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Bobojonov Oybek Abdujabbarovich, Ph D., students of Tashkent pediatric medical Institute of the MOH of RUz, Tashkent Karimov Hamid Yakubovich, professor, MD, Institute of Hematology and blood transfusion, MOH RUz, Tashkent Arifov Saidkosim Saidazimovich, MD, professor, Tashkent Institute of improvement of doctors of the MOH of RUz, Tashkent Boboev Kodirbek Tohtabayevich, MD, professor, Institute of Hematology and blood transfusion MOH of RUz, Tashkent E-mail: [email protected]
ANALYSIS OF THE ROLE rs1544410 POLYMORPHISM OF THE VDR GENE TO THE DEVELOPMENT OF ROSACEA ASSOCIATED WITH DEMODEX FOLLICULORUM MITES
Abstract: The role of the polymorphism rs1544410 of the VDR gene in the development of rosacea associated with mites of the genus Demodex folliculorum was studied. The studies were conducted on samples of 140 patients with rosacea and 145 conditionally healthy persons of Uzbek nationality. The results of the study showed the significant role of BsmI G > A polymorphism of the VDR gene in the development of rosacea associated with Demodex folliculorum. The relationship of the chances of developing rosacea in the presence of Demodex folliculorum is significantly higher in patients with heterozygous genotype G/A.
Keywords: rosacea, erythematous form, papular-pustular form, VDR gene, polymorphism rs1544410.
Rosacea is a recurrent chronic inflammatory skin disease exacerbates its clinical course, causing certain additional clini-
affecting mainly the Central part of the face and characterized cal signs, and makes torpid to the therapy [10]. by a stage course [1]. At present, it is important to study the genetic polymor-
The etiology and pathogenesis of the disease have not phism of various cytokines involved in the inflammatory pro-
been fully studied, and the method of treatment does not cess, in particular in the pathogenesis of rosacea. Until now, in
satisfy either doctors or patients. There are exogenous and scientific literature genetic aspects of rosacea, little studied. As
endogenous trigger factors that contribute to the develop- for the Association of causal factors (Demodex folliculorum,
ment of rosacea. Helicobacter pylori, microflora, etc.) with genotypic variants
Exogenous causes include alimentary factor, heat (show- of polymorphisms of different genes in patients with rosacea
er, bath, sauna), cold, solar insolation, etc., the presence of have not yet been studied.
Demodex folliculorum in the skin. Among the endogenous The aim of the present study was to analyze the role of
causes, the most often revealed are disorders of the gastroin- rs1544410 polymorphism of VDR gene in rosacea develop-
testinal tract, disorders of the immune system, psycho-vege- ment associated with Demodex folliculorum mites. tative disorders, vascular diseases, etc [2; 3; 4]. Material and methods of research. Under our clinical
In dermatology, the role of Demodex folliculorum in the supervision were 140 patients suffering from rosacea. There
development of this rosacea has been discussed for a long time were 103 women (74%) and 37 men (26%). According to
and many authors have found a high density of its contamina- the classification of the national society of rosacea (Wilkin J.,
tion in the lesion of patients compared to healthy individuals Dahl M., Detmar M., et al., 2002) erythemato-teleangiectatic
[5; 6; 7]. Thus, G. sh. Gulyamova (2007) found this infection the subtype was sick 100 (71.4%) patients, papules-pustular
in 86.3% of patients with rosacea [8]. R. S. Babayants et al. 40 (28.6%).
(1983) in 75.7% of patients [9]. The distribution of patients by clinical forms of rosacea
Despite numerous studies the question whether Demo- and sex is presented in (table 1). dex folliculorum is the cause of rosacea remains open. It is believed that the presence of this tick in the skin of patients
ANALYSIS OF THE ROLE rs1544410 POLYMORPHISM OF THE VDR GENE TO THE DEVELOPMENT OF ROSACEA ASSOCIATED WITH DEMODEX FOLLICULORUM MITES
Table 1. - Distribution of patients by clinical forms of rosacea and sex
Clinical subtitles rosacea Women Man Total
a6c % a6c % a6c %
Erythematosis -telangiectatic 74 52.85 26 18.6 100 50.6
papulo-pustular 29 20.7 11 7.9 40 28.6
Total 103 73.6 37 26.4 140 100
The material for the determination of Demodex folliculo-rum were morphological elements of the rash (papules, pustules, etc.), as well as the contents of the sebaceous glands. To take the material used a needle Banner, with which made a puncture in the morphological elements and scraped, or the skin was taken in the folds and by pressing it to get the material. The control group received only the contents of the sebaceous glands from the individuals. With a thick material, 1 drop of distilled water or saline was added. The obtained native drug was examined under the microscope "Leica" at 7x8 magnification. For a more in-depth study of mites used lenses with magnification of 10 x, 20 x and 40 x. When one or more ticks were found, Demodex was considered positive. The control group consisted of 30 healthy individuals.
All 140 patients with rosacea was determined by infection of mites of the genus Demodex. The frequency of patients with the presence of Demodex folliculorum tick was 69.3% (97/140). The remaining 43 patients (30.7%) did not have Demodex folliculorum. Of the 97 patients who were found Demodex folliculorum 66.0% (66/100) suffered erythematous telangiectatic subtype, 77.5% (31/40) with papulo-pustular rosacea. The remaining 43 patients (30.7%) did not have Demodex.
The control group consisted of 145 healthy unrelated individuals (Uzbek nationality), corresponding by sex and age of the examined group of patients (p > 0.05), and who had no history of skin pathology. Genomic DNA preparations were used as a material for molecular genetic screening. DNA isolation from peripheral blood was performed by phenol-chloroform extraction and using a set of rib-Sorb (AmpliSens, Russia). PCR genotyping of polymorphism rs1544410 of VDR gene was carried out using the device "Applied Biosystems" 2720 (USA), using a set of company "Litech" (Moscow), according to the instructions of manufacturers. PCR products were separated by electrophoresis in 1-2% agarose gel containing ethidium bromide.
The deviation of genotypes from the canonical distribution of hardy-Weinberg was estimated using a computer program "GenePop", available on the Internet [10]. The coefficient of deviation of the actual heterozygosity from the theoretical one was calculated by the following formula: D = (Hobs-Hexp)/Hexp, where hobs and hexp are the observed and expected heterozygosity, respectively.
As a tool of statistical calculations we used the application package "OpenEpi 2009, Version 2.3".
Research result. It must be emphasized that when comparing frequencies of alleles and genotypes for all genetic markers in patients with the presence of Demodex folliculorum and its absence in subgroups of patients erythemato-teleangiectatic and papules-pustular rosacea subtypes was not statistically significant differences (p > 0.05).
The comparative analysis of allele and genotype frequencies of rs1544410 polymorphism of VDR gene in rosacea patients associated with mites of Demodex folliculorum genus (i-subgroup) and patients with absence of Demodex folliculorum (II - subgroup) revealed a number of associations. As can be seen from tables 2 and 3, there was a statistically significant difference in the distribution of allele frequencies G and A between I and II subgroups: the frequency of adverse allele a was higher in the group of patients associated with Demodex folliculorum compared to the group of patients without Demodex (37.6% and 19.7%, respectively; x2 = 8.7; P = 0.003; OR = 2.4; 95% CI 1.34-4.48). The relative risk of rosacea + Demodex folliculorum combination for individuals with allele a increases 2.4 times compared to the alternative allele carriers. At the same time, the carrier of allele G is associated with a reduced risk of rosacea associated with Demodex (62.4% vs. 80.2%, respectively).
As expected, there was a significant decrease in the frequency of carrier of a favorable homozygous g/G genotype in patients associated with Demodex folliculorum compared to the group without Demodex folliculorum (33.0% vs. 65.1%, respectively; x2 = 12.5; P = 0.0001; or = 0.2; 95% CI0.123-0.56).
Among the carriers of heterozygous genotype G / a there is a significant predominance of the number of patients with rosacea associated with Demodex folliculorum compared to patients without Demodex folliculorum (58.8% versus 30.2%, respectively; tf = 9.7; P = 0.002).
The relative risk amounted to OR = 3.3 95% CJ1.529--7.07. At the same time, there was no significant difference in the distribution of frequencies of unfavorable genotype a / A between the comparative subgroups (x2 = 0.5; P = 0.4) although the frequency of this genotype in the first subgroup of patients was 8.2%, which is higher (OR = 1.8 at 95% CI0.374--9.06) compared to subgroup II (4.6%).
Table 2.- The frequency of distribution of alleles and genotypes of polymorphism rs1544410 of VDR gene in rosacea groups with presence and absence of Demodex folliculorum.
Allele frequency The frequency distribution of genotypes
Rosacea group G A G/G G/A A/A
n % n % n % n % n %
1 Major group (n = 140) 190 67.9 90 32.1 60 42.9 70 50.0 10 7.1
A associated with the mite Demodex n = 97 121 62.4 73 37.6 32 33.0 57 58.8 8 8.2
B with the absence of the mite Demodex n = 43 69 80.2 17 19.7 28 65.1 13 30.2 2 4.6
Table 3.- The differences of allele and genotype of the rs1544410 polymorphism of the VDR gene
Alleles and genotypes Group of patients with rosacea Statistical difference
associated with Demodex with no Demodex
Allel G 121 69 ^=8.7; P=0.003; OR=2.4; 95% CI 1.34-4.48
Allel A 73 17
genotyp G/G 32 28 X2=12.5; P=0.0001; OR=0.2; 95%CI 0.123-0.56
genotyp G/A 57 13 X2=9.7; P=0.002; OR=3.3; 95% CI1.529-7.07
genotyp A/A 8 2 X2=0.5; P=0.4; OR=1.8; 95% CI0.374-9.06
Thus, the results of this study indicate the significant role ratio of the chances of rosacea development in the presence of of polymorphism BsmI G > a of the VDR gene in the develop- Demodex folliculorum is significantly higher in patients with ment of rosacea associated with Demodex folliculorum. The heterozygous genotype G/A.
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10. URL: http://wbiomed.curtin.edu.au/genepop
