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Although there are a number of well-characterized genetic defects that lead to increased risk of thrombosis, little information is available on the relative importance of genetic factors in thrombosis risk in the general population. We performed a family-based study of the genetics of thrombosis in the Spanish population to assess the heritability of thrombosis and to identify the joint actions of genes on thrombosis risk and related quantitative hemostasis phenotypes. We examined 398 individuals in 21 extended pedigrees. Twelve pedigrees were ascertained through a proband with idiopathic thrombosis, and the remaining pedigrees were randomly ascertained. The heritability of thrombosis liability and the genetic correlations between thrombosis and each of the quantitative risk factors were estimated by means of a novel variance component method that used a multivariate threshold model. More than 60% of the variation in susceptibility to common thrombosis is attributable to genetic factors. Several quantitative risk factors exhibited significant genetic correlations with thrombosis, indicating that some of the genes that influence quantitative variation in these physiological correlates also influence the risk of thrombosis. Traits that exhibited significant genetic correlations with thrombosis included levels of several coagulation factors (factors VII, VIII, IX, XI, XII, and von Willebrand), tissue plasminogen activator, homocysteine, and the activated protein C ratio. This is the first study that quantifies the genetic component of susceptibility to common thrombosis. The high heritability of thrombosis risk and the significant genetic correlations between thrombosis and related risk factors suggest that the exploitation of correlated quantitative phenotypes will aid the search for susceptibility genes.

Lower levels of factor VIII and von Willebrand factor (vWF) have been reported in individuals with blood type O compared with individuals with other ABO blood types. However, this relationship has been demonstrated only by association studies and not by linkage studies. Also, it is not clear whether the ABO locus exerts a functional effect directly on these plasma factors or whether the ABO locus is in linkage disequilibrium with another locus that controls these factors. To distinguish between these 2 possibilities, we applied new statistical methods combining linkage and association tests in a pedigree-based sample. In contrast to most previous studies that used the ABO phenotypes, our study used the ABO genotypes, permitting us to distinguish AO from AA and BO from BB. Our results clearly showed significant linkage between the ABO locus and vWF antigen (P=0.00075). In addition, factor VIII coagulant activity and activated partial thromboplastin time showed suggestive linkage with the ABO locus (P=0.10 and P=0.13). All 3 plasma phenotypes showed significant differences between OO and non-OO genotypes. In addition, vWF antigen exhibited significant differences between O heterozygotes and non-OO homozygotes. This study is unique because it used a combined linkage and association test, which indicated that the ABO locus itself has a functional effect on these plasma phenotypes.

Association studies suggest that the G20210A mutation (G to A substitution at nucleotide position 20210) in the prothrombin gene (PT) is associated with increased plasma prothrombin activity and with increased risk for venous thromboembolism. To test directly for linkage between this PT variant and plasma prothrombin activity we performed a family-based study. The G20210A genotypes and plasma prothrombin activity levels were determined in 435 individuals belonging to 22 extended Spanish families. The sample was composed of 388 homozygous (G/G) normal individuals and 43 heterozygote (G/A) and 4 homozygote (A/A) carriers for the G20210A mutation. The results of variance-component linkage analysis yielded a highly significant lod score of 3.6 (P = 2.4 x 10(-5)) between this mutation and a quantitative trait locus (QTL) that influences prothrombin activity. Importantly, a conditional linkage analysis that simultaneously accounted for association with the G20210A variant completely eliminated the linkage signal, which indicates that this mutation affects the function of the prothrombin gene. Additionally, a bivariate linkage analysis of plasma prothrombin activity and thrombosis significantly improved the linkage signal for prothrombin activity (lod score = 4.7; P = 1.5 x 10(-6)) and provided strong evidence that this QTL has a pleiotropic effect on the risk of thrombosis (lod score = 2.43; P =.0004). These results represent the first direct genetic evidence that a QTL in the PT gene influences prothrombin activity levels and susceptibility to thrombosis and strongly support the conclusion that G20210A is a functional polymorphism. (Blood. 2000;95:2780-2785)

Transient pioneer neurons in the neocortical marginal zone generate an early corticofugal axonal projection at E12-E16 (Meyer et al. 1998). We have analysed the functional activity of glutamate and GABA receptors in such cells by measuring changes in intracellular calcium concentrations ([Ca2+]i). The activation of GABAA receptors with muscimol, as well as bath application of glutamate, lead to increases in [Ca2+]i in pioneer neurons. The stimulatory action of glutamate is mostly produced through the NMDA-type of ionotropic receptors. Metabotropic glutamate receptor activation has no effect on [Ca2+]i. Consistent with such results, immunocytochemical studies showed a prominent expression of GABAA and NMDA receptors in pioneer neurons. The activation of such receptors may be implicated in the remodelling of pioneer neurons during development.

Background and Objective: A new genetic risk factor for venous thromboembolism has recently been described which involves a G to A transition at position 20210 in the 3' untranslated region of the prothrombin gene. To date, only a few homozygotes for this mutation have been reported and in most of cases, they suffered from thrombotic disease. Here, we describe a pedigree including both heterozygous and homozygous subjects for prothrombin (PT) 20210 A., Design and Methods: This family was recruited in 1996 as part of our GAIT (Genetic Analysis of Idiopathic Thrombophilia) project. To qualify for the GAIT study, a pedigree was required to have at least 10 living individuals in three or more generations (i.e. extended pedigree). The pedigrees were selected through probands with idiopathic thrombophilia. A complete set of plasma and DNA determinations related to hemostasis was performed on this family., Results: The plasma studies yielded normal results in all of the individuals. The family members who had a history of thromboembolism were heterozygous carriers of the PT 20210 A variant. In addition, 4 relatives who were heterozygous, and two who were homozygous for this A allele, failed to show clinical manifestations. These two homozygotes were 51 and 19 years old., Interpretation and Conclusions: This case exemplifies the complexity of thrombotic disease since individuals homozygous for a mutant gene do not exhibit symptoms while heterozygous individuals often do exhibit the disease. This case suggests that the new genetic risk factor for thrombosis (i.e. PT 20210 A) may not be as strong as most of the previously described genetic risk factors.

Two major classes of early-born neurons are distinguished during early corticogenesis in the rat. The first class is formed by the cortical pioneer neurons, which are born in the ventricular neuroepithelium all over the cortical primordium. They appear at embryonic day (E) 11.5 in the lateral aspect of the telencephalic vesicle and cover its whole surface on E12. These cells, which show intense immunoreactivity for calbindin and calretinin, are characterized by their large size and axonal projection. They remain in the marginal zone after the formation of the cortical plate; they project first into the ventricular zone, and then into the subplate and the internal capsule. Therefore, these cells are the origin of the earliest efferent pathway of the developing cortex. Pioneer neurons are only present in prenatal brains. The second class is formed by subpial granule neurons, which form the subpial granular layer (SGL), previously considered to be found exclusively in the human cortex. SGL neurons are smaller than pioneer neurons. They are generated in a transient compartment of the retrobulbar ventricle between E12 and E14, and we propose the hypothesis that they invade the marginal zone, through tangential subpial migration, at different moments of fetal life. SGL neurons contain calbindin, calretinin, and gamma-aminobutyric acid (GABA), but the GABA-immunoreactive group becomes inconspicuous before birth. The extracellular matrix-like glycoprotein reelin, a molecule crucial for cortical lamination, is prenatally expressed by SGL neurons; postnatally, it is present in both Cajal-Retzius cells and subpial pyriform cells, both derivatives of SGL cells. In the rat, Cajal-Retzius cells are horizontal neurons that remain only until the end of the first postnatal week. They are located in layer I at a critical distance of approximately 20 microm from the pial surface and express reelin and, only occasionally, calretinin. Subpial pyriform cells coexpress reelin and calretinin and remain in layer I longer than Cajal-Retzius cells. Both pioneer neurons and subpial granule neurons are specific to the cortex. They mark the limit between the rudimentary cerebral cortex and olfactory bulb in the rat during early corticogenesis.

Nonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through "allelic exclusion" at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3' to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons.

We report the results of protein C gene (PROC) analysis in a Spanish family with hereditary PC deficiency characterized by the presence of three siblings with PC anticoagulant activity levels clearly below 50% of normal and PC antigen and amidolytic activities between 50 and 75% of normal. Their parents are first cousins and have PC levels between 50 and 80% of normal. Sequence analysis of the whole coding sequence of the PROC gene revealed that the three siblings are double homozygotes for a G to A transition at nucleotide 3203 that replaces arginine 87 by histidine (R87H) and for another G to A transition at nucleotide 7054, in intron 7 (7054G --> A). Both parents and one sister were found to be double heterozygotes for these two mutations. Screening for the intronic mutation in a control group and RT-PCR cDNA studies from ectopically transcribed mRNA indicated that 7054G --> A is most likely a rare but neutral DNA variant. These results and the fact that heterozygosity for the missense R87H mutation has also been found associated with a slightly decreased PC anticoagulant activity in another Spanish family, lead us to conclude that homozygosity for R87H is responsible for the PC deficient phenotype in these three siblings.

We report the genetic abnormalities in the protein C genes of a Spanish child with neonatal purpura fulminans and disseminated intravascular coagulation, associated with undetectable protein C levels. Direct sequencing of the nine protein C gene exons and their splice junctions indicated that the proband is a compound heterozygote with two mutant protein C gene alleles, Y124C and Q132X, that do not express protein C in plasma. The Y124C mutation was inherited from the mother and is due to a novel A to G transition at nucleotide 3416, which results in the substitution of cysteine for tyrosine 124, a highly conserved amino acid in EGF-like domains. The paternal inherited mutation (Q132X) is a C to T transition at nucleotide 3439, which replaces glutamine 132 with a Stop codon signal. This mutation, if expressed, should result in the synthesis of a truncated protein of 131 amino acids. Y124C or Q132X are present in the heterozygous state in the asymptomatic parents and siblings of the proband, all of which have half the normal plasma levels of protein C. Q123X has also been identified in families where type I PC deficiency is inherited as a clinically dominant trait. Therefore, the presence of the same mutation in a family showing a clinically recessive pattern of inheritance indicates that other factors, apart from the type of protein C gene mutation, are responsible for the clinical expression of protein C deficiency.

A novel polymorphism (6376 G/T) in intron 7 (17) of the human PROC gene has been identified by direct DNA sequencing. Restriction analysis with the use of mutagenic primers indicate that the allele frequencies are 0.17 (allele T) and 0.83 (allele G), with a calculated heterozygosity of 28%.

Single strand conformation polymorphism (SSCP) analysis of exon 7 of the protein C gene has identified a novel splice site missense mutation (184, Q-->H), in a newborn child with purpura fulminans and undetectable protein C levels. The mutations, seen in the homozygous state in the child and in the heterozygous state in her mother, was characterized and found to be a G to C nucleotide substitution at the -1 position of the donor splice site of intron 7 of the protein C gene, which changes histidine 184 for glutamine (184, Q-->H). According to analysis of the normal and mutated sequences, this mutation should also abolish the function of the donor splice site of intron 7 of the protein C gene. Since such a mutation is compatible with the absence of gene product in plasma and since DNA sequencing of all protein C gene exons in this patient did not reveal any other mutation, we postulate that mutation 184, Q-->H results in the absence of protein C gene product in plasma, which could be the cause of the severe phenotype observed in this patient.

An acceptor splice-site mutation (3318, A-->G) in the invariant AG of intron 5 of the human protein C gene has been identified in a Spanish family with heterozygous type I protein C (PC) deficiency and thromboembolic disease. Family studies confirmed cosegregation of the mutation with type I PC deficiency. Computer analysis of the mutated sequence predicted the normal splicing site to be abolished by this mutation, whereas a cryptic splice site located two nucleotides downstream, in exon 6, is probably activated. According to this, 3318, A-->G should result in a frameshift with a stop at codon 119, in agreement with the presence of a type I or quantitative PC deficient phenotype in the affected members of the family.

Background: In order to find alternatives for the diagnosis of hereditary protein C (PC) deficiency, we have studied the diagnostic informativity of the restriction fragment length polymorphism (RFLP) MI, located 7 kb upstream of the PC gene and detected with the restriction enzyme MspI., Methods: The RFLP MI has been analysed in 77 individuals belonging to 27 families with congenital PC deficiency, as well as in a control group of 46 healthy donors. The analysis has been performed by PCR amplification and MspI digestion of the polymorphic DNA fragment., Results: The allelic frequencies of the RFLP MI in the population studied are 0.69 for the allele A1, without the MspI restriction site, and 0.31 for the allele A2, with the MspI site. No differences have been found between the control and the PC deficient groups. The informativity of the polymorphism has been calculated to be 33.8%. Consegregation studies between this RFLP and PC deficiency have allowed the determination of the allele associated to the polymorphism in 21 out of the 27 studied families. Furthermore, an asymptomatic PC deficient carrier, with normal PC levels, has been identified., Conclusions: The study of this RFLP in families with hereditary PC deficiency may be useful for the identification of PC deficient carriers as well as for the prenatal diagnosis of the deficiency.

We have applied single-strand conformation polymorphism (SSCP) to the analysis of exon 7 of the anticoagulant protein C (PC) gene, in 13 PC-deficient Spanish families. Abnormal patterns were visualized in three samples from type I or quantitative PC deficient proposita. A previously undescribed mutation due to a TT insertion after nucleotide 6139, between codons Gly-142 and Arg-143 was found in one family. The mutation (6139,ins TT) should result in a frameshift with a stop at codon 156, which agrees with the presence of a type I or quantitative PC deficiency in the affected members of the family. The second mutation identified was a C to T transition at nucleotide 6274, 9 base pairs into intron G. This mutation (6274,C-->T), found for the first time in a Spanish family, is identical to the previously characterized PC Sant Louis. The third mutation was a G to A transition that replaces arginine 178 with glutamine (178,R-->Q). This is the third case of 178,R-->Q mutation in 17 apparently unrelated Spanish families with type I PC deficiency. Furthermore, SSCP analysis allowed the detection of another previously described mutation in a PC-deficient Spanish family (178,R-->W).