responders, while 23% of noncarriers didn’t have therapeutic response after 30 days of taking the clopidogrel. Patient who was CYP2C19*2 homozygous had no therapeutic response from the beginning of the treatment. Conclusions: Correlation between genotype and phenotype showed that CYP2C19*2 genotype was associated with diminished platelet response to clopidogrel treatment and poorer clinical outcomes.