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Keywords:

  • familial correlation;
  • haplotype;
  • polymorphism;
  • P-selectin;
  • serum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Summary. Background: P-selectin is an adhesion molecule known to be involved in the pathogenesis of several diseases through its major role in the initial phase of leukocytes recruitment during inflammation. However, genetic characterization of soluble P-selectin remains unclear. Objectives: In the STANISLAS cohort, we study the familial correlations of P-selectin levels and investigate the association of six P-selectin polymorphisms (C-2123G, A-1969G, S290N, N562D, V599L and T715P) and cardiovascular risk factors with P-selectin concentrations. Patients/Methods: Full phenotypic and genotypic information was available for 136 healthy families composed of both natural parents and at least one child (boys, n = 125; and girls, n = 139) aged more than 4 years. Results: While no correlation was observed between spouses, family correlations of P-selectin concentrations were highly significant for sibling (0.50 ± 0.12, P < 10−3) and child-parent pairs (0.42 ± 0.04, P < 10−3). P-selectin haplotypes explained about 25% of the variability of P-selectin concentrations, this effect being mainly due to the additive effects of two polymorphisms, V599L and T715P. After adjusting for the effect of the P-selectin polymorphisms, the sibling and child-parent correlations decreased to (0.39 ± 0.08, P < 10−4) and (0.32 ± 0.06, P < 10−4), respectively. Conclusions: In the present study, we showed that two P-selectin polymorphisms, V599L and T715P, explained about 25% of the variability of P-selectin concentrations and accounted for about 40% of their family resemblance. These results would suggest a genetic influence on P-selectin concentrations beyond the contribution of the P-selectin gene.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

P-selectin is a cellular adhesion molecule that belongs to the selectin family. It is localized within α-granules of platelets and Weibel-Palade bodies of endothelial cells. These molecules are rapidly mobilized at the plasma membrane level following cell activation [1]. P-selectin interacts with its natural ligand, P-selectin glycoprotein ligand-1 (SELPLG), present on neutrophiles and monocytes, and thus plays a major role in the initial phases of the adhesion of leukocytes to the endothelium, and in the interaction between leukocytes and platelets [1]. Consequently, P-selectin is involved in the recruitment of leukocytes on the activated vessel wall during inflammation and is suspected to play an important role in the early stages of atherosclerosis and its complications [2].

Although P-selectin is expressed as a functional membrane glycoprotein, a shorter soluble isoform has been reported. Human soluble P-selectin has been described as the product of alternative splicing of the exon containing the transmembrane domain [3,4]. In mice, a soluble form of P-selectin has also been suggested to result from proteolytic cleavage from activated platelets [5].

Soluble P-selectin concentration is increased in various cardiovascular (CV) disorders, including unstable angina [6], acute myocardial infarction [7–9], coronary artery spasm [10], hypercholesterolemia [11,12], peripheral vascular disease [13], hypertension [14] and congestive heart failure [15]. However, soluble P-selectin can also be measured in healthy subjects [16,17] and its circulating concentration was shown to be predictive of future vascular events in initially healthy women [18]. Several single nucleotide polymorphisms (SNPs) of the P-selectin gene (SELP), located on chromosome 1q21-1q24, have been reported [19]. Among those, the N562D (rs6127), S290N (rs6131) and T715P (rs6136) polymorphisms were found associated with susceptibility to myocardial infarction (MI) [19–21], whereas V599L (rs6133) polymorphism was related to thrombo-embolic stroke [22] and to asthma-associated phenotype [23]. Consistently with its protective effect for MI, the P715 allele was additionally associated with lower P-selectin concentrations in healthy subjects [24,25] and in patients with coronary heart disease [26,27]. Two SNPs, C-2123G (rs1800807) and A-1969G (rs1800805), located in the promoter region of the gene were also suspected to be associated with P-selectin concentrations [26].

The aim of the present study was to determine the extent to which SELP SNPs exert an influence on P-selectin serum variation in a sample of healthy nuclear families and the contribution of these polymorphisms to the familial resemblance.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

Subjects

Families were drawn from the STANISLAS cohort, a longitudinal family study initially recruited between 1994 and 1995 in the Centre de Médecine Préventive in Nancy, France [28], including 1006 families. In this investigation, we present data from the second examination, realized between 1998 and 1999 with 750 families coming back, obtained from a random sub-sample of 164 nuclear families composed of two parents between 33 and 64 years and at least two children between 4 and 24 years. Subjects were volunteers for a free health examination. They had to be born of French parents and grandparents and to live in the eastern part of France, in the two administrative divisions of Vosges and Meurthe et Moselle. Family members were free from serious or chronic diseases (mainly CV, hepatic or renal failure) and did not take lipid-lowering drugs. Exclusion criteria were as follow: aspartate aminotransferase (AST), alanine aminotransferase or γ-glutamyltransferase activities >200 U L−1; apolipoprotein (apo) E concentration >200 mg L−1; orosomucoid or haptoglobin >3 g L−1; cholesterol or triglyceride >10 mmol L−1; C-reactive protein (CRP) >30 mg L−1 or glucose >8 mmol L−1. In the present study, full phenotypic and genotypic information was available for 136 nuclear families composed of both natural parents and at least one child (boys, n = 125; and girls, n = 139) aged more than 4 years. The research protocol was approved by the local ethical committee (CCPRB Lorraine) and each subject gave a written informed consent.

Blood sample and data collection

Venous blood samples were collected by venipuncture after overnight fasting [28]. Blood samples were centrifuged (1500 × g for 15 min at 4 °C) within 2 h after collection, and resulting serum or plasma (EDTA) aliquots for P-selectin, tumor necrosis factor (TNF-α) and interleukin-6 (IL-6) determinations were promptly frozen at −196 °C in liquid nitrogen until analysis. Data collection included the measurements of basic blood constituents, functional tests, physical examinations, questionnaires on life-style description and medical history. Drug use, notably lipid-lowering and anti-hypertensive drugs, oral contraceptives and hormonal replacement therapies were assessed by interview during the blood sampling. Information about alcohol and tobacco consumption was collected by self-administered questionnaires. Body-mass index (BMI) was calculated according to the Quetelet’s formula: weight (kg)/[height2 (m)]. Blood pressure was calculated as the mean of three measurements taken under standardized conditions, in a supine position with a sphygmomanometer.

Analytical methods

Serum concentrations of glucose, total cholesterol and triglycerides were measured with commercially available kits (all from Merck, Darmstadt, Germany) on an AU5021 apparatus. Serum apolipoprotein A1 (Apo A1), apolipoprotein B (Apo B) and CRP were measured by immunonephelometry on a Behring Nephelometer Analyser (BN II; Dade-Behring, Marburg, Germany) with Behring reagents (Rueil-Malmaison, France). Concentrations of serum P-selectin, and EDTA plasma IL-6 and TNF-α were measured with commercially available enzyme-linked immunosorbant assays (R&D System, Abington, UK).

Genotyping of the SELP polymorphisms

Genomic DNA was extracted using the salting-out method [29]. Genotyping of the S290N and V599L SNPs was performed using a prototypic multilocus genotyping assay focused essentially upon inflammatory and immunomodulatory pathways as previously described [30,31]. The T715P polymorphism was detected using denaturing high-performance liquid chromatography (DHPLC). The fragment of the SELP gene was amplified using oligonucleotide primers, previously described [19]: 5′-AAATTGTACCTTGGCAGGTT-3′ and 5′-AGCTGTGAAATGCTCAGAAC-3′. Amplicons were screened using the WAVE DNA Fragment Analysis System using a DNASep column (Transgenomic, San Jose, CA, USA). The N562D polymorphism was genotyped using previously described [32] oligonucleotide primers and identified using a 2% agarose gel. Promoter polymorphisms were identified on an 8% acrylamide gel electrophoresis using the following primers sequences; C-2123G : 5’ GGC ATA AGG CTG ACA TCT CC -3’ and 5’- TGG AAC AAT ACC TAT CAC GCT GT -3’; A-1969G: 5’- CAC ACT TCC TCA TCC CCA TC -3’ and 5’- TGG AAC AAT ACC TAT CAC GCT GT -3’.

Statistical analysis

Basic statistical analyses were performed using the SAS statistical software (SAS Institute Inc., Cary, NC, USA). Because of their distributions, serum concentrations of triglycerides, CRP, IL-6, P-selectin, TNF-α and Apo A1 were loge-transformed before statistical analysis to reduce the skewness of the data. As individuals within a family are not independent, the association between P-selectin concentrations and any biological and clinical variable was investigated using the estimating equation (EE) technique [33] as implemented in the SAS GENMOD procedure.

Familial correlations of P-selectin concentrations were estimated by use of a maximum likelihood method [34]. Four types of correlations were of interest, between spouses, father and offspring, mother and offspring, and between siblings.

For each SNP, deviation from Hardy–Weinberg was tested among parents by a chi-squared test with one degree of freedom. Genotype parental data were used for estimating allele frequencies and pairwise linkage disequilibrium coefficients were estimated using the THESIAS software [35].

Associations between SNPs and P-selectin concentrations were first tested using familial measured genotype analysis [36]. The contribution of polymorphisms to P-selectin variability (R2: determination coefficient) was calculated as the proportion of variance explained by polymorphisms. Measured genotyped analyses were then followed by a measured haplotype analysis investigating the joint effect of the six SNPs on P-selectin variability [37]. For this purpose, the maximum likelihood program used in [36] was extended to haplotypes analysis according to the haplotypic methodology previously described for unrelated individuals [21]. Instead of using a standard normal distribution as in [21], a multivariate normal distribution as in [36,37] was modelled for estimating mean haplotype effects and residual familial random effects.

All family data analyses were adjusted for age, gender, tobacco consumption and oral contraceptive in females.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The means and standard deviations for P-selectin serum concentrations and potential covariates are given in Table 1, separately for fathers, mothers, sons and daughters, except for the smoking habit, which was expressed in percentages. In both generations, P-selectin serum concentrations were significantly higher in males than in females.

Table 1.   Descriptive characteristic according to the four sex-by-generation groups*
 Fathers n = 136Mothers n = 136PSons n = 125Daughters n = 139P
  1. *Arithmetic means (standard deviation) or per cent.

  2. Test for a difference between males and females in each generation. ns: not significant at 5% level.

  3. Tests were performed on loge-transformed values.

  4. §Levels were estimated by using the Friedewald formula: LDL-C = total cholesterol − (HDL-C + triglyceride/2.2), with measurements expressed in mmol L−1 and triglycerides <4.57 mmol L−1.

Age (years)44.7 (3.8)42.9 (4.0)0.000116.1 (3.7)16.2 (3.4)ns
Systolic BP (mmHg)125.3 (11.8)121.1 (14.8)0.0103119.3 (12.2)115.3 (10.0)0.0041
Diastolic BP (mmHg)75.4 (9.0)71.5 (10.2)0.000962.9 (10.4) 62 (10.4)ns
BMI (kg m−2)25.4 (2.9)24.1 (4.2)0.002120.1 (2.8)20.6 (2.8)ns
Alcohol consumption (g day−1)21.2 (25.5)6.0 (11.2)<0.00012.0 (6.9)0.8 (2.3)0.0347
Current smokers (%)29.9%18.8%0.024220.5%13.3%ns
Oral contraceptive use (%) 21.4%  24.9% 
Leukocyte count (109 L−1)6.7 (1.8)6.8 (1.5)ns6.4 (1.5)7.1 (1.7)0.0001
Platelet count (109 L−1)241.4 (54.4)245.6 (56.9)ns236.5 (46.2)254.4 (48.6)0.0008
Erythrocyte count (1012 L−1)5.0 (0.3)4.4 (0.3)<0.00015.0 (0.4)4.6 (0.3)<0.0001
Triglycerides (mmol L−1)1.4 (0.8)1.1 (0.6)<0.00010.9 (0.4)0.9 (0.5)ns
Total cholesterol (mmol L−1)5.9 (1.0)5.7 (0.9)0.01764.3 (0.7)4.7 (0.9)<0.0001
LDL cholesterol (mmol L−1)§3.8 (0.9)3.4 (0.8)<0.00012.5 (0.7)2.7 (0.8)0.0122
HDL cholesterol (mmol L−1)1.5 (0.4)1.8 (0.4)<0.00011.4 (0.3)1.6 (0.4)<0.0001
Apo A1 (g L−1)1.6 (0.2)1.7 (0.3)<0.00011.4 (0.2)1.5 (0.3)0.0001
Apo B (g L−1)1.1 (0.2)1.0 (0.2)<0.00010.7 (0.2)0.8 (0.2)0.0002
Glucose (mmol L−1)5.1 (0.6)4.8 (0.5)<0.00014.7 (0.5)4.7 (0.4)ns
CRP (mg L−1)1.4 (2.5)1.6 (2.0)ns0.9 (2.0)1.2 (2.3)0.0061
IL-6 (ng L−1)1.6 (2.2)1.1 (1.0)ns1.2 (1.7)1.5 (2.2)ns
TNF-α (ng L−1)2.3 (5.1)2.4 (4.1)ns2.8 (3.2)3.4 (4.2)ns
P-selectin (mg L−1)142.5 (45.8)124.5 (33.6)0.0001140.5 (41.8)123.8 (36.6)0.0002

Biological factors influencing serum P-selectin concentrations

No significant correlation between age and P-selectin concentration was found. In fathers, P-selectin concentrations were slightly higher in smokers (153.6 ± 51.5 mg L−1 vs. 137.7 ± 42.5 mg L−1, P = 0.05). P-selectin concentrations were lower in daughters using oral contraceptive than those who did not take oral contraceptives (112.0 ± 32.8 mg L−1 vs. 127.7 ± 37.1 mg L−1, P = 0.02). No significant difference was found in mothers. Glucose and platelet count were found significantly correlated with P-selectin concentrations in all groups of relatives (P < 0.05, data not shown) while leukocytes count were significantly correlated with P-selectin in fathers only (r = 0.38, P < 0.0001). Together, glucose, leukocyte counts and platelet counts explained about 19% (P < 10−3), 7% (P = 0.006), 8% (P < 10−3) and 11% (P < 10−3) of the variability of P-selectin concentrations in fathers, mothers, sons and daughters, respectively.

Familial correlations

While no correlation was observed between parents (r = 0.079 ± 0.085, P = 0.356), P-selectin concentrations were strongly correlated between biological relatives. The father-offspring, mother-offspring and sibling-sibling correlations were r = 0.394 (±0.123), r = 0.451 (±0.092) and r = 0.498 (±0.118), respectively, (P < 10−3 for each correlation). These three correlations were, however, not different from each other (χ= 2.482 with 2 df, P = 0.289), leading to a common correlation between biological relatives of 0.426 ± 0.042. All these correlations were similar according to the offspring gender (data not shown).

SELP polymorphisms

Allele frequencies and pairwise linkage disequilibrium (LD) coefficients between the six SNPs are given in Table 2. All parental genotype distributions were consistent with Hardy–Weinberg (HW) equilibrium except for the N562D (P = 0.031). As previously shown [19], these SNPs generate two blocks of strong LD, with very weak LD between blocks. The first block includes the C-2123G, A-1969G and S290N SNPs while the second consists of the N562D, V599L and T715P polymorphisms. The first block generates six haplotypes that accounted for 100% of the chromosomes, while in the second block 97% of the chromosomes were explained by only four haplotypes with frequency greater than 2% (Table 3). In particular, the D562, L599 and P715 alleles were each carried by a unique main haplotype.

Table 2.   Allele frequencies and pairwise linkage disequilibrium coefficients between SELP polymorphisms estimated in parents (n = 272)
PolymorphismReference SNP IDPositionNucleotide substitutionAllele FrequencyC-2123GA-1969GS290NN562DV599LT715P
  1. R2 are given above the diagonal while D’ values are shown below the diagonal.

  2. The sign in front of the coefficients indicates whether the linkage disequilibrium is positive (minor alleles preferentially associated) or negative (minor allele preferentially associated with major allele).

  3. ns, not significant; *P < 0.05; **P < 10−3.

C-2123Grs1800807PromoterC>G0.56/0.440.1610.1830.0030.0120.018
A-1969Grs1800805PromoterA>G0.65/0.35−0.62**0.0270.0170.0030.006
S290Nrs6131Exon 7G>A0.81/0.19−1.00**0.25*0.0020.0000.002
N562Drs6127Exon 11A>G0.47/0.53−0.07ns0.17*0.08ns0.1310.062
V599Lrs6133Exon 12G>T0.87/0.13−0.31ns0.11ns0.00 ns0.86**0.016
T715Prs6136Exon 13A>C0.90/0.10−0.38ns0.17 ns−0.26ns0.71**−1.00*
Table 3.   Main haplotype frequencies of the SELP gene estimated in parents (n = 272)
Block 1 polymorphismsBlock 2 polymorphisms
C-2123GA-1969GS290NHaplotype FrequencyN562DV599LT715PHaplotype frequency
CAS0.177NVT0.266
CAN0.106NVP0.079
CGS0.195NLT0.115
CGN0.068DVT0.508
GAS0.387    
GGS0.067    

Association of SELP polymorphisms with serum P-selectin concentrations

Figure 1 summarizes the association between SELP gene polymorphisms and P-selectin serum concentrations. A full description of association analyses is provided in Table 4. Two SNPs, V599L and T715P, were associated with P-selectin concentrations both in parents and offspring, the L599 and P715 alleles being associated with decreased P-selectin levels. The percentages of variance explained by these SNPs were, respectively, 10.3% and 20.7% in parents, and 12.7% and 16.6% in offspring (P < 10−3 for each of these four R2). The N562D was additionally found associated with P-selectin levels only in parents (R2 = 8.9%). Even if the contribution of the N562D was not significant in offspring (R2 = 2.3%, P = 0.32), no heterogeneity was observed between parents and offspring, as for any of the six SNPs (data not shown). As a consequence, further haplotype analyses would be carried out on the whole family data while additionally adjusting for generation.

image

Figure 1.  Association between SELP polymorphisms and serum P-selectin concentrations. *P < 10−3; R2: percentage of variance of P-selectin concentrations explained by a given polymorphism under a general genotypic model with two degrees of freedom.

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Table 4.   Association between serum concentrations of P-selectin and P-selectin gene polymorphisms*
ParentsOffspring
  1. *Mean (SD).

  2. Genotypic test for association (2 degrees of freedom) performed on values adjusted for gender, age, tobacco consumption and oral contraceptive use in women.

C-2123G
CCCGGGP-valueR2CCCGGGP-valueR2
127.3 (40.4), n = 84131.4 (37.6), n = 134143.9 (42.7), n = 54P = 0.0676.2%132.0 (42.7), n = 74132.9 (39.6), n = 134133.2 (43.3), n = 56P = 0.9851.5%
A-1969G
AAAGGGP-valueR2AAAGGGP-valueR2
138.9 (39.6), n = 116128.6 (39.2), n = 124125.2 (40.7), n = 32P = 0.0686.2%132.9 (43.2), n = 165139.4 (41.6), n = 33128.9 (35.1), n = 66P = 0.4812.0%
S290N
SSSNNNP-valueR2SSSNNNP-valueR2
134.1 (40.7), n = 179129.8 (39.3), n = 85129.2 (20.2), n = 8P = 0.7404.5%132.3 (40.6), n = 185133.7 (41.3), n = 71134.4 (54.8), n = 8P = 0.9441.5%
N562D
NNNDDDP-valueR2NNNDDDP-valueR2
120.5 (41.5), n = 50130.6 (40.5), n = 153145.7 (33.3), n = 69P = 0.0018.9%124.7 (43.0), n = 43132.6 (41.8), n = 155138.3 (37.9), n = 66P = 0.3202.3%
V599L
VVVLLLP-valueR2VVVLLLP-valueR2
137.9 (40.4), n = 204117.2 (32.0), n = 62108.6 (49.6), n = 5P = 0.000210.3%140.4 (40.5), n = 200107.5 (31.8), n = 61130.3 (62.5), n = 3P < 10−412.7%
T715P
TTTPPPP-valueR2TTTPPPP-valueR2
139.6 (37.7), n = 222104.2 (32.5), n = 4765.0 (42.5), n = 3P < 10−420.7%140.1 (39.3), n = 216100.6 (31.7), n = 4669.1 (29.9), n = 2P < 10−416.6%

Association of SELP haplotypes with serum P-selectin concentrations

The global test of association between the six haplotypes in block 1 and P-selectin levels was not significant (χ2 = 5.674 with 5 df, P = 0.339). However, the CGN haplotype tended to be associated with a slight decrease in P-selectin levels (−10.2 ± 4.44 mg L−1, P = 0.021) in comparison with the GAS haplotype (Fig. 2). Conversely, block 2 haplotypes were strongly associated with P-selectin levels (χ= 106.6 with 3 df, P < 10−7) (Fig. 2). In comparison with the DVT haplotype, both the NLT and NVP haplotypes were associated with a strong decrease in P-selectin levels (−24.3 ± 6.34 mg L−1 and −37.6 ± 5.05 mg L−1, respectively; both P < 10−4). As there was no difference in P-selectin levels between the DVT and NVT haplotypes that differed only by the position at the N562D locus, all these data are compatible with the additive decreasing effects of the L599 and P715 alleles. Further haplotype analysis revealed that, after adjusting for the effect of the L599 and P715 alleles, the effect of the block 1 CGN haplotype was no longer significant (−5.56 ± 2.96 mg L−1, P = 0.060).

image

Figure 2.  Association of SELP haplotypes with P-selectin serum concentrations. Polymorphisms are ordered according to their position on the genomic sequence; block 1: C-2123G, A-1969G and S290N; block 2: N562D, V599L, and T715P. Each square represents the mean effects (X-axis, in mg L−1) of SELP haplotypes on P-selectin concentrations by reference to the most common haplotype, separately in each block. The reference haplotypes are the GAS and DVT haplotypes in block 1 and block 2, respectively.

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Finally, the V599L and T715P polymorphisms explained together 23%, 27% and 25% of the variability of P-selectin levels in fathers, mothers and offspring, respectively. After adjusting for the effects of these polymorphisms, the correlation between biological relatives significantly (P = 0.041) decreased to 0.327 ± 0.051 but still remained highly significant (P < 10−4), SELP haplotypes then explaining 39% of the familial covariance (i.e ∼family correlations) of P-selectin concentrations.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

In this study, we investigated the familial correlations of serum P-selectin concentrations in a cohort of healthy French families and determined whether they could be partly explained by SELP polymorphisms. We showed that SELP haplotypes explained about 25% of the variability of P-selectin levels and about 40% of the family resemblance. Our haplotype analysis demonstrated that the effects of SELP polymorphisms were in fact due to the additive effects of two non-synonymous polymorphisms, V599L and T715P, both rare alleles being associated with decreased P-selectin levels.

The missense variant T715P is located in exon 13, which encodes the last consensus repeat of P-selectin. This is the last exon before the one that encodes for the transmembrane domain that is lacking in the soluble form produced by alternative splicing [4]. This missense mutation could affect the stability of mRNA and its efficiency of translation into a functional protein and could therefore explain the significant decrease of P-selectin concentrations observed in individual carriers of this mutation. On the other hand, experimental studies on mice have suggested that proteolytic cleavage could also explain the presence of shorter P-selectin molecules in plasma [5]. The production of soluble isoforms of cellular selectin by proteolytic cleavage has been reported for E-selectin and L-selectin [38]. If a fraction of the soluble P-selectin molecules is indeed produced by proteolytic cleavage, then the presence of the proline residue at position 715, in close proximity to the membrane, may modify the conformational state and/or sequence of the cleavage site of the membrane-bound P-selectin and affect the ability of P-selectin to be released from activated cells. Interestingly, given that high P-selectin levels are associated with increased risk of CV disorders [7–15], the observed association of the P715 allele with decreased P-selectin levels is then consistent with the observed protective effect of this allele on MI [19–21]. Recently, among 29 SELP SNPs examined, only this polymorphism was highly significantly associated with decreased P-selectin concentrations (P = 5.2 × 10−39), explaining 9.7% of variation after adjustment for clinical factors [39]. Our study suggests that a second non-synonymous polymorphism that leads to substitution of valine for leucine at amino acid 599 and is located within the eighth consensus repeat of the P-selectin protein is also associated with decrease in SELP levels. This consensus repeat domain has been shown to be of particular importance for the binding of P-selectin to its ligand on leukocytes [38] but the exact functional relevance of this mutation remains incompletely understood. Like the codon 715, the position 599 is near the transmembrane domain. Therefore, we could speculate that the V599L mutation influences P-selectin release through conformational modification.

Interestingly, several studies have previously investigated the relationships between SELP polymorphisms and P-selectin concentrations in unrelated subjects [24–26]. The T715P SNP, and to a lesser extent the C-2123G and A-1969G, were found associated with P-selectin levels. We observed similar effects for these SNPs in our healthy French sample population, but we were able to conclude that the effects of the promoter SNPs were likely to be the consequence of their low LD with the SNPs located in the coding regions. However, contrary to other authors [24,25], we did not show any interaction between smoking status and T715P genotype on P-selectin concentration that may be due to the lower tobacco consumption in our younger healthy population.

Finally, the S290N polymorphism, located within the third consensus repeat of the P-selectin protein of potential importance for the binding to ligands [40,41], has also been suspected to be associated with CV diseases, either in single locus or in haplotype analyses [21,42]. In this report, we did not observe any effect of the S290N, either in univariate or haplotypes analysis, on P-selectin concentrations, suggesting that the effect of this SNP on CV diseases, if any, could be through a conformational change of the protein rather than through a quantitative effect.

In conclusion, we showed that SELP polymorphisms strongly influence the concentration and the familial aggregation of P-selectin. After adjusting for the effect of SELP polymorphisms, the familial correlation of P-selectin levels between biological relatives still remained of great magnitude (r = 0.33), suggesting that other genetic or shared environmental factors could be involved in the determinism of P-selectin variability.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

This work was supported by an INSERM IDS Grant 4D00SE/1999, the Caisse Nationale d’Assurance Maladie des Travailleurs Salariés (CNAM), the Région Lorraine, the Communauté Urbaine de Nancy, and the Université Henri Poincaré Nancy I. We thank G. Siest, the staff of the Centre of Preventive Medicine of Vandoeuvre-lès-Nancy, France, and the participants who made this study possible. We are deeply grateful to A. Ponthieux, N. Haddy and M. Pfister (INSERM) for their technical assistance and S. Cheng (Roche Molecular Systems, USA) for providing genotypes tools and useful comments on the manuscript.

Disclosure of Conflict of Interests

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References

The authors state that they have no conflict of interests.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
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