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Results for “Haplotype structures”

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PubMed2004

Genotyping and haplotyping of the dopamine D4 receptor gene by capillary electrophoresis.

In this paper we report on simultaneous genotyping of adjacent polymorphisms (referred to as haplotyping) by combining double-tube allele-specific polymerase chain reaction, restriction fragment length polymorphism and capillary gel electrophoresis analysis of the resulting fragments. Direct molecular haplotyping is of particular importance in the case of double heterozygote samples, since in these instances the haplotype structure cannot be constructed based on genotype data. Our approach provided a powerful tool for coincidental genotype analysis of the 48 base pair (bp) variable number of tandem repeats of the third exon and haplotype investigation of the -616CG and -521CT single nucleotide polymorphisms of the dopamine D4 receptor (DRD4) gene. The linear polyacrylamide sieving matrix was optimized for the size range of the double-stranded DNA fragments of interest varying from 35 to 763 bp. We demonstrated that capillary gel electrophoresis in combination with laser induced fluorescence detection offers a sensitive and accurate tool for automated haplotyping in clinical settings.

Base Sequence↗

PubMed2006

Resampling-based multiple hypothesis testing procedures for genetic case-control association studies.

In case-control studies of unrelated subjects, gene-based hypothesis tests consider whether any tested feature in a candidate gene--single nucleotide polymorphisms (SNPs), haplotypes, or both--are associated with disease. Standard statistical tests are available that control the false-positive rate at the nominal level over all polymorphisms considered. However, more powerful tests can be constructed that use permutation resampling to account for correlations between polymorphisms and test statistics. A key question is whether the gain in power is large enough to justify the computational burden. We compared the computationally simple Simes Global Test to the min P test, which considers the permutation distribution of the minimum p-value from marginal tests of each SNP. In simulation studies incorporating empirical haplotype structures in 15 genes, the min P test controlled the type I error, and was modestly more powerful than the Simes test, by 2.1 percentage points on average. When disease susceptibility was conferred by a haplotype, the min P test sometimes, but not always, under-performed haplotype analysis. A resampling-based omnibus test combining the min P and haplotype frequency test controlled the type I error, and closely tracked the more powerful of the two component tests. This test achieved consistent gains in power (5.7 percentage points on average), compared to a simple Bonferroni test of Simes and haplotype analysis. Using data from the Shanghai Biliary Tract Cancer Study, the advantages of the newly proposed omnibus test were apparent in a population-based study of bile duct cancer and polymorphisms in the prostaglandin-endoperoxide synthase 2 (PTGS2) gene.

Algorithms↗

PubMed2003

Measured haplotype analysis of the aldosterone synthase gene and heart size.

Gene-association studies of heart size and the aldosterone synthase (CYP11B2) gene have produced inconsistent results, possibly because of limitations in the sample size and/or the number and location of the polymorphisms. An analysis of six polymorphisms spanning 6 kb of the CYP11B2 gene in Caucasian British families revealed a limited number of haplotypes because of strong linkage disequilibrium over this small region. The genotype and haplotype information was used in an association study involving 955 members of 229 families phenotyped for echocardiographic measures of heart size. In a mixed effects linear modelling analysis, the G5937C polymorphism was associated with cardiac wall thickness (P=0.02), and the intron conversion and A4550C polymorphisms were associated with left ventricular cavity size (P=0.02 and 0.002, respectively). Measured haplotype analyses confirmed the association of alleles at the intron conversion and G5937C polymorphisms with cardiac wall thickness (P=0.02), and alleles at the intron conversion polymorphism with left ventricular cavity size (P=0.04). The polymorphisms contributed to 2.0-3.4% of the variability in these traits. In summary, genetic polymorphisms at the CYP11B2 gene make a small contribution to quantitative variation in echocardiographic measures of heart size. These results point to the importance of analysing the full extent of genetic variation that captures the haplotype structure of a locus in gene association studies.

Cytochrome P-450 CYP11B2↗

PubMed2005

A natural CYP2B6 TATA box polymorphism (-82T--> C) leading to enhanced transcription and relocation of the transcriptional start site.

We investigated the impact of promoter polymorphisms on transcription of the human CYP2B6 gene. In total, 98 DNA samples from white persons from a previously characterized liver bank were sequenced throughout 2.3 kilobases of upstream sequence and haplotype structures were determined using additional coding sequence information. HepG2 cells and primary rat and human hepatocytes were transfected with luciferase reporter gene constructs driven by 2033 base pairs (bp) of the most frequent promoter variants. The novel haplotype *22 (-1848C--> A, -801G--> T, -750T--> C, and -82T--> C) showed 3- to 9-fold enhanced transcriptional activity in all transfected cells. Constructs containing single mutations surprisingly revealed -82T--> C, predicted to disrupt a putative TATA box, to be alone responsible for this effect. In silico analysis and electrophoretic mobility shift assay demonstrated conversion of the putative TATA box into a functional CCAAT/enhancer-binding protein binding site. Analysis of transcriptional start sites showed the mutant promoter to be transcribed from a start site located approximately 30 bp downstream of the wild-type start site, consistent with the use of a noncanonical TATA box at -55 bp. Median CYP2B6 mRNA expression and bupropion hydroxylase activity as a selective marker of CYP2B6 catalytic activity were approximately 2-fold higher in livers genotyped -82TC as in those genotyped -82TT (20.4 versus 9.8 arbitrary units, p = 0.007, and 201.8 versus 106.7 pmol/mg/min, p = 0.042, respectively). This promoter polymorphism thus contributes to CYP2B6 functional variability and represents a novel mechanism by which mutations can enhance transcription. Furthermore, a detailed interspecies comparison of CYP2B promoters and transcriptional start sites provided novel insights into evolutionary relationships.

Animals↗

PubMed2005

Tumor necrosis factor promoter haplotype associated with schizophrenia reveals a linked locus on 1q44.

Using restriction fragment length polymorphism and pyrosequencing methods, we genotyped two TNFA gene promoter SNPs (-G308A, -G238A) and analyzed the haplotype structure in 24 Canadian families of primarily Celtic origin. Our results demonstrate that after correction for multiple testing based on simulations of 10 000 replicates of unlinked/unassociated data, there is evidence for association (P=0.026) of a specific haplotype (-308A, -238G) with schizophrenia and schizophrenia spectrum disorders with a family-based trimmed haplotype linkage disequilibrium test (Trimhap). Stratifying the 22 families with genome scan data by TNFA promoter haplotypes followed by reanalysis of linkage to schizophrenia throughout the genome, we identified few loci that exhibit a considerable increase in LOD/HLOD scores. A locus on chromosome 1q44 (D1S1609) demonstrated a significant increase (P=0.025) in LOD score from 0.15 to 3.01 with a broad definition of the schizophrenia phenotype and a dominant mode of inheritance. This result replicates a previously reported positive result of linkage of schizophrenia spectrum disorders to this area of the genome. We also illustrated that simulation studies are pivotal in evaluating the significance of results obtained with newer statistical methods, when multiple, but not independent, tests are performed, and when sample stratification is utilized to reduce the impact of heterogeneity or assess the interaction between loci.

Chromosomes, Human, Pair 1↗

PubMed2002

Entropy as a measure for linkage disequilibrium over multilocus haplotype blocks.

OBJECTIVE: The presence of linkage disequilibrium (LD) forms the basis for a range of uses, including the fine-mapping of diseases and studies on human genealogy. Recent findings indicate that single nucleotide polymorphisms (SNP) can occur in blocks of limited haplotypic diversity with high degrees of LD. Commonly used measures for LD, such as r(2) and D', consider only two loci and might miss information to appropriately describe LD in larger haplotypic structures. METHODS: We introduce the Normalized Entropy Difference, epsilon, as a new multilocus measure for LD. A related quantity, deltaS, provides an approximate chi(2) test for the significance of LD. The ability of the measure to detect haplotype blocks is investigated using simulated data sets as well as a real data set previously analyzed by Daly et al. (2001). RESULTS: epsilon allows for arbitrary numbers of loci, describes LD with regard to the loci sequence, and can be interpreted as a multilocus extension of r(2). The application of epsilon to the data sets demonstrated the measure's ability to appropriately describe simultaneous multilocus LD and to detect haplotype blocks. CONCLUSIONS: epsilon is a reasonable multilocus LD measure and might be of potential use in the construction of the human haplotype map.

Chromosome Mapping↗

PubMed2004

Haploview: analysis and visualization of LD and haplotype maps.

UNLABELLED: Research over the last few years has revealed significant haplotype structure in the human genome. The characterization of these patterns, particularly in the context of medical genetic association studies, is becoming a routine research activity. Haploview is a software package that provides computation of linkage disequilibrium statistics and population haplotype patterns from primary genotype data in a visually appealing and interactive interface. AVAILABILITY: http://www.broad.mit.edu/mpg/haploview/ CONTACT: jcbarret@broad.mit.edu

Algorithms↗

PubMed2006

SNP identification, linkage disequilibrium, and haplotype analysis for a 200-kb genomic region in a Korean population.

Understanding patterns of linkage disequilibrium (LD) across genomes may facilitate association mapping studies to localize genetic variants influencing complex diseases, a recognition that led to the International Haplotype Mapping Project (HapMap). Divergent patterns of haplotype frequency and LD across global populations require that the HapMap database be supplemented with haplotype and LD data from additional populations. We conducted a pilot study of the LD and haplotype structure of a genomic region in a Korean population. A total of 165 SNPs were identified in a 200-kb region of 22q13.2 by direct sequencing. Unphased genotype data were generated for 76 SNPs in 90 unrelated Korean individuals. LD, haplotype diversity, and recombination rates were assessed in this region and compared with the HapMap database. The pattern of LD and haplotype frequencies of Korean samples showed a high degree of similarity with Japanese data. There was a strong correlation between high LD and low recombination frequency in this region. We found considerable similarities in local LD patterns between three Asian populations (Han Chinese, Japanese, and Korean) and the CEPH population. Haplotype frequencies were, however, significantly different between them. Our results should further the understanding of distinctive Korean genomic features and assist in designing appropriate association studies.

Asian People↗

PubMed2004

In-depth haplotype analysis of ABCA1 gene polymorphisms in relation to plasma ApoA1 levels and myocardial infarction.

OBJECTIVE: By regulating the cellular cholesterol efflux from peripheral cells to high-density lipoprotein, the ABCA1 protein is suspected to play a key role in lipid homeostasis and atherosclerosis. Twenty-six polymorphisms of the ABCA1 gene were genotyped and tested for association with plasma levels of ApoA1 and myocardial infarction (MI) in the ECTIM study. METHODS AND RESULTS: In addition to single-locus analysis, a systematic exploration of all possible haplotype effects was performed, with this exploration being performed on a minimal set of "tag" polymorphisms that define the haplotype structure of the gene. Two polymorphisms were associated with plasma levels of ApoA1, 1 in the promoter (C-564T) and 1 in the coding (R1587K) regions, whereas only 1 polymorphism (R219K) was associated with the risk of MI. However, no haplotype effect was detected on ApoA1 variability or on the risk of MI. CONCLUSIONS: ABCA1 gene polymorphisms but not haplotypes are involved in the variability of plasma ApoA1 and the susceptibility to coronary artery disease.

ATP Binding Cassette Transporter 1↗

PubMed2025

Polaris: Polarization of ancestral and derived polymorphic alleles for inferences of extended haplotype homozygosity in human populations.

SUMMARY: Statistical methods that measure the extent of haplotype homozygosity on chromosomes have been highly informative for identifying episodes of recent selection. For example, the integrated haplotype score (iHS) and the extended haplotype homozygosity (EHH) statistics detect long-range haplotype structure around derived and ancestral alleles indicative of classic and soft selective sweeps, respectively. However, to our knowledge, there are currently no publicly available methods that classify ancestral and derived alleles in genomic datasets for the purpose of quantifying the extent of haplotype homozygosity. Here, we introduce the Polaris package, which polarizes chromosomal variants into ancestral and derived alleles and creates corresponding genetic maps for analysis by selscan and HaploSweep, two versatile haplotype-based programs that perform scans for selection. With the input files generated by Polaris, selscan and/or HaploSweep can produce the appropriate sign (either positive or negative) for outlier iHS statistics, enabling users to distinguish between selection on derived or ancestral alleles. In addition, Polaris can convert the numerical output of these analyses into graphical representations of selective sweeps, increasing the functionality of our software. RESULTS: To demonstrate the utility of our approach, we applied the Polaris package to Chromosome 2 in the European Finnish, Middle Eastern Bedouin, and East African Maasai populations. More specifically, we examined the regulatory sequence in intron 13 of the MCM6 gene associated with lactase persistence (i.e. the ability to digest the lactose sugar present in fresh milk), a region of intense interest to human evolutionary geneticists. Our analyses showed that derived alleles (at known enhancers for lactase expression) sit on an extended haplotype background in the Finnish, Bedouin, and Maasai consistent with a classic selective sweep model as determined by iHS and EHH statistics. Importantly, we were able to immediately identify this target allele under selection based on the information generated by our software. We also explored outlier statistics across Chromosome 2 in two distinct datasets from these populations: (i) one containing polarized alleles generated with Polaris and (ii) the other containing unpolarized alleles in the original phased vcf file. Here, we found an excess of outlier statistics on Chromosome 2 in the unpolarized datasets, raising the possibility that a subset of these "hits" of selection may be unreliable. Overall, Polaris is a versatile package that enables users to efficiently explore, interpret, and report signals of recent selection in genomic datasets. AVAILABILITY AND IMPLEMENTATION: The Polaris package is free and open source on GitHub (https://github.com/alisi1989/Polaris) and DropBox (https://www.dropbox.com/scl/fo/mlxizft5267vem9u62qkn/AAnM0qX923zPzQBlPX8iteM?rlkey=uezrp4t2waffpj0nmo1evr320&e=1&st=jaodccws&dl=0).

Haplotypes↗

PubMed2006

Mutation and evolutionary analyses identify NR2E1-candidate-regulatory mutations in humans with severe cortical malformations.

Nuclear receptor 2E1 (NR2E1) is expressed in human fetal and adult brains; however, its role in human brain-behavior development is unknown. Previously, we have corrected the cortical hypoplasia and behavioral abnormalities in Nr2e1(-/-) mice using a genomic clone spanning human NR2E1, which bolsters the hypothesis that NR2E1 may similarly play a role in human cortical and behavioral development. To test the hypothesis that humans with abnormal brain-behavior development may have null or hypomorphic NR2E1 mutations, we undertook the first candidate mutation screen of NR2E1 by sequencing its entire coding region, untranslated, splice site, proximal promoter and evolutionarily conserved non-coding regions in 56 unrelated patients with cortical disorders, namely microcephaly. We then genotyped the candidate mutations in 325 unrelated control subjects and 15 relatives. We did not detect any coding region changes in NR2E1; however, we identified seven novel candidate regulatory mutations that were absent from control subjects. We used in silico tools to predict the effects of these candidate mutations on neural transcription factor binding sites (TFBS). Four candidate mutations were predicted to alter TFBS. To facilitate the present and future studies of NR2E1, we also elucidated its molecular evolution, genetic diversity, haplotype structure and linkage disequilibrium by sequencing an additional 94 unaffected humans representing Africa, the Americas, Asia, Europe, the Middle East and Oceania, as well as great apes and monkeys. We detected strong purifying selection, low genetic diversity, 21 novel polymorphisms and five common haplotypes at NR2E1. We conclude that protein-coding changes in NR2E1 do not contribute to cortical and behavioral abnormalities in the patients examined here, but that regulatory mutations may play a role.

Animals↗

PubMed2006

Coverage and characteristics of the Affymetrix GeneChip Human Mapping 100K SNP set.

Improvements in technology have made it possible to conduct genome-wide association mapping at costs within reach of academic investigators, and experiments are currently being conducted with a variety of high-throughput platforms. To provide an appropriate context for interpreting results of such studies, we summarize here results of an investigation of one of the first of these technologies to be publicly available, the Affymetrix GeneChip Human Mapping 100K set of single nucleotide polymorphisms (SNPs). In a systematic analysis of the pattern and distribution of SNPs in the Mapping 100K set, we find that SNPs in this set are undersampled from coding regions (both nonsynonymous and synonymous) and oversampled from regions outside genes, relative to SNPs in the overall HapMap database. In addition, we utilize a novel multilocus linkage disequilibrium (LD) coefficient based on information content (analogous to the information content scores commonly used for linkage mapping) that is equivalent to the familiar measure r2 in the special case of two loci. Using this approach, we are able to summarize for any subset of markers, such as the Affymetrix Mapping 100K set, the information available for association mapping in that subset, relative to the information available in the full set of markers included in the HapMap, and highlight circumstances in which this multilocus measure of LD provides substantial additional insight about the haplotype structure in a region over pairwise measures of LD.

Chromosome Mapping↗

PubMed2002

Sequence polymorphism at the human apolipoprotein AII gene ( APOA2): unexpected deficit of variation in an African-American sample.

A 3.3-kb region, encompassing the APOA2 gene and 2 kb of 5' and 3' flanking DNA, was re-sequenced in a "core" sample of 24 individuals, sampled without regard to the health from each of three populations: African-Americans from Jackson (Miss., USA), Europeans from North Karelia (Finland), and non-Hispanic European-Americans from Rochester, (Minn., USA). Fifteen variable sites were identified (14 SNPs and one multi-allelic microsatellite, all silent), and these sites segregated as 18 sequence haplotypes (or nine, if SNPs only are considered). The haplotype distribution in the core African-American sample was unusual, with a deficit of particular haplotypes compared with those found in the other two samples, and a significantly (P<0.05) low level of nucleotide diversity relative to patterns of polymorphism and divergence at other human loci. Six of the 14 SNPs, whose variation captured the haplotype structure of the core data, were then genotyped by oligonucleotide ligation assay in an additional 2183 individuals from the same three populations (n=843, n=452, and n=888, respectively). All six sites varied in each of the larger "epidemiological" samples, and together, they defined 19 SNP haplotypes, seven with relative frequencies greater than 1% in the total sample; all of these common haplotypes had been identified earlier in the core re-sequencing survey. Here also, the African-American sample showed significantly lower SNP heterozygosity and haplotype diversity than the other two samples. The deficit of polymorphism is consistent with a population-specific non-neutral increase in the relative frequency of several haplotypes in Jackson.

Alleles↗

PubMed2005

Sequencing drug response with HapMap.

The information about how DNA sequence varies across the human genome is crucial for unravelling the genetic basis of drug response. A haplotype map, or HapMap, intended to reveal such a variation pattern, has been recently developed by the International HapMap Consortium. Here, we present a conceptual model for directly characterizing specific DNA sequence variants that are responsible for drug response based on the haplotype structure provided by HapMap. Our model is developed in the maximum likelihood context, incorporated by clinically meaningful mathematical functions that model drug response and implemented with the EM algorithm. Our model is employed to a pharmacogenetic study of cardiovascular disease with 107 patients. We found that the haplotype constituted by allele Gly16 (G) at codon 16 and allele Glu27 (G) at codon 27 genotyped within the beta2AR candidate gene exhibits a different effect on heart rate curve from the rest haplotypes. Parents with the diplotype consisting of two copies of haplotype GG are more sensitive in heart rate to increasing dosages of dobutamine than those with other haplotypes. This model provides a powerful tool for elucidating the genetic variants of drug response and ultimately designing personalized medications based on each patient's genetic constitution.

Algorithms↗

PubMed2006

ABCB1/MDR1 gene determines susceptibility and phenotype in ulcerative colitis: discrimination of critical variants using a gene-wide haplotype tagging approach.

Several lines of evidence suggest a role for the multidrug resistance gene (ABCB1/MDR1) and its product, P-glycoprotein 170, in the pathogenesis of inflammatory bowel disease (IBD). In addition, P-glycoprotein activity determines bioavailability of many drugs used regularly in many medical specialties, and ABCB/MDR1 variation appears to be a critical pharmacogenetic determinant. We have utilized a gene-wide haplotype tagging approach to further define the identity of germ-line variations in the ABCB1/MDR1 gene contributing to IBD susceptibility. Six haplotype tagging single nucleotide polymorphisms (tSNPs) representing the haplotypic variations of the ABCB1/MDR1 gene were identified initially following the characterization of the haplotype structure of this gene in 24 Centre d'Etude du Polymorphisme Humain Caucasian trios. Genotyping was performed in 249 ulcerative colitis (UC) and 179 Crohn's disease (CD) patients and 260 healthy controls. Using log-likelihood analysis, we observed a highly significant association between the common haplotypes and UC (P=4.22 x 10(-7)) but not CD (P=0.22). This significant association was critically dependent on one tSNP, intronic variant rs3789243. All haplotypes with this variant retained a highly significant association (P=3.2 x 10(-7)-3.6 x 10(-12)), whereas significance was lost when rs3789243 was dropped in systematic haplotypic analysis. The effect of this tSNP was independent of C3435T SNP, previously suggested to be the critical variant in disease susceptibility and drug transport. The association with UC was shown to be strongest with the phenotype of extensive disease (P=1.7 x 10(-7)). This 'candidate gene' approach provides compelling evidence to support the contribution of the ABCB1/MDR1 gene in determining risk to UC but not to CD and provides new insights into the localization of the critical susceptibility determinants within the gene. In addition, these findings have potentially important implications in the application of pharmacogenetics across a range of common diseases, including HIV, epilepsy and colorectal cancer.

Adult↗

PubMed2004

Segmental phylogenetic relationships of inbred mouse strains revealed by fine-scale analysis of sequence variation across 4.6 mb of mouse genome.

High-density SNP screening of panels of inbred mouse strains has been proposed as a method to accelerate the identification of genes associated with complex biomedical phenotypes. To evaluate the potential of these studies, a more detailed understanding of the fine structure of sequence variation across inbred mouse strains is needed. Here, we use high-density oligonucleotide arrays to discover an extremely dense set of SNPs in 13 classical and two wild-derived inbred strains in five genomic intervals totaling 4.6 Mb of DNA sequence, and then analyze the segmental haplotype structure defined by these high-density SNPs. This analysis reveals segments ranging from 12 to 608 kb in length within which the inbred strains have a simple and distinct phylogenetic relationship with typically two or three clades accounting for the 13 classical strains examined. The phylogenetic relationships among strains change abruptly and unpredictably from segment to segment, and are distinct in each of the five genomic regions examined. The data suggest that at least 12 strains would need to be resequenced for exhaustive SNP discovery in every region of the mouse genome, that approximately 97% of the variation among inbred strains is ancestral (between clades) and approximately 3% private (within clades), and provides critical insights into the proposed use of panels of inbred strains to identify genes underlying quantitative trait loci.

Animals↗

PubMed2001

West Mediterranean islands (Corsica, Balearic islands, Sardinia) and the Basque population: contribution of HLA class I molecular markers to their evolutionary history.

The genetic structure of Balearic islands (Corsica and Sardinia), situated on the same trans-Mediterranean maritime routes and having very similar histories, were compared and their position among the neighbouring Caucasian populations was inferred. For this purpose, three HLA loci (HLA-A, -B and -Cw) were typed at the DNA level in these populations and the allelic and haplotypic frequencies were estimated. Because previous studies have shown common genetic features in the Sardinians and Basques, HLA-Cw molecular typing was also performed in a sample of French Basques in order to establish the haplotypic structure of this population for a more accurate comparison with the three others. By its allelic composition, the Corsican population has an intermediate position between the two other islander populations. Its close relationship with the Sardinian population, however, was clearly revealed by the phylogenetic analysis which also suggests a proximity with eastern Mediterranean peoples, whereas the Balearic islands are more narrowly related to Spain and western Europe. Peculiarities were observed in the distributions of some common haplotypes in the populations of the islands that confirm the results of the phylogenetic analysis and could be related to their history. Noteworthy is the presence of the HLA-A30-Cw*0501-B18 haplotype at frequencies approximately 2% in Corsica and the Balearic islands, yet the estimated frequencies of this haplotype are much lower than in the Sardinian and Basque populations.

Evolution, Molecular↗

PubMed2004

Candidate gene approach in association studies: would the factor V Leiden mutation have been found by this approach?

A re-emerging strategy in the search for disease susceptibility genes is the evaluation of candidate genes, which are thought to play a role in disease pathogenesis. Candidate genes are screened for single nucleotide polymorphisms (SNPs) in a case-control study. The factor V Leiden (FVL) mutation (1691G --> A in the F5 gene) is an important risk factor for venous thrombosis. We asked ourselves whether the FVL mutation would have been found using the candidate gene approach in the absence of prior knowledge of the haplotype structure of the F5 gene. We typed four SNPs in the F5 gene in the Leiden Thrombophilia study, that is, promoter (99930G --> A), exon 13 (55907A --> G), exon 16 (42855A --> G), and intron 19 (37833T --> G). These SNPs were known to have different population frequencies, making their presence in distinct haplotypes likely. None of these SNPs has previously been associated with venous thrombotic risk. Subsequently we derived haplotypes. One haplotype was clearly more frequent in patients than controls (GAAT; 20 versus 9%), suggesting that a polymorphism in or near the F5 gene in this haplotype is associated with an increased thrombotic risk. If we had sequenced the F5 gene in patients homozygous for this haplotype, in order to locate the possible causal polymorphism, we would have found that 16 (76%) patients were homozygous or heterozygous for a missense mutation in exon 10 (1691G --> A), which predicts the replacement of Arg506 by Gln in one of the cleavage sites for activated protein C, a mutation that we now know as the FVL mutation.

Case-Control Studies↗