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Study of gene — candidate marker's levels in patients with non-burdened and burdened familial history of NFPA...
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Kholova Dilorom Sharifovna, Khalimova Zamira Yusufovna, The Republican Specialized Scientific-Practical Medical Center of Endocrinology of the Republic of Uzbekistan, Tashkent.
E-mail: [email protected]
Study of gene — candidate marker's levels in patients with non-burdened and burdened familial history of NFPA (non-functioning pituitary adenomas)
Abstract: Early diagnostics of NFPA relates to difficult questions of modern neuroendocrinology that even high-informative computer tomographs (CT) and magnetic resonance imaging (MRI), and also clinical-laboratory, immunofermental — hormonal researches do not provide the right answer in 30-55% of cases, and at microadenomas (the tumor sizes is up to 10 mm) in 90 100% of cases. At a stage of microadenoma NFPA s are seldom diagnosed and often accidentally discovering during diagnostic. The diagnosis is defined, as a rule, when adenoma has already reach a considerable size, causing occur sight violations, headache and other neurologic symptoms connected with an invasion of tumor in the cavity of skull and structure of the basis of skull. As references showed NFPA is met in 25-43% of pituitary adenomas and up to 10% of all intra cranial tumors. Due to the progress of medical genetics, molecular biology there was a possibility to expand the pathogenesis representation of NFPA. Molecular and genetic researches showed that up to 5% of NFPA cases refer to genetically predisposed people. At the same time in literature there is practically no comparative data on clinical course and disease diagnostics in populations between sporadic and family NFPA disease. Researches implemented in this direction will allow improving preclinical diagnostics, to differentially approach to tactics of treatment of NFPA patients.
Keywords: non-functioning pituitary adenomas, the family anamnesis, molecular and genetic researches, early diagnostics.
In recent years, the efforts of researchers about study of the etiopathogenesis of NFPA directed to find out the molecular and genetic factors. Introduction of modern achievements of genetics and molecular biology in all basic and clinical medicine areas has significantly changed our understanding of the etiopathogenesis and, consequently, the ability to diagnose, treatment and prevention of many diseases. In endocrine system, genes have a relation to the realization ofvarious functions, encoding protein hormones, receptors, enzymes, steroid biosynthesis, intracellular signaling molecules, transport proteins, ion channels, transcription factors and other molecules. In present it is known that many endocrine diseases have a hereditary nature, as NFPA [9], associating with a defect of a particular gene or being characterized polygenic mode of inheritance [5; 6]. At the same time in the development of NFPA
there are not data about inheritance causes at the level of groups of genes derived from the structure and the biological function of the encoded protein molecules [3; 6].
On the strength of experience of the world genetics it can be confirmed that the majority of NFPA are genetically determined, although the disease in close relatives is not always possible to identify [1,2,4]. Identified familial history of NFPA in our study consisted of 25 cases, including panmixia — 11 and inbreeding — 14 patients.
Based on the above, the purpose of our study was to determine the level of gene markers — candidates in patients with non-burdened and burdened familial history of NFPA.
Materials and methods. We carried out the study to identify in 20 patients with non-burdened familial history (1 group) and in 25 patients with burdened familial history of NFPA (2 group)
Section 6. Medical science
polymorphic markers of genes — candidates inherited predisposition: p53, bcl-2, FNO-a, eNOS3, VEGF and VEGFR-2 as well as the activity of the NO-system. These patients were identified during the clinical examination in the clinic of the RSSPMCE in the period from 2010 to 2014 years, whose age were between 18 and 70 years (44,5 ± 3,85goda). In addition, studies have been conducted in healthy individuals — control group of 20 volunteers, gave an informative consent to research. The average age of the control group was 43,6 ± 4,5 years — 10 men and 10 women.
Verification of the diagnosis carried out due to data of the clinical symptoms, the results of magnetic resonance imaging (MRI) and/or computed tomography (CT); RIA- (Radioimmunoassay) serum study for determine the level of tropic and peripheral hormones (Hormone Research carried out on the counters «Gam-ma-12» and «Strantg 300»), and for study polymorphic markers of the genes candidate inherited predisposition (p53, bcl-2, FNO-a, VEGF and VEGFR-2 polymorphism NOS3, activity of NO-system functions). For this purpose was conformed Immunoassay Analyzer of the firm «Stat-Fax» (USA) with using a set of test-systems (OOO «Cytokine», St. Petersburg).
Results and its discussion. Studies have shown that NFPA patients in comparison with control data indicated a significant increased serum factors of regulation of the apoptosis — increasing the level of proapoptotic protein p53 to 87.5% (p <0.001); the activation of anti-apoptotic processes — the expression of proteins bcl-2 and FNO-a — 15,9% (p <0.05) and 24.0% (p <0.001) on the background of depressed basal eNOS3-vascular relaxing factor to 25.1% (p <0.01) and the absence of reaction factors of the regulation of angiogenesis — vascular endothelial growth factor (VEGF) and its soluble receptors VEGFR-2 that is not much different from those of the values in the control group — 3.8 and 4.6% (p> 0.05). At the same time, we found a direct dim correlation between levels of bcl-2 and FNO-a (0,596; p <0.05), absence of communication between the FNO-a and eNOS3 (0,029; p <0.5), and a strong relation between FNO- a and indicators of VEGF (0,81; p <0,001), VEGFR-2 (0.85; p <0.001) and P53 (0.80; p <0.001). The high rate
ofbcl-2 had little relation to such factors as VEGF, VEGFR-2 (0.64; p <0.001 and 0.54, p <0.001) and unessential relation with level of P53 and eNOS3 (0,35; p> 0 25 and 0.36; p> 0.25). Revealed a strong link between the level of VEGF and VEGFR-2 and P53 (0.87; p <0.001 and 0.80, p <0.001), an indicator of VEGFR-2 with p53 (0.77; p <0.001) and almost absence of communication between the index p53 and the level of eNOS 3 (0.13, p> 0.5).
Thus, the obtained data in patients NFPA in distinct from control values are concluded in that NFPA patients communication parameters FNO-a with VEGFR-2 reverses sign «positive» to «negative». Decreased of the eNOS3 activity does not depend on changing values of FNO-a, bcl-2, VEGF, VEGFR-2, since we have found a correlation between these indicators. At the same time attention draws to the fact that in patients with NFPA increasing of the anti-apoptotic factors FNO-a and bcl-2 and proapoptotic factor p53 comes on the maintain of the high correlation FNO-a p53 and decreased with bcl-2, that is falling of the control anti-apoptotic processes and high anti- proliferative activity of the FNO-a and which inhibits apparently tumor process progression in patients NFPA.
In determining of the NO-system activity in erythrocyte membranes in patients with NFPA have found that levels of major stable NO metabolites and the activity of eNOS were statistically significantly lower than the control and the activity of iNOS and ONO2-content, on the contrary, more than control.
Therefore, the content of NO, measured by its major stable metabolites NO2-and NO3, in patients with NFPA with non-burdened family history was lower than 18.9% of control (p <0.05), the activity of eNOS — 19.5% (p <0.05), and the activity of iNOS, on the contrary, higher by 45.5% (p <0.001), and ONO2- — by 36.4% (p<0.02) (Fig.1).
However, in patients with burdened familial history NO concentration was below the reference values to 36,7% (p <0,01), the activity of eNOS — by 44.1% (p <0.001), and indicators such as the NO-system iNOS ONO2-and, on the contrary were higher respectively by 109.1% and 72.7 (p <0.001).
Figure 1. The state of the activity of NO-system in erythrocytes of the patients with NFPA
* - P <0.05 versus control.
Note that in erythrocyte membranes in patients somatic NFPA group content of NO and eNOS activity was significantly lower than 22.0 and 30.5% (p <0.05 and p <0.01) and the activity
of iNOS and concentration ONO2--upper on 53.3 and 35.7%
(p <0.001 and p <0.001) than in patients with burdened familial history of NFPA.
NO reduction in patients with NFPA can be explained by inhibition of the activity of eNOS. However, high reaction rate of the iNOS supposes expression of NO tens or hundreds of times
greater than its synthesis with involving eNOS [7; 8]. About these processes also evidenced by our high level of data ONO2-.
However, on the level of activity of the NO-system in tissues can affect the degree of tissue destruction due to the aggressiveness of the pathological process. In our studies, it could be achieved in tumor size in patients NFPA. Analysis of the research showed that in patients NFPA with increased size of the tumor concentration of the main stable metabolites and eNOS activity progressively decreases, and the enzyme iNOS and content of ONO2-increases. In these
Study of gene — candidate marker's levels in patients with non-burdened and burdened familial history of NFPA.
patients of the somatic group violations of the indicators, characterizing the NO-system activity in membranes of erythrocytes is higher than in the burdened group. In assessing the activity of eNOS revealed similar dynamics — its activity decreased with increasing tumor size. In patients NFPA of the somatic groups eNOS activity with tumor size of10 mm was lower than in patients burdened group by 18.3% (p <0.01), with a 10 mm — 26.0% (p <0.01) giant — by 36.4% (p <0.001). At the same time assessing indicator of the iNOS activity found that NFPA patients of somatic group tumor size to 10 mm, it was higher than in burdened group ofpatients by 23.1% (p <0.01), with tumor size more than 10 mm — by 46.7% (p <0.001), and the giant — by 34.8% (p <0.001) (Fig.1).
Therefore, the degree of violation of NO-system activity in the erythrocyte membranes in the patients of the 1st, and 2nd groups depended upon the size of the tumor, and as larger it is, the violations will be more pronounced. More pronounced impairment in the activity of NO-system we found in patients of the burdened group compared to a somatic group, which is probably due to genetic polymorphism, the existence of violations of the genetic code in the NOS in NFPA patients with a genetically associated family history.
In connection with the findings, we can conclude that in patients with burdened anamnesis revealed more severe violations of NO-system activity than in patients without burdened anamnesis, which caused by a genetic defect of the ecNOS3, as consequence of predisposed of the patients with NFPA hereditary — burdened familial history to form dysfunction of the endothelium in the vascular wall.
Conclusions:
1. In patients with NFPA the eNOS3 activity is inhibits, that may underlie endothelial dysfunction. Inhibition of the eNOS3 activity occurs on the background of the activation of pro- and anti-apoptotic factors enhance level of p53, the content FNO-a and bcl-2 in the blood plasma, slight variations in the factors regulating angiogenesis (VEGF, VEGFR-2). There have revealed the absence of correlation relation between decreasing the activity of the enzyme eNOS3 and performance pro- and anti-apoptosis, angiogenesis regulation, indicating that the independence of these processes in the mechanisms of formation of NFPA.
2. It has found that in patients with NFPA was increasing indicator of correlation relation of FNO-a with VEGFR-2 that indicates about increasing the inhibition processes of the angiogenesis and proliferative activity of tissues. It has established the presence of high-performance connection ofVEGF and VEGFR-2 with indicators of apoptosis protein p53, which testifies to their importance in the mechanisms of inhibition of tumor. This is achieving, apparently at the expense of conservation within the control values of angiogenesis regulators VEGF and VEGFR-2 in the blood plasma of patients with NFPA.
3. It has found that in plasma of the patients with NFPA was observing an imbalance in the content factors of endothelial function (eNOS3), angiogenesis (VEGF and VEGFR-2), pro-apoptosis (p53) and anti-apoptosis (FNO-a and bcl-2). These data demonstrate its importance in the mechanisms of formation of NFPA and importance of the registration for monitoring, differential diagnosis, prognosis and the development of individualized treatment tactics.
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