Alfonso Buil

Protein C Levels Are Regulated by a Quantitative Trait Locus on Chromosome 16: Results from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project

Objective—Protein C (PC) is a component of the protein C anticoagulant pathway. PC deficiency is a risk factor associated with venous thromboembolism. As part of the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project, we conducted a genome-wide linkage scan to localize genes that influence variation in PC plasma levels. Methods and Results—PC levels were measured in 398 individuals belonging to 21 Spanish families. A total of 485 DNA microsatellite markers were genotyped to provide a 7.1-cM genetic map. Variance component linkage methods were used to evaluate linkage and to detect quantitative trait loci (QTL). A region on chromosome 16 (16q23), flanked by markers D16S3106 and D16S516, showed strong evidence of linkage with PC levels (LOD=3.69). This region contains 1 positional candidate gene, the NAD(P)H:dehydrogenase quinone 1 (NQO1), involved in vitamin K metabolism. The association of 1 SNP of this gene with PC levels (P =0.005) strongly supports the implication of NQO1 gene in the variability of PC levels. Conclusions—These results illustrate the application of genomic scans to identify the genetic determinants of quantitative variation in a component of the hemostatic pathways. They provide strong evidence for a locus (QTL) on chromosome 16 that influences PC levels.

A Genome Search for Genetic Determinants That Influence Plasma Fibrinogen Levels

Background—Fibrinogen levels are a widely accepted risk factor for cardiovascular disease, but the extent of the genetic component is unknown. Materials and Results—To search for these genes, we conducted a genome-wide scan using 21 Spanish families from the Genetic Analysis of Idiopathic Thrombophila (GAIT) Project. Two loci were detected: 1 on chromosome 12 and another on chromosome 14. There are no cardiovascular-related candidate genes on chromosome 14, which implies that this locus represents a novel cardiovascular risk factor. Importantly, the locus on chromosome 12 contains the hepatocyte nuclear factors (TCF1), a candidate gene involved in the hepatocyte-specific transcription of the fibrinogen α-chain and β-chain genes. Three polymorphisms in TCF1 showed significant association with fibrinogen levels, supporting the implication of TCF1 in the determination of this phenotype. Conclusions—Two loci, 1 on chromosome 12 (most likely the TCF1) and another on chromosome 14, are important determinants of fibrinogen levels in Spanish families. These data should help define the relationship between fibrinogen levels and the risk of cardiovascular disease.

Genome-wide Linkage Analysis for Identifying Quantitative Trait Loci Involved in the Regulation of Lipoprotein a (Lpa) Levels

Lipoprotein Lp(a) levels are highly heritable and are associated with cardiovascular risk. We performed a genome-wide linkage analysis to delineate the genomic regions that influence the concentration of Lp(a) in families from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. Lp(a) levels were measured in 387 individuals belonging to 21 extended Spanish families. A total of 485 DNA microsatellite markers were genotyped to provide a 7.1u2009cM genetic map. The variance component linkage method was used to evaluate linkage and to detect quantitative trait loci (QTLs). The main QTL that showed strong evidence of linkage with Lp(a) levels was located at the structural gene for apo(a) on chromosome 6 (LOD score=13.8). Interestingly, another QTL influencing Lp(a) concentration was located on chromosome 2 with an LOD score of 2.01. This region contains several candidate genes. One of them is the tissue factor pathway inhibitor (TFPI), which has antithrombotic action and also has the ability to bind lipoproteins. However, quantitative trait association analyses performed with 12 SNPs in TFPI gene revealed no association with Lp(a) levels. Our study confirms previous results on the genetic basis of Lp(a) levels. In addition, we report a new QTL on chromosome 2 involved in the quantitative variation of Lp(a). These data should serve as the basis for further detection of candidate genes and to elucidate the relationship between the concentration of Lp(a) and cardiovascular risk.

A locus on chromosome 2 influences levels of tissue factor pathway inhibitor: results from the GAIT study.

Objective—Levels of tissue factor pathway inhibitor (TFPI) have been associated with arteriosclerosis and thrombotic disease. Although a genetic component to variation in TFPI levels is well-documented, no systematic genome-wide screens have been conducted to localize genes influencing levels of TFPI. Methods and Results—We studied TFPI levels in 397 individuals in 21 Spanish families participating in the Genetic Analysis of Idiopathic Thrombosis (GAIT) study. Twelve families were selected through a proband with idiopathic thrombosis and 9 were ascertained without regard to phenotype. A genome scan was performed using microsatellite markers spaced at approximately 10 cM intervals. Standard multipoint variance component linkage methods were used. The heritability of TFPI levels was 0.52 (P<0.0001), with no evidence for shared household effects. In the genome screen, only 1 LOD score >2 was observed. On chromosome 2q, the maximum multipoint LOD score was 3.52 near marker D2S1384. This is near the structural gene for TFPI, which is located at 2q32. In follow-up association analyses, marginal evidence of association (P=0.04) was observed with the TFPI promoter variant C-399T. Conclusion—These results suggest that polymorphisms in and around the TFPI structural gene may be the major genetic determinants of variation in TFPI levels.

Genetic determinants of normal variation in coagulation factor (F) IX levels: Genome-wide scan and examination of the FIX structural gene

Summary.u2002 Background:u2002High‐normal and elevated plasma FIX activity (FIX:C) levels are associated with increased risk for venous‐ and possibly arterial‐thrombosis. Objective:u2002Because the broad normal range for FIX:C involves a substantial unknown genetic component, we sought to identify quantitative‐trait loci (QTLs) for this medically important hemostasis trait. Methods:u2002We performed a genome‐wide screen and a resequencing‐based variation scan of the known functional regions of every distinct FIX gene (F9) in the genetic analysis of idiopathic thrombophilia project (GAIT), a collection of 398 Spanish‐Caucasians from 21 pedigrees. Results:u2002We found no evidence for linkage (LOD scores <1.5) despite genotyping more than 540 uniformly‐spaced microsatellites. We identified 27 candidate F9 polymorphisms, including three in cis‐elements responsible for the increase in FIX:C that occurs with aging, but found no significant genotype‐specific differences in mean FIX:C levels (P‐valuesu2003≥u20030.11) despite evaluating every polymorphism in GAIT by marginal multicovariate measured‐genotype association analysis. Conclusions:u2002The heritable component of interindividual FIX:C variability likely involves a collection of QTLs with modest effects that may reside in genes other than F9. Nevertheless, because the alleles of these 27 polymorphisms exhibited a low overall degree of linkage disequilibrium, we are currently defining their haplotypes to interrogate several highly‐conserved non‐exonic sequences and other F9 segments not examined here.

Genomewide linkage analysis of soluble transferrin receptor plasma levels

Genetic control of soluble transferrin receptor (sTfR) levels was demonstrated using family-based studies (GAIT, Genetic Analysis of Idiopathic Thrombophilia project); moreover, a genetic relationship was observed between sTfR and the risk for thrombosis, suggesting that these phenotypes shared genetic determinants. We studied the regions that control sTfR. To assess such regions, a full genome scan was carried out using 604 highly polymorphic deoxyribonucleic acid markers (resolution 7.3xa0cM) in 21 extended pedigrees (358 individuals). Then, a quantitative trait linkage analysis was performed using variance components methods. The genomewide scan linkage analysis showed two regions (quantitative trait locus or QTL) with significant limit of detection (LOD) scores (2q23.14, LOD score=2.64, nominal p=0.00024; 3q21.2, LOD score=1.94, nominal p=0.0014). There were no obvious candidate genes in these regions. In conclusion, this linkage analysis suggested the existence of a QTL in 2q23.14 that probably harbored a gene (or genes) controlling sTfR levels. Moreover, a second linkage signal was observed in 3q21.2; albeit the evidence for this second locus was lower. The next step will be to identify the gene(s) and its possible involvement in thrombosis and iron homeostasis.

A genomewide study of body mass index and its genetic correlation with thromboembolic risk. Results from the GAIT project.

Thrombosis and obesity are complex epidemiologically associated diseases. The mechanism of this association is not yet understood. It was the objective of this study to identify genetic components of body mass index (BMI) and their possible role in the risk of thromboembolic disease. With the self-reported BMI of 397 individuals from 21 extended families enrolled in the GAIT (Genetic Analysis of Idiopathic Thrombophilia) Project, we estimated the heritability of BMI and the genetic correlation with the risk of thrombosis. Subjects were genotyped for an autosomal genome-wide scan with 363 highly-informative DNA markers. Univariate and bivariate multipoint linkage analyses were performed. The heritability for BMI was 0.31 (p=2.9×10⁻⁵). Thromboembolic disease (including venous and arterial) and BMI had a significant genetic correlation (ρG=0.54, p=0.005). Two linkage signals for BMI were obtained, one at 13q34 (LOD=3.36, p=0.0004) and other at 2q34, highly suggestive of linkage (LOD=1.95). Bivariate linkage analysis with BMI and thrombosis risk also showed a significant signal at 13q34 (LOD=3), indicating that this locus influences at the same time normal variation in the BMI phenotype as well as susceptibility to thrombosis. In conclusion, BMI and thrombosis are genetically correlated. The locus 13q34, which showed pleiotropy with both phenotypes, contains two candidate genes, which may explain our linkage pleiotropic signal and deserve further investigation as possible risk factors for obesity and thrombosis.