Ana Isabel Antón

Pharmacogenetic relevance of CYP4F2 V433M polymorphism on acenocoumarol therapy

VKORC1 and CYP2C9 polymorphisms are used to predict the safe dose of oral anticoagulant therapy. A new variant of CYP4F2 (V433M) has recently been related to the required warfarin dose. We evaluated its influence in earliest response to acenocoumarol in 100 selected men who started anticoagulation (3 mg for 3 consecutive days). V433M genotype exerted a gene dosage-dependent effect on the decrease of factors II, VII, IX, and X in the earliest response to acenocoumarol, with homozygous 433V subjects being the most sensitive. Similarly, after the initiation of therapy, international normalized ratio also experienced a gene dosage-dependent effect (P = .015), and 433V subjects needed 4 mg/week less than 433M carriers to achieve a steady anticoagulation (P = .043). Multivariate linear regression analysis revealed a significant contribution of V433M polymorphism to variability of both early international normalized ratio value (R(2) = 0.14) and dose requirements (R(2) = 0.19). Our data underline the relevant role of CYP4F2 V433M polymorphism in the pharmacogenetics of coumarin anticoagulants.

Functional consequences of the prothrombotic SERPINC1 rs2227589 polymorphism on antithrombin levels

This study examines the functional consequences of a SNP in the antithrombin SERPINC1 gene. The results suggest a biological explanation for the relationship between the SNP and venous thrombosis. Genetic factors involved in the interindividual variability of antithrombin have not been identified. We studied two polymorphisms of the gene coding for antithrombin (SER-PINC1) in 298 Spanish Caucasian blood donors: rs3138521, a DNA length polymorphism located on the promoter region and rs2227589, a SNP located on intron 1 that has been described as a mild thrombotic risk factor. We detected a complete linkage disequilibrium between these polymorphisms (D’=0.999). The rs3138521 polymorphism has no functional consequences. However, the rs2227589 SNP significantly associated with plasma anti-FXa activity and antithrombin levels: carriers of the A allele had slightly but significantly lower anticoagulant activity and levels than GG subjects (97.0±7.3% vs. 94.6±8.4%; p=0.032; 99.5±5.8% vs. 94.8±5.6%; p=0.001; respectively). Our results identified a functional effect of the rs2227589 polymorphism not explained by its linkage with the promoter polymorphism that support the moderate thrombotic risk associated with the A allele.

Creating a genotype-based dosing algorithm for acenocoumarol steady dose

Acenocoumarol is a commonly prescribed anticoagulant drug for the prophylaxis and treatment of venous and arterial thromboembolic disorders in several countries. In counterpart of warfarin, there is scarce information about pharmacogenetic algorithms for steady acenocoumarol dose estimation. The aim of this study was to develop an algorithm of prediction for acenocoumarol.The algorithm was created using the data from 973 retrospectively selected anticoagulated patients and was validated in a second independent cohort adding up to 2,683 patients. The best regression model to predict stable dosage in the Primary Cohort included clinical factors (age and body mass index, BSA) and genetic variants (VKORC1, CYP2C9* and CYP4F2 polymorphisms) and explained up to 50% of stable dose. In the validation study the clinical algorithm yielded an adjusted R²=0.15 (estimation´s standard error=4.5) and the genetic approach improved the dose forecast up to 30% (estimation´s standard error=4.6). Again, the best model combined clinical and genetic factors (R² = 0.48; estimation´s standard error=4) which provided the best results of doses estimates within 20% of the real dose in patients taking lower (≤ 7 mg/week) or higher (≥ 25 mg/week) acenocoumarol doses. In conclusion, we developed a prediction algorithm using clinical data and three polymorphisms in VKORC1, CYP2C9* and CYP4F2 genes that provided a steady acenocoumarol dose for about 50% of patients in the Validation Cohort. Such algorithm was especially useful to patients who need higher or lower acenocoumarol doses, those patients with higher time required until their stabilisation and are more prone to suffer a treatment derived complication.

Regulatory regions of SERPINC1 gene: Identification of the first mutation associated with antithrombin deficiency

Antithrombin is the main endogenous anticoagulant. Impaired function or deficiency of this molecule significantly increases the risk of thrombosis. We studied the genetic variability of SERPINC1 , the gene encoding antithrombin, to identify mutations affecting regulatory regions with functional effect on its levels. We sequenced 15,375 bp of this gene, including the potential promoter region, in three groups of subjects: five healthy subjects with antithrombin levels in the lowest (75%) and highest (115%) ranges of our population, 14 patients with venous thrombosis and a moderate antithrombin deficiency as the single thrombophilic defect, and two families with type I antithrombin deficiency who had neither mutations affecting exons or flanking regions, nor gross gene deletions. Our study confirmed the low genetic variability of SERPINC1 , particularly in the coding region, and its minor influence in the heterogeneity of antithrombin levels. Interestingly, in one family, we identified a g.2143 C>G transversion, located 170 bp upstream from the translation initiation codon. This mutation affected one of the four regions located in the minimal promoter that have potential regulatory activity according to previous DNase footprinting protection assays. Genotype-phenotype analysis in the affected family and reporter analysis in different hepatic cell lines demonstrated that this mutation significantly impaired, although it did not abolish, the downstream transcription. Therefore, this is the first mutation affecting a regulatory region of the SERPINC1 gene associated with antithrombin deficiency. Our results strongly sustain the inclusion of the promoter region of SERPINC1 in the molecular analysis of patients with antithrombin deficiency.

Novel loci involved in platelet function and platelet count identified by a genome-wide study performed in children.

Background Genome-wide association studies are currently identifying new loci with potential roles in thrombosis and hemostasis: these loci include novel polymorphisms associated with platelet function traits and count. However, no genome-wide study performed on children has been reported to date, in spite of the potential that these subjects have in genetic studies, when compared to adults, given the minimal degree of confounders, i.e., acquired and environmental factors, such as smoking, physical activity, diet, and drug or hormone intake, which are particularly important in platelet function. Design and Methods To identify new genetic variants involved in platelet reactivity and count, we performed a genome-wide association study on 75 children (8.5±1.8 years) using the Illumina Sentrix Human CNV370-Quad BeadChip containing 320,610 single nucleotide polymorphisms. Functional analyses included assessment of platelet aggregation and granule secretion triggered by different agonists (arachidonic acid, collagen, epinephrine, ADP), as well as platelet count. Associations were selected based on statistical significance and physiological relevance for a subsequent replication study in a similar sample of 286 children. Results We confirmed previously established associations with plasma levels of factors XII, VII and VIII as well as associations with platelet responses to ADP. Additionally, we identified 82 associations with platelet reactivity and count with a P value less than 10−5. From the associations selected for further replication, we validated two single nucleotide polymorphisms with mildly increased platelet reactivity (rs4366150 and rs1787566) on the LPAR1 and MYO5B genes, encoding lisophosphatidic acid receptor-1 and myosin VB, respectively; and rs1937970, located on the NRG3 gene coding neuroregulin-3, associated with platelet count. Conclusions Our genome-wide association study performed in children, followed by a validation analysis, led us to the identification of new genes potentially relevant in platelet function and biogenesis.

Pharmacogenetics of acenocoumarol in patients with extreme dose requirements.

Summary.  Background: There is currently intense debate as to whether pharmacogenetic algorithms for estimating the initial dose of coumarins provide a more accurate dose than the fixed‐dose approach. Recently, it has been suggested that the greatest benefit of pharmacogenetic algorithms is observed in patients with extreme dose requirements. Objectives: To identify clinical and genetic factors that better characterize patients who need extreme acenocoumarol doses for steady anticoagulation state. Patients/methods: We reviewed 9538 patients with a steady acenocoumarol dose from three Spanish hospitals, selecting 83 who took ≤ 5.00 mg week−1 (percentile 5, p5) and 203 taking ≥ 30.00 mg week−1 (p95). We also selected patients matched by gender and age taking 13.50–14.00 mg week−1 (p50). We genotyped VKORC1 (rs9923231), CALU (rs1043550), GGCX (rs699664), CYP2C9 (rs1799853; rs1057910), CYP4F2 (rs2108622) and F7 (rs5742910) single‐nucleotide polymorphisms (SNPs). Results: Comparison between p5 and p95 revealed five parameters with significant differences: body surface area (BSA) (P = 0.006), age, VKORC1, CYP2C9 and CYP4F2 genotypes (all P < 0.001). First VKORC1, and second, CYP2C9 SNPs played a strong effect by determining extreme doses, particularly in p95. Only one out of 203 p95 had the VKORC1 A‐1639A genotype, but this subject was CYP2C9*1/*1. In contrast, nine out of 83 p5 carried the VKORC1 G‐1639G genotype, although six of them were CYP2C9*3 homozygotes and another two were heterozygotes. Surprisingly, CYP4F2 V433M SNP displayed prevalences that suggest that its influence might only be evident when patients are treated with high doses. Conclusion: Two clinical data, age and BSA, and three SNPs in the VKORC1, CYP2C9 and CYP4F2 genes strongly predict outlier patients treated with acenocoumarol.

Rare homozygous status of P43 β1-tubulin polymorphism causes alterations in platelet ultrastructure

β1-tubulin is the main constituent of the platelet marginal band and studies with deficient mice showed that it maintains discoid shape and it is required for normal platelet formation. TUBB1 Q43P polymorphism is associated with decreased β1-tubulin expression, diminished platelet reactivity, and partial loss of discoid shape in heterozygous carriers. However, to date no studies have been carried out on homozygous PP individuals. Our study included 19 subjects genotyped for TUBB1 Q43P polymorphism (4 QQ, 4 QP, and 2 PP). The two PP individuals were recruited after genotyping of 2073 individuals. Biochemical, microscopy, and molecular studies were performed. Real-time PCR showed a ~40% decrease in TUBB1 mRNA in the two PP individuals compared to four QQ subjects. Western blot analysis confirmed this reduction. Electron microscopy revealed a majority of normal discoid platelets in PP individuals, although platelets with loose, re-orientated or invaginated protofilaments, and an over-developed open canalicular system were observed. Such abnormalities were not observed in QQ subjects. Morphometric analyses showed no differences between PP and QQ individuals. Immunofluorescence confirmed the presence of a normal marginal band in a majority of platelets from PP subjects. Interestingly, both PP subjects had a 40% lower platelet count than QP and QQ. TUBB1 Q43P polymorphism in homozygosity mildly affects platelet ultrastructure and our data further suggest that high levels of β1-tubulin might not be critical to sustain platelet discoid shape.

Chediak-Higashi syndrome: description of two novel homozygous missense mutations causing divergent clinical phenotype

Chediak–Higashi syndrome (CHS) is a rare autosomal recessive disease resulting from mutations in the LYST/CHS1 gene, which encodes for a 429 kDa protein, CHS1/LYST, that regulates vesicle trafficking and determines the size of lysosomes and other organelles. To date, 60 different mutations have been characterized, and a reasonably straightforward phenotype–genotype correlation has been suggested. We describe two patients on opposite ends of the CHS clinical spectrum with novel missense mutations. We characterized these patients in terms of their mutations, protein localization and expression, mRNA stability, and electrostatic potential. Patient 1 is the first report of a severe early‐onset CHS with a homozygous missense mutation (c.11362 G>A, p.G3725R) in the LYST/CHS1 gene. This molecular change results in a reduction at the CHS1 protein level, not due to an mRNA effect, but maybe a consequence of both, a change in the structure of the protein and most likely attributable to the remarkable serious perturbation in the electrostatic potential. Patient 2, who exhibited the adolescence form of the disease, was found to be homozygous for a novel missense mutation c.961 T>C, p.C258R, which seemed to have minor effect on the structure of the CHS1/LYST protein. Reexamining accepted premises of missense mutant alleles being reported among patients with clinically mild forms of the disorder should be carried out, and attempts to link genotype and clinical phenotype require identifying the actual molecular effect of the mutation. Early and accurate diagnosis of the severity of the disease is extremely important to early differentiate patients who would benefit from premature enrollment into a transplantation protocol.

Functional and molecular characterization of inherited platelet disorders in the Iberian Peninsula: results from a collaborative study

BackgroundThe diagnostic evaluation of inherited platelet disorders (IPDs) is complicated and time-consuming, resulting in a relevant number of undiagnosed and incorrectly classified patients. In order to evaluate the spectrum of IPDs in individuals with clinical suspicion of these disorders, and to provide a diagnostic tool to centers not having access to specific platelets studies, we established the project “Functional and Molecular Characterization of Patients with Inherited Platelet Disorders” under the scientific sponsorship of the Spanish Society of Thrombosis and Haemostasis.Patients/methodsSubjects were patients from a prospective cohort of individuals referred for clinical suspicion of IPDs as well as healthy controls. Functional studies included light transmission aggregation, flow cytometry, and when indicated, Western-blot analysis of platelet glycoproteins, and clot retraction analysis. Genetic analysis was mainly performed by sequencing of coding regions and proximal regulatory regions of the genes of interest.ResultsOf the 70 cases referred for study, we functionally and molecularly characterized 12 patients with Glanzmann Thrombasthenia, 8 patients with Bernard Soulier syndrome, and 8 with other forms of IPDs. Twelve novel mutations were identified among these patients. The systematic study of patients revealed that almost one-third of patients had been previously misdiagnosed.ConclusionsOur study provides a global picture of the current limitations and access to the diagnosis of IPDs, identifies and confirms new genetic variants that cause these disorders, and emphasizes the need of creating reference centers that can help health care providers in the recognition of these defects.

Genotype-phenotype relationship for six common polymorphisms in genes affecting platelet function from 286 healthy subjects and 160 patients with mucocutaneous bleeding of unknown cause

Polymorphisms affecting platelet receptors and intracellular proteins have been extensively studied in relation to their potential influence in thrombosis and haemorrhages. However, few reports have addressed their impact on platelet function, with contradictory results. Limitations of these studies include, among others, small number of patients, the platelet functional parameters analyzed and their known variability in the healthy population. We studied the effect of six polymorphisms [ITGB3 1565T > C (HPA‐1), GPIBA variable number tandem repeat and 524C > T (HPA‐2), ITGA2 807C > T, ADRA2A 1780A > G, and TUBB1 Q43P] on platelet function in 286 healthy subjects and their potential pathogenetic role in 160 patients with hereditary mucocutaneous bleeding of unknown cause. We found no effect of any of these polymorphisms on platelet aggregation, secretion, PFA‐100®, and thrombin generation in platelet rich plasma. Furthermore, patients and controls showed no significant differences in the frequency of any of these polymorphisms. Thus, our study demonstrated that polymorphisms in genes affecting platelet function do not influence significantly major platelet functions and appear irrelevant in the pathogenesis of bleeding disorders.