The role of арое gene l28/28p polymorphisms in prenosological diagnostics of cardiovascular diseases in elite athletes

Фотографии: 

D.V. Muzhenya, postgraduate
A.R. Tuguz, professor, Dr.Biol.
A.S. Doroshenko, associate professor, Ph.D.
K.A. Rudenko, postgraduate
E.N. Anokhina, postgraduate
Adygeya state university, Maikop

Key words: gene, polymorphism, alleles, athletes, atherogenesis, apolipoprotein, SNP, cardiovascular system.

Introduction. In the modern world practice of researches there exists a high level of heritability of human morphofunctional characteristics, which are in demand in elite sport and stipulated by single nucleotide polymorphisms (SNP) - polymorphisms in the genes involved in the regulation of cardiovascular system, increasing variabilities of adaptive mechanisms to strenuous physical loadings [1-6].

SNP, determined with the use of molecular genetic methods, are highly informational in estimation of the potential of development of physical qualities, limiting factors of human working capacity, at early prenosological diagnostics of hereditary predisposition to development of industrial diseases [3-6].

The genetic markers determining the CVS ability to physiological adaptation at strenuous exercise are in demand in the modern sport in training world-class athletes and correcting a training process [5-9].

The apolipoprotein Е (АРОЕ) gene, regulating lipid metabolism and work of the cardiovascular system, can be one of such markers [10].

The apolipoprotein Е is synthesized in the liver and brain and is a part of chylomicrons and low-density lipoproteins (LDLP) with more pronounced atherogenic effect (resulting in artery disease). АРОЕ initiates the uptake and removal of LDLP by interacting with a specific receptor on the hepatocyte surface; contributes to the processes of: immunoregulation, nerve regeneration and activation of lipolytic enzymes (liver lipases, lipoprotein lipases and lecithin-cholesterol acyltransferase) [11-14].

АРОЕ gene mutations change the apolipoprotein molecular composition, provoking lipidosis. The АРОЕ genetic polymorphism is an essential precondition of hyperlipoproteinemia, decreasing functional capabilities of the CVS, the risk of coronary and peripheral artery diseases and cardiovascular disease continuum. The human АРОЕ gene is located in the chromosome 19 19-q13.32 (Fig.) [15].

Fig. Cytogenetic structure of the chromosome 19 (HuGENavigator data)

The most popular 6 of the АРОЕ gene polymorphisms are: three homozygous (Apo Е4/4, Apo ЕЗ/3, Apo Е2/2) and three heterozygous (Apo Е4/3, Apo Е4/2, Apo ЕЗ/2) [16]. The АРОЕ gene Leu28Pro polymorphism (ApoE4Freiburg) was first described by Orth M. et.all. (1999) and according to the HuGeNET data, is one of the least studied among the apolipoprotein E mutations, but perspective in predictive medicine [17].

The distribution of the АРОЕ gene Leu28Pro polymorphism in the world population, in accordance with the dbSNP (date base Single Nucleotide Polymorphism), is stipulated in Table 1 [18].

Table 1. The analysis of the distribution of the АРОЕ Leu28Pro polymorphism by the data dbSNP

Populations

Year

sampling

Allele

Leu

Pro

North America

2000, 2000

84/48

1.000/0.980

0.000 /0.020

Europe

2000, 2001

184/456

1.000

0.000

North America

2003

4520

0.999

0.001

Central and South America

2003

48

1.000

0.000

East Asia

2004

84

1.000

0.000

The Leu28Pro association of the АРОЕ gene polymorphism with the risk of lipidosis and, consequently, cardiovascular diseases was analyzed in a few researches. The patients had higher frequency of the “mutant” allele 28Pro, than in the random sampling of the population. Given the 28Pro the risk of ischemic heart disease (IHD) is 5,3 times higher, and in case of the co inheritance of the 28Pro allele of the АРОЕ gene and its isoform ApoE*4 - 20 times higher [16;19].

The Leu28Pro association of АРОЕ gene polymorphisms with atherogenesis is hardly studied, but it could be of both scientific and practical value in respect to prenosological diagnosis of cardiovascular diseases among different contingents of the population and, specifically, elite athletes.

The purpose of the study was to examine the frequency distribution and Lеu28/28Pro associations of the АРОЕ gene polymorphism at the risk of atherogenesis in elite athletes of the Republic of Adygeya.

Materials and methods. 94 males ages 18-60, including 39 athletes, 25 healthy young men not engaged in sports and 30 patients with CVD were involved in the experiment. The group of elite athletes (16 candidates for master of sport, 6 masters of sport, 18 first-grade athletes) were football players of the “Druzhba” club, basketball players of the Maikop “Dynamo” team (n=28) and the members of the RA athletic national team (middle distance running, n=11) aged 18-25 (mean age 22,7±3,85) with different physical loading on the CVS within training and competitive activities. The athletes’ contingent was chosen in compliance with the suggested by V.S. Farfel (1975) classification of speed and strength sports with maximal and submaximal intensity and with over 8 years experience.

The monitoring was presented in the random sample of healthy males (n=25) aged 18-24 (mean age 21±3,15), not involved in sport and without hereditary loading in respect to CVD. Patients with CVD (n=30) – patients of the cardiological care unit of ARCH (Maikop town, the Republic of Adygeya) aged 47-60 (55,4 ± 7,7) with CVD, developed secondary to coronary (n=19) and peripheral artery diseases (n=11). Patients with coronary artery disease had various types of ischemic heart disease, myocardial infarction (MI), heart failure (HF) etc.; in case of peripheral artery disease – complications referred to thromboses, aortic occlusion and lower and upper limb arterial occlusive disease, primary hypertension etc. Cardiological patients were diagnosed in compliance with: standard biochemical blood tests; instrumental methods of examination: ECG, ECG Holter monitoring, echocardiography (ECHO-CS).

Materials and methods. The distribution of the APOE gene Leu28Pro polymorphisms was studied using the SNP – the method with electrophoretic detection of results and the dual priming system (Lytech Co. Ltd.);

Table 2. The distribution analysis of АРОЕ Leu28Pro polymorphism by dbSNP

Gene

Alleles

Symbol of allele

Primer

APOE

Leu

C

5'-atgccgatgacctgcagaag Cgcctggcagt gtaccaggcc-3'

Pro

T

5'-atgccgatgacctgcagaag Tgcctggcagt gtaccaggcc-3'

The quality of genomic DNA derived from the peripheral blood was tested on the spectrophotometer NanoDrop 2000c (Termo Scientific, USA). The findings were processed at UV irradiation (wave length 310 nm) in the transilluminator Gel Doc (Bio-Rad) using the Quantity One software (Bio-Rad).

Blood cholesterol was measured on the semi-automatic biochemistry analyzer (BS3000P, China), with the reagent kit Cholesterol DiaS (JCV Diakon-DS, Russia) for the reference values 3,0-5,2 mmol/l.

Statistical analysis of test data. Significant differences (р<0,05) were calculated using the Fischer’s nonparametric method, χ2 (xi2 analysis) for Yates continuity correction in 2x2 contingency tables and calculation of the odds ratio (OR – odds-ration), 95% confidence interval (95% СI).

Results and discussion. The predictive value of the АРОЕ gene Leu28Pro polymorphisms in lipidosis and atherogenesis was estimated using the comparative distribution analysis of the genotype and allele frequencies in the control group, in the athletes and patients with CVD. The data are adduced in Tables 3, 4.

Table 3. The frequencies of genotypes and L28/28P alleles of the АРОЕ gene in athletes in the control group and the group with CVD

Alleles/ genotypes

Genotype and allele frequencies

 

Genotypes

Athletes (n=39)

Patients with CVD (n=30)

Control (n=25)

 

L28L

92,3%

73,3%

100%

 

L28P

7,7%

26,7%

0%

 

P28P

0%

0%

0%

 

р,  OR CI (95%)

р * = 0,03; 4,36 (0,94-88,5)

р **= 0,005 ; 19.27 (0.92 – 115)

 

Alleles

 

 

L28

0.962

0,866

1,000

 

28P

0.038

0,134

0

 

р,

OR (CI 95%)

р* = 0,04

3,85 (0,94-88,5)

р** = 0,007

16,35(0,92 – 115)

 
 
         

 

Note: р- statistical significance in genotype and allele frequencies:

*athletes and experimental groups;  **  control and experimental groups.

“Normal” homozygous Leu28 polymorphism and Leu28Leu genotype prevail in all the examined contingents. The “mutant” allele 28Рro of the АРОЕ gene was detected in the athletes and the group with CVD in the heterozygous state only (Tab. 3). The statistically significant excess of Leu28Pro genotype frequencies was detected in the ones with coronary and peripheral artery diseases: IHD, MI, stroke, ischemia, thrombosis of lower limb etc. (Tab. 3).

Higher frequency of mutant 28P allele of apolipoprotein E was detected in the alleles Leu28/28Pro in the groups of subjects with CVD as opposed to athletes and the control group (Tab. 3). The biochemical blood test of the patients with the АРОЕ heterozygous genotype compared to the “norm” revealed the increase of blood cholesterol (>7,5 mmol/l), what proves its association with lipidosis at atherosclerotic vascular diseases of different circulation systems. The distribution analysis of genotype and Leu28Pro of АРОЕ alleles was made in the group of elite athletes to estimate the risk of lipidosis and atherogenesis (Tab. 4).

Table 4. Frequencies of genotypes and Leu28/28Pro alleles of APOE gene in the groups of athletes

Alleles/ genotypes

Genotype and allele frequencies

Genotypes

All (n=39)

Football players (n=19)

Basketball players (n=9)

Athletics (n=11)

*

L28L

92,3%

100%

100%

72,7%

р < 0,05

L28P

7,7%

0

0

27,3%

P28P

0

0

0

0

 

Alleles

 

**

L28

0.962

1,000

1,000

0.864

p<0,05

28P

0.038

0

0

0.136

Note: р- statistical significance: * in genotype frequencies; ** in allele frequencies.

Football and basketball players do not have the allele 28Pro and the genotype Leu28Pro of the APOE gene. In the group of athletes the predictively unfavourable Leu28Pro heterozygous genotype, associated with the risk of CVD (OR=3,85; CI (0,94-88,5) was noted in 27,3% of cases, which is comparable for patients with CVD (Tab. 3). In conditions of strenuous physical loadings the hereditary predisposition to CVD of these athletes combined with other risk factors can result in the physiological deadaptation of the body and development of distress. The examinations have not revealed any significant variance in the distribution of the АРОЕ gene 28Pro polymorphism subject to ethnicity (Adygei and Russian people) among elite athletes, donors, patients (the data are not adduced). 

The 28Pro АРОЕ gene alleles, associated with the high (over 5 times) risk of IHD by the data of Nguen Thi Chang (2012), were detected in the healthy Adygei, which is comparable with the results for world populations (Tab.1).

The allocation in the group of athletes of higher frequencies of the 28Pro (0,136) polymorphism, associated with lipidosis and increasing the risk of atherosclerotic vascular disease is an unfavourable predictor of the CVD risk in these athletes (OR = 3,85; CI (0,94-88,5). 

The findings along with additional instrumental methods of examination of the CVS functional state can be of great value in sports medicine in respect to the prenosological diagnostic of CVD.

Conclusions.

  • Patients with cardiovascular diseases were proved to have significantly higher frequencies of the Leu28Pro (heterozygous) genotype and the mutant 28Р allele of АРОЕ.
  •  The Leu28Pro genotype and АРОЕ gene 28Pro polymorphism carrier state is a predictively unfavourable factor of lipidosis, atherogenesis and, consequently, higher risk of CVD.
  • The АРОЕ gene 28Pro polymorphism can be used as a specific marker of hereditary predisposition to CVS functional abnormalities, which is to be taken into account when organizing a training process and qualifying children in sport schools.

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Author’s contacts: lab_genetic@mail.ru