Резюме
Іванова Н.М., Кутасевич Я.Ф., Савенкова В.В., Реміз О.М. Взаємозв’язок між однонуклеотидним поліморфізмом RS7574865 гену stat4 та розвитком червоного вовчаку.
У результаті досліджень було виявлено тенденцію до превалювання мутантного алелю Т у хворих на хронічний червоний вовчак. У групі хворих чоловіків було знайдено превалювання генотипу GT (мутантна гетерозигота) у 57,11 ± 6,9%. У жінок різниці між хворими та контрольною групою виявлено не було. Було знайдено взаємозв’язок між однонуклеотидним поліморфізмом rs7574865 гену STAT4 та розвитком червоного вовчака у хворих. Наявність однонуклеотидної заміни гуаніну на тимін у третьому інтроні є фактором ризику для розвитку хронічного червоного вовчака.
Ключові слова: хронічний червоний вовчак, ген STAT4, поліморфізм rs7574865.
Резюме
Иванова Н.Н., Кутасевич Я.Ф., Савенкова В.В., Ремиз А.Н. Взаимосвязь между однонуклеотидным полиморфизмом RS7574865 гена stat4 и развитием красной волчанки.
В результате исследований наблюдалась тенденция превалирования мутантного алеля Т у больных на хроническую красную волчанку. В группе больных мужчин было найдено превалирование генотипа GT (мутантная гетерозигота) в 57,11 ± 6,9%. У женщин различия между больными и контрольной группой выявлено не было. Была найдена взаимосвязь между однонуклеотидным полиморфизмом rs7574865 гена STAT4 и развитием красной волчанки у больных. Наличие однонуклеотидной замены гуанина на тимин в третьем интроне свидельствовала о факторе риска для развития хронической красной волчанки.
Ключевые слова: хроническая красная волчанка, ген STAT4, полиморфизм rs7574865.

Summary
Ivanova N.N., Kutasevich Ya.F.,Savenkova V.V., Remiz O.M. Relationship between single nucleotide polymorphisms RS7574865 of STAT4 gene and development of lupus erythematosus.
The studies tended prevalence of mutant Hallel T in patients with chronic lupus erythematosus. In the group of men with chronic lupus erythematosus have been found the prevalence of genotype GT (the mutant heterozygotes) in 57,11 ± 6,9%. In women the differences between patients and controls have been identified. Correlation was found between single nucleotide polymorphisms rs7574865 stat4 gene and development of lupus erythematosus in patients. Availability of single nucleotide replacement of guanine to thymine in the third intron evidence of the risk factor for development of lupus erythematosus.
Key words: lupus erythematosus, stat4 gene, polymorphisms rs7574865.
 
Рецензент: д.мед.н., проф. Е.М. Солошенко

УДК 616.5-002.525.2:575.174.015.3

ДУ «Інститут дерматології та венерології НАМН України» (Харків)

ГУ "Институт дерматологии и венерологии НАМН Украины" (Харьков)

GI "Institute of Dermatology and Venereology of the NAMS of Ukraine" (Kharkiv)

jet-74@mail.ru

Autoimmune connective tissue diseases are becoming increasingly pressing problem in modern medicine. [5]. Systemic lupus erythematosus (SLE) is a type of  immune system disorders known as autoimmune disease. In autoimmune diseases the immunoglobulins to organism’s own proteins are synthesized, that's why the immune system considers cells of the organizm as foreign antigens and attacks them. This leads to inflammation and damage of various body tissues. Lupus is a chronic autoimmune disease that manifests itself in several forms and can cause inflammation of the joints, muscles and various other parts of the body [6].

Women are more likely than men to suffer from SLE and this value according to different research centers range from 1:9 to 1:11. Moreover, family histories of the disease are known. [12]. Nearly one third of women giving birth in Ukrainian population is established to have a genetically determined medium or severe abnormalities of connective tissue [9].

Chronic lupus erythematosus is not only an inherited disorder, because the disease itself is not inherited directly, but rather the tendency to its development [8, 10]. The fact that lupus can run in families, shows that the disease has a genetic basis. The role of genetic factors indicate higher concordance for FLE in monozygotic than in dizygotic twins and prevalence of FLE (5-12%) among blood relatives of patients [15].

Abnormalities in the structure of the STAT4 gene may lead to the development of autoimmune processes. Many studies have shown the role of different single nucleotide polymorphisms of the STAT4 gene in the development of pathologies such as chronic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, allergies, asthma, etc. [4, 3, 14, 2]. Polymorphism rs7574865, that was shown to have the most significant correlation with these abnormalities, is a single nucleotide substitution of guanine (G) to thymine (T) in the third intron of the gene. Normally this intron comprises guanine, but mutations (transitions) replaces it by thymine [11, 13]. Regarding chronic lupus erythematosus, mainly affecting the skin, these processes have not been sufficiently studied.

Gene STAT4 (signal transducer and activator of transcription 4) is located in human chromosome 2q and encodes a protein that belongs to the transcription factors STAT [16]. STAT proteins are phosphorylated by receptor-associated kinases under the influence of growth factors and cytokines, form homo- or heterodimers and are transferred to the cell nucleus. STAT proteins function in the nuclies as activators of transcription. They are important in the differentiation of T-helper cells and in the regulation of Th1/Th2 balance. STAT4 proteins transfer cytokine signals from the IL-13, IL-23, IFN-I to T-helper cells and monocytes, which is a signal for their differentiation and production of interferon γ. However, the detailed mechanism of this process remains quite unclear [17, 1].

To sum up, STAT4 gene can be considered as one of the important factors in maintaining normal immune status, so that studying the correlation of one-nucleotide polymorphism with SLE is of interest.

Materials and Methods

Patients suffering from SLE, who were receiving medical treatment at non-emergency hospital and at policlinic of the SE “Institute of Dermatology and Venereology of National Academy of Medical Sciences of Ukraine”, were studied.

The article deals with the polymorphism rs7574865 of STAT4 gene. This polymorphism was studied in two groups of patients.

Main group – 13 patients aged 22 to 73 years (6 wemen, 7 men, mean age of patients – 47,3 ± 6,9 years).

Control group consisted of 23 healthy individuals aged 20 to 72 years (9 men and 14 women), mean age - 42,6 ± 7,2 years.

Statistical analysis was performed using Fisher's F test to evaluate the difference between the percentage of different samples, test χ ² [17].

DNA isolation was carried out using phenol method from peripheral blood leukocytes by the standard method [18]. Research of genotype on STAT4 gene in patients was carried out by site-specific polymerase chain reaction (PCR). PCR on mutant (TT - mutant homozygote, GT - mutant heterozygote) and normal (GG) genotypes using two pairs of primers was performed. Primers FWrs757 (GAA AAG TTG GTG ACC AAA ATG CG) and Rrs757 (GAC ACT TTG AGA GTT GCT TCG T) were used for normal genotype. Primers FMrs757 (GAA AAG TTG GTG ACC AAA ATG GT) and Rrs757 (GAC ACT TTG AGA GTT GCT TCG T) were used for mutant genotype.

Determination of genotypes was carried out after PCR by electrophoresis in 1.5% agarose gels. The size of DNA fragments generated of STAT4 gene - 366 n.p. for both (normal and mutant) DNA fragments.

Results and discussion

Examination of rs7574865 polymorphysm of STAT4 gene in patients, suffering of chronic lupus erhymatosus, was made (table 1). As a result, tendency for prevaling of mutant genotype (T) in patients of booth genders was observed. Moreower, frequency of rs7574865 genotypes of STAT4 gene in patients, suffering from chronic lupus erhymatosus, and in control group was analysed (table 1).

Table 1 – The distribution of alleles and genotypes of rs7574865 STAT4 in patients with chronic lupus erythematosus

n – amount of all patients, f – quantities.

In men of control group significant prevalence of normal genotype (GG) was shown – 100,00 ± 12%. In men with chronic lupus erythematosus the normal GG genotype was observed only in 42,86 ± 5,7% (p <0,01). Genotype GT (mutant heterozygote), which was completely absent in the control (0%), in male patients was observed in 57,11 ± 6,9% (p <0,01). In wemen there was no difference between patients and control group.

When comparing the percentage of patients suffering on chronic lupus erythematosus with normal genotype (GG) and mutant genotype (mutant heterozygote GT), a statistically significant correlation (significance level 0.001) between the frequency of SLE and gene polymorphism rs7574865 STAT4 was found. Presence of this mutation increased risk of chronic lupus erythematosus.

Thus, correlation between single nucleotide polymorphism rs7574865 in STAT4 gene and the development of chronic lupus erythematosus in men was found. The presence of single nucleotide substitution of guanine (normal genotype GG) to thymine (GT mutant heterozygote) in the third intron of the gene increases a risk for the development of chronic lupus erythematosus, as normal intron comprises guanine, but mutation (transition) replaces it by thymine, that interferes gene transcription.

Development of modern affordable to use genetic methods of diagnostics will determine the risk group, predict the course and help to recommend appropriate tactics of treatment and methods of primary prevention.

Литература

1. Гланц С. Медико-биологическая статистика / С. Гланц. – М.: Практика, 1998. – 459 с.
2. Дядык А.И. Патогенез системной красной волчанки: настоящее и будущее / А.И. Дядык, А.Э. Багрий, И.В. Ракитская // Український ревматологічний журнал. – 2003. – № 1 (11). – С. 3–9.
3. Клинико-морфологическая диагностика и принципы лечения кожных болезней: рук-во для врачей / М.А. Пальцев, Н.Н. Потекаев, И.А. Казанцева, С.С. Кряжева. – М. : Медицина, 2006. – 512 с.
4. Коваленко В.М. Медико-соціальні аспекти хвороб системи кровообігу (аналітично-статистичний посібник) / В.М. Коваленко, В.М. Корнацький. – Київ : МОЗУ, 2009. – С. 108–121.
5. Коваленко В.Н. Ревматология как одна из важнейших проблем медицины / В.Н. Коваленко, А.Г. Каминский // Укр. ревматол. журн. – 2000. – Т. 1, № 1. – С. 3–8.
6. Мавров И.И. Основы диагностики и лечения в дерматологии и венерологии: пособие для врачей, интернов и студентов / И.И. Мавров, Л.А. Болотная, И.М. Сербина. – Харьков: Факт, 2007. – 792 с.
7. Маниатис Т. Методы генной инженерии. Молекулярное клонирование / Т. Маниатис, Э. Фрич, Дж. Сэмбрук; пер. с англ. - М.: Мир, 1984. - 480 с.
8. Мордовцев В.Н. Заболевания кожи с наследственным предрасположением / В.Н. Мордовцев, П.М. Алиева, А.С. Сергеев. – Махачкала : изд-во типографии ДНЦ РАН, 2002. – 260 с.
9. Неєлова О.В. Прогнозування та профілактика перинатальних ускладнень у жінок зі сполучнотканинними дисплазіями: автореф. дис. на здобуття наук. ступеня канд. мед. наук: спец. 14.01.01 «Акушерство та гінекологія» / О.В. Неєлова. – Харків, 2008. – 21 с.
10. Ardoin S.P. Developments of the scientific understanding of lupus / S.P. Ardoin, D.S. Pisetsky // Athr. Res. Ther. – 2008. – Vol. 10. – P. 218–226.
11. Association of genetic variations in the STAT4 and IRF7/KIAA1542 regions with systemic lupus erythematosus in a Northern Han Chinese population / Р. Li, С. Cao, Н. Luan [et al.] // Hum. Immunol. – 2011. - Vol. 72, № 3. – P. 249-255.
12. Current status of lupus genetics / A.L. Sestak, S.K. Nath, A.H. Sawalha, J.B. Harley // Arthr. Res. Ther. – 2008. – Vol. 9. – P. 210–219.
13. Hinks A. Overlap of disease susceptibility loci for rheumatoid arthritis and juvenile idiopathic arthritis / A. Hinks, S. Eyre, X. Ke // Ann. Rheum. Dis. – 2010. – Vol. 69, № 6. – P. 1049–1053.
14. Palacios R. IgG antibody reactivity to Borrelia burgdorferi sensu stricto antigens in patients with morphea in Colombia / R. Palacios, A. Torres, R. Trujillo // Int. J. Dermatol. – 2003. – Vol. 42, № 11. – P. 882–886.
15. Rahman A. Mechanisms of the disease. Systemic lupus erythematosus / A. Rahman, D.A. Isenberg // N. Engl. J. Med. – 2008. – Vol. 358. – P. 929–939.
16. STAT4: genetics, mechanisms, and implications for autoimmunity review for current allergy and asthma reports / B.D. Korman, D.L. Kastner, P.K. Gregersen, E.F. Remmers // Curr. Allergy Asthma Rep. – 2008. - Vol. 8, № 5. - P. 398–403.
17. The STAT4 gene influences the genetic predisposition to systemic sclerosis phenotype / B. Rueda, J. Broen, C.  Simeon [et al.] // Human Molecular Genetics. – 2009. - Vol. 18, № 11. – P. 2071–2077.