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National *Kozovyi R., Vakalyuk I., Yatsyshyn R.
Ivano-Frankivsk Medical University, Ivano-Frankivsk, Ukraine
FREQUENCY OF POLYMORPHIC VARIANTS OF THE GSTT1 AND GSTM1 GENES AMONG LONG LIVERS WITH ARTERIAL HYPERTENSION AND OSTEOARTHRITIS RESIDING IN THE
CARPATHIAN REGION
Abstract
Objectives: The objective of the research was to study the relationships between the frequency of polymorphic variants of the GSTT1 and GSTM1 genes among long livers with arterial hypertension and osteoarthritis residing in the Carpathian region.
Methods: Molecular genetic testing of genes of phase II biotransformation of xenobiotics was performed in 166 long livers residing in Ivano-Frankivsk region. To compare the results, the patients were divided into four groups: Group I included 53 long livers with stage II arterial hypertension and stage II osteoarthritis; Group II comprised 41 long livers with stage II arterial hypertension without osteoarthritis; Group III included 34 long livers with stage II osteoarthritis without arterial hypertension; Group IV comprised 38 apparently healthy long livers (without any diagnosed disease).
Results: The analysis of the obtained data has revealed that the frequency of the functionally inactive GSTT1 and GSTM1 alleles among the all long livers of Ivano-Frankivsk region was 24.70% and 46.99%, respectively. In long livers of four comparison groups, the functionally active GSTT1 and GSTM1 alleles predominated. There were no statistically significant differences between patients with arterial hypertension and osteoarthritis and healthy long livers with the GSTM1 +/GSTT1+, GSTM1 +/GSTT1 -, GSTM1 -/GSTT1 +, GSTM1-/GSTT1 -genotypes. When studying the peculiarities of the onset and clinical course of arterial hypertension and osteoarthritis in long livers, we have revealed that in 76% of cases, clinical manifestations of the disease were detected in senile patients (over 75 years of age). A relatively slow worsening of symptoms was observed; the transition from the first to the second stage continued for 10-20 years.
Keywords
Polymorphic variants of genes, GSTM1, GSTT1, long livers
Introduction.
The development of pathological conditions depends on individual hereditary traits determining different sensitivity to the same exogenous factors. The xe-nobiotic detoxification system plays the key role in the response of a particular person to environmental exposure. Therefore, the study of individual peculiarities of the detoxification system functioning being relevant
and necessary under conditions of modern anthropogenic load is essential when determining the individual risk of developing pathological processes [1, 2]. There are typically three stages involved in the system of xe-nobiotic transformation, namely the activation of xeno-biotics (phase I), the detoxification of xenobiotics (phase II) and the elimination of xenobiotics from the body (phase III). Phase I - the activation of xenobiotics or metabolic transformation - involves the attachment
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of modifying functional groups (-OH, -SH, -NH3) to xenobiotics with subsequent oxidation reactions, repair reactions and hydrolysis reactions resulting in the formation of intermediate metabolites. This process is catalyzed by the cytochrome P450 microsomal enzyme system (the family of cytochrome enzymes) and some other enzymes (oxidases, reductases, hydrolases and dehydrogenases) [3, 4]. Phase II biotransformation -the detoxification - involves the conjugation, that is, the attachment of intermediate metabolites to endogenous ligands which increase the hydrophilic nature of these compounds thereby contributing to eliminating them from the body. Phase III consists in the evacuation or the elimination of water-soluble, non-toxic substances from the body. This function is performed by the transporters of the compounds that were formed -P-glycoproteins which promote the excretion of xeno-biotics into the bile or blood.
A large number of studies are dedicated to the study of the phase II enzymes involved in the detoxification of xenobiotics, glutathione S-transferases in particular [5, 6]. The multigenic family of glutathione-S-transferases detoxicates various aliphatic, aromatic and heterocyclic compounds through their conjugation with glutathione. Cytosolic glutathione-S-transferases are divided into seven classes: a, ra, n, c, 0, <;. A large number of polymorphic variants of the glutathione-S-transferase genes have been found. Several deletion polymorphisms of the glutathione-S-transferase^ (GSTM 1) and 0 (GSTT 1) genes determining the absence of functional enzyme activity have been described. The current belief is that carriers of such deletions are genetically predisposed to some multifactorial diseases. The role of some genes including polymorphic variants of detoxification system in the development of aging is now under consideration [7, 8, 9].
Due to the fact that the median age of the world's population has increased, great attention is paid to the study of primary mechanisms of the body and population aging as well as factors responsible for life expectancy. Since in most cases, senile patients develop comorbid pathology, the works dealing with the relationships between exogenous and endogenous factors and longevity as well as the molecular and genetic mechanisms of developing different diseases in long livers are relevant. Our previous research has shown that the most prevalent pathological conditions found in long livers of the Carpathian region were arterial hypertension (AH) and osteoarthritis (OA) [10]. A group of scientists - Beihai Ge, Yadong Song, Yi Zhang, Xiaowen Liu, Yuxiang Wen, Xiaomei Guo - conducted a meta-analysis of 185 studies concerning glutathione S-transferase M1 (GSTM1) and T1 (GSTT1) null polymorphisms and the risk of hypertension [11]. A detailed analysis of 14 studies was carried out. The results were controversial. The authors stated that in that study, although they had pooled all published studies currently available on that topic, they believed their study was still far from conclusive. The association of genetic polymorphisms with hypertension was strongly influenced by differences in the selection of cases and controls, ethnicity, sample size, environmental factors and
other ecological factors. Selection bias was also possible in hospital-based studies because the GSTs might be related to the risk for chronic diseases. And most studies had not been clear about whether cases had represented the first diagnosis of hypertension. Further studies with unbiased-matched homogeneous patients and well matched controls were required to examine associations between the GSTM1 and GSTT1 polymorphisms and hypertension risk. Furthermore, the negative result also might be caused by interethnic differences [11].
Therefore, we were interested in conducting such research on long livers of the Carpathian region.
Objectives
The objective of the research was to study the relationships between the frequency of polymorphic variants of the GSTT1 and GSTM1 genes among long livers with arterial hypertension and osteoarthritis residing in the Carpathian region.
Materials and methods
At the study initiation, in Ivano-Frankivsk region (Ukraine) there were 4,586 people at the age of 90 years and over (long livers). DNA samples taken from 166 long livers (the average age - 95 years) permanently residing in Ivano-Frankivsk region served as a material for study. In order to compare the results, the patients were divided into four groups: Group I included 53 long livers with stage II AH and stage II OA; Group II comprised 41 long livers with stage II AH without OA; Group III included 34 long livers with stage II OA without AH; Group IV comprised 38 apparently healthy long livers (without any diagnosed disease). Selection criteria included age, ethnicity, length of residence, health status (AH and/or OA without other diseases). Inclusion criteria - patients must meet all of the following criteria: 1) Male or female patient aged 90 years or older; 2) patients: with arterial hypertension (AH) stage II and/or osteoarthritis (OA) stage II and without other diseases, or healthy; 3) ethnicity - Ukrainian; 4) length of residence - all lifetime; 5) patients who, after the nature of the study and the disclosure of their data have been explained to them, have freely given Informed Consent in writing. Exclusion criteria - patients will be excluded for any one of the following criteria: 1) Male or female patient aged have not 90 years or older; 2) patients: with arterial hypertension (AH) stage I, III, IV and/or osteoarthritis (OA) stage I, III, IV 3) patients with other diseases; 4) ethnicity - non Ukrainian; 5) length of residence - some time; 6) patients who, after the nature of the study and the disclosure of their data have not been explained to them, and have not freely given Informed Consent in writing.
The confirmation of medical information including the diagnosis and clinical features of the disease was made by family doctors or gerontologists; all the information was provided to us with the patients' consent. Thus, we selected the patients without comorbid-ities.
The glutathione-S-transferase gene (GSTT1 and GSTM1) polymorphisms were determined using the multiplex polymerase chain reaction method with the detection in 1.5% agarose gel. The primers for the detection of polymorphic variants of the GSTT1
andGSTM1 genes were sequenced and the conditions for amplifications were designed according to the protocol [12]. The obtained results were statistically analyzed using the %2method (Statistika 10.0), the Fisher's method and the odds ratio (OR).
Results
The analysis of the obtained data has revealed that the frequency of the functionally inactive GSTT1 and GSTM1 alleles among the all long livers of Ivano-
Frankivsk region was 24.70% and 46.99%, respectively. The frequencies of the combinations of GSTM1+/GSTT1 +, GSTM1+/GSTT1-, GSTM1-/GSTT1+, GSTM1 -/GSTT1 -allelic variants were primarily studied in the group of long livers with AH and OA in comparison with healthy individuals. There were no significant differences in the percentage values of the distribution of the genotype frequencies of the GSTM1 and GSTT1 genes between these patients and healthy long livers (Table 1).
Table 1. Distribution of genotype frequencies of the GSTM1 and GSTT1 genes in patients with AH and OA compared to healthy long livers
Gene Genotype Group I, n = 53 Group IV, n= 38 x2 OR 95%CI P
n % n %
GSTM1 GSTM1- 25 47.2 18 47.3 0.04 1.01 0.44-2.30 >0.05
GSTM1+ 28 52.8 20 52.7 0.04 1.01 0.44-2.32 >0.05
GSTT1 GSTT1- 13 24.5 9 23.7 0.02 1.05 0.39-2.78 >0.05
GSTT1+ 40 75.4 29 76.3 0.02 0.95 0.36-2.53 >0.05
GSTM1 +/GSTT1+ 21 39.6 15 39.5 0.04 1.00 0.43-2.33 >0.05
GSTM1 +/GSTT1 - 7 13.2 5 13.2 0.09 1.00 0.30-3.22 >0.05
GSTM1-/GSTT1+ 19 35.9 14 36.8 0.02 0.96 0.40-2.28 >0.05
GSTM1 -/GSTT1 - 6 11.3 4 10.5 0.05 1.09 0.28-4.14 >0.05
The frequency of the inactive GSTTM1 allele in Group I was 47.2% and in Group IV it was 47.3%, while the frequency of the inactive GSTT1 allele was 24.5 and 23.7%, respectively. However, the highest risk of developing AH and OA was observed in carriers of the GSTM1-/GSTT1- deletion genotype (OR=1.09,
^>0.05). The analysis of the obtained data concerning the genotype frequencies of the GSTM1 and GSTT1 genes among long livers of Group II compared to the data of Group IV has not revealed any statistically significant differences (x2 0.02 - 0.04, OR- 1.0) (Table 2).
Table 2. Distribution of genotype frequencies of the GSTM1 and GSTT1 genes in patients with AH and healthy long livers
Gene Genotype Group II, n = 41 Group IV, n= 38 x2 OR 95%CI P
n % n %
GSTM1 GSTM1- 19 46.3 18 47.3 0.02 0.96 0.40-2.32 >0.05
GSTM1+ 22 53.7 20 52.7 0.02 1.04 0.43-2.52 >0.05
GSTT1 GSTT1- 10 24.4 9 23.7 0.04 0.96 0.34-2.70 >0.05
GSTT1+ 31 75.6 29 76.3 0.04 1.04 0.37-2.92 >0.05
GSTM1 +/GSTT1+ 16 39 15 39.5 0.04 0.98 0.40-2.42 >0.05
GSTM1 +/GSTT1 - 6 14.6 5 13.2 0.02 1.13 0.32-4.06 >0.05
GSTM1-/GSTT1+ 15 36.6 14 36.8 0.04 0.99 0.40-2.47 >0.05
GSTM1 -/GSTT1 - 4 9.8 4 10.5 0.07 0.92 0.21-3.96 >0.05
In long livers of four comparison groups, the functionally active GSTT1 and GSTM1 alleles predominated. At the same time, the GSTT1 gene deletion was less common as compared to the GSTM1 gene deletion - by 1.89 times in long livers with AH and by 1.99 times in healthy individuals. The combinations of
GSTM1+/GSTT1+ allelic variants were most commonly detected in long livers of Group II (38.2%) and Group IV (39.5%). Similar results of the distribution of GSTM1 +/ GSTT1+ allelic variants were obtained when comparing long livers of Group III and Group IV (Table 3).
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Table 3. Distribution of genotype frequencies of the GSTM1 and GSTT1 genes in patients with OA and healthy
long livers
Gene Genotype Group II, n = 34 Group IV, n= 38 x2 OR 95%CI P
n % n %
GSTM1 GSTM1- 16 47 18 47.3 0.04 1.01 0.40-2.56 >0.05
GSTM1+ 18 53 20 52.7 0.04 0.99 0.39-2.50 >0.05
GSTT1 GSTT1- 9 26.5 9 23.7 0.00 0.86 0.30-2.51 >0.05
GSTT1+ 25 73.5 29 76.3 0.00 1.16 0.40-3.37 >0.05
GSTM1+/GSTT1+ 13 38.2 15 39.5 0.02 0.95 0.37-2.45 >0.05
GSTM1+/GSTT1- 5 14.7 5 13.2 0.02 1.14 0.30-4.33 >0.05
GSTM1-/GSTT1+ 12 35.3 14 36.8 0.01 0.94 0.36-2.45 >0.05
GSTM1 -/GSTT1 - 4 11.8 4 10.5 0.04 1.13 0.26-4.93 >0.05
Thus, there were no statistically significant differences between patients with AH and OA and healthy long livers with the GSTM1+/GSTT1+, GSTM1 +/GSTT1 -, GSTM1 -/GSTT1+, GSTM1-/GSTT1 - genotypes.
Discussion
When studying the peculiarities of the onset and clinical course of AH and OA in long livers, we have revealed that in 76% of cases, clinical manifestations of the disease were detected in senile patients (over 75 years of age). A relatively slow worsening of symptoms was observed; the transition from the first to the second stage continued for 10-20 years. The study of epige-netic mechanisms of detoxification in these long livers, that is the study of direct functional activity of enzymes encoded by these genes is of great importance as well.
Our results concerning the frequency of the functionally inactive GSTT1 and GSTM1 alleles among long livers of Ivano-Frankivsk region were similar to those obtained by Lee [12] and Wang R [13]. We could not reliably confirm the association of glutathione-S-transferase (GSTT1 and GSTM1) gene polymorphism and arterial hypertension as well. However, in contrast to Klein T [14] who revealed that the normal distribution of the GSTM1 negative genotype in patients with indication for hip or knee replacement indicated that the role GSTM1 in those patients was different from that in other aseptic inflammatory diseases such as ozone-related inflammatory reactions of the respiratory tract, we have not detected any statistically significant differences between patients with OA and healthy individuals. The study carried out by Dae Jung Choi indicated that the polymorphisms of GST gene might be a susceptibility factor in the development of osteoarthritis in Korean population [15].
A number of authors have already analyzed the association between polymorphic variants of different genes including genes of the xenobiotic detoxification system and life duration. For example, Pesch B. et al. studied polymorphism of the CYP1A1, CYP1B1 cytochrome P450 genes and the glutathione S-transferase genes (GSTM1, GSTT1, GSTP1) in 205 Germans at the age of 80 years and older as well as 294 Germans under 80 years of age. The most pronounced association between the GSTM1 gene deletion and longevity was found in 14% of elderly Germans and 21% of individuals of the control group [16]. Structural differences in
isoenzymes lead to different ability to metabolize xeno-biotics resulting in different degrees of susceptibility to multifactorial diseases [17].
For the first time ever, the glutathione-S-transfer-ase gene (GSTT1 and GSTM1) polymorphism has been studied in long livers of the Carpathian region. Furthermore, the peculiarities of the distribution of the GSTT1 and GSTM1 gene deletions in patients with AH and OA have been investigated. Some authors have studied the role of the GSTT1 and GSTM1 gene polymorphism in predicting the development of comorbid pathologies (AH and chronic obstructive pulmonary disease) and the severity of their clinical course in Ivano-Frankivsk region residents [18]. The GSTM1-/GSTT1- genotype has been proven to be predominant in patients with family history of such diseases. In contrast to our study genetic polymorphism has been analyzed in elderly people. Another author who studied the child population of Ivano-Frankivsk region has found that the severity of placental dysfunction, gestosis and the risk of intrauterine growth restriction increase in the GSTM1-/GSTT1 genotype [19].
Conclusions
1. The frequency of the functionally inactive GSTT1 and GSTM1 alleles among the all long livers of Ivano-Frankivsk region was 24.70% and 46.99%, respectively.
2. In long livers of four comparison groups, the functionally active GSTT1 and GSTM1 alleles predominated.
3. There were no statistically significant differences between patients with AH and OA and healthy long livers with the GSTM1 +/GSTT1 +, GSTM1 +/GSTT1 -, GSTM1 -/GSTT1 +, GSTM1-/GSTT1 - genotypes.
Prospects for further research
The development of any multifactorial pathology (AH and OA) requires the combinations of three factors, namely genetic factor, external environmental factor and stochastic factor. Therefore, the study of living conditions and lifestyle of studied long livers is promising.
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