Exome analysis and functional classification of identified variants in racing Quarter Horses.

The main objectives of this study were to identify and functionally classify SNPs and indels by exome sequencing of animals of the racing line of Quarter Horses. Based on the individual genomic estimated breeding values (GEBVs) for maximum speed index (SImax) obtained for 349 animals, two groups of 20 extreme animals were formed. Of these individuals, 20 animals with high GEBVs for SImax and 19 with low GEBVs for SImax had their exons and 5' and 3' UTRs sequenced. Considering SNPs and indels, 105 182 variants were identified in the expressed regions of the Quarter Horse genome. Of these, 72 166 variants were already known and 33 016 are new variants and were deposited in a database. The analysis of the set of gene variants significantly related (Padjusted  < 0.05) to extreme animals in conjunction with the predicted impact of the changes and the physiological role of protein product pointed to two candidate genes potentially related to racing performance: SLC3A1 on ECA15 and CCN6 on ECA10.

[Spinal cord infarction and recurrent venous thrombosis in association with estrogens and the 20210A allele of the prothrombin gene]. / Mielopatía aguda isquémica y trombosis venosa recurrente asociada a estrógenos y alelo 20210A del gen de la protrombina.

The acute spinal cord infarction is a rare cause of acute-onset paraplegia. Furthermore, it is specially uncommon that the infarction occurs in patients with apparent low predisposition to vascular disease. The 20210A allele of the prothrombin gene (causing a threefold-increased risk in venous thromboembolism) was recently associated with unexplained spinal cord infarction in young women under treatment with estrogens (contraceptive pill). We report a case of anterior spinal artery syndrome resulting from an ischaemic infarction at the anterior aspect of the spinal cord in a healthy 50-year-old woman, carrying this mutation, being the first published case under treatment with transdermal estradiol. She referred the typical sudden-onset back pain associated to clinical anterior spinal artery syndrome with sphincter dysfunction and nontraumatic paraplegia. A possible multiple sclerosis was ruled out and the steroids or immunoglobulin therapy induced no clinical improvement. Cerebrospinal fluid and other investigations were all negative. Sequential MRI scans revealed development of spinal cord infarction from T10 to T11, with increased signal in T2-weighted image (T2). Because she referred a previous thrombophlebitis and suffered a deep-vein thrombosis one month after paraplegia, a complete coagulation study was performed. Antithrombin, proteins C and S, homocysteine, factor V Leiden, lupus anticoagulant and anticardiolipin antibodies were all normal or negatives. In opposite, the 20210A variation was positive (heterozygous) and the factor VIIIc level was very high (280 U/dl eight months later). We argue the relative importance of both findings. The patient had no a substantial recovery over a period of 20 months.Certainly, the prothrombin 20210A seems to be associated with unexplained ischemic myelopathy among the young women with estrogens.

Organization of the glycoprotein (GP) IIb/IIIa heterodimer on resting human platelets studied by flow cytometric energy transfer.

Glycoprotein IIb/IIIa is a heterodimer of glycoproteins IIb and IIIa which serves as the inducible receptor for fibrinogen and other adhesive proteins at the surface of platelets. Although a model of the quaternary structure of the GPIIb/IIIa molecule has been constructed in solution by Calvete et al. [Biochem. J. 282 (1992) 523], a corresponding model at the surface of intact platelets is still missing. In the present work conformation and lateral distribution of the GPIIb/IIIa heterodimer were studied at a nanometer resolution on the surface of resting human platelets under physiological conditions. The experiments were based on dual wavelength flow cytometric detection of fluorescence resonance energy transfer and application of a panel of monoclonal antibodies raised against well described binding sites. Monodisperse distribution of the GPIIb/IIIa heterodimer has been observed and a detailed three-dimensional proximity map of antibody binding sites was constructed on the platelet membrane, under physiological conditions, for the first time. Our data support the view that the GPIIb subunit is in a bent conformation. A detailed analysis of the K(d)-values and the number of binding sites for a set of monoclonal antibodies was also carried out giving supplementary data for the topology of the binding sites. Our results provide a refinement of the membrane-topology of the GPIIb/IIIa heterodimer.

Normal frequencies of the C677T genotypes on the methylenetetrahydrofolate reductase (MTHFR) gene among lymphoproliferative disorders but not in multiple myeloma.

The folate availability seems to be critical for the DNA integrity since it is required for the transfer of methyl groups in the biosynthesis of thymidilate. Although the excessive incorporation of uracils to the DNA can be efficiently removed, this mechanism of reparation produces many double-strand breaks from two opposing nicks. Several chromosomal abnormalities (mainly translocations and deletions perhaps not well understood) are involved in the origin of lymphoproliferative disorders. The TT homozygosity at nucleotide 677 in the gene of methylene tetrahydrofolatereductase (MTHFR), a key enzyme in folate metabolism, was recently linked to a significant protection against colon carcinoma and acute lymphoblastic leukaemia in adults. We analysed the genotype frequencies of C677T-MTHFR in a group of 143 patients with lymphoproliferative disorders (REAL classification) and 200 controls. Overally, the frequencies of the polymorphic allele were similar (35.3% and 32.0% respectively)(P=0.6). We did not find differences between patients and controls except for myeloma/plasmacytoma group (n=26) which showed a CC genotype less than expected (19% vs 46%) (p=0.01) with a frequency ratio of 0.28 (0.10-0.77). Even among the IgG myeloma cases only one patient showed a common genotype (CC) (1/15, 7%) (P=0.003). If these preliminary data are validated with prospective studies, the 677C allele of MTHFR gene could be confirmed as an effective multiple myeloma protective factor (specially for the IgG cases).

Increased expression of vascular endothelial growth factor in pyogenic granulomas.

The expression of vascular endothelial growth factor (VEGF) was analysed in biopsy samples from patients with pyogenic granuloma. The results disclosed the presence of a strong VEGF signal in pyogenic granulomas, which are constituted by a vast majority of cells of endothelial lineage. A marked positivity was evident in areas of proliferating endothelial cells without vessel lumen formation. In the same respect, staining for VEGF was less marked in the vessels with a well-developed lumen. The fact that VEGF production appears to be limited to endothelial cell precursors or immature endothelial cells prior to the complete development of the vessels, leads to the possibility that VEGF may act as an autocrine factor in circumstances of endothelial cell stimulation.

Molecular genetic analysis of a compound heterozygote for the glycoprotein (GP) IIb gene associated with Glanzmann's thrombasthenia: disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIa complexes.

This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann's thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)-single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient's GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C-->A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C-->A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband's GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113-->C transition that changes Cys674-->Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.

Cardiac troponin I (cTn I) and the postmortem diagnosis of myocardial infarction.

In clinical practice several biochemical markers are used for the diagnosis of myocardial infarction. Because of its extremely high specificity for myocardial damage, cardiac troponin I (cTn I) is frequently used. The aim of this study was to evaluate the diagnostic efficacy of postmortem cTn I determinations in pericardial fluid and serum and to compare these results with other biochemical markers and with structural findings used to diagnose acute myocardial ischaemia. We studied 89 cadavers with a mean age of 51.38 +/- 2.04 (SD 19.27 years). Cases were allocated to 1 of 4 diagnostic groups depending on the probable intensity of myocardial damage and cause of death. In pericardial fluid we obtained statistically significant differences for the four biochemical parameters, while in serum myosin heavy chains and myoglobin showed statistically significant differences. The highest levels of biochemical markers in pericardial fluid were observed in subjects who had died from definite myocardial infarction.

Functional characterization of GPIIb- and GPIIIa-specific monoclonal antibodies. Further evidence for the existence of agonist-specific activated states of the platelet fibrinogen receptor.

In this work human platelet aggregation induced in vitro by ADP, collagen, arachidonic acid and U-46619 (a thromboxane A(2) analogue) was used as a functional test to characterize 19 anti-GPIIb (M series) and anti2 GPIIIa (P series) monoclonal antibodies whose epitope location is known for most of them. Additionally, flow cytofluorimetry was applied to study the epitope expression of these antibodies in resting, EDTA-treated and SFLLRN peptide (thrombin receptor agonist)-activated platelets. Antibodies M6 (epitope located at GPIIbH 657-665), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-303) bind weakly to only 43%, 70% and 66%, respectively, of the resting platelet population. This binding was enhanced in EDTA-treated and in activated platelets. Platelet activation enhances the apparent binding of most of the other antibodies. Further evidence on the existence of agonist-specific activated states of GPIIb/IIIa was provided by the agonist-dependent immunochemical inhibition in vitro of platelet aggregation by some of the anti-subunit antibodies studied here. The most notable cases are those of P40 and M6, which at 140 nM inhibit most, the platelet aggregation induced by arachidonic acid and U-46619. On the other hand, three of the most strong and agonist-independent inhibitors, P37 (GPIIIa 101-109), P97 and P95-2 (GPIIIa N-terminal half) bind to resting platelets with high affinity (5-8 nM), compete with each other for binding to GPIIb-IIIa and their epitopes are located at the N-terminal domain of GPIIIa, where the receptor ligand binding site(s) have been found. Given that the formation of activated GPIIb-IIIa (GPIIb-IIIa*) is the first step at which the anti-subunit antibodies can intervene as inhibitors and that agonist-specific inhibitors should block only agonist-specific steps, while nonspecific inhibitors should block steps common to all the agonists, then our present work support the hypothesis that there are different agonist-specific GPIIb-IIIa*s or, alternatively, different receptor environments, that can be specifically blocked by some of the antibodies. These results add to earlier evidence on agonist-dependent ligand specificity and activated states found for this and other integrins. Finally, the correlation between the in vitro inhibition of platelet aggregation and the antithrombotic activity in vivo is discussed for these antibodies.

HLA-B27 polymorphism and worldwide susceptibility to ankylosing spondylitis.

HLA-B27 is strongly associated to ankylosing spondylitis (AS) and represents a family of eleven B27 alleles (B*2701-11). Our aim was to analyze the distribution of B27 subtypes by PCR/SSOP and genomic sequencing in a large group of populations (n = 17). 711 B27-positive samples from Caucasoid, Asian, African, Amerindian and Polynesian populations were selected to ascertain transracial gene mapping of the B27 subtypes. 476 of these were AS patients, chosen to investigate the contribution of B27 alleles to AS susceptibility. Some significant new findings have arisen from this study: 1) B*2705 was the predominant subtype in circumpolar and subarctic areas. B*2702 was found to be practically restricted to Caucasian populations, showing a higher frequency in Middle-East (Jews) and North Africa (Arabs/Berbers) groups. 2) B*2703 appears associated with AS in Western Africans. This is of remarkable interest since it was suggested that B*2703 would be negatively disease-associated. 3) Although B*2706 appears negatively associated with AS in Thais, we identified two patients from northern China carrying it. This may be a reflection of a disease heterogeneity and could indicate that more than one pathogenic agent can be involved in AS. B*2709 has been recently described as negatively associated with AS in Sardinians. The molecular changes His114Asp (B*2706) and Asp116His (B*2709) could modify the genetic susceptibility to AS.

Inhibition of the intimal hyperplasia in an arterial autograft model by blockade of the N-terminal of the integrin beta3 subunit by monoclonal antibody P37.

Myointima formation or intimal hyperplasia is a major undesirable problem at the anastomotic ends of narrow bore arterial autografts and in other arterial wall injuries, which often leads to late restenosis and thrombosis and whose pathogenesis is still not understood. Platelets are suspected to intervene at some stages of its development, together with endothelial and muscle cells, the extracellular matrix and, most probably, adhesion receptors. To ascertain whether and at what stage beta3 integrins are involved, a rat arterial autograft model was used, together with monoclonal antibody P37, which is directed to the sequence 101-109 of the beta3 subunit of the human platelet fibrinogen receptor (integrin alphaIIbbeta3) and inhibits platelet aggregation in vitro and acute thrombosis in vivo. Three groups of animals were used: group I underwent an arterial autograft of a 5-mm segment of the right common iliac artery; group II received, intravenously, a single dose (0.8 mg/kg) of P37 at 15 min before the graft implantation; and group III was treated as group II but a similar dose of antibody was additionally given on day 14 after the operation. Animals in each group were sacrificed on days 7, 14, 21, 30 and 50 after the operation, and the grafts were removed for light and electron microscopy observation and further time-dependent morphometric analysis. By day 14, group I autografts already showed intimal hyperplasia and secretory smooth muscle cells, while group II and II autografts presented only some degenerative changes in the medial layer, with no signs of hyperplasia. Intimal hyperplasia was observed on day 21 in group II and on day 30 in group III, although less pronounced than in the corresponding controls. However, by day 50, the three groups had the same thickness of myointima. The immunohistochemical determination of metalloproteases suggests no role for these enzymes in the immunoinhibition of myointima formation. We conclude that P37 inhibits the onset of the intimal hyperplasia in the arterial autografts and that this onset in treated animals seems to be related to the decay of the circulating antibody. Further work is required to decide whether a higher or longer presence of circulating P37 can definitively prevent the development of intimal hyperplasia, as well as to ascertain which cells and which beta3 integrin receptors intervene.

Antithrombotic map of the N-terminal domain of the GPIIIa subunit of the human platelet fibrinogen receptor (GPIIb-IIIa) determined in vivo by monoclonal, immunochemical inhibition of acute arterial thrombosis.

The inhibition of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa (GPIIb-IIIa) or integrin alphaIIbbeta3, has recently became an accepted practice in clinical cardiology. The interest lies now in the improvement of the antithrombotic activity and the minimization of the secondary effects of the receptor inhibitors, by their evaluation in vivo in the different dynamic conditions and pathological states under which these inhibitors have to perform. In this paper, we functionally map in vivo the N-terminal domain of the GPIIIa subunit, using the antithrombotic activity of five murine monoclonal antibodies (mabs) (P37, P40, 95-1, P95-2 and P97), all of them inhibitors of platelet aggregation in vitro and directed to this ligand binding domain of the human fibrinogen receptor. Competition experiments have shown that these mabs bind with high affinity (5-7 nM) and compete very strongly among themselves for binding to human resting platelets, except P40, which neither binds nor competes. These antibodies were assayed in a dog model of acute thrombosis in the carotid artery, which were induced 15 min after their intravenous administration (0.8 mg/kg). The antithrombotic activity was quantified by the measurement of the [111In]oxine-labelled platelet deposition at the site of the arterial lesion and was expressed as the percentage of the total circulating platelets. Antibody P37, directed to the GPIIIa 101-109 sequence, decreased the platelet deposition 630-fold with respect to control animals. P95-2, P97 and P95-1 decreased the platelet deposition 160-, 32- and 25-fold, respectively, while P40, directed to the GPIIIa 260-302 sequence, did not show any antithrombotic activity. We conclude that all the mabs directed to the N-terminal domain of GPIIIa, which inhibit platelet aggregation in vitro and whose epitopes are very close to each other and exposed in resting platelets, have high antithrombotic activity in vivo, which varies depending on the actual location of the epitopes in the receptor topography. Among these antibodies, P37, the strongest receptor inhibitor in vivo and whose epitope is most probably the closest to the fibrinogen binding site(s), seems the best candidate for comparative studies in animal models with today's best GPIIb-IIIa inhibitors and for clinical trials in humans in order to arrest or prevent thrombosis, reocclusion and late restenosis.

[Cesarean section in a patient with a congenital deficit of antithrombin III: apropos of a case]. / Cesárea en una paciente con déficit congénito de antitrombina III: a propósito de un case.

Antithrombin III (AT III) is a physiological inhibitor of coagulation. AT III deficit, whether congenital or acquired, results in a state of hypercoagulability characterized by recurring instances of venous thrombosis in young people. Although AT III levels normally change little during pregnancy, a deficit can be associated to the appearance of recurring thromboembolism and the need to perform cesarean section increases the risk. We report a cesarean section under general anesthesia in a patient with congenital AT III, reviewing the etiology and pathophysiology of this entity as well as its treatment.

Twofold symmetry of human fibrinogen proved at the beta chain distal domains by monoclonal-immunoelectron microscopy and image analysis.

Using a murine antibody (F7) specific for the C-terminal domain of the beta chain of human fibrinogen combined with electron microscopy and image analysis, we show unequivocally that the epitopes for F7 are at the distal nodules of fibrinogen, equidistant from the center of the molecule and arranged not colinearly with the long axis of the molecule but at opposite sides of it, i.e., following twofold symmetry. Thus, given the monoclonality of the immunochemical probe used and the dimeric nature of the fibrinogen molecule, we can conclude that the distal domains of the two beta chains are arranged in the same manner as these epitopes and, therefore, that the fibrinogen molecule has twofold symmetry. This symmetry pattern found here for F7 is the same as that found recently for the platelet fibrinogen receptor binding sites [Weisel, J. W., Nagaswami, C., Vilaire, G., & Bennett, J. S. (1992) J. Biol. Chem. 23, 16637-16643], located almost certainly at the C-terminal end of the gamma chains, and gives further support to the most accurate model of fibrinogen available so far. We discuss the consequences of this symmetry pattern and of the molecular rigidity of fibrinogen in the actual models of fibrin polymerization and platelet aggregation and adhesion.