Polymorphism C373G of the PECAM-1 Gene in chilean subjects with coronary disease and controls/Polimorfismo C373G del Gen PECAM-1 en individuos chilenos con enfermedad coronaria y controles.
KEY WORDS: Aterosclerosis; PECAM-1; Polymorphism.
RESUMEN: La molecula de adhesion celular endotelial plaquetaria-1 (PECAM-1) es una glicoproteina de membrana expresada por celulas endoteliales, plaquetas, monocitos, neutrofilos y algunos tipos de linfocitos T. Constituye una pieza clave en la extravasacion de leucocitos a traves de las uniones intercelulares del endotelio vascular durante el proceso inflamatorio. El objetivo de nuestro estudio fue determinar la asociacion entre el polimorfismo C373G (Leu125Val) del gen PECAM-1 y enfermedad coronaria, en individuos chilenos. Se estudio un total de 220 individuos (112 casos y 108 controles). La presencia de enfermedad coronaria fue confirmada mediante angiografia (estenosis > 70%). El polimorfismo C373G, fue detectado mediante la tecnica de reaccion en cadena de polimerasa seguida de restriccion enzimatica. Las frecuencias genotipicas observadas en ambos grupos cumplen con la ley de Hardy-Weinberg. La distribucion de genotipos como la frecuencia relativa de alelos para el polimorfismo C373G del gen PECAM-1, fueron similares entre casos y controles (p = 0.820 y p = 0.739, respectivamente). Ademas, la OR asociada al alelo mutado G fue 0.92 (I.C. 95%, 0.54 - 1.57; p= NS). Los datos obtenidos sugieren que el polimorfismo C373G del gen PECAM-1 no esta asociado a enfermedad coronaria en la poblacion analizada.
PALABRAS CLAVE: Aterosclerosis; PECAM-1; Polimorfismo.
Actually, the atherosclerosis is considered an inflammatory disease (Libby, 2002; Meng, 2006), due to that the inflammation participates centrally in all stages of this disease, from the initial lesion to the end-stage thrombotic complications (Libby, 2006).
One of the characteristics of the inflammation is the leukocyte recruitment to the damaged tissue, in this case the vascular endothelium (Price & Loscalzo, 1999; Libby, 2002). The recruitment of these inflammatory cells and their transendothelial migration is mediated by the cellular adhesion molecules (CAMs), which are expressed by endothelial cells and circulating leukocytes (monocytes and linfocytes) in response to different inflammatory stimuli (Davies et al., 1993).
Three families of CAMs participate in the leukocyteendothelium interaction: a) the selectines that facilitate rolling of leukocytes over the endothelial surface; b) the integrines, basically for the adherence of the leukocytes to the endothelium and c) the superfamily of immunoglobulines that have importance in the adhesion and transmigration of the leukocytes (Davies et al.; Muller et al., 1993; Albelda et al., 1994; Blankenberg et al., 2003).
PECAM-1 is a 130-kDa membrane glycoprotein and a member of immunoglobulin (Ig) superfamily; their gene is located in the long arm of the chromosome 17 (17q23) (Gumina et al., 1996). PECAM-1 is constituted by 6 Iglike (homology) extracellular domains, a short transmembrane domain and a citoplasmatic tail of 118 aminoacids, is expressed on the surface of platelets, monocytes, neutrophils and some types of T lymphocytes (Newman et al., 1990; Newman, 1997). It is an important constituent in the intercellular unions between the endothelial cells and a key element in the transendothelial migration of leukocytes during the inflammatory process (Albelda et al., 1991; Muller et al.).
Considering the role of PECAM-1 in the development of atherosclerosis and other diseases such as cancer, HIV infection and multiple sclerosis (Bergom et al., 2005; Maas et al., 2005; Eugenin et al., 2006; Nelissen et al., 2006), there are a great interest to study its normal function, its genetic variations and its relation with these diseases.
Several polymorphisms have been identified in the PECAM-1 gene: C373G (Leul25Val, exon 3), Gl688A (Ser563Asn, exon 8), A2212G (Arg670Gly, exon 12) and G53A (5'UTR region) (Behar et al., 1996; Wenzel et al., 1999; Gardemann et al., 2000; Sasaoka et al., 2001; Elrayess et al., 2003; Song et al., 2003; Elrayess et al., 2004; Listi et al., 2004; Wei et al., 2004; Fang et al., 2005).
In particular, the C373G polymorphism of the PECAM-1 gene has been widely investigated, specially their association with coronary artery disease - CAD (Wenzel et al.; Gardemann et al.; Song et al.; Wei et al.; Fang et al.) and acute myocardial infarction - AMI (Sasaoka et al.; Listi et al.). However, there are no studies to date regarding the association of CAD with C373G polymorphism in American population, so we performed genotyping analyses of C373G polymorphism of PECAM-1 gene in Chilean patients with coronary artery disease and controls, and analyzed its association with the presence of CAD.
MATERIAL AND METHOD
Subjects. A total of 112 unrelated patients (83 men and 29 women), with diagnosis of coronary artery disease (CAD), defined as severe coronary artery stenosis by angiography (Stenosis >70%), admitted to the Cardiology Service of the Hernan Henriquez Hospital of Temuco City, Chile, were included in this study.
The control group, included 108 unrelated individuals (59 men and 49 women) without history or clinical evidence of CAD at the moment of participated on this study.
In both groups, there was no preselection of serum lipid levels. Demographic data and history of hypertension, diabetes mellitus, cigarette smoking, and hypercholesterolemia were assessed in each subject during a personal interview. Subjects with a history of diabetes or basal glycemia ?126mg/dl were defined as diabetic (American Diabetes Association, 2006).
The study protocol was approved by the Ethics Committee of the University of La Frontera, and all subjects gave written informed consents.
Biochemical determinations. Blood samples were obtained by venipuncture after overnight fast (>12h). Total Cholesterol and triglycerydes were determines by enzymatic-colorimetric assay (Fossati et al., 1982; Fossati et al., 1987). High-density lipoprotein cholesterol (HDL-C) concentrations were measured by enzymatic assay after phosphotungstic acid and magnesium precipitation of Low density lipoprotein cholesterol (LDL-C) and Very low density lipoprotein cholesterol VLDL-C (Burstein et al., 1970). LDL-C concentration was calculated using the Friedewald equation (Friedewald et al., 1972). Serum levels of glucose and uric acid were determined by enzymatic colorimetric methods (Barham et al., 1972; Fossati et al., 1980).
Genotypification of C373G polymorphism of the PECAM-1 gene. Genomic DNA was extracted using a procedure described by Salazar et al. (2001). The C373G polymorphism of PECAM-1 gene was detected by polymerase chain reaction followed restriction fragments length polymorphism (PCR-RFLP). A 504-bp fragment was amplified using the following oligonucleotide primers: sense 5'-CTA TCA GCC TGG CCC TGT AG-3' and antisense 5'TAT TCA CGC CAC TGT GTG CT-3', previously described (Wei et al.).
The PCR conditions were: initial denaturation at 98?C for 3 min; the amplification was performed in 30 cycles consisting of 1 min at 94?C, 1 min at 60?C and 1 min at 72?C, and additionally a final extension of 10 min. PCR products were evaluated on 1% agarose gel, stained with ethidium bromide (0.5 mg/dl) and visualized on a UV transilluminator. PCR products were digested with PvuII (Fermentas, Lithuania), in a total reaction volume of 20 ml. The enzymatic digestions were carried out at 37?C for 12 hours, and the restriction fragments were separated on 2% agarose gel and visualized on a UV transilluminator. The wild type genotype (CC) was identified by the presence of two fragments of 430 and 74 bp; mutated heterozygote genotype (CG) by three fragments of 504, 430 and 74 bp, and mutated homozygote genotype (GG) by a fragment of 504 bp (Fig. 1).
Quality control of biochemical and molecular determinations. The accuracy of biochemical determinations were evaluated by the use of normal and pathological commercial controls (Human, Germany). The possibility of contamination on molecular analysis was excluded by use of reactive controls on each amplification series. The correct assessment of genotype for C373G polymorphism at the PECAM-1 gene was confirmed by the randomly repetition of 10% of carry out analyses.
Statistical analysis. Statistical analysis was done using the Sigma Stat Software, version 2.0 (Jandel Sci., San Rafael, CA, USA). Data are presented as mean [+ or -] SD. Differences between the means of the two continuous variables were evaluated by Student t-test. The allelic frequencies and genotype distribution were estimated by gene counting. Differences between noncontinuous variables, genotype distribution, allele frequency, and Hardy-Weinberg equilibrium were tested by chi-square ([x.sup.2]) analysis. The Odds ratio (OR) for CAD and their 95% confidence interval (CI) associated with the G variant was also calculated. Statistical significance was at p < 0.05.
[FIGURE 1 OMITTED]
Table I show the biodemographic characteristics of individuals enrolled in the study. The frequency of men and diabetes mellitus were higher in CAD group (p <0.001). In relation to biochemical parameters, we observed that CAD patients presented a lower HDL-cholesterol levels (p <0.001) and higher glucose and uric acid concentrations (p <0.001). No significant differences were found between the groups for total cholesterol (p = 0.152), triglycerides (p=0.093) and LDL-cholesterol (p=0.868) concentrations.
The genotype frequencies were in agreement with those predicted by the HardyWeinberg equilibrium (Table II). The genotype distribution and the relative allele frequencies for C373G polymorphism of PECAM-1 gene were similar between cases and controls (p=0.820 and p=0.739, respectively). Moreover, the OR associated to the mutated G allele was 0.92 (C.I. 95%, 0.54 - 1.57; p >0.05), confirming the absence of association.
The multifactorial origin of cardiovascular diseases is widely documented (Mensah et al., 2005). The contribution of abnormal concentrations of lipids, glucose and uric acid in the development of coronary disease has been confirmed by several authors, although in the case of uric acid the degree of the contribution remains controversial (Austin et al., 1998; Bickel et al., 2002; Bauters et al., 2003; Tsouli et al., 2006).
When we evaluated the laboratory parameters, was observed that the serum level of glucose and uric acid were higher in the subjects with CAD than controls. In addition, the HDL cholesterol concentrations were lower in this group. These results corroborate the association between these parameters and CAD. Contradictorily, the concentrations of total cholesterol, triglycerides and LDL cholesterol were similar in CAD patients and controls, probably due to use cholesterol-lowering therapy in CAD patients.
In relation to C373G polymorphism of the PECAM1 gene, we not found association between this genetic variant and the presence of CAD in Chilean subjects. The frequency of the mutated G allele was similar between cases and controls (0.549 vs. 0.569, p=0.739). In addition, the OR associated to G allele was not significant (OR= 0.9, CI 95%; 0.54 -1.57). On the other hand, Wenzel et al. (1999) suggest that C373G polymorphism is a risk factor for the development of coronary disease in German subjects. However, one year later in the same population, but with a mayor number of subjetcs, no evidence was found that permits indicate that the C373G polymorphism is an independent risk factor for CAD (Gardemann et al.).
During the year 2003, another investigation realized in subjects from Finnish and German population demonstrates that C373G polymorphism was not associated with the degree of progression of atherosclerosis in patients submitted to coronary bypass surgery (Elrayess et al., 2003). On the other hand, in the same year, the C373G polymorphism was strongly associated with CAD in Chinese population (Song et al., 2003). Similarly, in the years 2004 and 2005, was demonstrated that the allele G (Val) and the homozygous genotype GG (Val/Val) are significantly associated with CAD in subjects from Asia and India (Wei et al.; Fang et al.).
In addition, the possible association between C373G polymorphism of the PECAM-1 gene and AMI has been investigated, and in a similar way to observed with CAD, the findings of these studies are conflicting. In Italian population was not found association between this polymorphism and AMI (Listi et al.). However, in Japanese male a weak association was found, indicating that C373G polymorphism could not be the primary determinant of PECAM-1 association with AMI. This weak association may be in part explained by the linkage disequilibrium with others polymorphisms at the PECAM-1 gene, such as G1688A and/or A2212G which would have a genuine association with AMI. Furthermore, this investigation suggest that another possible explanation for the association of AMI with polymorphisms of PECAM-1 is the interaction with other genes localized near to PECAM1 gene within the chromosome 17, an example of this is the angiotensin converting enzyme gene (Sasaoka et al.).
These ambiguous results may be explained, in some measure, by interethnic differences. Is important to emphasize the mestizo character of Chilean population. The ethnical origin is represented principally by the native (Mapuche) and Spaniard population; furthermore other important ethnic groups (German, French, Italian and Swiss, among others) have been contributed to the genetic background of our population. Thus, is not absurd believe that the frequencies of alleles for the investigated polymorphism and others, have unique characteristics that correspond to the mixture of races previously mentioned.
Other important factor that may be related to the inconsistency in the results from genetic association studies is the sample size. Sample sizes of thousands are required to have adequate power to detect genes conferring ORs as low as 1.2, with minor allele frequencies in the range of 5-10% (Hingorani, 2004). Finally, is important keep in mind that CAD have a multifactorial origin, thus the role of a single nucleotide polymorphism (SNP) can be influenced by the interaction with other genes, in addition to the modulatory effects of the environment.
In summary, our data revealed that C373G polymorphism of PECAM-1 gene is not associated with coronary artery disease in the studied individuals. However, this result cannot be generalized because the heterogeneity of Chilean population.
This study was supported by grants from Direccion de Investigacion y Desarrollo, Universidad de La Frontera (DIDUFRO EP 120541 and PPF 140611).
Received: 01-03-2007 Accepted: 30-03-2007
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Prof. Dr. Luis A. Salazar
Departamento de Ciencias Basicas
Facultad de Medicina, Universidad de La Frontera
Av. Francisco Salazar 01145
Casilla 54-D, Temuco - CHILE
*,**,**** Christian L. Herrera; *,**** Priscilla C. Jaramillo; *** Fernando Lanas & * Luis A. Salazar
* Laboratorio de Biologia Molecular y Farmacogenetica, Depto. de Ciencias Basicas, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
** Departamento de Ciencias Preclinicas, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
*** Departamento de Medicina Interna, Facultad de Medicina, Universidad de La Frontera, Temuco, Chile.
**** Alumnos Programa Doctorado en Ciencias c/m Biologia Celular y Molecular Aplicada, Universidad de La Frontera, Temuco, Chile.
Table 1. Clinical characteristics of the study population Cases Controls (112) (108) Age, years 59.7 [+ or -] 9.9 * 41.7 [+ or -] 9.5 Sex: Male (%) 74.1 * 54.6 Diabetes mellitus (%) 21.4 * 2.8 Total cholesterol 184.2 [+ or -] 54 194.3 [+ or -] 48.9 (mg/dL) LDL cholesterol (mg/dL) 116.3 [+ or -] 37.1 115.4 [+ or -] 41.5 HDL cholesterol (mg/dL) 32.2 [+ or -] 7.8 * 52.1 [+ or -] 12.1 Triglycerides (mg/dL) 182.8 [+ or -] 193.8 144.2 [+ or -] 136.3 Glucose (mg/dL) 111.9 [+ or -] 42.5 * 93.4 [+ or -] 40.3 Uric acid (mg/dL) 5.6 [+ or -] 1.6 * 4.5 [+ or -] 1.5 Number of subjects in parenthesis: * p<0.001 Table II. Genotype distribution and relative allele frequencies of C373G polymorphism at the PECAM-1 gene in Chilean cases and controls. Genotypes Allele CC CG GG C G Cases (112) 16.9% 56.3% 26.8% 0.451 0.549 (19) (63) (30) Controls (108) 16.7% 52.8% 30.5% 0.431 0.569 (18) (57) (33) [chi square] = 0.397 ; 72 = 0.111; 1 df, 2 df; p = 0.820 p = 0.739 Number of individuals in parenthesis: df, degree of freedom. Hardy-Weinberg Equilibrium: Cases: [chi square] = 2.07 (p=NS) Controls: [chi square] = 0.63 (p=NS)