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MEDICAL SCIENCES
RELATIONSHIP BETWEEN HEMODYNAMIC PARAMETRES AND NPPA, NPPB, NPR3 GENES POLYMORPHISM IN PATIENTS
WITH ISCHEMIC HEART DISEASE Alyavi A.L.1, Tulyaganova D.K.2, Sabirjanova Z.T.3, Radjabova D.I.4, Uzokov J.K5, Shodiev J.D.6, Toshev B.7 (Republic of Uzbekistan) Email: Alyavi538@scientifictext.ru
1Alyavi Anis Lyutfullayevich - Professor, Doctor of Medical Science, General Director; 2Tulyaganova Dildora Karimovna - Doctor of Medical Science, Senior Researcher, REPUBLICAN SPECIALIZED SCIENTIFIC-PRACTICAL MEDICAL CENTER OF THERAPY AND MEDICAL REHABILITATION; 3Sabirjanova Zulfiya Talgatovna - Docent, Candidate of Medical Sciences, THERAPY DEPARTMENT OF THE TASHKENT PEDIATRIC MEDICAL INSTITUTE; 4Radjabova Diyora Iskandarovna - Junior researcher; 5Uzokov Jamol Kamilovich - Junior researcher; 6Shodiev Jasur Davlatovich - Junior researcher; 1 Toshev Berkzod - Junior researcher, REPUBLICAN SPECIALIZED SCIENTIFIC-PRACTICAL MEDICAL CENTER OF THERAPY AND MEDICAL REHABILITATION, TASHKENT, REPUBLIC OF UZBEKISTAN
Abstract: in this study it has been shown the relationship of NPPA genotypes and allelic NPPB gene SNP with echocardiographic and angiographic parameters that play an important role in the pathogenesis of the ischemic heart disease. As well, it has been shown that some clinical features of the coronary heart disease were associated with above mentioned gene alleles. These genetic relationships are essential for the management of patients with coronary artery disease and these results open new perspectives for the understanding of the genetic mechanisms of development and progression of cardiovascular disease. Keywords: ischemic heart disease, gene polymorphism.
ВЗАИМОСВЯЗЬ МЕЖДУ ГЕМОДИНАМИЧЕСКИМИ ПАРАМЕТРАМИ И ПОЛИМОРФИЗМОМ NPPA, NPPB, NPR3 ГЕНОВ У ПАЦИЕНТОВ С ИШЕМИЧЕСКОЙ БОЛЕЗНЬЮ СЕРДЦА Аляви А.Л.1, Туляганова Д.К.2, Сабиржанова З.Т.3, Раджабова Д.И.4, Узоков Ж.К.5, Шодиев Ж.Д.6, Тошев Б.7 (Республика Узбекистан)
1Аляви Анис Лутфуллаевич - профессор, доктор медицинских наук, генеральный директор;
2Туляганова Дилдора Каримовна - доктор медицинских наук, старший научный сотрудник, Республиканский специализированный научно-практический медицинский центр терапии и медицинской реабилитации;
3Сабиржанова Зулфия Талгатовна - доцент, кандидат медицинских наук, кафедра терапии, Ташкентский педиатрический медицинский институт,
4Раджабова Диёра Искандаровна - младший научный сотрудник;
5Узоков Жамол Камилович - младший научный сотрудник;
6Шодиев Жасур Давлатович - младший научный сотрудник;
7Тошев Бекзод - младший научный сотрудник, Республиканский специализированный научно-практический медицинский центр терапии и медицинской реабилитации, г. Ташкент, Республика Узбекистан
Аннотация: в данном исследовании показана связь NPPA гентопиов и SNP аллеля NPPB гена с эхокардиографическими и ангиографическими параметрами, играющими важную роль в патогенезе ишемической болезни сердца. Кроме того, было показано, что некоторые клинические признаки ишемической болезни сердца связаны с вышеупомянутыми аллелями генов. Эти генетические связи имеют важное значение для лечения пациентов с ишемической болезнью сердца, и данные результаты открывают новые перспективы для понятия генетических механизмов развития и прогрессирования кардиоваскулярных заболеваний.
Ключевые слова: ишемическая болезнь сердца, полиморфизм генов.
Relevance. Coronary heart disease (CHD) is referred to multifactorial diseases, the development of which is the result of a complex interaction of hereditary predisposition and environmental factors. Since heredity is an independent risk factor, CHD is now becoming apparent [1]. Genetic associative studies and analysis of candidate genes have revealed a number of polymorphic variants of genes predisposing to the development of CHD [1, 2]. The results of these studies are contradictory and there is no consensus on their application. In addition, there are racial and ethnic differences in gene polymorphism [1], which explains the relevance of research in the Uzbek population. Analysis of gene associations with the disease and subsequent evaluation of individual genetic risk, which is essential for developing a differentiated approach to the prevention and treatment of diseases, in particular CHD and its complications - CHF, depending on the hereditary predisposition of the individual patient. The development of strategies for early preclinical diagnostics is one of the most relevant and progressive approaches that determine the prospects and opportunities for predicting and conducting preventive therapy by using genetic predictors.
Objective. To study the relationship between clinical, echocardiography and angiographic parameters with polymorphisms of NPPA, NPPB genes in patients with coronary heart disease.
Materials and methods of research. The study included 96 patients with CHD with stable angina pectoris of FC II-III. The mean age of the patients was 56.8 ± 5.3 years. All patients underwent echocardiography, coronary angiography and the study of polymorphism of the NPPA and NPPB genes. The control group comprised 20 healthy donors (mean age 53.6 ± 4.8 years) without cardiovascular pathology and severe chronic diseases.
EchoCG and Doppler EchoCG were used to study cardiac remodeling, systolic and diastolic LV functions. Studies carried out on the Samsung medison "Accuvix V20" ultrasound device (Korea) using a sector sensor with a color mode and pulse-wave, continuous-wave mode with a frequency of 2-4 MHz in standard echocardiography positions. It was studied next structural parameters: the end systolic diameter (ESD), the end diastolic diameter (EDD), the thickness of interventricular septum (IVS) and the posterior wall thickness (PWT) of the left ventricle (LV), the thickening of the IVS, the thickening of the PW. It was calculated the indicators, that characterize the functional state of the left ventricle: end systolic volume (ESV), end diastolic volume (EDV). The value of the ejection fraction (EF), calculated by the method of L. Teichholtz. Coronary angiography was performed in a catheterization laboratory on General Electrics devices (USA), performed according to the Judkins method [Judkins M.P. 1967] or Amplatz [Amplatz K. ettal. 1967] with diagnostic catheters 5F, 6F (USCI, Cordis,) by transfemoral or transradial access using contrast agent "Unigexol". The study of gene polymorphisms was carried out at the Center for High Technologies of the Uzbekistan Academy of Sciences. It were taken 96 samples for genotyping in patients with CHD. To study single nucleotide mutations (SNP) of the NPPA and NPPB genes, 48 polymorphisms were selected. The material for DNA was venous blood from the ulnar vein of 1 ml. To collect, store and transport blood used disposable plastic tubes with a volume of 2.5 ml with an anticoagulant (conservative) of 0.5 ml.
Statistical processing of survey results using variational statistics methods recommended for biomedical research on IBM PC AT Pentium IV.
Results of the study. During the study by EchoCG, it was revealed that with preserved LV systolic function, most parameters of transmittal blood flow (TMBF) before coronary angiography differed from the norm, clear violations of diastolic LV function were determined. Speed .A was
51
greater than the speed E, the ratio E / A - less than 1, which indicates diastolic dysfunction in the type of violation of relaxation. The fraction of early filling of the left ventricular (EFF LV) was 42,030 ± 1,751%, the filling fraction during the systole of left atrium (AFF LA) - 57,970 ± 1,756%. The duration of the DT interval normalized at the heart rate was 200.790 ± 10.554 conv. Unit, The duration of the IVRT interval normalized at heart rate is 139,570 ± 4,450 conv. Units In the temporal structure of the cardiac cycle, the ET period prevailed over the MM period. Dopplerographic characteristics of systolic ejection into the aorta (Vао, VTIao, ETLV) were within the limits of normal values (Table 1).
Table 1. Features of dopplerography in patients with stable forms of chronic coronary heart disease who
underwent coronary stenting
Indicator The value of the indicator ^±m) Control group
E, cm/s 37,500± 2,681 83,050± 1,537
A, cm/s 82,140± 2,516 64,820± 1,011
E/A 0,460± 0,031 1,280± 0,014
VTIE, cm 4,850± 0,320 7,410± 0,199
VTIA, cm 8,080± 0,227 4,870± 1,175
EFF, % 36,960± 1,980 60,430± 0,449
AFF, % 63,040± 1,976 39,570± 0,454
DT/RR, cond 233,80± 24,09 176,03± 5,78
IVRT/RR, cond 139,57± 4,45 83,32± 25,65
Vao, m 0,935± 0,020 1,227± 0,025
VTLao, m 18,980± 0,291 23,410± 0,349
ETLV/RR, cond 303,24± 10,16 378,35± 20,19
ETtm/RR, cond 582,81± 20,11 561,13± 15,45
MM/RR, cond 470,49± 13,14 485,99± 21,15
In patients who underwent myocardial revascularization with the method of planned coronary stenting, volume LV values (EDV, ESV) were significantly higher, and LV EF was lower than in the control group, in whom these values were within the norm (Table 2). Thus, in patients who were subsequently intervened, during the initial examination, clear evidence of intracardiac hemodynamic disturbances and all signs of diastolic LV dysfunction were documented.
Table 2. Indices of intracardiac hemodynamics ofpatients undergoing myocardial revascularization
Indicator Main group (n=29) ^±m) Control group
EDV LV, ml 210,96±5,15 103,89±2,87
ESV LV, ml 122,58±2,22 37,26±1,6
EF, % 41,76±1,03 64,30±0,63
Further, to achieve this goal there were chosen 48 polymorphisms of single nucleotide mutations (SNP) of the NPPA and NPPB genes presented in Table 3.
SNP # rs # Position (hg17)* HW p- value Call rate % The frequency of minor alleles Alleles NPPA NPPB
1 rs198402 11818932 1 97.8 0.17 A:G 3' 3'
2 rs2075538 11830868 1 98.9 0.10 A:G 3' 3'
3 rs198403 11830920 1 100 0.09 C:T 3' 3'
4 rs198404 11831093 1 97.8 0.09 A:T 3' 3'
5 rs198405 11831537 1 95.7 0.14 A:G 3' 3'
6 rs198406 11831858 1 93.5 0.45 T:C 3' 3'
7 rs2075539 11832024 1 98.9 0.05 C:T 3' 3'
8 rs198408 11832396 1 80.6 0.44 A:T 3' 3'
9 rs169158 11832494 1 92.5 0.14 A:G 3' 3'
10 rs2272803 11833055 1 97.8 0.05 C:A 3' 3'
11 rs198409 11833115 1 98.9 0.17 A:C 3' 3'
12 rs1023252 11833299 1 95.7 0.26 C:A 3' 3'
13 rs198411 11834651 1 98.9 0.16 G:A 3' 3'
14 rs198412 11834703 1 100 0.09 A:G 3' 3'
15 rs198413 11835068 1 94.6 0.14 A:G 3' 3'
16 rs198415 11835858 1 97.8 0.17 A:G 3' 3'
17 rs14078 11837276 1 92.5 0.10 C:T 3' 3'
18 rs198357 11838151 1 96.8 0.16 G:T 3' 3'
19 rs198358 11838342 0.13 100 0.27 A:G 3' 3'
20 rs198359 11838450 1 79.6 0.15 A:G 3' 3'
21 rs198360 11838835 1 97.8 0.17 A:G 3' 3'
22 rs198361 11839899 0.97 100 0.19 A:G 3' 3'
23 rs5068 11840240 1 89.2 0.06 T:C 3' UTR 3'
24 rs5067 11840247 1 83.9 0.15 T:C 3' UTR 3'
25 rs5066 11840261 0.37 78.5 0.08 G:T 3' UTR 3'
26 rs5065 11840334 1 75.3 0.15 T:C Nonsense 3'
27 rs5063 11841914 1 98.9 0.05 G:A Missense 3'
28 rs198372 11843780 1 97.8 0.17 C:T 5' 3'
29 rs198373 11843801 1 96.8 0.17 T:C 5' 3'
30 rs632793 11844943 1 97.8 0.47 T:C 5' 3'
31 rs577040 11845452 1 100 0.18 G:T 5' 3'
32 rs2981953 11846110 0.06 80.6 0.41 A:G 5' 3'
33 rs2981954 11846718 0.94 98.9 0.19 C:T 5' 3'
34 rs198374 11847835 1 97.8 0.18 C:T 5' 3'
35 rs198375 11848023 1 100 0.47 A:G 5' 3'
36 rs198378 11848462 1 95.7 0.19 T:C 5' 3'
37 rs6668352 11849095 0.75 100 0.29 C:T 5' 3'
38 rs198381 11850015 1 98.9 0.18 T:C 5' 3'
39 rs616308 11850152 0.94 94.6 0.19 C:T 5' 3'
40 rs198387 11851184 1 97.8 0.18 T:C 5' 3'
41 rs198388 11851606 0.82 93.5 0.50 G:A 5' 3'
42 rs198389 11853537 0.94 97.8 0.50 T:C 5' 5'
43 rs3753581 11854455 0.45 77.4 0.38 G:T 5' 5'
44 rs3753580 11855314 0.99 97.8 0.38 A:G 5' 5'
45 rs12406089 11855447 1 83.9 0.37 G:C 5' 5'
46 rs6668659 11856564 0.97 94.6 0.36 A:C 5' 5'
47 rs6676300 11859566 0.66 89.2 0.37 T:C 5' 5'
48 rs1009592 11862980 1 94.6 0.38 G:C 5' 5'
As a result of the genotyping of 96 samples of CHD patients, it was found that in 40% of the samples were detected no mutated alleles. In the remaining samples were detected 13 SNPs, shown in Table 4.
SNP NPPA position NPPB position The frequency of minor alleles Association with ANP p-value Association with BNP p-value
rs169158 3 3 0.14 2x10-5 6x10-4
rs1023252 3 3 0.25 5x10-7 0.07
rs198412 3 3 0.08 0.62 0.08
rs5065 3 3 0.14 5x10-5 0.003
rs198358 3 3 0.19 3x10-5 0.001
rs5068 3' UTR 3 0.04 6x10-6 0.18
rs5063 Missense 3 0.04 6x10-55 0.003
rs632793 5 3 0.39 0.05 2x10-8
rs577040 5 3 0.13 0.01 6x10-4
rs198375 5 3 0.40 0.07 2E-06
rs198387 5 3 0.14 0.001 4x10-5
rs198388 5 3 0.42 0.39 2x10-6
rs198389 5 5 0.36 0.07 0.007
In the studied group of patients, 91 patients had homozygous CC genotype by rs5068 allele of NPPA gene, in 5 samples heterozygous CT genotype was found, homozygous genotype of TT was not found in any sample. There was no statistically significant association in the t-test between the two genotypes with respect to sex, body mass index, age (p>0.05). Evaluation of the influence of the NPPA and NPPB genes on the cerebral natriuretic peptide on the LV structural and functional state according to Echo-CG data in patients with CHD revealed the association of SNP with LV hypertrophy in CHD patients with arterial hypertension with an interventricular septum thickness of more than 12 mm (r = 0.43, p <0.05) (table 5). In the study of coronary angiography data, the association of these genes with multiple (two or more) lesions of the vessels (LCA, LAD) (r = 0.44, p <0.05) was revealed.
Table 5. Correlation of indices of the LV structural and functional state with polymorphisms of NPPA and
NPPB brain natriuretic peptide genes
EchoCG indicators NPPA gene genotypes NPPB gene genotypes
C/T T/C C/C A/G G/G C/C
EDD LV (mm) 59,8±2,9 56,2±3,4 53,1±2,4 59,2±3,8 55,1±3,1 57,3±4,7
ESD LV (mm) 39,6±2,8 37,6±3,6 32,8±2,9 39,9±3,2 35,3±3,9 38,9±4,1
SF % 27,9±3,2 30,0±3,8 32,2±3,6 28,2±3,1 30,7±3,0 29,4±3,4
EF % 38,7±4,1 44,5±5,8 49,2±4,8 39,6±4,9 46,1±5,9 43,4±6,2
LA, mm 51,2±4,8 47,6±5,3 42,3±3,2 50,6±3,6 46,1±4,2 48,3±5,1
IVS LV, mm 14,1±1,1 12,5±2,5 11,7±1,1 13,5±1,9 12,6±2,5 13,1±2,2
PWT LV, mm 13,8±1,2 12,2±1,9 10,9±1,0 13,1±1,8 12,4±2,4 12,6±2,0
The allele rs198388 of the NPPB gene was found mostly in patients with coronary artery disease in combination with hypertension of the third degree, but with normal left ventricular function, which suggests that most likely this mutation has no effect on left ventricular dysfunction or acts as a Protective effect in the formation of chronic heart failure. It is necessary to conduct an additional study of patients to determine the frequency of this genotype.
Despite the detection of the mutant allele T381C (rs198389), no statistically significant associations of this genotype were found with any of the parameters studied, however, there is evidence in the literature that for the European population this polymorphism is strictly associated with hypertension, diabetes and coronary heart disease [4]. Since the entire study group has a history of either chronic heart failure or myocardial infarction, we can not judge the association of
this genotype without additional studies of the control population sample. Data on all polymorphisms with identified associations are presented in Table 6.
Table 6. Frequency of occurrence of allelic genotypes ofSNP genes NPPA and NPPB in the studied group
SNP Heterozygous allele Dominant allele Homozygous minor allele
rs5068 СТ (5) - 5,2% CC (91) - 94,8% TT (0)
rs198388 CT (8) - 8,3% CC (87) - 90,6% TT (1) - 1,1%
rs198389 TC (7) - 7,3% TT (89) - 92,7% CC (0)
rs5065 TC (10) - 10,4% CC (85) - 85,4% TT (3) - 3,1%
rs632793 AG (3) - 3,1% AA (93) - 96,9 % GG (0)
rs198358 AG (18) - 18,8% AA (77) -80,2 % GG (1) - 1,1%
Discussion. The results obtained by us in the surveys of patients of the Uzbek population can be compared with the results of studies carried out in persons of the Italian population, where it was shown that the markers of the NPR1 gene are largely related to the left ventricular myocardial mass index and the thickness of the IVF. The polymorphism of the GT allele was associated with essential hypertension, while the I / D deletion did not show a significant relationship with hypertensive disease. It was suggested that mutant alleles may be associated with a decrease in the activity of the A-receptor NUP [6]. It is interesting to compare the data on the relationship between the echo indicators and the polymorphism of the BNP genes with the results of a prospective study (Amanda A. Fox et al.). Amanda A. Fox investigated the relationship between the system of natriuretic peptide genes with left ventricular dysfunction after CABG and showed genetic variability in NPPA / NPPB and NPR3 genes associated with the risk of developing and progressing of ventricular dysfunction after primary CABG [3]. Knowledge of such genotypic predictors obtained in different populations can lead to a better understanding of the molecular mechanisms underlying the development of ventricular dysfunction, and, consequently, will allow them to optimize their prevention.
Conclusion. During our research, it were established associative relationships of allelic genotypes of NPPA and NPPB genes SNP with the structural and functional state of the myocardium in patients with CHD, which opens new prospects for studying and understanding genetic mechanisms of the development and progression of the disease.
References / Список литературы
1. Всемирная организация здравоохранения. Информационный бюллетень. Десять причин смерти в мире, 2014.
2. Торшин И.Ю., Громова О.А. Сосудистые заболевания сердца, мозга и молекулярные гены. Ассоциативные исследования и патофизиология сосудистых заболеваний // Трудный пациент, 2008. № 4.
3. Fox Amanda A., Collard Charles D. et al. Natriuretic peptide system gene variants are associated with ventricular dysfunction after coronary artery bypass grafting // Anesthesiology, 2009. April. 110 (4): 738-747.
4. Meirhaeghe A., Sandhu M.S., McCarthy M.I., de Groote P., Cottel D., Arveiler D. et al. Association between the T-381C polymorphism of the brain natriuretic peptide gene and risk of type 2 diabetes in human populations // Hum Mol Genet, 2007; 16: 1343-50.
5. Potter L.R., Abbey-Hosch S., Dickey D.M. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions // Endocr Rev., 2006; 27: 47
6. Rubattu S., Bigatti G., Evangelista A., Lanzani C., Stanzione R., Zagato L. et al. Association of atrial natriuretic peptide and type A natriuretic peptide receptor gene polymorphisms with left ventricular mass in human essential hypertension // J Am Coll Cardiol., 2006; 48: 499-505.
7. Weber M., Burian M., Dragutinovic I., Moellmann H., Nef H., Elsaesser A. et al. Genetic polymorphism of the type A human natriuretic peptide receptor (NPR-A) gene contributes to the interindividual variability in the BNP system // Eur J Heart Fail, 2008; 10: 482-9.
THE ISOLATION FROM LIVER OF SAPONINS SOLOMON'S SEAL
SMOOTH 1 2 Iskenderov G.B. , Pashayeva S.A. (Republic оf Azerbaijan)
Email: Iskenderov538@scientifictext.ru
1Iskenderov Gaibverdi Bashirovich - Doctor of Pharmacy, Professor; 2Pashayeva Sara Agakishievna - Assistant, DEPARTMENT OF GENERAL AND TOXICOLOGICAL CHEMISTRY, AZERBAIJAN MEDICAL UNIVERSITY, BAKU, REPUBLIC OF AZERBAIJAN
Abstract: goal. Development of methods of chemical-Toxicological analysis of saponins Solomon's seal smooth.
Methods. Isolation of saponins of plants of the biological material depends on several factors. Polygonatum glaberrimum C. Koch, as a poisonous plant was investigated in chemical-Toxicological aspects. We studied the factors influencing the isolation to establish the optimal conditions for maximal isolation of saponins from biological material. For the manifestation of the chromatograms was used reagent Sanya, 2-3 min. then dried in a drying Cabinet at a temperature of80-900 C. the Saponin in the solution were quantitatively determined with a spectrophotometer "Jenway 7315".
Results. The best solvent is not pure n-butanol and n-butanol saturated with water. This solvent is the most isolates of the liver saponins and extractive substances is minimal. the correlation of the biological object, and an insulating solvent significantly affects the yield of saponins of biological material. Extraction of saponins from the biological object of considerable importance is the time used for isolation. Experiments have shown that this factor is not less than 3 hours. Triple insulation creates maximum output of saponins from the biological object. When you isolate saponins from biological material is the most appropriate room temperature. On the basis of their studies suggested that the optimal method that can be used in laboratory diagnosis of acute poisoning, as well as in forensic chemical practice.
Conclusion. The optimal parameters conditions isolation of saponins from biological material: solvent - n-butanol saturated with water, the ratio of a biomaterial-extracting solvent of 1:2.5, contact time of the solvent with the object of not less than 3 hours, the ratio of extraction 3 times, pH neutral, temperature room.
Keywords: Polygonatum glaberrimum C. Koch, the chemical-Toxicological analysis.
ИЗОЛИРОВАНИЕ ИЗ ПЕЧЕНИ САПОНИНОВ КУПЕНЫ ГЛАДКОЙ 12 Искендеров Г.Б. , Пашаева С.А. (Азербайджанская Республика)
1Искендеров Гаибверди Баширович - доктор фармацевтических наук, профессор; 2Пашаева Сара Агакишиевна - ассистент, кафедра общей и токсикологической химии, Азербайджанский медицинский университет, г. Баку, Азербайджанская Республика
Аннотация: цель. Разработка методов химико-токсикологического анализа сапонинов купены гладкой.
