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"Child`s Health" 6 (57) 2014

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Significance of the deletion polymorphism of glutathione transferases (GSTM1 and GSTT1) genes under different inflammatory phenotypes of childhood bronchial asthma

Authors: Koloskova O.K., Bezrukov L.O., Bilous T.M., Grygola O.G., Ortemenka Ye.P. - Bukovinian State Medical University, Chernivtsy city, Ukraine

Categories: Pediatrics/Neonatology

Sections: Clinical researches

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Chronic airway inflammation is one of the key phenomena of bronchial asthma. Currently, an inefficiency of controlling therapy of bronchial asthma has been associating with heterogeneity of the airway inflammation, namely, with the existence of eosinophilic, neutrophilic and paucigranulocytic types of the bronchial inflammatory process. At the same time, it has been noticed a tendency toward increased incidence of non-allergic bronchial asthma, associated with noneosinophilic airway inflammation, which often characterized by a therapeutic resistance leading to a suboptimal asthma control. However, nowadays the diagnosis of various inflammatory childhood asthma phenotypes remains imperfect because of instability of the results of a sputum cytology analysis in children. Thus, a specific type of airway inflammation in asthmatic patients quite often has not constant, but transient, discrete character.

Given this, we can assume that the reduction of diagnostic errors may be achieved by defining the inflammatory pattern of bronchi on base of the complex of asthma features and, as well, relying on genetic markers of predisposition to their development Indeed, bronchial asthma is considering as a classic multifactorial disease, in which development an important role plays environmental factors as well as genetic predisposition to the development of this pathology. But nowadays mechanism of this disease is treated with molecular-genetic point of view, taking into account the polymorphism of genes that influence the development and clinical manifestations of asthma.

Formation of bronchial inflammation is determined to some extent by genetic components, which, in particular, is modifying the ability of airways to protect themselves from inhaled environmental pathogenic substances. It has been noted that the polymorphism of genes encoding enzymes of the second phase of detoxication of xenobiotics, affect the functionality of these enzymes in the lungs and other organs, which increases genetic susceptibility to oxidative stress and bronchial asthma. These genes-modifiers include such genes of glutathione-S-transferases as GSTM1, GSTT1, and, at the same time, some researches have been showed the association of genotypes GSTT1-/GSTM1 with significantly increased risk of bronchial asthma in children. However, we should admit that a predictor role of genetic factors in the development of airway inflammation of the bronchi remains understudied.

The aim of research. To study peculiarities of a heterogeneous airway inflammation in children with bronchial asthma under the deletion polymorphism of the glutathione-S-transferase genes (GSTM1 and GSTT1).

Materials and Methods. At the allergological department of the Chernovtsy Regional Children Clinical Hospital (Ukraine) 102 school-aged children with bronchial asthma have been examined, of whom in 46 patients (I-st clinical group) the eosinophilic type of bronchial inflammation was verified, and in 33 patients (II-nd clinical group) the neutrophilic airway inflammation was recognized, but in 23 cases (ІІІ-d comparison group) the patterns of paucigranulocytic inflammation of bronchi were identified.

The comparison groups did not differ significantly on the main clinical characteristics (sex, age, place of residence, severity of the disease).

The types of airway inflammation has been detected by the results of cytological analysis of sputum, induced by inhalation of hypertonic solutions (3%, 5%, 7%) of sodium chloride (by the method of Pavord I.D. in the modification of Pizzichini M.M., 1996). The eosinophilic type of bronchial inflammation has been identified in patients who had 3% or more of eosinophils in induced sputum. The local neutrophilic airway inflammation has been verified in school-aged children under the presence of the total number of neutrophils 59% and more, but 2% and less of eosinophils in cells sediment of sputum. The paucigranulocytic inflammation of bronchi has been proved under the lower content of these granulocytes in induced sputum: less than 3% of eosinophils and less than 59% of neutrophils.

Determination of deletions in the genes GSTM1 and GSTT1 was conducted using multiplex polymerase chain reaction (PCR), and an amplification of fragments of BRCA1 was used as a positive control of successfulness of PCR.  To visualize the DNA fragments, a gel was stained with ethidium bromide and then photogravured under ultraviolet light by the device GelDoc 2000 (BioRad, USA). To determine the length of the fragments, their electrophoretic mobility compared with the mobility of such DNA-marker as Gene Ruler DNA Leader Mix (Fermentas, Lithuania).

Homozygous forms with deletion of both copies of genes GSTT1 and GSTM1 were identified by the absence of the corresponding fragment, visualized by the diaelectrophoresis. These genotypes are designated as T1- and M1-. Accordingly, the presence of these fragments testified homo- or heterozygosity by a normal copy of the gene. The genotype of such patients designated as T1+ and M1+.

These research results were analyzed by the methods of biostatistics and clinical epidemiology, and using the software package "STATISTICA 7.0" StatSoft Inc. and Excel XP for Windows on a PC, by parametric (Student’s Pt criterion) and nonparametric  (Fisher’s Pφ criterion) methods of calculation. The survey was carried out with a strict observance of the bioethical requirements, in parallel clinical comparison groups formed on the basis of a simple randomization by the "case-control" method. 

Results. In patients with bronchial asthma the homozygous deletion of gene GSTT1 has been detected in every fifth child independently of type of airway inflammation. However, the deletion of gene GSTMdel has been identified in 32,6% children with the eosinophilic asthma phenotype and in 51,5% patients with neutrophilic asthma, but only in 21,7% cases of paucigranulocytic subtype of the disease (Р I:II:III<0,05). At that, in children with genotype T1delMthe severe course of BA has been diagnosed in 37,5% patients of the I-st clinical group as well as in 50,0% children of the II-nd comparison group, but in 100% of observations in the III-d group (Р III:I,II<0,05).

In patients with neutrophilic type of airway inflammation under the T1delM1 genotype contaminant atopic diseases has been diagnosed more often. Thus, these patients have signs of allergic rhinitis and atopic dermatitis in 75,0% and 50,0% cases correspondingly, but only in 12,5% and 50% observations, correspondingly, in children with eosinophilic asthma phenotype under the same polymorphism of glutathione-S-transferases (Р <0,05). At the same time, these allergic disorders were not recognized in any case of paucigranulocytic asthma. 

Conclusions. Thus, the certain specificity of clinical course of the disease have been determined in children with heterogeneous inflammatory asthma phenotypes in the presence of deletion polymorphism of GSTM1 and GSTT1 genes,  that should be considered when choosing the patients’ treatment strategy.


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