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The system of interferons is the integral part of the immune system which provides coordination of proliferation, differentiation and activation of effectory cells of the immunity. The interferon status is the assessment of the functional state of the interferon system [7]. It is known that IFN-a, playing one of the main roles in the viral elimination, has direct antiviral effect as well as indirect immunomodulationg activity. IFN-a is produced almost by all the cells of the body, but most of all by macrophages and lymphocytes. IFN-a is the strong antiviral protein [8]. Analysis of the results showed that the serum content of IFN-a in the group of children with primary papillomatosis of the larynx was reliable increased in comparison with data of the group of children with frequent recurrences and children of control group. Thus, in group of children with primary recurrence the level of IFN-a was increased 3.5 times in comparison with control group
and 2.4 times — with values of children with frequent recurrences. Evidently in cases of frequent recurrences of disease the exhaustion of the reserves of the interferon system that is accompanied by inhibition of the IFN-a production.
Thus, our investigations allowed revealing reliable changes in the functioning of the immune system of the children with laryngeal papillomatosis. Our purpose was to find the most vulnerable indicators of the immunity which may be served in the further as important diagnostic, therapeutic and prognostic criteria in laryngeal papillomatosis in children. We managed to establish deep T-cellu-lar immune deficit which expressed in inhibition of the number of T-lymphocytes and T-helpers/inductors on the basis of increase in T-cytotoxic lymphocytes, natural killers, serum IgA and activation of IL-6, which is important prognostic criterion in the formation and development of tumors ofviral origin.
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3. VasilievM. Yu. J. Eksperimentalnoyoncologii. - 1989. - 3: 8-12.
4. Voznesenskaya I. A. Papillomas of the upper respiratory ways. - L., 1974. - P. 63-91.
5. Galkina O. V., Katinas E. B., Lavreneva G. V. et al. Medicinskayaimmunologia. - 2001. - 3: 311-312.
6. Grigoryan S. S., Ershov F. I. Materials of the Scientific conference: "Inductors and other immunomodulators in the radiology and oncology". - Obninsk, 1989. - P. 4-10.
7. Ershov F. I. Interferon system in norm and pathology. - M.: Medicina, 1996. - P. 36-40.
8. Ivanchenko G. F., Karimova F. S. In: diseases of the vocal apparatus of the upper respiratory tract. - M., 2001. - P. 111-113.
9. Nurmukhametov R. Kh., Onufrieva E. K., Soldatskiy Yu. L., Brodskiy M. Yu. Effect ofthe type ofhuman papilloma virus on the development ofthe juvenile respiratory papillomatosis and efficacy ofinterferon therapy in children//Vestnik ofotorhinolaryngology. - 2000. - 2: 37-40.
10. Nurmukhametov R. Kh., Onufrieva E. K. et al. Assessment of the formation of antibodies to interferon and their effect on the efficacy of interferon therapyh in children with juvenile respiratory papillomatosis//Vestnik otorhinolaryngol. - M., 2000. - 4: 22-25.
11. Petukhova L. I., et al. Ultrasound aspects of diagnosis of the laryngeal diseases in young children//Echography. - 2000. - 1: 3: 330-334.
12. Semenov A. V. Papillomavirus infection. Some aspects of the therapy of infectious patients. Mannual Book for Physicians, ed. By Yu. V. Lobsin. - SPb.: Foliant, 2005. - P. 777-786.
13. Feltkamp M. C., Broer R., Summa F. M. et al. Cancer Res. - 2003. - 63: 10: 2695-2700.
14. Koutsky L. A., Ault K. A., Wheeler C. M. et al. N Engl J Med. - 2002. - 347: 21: 1645-1651.
15. Mellin H., Friesland S., Auer G. et al. Anticancer Res. - 2003. - 23.
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18. Soldatski I. L. Subglottic hemangioma: experience of treatment/II International Congress on Malformations and Rare Tumors of Head and Neck.Avellino (Italy), 18-21 October, 2000. - P. 144.
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Makhmudova Aziza,
Research Scientific Institute of Hematology and Blood Transfusion, MoH of Uzbekistan Republic Uzbekistan, Tashkent E-mail: [email protected] Karimov Khamid Yakubovich, Makhmudova Aziza Djumanovna, Shamsutdinova Dildora Bakhtiyarovna, Boboev Kodirjon Tuxtaboevich, Berger Inna Viktorovna
The role of thrombophilia geneticdeterminants in the clinical course of hemophilia
Abstract: In the article studied the effect of thrombophilia genetic markers (FII, FV and MTHFR) on the clinical course of hemophilia A. For this purpose we studied DNA samples from 75 apparently healthy donors (control group), 36 patients with
severe and 38 patients with mild form of hemophilia A. The obtained results did not support the version of the development of the genetic-compensatory mechanism in the organism of patients with hemophilia in the form appearance of the defect in the genes of thrombophilia and do not allow to make a definitive conclusion about the patterns governing the course of hemophilia in the persistence of the combined forms of thrombophilic mutations.
Keywords: thrombophilia, hemophilia, genes FII, FV, MTHFR.
Until now, the severity of hemorrhagic syndrome was explained only by the low concentration of the coagulation factors (hemophilia A and von Willebrand's disease, etc.) in patients with the hemostatic system disorders. However, recently, several authors noted that in the same level of the clotting factors deficiency, the clinical manifestations of the disease can greatly vary-ated, especially in patients with severe and very severe forms of hemophilia [3; 5-11; 13].
In this regard, an active search were conducted for the causes of the high variability of phenotypic manifestations of hemophilia, among which, it was not possible underestimate the contribution to the mutations or polymorphisms of some genes anticlotting hemostatic system under which there is a significant reduction in clinical severity of the disease. Of these, the most significant point mutations are:
a) G1691A — in the gene for factor V - Leiden mutation (rs6025);
b) G20210A — in the prothrombin gene II (rs1799963);
c) C677T — in the gene of methylenetetrahydrofolatereduc-tase (MTHFR, rs1801133). However, the results of some studies on the contribution of these mutations in the clinical course of hemophilia A (HA) are quite controversial [3; 4; 12; 13].
Taking into consideration the novelty and the lack of information on such issues, the aim of this work was to study the relationship between gene mutations FII, FV (whose products are in the same biochemical pathway wi th FVIII), and the enzyme MTHFR c clinical course of the GA.
Materials and methods
Materials for the study were the DNA samples from 74 patients with HA and 75 apparently healthy donors (control group). GA group patients were divided into 2 groups:
a) the subgroup ofpatients with a severe form - factor VIII level was 3.1 % (36 patients, 5 ofthem intron 22) with inversion mutation;
b) the subgroup of patients with mild form - the level of factor VIII was more than 5 % (38 patients). The average age of the patients was 22.6 ± 4.6 years.
The clinical diagnosis of HA were applied clinical, genealogical and hemostasis standard survey techniques.
Samples of genomic DNA were isolated from leukocyte fraction according to standard procedures. MTHFR C677T gene polymorphism, the FV G1691A and FII G20210A were determined by PCR (Applied Biosistems-2720, USA) and PCR in a "real time" (Rotor-Gene 6000, Australia). Visualization of DNA fragments was performed in transmitted UV light after staining the gel with ethidium bromide. Interpretation of the results was carried out
according to the instructions for the test systems manufacturer (OOO "AmpliKit" of St.-Petersburg and OOO "GenoTehnologi-ya", Moscow).
Statistical analysis of the results carried out by using statistical software package «OpenEpi 2009, Version 2.3». The differences between the control and the test groups were calculated with odds ratio (OR) with 95 % confidence interval (CI). The predictive efficacy (AUC-qualifier) of the genetic markers was determined by the standard formula:
AUC = (Se + Sp)/2, where Se and Sp — the sensitivity and specificity of a genetic marker, respectively.
If the index AUC < 0.5, the marker — the occasional qualifier; AUC = 0.5-0.6 — bad; AUC = 0.6-0.7 — medium; AUC = 0.7-0.8 — good; AUC > 0.8 — great classifier [14].
Results and discussion
At the beginning of the study, we investigated the prevalence of allelic variants of genes inherited thrombophilia — factors FII prothrombin (G20210A) and FV-Leiden, and MTHFR (C677T) in 75 apparently healthy donors, which had not the history of throm-botic episodes.
The frequency of allelic polymorphism G20210A prothrombin gene FII in the studied group of healthy Uzbek nationality donors had a relatively low rate — 1.3 % (1/75), indicating the particular genetic marker of population (Table. 1).
It is appropriate to emphasize that the G20210A polymorphism in the vast majority is found only in some populations of Europe and the white Americans. Among the Asian population this polymorphism is very rare or absent.
In the HA group of patients, the frequency of this marker was 2.7 % (2/74). In this case, the difference in the frequency distribution of the polymorphism among the studied group of general patients and healthy donors was not statistically significant (x2 = 0.3; P = 0.5; OR = 2.1; 95 % CI 0.1824, 23.17). It was found that in the subgroup of patients with mild GA, this mutation is detected in 2 times more often than in the control group (2.6 % and 1.3 %, respectively, x2 = 0.2; P = 0.6; OR = 2.0; 95 % CI 0.1217, 32.88). However, this difference was not statistically significant. In addition, the difference in this indicator between this subgroup of patients, and the subgroup of patients with severe HA also was insignificant (2.6 % and 2.8 %, respectively, P > 0.05).
The calculated rate sensitivity of this marker has been very low and consistent with SE = 0.027, the value of specificity, on the contrary — very high — SP = 0.99 (Table 2).
Genes
Studied groups and subgroups n Factor II Factor V MTHFR
n % n % n %
Control group 75 1 1.3 2 2.7 31 41.3
The core group, including: 74 2 2.7 3 4.0 34 46.0
Subgroup with severe HA 36 1 2.8 1 2.8 18 50.0
Subgroup with mild HA 38 1 2.6 2 5.3 16 42.1
Table 1. — Distribution of thrombophilia gene polymorphism in hemophilia patients and hemophiliagene carriers
Table 2. - Indicators of prognostic efficiency of thrombophilia markers
Genetic marker SE SP AUC OR (95 %CI) *р
FII G20210A 0.027 0.99 0.51 2.1 (0.1824-23.17) 0.9
FV G1691 A 0.040 0.97 0.51 1.5 (0.2502-9.506) 0.9
MTHFR "C677 T" 0.460 0.59 0.52 0.8 (0.4335-1.585) 0.2
Note: SE — sensivity; SP — specifity; AUC — prognostic efficiency, *p — Fisher's accuracy test.
Evaluating the effectiveness of the labeling marker was also very low, and was AUC = 0.51. These data suggest that the rare mutation of G20210A prothrombin gene FII is ineffective classifier to label the clinical course of hemophilia, even when OR=2.1.
In turn, the mutant allele — Leiden gene FV among controls was found in 2/75 cases (2.7 %) and was present in both cases, the heterozygous genotype. In the total group of patients with hemophilia frequency of this polymorphism was 4.0 % (3/74). In this case, the difference in the frequency of the carrier of genetic markers for these groups also was statistically significant (x2 = 0.2, P = 0.6; OR = 1.5; 95 % CI 0.2502-9.506).
In the studied subgroup of patients with a mild form of HA mutant allele — Leiden was met by more than 2 times higher than in the control group (5.3 % and 2.7 %, respectively). However, this difference also did not reach statistical significance level (x2 = 0.5; P = 0.5; OR=2.03; 95 % CI 0.2744, 14.98). Although OR = 1.9 comparative analysis of the frequency of this mutation in the subgroups of patients with mild and severe forms of HA also showed statistically insignificant differences (5.3 % and 2.8 %, respectively, x2 = 0.3; P = 0.6; OR = 1.9; 95 % CI 0.1686-22.42).
SE and SP indicators were equal to 0.04 and 0.97 respectively, and the evaluation of AUC efficiency was equal to 0.51 (Table 2). These figures also indicated a low degree ofpredictive value ofpoly-morphism G1691AFV gene as an independent marker.
It is interesting to note that the highest frequency of occurrence in the groups studied had the mutation of "C677T" MTGFR gene. The frequency of this mutation among healthy individuals was 41.3 % (31/75), including 29 individuals (97.3 %) had heterozygous and 2 (2.7 %) homozygous genotype.
In the core group of patients the incidence of mutation C677T MTHFR gene was 46.0 % (34/74), among them 91.2 % had heterozygous, and 8.8 % — homozygous genotype. In this case, the detected difference between the main and control groups did not reach the limits of statistical significance (x2 = 0.3, P = 0.6; OR = 0.8; 95 % CI 0.4335-1.585). The difference in the frequency of the carrier of the genetic marker among the surveyed subgroups with severe and mild forms of hemophilia and was statistically significant (50.0 % vs. 42.1 %, respectively, x2 = 0.5; P = 0.5; OR = 1.4; 95 % CI 0.5494, 3.441).
For this polymorphism the indicators of sensitivity and specificity showed an average values and correlated SE = 0.46 and SP = 0.59 (Table. 2). The calculated ratio of AUC (0.52) also demonstrated the low level of efficiency polymorphism "C677T" MTGFR gene as an independent candidate gene.
Given the high correlation between homozygous form carrier C677T MTHFR gene mutation with hyperhomocysteinemia and followed by hypercoagulability, we hypothesized that there may be mild developmental disorders of hemostasis hypercoagulable character in individuals with the homozygous form of polymorphism, even if the GA. However, in our case, in patients with the presence of homozygous mutations was not observed the clinical manifestations of thrombosis and complications. Perhaps this is due to the fact that genetically determined procoagulant activity of these individuals, and trombofilic phenomenon could not be
fully realized due to a defect in the coagulation cascade, caused by deficiency of factor VIII. In these patients, were not excluded the possible absence of exogenous provoked factors, that could activate the trigger mechanisms of prothrombotic changes in the hemostatic system.
One important aspect in the study of the genetic bases of forecasting the severity of the particular disease, is the interaction of genes, so-called gene-gene interactions.
Among studied 74 patients, only in 2 cases were detected the simultaneous carriage of alleles "FV + MTHFR" (2.7 %) and one "FII + FV" (1.35 %), whereas in the control group of such combinations was not observed. However, due to the relatively small number of investigated persons in the studied groups, the obtained data doesn't allow to make a definitive conclusion about the governing patterns on the hemophilia course, and incombined thrombophilia mutations gene carriers.
Discussion
Despite a number of ongoing and fairly intensive works, associated with the study of the effect of thrombophilic manifestations on the course of coagulopathy, the data on the formation of individual predisposition to certain thrombotic manifestations in patients with bleeding diathesis remains highly controversial.
In our studies, it was shown that the presence of polymorphic loci of genes FII, FV, MTHFR, predisposing to thrombosis, does not make an independent contribution to the development of a hypercoagulable state in patients with GA, confirming the version of some authors [3; 4; 12].
It should be noted the absence of significant differences between our results and those of Turkish colleagues. Thus, two groups of Turkish researchers have shown the absence of any effect on thrombotic factors for hemophilia varying severity [3; 12]. Taking into account the population and ethnic characteristics of genetic polymorphisms, these data may indicate high genetic affinity between our two Turkic peoples.
However, in the most of such studies were found association of prothrombotic gene alleles with clinical course of hemophilia. For example, Nowak-Gottl U. S. et al. (2003) and Kurnik K. et al. (2007) in their studies emphasize that the clinical phenotype of hemophilia A in childhood depends from the presence of hereditary thrombophilia factor [8; 10].
Lopez-Jimenez J. J. et al. (2009) in their studies emphasize the association only FII 20210A and FV Leiden genetic markers with severe hemophilia clinical course [9]. Franchini M., et al. (2010) and Van Dijk K. et al. (2004) in their review analyzes showed the association between FV Leiden and severe form of HA, this the genetic factor significantly reduces the clinical severity of the disease. Thus, Van Dijk K. et al. (2004) considered the association thrombophilic other factorsinconclusive [13].
Other researchers showed the reduction of the number of spontaneous bleeding and arthropathy in carriers of a point mutation factor FII 20210A compared with patients who do not have this mutation [11].
There is an interesting hypothesis about the positive association between the thrombotic factors and the inversion mutation
gene intron 22 of FVIII [11]. According to investigation dates of K. T. Boboev (2011) in Uzbekistan, the proportion of the intron mutation inversion in patients with severe HA consists about 41 %. In this paper was investigated the subgroup of patients with severe HA and patients with the presence of the mutation in the gene for FVIII. However, the frequency of thrombophilic mutations among these patients was close to the common population index. A small number of such patients did not allow us to made final conclusion on the association between this form and thrombophilic mutations [1]. It should be noted that some authors in their studies found significant associations between different factors of thrombophilia with von Willebrand's disease — another representative of plasma hemostasis disorders. Thus, Franchini M. et al. (2006) and Ahmad F. et al. (2010) argued, that the prothrombotic markers FII, FV and MTHFR significantly affect the course ofvon Willebrand's disease [2; 7].
In our opinion, such contradictory data is primarily associated with population characteristics of the studied markers, also with the heterogeneity of the studied groups of patients and of healthy individuals, and showed the significant difference in the number of patients tested in all studies.
Thus, our results do not reliably prove versions of some of the authors of the presence of genetic compensatory mechanism in the organism of patients with severe hemophilia, that form a defect in the genes of anticoagulative hemostatic and fibrinolytic systems. As Franchini M. et al. (2009), we also consider that for the final evaluation of the prognostic value of mutations in determinants genes of hereditary thrombophilia, as well as dysfunction of the protein in a cascade of clinical manifestations of hemophilia, apparently, needs more study patients and longer follow-up of these patients [6]. In addition, it would be interesting to extend the range of the studied genes determinants of hemostasis systems.
Conclusions:
1. The impact of thrombophilic mutations on the pheno-typic variability of the manifestations of current hemophilia is not significant.
2. The thrombophilic markers are ineffective independent classifiers for marking the clinical course of hemophilia.
3. For the final evaluation of the correlation of gene-gene interactions hereditary thrombophilia with clinical manifestations ofhemo-philia is necessary to expand the range of genetic markers of thrombophilia and significantly increase the number of patients tested.
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6. Franchini M., Montagnana M., Targher G., Veneri D., Zaffanello M., Salvagno G. L. et al. Interpatient phenotypic inconsistency in severe congenital hemophilia: a systematic review of the role of inherited thrombophilia//Semin Thromb Hemost. -2009. - 35(3): 307-312.
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14. [Electronic resource]. - Available from: http://vigg.ru/fileadmin/user_upload/Rubanovich
