Polymorphism of C3 complement in association with myocardial infarction in a sample of central Tunisia
© Leban et al.; licensee BioMed Central Ltd. 2013
Received: 14 January 2013
Accepted: 4 March 2013
Published: 13 June 2013
Myocardial infarction (MI) is a major clinical problem because of its large contribution to mortality. The genetic bases of this disease have been widely studied in recent years to find a clear association with some genetic markers that increase the risk of its occurrence. In the present investigation, the correlation between MI and the C3 complement polymorphism was analyzed using a case–control study.
Our study ported on one hundred seventy survived myocardial infarction patients and ninety five healthy controls. The C3 allele identification was investigated using the amplification refractory mutation system PCR to determine the C3*S and the C3*F alleles of the C3 polymorphism.
Frequencies of C3*S and C3*F in patients are 0.59 and 0.41 respectively. Fisher test results showed a significant increase of C3*F allele in the sample of patients (0.41; odds ratio: 2.616; C.I [1.738-3.938]) compared to controls (0.21; odds ratio: 0.382; 95% CI [0.254-0.575]), p = 2.742 × 10-6.
A strong positive correlation was found between C3 polymorphism and MI estimating that the risk of myocardial infarction is significantly increased among patients with C3*F allele of this polymorphism.
The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1190484203893646
Myocardial infarction (MI) is the rapid development of myocardial necrosis caused by a critical imbalance between oxygen supply and demand of the myocardium. This usually results from plaque rupture with thrombus formation in a coronary vessel, resulting in an acute reduction of blood supply to a portion of the myocardium. Important risk factors are: previous cardiovascular disease, older age, tobacco smoking, high blood levels of certain lipids (triglycerides, low-density lipoprotein) and low levels of high density lipoprotein (HDL), diabetes, high blood pressure, obesity, chronic kidney disease, heart failure, excessive alcohol consumption, abuse of certain drugs, Thiamine deficiency (beriberi), and chronic high stress levels [1–3]. MI is a major clinical problem because of its large contribution to mortality. According to the World Health Organization, heart attacks are among the most important cause of death for both men and women all over the world.
List of genetic markers analyzed for the association with myocardial infarction
−8C > G
21993223 G > A
7543947C > G
−2518A > G
The C3 complement component polymorphism was often used as a powerful marker for genetic studies of populations , since it is biallelic and codominant single nucleotide polymorphism, the complement system has a critical role in both the innate and the adaptive immune responses . In humans, C3*F (C3102G) has been associated with autoimmune diseases [21, 22]. Few researches are carried out in the literature on the correlation between C3 polymorphism and MI. This relationship has been studied most often on the serum level. Previous studies showed that the C3 serum protein is correlated with a set of risk factors for MI . Considering the pathophysiological data, it seems that the carriers of the C3*F allele are able to control inflammatory diseases to a lesser extent than non-carriers may do. Therefore, in the case of C3*F carriers, there may be a higher likelihood of inflammation within the vulnerable plaques that may facilitate their rupture  and the consequential development of myocardial infarction. The more common C3*S allele may have an effect of opposite direction that protects against the development of MI . C3 could be considered as a factor screening primary prevention of myocardial infarction . In this context, we are interested to analyze the association between the single-nucleotide polymorphism in the C3 complement component gene and the risk of developing MI in the Tunisian population. To achieve this purpose, we carried out a case control study. This research falls within the framework of improving the strategy for prevention of this disease. By identifying the genetic markers that may increase the risk of its occurrence, it would be possible to carry out the interventions needed to reduce the possibility of future events.
This work was performed on 170 blood samples of patients aged between 44 and 83 years (mean age: 65,48 ±10,91 years) who have survived to myocardial infarction followed in the cardiology departments at the University Hospitals of Sousse, Monastir and Mahdia. For blood collect, we have obtained approval from the Hospital authority and the ethics committee after consent of patients. For each patients, we reported the body mass index, the triglycerides, HDL-c, LDL-c and cholesterol concentrations, the risk factors (mainly smoking), and the history of the disease (such as diabetes, arterial hypertension, dyslipidemia,…). Ninety-five healthy Tunisian adult blood donors were included in the study as control sample.
DNA extraction was performed on 5 ml of peripheral-blood samples collected on EDTA from patients and controls using the standard phenol/chloroform method. Amplification Refractory mutation System (ARMS) PCR  was used for the two groups to determine the C3*S and C3*F alleles. The technique requires that the terminal 3'-nucleotide only of a PCR primer be allele specific. Thus the primer is synthesized in two forms. The normal form (C3*S primer) is refractory to PCR on mutant template DNA (C3*F) and vice versa. Introducing additional deliberate mismatches near the 3' end of appropriate primers does not allow non specific amplification to proceed, that’s why we preferred this method which seems more convenient for genotyping than enzymatic digestion which requires two reactions to be performed.
In the ARMS reaction, three primes were used: one common reverse primer (5′-TGTTGACCATGACCGTCCGGCCCACGGTA-3′) and two forward specific primers (5′-CCAACAGGGAGTTCAAGTCAAAAGGTGG-3′ for C3*S and 5′-CCAACAGGGAGTTCAAGTCAGAAAAGGTGC-3′ for C3*F). For each DNA, the amplification is performed twice on a final volume of 25 μl containing 100 ng of DNA, 0.6 μM of each primer, 3 mM of MgCl2, 0.32 mM of each dNTP and 1.5 U of Taq polymerase (Promega). The reaction is performed on an Appligene Oncor thermocycler under the following conditions: 2 min at 94°C, 33 cycles of 30 s at 94°C, 1 min at 50°C, 1 min at 72°C and finally, one cycle of 10 min at 72°C. PCR products were revealed on 2% agarose gel electrophoresis and visualized under UV light after ethidium bromide staining. Genotypes are determined for all individuals and frequencies are calculated for the two alleles.
Allele frequencies are compared in the samples of MI patients and controls using Epi info 6 software (Fisher test).
Association test between C3 polymorphism and myocardial infarction by comparing genotype and allele frequencies of C3*S and C3*F in patients and controls
Odds ratio = 0.382
p = 2.742 × 10-6
C.I. = [0.254-0.575]
Odds ratio = 2.616
C.I. = [1.738-3.938]
C3*S allele frequency is higher than C3*F in the two populations (controls and patients). For the C3*F allele, the frequency found in patients (0.41) is 2 times higher than that found in controls (0.21). Thus, the C3*S allele frequency is reduced (0.59) in the sample of patients compared to that of controls (0.79).
Fisher test results showed a significant increase of C3*F allele in the sample of patients (p = 2.742 × 10-6). Therefore, it is estimated that the risk of myocardial infarction is significantly increased among patients with the C3*F polymorphism (Table 2).
Our results about the allelic frequencies of the donors sample are compared to those of previous study in which sera from 95 healthy donors (representative of the Tunisian general population) are phenotyped using agarose gel electrophoresis . The similarity observed between C3*S and C3*F frequencies in the two studies strengthens our findings.
Our results showed a strong association between C3*F polymorphism and the risk of the MI since the probability P is 2.742 × 10-6 (<0.05). Based on these results, homozygous for the C3*S allele are more protected against the occurrence of myocardial infarction. In the contrary, this risk is highly increased for homozygous C3*F individuals.
In the literature, only few associations between the C3 component polymorphism and myocardial infarction have been analyzed. In one of them, carried out on the territory of Delhi (India), the difference between MI cases and controls was found to be statistically non-significant indicating that, in this Indian population, the C3 marker is not associated with the risk of myocardial infarction . Another study was a C3 phenotype serum investigation in patients with heart infarct. Despite differences in the percentage distribution of various phenotypes between the patients’ sera and those of controls, no significant association could be established . However, a positive correlation was found in the Hungarian population in which the C3*F allele may be associated with an increased risk of developing myocardial infarction in coronary heart disease patients .
How explain the implication of the variant C3F in the diseases development?
Number of studies suggests that functional differences exist between C3 complement allotypes F and S in the pathogenesis of MI and other diseases with a higher risk found in individuals with allotype F. Hemolytic activity is slower for C3*F than for C3*S products . It has been shown that erythrocytes encoded with C3*F are surrounded by much more bearing monocytes than C3*S .
The C3F variant bound Factor H Complement (CFH) less than C3S, causing decreased Factor I Complement cofactor activity, and enhanced C3 alternative pathway amplification. By combining risk and protective alternative pathway variants (activators and regulators), it has been shown that C3, CFH, and Factor B variants collaborate to set levels of alternative pathway of systemic complement activity in plasma, thereby influencing risk in complement-dependent diseases . However, no difference between C3*F and C3*S forms in their binding to complement receptors types 1, 2 and 3 was shown . In addition, in vitro experimental evidence showing functional differences between the two forms of C3 is not conclusive .
In conclusion, our study showed a strong association between the C3*F variant and MI, (0.41 versus 0.21 with p = 2.742 × 10-6). C3*F appears to have a causal role in increasing the risk of occurrence of this disease. These results add to our understanding of the genetic factors of myocardial infarction and provide evidence that the C3 complement component may be a predisposing factor for this common disease. The determination of C3 polymorphism may help in the identification of patients at risk for developing MI.
We thank Professor Gérard Lefranc from the University Montpellier 2 for his help and for providing some reagents for the molecular analysis.
- Pearte CA, Furberg CD, O’Meara ES, Psaty BM, Kuller L, Powe NR, Manolio T: Characteristics and baseline clinical predictors of future fatal versus nonfatal coronary heart disease events in older adults: the Cardiovascular Health Study. Circ. 2006, 113 (18): 2177-2185. 10.1161/CIRCULATIONAHA.105.610352.View ArticleGoogle Scholar
- Bax L, Algra A, Mali WP, Edlinger M, Beutler JJ, van der Graaf Y: Renal function as a risk indicator for cardiovascular events in 3216 patients with manifest arterial disease. Atherosclerosis. 2008, 200: 184-10.1016/j.atherosclerosis.2007.12.006.View ArticlePubMedGoogle Scholar
- Bello S, Neri M, Riezzo I, Othman MS, Turillazzi E, Fineschi V: Cardiac beriberi: morphological findings in two fatal cases. Diagn Pathol. 2011, 6: 8-10.1186/1746-1596-6-8.PubMed CentralView ArticlePubMedGoogle Scholar
- Botto M, Fong KY, So AK, Koch C, Walport MJ: Molecular basis of polymorphisms f human complement component C3. J Exp Med. 1990, 172: 1011-1017. 10.1084/jem.172.4.1011. etView ArticlePubMedGoogle Scholar
- Agarwal DP, Srivastava LM, Benkmann HG, Goedde HW: Studies on the polymorphism of C3, Tf and Bg in Down’s syndrome and other diseases. Humangenetik. 1975, 29 (1): 23-28. 10.1007/BF00273347.PubMedGoogle Scholar
- Golabi P, Kshatriya GK, Kapoor AK: Association of genetic markers with coronary heart disease (myocardial infarction)–a case–control study. J Indian Med Assoc. 1999, 97 (1): 6-7.PubMedGoogle Scholar
- Császár A, Duba J, Melegh B, Kramer J, Szalai C, Prohászka Z, Karádi I, Kovács M, Méhes K, Romics L, Füst G: Increased frequency of the C3*F allele and the Leiden mutation of coagulation factor V in patients with severe coronary heart disease who survived myocardial infarction. Exp Clin Immunogenet. 2001, 18 (4): 206-212. 10.1159/000049199.View ArticlePubMedGoogle Scholar
- Börgel J, Bulut D, Hanefeld C, Neubauer H, Mügge A, Epplen JT, Holland-Letz T, Spiecker M: The CYP2J2 G-50 T polymorphism and myocardial infarction in patients with cardiovascular risk profile. BMC Cardiovasc Disord. 2008, 8: 41-10.1186/1471-2261-8-41.PubMed CentralView ArticlePubMedGoogle Scholar
- Douvaras P, Antonatos DG, Kekou K, Patsilinakos S, Chouliaras G, Christou A, Andrikou A, Kanavakis E: Association of VEGF gene polymorphisms with the development of heart failure in patients after myocardial infarction. Cardiology. 2009, 114 (1): 11-18. 10.1159/000210189.View ArticlePubMedGoogle Scholar
- Xie X, Ma YT, Fu ZY, Yang YN, Ma X, Chen BD, Wang YH, Liu F: Association of polymorphisms of PTGS2 and CYP8A1 with myocardial infarction. Clin Chem Lab Med. 2009, 47 (3): 347-352.View ArticlePubMedGoogle Scholar
- Pei F, Han Y, Zhang X, Yan C, Huang M, Huang L, Kang J: Association of interleukin-18 gene promoter polymorphisms with risk of acute myocardial infarction in northern Chinese Han population. Clin Chem Lab Med. 2009, 47 (5): 523-529.View ArticlePubMedGoogle Scholar
- Liu X, Wang X, Shen Y, Wu L, Ruan X, Lindpaintner K, Yusuf S, Engert JC, Anand S, Tan X, Liu L: The functional variant rs1048990 in PSMA6 is associated with susceptibility to myocardial infarction in a Chinese population. Atherosclerosis. 2009, 206 (1): 199-203. 10.1016/j.atherosclerosis.2009.02.004.View ArticlePubMedGoogle Scholar
- Yang XC, Zhang Q, Chen ML, Li Q, Yang ZS, Li L, Cao FF, Chen XD, Liu WJ, Jin L, Wang XF: MTAP and CDKN2B genes are associated with myocardial infarction in Chinese Hans. Clin Biochem. 2009, 42 (10–11): 1071-5.View ArticlePubMedGoogle Scholar
- Hanasaki H, Takemura Y, Fukuo K, Ohishi M, Onishi M, Yasuda O, Katsuya T, Awata N, Kato N, Ogihara T, Rakugi H: Fas promoter region gene polymorphism is associated with an increased risk for myocardial infarction. Hypertens Res. 2009, 32 (4): 261-264. 10.1038/hr.2009.2.View ArticlePubMedGoogle Scholar
- Xia DS, Guo QY, Liu YQ, Li C, Zhang F, Wei MX: Association of serotonin transporter gene linked polymorphic region polymorphism with early onset myocardial infarction and platelet membrane glycoprotein Ib. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2009, 26 (1): 31-34.PubMedGoogle Scholar
- Oztürk O, Oztürk U: Relation between angiotensin-converting enzyme I/D gene polymorphism and pulse pressure in patients with a first anterior acute myocardial infarction. Anadolu Kardiyol Derg. 2009, 9 (1): 9-14.PubMedGoogle Scholar
- Wiedmann S, Neureuther K, Stark K, Reinhard W, Kallmünzer B, Baessler A, Fischer M, Linsel-Nitschke P, Erdmann J, Schunkert H, Hengstenberg C: Lack of Association Between a Common Polymorphism Near the INSIG2 Gene and BMI, Myocardial Infarction, and Cardiovascular Risk Factors. Obesity (Silver Spring). 2009, 17 (7): 1390-5.Google Scholar
- Zhang X, Han Y, Kang J, Yan C: A monocyte chemoattractant protein-1 gene polymorphism is not associated with coronary artery disease in a Han Chinese population. Clin Chim Acta. 2009, 403 (1–2): 241-243.View ArticlePubMedGoogle Scholar
- Davrinche C, Rivat C: Polymorphism of the third component of human complement. Rev Fr Transfus Immuno hematol. 1982, 5 (2): 199-213.View ArticleGoogle Scholar
- Bickerstaff MCM, Botto M, Hutchinson WL, Herbert J: Tennent GA et Bybee A: Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity. Nat Med. 1999, 5: 694-697. 10.1038/9544.View ArticlePubMedGoogle Scholar
- Puttick AH, Briggs DC, Welsh KI, Williamson EA, Jacoby RK, Jones VE: Genes associated with rheumatoid arthritis and mild inflammatory arthritis. Association of HLA with complement C3 and immunoglobulin Gm allotypes. Ann Rheum Dis. 1990, 49: 225-8. 10.1136/ard.49.4.225.PubMed CentralView ArticlePubMedGoogle Scholar
- Persson U, Truedsson L, Westman KWA, Segelmark M: C3 and C4 allotypes in anti-neutrophil cytoplasmic autoqntibody (ANCA)-positive vasculitis. Clin Exp Immunol. 1999, 116: 379-382. 10.1046/j.1365-2249.1999.00889.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Muscari A, Massarelli G, Bastagli L, Poggiopollini G, Tomassetti V, Volta U, Puddu GM, Puddu P: Relationship between serum C3 levels and traditional risk factors for myocardial infarction. Acta Cardiol. 1998, 53 (6): 345-354.PubMedGoogle Scholar
- Kullo IJ, Edwards WD, Schwartz RS: Vulnerable plaque: Pathobiology and clinical implications. Ann Intern Med. 1998, 129: 1050-1060. 10.7326/0003-4819-129-12-199812150-00010.View ArticlePubMedGoogle Scholar
- Muscari A, Massarelli G, Puddu GM, Sangiorgi Z, Dormi A, Bagnoli M, Gaddi A, Puddu P: Serum C3 as a screening factor in the primary prevention of myocardial infarct. Cardiologia. 1995, 40 (7): 507-514.PubMedGoogle Scholar
- Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalshekerl N, Smith JC, Markham AF: Analysis of any point mutation in DNA. The ampliflcation refractory mutation system (ARMS). Nucleic Acids Res. 1989, 17 (7): 2503-2516. 10.1093/nar/17.7.2503.PubMed CentralView ArticlePubMedGoogle Scholar
- Chibani J, Lefranc G, Constans J: Serum protein polymorphism among Tunisian Berbers: Haptoglobulin, transferrin and group specific component subtypes C3 and BF types. Ann human Biol. 1985, 12: 449-462. 10.1080/03014468500008011.View ArticleGoogle Scholar
- Kristensen BP, Petersen GB: Association between coronary heart disease and the C3F-gene in essential hypertension. Circulation. 1978, 58 (4): 622-625. 10.1161/01.CIR.58.4.622.View ArticlePubMedGoogle Scholar
- Messias-Reason IJ, Urbanetz L, Pereira da Cunha C: Complement C3 F and BF S allotypes are risk factors for Chagas disease cardiomyopathy. Tissue Antigens. 2003, 62 (4): 308-312. 10.1034/j.1399-0039.2003.00101.x.View ArticlePubMedGoogle Scholar
- Suadicani P, Hein HO, Finn G: Complement C3 genotype variants and risk of lung cancer mortality. ISRN Pulmonology. 2011, doi:10.5402/2011/423571.Google Scholar
- Yates JRW, Sepp T, Matharu BK, Khan JC, Thurlby DA, Shahid H, Clayton DG, Hayward C, Morgan J, Wright AF, Armbrecht AM, Dhillon B, Deary IJ, Redmond E, Bird AC, Moore AT: Complement C3 variant and the risk of age-related macular degeneration. J Med. 2007, 357: 553-561.Google Scholar
- Despriet DD, van Duijn CM, Oostra BA, Uitterlinden AG, Hofman A, Wright AF, ten Brink JB, Bakker A, de Jong PT, Vingerling JR, Bergen AA, Klaver CC: Complement component C3 and risk of Age-Related Macular Degeneration. Ophtalmology. 2009, 116 (3): 474-480. 10.1016/j.ophtha.2008.09.055.View ArticleGoogle Scholar
- Park KH, Fridley BL, Ryu E, Tosakulwong N, Edwards AO: Complement Component 3 (C3) Haplotypes and Risk of Advanced Age-Related Macular Degeneration. Invest Ophthalmol Vis Sci. 2009, 50 (7): 3386-93. 10.1167/iovs.08-3231.View ArticlePubMedGoogle Scholar
- Welch TR, Beischel L, Kleesattel A: Functional consequences of the genetic polymorphism of the third component of complement. J Pediatr. 1990, 116 (Suppl): S92-S97.View ArticlePubMedGoogle Scholar
- Arvilommi H: Capacity of complement C3 phenotypes to bind on to mononuclear cells in man. Nature. 1974, 251: 740-741. 10.1038/251740a0.View ArticlePubMedGoogle Scholar
- Heurich M, Martínez-Barricarte R, Francis NJ, Roberts DL: Rodríguez de Cordoba S, Morgan BP and Harris CL: Common polymorphisms in C3, factor B, and factor H collaborate to determine systemic complement activity and disease risk. PNAS. 2011, 108 (21): 8761-8766. 10.1073/pnas.1019338108.PubMed CentralView ArticlePubMedGoogle Scholar
- Bartók I, Walport MJ: Comparison of the binding of C3S and C3F to complement receptors types 1, 2 and 3. J Immunol. 1995, 154: 5367-5375.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.