Genetic predisposition to fetal alcohol syndrome: association with congenital disorders of N-glycosylation.

BackgroundFetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy; although additional factors might be involved, as development and severity are not directly related to alcohol intake. The abnormal glycosylation caused by alcohol might play a role in FAS according to the clinical similarities shared with congenital disorders of glycosylation (CDG). Thus, mutations underlying CDG, affecting genes involved in glycosylation, could also be involved in FAS.MethodsA panel of 74 genes involved in N-glycosylation was sequenced in 25 FAS patients and 20 controls with prenatal alcohol exposure. Transferrin glycoforms were evaluated by HPLC.ResultsRare (minor allele frequency<0.009) missense/splice site variants were more frequent in FAS than controls (84% vs. 50%; P=0.034, odds ratio: 5.25, 95% confidence interval: 1.3-20.9). Remarkably, three patients, but no controls, carried variants with functional effects identified in CDG patients. Moreover, the patient with the most severe clinical phenotype was the only one carrying two variants with functional effects. Family studies support that the combination of a genetic defect and alcohol consumption during pregnancy might have a role in FAS development.ConclusionsOur study supports that the rare variants of genes involved in N-glycosylation could play a role in the development and severity of FAS under prenatal alcohol exposure.

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.

Study of 18 functional hemostatic polymorphisms in mucocutaneous bleeding disorders.

Hereditary disorders of primary hemostasis, characterized by mucocutaneous bleeding (MCB), are highly prevalent in children. Few cases are clearly monogenic, but the overwhelming majority are classified as mild bleeding disorders, with wide clinical and laboratory heterogeneity suggestive of complex polygenic diseases. In this framework, and by homology with venous thrombosis, some functional polymorphisms affecting the hemostatic system should be considered. We evaluated the role of 18 common hemostatic polymorphisms on the occurrence and severity of MCB in a case-control study including 269 patients and 286 matched controls consecutively recruited. FV Leiden was associated with milder bleeding severity, assessed by a standardized bleeding score (p = 0.013). Multivariate analysis revealed that three additional polymorphisms protected against MCB (F13 Leu34, OR = 0.66; 95% CI, 0.47-0.94; p = 0.024; VKORC1 1173T, OR = 0.59; 95% CI, 0.40-0.87; p = 0.009; and non-O blood group alleles, OR = 0.59; 95% CI, 0.41-0.86; p = 0.006). When combined, these polymorphisms showed an additive protection (OR = 0.24; 95% CI, 0.11-0.52), supporting the polygenic nature of MCB. Our data suggest that some common polymorphisms affecting hemostasis-related genes could protect from bleeding.

Functional consequences of the prothrombotic SERPINC1 rs2227589 polymorphism on antithrombin levels.

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.

Antithrombin controls tumor migration, invasion and angiogenesis by inhibition of enteropeptidase.

Antithrombin is a key inhibitor of the coagulation cascade, but it may also function as an anti-inflammatory, anti-angiogenic, anti-viral and anti-apoptotic protein. Here, we report a novel function of antithrombin as a modulator of tumor cell migration and invasion. Antithrombin inhibited enteropeptidase on the membrane surface of HT-29, A549 and U-87 MG cells. The inhibitory process required the activation of antithrombin by heparin, and the reactive center loop and the heparin binding domain were essential. Surprisingly, antithrombin non-covalently inhibited enteropeptidase, revealing a novel mechanism of inhibition for this serpin. Moreover, as a consequence of this inhibition, antithrombin was cleaved, resulting in a molecule with anti-angiogenic properties that reduced vessel-like formation of endothelial cells. The addition of antithrombin and heparin to U-87 MG and A549 cells reduced motility in wound healing assays, inhibited the invasion in transwell assays and the degradation of a gelatin matrix mediated by invadopodia. These processes were controlled by enteropeptidase, as demonstrated by RNA interference experiments. Carcinoma cell xenografts in nude mice showed in vivo co-localization of enteropeptidase and antithrombin. Finally, treatment with heparin reduced experimental metastasis induced by HT29 cells in vivo. In conclusion, the inhibition of enteropeptidase by antithrombin may have a double anti-tumor effect through inhibiting a protease involved in metastasis and generating an anti-angiogenic molecule.

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

BACKGROUND: The 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/METHODS: Subjects 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. RESULTS: Of 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. CONCLUSIONS: Our 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.

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.

Influence of the F12 -4 C>T polymorphism on hemostatic tests.

The common F12 -4 C>T polymorphism significantly regulates plasma levels of FXII, the first element of the intrinsic pathway of coagulation. Due to the robust effects that this pathway has on blood coagulation in vitro, the objective of our study was to evaluate the influence of this polymorphism on different hemostatic tests. We studied 46 hemostatic parameters in 566 participants: 280 patients with mucocutaneous bleeding and 286 controls. The F12 -4T allele, associated with reduced levels of FXII (P < 0.001), also significantly delayed the activated partial thromboplastin time (aPTT) expressed as aPTTr (ratio sample plasma/normal pooled plasma). Thus, both patients and controls carrying the T allele had higher aPTTr than C/C homozygous individuals (P < 0.001). Interestingly, 92% of healthy controls who had prolonged aPTTr carried the F12 -4T allele. Moreover, individuals with the F12 -4T allele also had less thrombin generation (assessed by endogenous thrombin potential, thrombin peak and time to achieve the peak of thrombin) using a test with low tissue factor concentration and explicit contact phase activation. Finally, both patients and controls carrying the F12 -4T allele also displayed significantly lower FIXc and FVIIc levels than C/C individuals (P < 0.01). For all associations except for FVIIc, a gene-dosage effect was observed, and homozygous TT individuals had the farthest values. Our study reveals a significant effect of the F12 -4 C>T polymorphism on hemostatic tests widely used in routine clinical practice.

CALU A29809G polymorphism in coronary atherothrombosis: Implications for coronary calcification and prognosis.

INTRODUCTION: Arterial calcification is a risk factor for atherosclerosis. Calumenin (CALU), a protein regulating proteins involved in coagulation and arterial calcification also has extracellular functions related to atherosclerosis. We recently described that CALU polymorphism A29809G was related to acenocoumarol requirements, and we wanted to evaluate its role in arterial calcification and prognosis. PATIENTS AND METHODS: A total of 374 consecutive patients with non-ST-elevation acute coronary syndrome (nSTACS). In 175 of them, who underwent percutaneous coronary intervention, we assessed calcification in each main coronary artery. Follow-up at 1 and 6 months was performed for adverse end-points. RESULTS: CALU 29809G carriers were more frequent in the low calcium group (P = 0.037). The presence of >or=3 cardiovascular risk factors and CALU polymorphism were associated with arterial calcification (OR 2.34, P = 0.049; and OR 0.34, P = 0.019, respectively). CALU 29809G allele was the only variable associated with events at 1 month (HR 0.42; P = 0.042). Multivariate analysis showed that, at 6 months, age and severe anginal symptoms were associated with worse prognosis (HR 2.13, P = 0.023; and HR 2.01, P = 0.011, respectively), whereas CALU 29809G allele associated with good prognosis (HR 0.59, P = 0.044). Our results suggest that CALU A29809G is associated with arterial calcification and short-term prognosis of the outcome of patients with nSTACS.

Novel cassette-based shuttle vector system for gram-positive bacteria.

Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.