Comparative research of c235t and c174t gene mutation frequencies for angiotensine gene and of C667t for methylthetrahydrofolate reductase in patients with cardiovascular diseasies and relatively healthy individuals in Azerabujan Republic Текст научной статьи по специальности «Биотехнологии в медицине»

Comparative research of C235T and C174T gene mutation frequencies for angiotensine gene and of C667T for methylthetrahydrofolate reductase in patients with cardiovascular

diseasies

and relatively healthy individuals in Azerabujan republic Guliyeva R. (Republic of Azerbaijan)

Определение разности частот встречаемости полиморфизмов С235Т и С174Т гена ангиотензиногена и С667тТ гена метилентетрагидрофолатредуктазы между больными с сердечно-сосудистыми заболеваниями и относительно здоровыми

лицами

из Азербайджанской республики Гулиева Р. Г. (Азербайджанская Республика)

Гулиева Рена Гейдаровна / Guliyeva Rana - магистр биологии, докторант по программе доктора философии по биологическим наукам, кафедра «Генетика и дарвинизм»,

Бакинский государственный университет, г. Баку, Азербайджанская Республика

Abstract: С174Т (МеП74ТИг), С235Т (Мет235ТИг) mutations polymorphism of AGTgene and С677Т (А1аб77Уа1) mutation of MTHFR were for the first time researched for patients with cardiovascular diseases from Azerbaijan Republic. Identified frequencies of these mutations in patients with cardiovascular diseases statistically obviously were higher than those in the control group. Homozygous, compound and heterozygous states for all the three mutations were identified in patients with severe arterial hypertension. Presence of mutation in heterozygous state was found mainly in patients with moderate type of disease. Depending on inherited predetermination of the certain patient, the following assessment of his/her individual genetic risk has important practical significance for the development of differentiated approach in prophylaxis and treatment of cardiovascular diseases and their consequences. The obtained results are planned to be used in the course of prophylactic events in the population of Republic.

Аннотация: впервые нами изучен полиморфизм мутаций С174Т (Мет174Тре), С235Т (Мет235Тре) гена АГТ и мутации С677Т (Ала677Вал) гена МТГФР у больных с сердечно-сосудистыми заболеваниями из Азербайджанской Республики. Идентифицировано наличие этих мутаций у больных с сердечнососудистыми заболеваниями, частота встречаемости которых значительно превышала значения таковых в контрольной группе. У больных с тяжелой формой артериальной гипертензии выявлены гомозиготные и гетерозиготные состояния всех трех мутаций. Наличие мутации в гетерозиготном состоянии обнаружено в основном у больных с умеренной формой артериальной гипертензии. Планируется использование полученных результатов в ходе проведения профилактических мероприятий среди населения Республики. Установление связи данных полиморфизмов с сердечно-сосудистыми заболеваниями и последующая оценка индивидуального генетического риска имеют важное значение для разработки дифференцированного подхода к профилактике и лечению сердечно-сосудистых заболеваний и их осложнений в зависимости от наследственной предрасположенности конкретного пациента.

Keywords: genetic polymorphism, gene frequency, phenotypic frequency, genotype, angiotensinogen,

methylthetrahydrofolate reductase, polymerase chain reaction, cardiovascular diseases.

Ключевые слова: генетический полиморфизм, ангиотензиноген, метилентетрагидрофолатредуктаза, полимеразно-цепная реакция, сердечно-сосудистые заболевания.

Introduction

Gene of angiotensin (AGT) encodes angiotensinogen protein - alpha globulin fraction serum globulin, developed mainly by lever cells, out of which angiotensin 1is formed under the impact of rennin. Angiotensin 1 does not possess any biologic activity and exists only as a predecessor of angiotensin 2, which in its turn raises the blood pressure, lessening the vessels diameter and increasing water and salts resorption. 15 point mutations, most of which lead to amino acids substitutions, were identified in the course of angiotensinogen gene study [1], [8]. Much work was devoted to analysis of relations between these mutations and cardiovascular diseases. Most widely there were researched variants related to amino acidic substitutions as cytosine nucleotide substitution for thymine nucleotide in the position 235 with the consequent amino acid substitution of methionine by threonine, and cytosine nucleotide by thymine nucleotide in the position 174 with consequent amino acid substitution of threonine for methionine. There exist controversial results on the presence of the given polymorphisms and their relations with cardiovascular diseases. Strict correlation exists betweenThr174Met and Met235Thr alleles and various forms of hypertension, mainly, in European populations and in Japans. At the same time there observed the absence of the said association in Afro-Americans [5]. It is also shown, that Met235Thr variant is an independent risk factor for myocardial infarct and ischemic heart disease (IHD) [3] development in Europeans, while in Japans there was identified no association of the given polymorphism with IHD [2]. Hence, the Met235Thr variant contributes some certain pathogenic effect, but it couldn’t be considered as a significant mutation, because its effect differs greatly in various ethnic groups’

representatives. Effect of ethno-polymorphic variant can define non-equal link with some pathogenesis variants of angiotensinogen gene or, on the other hand, it can manifest itself on specific population genetic background only. Frequency of mutant gene variant is in range of 34-43%.Mutation inheritance type: autosome dominant (encountered in men and women equally, it is enough to inherit one mutant gene variant from any of parents, and it gives 50% chance to children to get the disease) [8].

Methylthetrahydrofolate reductase (MTHFR) is the key enzyme of folate cycle and catalyses the reaction of folic acid (folate) transformation into active form, which participates in the synthesis of methionine amino acid responsible for DNA methylation at mitosis and removal of extra homocysteine amino acid, which possesses obvious toxic effect. Increased concentration of homocysteine in blood leads to early myocardial infarct development and to thrombosis-vascular disease, thrombosis development in deep and surface veins, thrombosis of carotid arteries, Crone disease, to some psychic diseases (epilepsy), etc. There exist different allelic variants of this enzyme. Practical significance has the allele: substitution of cytosine nucleotide for thymine nucleotide in the position 677 with consequent amino acid substitution of alanine by valine (Ala677Val). Activity reduction up to 35% from the mean value occurs by this mutation, resulting in non-favourable effects development as DNA methylation lack. Individuals inherited this genotype variant from both of parents are more (14-21%) exposed to cardiovascular diseases [6], [7], [9].

Taking into account the non-research of the issue, related to presence of C174T (Met174Thr) and C235T (Met235Thr) AGT gene mutations polymorphisms and C677T (Ala677Val) polymorphism of MTHFR gene in the population of the Republic, the goal of our studies was the identification of those polymorphism frequencies in patients with cardiovascular diseases (ischemic-heart disease, myocardial infarct and hypertension ) with assessment of mutation phenotypic and gene frequencies and comparison between those indications and the same indications in relatively healthy individuals.

Materials and methods of study

Material for the researches was 2ml of venous blood on EDTA (or heparin) anticoagulant from 72 people (39 men and 33 women) of 18 to 67 years of age with cardiovascular system disease. Control group was of practically healthy individuals: 30 men and 24 women, that is 54 people in the age between 20 and 52.

We have used in our work molecular-genetic methods based on polymerase chain reaction and electrophoresis of genomic DNA, DNA amplified fragments and separate fragments’ nucleotides to research genetic polymorphisms.

Genomic DNA was isolated from venous blood using ready QIAampgenomic DNA and RNA kits, manufactured by QIAGEN, Germany [4].

Intactness and the quantity of the isolated DNA as well as of amplificate (gene fragment) were defined by electrophoresis in 1.7% agarose gel using electrophoretic apparatus (PowerPacBasicGelDocIMEZ) Imager, BioRad company, USA. During electrophoresis the marker for the synthesized DNA fragments was DNALadder 100 bp [4].

Polymerase-chain reaction (PCR) was carried out in the following regime: 95оС for 2 minutes, (95оС for 30sec, 58оС for 30sec, 78оС for 2minutes for 25-30 cycles), 72оС for 10 minutes and pause at 4оС for 7 minutes in amplificator Professional Thermocycler manufactures by Biometra, Germany [4]. For the amplification of each out of 5 gene sites we used 2 primers (Forward and Reverse). Gene fragments for researches were five exons of AGT gene. For PCR of MTHFR gene fragment, two synthetic oligonucleotide primers were used as primers (Forward and Reverse).

The structure of synthetic nucleotide primers which we used for PCR of five exons of AGT gene is presented in Table 1.

To purify DNA fragments, a set of magnets was used after first stage of PCR: “Agencourt AMPure XP PCR purification” and SPRIPlate 96 Super Magnet Plate. Purified DNA fragments were used for the following researches. The second PCR were carried out in the regime: 95оС for 2 minutes, (95оС for 30sec, 58оС for 30sec, 78оС for 2minutes for 25-30 cycles), 72оС for 10 minutes and pause at 4оС for 7 minutes in amplificator.

Then the standard procedure on the apparatus GENOMELabGeXP™ Sequencing to identify nucleotide sequence of each DNA fragment.

Table 1. Name and structure of synthetic primers for AGT gene and MTHFR gene

Primer name Nucleotide sequence of a primer

1. Sequence- AGT F1 5'-TGC TTC TGT GTT TTC CCC AGT-3'

1.Sequence- AGT R1 5'-AGA GAC AAG ACC GAG AAG GAG C-3'

2.Sequence- AGT F2 5'-GGG CTA AAT GGT GAC AGG GA-3'

2. Sequence- AGT R2 5'-CCA GAG CCA GCA GAG AGG TTT-3'

3. Sequence- AGT F3 5I-CCT CAT TCC TGC CCC TGT CT-3I

3. Sequence- AGT R3 5'-GCT CAG GTG TGT CTA CTC CCC A-3'

4. Sequence- AGT F4 5'-AGC ACA GAG GTC CTG AGC C-3'

4. Sequence- AGT R4 5'-CCA AAG TCC AGG AAA GCA C-3!

5. Sequence- AGT F5 5‘-AGA TCA TAA GTC TTG GGC C-3!

5. Sequence- AGT R4 5‘-GCA TAG GCC AGG TTT CCA C-3!

6. Sequence- MTHFR- R 5‘-GCC TCC CTC CTCCTC TTC CCA CTG-3‘

6. Sequence- MTHFR- F 5‘-GCC AGC ACC GCC GTG AAC TAC TG-3‘

RESEARCH RESULTS

After isolation of DNA from white blood cells out of patients’ venous blood, mutations identification were carried out as follows. First of all the quality of the isolated genomic DNA was checked. Intactness of the isolated DNA as well as of amplificate (gene fragment) was defined by electrophoresis in 1.7% agarose gel using electrophoresis. DNALadder 100bp was as a marker.

Figure 1 presents a photo of amplified fragments of AGT gene

1 2 3 4 5

Figure 1. Electrophoregram of DNA size marker (band 1) and fragments of AGT gene after PCR (bands 2-5)

Results of genetic screening of C174T and C235T alleles of AGT gene as well as C677T MTHFR gene in practically healthy individuals from control group are presented in the Table 2.

In nine cases out of 54 studied individuals from control group (30 men and 24 women within the age range 2052 years), C174T (Met174Thr) mutation of AGT gene was found and it was heterozygous (C/T). Phenotype frequency, genotype frequency and allele frequency were as 16.7, 0.1667 and 0.833, respectively.

C235T (Met 235Thr) mutation of AGT gene is identified as heterozygous in 6 individuals in control group where phenotypic frequency was 11.1, genotype frequency - 0.1111 and allele frequency - 0.0556.

Table 2. Phenotype, genotype and gene frequencies of C174T, C235T alleles of AGT gene and C677T allele of MTHFR gene in

individuals in control group

Phenotype frequency (in %) Genotype frequency (in unity fractions) Alleles frequency (in unity fractions)

С174Т mutation of AGT (Met174Thr)

T/T - T/T - T0.0833

C/T16.7 C/T 0.1667 С 0.9167

C/C 83.3 C/C 0.8333

С235Т mutation of AGT (Met235Thr)

T/T - - T 0.0556

C/T 11.1 C/T 0.1111 C 0.9444

C/C 88.9 C/C 0.8889

С677Т mutation of MTHFR (Ala677Val)

T/T - T/T - T 0.1111

C/T 22.2 C/T 0.2222 C 0.8889

C/C 77.8 C/C 0.7777

In 12 cases out of 54, mutation C677T MTHFR (Ala677Val) in heterozygous state was identified with phenotype frequency 22.2%. Genotype and allele frequency were 0.2222 and 0.1111, respectively.

72 patients: 39 men and 33 women with cardiovascular diseases as: ischemic heart disease, myocardial infarct and hypertension were also included into research. Their age varied between 18 and 67 years of age. Duration of their arterial hypertension according to anamnesis was 2 to 6 years. 54 out of 72 (75%) had inherited burden on arterial hypertension.

When distributing patients on the severity of their arterial hypertension, 18 individuals out of 72 (25%) had mild form of arterial hypertension, 30 (41.67%) had moderate arterial hypertension, and 24 (33.33%) had severe form of arterial hypertension. In most of them the course of disease was stable. In 9 (12.5%) patients we observed hypertensic crisis in course of arterial hypertension, those crisis occurred not oftener than 3-4 times a year.

Results of genetic screening of C174T and C235T alleles of AGT gene and C677T of MTHFR gene among patients with cardiovascular diseases as ischemic heart disease, myocardial infarct and hypertension are presented in the table 3.

Result of AGT gene fragments sequencing has shown substitution of cytosine nucleotide by thymine which leads to amino acid substitution - methionine is changed by threonine in 24 patients. In three cases in men the said mutation was in homozygous state (T/T) - 4.17%, in the rest 21cases mutation was in heterozygous state (C/T) -29.16%. In the experimental group phenotype frequency of homozygous and heterozygous states was 16.63% higher than in control group.

In nine cases: six men and three women C235T mutation of AGT gene was in homozygous state (T/T), and in the rest eighteen cases it was in heterozygous state (С/T). (T/T), (С/T) и (С/С) phenotypes’ frequencies equaled 12.5%, 25.0% and 62.5%, respectively. Genotype frequencies corresponded as for T/T - 0.1250, С/T - 0.2500 и С/С - 0.6250. Consequently, T and C alleles’ frequencies in unity fractions were 0.2500, 0.7500, respectively. In patients with cardiovascular diseases: ischemic heart disease, myocardial infarct and hypertension the phenotypic frequency of the said mutation was 26.4% higher than the same indication in control group. Mutation frequency is similar to those obtained in population researches for different countries throughout the world [5], [6].

As a result of sequencing of MTHFR gene fragment from 72 patients, in 27 cases (37.5%) we got heterozygous C677T mutation of MTHFR gene (Ala677Val). The frequency of C677T mutation of MTHFR gene (Ala677Val) in patients’ group was 15.3% statistically higher than the same indication in individuals in control group.

Table 3. Phenotypic, genotypic and alleles frequencies of С174Т, С235Т of AGT gene, and С677Т of MTHFR among patients

with cardiovascular diseases

Phenotype frequency (в%) Genotype frequency (in unity fractions) Alleles frequency (in unity fractions)

С174Т mutation of AGT (Met174Thr)

T/T 4.17 T/T 0.0417 T0.1875

C/T29.16 C/T 0.2916 С 0.8125

C/C 66.67 C/C 0.6667

С235Т mutation of AGT (Met235Thr)

T/T 12.5 T/T 0.1250 T 0.2500

C/T 25.0 C/T 0.2500 C 0.7500

C/C 62.5 C/C 0.6250

С677Т mutation of MTHFR (Ala677Val)

T/T - T/T - T 0.1875

C/T37.5 C/T 0.3750 C 0.8125

C/C 62.5 C/C 0.6250

When looking on distribution of С174Т, C235T, С677Т mutations between patients with different severity degrees of arterial hypertension we got the following picture: all 24 patients with severe arterial hypertension form had those mutations. In 9 cases (12.5%) patients had homozygous С235Т mutation, and in 3 cases - homozygous C174T mutation (4.16%). In 12 cases compound mutations of С174Т and С677Т (16.7%) were observed.

In the group of 30 patients with moderate arterial hypertension the following heterozygous mutations were identified: 3 cases with C235T, in 12 cases mutation C677T and in 6 cases C174T mutation.

It should be mentioned, that in the group with mild form of arterial hypertension of 18 patients 6 mutations were found: C174T and C235T in heterozygous state.

As it is seen from our data, the difference in frequencies of mutations between groups of patients with cardiovascular diseases and relatively healthy individuals was 26.4% for C235T (Met235Thr) of AGT gene, 33.3% for C174T (Thr174Met) of AGT gene and 15.3% for C677T (Ala677Val) of MTHFR gene. Hence, statistically significant difference in frequencies was defined for all three mutations between experimental and control groups.

Thus, we have studied polymorphism of two polymorphisms for C174T (Met174Thr) and C235T (Met235Thr) of AGT gene mutations and C677T (Ala677Val) mutation of MTHFR gene for individuals with ischemic heart disease, myocardial infarct and hypertension by complex of modern molecular-genetic diagnostics methods. Statistically significant those mutations frequencies difference was identified in the group of patients with severe forms of cardiovascular system lesions. Homozygous and compound state was observed in people with severe disease form. Namely, heterozygous state of mutation was found in individuals with moderate form of arterial hypertension. Consequently, the obtained results of molecular-genetic researches in patients with cardiovascular diseases are of great importance. On-time prophylaxis through finding С174Т, С235Т of AGT mutations and C677T mutation of MTHFR gene in patients will allow doctors to carry out qualified treatment of cardiovascular diseases.

Conclusions:

1. The following frequencies of the given mutations are observed in the control group: C235T (Met235Thr) AGT - 11.11%, C174T (Thr174Met) AGT - 16.7%, C677T(Ala677Val) MTHFR - 22.2%.

2. In patients with severe arterial hypertension, homozygous and compound mutations were found for all three mutations.

3. The presence of mutation in heterozygous state has been mainly found in patients with moderate arterial hypertension.

4. The following frequencies of the given mutations were identified in patients with different degrees of severity

of cardiovascular diseases: C235T(Met235Thr) AGT - 37.5%, C174T(Thr174Met) AGT -33.3%,

C677T(Ala677Val) MTHFR - 37.5%.

5. Statistically significant difference in frequencies was defined for all three mutations between experimental and control groups.

6. On-time identification of mutations C174T, C235T of AGT gene and mutation C677T MTHFR gene in individuals will allow doctors to carry out prophylaxis with the following qualified treatment of cardiovascular diseases.

References

1. Введение в молекулярную диагностику. В 2-х томах. Том 2. / Под редакцией академика РАН и РАМН М. А. Пальцева. Москва, ОАО «Изд. Медицина», 2011. 504 с.

2. Нгуен Тхи Чанг, Шкурат Т. П. Исследование ассоциации Т174М и М235Т гена ангиотензиногена с

ишемической болезнью сердца в Ростовской популяции. [Электронный ресурс]: Биомедицинский журнал. Том 11. ст.7. Апрель, 2010, с.63-77. Режим доступа:

http://www.medline.ru/public/art/tom11/art007pdf.phtml. (дата обращения: 27.11.2015).

3. Ишемическая болезнь сердца. [Электронный ресурс]: Биология и медицина. Режим доступа: http://medbiol.ru/medbiol/biochem/001be90e.htm (дата обращения: 27.11.2015)

4. Application information. Purification of GENOMELABTM GeXP Sequencing Productions using SPRI Clean SEQr Magnetic Beards. CEQ 2000, CEQ 2000XL, CEQ 8000, CEQ 8800 &GeXP Instruments BECKMAN COULTER. Application Team Europe. URL:

http://www.beckman.cz/Media/Default/LS/DocGeXP/GeXP%20Applications%20Note%205%20CleanSeq.pdf. (дата обращения: 27.11.2015)

5. Ash GI, Scott RA, Deason M., Dawson TA, Wolde B., Bekele Z., Teka S., Pitsiladis YP. No Association between ACE Gene Variation and Endurance Athlete Status in Ethiopians. [Электронный ресурс]: Medicine and science in sport and exercise. 2011. Режим доступа: http://www.ncbi.nlm.nih.gov/pubmed/20798657 (дата обращения: 27.11.2015)

6. Gu W., Zhang F., Lupski J. R. Mechanisms for human genomic rearrangements. [Электронный ресурс]: European Journal Human Genetics, 2008, vol.1, p.4-12. Режим доступа: http://www.ncbi.nlm.nih.gov/pubmed/19715442 (дата обращения: 27.11.2015)

7. Kim SM, Oh SD, Jung IG, Lee J, Sim YJ, Lee JK, Kang BY. Distribution of the Trp64Arg polymorphism in the beta(3)-adrenergic receptor gene in athletes and its influence on cardiovascular function. Kardiologia Polska. August, 2010. 68(8), p.920-926.

8. Kurland L., Liljedahi U., Karlsson J. et al. Angiotensinogen gene polymorphism: relationship to blood pressure response to antihypertensive treatment. Results from the Swedish Irbesartan Left Ventricular Hypertrophy Investigation vs Atenolol (SILVHIA) trial // American Journal Hypertension. 2004. Vol.17, issue 1, 2004, p.813.

9. Liljedahi U., Karlsson J., Melhus H. et al. (2003) A microarray minisequencing system for pharmacogenetic profiling of antihypertensive drug response // Pharmacogenetics.Vol.13, № 1, p. 7-17.