Biomarkers and Applications (ISSN: 2576-9588)

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Polymorphisms in Acute Coronary Syndrome

Viviane do Carmo Vasconcelos de Carvalho1, Maria José Ribeiro Bezerra1, Lílian Caroliny Amorim Silva1, Alex José de Melo Silva1, Fábia Carla da Silva Soares1, Sávio Augusto Vieira de Oliveira1, Roberto Pereira Werkhauser1, Carlos Gustavo Regisda Silva2, Clarice Neuensch wander Lins de Morais3, Adriana Vieira Gomes4, Silvia Maria Lucena Montenegro1*

 *1Department of Immunology, Aggeu Magalhães Institute, Brazil

2 Laboratory of Parasitic Biology, Gonçalo Moniz Research Center, Brazil

3Department of Virology, Aggeu Magalhães Institute, Brazil

4 Faculty of Medical Sciences , University of Pernambuco, Brazil

*Corresponding author: Silvia Maria Lucena Montenegro, Department of Immunology, Aggeu Magalhães Institute, Brazil, Tel: +55 (81) 2101-2565; Email: silvia@cpqam.fiocruz.br

Received Date: 15 June, 2017; Accepted Date: , 18 June, 2017; Published Date: 22 June, 2017

Citation:  Vasconcelos de Carvalho VDC, Bezerra MJR, Silva LCA, Melo Silva AJD, Silva Soares FCD, et al. (2017) Polymorphisms in Acute Coronary Syndrome. Biomark Applic 2017:  BMAP104. DOI: 10.29011/BMAP-104. 100104

Background: Because of their effects on inflammatory processes, some genes are candidates for association with Acute Coronary Syndrome (ACS). While a relationship between Single Nucleotide Polymorphisms (SNP) and ACS has been suggested in some populations, but not in others, genotype characterization of genes related to inflammatory reactions, becomes necessary for specific populations.

Aim: In this study, we evaluate IL6 (rs1800795), LEPR (rs6700896) and IL1b (rs16944) SNPs in a healthy population and compare the frequencies with other populations to characterize them in a region of Brazil and investigate the role of SNPs as prognostic markers in the development of ACS in futures studies.

Methods: IL6, LEPR and IL1b genes were genotyped in 295 blood donors from Fundação de Hematologia e Hemoterapia de Pernambuco. Genotyping was carried out by polymerase chain reaction, followed by DNA sequencing or enzymatic cleavage. G Williams test and odds ratio with confidence intervals of 95% were used. The significance value was p < 0.05.

Results: There was no difference in genotype distribution between gender or age groups. Genotypic and allelic frequencies were similar between Brazilian populations but different from most other population’s analyzed (p < 0.05).

Conclusions: Genotypic characterization of genes that can influence the development of ACS is important, since it can provide a tool for future studies in the use of genetic markers in prevention and diagnosis of diseases.

Keywords: Acute Coronary Syndrome; Blood Donors; Prognostic Markers; Polymerase Chain Reaction; Single Nucleotide Polymorphisms

1. Discussion

 In this study, it is hypothesized that the presence of polymorphisms in the IL6, LEPR and IL1 genes may contribute to the development of ACS, considering that these polymorphisms may exacerbate inflammatory responses and contribute to the formation of atherosclerosis. Blood donors have been selected for control groups in genetic studies as it is assumed that this group represents the general population. Authors have observed [26,27] that most randomly recruited blood donors are male, in agreement with our study. Swirta et al. (2015) [27] studying the genetic influence on coronary artery disease (CAD),  recruited Polish blood donors as control group and verified that 85.3% were male with 34.0 years old of mean age, lower than our study (47 years old). Although our results indicated no risk association between gender and / or age in developing ACS with the studied polymorphisms, this possibility should not be excluded [28,29] Chiappelli et al. (2005) [30], in a study with Italian men over 67 years old, associated IL6 C allele with AMI and suggested that this association is age-dependent. Perhaps due to the fact that adults over 65 years were not included in the present study, we were unable to observe significant differences between the frequency of IL-6 gene polymorphism and gender (p = 0.63) or age (p = 0.20). In contrast, Jin et al. (2015) [22] found no association between LEPR polymorphism and susceptibility to cardiovascular disease in Chinese men and women of 59 years old, which support our finding, despite our mean age is lower. Besides, IL1 gene polymorphism has been associated with atherosclerosis in some populations [9,29]. Iacoviello et al. (2005) [29] stated that TT genotype conferred 64% less risk of AMI development in younger women. Also, Rios et al. (2010) [9] found that CC genotype increases the risk of CAD in individuals between 50 and 56 years old.

 When comparing the genotype frequencies among Brazilian individuals from different regions, we can observe that, despite the intense miscegenation between them, the genetic characteristics in relation to the SNPs evaluated remained similar. In the North East population of this study, the genotype distributions of IL-6 gene were different (p ≤ 0.0004) when compared to other populations, although a similarity was demonstrated in relation to the population from Amazon (p = 0.69). CC genotype was less frequent (5.3%) than in Portugal (12.5%), Malaysia (7.0%) and Italy (14.3%) populations. In a study with Pakistani patients, Satti et al. (2013) [30] demonstrated that the frequency of CC genotype, IL-6 and C - reactive protein plasma levels were higher in CAD patients than in control subjects (p = 0.0025). LEPR genotype distributions from our study were different when compared to China and Egypt populations (p < 0, 0001) [12,22]. T minor allele has been associated with a significant increase in the risk of CAD [8] which was not corroborated by Jin et al. (2015) [22], since they found no association between T allele and cardiovascular disease in Chinese patients. Interestingly, there was no evidence of this allele in the study by Swellan et al. (2012) [12] in Egyptian healthy individuals. Regarding to IL1 gene, our results were similar to genotype distributions in Rio de Janeiro (p = 0.08) [16] and CT genotype was the most frequent in both population (22.4% and 9.0%, respectively). 

 The CC genotype in IL1 gene was most frequent in Portugal (44.5%), Germany (45.8%) and Italy (43.9%) populations and is associated with an higher risk for CAD because it increases levels of the corresponding inflammatory cytokine, contributing  to the progression of atherosclerotic plaque [29].The genotypic distributions evaluated in the blood donor of the present study, when compared to other populations, showed the diversity of the SNPs frequencies in other countries, evidencing the importance of genetic studies in specific population groups to establish a cause-effect relationship between genes and diseases.     

 2. Conclusions

 Knowledge regarding the frequencies of genetic polymorphisms in a particular population may be a very useful tool for understanding and assessing the risk of development of several diseases. The investigation of risk factors, as well as genetic markers for ACS, is essential for prevention, diagnosis and treatment, and can reduce morbidity and mortality. These findings may be employed for future association studies regarding these polymorphisms with the inflammatory nature of diseases. One further explanation for the differences found with other populations may be the different cultural and ethnic backgrounds, which were not analyzed. Results showed no future risk of ACS development when using IL6, LEPR and IL1 markers. However, it was not possible to identify the presence of other risk factors for ACS, apart from hypertension and diabetes, in recruited blood donors.

A study of data of Dyslipidemia, smoking, alcohol, physical inactivity and family history would be important in order to relate with genetic polymorphisms, and thus provide a better assessment of the risk of developing ACS.

 3. Acknowledgments

The authors thank the Instituto Aggeu Magalhães/Fiocruz core-facilities laboratories for the use of its technologies, the blood donors from Hemope who agreed to participate in this study and Audrey Violeta Martins de Vasconcelos for allowing access to Hemope.


Polymorphisms

Primers

Amplification conditions

Fragments sizes (bp)

References

IL6 (rs1800795)

F: 5’ AGC CTC AAT GAC GAC CTA AGC 3’

R: 5’ ACT GGA GAT GTC TGA GGC TCA TT 3’

94°C – 2 min

94°C – 1 min

 

35X

65°C – 1 min

68°C – 1 min

65°C – 5 min

 

226

 [10] Adapted

LEPR (rs6700896)

F: 5´ GCC CTT CTT TCC TCA AGC CTT CC 3’

 

R: 5’ GCT CCA AAG CCA GAC AAA CTG GT 3’

95°C – 5 min

 95°C – 30 seg

 68°C – 30 seg 55°C – 30 seg

68°C – 5 min

 

515

[12] Adapted

IL1 (rs1694)

F: 5’ TGG CAT TGA TCT GGT TCA TC 3’

 

R: 5’ GTT TAG GAA TCT TCC CAC TT 3’

94°C – 5 min

 

35X

 94°C – 60 seg

 55°C – 40 seg

 72°C – 40 seg

74°C – 7 min

 

304

 

[13] Adapted


Table 1: Sequence of primers and amplification conditions for each polymorphism

 

Polymorphisms

Gender n = 295

p

Age n = 295

p

Genotypes

Male                 n = 244 (%)

Female          n = 51 (%)

47 years            n = 179 (%)

> 47 years           n = 116 (%)

IL6  (rs  1800795)

 

 

 

 

 

 

GG

148 (60.6)

28 (55.0)

0.63

103 (57.5)

75 (64.6)

0.2

GC

84 (34.4)

19 (37.2)

68 (38.0)

33 (28.5)

CC

12 (5.0)

4 (7.8)

8 (4.5)

8 (6.9)

Alleles

 

 

 

 

 

 

G

380 (77.9)

75 (73.5)

 

274 (76.5)

183 (78.9)

 

C

108 (22.1)

27 (26.5)

 

84 (24.5)

49 (21.1)

 

LEPR  (rs 6700896)

 

 

 

 

 

 

CC

73 (29.9)

23 (45.1)

0.09

60 (33.5)

36 (31.0)

0.96

CT

133 (54.5)

20 (39.2)

94 (52.5)

59 (50.9)

TT

38 (15.6)

08 (15.7)

25 (14.0)

21 (18.1)

Alleles

 

 

 

 

 

 

C

279 (57.2)

66 (64.7)

 

214 (67.3)

131 (56.5)

 

T

209 (42.8)

36 (35.3)

 

144 (40.2)

101 (43.5)

 

 IL1                (rs 16944)

 

 

 

 

 

 

CC

75 (30.8)

13 (30.2)

0.96

57 (31.8)

33 (28.4)

0.44

CT

114 (46.7)

21 (48.8)

87 (48.6)

53 (45.7)

TT

55 (22.5)

9 (21.0)

35 (19.6)

30 (25.9)

Alleles

 

 

 

 

 

 

C

264 (54.1)

47 (46.1)

 

201 (56.1)

119 (53.3)

 

T

224 (45.9)

39 (38.2)

 

157 (43.8)

113 (48.7)

 

 

                                                                                                                p - G Williams Test

Table 2: Genotypic frequencies of polymorphisms in IL-6, IL-1, LEPR and IL-1 according to gender and age

 

 

Polymorphisms

Country

n

Genotype frequencies   n(%)

p

Allele frequencies n(%)

p

 

 

 

GG

GC

CC

 

G

C

 

 IL6 (rs1800795)

Brazil - NorthEast

295

176 (59.4)

103 (35.3)

16 (5.3)

Reference

455 (77.1)

135 (22.9)

Reference

Brazil - Amazon

77

49 (63.64)

23 (29.87)

5 (6.49)

0.6934

121 (78.6)

33 (21.4)

0.7005

Portugal

735

319 (43.4)

324 (44.1)

92 (12.5)

< 0.0001

962 (65.4)

508 (34.6)

< 0.0001

Malaysia

100

12 (12.0)

81 (81.0)

7 (7.0)

< 0.0001

105 (52.5)

95 (47.5)

< 0.0001

Italy

112

45 (40.2)

51 (45.5)

16 (14.3)

0.0005

141 (62.9)

83 (37.1)

< 0.0001

China

331

329 (99.4)

1 (0.3)

1 (0.3)

< 0.0001

659 (99.5)

3 (0.5)

< 0.0001

Mexico

102

80 (78.4)

20 (19.6)

2 (2.0)

0.002

180 (88.2)

24 (11.8)

0.0004

 

 

 

CC

CT

TT

 

C

T

 

LEPR (rs6700896)

Brazil - NorthEast

295

96 (32.5)

153 (51.9)

45 (15.6)

Reference

345 (58.5)

243 (41.2)

Reference

China

109

5 (4.6)

24 (22.1)

80 (73.4)

< 0.0001

34 (15.6)

184 (84.4)

< 0.0001

Egypt

30

30 (100.0)

0 (0.0)

0 (0.0)

< 0.0001

60 (100.0)

0 (0.0)

< 0.0001

 

 

 

CC

CT

TT

 

C

T

 

IL1   (rs16944)

Brazil - NorthEast

295

91 (30.8)

138 (46.8)

66 (22.4)

Reference

320 (54.2)

270 (45.8)

Reference

Brazil - Rio de Janeiro

44

17 (38.7)

23 (52.3)

4 (9.0)

0.0875

57 (64.8)

31 (35.2)

0.062

Portugal

735

327 (44.5)

309 (42.0)

99 (13.5)

< 0.0001

963 (65.5)

507 (34.5)

< 0.0001

Germany

94

43 (45.8)

41 (43.6)

10 (10.6)

0.0051

127 (67.5)

61 (32.5)

0.0012

Italy

205

90 (43.9)

89 (43.4)

26 (12.7)

0.002

269 (65.6)

141 (34.4)

0.0003

Malaysia

60

13 (21.6)

22 (36.7)

25 (41.7)

0.01

48 (40.0)

72 (60.0)

0.0045

Korea

364

61 (16.8)

194 (53.3)

109 (29.9)

< 0.0001

316 (43.4)

412 (56.6)

< 0.0001


n - Number of subjects; p - G Williams Test

Table 3: Distribution of genotype frequencies of polymorphisms in different healthy populations 

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