The Role and Significance of Polymorphisms of Certain Genes in Patients with Abdominal Aortic Aneurysm

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LIST OF ABBREVIATIONS

АAА ― abdominal aortic aneurysm

АCE ― angiotensin converting enzyme

AGT ― angiotensin

АH ― arterial hypertension

Ala ― alanin

APOЕ ― apolipoprotein E

Asn ― asparagine

CHD ― coronary heart disease

CI ― confidence interval

COPD ― chronic obstructive pulmonary disease

EDN1 ― endothelin-1

GPs ― gene polymorphisms

ITGA2 ― integrin alpha-2

Leu ― leucine

LpL ― lipoprotein lipase

Lys ― lysine

Met ― methionine

NOS3 ― nitric oxide synthase 3

OATP1B1 ― organic anion transporting polypeptide, 1B1

OR ― odds ratio

Pro ― proline

SELP ― P-selectin

Ser ― serin

SLC01B1 ― solute carrier organic anion transporter family member 1B1

Ter ― termination codon

Thr ― tryptophan

Val ― valinl

INTRODUCTION

Despite a long history of investigation of the etiopathogenesis of the abdominal aortic aneurysm (AAA), many factors that influence the risk and course of the disease, have not been studied up to the present moment. It has been shown in some works that patients with AAA have a number of concomitant diseases mostly associated with disorders of lipid metabolism, vascular endothelial dysfunction, arterial hypertension, diabetes mellitus [1, 2]. However, their role in the pathogenesis of AAA is still being studied, and the results are controversial. Besides, increasing significance in the etiology and pathogenesis of AAA is assigned to some genetic polymorphisms, especially to the risk factors of the development of AAA [3–12]. With this, very few scientific works are devoted to study of gene polymorphisms (GPs) playing a definite role in the development of a number of concomitant pathologies in AAA. In this context, we studied some GPs in patients with AAA in the aspect of their probable influence on the pathogenesis of the disease.

The aim of this study was analyze the existence of association of the abdominal aortic aneurysm with some gene polymorphisms.

MATERIALS AND METHODS

The study was approved by the local Ethics Committee of Pavlov Ryazan State Medical University (Protocol No. 11 of 2021, May 11) and registered on ClinicalTrials.gov platform. All the patients signed a written informed consent to participate in this study.

GPs were analyzed in 20 patients with AAA (study group) and in 5 patients without AAA (control group). Of the total number of patients with AAA (study group) there were 18 (90%) of men, 2 (10%) women. The control group included 4 (80%) men and 1 (20%) woman. The mean age was 68.1 ± 7.3 and 64.2 ± 7.2 years in the study group and the control group, respectively.

In the study group there were 17 (85%) smokers, the main concomitant diseases were:

- coronary heart disease (CHD) in 11 (55%) patients;

- diabetes mellitus in 1 (5%) patient;

- carotid artery atherosclerosis/stroke in 4 (20%) patients;

- peripheral artery diseases in 9 (45%) patients;

- arterial hypertension (AH) in 18 (90%) patients;

- aneurysms of other locations in 6 (30%) patients;

- chronic obstructive pulmonary disease in 1 (5%) patient;

- arrhythmia in 4 (20%) patients.

In the control group of 5 volunteers, there was only one case of AH, no other concomitant diseases were identified.

The genetic status of the patients was studied by a molecular genetic method. Blood was taken from the peripheral vein. The genomic DNA was isolated from the whole blood leukocytes using ‘DNA-ekspresskrov’ reagent (Litekh, Russian Federation) and was analyzed. With the sample of isolated DNA two amplification reactions were performed with two pairs of allele-specific primers, and three conclusions were made: homozygosity for allele1, heterozygosity, homozygosity for allele 2. The choice of genes was based on the integral approach used in the analysis of the etiology and pathogenesis of AAA [3–14].

Polymorphisms of the following genes were analyzed:

- lysine198asparagine (Lys198Asn) in the endothelin 1 (DN1) gene;

- С-786Т in the nitric oxide synthase 3 (NOS3) gene;

- leucine28proline (Leu28Pro) in the apolipoprotein E (APOЕ) gene;

- valin174alanin (Val174Ala) in the gene of solute carrier organic anion transporter family member 1B1 (SLC01B1);

- tryptophan715 proline (Thr715Pro) in the P-selectin (SELP) gene;

- C807T in the integrin alpha-2 (ITGA2) gene;

- serin 447 termination codon (serin447Ter) in the lipoprotein lipase (LpL) gene;

- tryptophan174methionine (Thr174Met) in the angiotensin 1 (AGT) gene;

- Met235Thr in thе AGT gene.

Statistical analysis was performed on a PC using IBM SPSS Statistics 21 (IBM Corp., 1989–2012, USA). In the work, qualitative parameters (risk factors and alleles) are presented as fractions. Qualitative parameters were compared using the chi square (÷2) test for arbitrary tables. Method of logistic regression (the results are given in the form of odds ratio (OR) with confidence interval (CI)), and Pearson correlation analysis (the result is presented as correlation coefficient, r) were used. The differences between the groups were considered statistically significant at p < 0.05.

RESULTS

Comparison of the frequency of occurrence of homo- and heterozygotes in the study groups are given in Table 1.

On the basis of the results of Table 1, no statistically significant differences in GPs and their frequency were found by us between the study and control groups.

Table 2 presents the results of the analysis of the existence of homozygous and heterozygous alleles in the study and control groups.

At the next stage of the study, a probable influence of GPs on the development of AAA was analyzed (Table 3).

 

Table 1. Frequency of Homozygous and Heterozygous Alleles in the Study and Control Groups

Polymorphism in Gene	Group	Homozygote for Allele 1, % (n)	Heterozygote, % (n)	Homozygote for Allele 2, % (n)
Lys198Asn in the EDN1 gene	Study group, n = 20	80 (16)	15 (3)	5 (1)
	Control group, n = 5	60 (3)	40 (2)	0
	р	> 0.05 (df = 2; ÷2 = 1.71)
С-786Т in the NOS3 gene	Study group, n = 20	10 (2)	55 (11)	35 (7)
	Control group, n = 5	0	100 (5)	0
	р	> 0.05 (df = 2; ÷2 = 3.52)
Leu28Pro in the APOЕ gene	Study group, n = 20	100 (20)	0	0
	Control group, n = 5	100 (5)	0	0
	р	> 0.05 (df = 2; ÷2 = NaN)
Val174Ala in the SLC01B1 gene	Study group, n = 20	40 (8)	60 (12)	0
	Control group, n = 5	60 (3)	40 (2)	0
	р	> 0.05 (df =2; ÷2 = NaN)
Thr715Pro in the SELP gene	Study group, n = 20	75 (15)	20 (4)	5 (1)
	Control group, n =5	60 (3)	40 (2)	0
	р	> 0.05 (df =2; ÷2 =1.04)
C807T in the ITGA2 gene	Study group, n = 20	25 (5)	55 (11)	20 (4)
	Control group, n = 5	40 (2)	60 (3)	0
	р	> 0.05 (df = 2; ÷2 = 1.34)
Ser447Ter in the LpL gene	Study group, n = 20	80 (16)	20 (4)	0
	Control group, n = 5	100 (5)	0	0
	р	> 0.05 (df = 2; ÷2 = NaN)
Thr174Met in the AGT gene	Study group, n = 20	10 (2)	90 (18)	0
	Control group, n = 5	0	100 (5)	0
	р	> 0.05 (df = 2; ÷2 = NaN)
Met235Thr in the AGT gene	Study group, n = 20	40 (8)	40 (8)	20 (4)
	Control group, n = 5	60 (3)	20 (1)	20 (1)
	р	> 0.05 (df = 2; ÷2 = 0.81)

 

Table 2. Presence of Homozygous and Heterozygous Alleles in Study and Control Groups

Allele	Study Group, % (n)	Control Group, % (n)	р
Homozygous 1	100 (9)	77.8 (7)	< 0.001 (df = 2; ÷2 = 16.7)
Heterozygous	88.9 (8)	77.8 (7)
Homozygous 2	55.6 (5)	11.1 (1)

 

Table 3. Influence of Gene Polymorphisms on Development of Abdominal Aortic Aneurysm

Allele	OR	95% CI for OR		р
		lower	upper
Lys198Asn in the EDN1 gene
Homozygote for allele 1	2.667	0.327	21.733	> 0.05
Heterozygote	0.265	0.030	2.318	> 0.05
Homozygote for allele 2	–	–	–	–
Val174Ala in the SLC01B1 gene
Homozygote for allele 1	0.444	0.060	3.285	> 0.05
Heterozygote	2.250	0.304	16.632	> 0.05
Homozygote for allele 2	–	–	–	–
Thr715Pro in the SELP gene
Homozygote for allele 1	2.000	0.256	15.623	> 0.05
Heterozygote	0.375	0.046	3.056	> 0.05
Homozygote for allele 2	–	–	–	–
C807T in the ITGA2 gene
Homozygote for allele 1	0.500	0.064	3.906	> 0.05
Heterozygote	0.815	0.111	5.987	> 0.05
Homozygote for allele 2	–	–	–	–
Met235Thr in the AGT gene
Homozygote for allele 1	0.444	0.060	3.285	> 0.05
Heterozygote	2.667	0.250	28.438	> 0.05
Homozygote for allele 2	1.000	0.086	11.588	> 0.05

Note: OR ― odds ratio CI ― confidence interval. Influence of the parameters was determined by OR calculated by logistic regression method, and the dependence between parameters ― by Pearson correlation analysis

 

Interesting data were obtained in the correlation analysis (Figures 1, 2).

 

Fig. 1. Results of Pearson correlation analysis of gene polymorphisms in patients with abdominal aortic aneurysm (study group).

Note: the first number ― correlation coefficient (r), the second number ― statistical significance (p).

 

Fig. 2. Results of Pearson correlation analysis of gene polymorphisms in patients without abdominal aortic aneurysm (control group).

Note: the first number ― correlation coefficient (r), the second number ― statistical significance (p).

 

From the data presented in Figures 1 and 2 it follows that significant correlations of GPs coincided in the study and control groups for the interrelation of Ser447Ter in the LpL gene and Leu28Pro (r = 0.70; p < 0.001) in the APOE gene.

In the study group of patients, the correlation relationships were identified with Ser447Ter polymorphism in the LpL gene:

- direct correlation with Lys198Asn (r = 0.63; p < 0.001) polymorphism in the EDN1 gene, Leu28Pro (r = 0.70; p < 0.001) in the APOE gene and Thr715Pro (r = 0.63; p < 0.001) in the SELP gene;

- inverse correlation with С786Т polymorphism (r = -0.35; p = 0.006) in the NOS3 gene.

Similar results were obtained for Leu28Pro polymorphism in the APOE gene:

- direct correlation with Ser447Ter in the LpL gene, Lys198Asn (r = 0.70; p < 0.001) in the EDN1 gene and Thr715Pro (r = 0.63; p < 0.001) in the SELP gene;

- inverse correlation with С786Т (r = -0.35; p = 0.006) in the NOS3 gene.

Thr715Pro polymorphism in the SELP gene, along with correlations with Ser447Ter (r = 0.63; p < 0.001) in the LpL gene and Leu28Pro in the APOE gene, has an additional direct correlation with Lys198Asn (r = 0.55; p < 0.001) in the EDN1 gene.

For Thr174Met polymorphism in the AGT gene there were obtained:

- direct correlation with Val174Ala (r = 0.40; p = 0.002) in the SLC01B1 gene;

- inverse correlation with Leu28Pro (r = -0,35; p = 0,006) in the APOE gene.

To note, polymorphism Met235Thr in the AGT gene is:

- in direct correlation with Val174Ala (r = 0.33; p = 0.011) in the SLC01B1 gene;

- in inverse correlation with C807T in the ITGA2 gene.

In the control group, the following correlation relationships were identified:

- C807T polymorphism in the ITGA2 gene directly correlates with Lys198Asn (r = 0.70; p = 0.004) in the EDN1 gene and Val174Ala in the SLC01B1 gene.

- Thr174Met polymorphism in the AGT gene directly correlates with С786Т (r = 1.00; p < 0.001) in the NOS3 gene, Ser447Ter in the LpL gene and Leu28Pro (r = 1.00; p < 0.001) in the APOE gene.

DISCUSSION

The analysis of the presented data revealed statistically significant differences in the dominance of homozygous and heterozygous alleles in the main and control groups, which probably influences the occurrence of AAA. There was also found the absence of polymorphism for the 2^nd allele in the control group except for the AGT gene (Met235Thr polymorphism). In the meantime, there is little information in the literature about dominance of alleles, their role in the development of AAA and related risk factors.

A. Sethi, et al. found mutations of Thr235 and Met174 among 9100 women and men of the general population of Denmark (54% had AH) in 41% and 12% of cases, respectively; mutation of Met174 always occurred on the same allele as mutation of Thr235. In the multifactorial logistic regression analysis it was found that in women homozygous for Thr235, in comparison with women who are not carriers, OR for AH was 1.29 (95% CI 1.05–1.58 ); in women homozygous also for Thr174 (and non-carriers of Мет174), OR increased to 1.5 (from 1,15 to 1.96). Women, homozygous for Thr235, also had an increased risk of isolated elevation of systolic arterial pressure (ОR 1.37; 95% CI 1.02–1.84) and moderate elevation of arterial pressure (ОR 40; 95% CI 1.10–1.77). Here, the authors did not reveal any statistically significant correlation between the elevated arterial pressure and genotype in men or between genotype and systolic arterial pressure, diastolic arterial pressure or pulse pressure in both genders. Homozygosity both for Thr235 and Thr174 was associated with 10% increase in the level of angiotensin in plasma in both genders compared to homozygosity for Met235 and Thr174 [6].

J. A. Staessen, et al. showed in their work that, compared to MM homozygotes, TT homozygotes and M heterozygotes had excessive risk for AH in 31% and 11% of cases, respectively [7]. As noted in the work of J. C. Bis, et al., in patients with AH receiving pharmacological therapy, angiotensinogen genotype modified relationship of angiotensin converting enzyme (ACE) inhibitors with the development of stroke, and the risk of stroke associated with use of ACE inhibitor in participants with ThrThr genotype (ОR 0.37; 95% CI 0.14–0.99), was about a quarter lower than in participants with a copy of Met235 allele (ОR 1.44; 95% CI 0.88–2.35). The risk of myocardial infarction associated with the use of ACE inhibitor, did not depend on Met235Thr genotype of angiotensinogen [8]. This aspect has a very important role, in a multicenter study earlier published by us, is was established that the frequency of AH and CHD in patients with AAA reached 80% and 77%, while the optimal drug therapy was given to less than half the patients [2], and only a part of them received combined therapy including ACE inhibitors.

Statistically significant correlations of polymorphisms in the gene between the study and control groups coincided in the dependence of Ser447Ter in the LpL gene and Leu28Pro in the APOE gene. Besides, in the study group of patients, the greatest relationships were found with Ser447Ter polymorphism in LpL gene: direct relationships with Lys198Asn polymorphisms in the EDN1 gene. Meta-analysis of C. Wang, et al. showed that LPL Ser447Ter polymorphism was associated with a considerably lower risk of ischemic stroke, especially of atherosclerotic stroke subtype, both in the representatives of the Caucasian race and in the population of East Asia. Nevertheless, the authors made a suggestion about the association of Lys198Asn polymorphism of the EDN1 gene with increased risk of ischemic stroke [9].

Correlation of these parameters in our study in the patients with AAA may evidence increased risk of cardiovascular complications, but requires further study. To add, in patients of the main group atherosclerosis of brachiocephalic arteries/stroke was found in 20% of cases.

An important direct correlation of Leu28Pro in the APOE gene and Thr715Pro in the SELP gene was obtained. Here it is necessary to mention the results of meta-analysis by G. Herrera–Maya, et al. [10], which provide empirical evidence that genetic polymorphisms of SELP may promote development of CHD, in particular, myocardial infarction. Thus, genetic polymorphisms of SELP may be potential and practical biomarkers for early diagnosis of CHD and myocardial infarction. In this connection it should be noted that in the group with the obtained correlations, CHD was diagnosed in 11 (55%) cases.

The data obtained by us demonstrate that along with Ser447Ter in the LpL gene there is also a direct correlation with Lys198Asn in the EDN1 gene and Thr715Pro in the SELP gene, which, according to some studies, is associated with diabetes mellitus and development of stroke [10, 11]. At the same time, there exist rather contraversial data on the influence of diabetes on the pathogenesis of AAA [2].

Thr174Met polymorphism in the AGT gene directly correlates with Val174Ala in the SLC01B1 gene. According to A. Kalliokoski, et al., genetic variability of genes can lead to the interindividual differences in the pharmacokinetics. In particular, single-nucleotide polymorphism (c.521T > C, p.Val174Ala) in the SLCO1B1 gene encoding the organic anion transporting polypeptide, 1B1 (OATP1B1), reduces the ability of OATP1B1 to transport the active simvastatin acid to the liver leading to increase in its concentration in plasma, which, in turn, increases the risk of development of simvastatin-induced myopathy. Besides, it is shown in the same review that SLCO1B1 polymorphism also affects pharmacokinetics of many statins and of repaglinide antidiabetic drug, that are used in treatment of atherosclerosis and diabetes in patients with AAA to reduce the risk of cardiovascular complications. With that, Met235Thr polymorphism in the AGT gene has a direct relationship with Val174Ala in the SLC01B1 gene [12].

CONCLUSION

Based on the results of our study, statistically significant differences in the dominance of homozygous and heterozygous alleles in the main and control groups were established. The existence of direct correlations of some polymorphisms of a number of genes in patients with abdominal aortic aneurysm has been established, which shows their probable role in the development of this pathology and may be a screening test for determination of the probability for its development.

ADDITIONAL INFORMATION

Funding. This study was not supported by any external sources of funding.

Conflict of interests. The authors declare no conflicts of interests.

Contribution of the authors: E. L. Kalmykov ― study concept and design, statistical analysis, data analysis and interpretation, text writing; I. A. Suchkov ― the concept and design of the study, checking the critical intellectual content of the work, final approval for publication of the manuscript; R. E. Kalinin ― concept and design of the study, verification of the critical intellectual content of the work, final approval for publication of the manuscript; O. Ne’matzoda ― analysis and interpretation of data; D. S. Dodkhoyev ― statistical analysis, analysis and interpretation of data.. All authors made a substantial contribution to the conception of the work, acquisition, analysis, interpretation of data for the work, drafting and revising the work, final approval of the version to be published and agree to be accountable for all aspects of the work.

About the authors

Egan Kalmykov

Clinic for Vascular and Endovascular Surgery, Theodor Fontaine Medical Institute

Author for correspondence.
Email: egan0428@mail.ru
ORCID iD: 0000-0001-6784-2243
Scopus Author ID: 8623-8897

MD, Dr. Sci. (Med.)

Germany, Brandenburg

Igor’ A. Suchkov

Ryazan State Medical University

Email: suchkov_med@mail.ru
ORCID iD: 0000-0002-1292-5452
SPIN-code: 6473-8662

MD, Dr. Sci. (Med.), Professor

Russian Federation, Ryazan

Roman E. Kalinin

Ryazan State Medical University

Email: kalinin-re@yandex.ru
ORCID iD: 0000-0002-0817-9573
SPIN-code: 5009-2318

MD, Dr. Sci. (Med.), Professor

Russian Federation, Ryazan

Okildyhon Ne’matzoda

Republican Scientific Center for Cardiovascular Surgery

Email: sadriev_o_n@mail.ru
ORCID iD: 0000-0001-7602-7611
SPIN-code: 2408-9107

MD, Cand. Sci. (Med.)

Tajikistan, Dushanbe

Jamshed S. Dodkhoev

Avicenna Tajik Medical University

Email: jamshedsd@yandex.com
ORCID iD: 0000-0002-9228-8544
SPIN-code: 6609-4501

MD, Dr. Sci. (Med.), Associate Professor

Tajikistan, Dushanbe

References

Supplementary files