RU2757936C1 - Method for predicting the risk of developing chronic true eczema based on molecular genetic data - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to the field of medicine and biotechnology, can be used to identify the risk of developing chronic true eczema in individuals of Russian nationality, born and living in the Central Black Earth Region of Russia. The method includes isolation of DNA from peripheral venous blood, analysis of genetic polymorphisms rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene. The GCATA haplotype at loci rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 of the FLG gene is a risk factor for the development of chronic true eczema (OR=5.48).EFFECT: use of this method will allow at the preclinical stage forming risk groups among individuals and timely implement in these groups the necessary therapeutic and prophylactic measures to prevent the development of CTE.1 cl, 5 dwg, 4 ex

Description

Eczema is an acute or chronic recurrent allergic skin disease that forms under the influence of exogenous and endogenous trigger factors and is characterized by the appearance of a polymorphic rash, an acute inflammatory reaction caused by serous inflammation of the skin, and severe itching (Federal Clinical Guidelines, 2016).

At different stages of the development of the doctrine of eczema, great importance in the etiology and pathogenesis of eczema was attached to the neurogenic and allergic theories, the role of the endocrine system, and hereditary factors.

The disease has a serious negative impact on the quality of life of patients (Wallmeyer L., Dietert K., Sochorová M., et al. TSLP is a direct trigger for T cell migration in filaggrin-deficient skin equivalents // Sci Rep. 2017; 7 (1 ): 774). Eczema is common in 10-20% of children, often manifests in early childhood, and up to 45% of all cases of the disease manifest in the first 6 months of life (Tanjung C., Rzehak P., Mansyur M., et al. Study protocol to investigate the environmental and genetic aetiology of atopic dermatitis: the Indonesian Prospective Study of Atopic Dermatitis in Infants (ISADI) // BMJ Open. 2017; 7 (3): e012475).

Eczema is a multifactorial disease, the development of which is determined by a polygenic basis (hereditary predisposition predetermines various disorders of the skin, nervous, endocrine, immune systems) and the action of provoking exogenous factors (bacterial and fungal infectious agents, chemicals, physical factors, drugs, food products) and endogenous (antigens of microorganisms from foci of chronic infection) nature (Federal clinical recommendations, 2016; Zavadsky V.N. ): 100-107). Twin and family studies indicate a significant role of hereditary factors (72-90%) in the formation of eczema (Al-Afif KAM, Buraik MA, Buddenkotte J., et al. Understanding the Burden of Atopic Dermatitis in Africa and the Middle East // Dermatol Ther (Heidelb). 2019; 9 (2): 223-241). The probability of developing the disease is about 40% in the presence of eczema in one of the parents, and this indicator reaches 50-60% in the presence of the disease in both parents (Federal Clinical Recommendations, 2016).

A fairly large number of works are devoted to the study of the molecular genetic basis of eczema. The search for genetic determinants of the disease is being actively pursued using the methods of genome-wide association analysis (GWAS). To date, according to the GWAS catalog (https://www.ebi.ac.uk/gwas/efotraits/EFO_0000274), various scientific teams have conducted eleven full-genomic studies of eczema worldwide (in foreign literary sources, eczema is synonymous with atopic dermatitis [MIM 603165], which revealed more than 100 GWAS-significant polymorphic loci involved in the formation of the disease.

An important contribution to the formation of susceptibility to the development of eczema is made by mutations in the filaggrin gene (Marenholz I., Esparza-Gordillo J., Rüschendorf F., et al. Meta-analysis identifies seven susceptibility loci involved in the atopic march // Nat Commun. 2015; 6: 8804). Various scientific teams are actively studying the associations of mutations in the filaggrin gene associated with loss-of-function variants with the formation of eczema. Also shown are the associations of a number of polymorphic loci of the filaggrin gene with the development of eczema and according to the data of full-genomic studies. At the same time, it should be noted that the overwhelming number of works aimed at searching for molecular genetic markers associated with the development of eczema have been carried out abroad, and in Russia such studies are rare (Gimalova G.F., Karunas A.S., Fedorova Yu. Yu., Gumennaya E.R., Levasheva S.V., Etkina E.I., Khusnutdinova E.K. Replication of data from genome-wide analyzes of the association of atopic dermatitis in the Republic of Bashkortostan // Medical Genetics. 2016; 15 (4): 25- 28).

Filaggrin is a multifunctional, histidine-rich, insoluble epidermal protein synthesized as a large precursor (400 kDa) called profilaggrin and stored in keratohyalin granules (Cau L., Pendaries V., Lhuillier E., et al. Lowering relative humidity level increases epidermal protein deimination and drives human filaggrin breakdown // Dermatol Sci. 2017; 86 (2): 106-113.). Profilaggrin is post-translationally cleaved into separate filaggrin peptides that bind to keratin filaments and aggregate in the cytoskeleton of keratinocytes, condensing into dense bundles (Pin D., Pendaries V., Keita Alassane S., et al. Refined Immunochemical Eprmidealization in Healthy Dog Skin of Cornification Proteins, Filaggrin, and Corneodesmosin // J Histochem Cytochem. 2019; 67 (2): 85–97.). This helps to maintain adhesion and constancy between corneocytes, which form the skin barrier, thereby preventing transepidermal water loss and protecting against adverse effects of external factors such as pathogens and allergens (Goleva E., Berdyshev E., Leung DY Epithelial barrier repair and prevention of allergy // J Clin Invest. 2019; 129 (4): 1463-1474.). In addition, individual filaggrin peptides are further degraded into free amino acids, which, in turn, are degraded into urocanic and pyrrolidone carboxylic acids, which protect the skin from ultraviolet radiation and promote its natural hydration and maintain the pH gradient of the skin (McAleer MA, Jakasa I., Raj N ., et al. Early-life regional and temporal variation in filaggrin-derived natural moisturizing factor, filaggrin-processing enzyme activity, corneocyte phenotypes and plasmin activity: implications for atopic dermatitis // Br J Dermatol. 2018; 179 (2): 431 –441).

Thus, filaggrin plays a key role in the formation of the skin barrier, it is involved in maintaining the level of hydration in the epidermis, regulation of skin pH, antibacterial protection and skin resistance to ultraviolet radiation (Margolis DJ, Mitra N., Gochnauer H., et al. Uncommon Filaggrin Variants Are Associated with Persistent Atopic Dermatitis in African Americans // J Invest Dermatol. 2018; 138 (7): 1501-1506). According to the literature, the lack of filaggrin underlies the pathogenesis of various skin diseases (eczema, allergic contact dermatitis, ichthyosis vulgaris), and is also associated with the formation of non-skin diseases (diabetes mellitus, inflammatory diseases of the gastrointestinal tract) (Ziyab AH, Ewart S ., Lockett GA, et al. Expression of the filaggrin gene in umbilical cord blood predicts eczema risk in infancy: A birth cohort study // Clin Exp Allergy. 2017; 47 (9): 1185-1192.).

To date, about 60 mutations have been identified in the FLG gene associated with loss-of-function variants. It was found that mutations leading to loss of function or null mutations in the filaggrin gene cause the formation of an inactive form of the synthesized polypeptide (due to premature termination of synthesis, the signal for which is the stop codons that appeared as a result of mutations). This ultimately leads to a low concentration of profilaggrin in the granular layer of the epidermis, which subsequently determines the formation of an abnormally thin layer of keratinocytes, and is a morphological substrate underlying the predisposition to chronic skin diseases (dermatitis, eczema, psoriasis, etc.) (Čepelak I ., Dodig S., Pavić I. Filaggrin and atopic march // Biochem Med (Zagreb). 2019; 29 (2): 020501).

The frequency of mutations of the FLG gene in the general population is 8-10%, and there are significant interethnic differences in their prevalence (Park KD, Pak SC, Park KK The Pathogenetic Effect of Natural and Bacterial Toxins on Atopic Dermatitis // Toxins (Basel). 2016; 9 (1): 3).

According to the literature, mutations in the filaggrin gene are associated with the development of various skin diseases such as eczema, allergic contact dermatitis, atopic dermatitis, ichthyosis vulgaris, herpetic eczema, etc., and are also risk factors for the development of allergic rhinitis, bronchial asthma, diabetes mellitus and dr.

Mutations in the FLG gene associated with loss of function are known risk factors for the development of eczema and occur in 10-50% of patients (Leitch CS, Natafji E., Yu C., et al. Filaggrin-null mutations are associated with increased maturation markers on Langerhans cells // J Allergy Clin Immunol. 2016; 138 (2): 482-490.e7). They reduce the expression of filaggrin and contribute to the appearance of functional defects of the epidermal barrier (Wallmeyer L., Dietert K., Sochorová M., et al. TSLP is a direct trigger for T cell migration in filaggrin-deficient skin equivalents // Sci Rep. 2017; 7 (1): 774). There is evidence that patients with eczema with mutations in the FLG gene have a more severe course of the disease and increased allergic sensitization than patients with eczema who do not have these mutations. In addition, it should be noted that the presence of mutations in the FLG gene in a woman increases the risk of developing eczema in her child by 1.5 times (p = 8.4x10 ^-8 ), even if he did not inherit this genetic defect from the mother (Esparza- Gordillo J., Matanovic A., Marenholz I., et al. Maternal filaggrin mutations increase the risk of atopic dermatitis in children: an effect independent of mutation inheritance // PLoS Genet. 2015; 11 (3): e1005076).

In the Russian Federation, studies of the involvement of the filaggrin gene in the formation of a predisposition to chronic true eczema and its complications are sporadic and fragmentary, and there are no data on the role of the genetic variants rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 FLG in the development of CIE.

To assess the current patent situation, a search was carried out on titles of protection for the period from 1990 to 2020. The analysis of the documents was carried out in the direction: a method for predicting the risk of developing chronic true eczema based on molecular genetic data depending on polymorphic markers of the filaggrin gene. Sources of information: sites of the Federal Institute of Industrial Property http://fips.ru.

In the studied medical scientific and available patent literature, the authors did not find a way to predict the risk of developing CIE based on data on the GCATA haplotype of genetic polymorphisms rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene, which are in linkage disequilibrium.

There is a known method for predicting the risk of early development of occupational allergic dermatoses according to RF patent No. 2467330 (published 2012.11.20), including the collection of venous blood, isolation of genetic material, carrying out a polymerase chain reaction with specific primers, determining the nucleotide sequence and, based on this, determining the polymorphic variant A4889G of cytochrome P-450 1A1, characterized in that when carrying out the polymerase chain reaction, specific primers C1A1-F Biotin are used - ggT gTT Aag TgA gAA ggT gAT T C1A1-R Cag gAT AgC Cag gAA gAg AAA, and after the polymerase chain reaction, a pyrosequencing reaction is carried out in real time using a specific primer C1A1-S ACC TCC Cag Cgg gCA A and detection of the nucleotide sequence using a chemiluminescent signal, then the obtained nucleotide sequence at position 4889 is compared with the reference sequences, if The pyrogram of the absence of replacement of adenine for guanine and when the body is exposed to a harmful production factor of irritating and sensitizing action predict the risk of early development of occupational allergic dermatoses less than 40%, when a heterozygous variant Cyp1A1 * 2C is detected on the obtained pyrogram, due to the replacement of adenine with guanine in the nucleotide sequence in position 4889, and when the body is exposed to a harmful production factor of irritating and sensitizing action, 40 ÷ 80% of the risk of early development of occupational allergic dermatoses is predicted. The disadvantage of the patent is that the influence of other genetic polymorphisms and their combinations on the development of CIE is not taken into account.

There is a known method for predicting the course and evaluating the effectiveness of treatment of atopic dermatitis, which consists in conducting a clinical and laboratory examination of patients with verified atopic dermatitis with the determination of the serum protein content - lactoferrin (Lf) in μg / ml, alpha-1-antitrypsin (a1-AT ) in g / l, alpha-2-macroglobulin (a2-MG) in g / l, then the K coefficient is calculated according to a certain formula and, with its values from 6 to 15, a mild severity of the disease is predicted, and at 15 or more, the average and severe severity of the disease, a month after treatment, a second examination is carried out with the calculation of the K coefficient and when it decreases by 30% or more from its initial value, treatment is considered effective, long-term remission is predicted, and if the K coefficient decreases to less than 30% of its initial value, treatment consider it ineffective, predict a short period of remission and a high risk of relapse [patent RU 2603463, 2016]. The disadvantages of this method are the delay in assessing the effectiveness of treatment and laboriousness, as well as the fact that the effect of genetic polymorphisms and their combinations on the development of HIE is not taken into account.

A method for predicting a moderate course of chronic true eczema in individuals of Russian nationality, natives of the Central Black Earth Region of the Russian Federation (RF Patent No. 2578441) is known from the prior art. The essence of the method lies in the fact that DNA is isolated from peripheral venous blood, analysis of polymorphisms of genes -308G / A TNFα, + 250A / G Ltα, + 1663A / G TNFR2 and their combinations is carried out. An increased risk of developing a moderate course of chronic true eczema is predicted in the presence of the + 250G Ltα allele or the combination of the + 250G Ltα allele with the + 1663G TNFR2 allele, or the combination of the -308A TNFα allele with the + 1663G TNFR2 allele, or the combination of the -308G TNFα allele with the + 250 allele ... A reduced risk of developing a moderate course of CIE is predicted when the -308G TNFα allele is combined with the + 250АА Ltα genotype. However, the known method estimates only the genetic risk of developing chronic true eczema, since the genes of the tumor necrosis factor receptor system, reflecting the events of apoptosis, are investigated, but the influence on the development of CIE of other genetic polymorphisms and their combinations is not taken into account.

For the prototype, an article identified in the scientific and medical literature is selected, which describes a method for predicting the risk of developing chronic true eczema (hereinafter CIE) in males based on data on genetic polymorphism + 250A / G Ltb [Association of polymorphism of lymphotoxin b (+250 A / G Ltb) with the formation of chronic true eczema [Electronic resource] / Ya.E. Denisova, M.I. Churnosov // Modern problems of science and education. - 2014. - No. 3. - Access mode: http://www.science-education.ru/117-13677]. The method includes taking venous blood, isolating genomic DNA from peripheral blood and analyzing the +250 A / G Ltb locus by the polymerase chain reaction (hereinafter PCR) DNA synthesis method. The genotype + 250GG Ltb is a risk factor for the development of this disease in men, and the allele + 250A Ltb is a protective factor. The disadvantage of the prototype is the narrow focus, since the method is applicable only for predicting the risk of developing CIE in male individuals, as well as the fact that the influence of other genetic polymorphisms and their combinations on the development of CIE is not taken into account.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the risk of developing CIE based on data on the GCATA haplotype of polymorphic loci rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 of the FLG gene, which are in linkage disequilibrium.

The technical result of using the invention is to obtain criteria for assessing the risk of developing CIE, born and living in the Central Black Earth Region of Russia, having Russian nationality and not being relatives, based on data on polymorphic loci rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene, including:

- isolation of DNA from peripheral venous blood;

- analysis of polymorphisms rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene;

- predicting a high risk of developing CIE when detecting the GCATA haplotype of genetic polymorphisms rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene.

The novelty and inventive step lie in the fact that the prior art does not know the possibility of predicting the development of CIE based on data on the GCATA haplotype of polymorphic loci rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene, which are in linkage disequilibrium.

The method is carried out as follows:

Isolation of genomic DNA from peripheral blood is carried out by phenol-chloroform extraction (Mathew, 1984) in two stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl ₂ , 10 mM Tris-HCl (pH = 7.6) are added to 4 ml of blood with EDTA. The resulting mixture is stirred and centrifuged at 4 ° C, 4000 rpm. within 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA (pH = 8.0) and 75 mM NaCl are added to the sediment and resuspended. Then add 0.4 ml of 10% SDS, 35 µl of proteinase K (10 mg / ml) and incubate the sample at 37 ° C for 16 hours.

At the second stage, DNA extraction is carried out sequentially from the obtained lysate with equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm. within 10 minutes. After each centrifugation, the aqueous phase is sampled. DNA is precipitated from solution with two volumes of chilled 96% ethanol. After lyophilization, the resulting DNA is dissolved in bidistilled, deionized water and stored at -20 ° C.

Analysis of the polymorphic markers rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 of the FLG gene was carried out by polymerase chain reaction (PCR) on a CFX-96 Real-Time System (Bio-Rad) thermal cycler using standard oligonucleotide primers and probes 8edePede myelorerohidаse сonsentrаtiоns аssосiаte signifiсantly with thе risk fоr аthеrоsсlеrоsis disеаse аnd аbdоminаl аоrtiс аnеurysm [Techt] / P. Prodhan. J. Immunol. - 2010. - Vol. 72, No. 2. - P. 150-157.] (Synthesized at Test-Gen LLC (Ulyanovsk)).

Genomic DNA was amplified in a reaction mixture with a total volume of 10 μL, including a mixture for MMP PCR - 4 μL, Taq polymerase - 2 μL, a test sample (~ 30 ng DNA / μL) - 1 μL, deionized water - 3 μL.

The genotyping of the studied samples was carried out using the CFX-Manager ™ software by the method of allele discrimination by the values of relative fluorescence units (hereinafter RFU).

The invention is characterized by the following figures:

- AA,

- GA,

- GG, ■ - отрицательный контроль).FIG. 1. Discrimination of alleles by the TaqMan probe detection method according to the RFU values of each probe on the CFX96 amplifier with a real-time detection system of the rs3126085 FLG (

- AA,

- GA,

- GG, ■ - negative control).

- TT,

- CC,

- TC, ■ - отрицательный контроль).FIG. 2. Discrimination of alleles by the TaqMan probe detection method according to the RFU values of each probe on the CFX96 amplifier with a real-time detection system of rs 12144049 FLG (

- TT,

- CC,

- TC, ■ - negative control).

- AA,

- TT,

- AT, ■ - отрицательный контроль).FIG. 3. Discrimination of alleles by the TaqMan probe detection method according to the RFU values of each probe on the CFX96 amplifier with a real-time detection system of rs 6661961 FLG (

- AA,

- TT,

- AT, ■ - negative control).

- TT,

- GG,

- TG, ■ - отрицательный контроль).FIG. 4. Discrimination of alleles by the TaqMan probe detection method according to the RFU values of each probe on the CFX96 amplifier with a real-time detection system of rs rs471144 FLG (

- TT,

- GG,

- TG, ■ - negative control).

- AA,

- CC,

- AC, ■ - отрицательный контроль).FIG. 5. Discrimination of alleles by the TaqMan probe detection method according to the RFU values of each probe on the CFX96 amplifier with a real-time detection system of the rs10888499 FLG (

- AA,

- CC,

- AC, ■ - negative control).

Using the Haploview v.4.2 program (https://www.broadinstitute.org/haploview/haploview), the linkage disequilibrium between pairs of SNPs was analyzed and haploblocks were constructed. Linkage disequilibrium between adjacent SNPs was assessed using Lewontin's D 'coefficient and Pearson's r2 correlation coefficient. In accordance with the algorithms "Solid Spine" and "Four gamete frequencies" with a predetermined threshold D '> 0.8, the block structure was determined. (Belyaeva TM. Study of associations of haplotypes of the FLG gene polymorphism with the development of chronic true eczema in men. Scientific results of biomedical research. 2020; 6 (2): 160-171).

The calculation of the haplotype frequencies and the analysis of their associations with the formation of CIE was carried out using the PLINK v. 2.050 (http://zzz.bwh.harvard.edu/plink/) using the EM algorithm. _{Р рerm} <0.05 was taken as a statistically significant level.

The possibility of using the proposed method for predicting the risk of developing CIE is confirmed by the analysis of the observation results of 656 patients, of which 350 patients with chronic true eczema and 306 individuals of the control group (CIE was absent). Among patients, the average age is 42.73 ± 17.53 years, from 18 to 86 years, in the control group, the average age is 42.56 ± 15.42 years, from 17 to 88 years. The studied groups included individuals born and living in the Central Black Earth Region of Russia, having a Russian nationality and not being related. Informed consent was obtained from all patients to conduct this study. The work was carried out under the supervision of the Ethics Committee of the Medical Institute of the National Research University "BelSU" in the period from 2013 to 2018. For each patient, a specially developed questionnaire was filled out, which included the following data: age, gender, place of birth, presence of environmental risk factors, complaints ( rashes, itching, sleep disturbance), anamnestic data (age, onset of the disease, number of exacerbations per year, seasonality of exacerbations, the presence of hereditary burden), complications of the disease, concomitant pathology, results of clinical and laboratory examinations.

Clinical and clinical and laboratory examination of patients was carried out at the bases of the Kursk Regional Clinical Dermatovenerologic Dispensary and OGBUZ Dermatovenerologic Dispensary in Belgorod. The study group of patients included patients with a diagnosis of chronic true eczema, which was established on the basis of complaints, anamnesis, clinical manifestations, the course of the disease and laboratory research methods (Federal Clinical Recommendations, 2016). The control group included individuals without skin diseases and somatic pathology leading to secondary skin lesions. The control group was formed during preventive examinations of the population.

The typing of molecular genetic markers was carried out at the Department of Biomedical Disciplines, Faculty of General Medicine and Pediatrics, Medical Institute, Belgorod State National Research University.

When calculating the frequencies of haplotypes and analyzing their associations with the formation of CIE, a connection was established with the formation of a disease of the GCATA haplotype of polymorphic loci rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 of the FLG gene, which are in linkage disequilibrium. The GCATA haplotype rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 is a risk factor for the development of CIE (OR = 5.48; p = 0.003).

As examples of the specific application of the developed method, a survey of patients born and living in the Central Black Earth Region of Russia who have Russian nationality and are not relatives is given: a genetic examination was carried out at the loci rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene.

Example 1

Venous blood was taken from patient S., during genotyping of DNA markers, the GCATA haplotype was identified at the locus rs3126085, rs12144049, rs6661961, rs471144 and rs10888499 of the FLG gene, which allowed the patient to be assigned to the group of patients with a high risk of developing CIE. Further observation confirmed the diagnosis of chronic true eczema in the patient.

Example 2

Venous blood was taken from patient I., genotyping of DNA markers revealed the GCATA haplotype at the locus rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 of the FLG gene, which allowed the patient to be assigned to the group of patients with a high risk of developing CIE. Further observation confirmed the diagnosis of chronic true eczema.

Example 3

Venous blood was taken from patient Z. Further observation did not confirm the diagnosis of chronic true eczema.

Example 4

Venous blood was taken from patient F., genotyping of DNA markers revealed the ACATA haplotype at the locus rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 of the FLG gene, which allowed the patient to be assigned to the group with a low risk of developing CIE. Further observation did not confirm the diagnosis of chronic true eczema.

The use of this method will allow at the preclinical stage to form risk groups among individuals and timely implement in these groups the necessary therapeutic and prophylactic measures to prevent the development of CIE.

Claims (1)

A method for predicting the risk of developing chronic true eczema in individuals of Russian nationality born and living in the Central Chernozem region of Russia, including DNA extraction from peripheral venous blood, analysis of genetic markers of the filaggrin gene, characterized in that a high risk of developing chronic true eczema is predicted when the GCATA haplotype is detected by loci rs3126085, rs12144049, rs6661961, rs471144, and rs10888499 of the FLG gene, which are in linkage disequilibrium.

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