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Prepublished online May 16, 2006; doi:10.1182/blood-2006-01-008094

Fine mapping of quantitative trait nucleotides underlying thrombin activatable fibrinolysis inhibitor antigen levels by a trans-ethnic study Corinne Frere, David-Alexandre Tregouet, Pierre-Emmanuel Morange, Noemie Saut, Dinar Kouassi, Irene Juhan-Vague, Laurence Tiret and Marie-Christine Alessi

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Blood First Edition Paper, prepublished online May 16, 2006; DOI 10.1182/blood-2006-01-008094

1

Fine Mapping of Quantitative Trait Nucleotides underlying Thrombin Activatable Fibrinolysis Inhibitor Antigen levels by a trans-ethnic study Corinne Frère1*, David-Alexandre Tregouet2*, Pierre-Emmanuel Morange1,2, Noémie Saut1, Dinar Kouassi1, Irène Juhan-Vague1 Laurence Tiret2, and Marie-Christine Alessi1. 1

INSERM, UMR 626,Marseille, F-13385 France; Faculté de Médecine CHU Timone,

Marseille, F-13385 France. 2

INSERM, U525, IFR 14, Paris, F-75634 France; Université Pierre et Marie Curie, Paris, F-

75634 France. * The authors equally contributed to the work

Correspondence should be addressed to: Pr. Marie-Christine ALESSI INSERM, UMR 626,Marseille, F-13385 France; Faculté de Médecine CHU Timone, Marseille, F-13385 France. Telephone : 33-491324504 Fax : 33-491254336 E-mail : [email protected]

Copyright © 2006 American Society of Hematology

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2 Abstract Recent studies revisiting the association between plasma Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) Ag levels and polymorphisms of the CPB2 gene (coding for TAFI) suggested that TAFI Ag levels were influenced by two major quantitative trait nucleotides (QTNs) in Caucasians. However, the strong linkage disequilibrium (LD) between CPB2 polymorphisms in Caucasians did not allow to distinguish which polymorphisms could be the putative QTNs. To get a better insight into the identification of QTNs, a trans-ethnic haplotype analysis contrasting two populations of African and European subjects was performed using 13 CPB2 polymorphisms. Results of the haplotype analyses suggested that three QTNs had independent effects and explained about 15% of the TAFI variability, consistently in the two populations. The lower LD observed in the African population enabled us to identify the g.1583T>A SNP located in 3'UTR as one of these QTNs, whereas the g.2599C>G and g.-2345_-2344insG SNPs located in the 5' region might be the two other QTNs. A phylogenetic study suggested that these three polymorphisms occurred before the period of migration "out of Africa". Although this trans-ethnic comparison contributed to better map the putative CPB2 QTNs, further studies are required to clarify the role of the promoter region.

196 words

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3 Introduction The

thrombin-activatable

procarboxypeptidase B

1,2

fibrinolysis

inhibitor

, procarboxypeptidase U

3

(TAFI),

also

known

as

plasma

or procarboxypeptidase R, is a plasma

zygomen that can be activated by the thrombin-thrombomodulin complex into an active enzyme (TAFIa)

4-6

. TAFIa can then potently attenuate fibrinolysis by removing

carboxyterminal lysine residues from partially degraded fibrin during the clot lysis process which results in a decreased plasminogen binding on fibrin surface and therefore in a decrease of fibrinolytic activity

7,8

. Because it plays an important role in the balance between the

coagulation and the fibrinolytic system, TAFI has been suspected to constitute a marker for atherothrombotic diseases. Supporting this hypothesis, elevated plasma TAFI concentrations have been reported in patients with deep-vein thrombosis 9 and were found more frequently in patients with stable angina pectoris than in a healthy population

10

. Plasma TAFI

concentrations have also been shown to be higher in patients with type II diabetes mellitus than in healthy controls 11. In healthy individuals, plasma levels of TAFI antigen (TAFI Ag) have been shown to exhibit a large interindividual variability poorly explained by environmental factors

12-14

.

Plasma levels of TAFI Ag were then suspected to be under a strong genetic influence, hypothesis supported by several studies showing that polymorphisms of the gene encoding TAFI (named CPB2) were strongly associated with TAFI Ag circulating levels

15,16

. The

results of a series of combined segregation-linkage analyses strengthened this hypothesis: plasma levels of TAFI Ag were shown to exhibit a strong familial resemblance totally explained by the likely existence of two CPB2-linked functional variants

17

. These two

putative variants were postulated to explain more than 70% of the total variability of plasma TAFI Ag levels in healthy adults of Caucasian origin 17.

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4 The CPB2 gene has been mapped to 13q14.11 and consists of 11 exons spanning approximately 48kb of genomic DNA

18

. A large number of single nucleotide polymorphisms

(SNPs) within the CPB2 gene have been described, among which three substitutions in the coding sequence, a G to A at nucleotide 505 leading to an Ala to Thr substitution at amino acid 147 (p.A147T), a C to T at nucleotide 678 leading to a silent mutation (c.678C>T), and a C to T at nucleotide 1040 leading to a Thr to Ile substitution at amino acid 325 (p.T325I) 19-21. The two non synonymous polymorphisms were shown to be associated with TAFI Ag levels 15

. Moreover, the p.T325I was proven to be functional, the Ile325 allele having increased

antifibrinolytic properties by comparison to the Thr325 allele 19. Molecular screening of the 5' and the 3’ untranslated (3'UTR) regions of CPB2 later identified several SNPs that were all strongly associated with plasma TAFI Ag levels 22,23

15,16

. However, it was recently demonstrated

that, in some enzyme-linked immunosorbent assays (ELISA) used for TAFI Ag levels

determination, decreased antibody reactivity toward the TAFI Ile325 isoform led to erroneous TAFI Ag levels that were likely to have overestimated TAFI heritability and effects associated with CPB2 polymorphisms in previous studies

15-17

. Two subsequent studies re-

addressed the association between CPB2 polymorphisms and plasma TAFI Ag levels using assays free of isoform-dependent artefact in Caucasian individuals

24,25

. Even though the

conclusions of both reports were still in agreement with the likely existence of two CPB2 polymorphisms underlying TAFI Ag variability, these new analyses suggested that the two polymorphisms would explain less than 20% of the total variability of plasma TAFI Ag levels in healthy adults of Caucasian origin, as compared to the 70% previously estimated

17

.

However, due to the strong linkage disequilibrium (LD) between all the CPB2 polymorphisms in the Caucasian population, it was not possible to determine which SNPs could be the putative quantitative trait nucleotides (QTNs). In particular, the strong LD between the p.A147T and the g.1583T>A polymorphisms did not allow one to disentangle their effects 24.

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5 The lower LD and the greater haplotype diversity observed in African-descent populations 26-29

argued that the study of such a population could help to better map the QTNs by

identifying informative contrasts between polymorphisms that, in Caucasian populations, could not be discriminated because of strong LD

30,31

. In order to get a better insight into the

identification of the putative QTNs underlying TAFI Ag variability, we carried out a transethnic association analysis contrasting two populations of African and European origins. Since the coding sequence of the CPB2 gene had not been extensively screened, we first performed a molecular screening to identify all common exonic polymorphisms. The detected polymorphisms, as well as those previously identified by the screening of the 5' and the 3'UTR regions, were studied in relation to plasma TAFI Ag levels by means of haplotype analysis. Finally, a phylogenetic analysis of the CPB2 haplotypes was performed in the two populations.

Material and Methods Study population The African study sample (170 subjects, 144 men and 26 women) was composed of healthy blood donors aged 19-52 years (mean 29±7) recruited in a health care center in Abidjan (Republic of the Ivory Coast). All participants were asked to complete a medical questionnaire and were examined using a standardized epidemiologic protocol. The European study population was composed of 123 healthy men living in the Marseilles area (France). Individuals aged 40-60 years (mean 53±9) were recruited on the occasion of a health checkup in a health care center 15. All the subjects gave their informed consent to participate in the study. Approval was obtained from our hospital institutional review board for this study.

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6 TAFI antigen determination Blood samples were obtained from the antecubital vein in the morning, after overnight fasting, collected into 3.8% trisodium citrate (0.129 M) (9:1, v/v). Platelet-poor plasma was obtained after centrifugation (2500g for 30 minutes at 4°C) and kept frozen below –80°C until analysis. Antigen (Ag) determination of TAFI was performed using the Asserachrom TAFI ELISA (Diagnostica Stago, Asnières, France) according to the manufacturer. This assay is based on two monoclonal antibodies raised against TAFI purified from plasma. These antibodies recognize the TAFI 325Thr as well as the TAFI 325Ile isoform and this assay is free of isoform-dependent artefact as previously described

25 32

. Results were expressed as

µg/ml.

Detection of new TAFI gene polymorphisms and genotyping The molecular screening of the entire exonic CPB2 sequence (NM_001872) was performed by comparing 80 chromosomes from 40 European subjects recruited from a systematic screening of a healthy population

15

. Genomic DNA was extracted from peripheral blood

leukocytes by the salting-out method

33

. DNA-sequence variations were identified by

PCR/SSCP and followed by sequencing as previously described 15. The identified SNPs as well as all previously described SNPs of the coding sequence and the 5' and 3’UTR regions were then genotyped in the present study. Genotyping was performed using polymerase chain reactions (PCR) amplification followed by restriction digestion or using allele-specific PCR.

Statistical analysis Allele frequencies were estimated by gene-counting and departure from Hardy-Weinberg equilibrium was tested using a χ2 with 1 degree of freedom (df). Allele frequencies were

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7 compared between the African and Caucasian populations by a χ2 test with 1 df. Association between each CPB2 polymorphism and TAFI Ag plasma levels was first investigated by use of classical linear model assuming additive allele effects after having tested for the deviation from additivity. Pairwise LD was estimated using the THESIAS software based on the SEM algorithm

34

and

the extent of disequilibrium was expressed in terms of D’ which is the ratio of the unstandardized coefficient to its maximal/minimal value

35

. Haplotype analysis was

performed using the THESIAS software which allows one to simultaneously estimate haplotype frequencies and haplotype effects by comparison to a reference haplotype under the assumption of additive effects of haplotypes on phenotype. The phenotypic mean associated with one dose of each haplotype was reported with its 95% confidence interval. The mean TAFI Ag level of an individual is then the sum of the mean levels associated with his two haplotypes. A global test of association between haplotypes and the phenotype was performed by a likelihood ratio test (LRT) (χ2 with m-1 df in the case of m haplotypes). By setting appropriate constraints on parameters, the LRT statistic also allowed us to compare the effects between pairs of haplotypes, in particular those differing by only one nucleotide substitution. Homogeneity of the haplotype effects across populations was tested by introducing corresponding interaction parameters in the haplotypic model. Effects of rare haplotypes (frequency < 3%) were set to 0. Finally, the evolutionary relatedness of the inferred CPB2 haplotypes was investigated using the Maximum Parsimony method implemented in the TCS program 36.

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8 Results Description of CPB2 polymorphisms Our molecular screening of the coding region in 40 European subjects identified three new silent substitutions (a C to T at nucleotide 310 in exon 3B , a T to A at nucleotide 499 in exon 4 and a G to A at nucleotide 663 in exon 6), in addition to the three previously described SNPs (c.678C>T, p.A147T and p.T325I). All the SNPs of the coding region as well as those previously described in the 5' and 3'UTR (g.-2599C>G, g.-2345_-2344insG, g.-1925T>C, g.-1690A>G, g.-1102G>T, g.530C>T, g.-152A>G, g.-438G>A and g.1542C>G, g.1583T>A) were genotyped in the Caucasian and African samples. No carrier of the T499A mutation was detected in either population. The g.-530C>T, g.-152A>G and g.-1925T>C polymorphisms were in complete association in the African population while no mutant was detected in Caucasians. As a consequence, the present analysis included thirteen SNPs, six in the 5' region (g.-2599C>G, g.-2345_-2344insG, g.-1925T>C, g.-1690A>G, g.-1102G>T, g.-438G>A), five in the coding region (c.310C>T, p.A147T, c.663G>A, c.678C>T, p.T325I) and two in the 3’UTR region (g.1542C>G and g.1583T>A).

Allele frequencies and linkage disequilibrium between polymorphisms Allele frequencies of the thirteen SNPs in Africans and Caucasians are reported in Table 1. All genotype distributions were compatible with Hardy-Weinberg equilibrium in both populations. Most of the SNPs exhibited significant difference in allele frequency between populations. The pattern of LD is reported in Table 2. In Caucasians, all SNPs were in strong LD one with each other (the g.-1690A>G, g.-1102G>T and g.-438G>A SNPs being in complete association). By contrast, SNPs in Africans were distributed into three LD blocks, the first one

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9 comprising the SNPs of the 5' region, the second one comprising the SNPs of the coding region and the g.1542C>G, and the third one composed of the g.1583T>A alone.

Association of CPB2 polymorphisms with plasma TAFI Ag levels African individuals exhibited lower levels of plasma TAFI Ag than Caucasians (mean (SE) : 11.23 (2.25) vs 12.37 (2.99) µg/ml; p 3%) accounting for 86% of the sampled chromosomes, whereas in Africans, they generated six common haplotypes accounting for 94% of the chromosomes. Although the haplotype structure was not identical in the two populations, the common haplotypes could be tagged by the same subset of SNPs consisting of the p.A147T (G>A), p.T325I (C>T), g.1542C>G and g.1583T>A SNPs (Figure 3). These haplotypes explained 6.7% (p = 0.11) and 14.6% (p = 0.0001) of the variability of plasma TAFI levels in Caucasians and Africans, respectively. In Caucasians, the two haplotypes carrying the +1583A allele were associated with the highest TAFI levels. These two haplotypes, only differing at the p.A147T locus, did not show

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11 difference in TAFI levels (6.61 vs 7.10 µg/ml, p = 0.66) suggesting that the effect of the p.A147T polymorphism observed in univariate analysis was the consequence of its LD with the g.1583T>A SNP. No difference in TAFI levels was observed between haplotypes carrying the +1583T allele (χ2 = 0.11 with 2 df, p = 0.94) indicating that, after adjusting for the effect of the g.1583T>A SNP, no other polymorphism was associated with TAFI levels. In Africans, comparisons of the three pairs of haplotypes that differed at only the g.1583T>A site revealed that the effect of the +1583A allele was homogeneous (χ2 = 1.56 with 2 df, p = 0.46 for the test of homogeneity) across the three pairs, the +1583A allele being associated with increased TAFI levels (pC SNP observed in univariate analysis was also the consequence of its LD with the g.1583T>A SNP. Pooled analysis In order to cope with the LD between the 5' and coding/3' UTR regions, haplotype analysis was performed on the SNPs identified as potentially interesting from the two previous sub-analyses, i.e. the g.-2599C>G, g.-2345_-2344insG, g.-1690A>G and g.1583T>A polymorphisms. A first analysis demonstrated that, in both populations, the two haplotypes that differed only at position -1690 did not differ in TAFI levels, precluding any effect of this SNP. Therefore, for ease of presentation, Figure 4 illustrates the association between TAFI levels and the main haplotypes generated by the g.-2599C>G, g.-2345_2344insG and g.1583T>A SNPs. In both populations, TAFI gene haplotypes explained about 14% of plasma TAFI variability. Except the fact that the G2A haplotype was not present in the European population, the patterns of haplotypic association were very similar in the two populations (Figure 4). Haplotype analysis was therefore carried out on the pooled sample of African and European subjects while adjusting for ethnicity, after having checked for the homogeneity of the haplotype effects between populations (test for homogeneity: χ2 = 4.09

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12 with 3 df , p = 0.25). The haplotypes carrying the +1583A allele (C1A and G2A) were associated with higher TAFI levels than their counterparts carrying the +1583T allele (C1T and G2T). The +1583A allele was associated with a population-adjusted average increase of +0.79 [0.28 – 1.31] µg/ml (p=0.003). Besides, the comparison of the C2T vs C1T haplotypes suggested an increasing effect of the –2345 1G allele (+0.93 [0.17 – 1.69] µg/ml, p = 0.016), and the comparison of the C2T vs G2T haplotypes additionally suggested an increasing effect of the –2599G allele (+0.70 [0.09 - 1.32] µg/ml; p = 0.025). In summary, haplotype analyses indicated that three SNPs had independent effects on TAFI levels, g.-2599C>G, g.-2345_2344insG and g.1583T>A.

Phylogenetic analysis of CPB2 haplotypes Phylogenetic analysis was performed to infer the mutational steps among haplotypes. Due to the low to moderate LD between SNPs of the 5' region and those of the coding/3'UTR region, analyses were performed separately for the two regions. Although some haplotypes were population-specific, the consensus gene trees were very similar in the two populations, supporting the robustness of the results (Figure 5). The reconstructed gene tree for the 5' region suggested that all haplotypes derived one from another by single nucleotide changes. Haplotypes carrying the different alleles at the –2599 site were divided into two distinct clades, whereas the haplotype carrying the 1G allele at the –2345 site derived from a single haplotype. For the coding/3'UTR region, phylogenetic ambiguity was present in the parsimony gene trees reconstructed for both populations, as shown by the loops (Figure 5). However, haplotypes carrying the different alleles at the +1583 site appeared clearly divided into two distinct clades.

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13 Discussion Recent studies revisiting the association between plasma TAFI Ag levels and CPB2 polymorphisms have suggested that two polymorphisms accounted for about 20% of the plasma TAFI Ag variability in the Caucasian population

24,25

. One of these polymorphisms

was likely to be located in the promoter region of the TAFI gene while the second would be located in the coding or 3' region. However, due to the strong LD between all CPB2 polymorphisms in Caucasians, it was difficult to distinguish those that have a true functional role from those that are only markers in LD with the CPB2-linked QTNs. One possible strategy to get a better insight into the identification of QTNs is a trans-ethnic comparison in which differences of LD patterns between populations can help to better characterize QTNs, as it was recently shown for angiotensin-1 converting enzyme

31

. Indeed, it has been

demonstrated that blocks of LD are smaller in subjects of African origin than in subjects of Caucasian origin, which may allow a finer mapping of putative functional polymorphisms 39

37-

. This report is to our knowledge the first one to investigate the association between CPB2

polymorphisms and plasma TAFI Ag levels in an African population and to compare the results to those obtained in a Caucasian population. All known CPB2 polymorphisms as well as three new silent coding mutations, c.310C>T, T499A and c.663G>A, were investigated by mean of haplotype analysis. Haplotype analysis suggested that among the 3 polymorphisms in the coding and 3'UTR regions associated with plasma TAFI levels in univariate analysis in both populations, the g.1583T>A polymorphism was the only one independently associated with the phenotype. While this polymorphism was in relative strong LD with the SNPs located in the 5' region in the Caucasian population, the absence of LD in the African population enabled us to clearly demonstrate that the increasing effect of the +1583A allele was independent of any other polymorphism. In addition, two SNPs in the 5' region were

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14 found associated with TAFI levels. In both populations, the –2599G and –2345 1G alleles were associated with increased TAFI levels, the effect of these SNPs being independent from each other and from the g.1583T>A SNP. The present data confirmed that CPB2-linked QTNs located in the 5' and 3'UTR regions independently influenced plasma TAFI levels attributable to CPB2 haplotypes

25

24,25

. The proportion of TAFI variability

was remarkably similar (around 15%) in the two

populations despite differences in allele frequencies and environmental backgrounds. The phylogenetic trees were also quite similar between the two populations despite the existence of population-specific haplotypes. The fact that the three putative QTNs occurred on the same branches of the trees in both populations is in favor of mutations dating from the period before the migration "out of Africa". As already suggested from our previous study

25

, the 3'UTR QTN might be the

g.1583T>A, an hypothesis which is consistent with a recent work showing that haplotypes containing the +1583T allele result in a transcript with decreased stability 40. Unexpectedly, the p.A147T polymorphism did not appear to have an own effect in haplotype analysis, although it exhibited the strongest association with TAFI levels in univariate analysis. This is probably explained by the fact that it was in LD with the three putative QTNs and cumulated their effects in univariate analysis. Importantly, as already observed

25

, the p.T325I

polymorphism was no longer associated with TAFI levels when using the new method free of artifact. Our analysis additionally suggested the existence of two QTNs in the 5' region, g.2599C>G and –2345 2G/1G, instead of only one suggested by our previous studies –2345 2G/1G SNP was however not investigated in the first study

24 25

24,25

. The

, while in the second

study, the strong LD in the 5' region might have hampered a correct identification of the genetic effects 25.

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15 While the greater diversity of the CPB2 gene in Africans subjects helped to identify the g.1583T>A SNP as one of the QTLs underlying TAFI levels, the strong LD observed in the 5' region did not allow us to get a complete insight into the identification of the putative QTNs located in this region. Since our molecular screening was performed only on chromosomes from European subjects, it cannot be ruled out that we missed some SNPs specific to the African population that could have contributed to better map the QTNs. Our screening strategy had about 100% power of detecting any mutation with frequency ≥0.05 and 56% ≥0.01. We cannot exclude that some rare mutations have been missed. Similarly, the search for additional CPB2 SNPs into public database, such as that of the HAPMAP project (www.hapmap.org), reveals that except several intronic SNPs, only one synonymous SNP located in the 3’UTR region with frequency less than 3.8%, was not studied in our analysis. However, our sample size would have been too relatively low to be able to demonstrate any low/moderate effect of this SNP. Would this SNP have a strong effect on TAFI Ag levels, our screening strategy based on comparing 40 DNA from individuals with low TAFI levels to 40 DNA from individuals with high TAFI levels 15 would have likely detected this SNP. Despite a poor influence of environment on TAFI levels

12,24

, we also can not exclude an interaction

between genotype and environment on determination of TAFI plasma levels. Because of the LD among the 13 SNPs studied, a standard Bonferroni correction for multiple testing could not be applied since it would have been too conservative. Using the method proposed by Li and Ji 41, the number of independent components underlying the LD structure of the set of SNPs was estimated to be ~6. After correcting for this number, which corresponds to consider significant any p-value ≤ 0.008, the effect of the g.1583T>A SNP remained significant whereas that of the two 5' QTNs was no longer significant. Larger studies conducted in populations of different origins are needed to refine the role of the 5' region.

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16 In conclusion, this trans-ethnic study helped to better map the CPB2-linked QTNs responsible for the genetic control of TAFI Ag levels. Data from Africans and Europeans converged to identify the g.1583T>A polymorphism as one of the QTNs whereas larger epidemiological studies and functional experiments are required to better clarify the contribution of the promoter region.

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17 References

1. Tan AK, Eaton DL. Activation and characterization of procarboxypeptidase B from human plasma. Biochemistry. 1995;34:5811-5816 2. Eaton DL, Malloy BE, Tsai SP, Henzel W, Drayna D. Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma. J Biol Chem. 1991;266:21833-21838. 3. Hendriks D, Scharpe S, van Sande M, Lommaert MP. Characterisation of a carboxypeptidase in human serum distinct from carboxypeptidase N. J Clin Chem Clin Biochem. 1989;27:277-285 4. Bajzar L, Morser J, Nesheim M. TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. J Biol Chem. 1996;271:16603-16608 5. Boffa MB, Wang W, Bajzar L, Nesheim ME. Plasma and recombinant thrombinactivable fibrinolysis inhibitor (TAFI) and activated TAFI compared with respect to glycosylation, thrombin/thrombomodulin-dependent activation, thermal stability, and enzymatic properties. J Biol Chem. 1998;273:2127-2135 6. Kokame K, Zheng X, Sadler JE. Activation of thrombin-activable fibrinolysis inhibitor requires epidermal growth factor-like domain 3 of thrombomodulin and is inhibited competitively by protein C. J Biol Chem. 1998;273:12135-12139 7. Wang W, Boffa MB, Bajzar L, Walker JB, Nesheim ME. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor. J Biol Chem. 1998;273:27176-27181 8. Sakharov DV, Plow EF, Rijken DC. On the mecanism of the antifibrinolytic activity of plasma carboxypeptidase B. J Biol Chem. 1997;272:14477-14482. 9. van Tilburg NH, Rosendaal FR, Bertina RM. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis. Blood. 2000;95:2855-2859 10. Silveira A, Schatteman K, Goossens F, Moor E, Scharpe S, Stromqvist M, Hendriks D, Hamsten A. Plasma procarboxypeptidase U in men with symptomatic coronary artery disease. Thromb Haemost. 2000;84:364-368 11. Hori Y, Gabazza EC, Yano Y, Katsuki A, Suzuki K, Adachi Y, Sumida Y. Insulin resistance is associated with increased circulating level of thrombin-activatable fibrinolysis inhibitor in type 2 diabetic patients. J Clin Endocrinol Metab. 2002;87:660-665 12. Chetaille P, Alessi MC, Kouassi D, Morange PE, Juhan-Vague I. Plasma TAFI antigen variations in healthy subjects. Thromb Haemost. 2000;83:902-905 13. Stromqvist M, Schatteman K, Leurs J, Verkerk R, Andersson JO, Johansson T, Scharpe S, Hendriks D. Immunological assay for the determination of procarboxypeptidase U antigen levels in human plasma. Thromb Haemost. 2001;85:12-17 14. Guo X, Morioka A, Kaneko Y, Okada N, Obata K, Nomura T, Campbell W, Okada H. Arginine carboxypeptidase (CPR) in human plasma determined with sandwich ELISA. Microbiol Immunol. 1999;43:691-698 15. Henry M, Aubert H, Morange PE, Nanni I, Alessi MC, Tiret L, Juhan-Vague I. Identification of polymorphisms in the promoter and the 3' region of the TAFI gene: evidence that plasma TAFI antigen levels are strongly genetically controlled. Blood. 2001;97:20532058 16. Franco RF, Fagundes MG, Meijers JC, Reitsma PH, Lourenco D, Morelli V, Maffei FH, Ferrari IC, Piccinato CE, Silva WA, Jr., Zago MA. Identification of

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18 polymorphisms in the 5'-untranslated region of the TAFI gene: relationship with plasma TAFI levels and risk of venous thrombosis. Haematologica. 2001;86:510-517 17. Tregouet DA, Aubert H, Henry M, Morange P, Visvikis S, Juhan-Vague I, Tiret L. Combined segregation-linkage analysis of plasma thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels with TAFI gene polymorphisms. Hum Genet. 2001;109:191197 18. Boffa MB, Reid TS, Joo E, Nesheim ME, Koschinsky ML. Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B). Biochemistry. 1999;38:6547-6558 19. Schneider M, Boffa M, Stewart R, Rahman M, Koschinsky M, Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme. J Biol Chem. 2002;277:10211030 20. Zhao L, Morser J, Bajzar L, Nesheim M, Nagashima M. Identification and characterization of two thrombin-activatable fibrinolysis inhibitor isoforms. Thromb Haemost. 1998;80:949-955 21. Brouwers GJ, Vos HL, Leebeek FW, Bulk S, Schneider M, Boffa M, Koschinsky M, van Tilburg NH, Nesheim ME, Bertina RM, Gomez Garcia EB. A novel, possibly functional, single nucleotide polymorphism in the coding region of the thrombin-activatable fibrinolysis inhibitor (TAFI) gene is also associated with TAFI levels. Blood. 2001;98:19921993 22. Guimaraes AH, van Tilburg NH, Vos HL, Bertina RM, Rijken DC. Association between thrombin activatable fibrinolysis inhibitor genotype and levels in plasma: comparison of different assays. Br J Haematol. 2004;124:659-665 23. Gils A, Alessi MC, Brouwers E, Peeters M, Marx P, Leurs J, Bouma B, Hendriks D, Juhan-Vague I, Declerck PJ. Development of a genotype 325-specific proCPU/TAFI ELISA. Arterioscler Thromb Vasc Biol. 2003;23:1122-1127 24. Morange PE, Tregouet DA, Frere C, Luc G, Arveiler D, Ferrieres J, Amouyel P, Evans A, Ducimetiere P, Cambien F, Tiret L, Juhan-Vague I. TAFI gene haplotypes, TAFI plasma levels and future risk of coronary heart disease: the PRIME Study. J Thromb Haemost. 2005;3:1503-1510 25. Frere C, Morange PE, Saut N, Tregouet DA, Grosley M, Beltran J, Juhan-Vague I, Alessi MC. Quantification of thrombin activatable fibrinolysis inhibitor (TAFI) gene polymorphism effects on plasma levels of TAFI measured with assays insensitive to isoformdependent artefact. Thromb Haemost. 2005;94:373-379 26. Crawford DC, Carlson CS, Rieder MJ, Carrington DP, Yi Q, Smith JD, Eberle MA, Kruglyak L, Nickerson DA. Haplotype diversity across 100 candidate genes for inflammation, lipid metabolism, and blood pressure regulation in two populations. Am J Hum Genet. 2004;74:610-622 27. Shifman S, Kuypers J, Kokoris M, Yakir B, Darvasi A. Linkage disequilibrium patterns of the human genome across populations. Hum Mol Genet. 2003;12:771-776 28. Lonjou C, Zhang W, Collins A, Tapper WJ, Elahi E, Maniatis N, Morton NE. Linkage disequilibrium in human populations. Proc Natl Acad Sci U S A. 2003;100:60696074 29. Armour JA, Anttinen T, May CA, Vega EE, Sajantila A, Kidd JR, Kidd KK, Bertranpetit J, Paabo S, Jeffreys AJ. Minisatellite diversity supports a recent African origin for modern humans. Nat Genet. 1996;13:154-160 30. Cox R, Bouzekri N, Martin S, Southam L, Hugill A, Golamaully M, Cooper R, Adeyemo A, Soubrier F, Ward R, Lathrop GM, Matsuda F, Farrall M. Angiotensin-1-

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19 converting enzyme (ACE) plasma concentration is influenced by multiple ACE-linked quantitative trait nucleotides. Hum Mol Genet. 2002;11:2969-2977 31. McKenzie CA, Abecassis GR, Keavney B, Forrester T, Ratcliffe PJ, Julier C, Connel JMC, Bennett F, McFarlane-Anderson N, Lathrop GM, Cardon LR. Trans-ethnic fine mapping of a quantitative trait locus for circulating angiotensin I-converting enzyme(ACE). Hum Mol Genet. 2001;10:1077-1084. 32. Cost H, Grimaux M, Grosley M, Woodhams B. A new antigen assay independent of polymorphisms 325Thr/Ile and 147 Ala/Thr. Pathophysiol Haemost Thromb. 2004;33:64 (abstract) 33. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215 34. Tregouet DA, Escolano S, Tiret L, Mallet A, Golmard JL. A new algorithm for haplotype-based association analysis: the Stochastic-EM algorithm. Ann Hum Genet. 2004;68:165-177 35. Nei M. Molecular Evolutionary Genetics. New York: Columbia University Press; 1987 36. Clement M, Posada D, Crandall KA. TCS: a computer program to estimate gene genealogies. Mol Ecol. 2000;9:1657-1659 37. Sawyer SL, Mukherjee N, Pakstis AJ, Feuk L, Kidd JR, Brookes AJ, Kidd KK. Linkage disequilibrium patterns vary substantially among populations. Eur J Hum Genet. 2005;13:677-686 38. Altshuler D, Brooks LD, Chakravarti A, Collins FS, Daly MJ, Donnelly P. A haplotype map of the human genome. Nature. 2005;437:1299-1320 39. Reich DE, Cargill M, Bolk S, Ireland J, Sabeti PC, Richter DJ, Lavery T, Kouyoumjian R, Farhadian SF, Ward R, Lander ES. Linkage disequilibrium in the human genome. Nature. 2001;411:199-204 40. Maret D, Brien DF, Franco RF, Nesheim ME, Koschinsky ML, Boffa MB. Polymorphisms in the 3-untranslated region of the TAFI mRNA modulate TAFI gene expression through control of mRNA stability. J Thromb Haemost. 2003;1 Supplement 1 : ISTH 2003 Congress Abstracts:(Abstract) 41. Li J, Ji L. Adjusting multiple testing in multilocus analyses using the eigenvalues of a correlation matrix. Heredity. 2005;95:221-227

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20 Table 1. Allele frequencies of the CPB2 polymorphisms in the Caucasian and African samples Polymorphisms

Test(1)

Caucasians

Africans

N = 123

N = 170

g.-2599C>G

0.51 / 0.49

0.43 / 0.57

p = 0.041

g.-2345_-2344insG

0.69 / 0.31

0.71 / 0.29

p = 0.604

g.-1925T>C

-

0.92 / 0.08

g.-1690A>G

0.71 / 0.29

0.77 / 0.23

p = 0.121

g.-1102G>T

0.71 / 0.29

0.79 / 0.21

p = 0.017

g.-438G>A

0.71 / 0.29

0.75 / 0.25

p = 0.239

c.310C>T

0.69 / 0.31

0.88 / 0.12

p < 10-4

p.A147T

0.75 / 0.25

0.67 / 0.33

p = 0.035

c.663G>A

0.71 / 0.29

0.89 / 0.11

p < 10-4

c.678T>C

0.71 / 0.29

0.66 / 0.34

p = 0.196

p.T325I

0.67 / 0.33

0.89 / 0.11

p < 10-4

g.1542C>G

0.70 / 0.30

0.89 / 0.11

p < 10-4

g.1583T>A

0.70 / 0.30

0.60 / 0.40

p = 0.013

(1)

Test for a difference in allele frequency between Caucasians and Africans

21 Table 2. Pairwise linkage disequilibrium between CPB2 polymorphisms

-2345_2G1G

-1.000

-2345_2G1G

g.-1690A>G

1.000

-1.000

g.-1690A>G

g.-1102G>T

0.896

-0.820

1.000

g.-1102G>T

g.-438G>A

1.000

-1.000

1.000

1.000

c.310C>T

0.900

-0.916

0.957

0.919

0.957

c.310C>T

p.A147T

-0.857

0.844

-1.000

-0.716

-1.000

-1.000

p.A147T

c.663G>A

0.807

-0.814

0.828

0.821

0.827

0.910

-1.000

c.663G>A

c.678C>T

-0.844

0.825

-1.000

-0.795

-1.000

-1.000

0.976

-1.000

c.678C>T

p.T325I

0.734

-0.782

0.766

0.706

0.766

0.764

-0.898

0.909

-0.919

g.1542C>G

0.826

-0.733

0.850

0.793

0.850

0.876

-0.877

0.867

-0.913

0.914

g.1542C>G

g.1583T>A

-0.779

0.766

-0.744

-0.576

-0.743

-0.761

0.926

-0.829

0.938

-0.928

-0.921

g.-438G>A

p.T325I

D' in African population g.-2599C>G -2345_2G1G

0.917

-2345_2G1G

g.-1925T>C

-0.861

-1.000

g.-1690A>G

-0.902

-0.843

-1.000

g.-1690A>G

g.-1102G>T

-0.896

-0.829

-1.000

0.962

g.-1102G>T

g.-1925T>C

g.-438G>A

-0.628

-0.721

-1.000

0.861

0.961

g.-438G>A

c.310C>T

-0.492

-1.000

0.186

0.598

0.581

0.882

c.310C>T

p.A147T

0.401

0.530

-0.238

-0.274

-0.308

-0.248

-0.729

p.A147T

c.663G>A

-0.452

-1.000

0.167

0.645

0.659

0.956

1.000

-1.000

c.663G>A

c.678C>T

0.413

0.547

-0.679

-0.247

-0.272

-0.218

-0.736

0.960

-1.000

c.678C>T

p.T325I

-0.219

-0.521

-1.000

0.472

0.497

0.783

0.874

-0.828

0.905

-0.828

g.1542C>G

-0.058

-0.289

-1.000

0.410

0.444

0.732

0.838

-0.812

0.870

-0.818

0.969

g.1542C>G

g.1583T>A

0.041

0.201

-0.337

0.150

0.182

0.115

-0.305

0.630

-0.296

0.619

-0.411

-0.396

All pairwise LD were significantly different from 0 except those in italics

p.T325I

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D' in Caucasian population g.-2599C>G

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22 Figure 1 Association between CPB2 polymorphisms and plasma TAFI Ag levels

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23 Figure 2

Association between plasma TAFI Ag levels and CPB2 haplotypes derived from the g.-2599C>G, g.-2345_-2344insG and g.-1690A>G polymorphisms: polymorphisms are ordered according to their position on the genomic sequence. Each bar corresponds to the expected mean of TAFI Ag levels associated with one dose of haplotype under the assumption of additive haplotype effects

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24 Figure 3

Association between plasma TAFI Ag levels and CPB2 haplotypes derived from the p.A147T (G/A),p.T325I (C/T), g.1542C>G and g.1583T>A polymorphisms: polymorphisms are ordered according to their position on the genomic sequence. Each bar corresponds to the expected mean of TAFI Ag levels associated with one dose of haplotype under the assumption of additive haplotype effects

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25 Figure 4

Association between plasma TAFI Ag levels and CPB2 haplotypes derived from the G.2599C>G, g.-2345_-2344insG, and g.1583T>A polymorphisms: polymorphisms are ordered according to their position on the genomic sequence. Each bar corresponds to the expected mean of TAFI Ag levels associated with one dose of haplotype under the assumption of additive haplotype effects

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26 Figure 5

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27 Consensus parsimony gene trees inferred from CPB2 haplotypes in Caucasians and Africans. A) Gene tree constructed from 6 variant sites in the 5' region (g.-2599C>G, g.2345_-2344insG, g.-1925T>C, g.-1690A>G, g.-1102G>T and g.-438G>A); B) Gene tree constructed from 7 variant sites in the coding/3'UTR region (c.310C>T, p.A147T (G>A), G633A, c.678T>C, p.T325I (C>T), g.1542C>G and g.1583T>A). The relative frequency of each haplotype is indicated by the area of the circle. Haplotypes with frequency < 0.02 are not taken into consideration. Full black circles correspond to haplotype not present in the samples. Loops indicate phylogenetic ambiguity due to recombination or to parallel, convergent or reverse changes. Alleles in underlined bold correspond to the putative QTNs (g.-2599C>G, g.-2345_-2344insG and g.1583T>A).