RU2585960C1 - Method for detection of genetic predisposition to disruption of skin barrier function - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to method for detection of genetic predisposition to disruption of skin barrier function. Method summary consists in fact that genetic material is recovered from venous blood. DNA synthesis polymerase chain reaction is carried out followed by analysis of polymorphism of restriction fragment lengths, determination of nucleotide sequence and determination of filaggrin 2282del4 polymorphic variant. When carrying out polymerase chain reaction using specific primers: “FLG-2 F1” 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3'; “FLG-2 R1” 5'CAA-gTg-CAg-gAg-AAA-gAC-ATg-gAT3', amplicon size is 205 base pairs, and after carrying out polymerase chain reaction pyrosequencing reaction is carried out in real time using specific primer “FLG-2 S1” 5'gAC-ATT-CAg-AAg-ACT-CAg-AC3'. Nucleotide sequence detecting is performed by chemiluminescent signal, it is followed by comparing obtained nucleotide sequence in position 2282 with reference sequences. If obtained pyrogram shows absence of deletion in position 2282 of filaggrin gene at action on body of harmful production factors of irritant and sensitising action low hereditary predisposition caused skin barrier function is defined. If obtained pyrogram shows heterozygous version ins/del 2282 of filaggrin gene high inherited predisposition caused skin barrier function is defined.

EFFECT: using declared method allows to increase efficiency of detection of genetic predisposition to disruption of skin barrier function at action of irritant and sensitising factors action on body.

1 cl, 2 dwg, 1 tbl, 2 ex

Description

The invention relates to medicine and can be used to determine a hereditary predisposition to a violation of the barrier function of the skin.

A known method for determining a hereditary predisposition to impaired barrier function of the skin, including venous blood sampling, isolation of genetic material, polymerase chain reaction of DNA synthesis, followed by analysis of restriction fragment length polymorphism, determination of the nucleotide sequence and based on this determination of the polymorphic variant 2282del4 of the filaggrin gene (see Khantimerova E.F., Nurtdinova G.M., Karunas A.S., Gimalova G.F. Clinical and genetic characteristics of patients with atopic dermatitis volume and urticaria with mutations in the filaggrin gene // Fundamental Research. - 2014. - No. 10 (4). - S. 752-756).

However, this method has a low throughput due to the low speed of the examination of patients (analysis time 1-2 days), due to the complexity, the complexity of the used laboratory technologies, which are harmful to the medical staff conducting them. This makes it difficult to determine a hereditary predisposition to a violation of the barrier function of the skin, for example, during medical examinations or periodic medical examinations of a large number of people working in harmful and / or dangerous working conditions for short (short) periods of time.

The objective of the present invention is to enable the determination of a hereditary predisposition to a violation of the barrier function of the skin in a short (compressed) time period (3 ÷ 5) hours, for example, during medical examinations or periodic medical examinations of a large number of workers in harmful and / or dangerous working conditions.

The technical result of the invention is to increase the speed of determining the hereditary predisposition to a violation of the barrier function of the skin when exposed to factors of an irritating and sensitizing effect on the body.

This task is achieved by the fact that in the known method for determining a hereditary predisposition to impaired barrier function of the skin, including venous blood sampling, isolation of genetic material, polymerase chain reaction of DNA synthesis followed by analysis of restriction fragment length polymorphism, determination of the nucleotide sequence and, based on this, the determination of polymorphic variant 2282del4 filaggrin, when carrying out the polymerase chain reaction, specific primers are used: “FLG-2 F1” 5'Biot in-TgA-CCA-gCC-TgT-CCA-Tgg-C3 '; "FLG-2 R1" 5'CAA-gTg-CAg-gAg-AAA-gAC-ATg-gAT3 ', the amplicon size is 205 base pairs, and after the polymerase chain reaction, the pyrosequencing reaction is carried out in real time using a specific primer " FLG-2 S1 "5'gAC-ATT-CAg-AAg-ACT-CAg-AC3 'and the detection of the nucleotide sequence using a chemiluminescent signal, then compare the obtained nucleotide sequence at position 2282 with reference sequences, after which detection on the resulting pyrogram no deletion at position 2282 of the fil gene when exposed to a harmful production factor of an irritating and sensitizing effect on the body, ggrina determines a low hereditary predisposition to a violation of the barrier function of the skin; when a heterozygous ins / del variant at position 2282 of the filaggrin gene is detected on the resulting pyrogram, it determines a high production factor of an irritating and sensitizing effect hereditary predisposition to impaired barrier function of the skin.

Studies on patent and scientific and technical information sources have shown that the proposed method is unknown and does not follow explicitly from the studied prior art, i.e. meets the criteria of "novelty" and "inventive step".

The proposed method can be applied in clinical, biochemical, molecular genetic laboratories equipped with widely used equipment manufactured by domestic or foreign industry.

Therefore, the claimed method is affordable and practically applicable.

Thus, a set of techniques has been proposed to increase the speed of determining the hereditary predisposition to impaired skin barrier function, which, in turn, will increase the reliability of determining a low hereditary predisposition to impaired skin barrier function when the organism is exposed to harmful production factors of an irritating and sensitizing effect , for example, during medical examinations or periodic medical examinations of a large number of and working in harmful and / or dangerous conditions for short (short) periods of time.

Table 1 presents the sequence of primers used.

The proposed method is illustrated in the drawing.

In FIG. 1 shows a pyrogram with a variant of the heterozygous genotype for a detectable allelic variant having a deletion at position 2282 (ins / del) (Example 1). In FIG. 2 shows a pyrogram with a variant of the homozygous genotype for the determined allelic variant, which does not have a deletion substitution, at position 2282 (ins / ins) in the nucleotide sequence — it corresponds to the norm (example 2).

The proposed method is as follows.

Venous blood sampling is performed in patients. DNA sorbent is isolated from 100 μl of whole blood using a single-tube method using the DNA sorb-B isolation kit (TU 9398-071-01897593-2008) in accordance with the instructions for the kit used.

A series of dilutions in TE buffer is prepared from the obtained DNA preparation (50 μl) to a final concentration of 1-3 ng / μl, 10 μl of the final DNA solution is added to the tube for polymerase chain reaction with specific primers.

The volume of the mixture for the polymer chain reaction is 25 μl, using reagents manufactured by the Central Scientific Research Institute of Epidemiology FBUN: “2.5 × PCR buffer-2-blue”, “dNTPs mixture”, primers “FLG2-F1” and “FLG2 -R1. " The number of primers in the reaction is 7 pmol. For amplification of the studied genetic locus, specific oligonucleotide primers “FLG-2 F1” 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3 'and “FLG-2 R1” 5'CAA-gTg-CAg-gAg- are used AAA-gAC-ATg-gAT3 ', one of which is labeled with biotin (the sequence of primers used is presented in Table 1). The number of primers "FLG-2 Fl" 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3 'and "FLG-2 R1" 5'CAA-gTg-CAg-gAg-AAA-gAC-ATg-gAT3 'in the polymerase chain reaction - 7 pmol.

The amplification reaction is carried out according to the following program.

When using, for example, the “T 100 Thermal Cycler” thermocycler (Bio Rad, USA), when the temperature reaches 95 ° C (pause mode), put the tubes in the cells of the thermocycler and maintain them at a temperature of 95 ° C for 15 minutes. Then test tubes withstand 45 cycles of 95 ° C for 15 seconds, 65 ° C for 15 seconds, 72 ° C for 20 seconds.

After amplification, samples are prepared for sequencing. The amplification product is immobilized with 1 μl of Streptavidin Sepharose particles (GE Healthcare, Sweden) in 40 μl of Binding Buffer (FBUN Central Research Institute of Epidemiology). After immobilization, a series of washes is carried out using the sample preparation station PyroMark Q24 Workstation)) (Qiagen, Germany) and the Piro-prep reagent kit (FBUN Central Research Institute of Epidemiology, registration certificate No. ФСР 2012/13246 dated March 19, 2012 g.). As a result of sample preparation, a single-chain PCR fragment amplified from a biotinylated primer is obtained, which is used as a matrix in the pyrosequencing reaction.

Pyrosequencing is carried out using a primer “FLG2-S1” in an amount of 7.5 pmol and a reagent kit “PyroMark® Gold Q96 Reagents” (“Qiagen”, Germany) on a device “PyroMark Q24” (“Qiagen”, Germany). The sequence of the primer for sequencing "FLG-2 S1" is as follows: 5'gAC-ATT-CAg-AAg-ACT-CAg-AC3 '. The sequence of adding nucleotides to the pyrosequencing reaction: GACACAGATCAGTGTCA.

Next, the obtained nucleotide sequence is compared with reference sequences using the PyroMark Q24 2.0.6 software and the resulting pyrogram determines which variant of the mutation takes place in the nucleotide sequence (see, for example, Figs. 1 and 2).

If there is a lack of deletion at position 2282 of the FLG gene on the resulting pyrogram, and when the body is exposed to a harmful production factor of irritating and sensitizing effects, a low hereditary predisposition to impaired skin barrier function is determined, and if the heterozygous ins / del variant is detected at position 2282 of the FLG gene and when the organism is exposed to a harmful production factor of an irritating and sensitizing effect, a high hereditary predisposition is determined to disruption of the skin barrier function at which facilitates the penetration of allergens and infectious agents through the epidermis, the latter initiated the development of local and systemic allergic reactions of the organism, that leads to the development of professional allergic dermatoses.

Example 1. Patient T., 48 years old

When analyzing the patient’s medical history and professional route, it was revealed that patient T. worked from 1986 to the present time as a galvanic in contact with chromium, nickel, and cobalt compounds. Considers herself a patient since 1987, when the rash on the back surfaces of the hands, accompanied by itching, first appeared. Appealed to the dermatologist at the place of residence, received treatment with a temporary effect. On weekends, during the holidays notes improvement. For the first time she was sent to the clinic of the institute of the Federal State Budget Scientific Institution Scientific Research Institute of MT to solve the question of the relationship between skin disease and the profession in 2002. The examination revealed an increased sensitivity to chromium, nickel, cobalt salts and an opinion was made on the professional nature of the skin disease.

A biochemical analysis of blood revealed a moderate increase in the level of liver enzymes ALT 56 U / L (N 0-35 U / L), ACT 45 U / 1 (N 0-35 U / 1), an excess of serum cholesterol of 6.8 mmol / l (N 3.0-6.2 mmol / l). An immunological study determined a moderate elevated Ig E content of Trichophyton rubrum of 1.42 kU / L (N up to 0.35 kU / L). When studying the state of the barrier function of the skin using the "Skin-o-mat" device manufactured by Cosmomed GmbH, Germany, the hydration indicators were reduced by 25.54 (N from 35), the pH level was increased by 6.54 (N 4.5-5 5), the lipid content on the surface of the skin is reduced 2.83 μg / cm ² (N 6.0-6.5 μg / cm ² ).

Blood was examined according to the proposed method.

The total DNA was isolated from 100 μl of whole blood using a single-tube method using the DNA-Sorb-B isolation kit (TU 9398-071-01897593-2008) in accordance with the instructions for the kit used.

A series of dilutions in TE buffer was prepared from the obtained DNA preparation (with a volume of 50 μl) to a final concentration of 1–3 ng / μl, 10 mg of the final DNA solution was added to a tube for polymerase chain reaction. The volume of the mixture for the polymer chain reaction was 25 μl, using reagents manufactured by the Central Scientific Research Institute of Epidemiology FBUN: 2.5 × PCR buffer-2-blue, dNTPs mixture, primers FLG2-F1 and FLG2 -R1. " The number of primers in the reaction is 7 pmol. For amplification of the studied genetic locus, specific oligonucleotide primers “FLG-2 F1” 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3 'and “FLG-2 R1” 5'CAA-gTg-CAg-gAg- were used AAA-gAC-ATg-gAT3 ', one of which is labeled with biotin (the sequence of primers used is presented in Table 1). The number of primers "FLG-2 F1" 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3 'and "FLG-2 R1" 5'CAA-gTg-CAg-gAg-AAA-gAC-ATg-gAT3 'in the polymerase chain reaction - 7 pmol.

The amplification reaction was carried out according to the following program.

When using the T 100 Thermal Cycler thermocycler (Bio Rad, USA), when the temperature reached 95 ° C (pause mode), put tubes in the cells of the thermocycler and maintain them at a temperature of 95 ° C for 15 minutes. Then the tubes were kept for 45 cycles of 95 ° C for 15 seconds, 65 ° C for 15 seconds, 72 ° C for 20 seconds.

After amplification, samples were prepared for sequencing. The amplification product was immobilized with 1 μl of Streptavidin Sepharose particles (GE Healthcare, Sweden) in 40 μl of Binding Buffer (FBUN Central Research Institute of Epidemiology). After immobilization, a series of washes was carried out using the PyroMark Q24 Workstation sample preparation station (Qiagen, Germany) and the Piro-prep reagent kit (FBUN Central Research Institute of Epidemiology, registration certificate No. ФСР 2012/13246 dated March 19, 2012 ) As a result of sample preparation, a single-chain PCR fragment amplified from a biotinylated primer was obtained, which was used as a matrix in the pyrosequencing reaction.

Pyrosequencing was performed using a FLG2-S1 primer in an amount of 7.5 pmol and a PyroMark® Gold Q96 Reagents reagent kit (Qiagen, Germany) using a PyroMark Q24 instrument (Qiagen, Germany). The sequence of the primer for sequencing "FLG-2 S1" is as follows: 5'gAC-ATT-CAg-AAg-ACT-CAg-AC3 '. The sequence of adding nucleotides to the pyrosequencing reaction: GACACAGATCAGTGTCA.

Next, we compared the obtained nucleotide sequence with reference sequences using the PyroMark Q24 2.0.6 software and determined the pyrogram to determine the deletion in the heterozygous state at position 2282 (ins / del) of the filaggrin gene (see Fig. 1), i.e. . filaggrin protein synthesis is impaired.

Conclusion: in patient B., a heterozygous variant of 2282 (ins / del) filaggrin gene was detected, having a deletion in the heterozygous state, indicating a violation of the synthesis of filaggrin protein, which leads to a violation in the process of final differentiation of the epidermis, leading to a pronounced violation of the skin barrier function ( reduced 25.54 (N from 35), pH increased 6.54 (N 4.5-5.5), the lipid content on the surface of the skin was reduced 2.83 μg / cm2 (N 6.0-6.5 μg / cm2)).

This, in turn, led to the development of occupational allergic dermatosis 1 year after the start of work with irritating and sensitizing substances. The time to determine the hereditary predisposition of patient T. to a violation of the barrier function of the skin was 4.5 hours.

Example 2. Patient K., 64 years old

When analyzing the patient’s medical history and professional route, it was revealed that patient K. worked from 1969 to 2007 as a turner in contact with chromium, nickel, and cobalt compounds. Considers herself ill since 2005, when the rash on the back surfaces of the hands and forearms, accompanied by itching, first appeared, then the rash spread to the lower legs. Appealed to the dermatologist at the place of residence, received treatment with a temporary effect. On weekends, during the holidays noted improvement. For the first time he was sent to the clinic of the institute of FSBI “Research Institute of MT” to solve the question of the relationship of skin disease with the profession in 2008. The examination revealed an increased sensitivity to chromium, nickel, cobalt salts and an opinion was made on the professional nature of the skin disease.

The biochemical analysis of blood revealed no deviations from the norm.

An immunological study did not determine an elevated IgE content of Trichophyton rubrum 0.1 kU / L (N up to 0.35 kU / L).

When studying the state of the barrier function of the skin using the "Skin-o-mat" device manufactured by Cosmomed GmbH, Germany, the hydration indicators were normal 36.05 (N from 35), the pH level was within the normal range of 5.3 (N 4 5-5.5), the lipid content on the surface of the skin is reduced to 5.5 μg / cm ² (N 6.0-6.5 μg / cm ² ).

Blood was examined according to the proposed method.

The total DNA was isolated from 100 μl of whole blood using a single-tube method using the DNA-Sorb-B isolation kit (TU 9398-071-01897593-2008) in accordance with the instructions for the kit used.

A series of dilutions in TE buffer was prepared from the obtained DNA preparation (with a volume of 50 μl) to a final concentration of 1–3 ng / μl, 10 mg of the final DNA solution was added to a tube for polymerase chain reaction. The volume of the mixture for the polymer chain reaction was 25 μl, using reagents manufactured by the Central Scientific Research Institute of Epidemiology FBUN: 2.5 × PCR buffer-2-blue, dNTPs mixture, primers FLG2-F1 and FLG2 -R1. " The number of primers in the reaction is 7 pmol. For amplification of the studied genetic locus, specific oligonucleotide primers “FLG-2 F1” 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3 'and “FLG-2 R1” 5'CAA-gTg-CAg-gAg- were used AAA-gAC-ATg-gAT3 ', one of which is labeled with biotin (the sequence of primers used is presented in Table 1). The number of primers "FLG-2 F1" 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3 'and "FLG-2 R1" 5'CAA-gTg-CAg-gAg-AAA-gAC-ATg-gAT3 'in the polymerase chain reaction - 7 pmol.

The amplification reaction was carried out according to the following program.

When using the T 100 Thermal Cycler thermocycler (Bio Rad, USA), when the temperature reached 95 ° C (pause mode), put tubes in the cells of the thermocycler and maintain them at a temperature of 95 ° C for 15 minutes. Then the tubes were kept for 45 cycles of 95 ° C for 15 seconds, 65 ° C for 15 seconds, 72 ° C for 20 seconds.

After amplification, samples were prepared for sequencing. The amplification product was immobilized with 1 μl of Streptavidin Sepharose particles (GE Healthcare, Sweden) in 40 μl of Binding Buffer (FBUN Central Research Institute of Epidemiology). After immobilization, a series of washes was carried out using the PyroMark Q24 Workstation sample preparation station (Qiagen, Germany) and the Piro-prep reagent kit (FBUN Central Research Institute of Epidemiology, registration certificate No. ФСР 2012/13246 dated March 19, 2012 ) As a result of sample preparation, a single-chain PCR fragment amplified from a biotinylated primer was obtained, which was used as a matrix in the pyrosequencing reaction.

Pyrosequencing was performed using a FLG2-S1 primer in an amount of 7.5 pmol and a PyroMark® Gold Q96 Reagents reagent kit (Qiagen, Germany) using a PyroMark Q24 instrument (Qiagen, Germany). The sequence of the primer for sequencing "FLG-2 S1" is as follows: 5'gAC-ATT-CAg-AAg-ACT-CAg-AC3 '. The sequence of adding nucleotides to the pyrosequencing reaction: GACACAGATCAGTGTCA.

Next, we compared the obtained nucleotide sequence with reference sequences using the PyroMark Q24 2.0.6 software and determined the absence of deletion at position 2282 (ins / ins) of the filaggrin gene (see Fig. 2) corresponding to the norm using the obtained pyrogram. e. filaggrin protein is synthesized normally.

Conclusion: in patient K., a homozygous variant of FLG (ins / ins) polymorphism 2282del4 was detected, which did not have a deletion at position 2282, indicating normal synthesis of this protein, while a moderate decrease in skin barrier function was observed, which manifests itself only in a decrease in lipid content skin surface, which may be caused by a long experience with irritating and sensitizing factors (chromium, nickel, cobalt). At the same time, professional allergic dermatosis developed 38 years after the start of work with irritating and sensitizing factors (chromium, nickel, cobalt). The time to determine the hereditary predisposition of patient K. to a violation of the barrier function of the skin was 3.0 hours.

The proposed method for predicting a hereditary predisposition to impaired barrier function of the skin can improve the accuracy of the results by minimizing errors in interpreting the results by using computer processing of the results and the speed of analysis (1-2 days in the known method and 3-5 hours in the proposed method ) compared with the prototype due to the use of new laboratory technology - the pyrosequencing method, the use of which allows per unit time and conduct a greater number of tests, the results of which define a hereditary predisposition to a breach of the barrier function of the skin, for example, during the clinical examination or periodic medical examinations of a large number of workers in hazardous and / or hazardous conditions for short (short) periods of time.

The proposed method for determining a hereditary predisposition to impaired skin barrier function can be used in clinical, biochemical, molecular genetic laboratories equipped with equipment manufactured by domestic or foreign industry.

Claims (1)

A method for determining a hereditary predisposition to a violation of the barrier function of the skin, including venous blood sampling, isolation of genetic material, polymerase chain reaction of DNA synthesis followed by analysis of restriction fragment length polymorphism, determination of the nucleotide sequence and based on this determination of the polymorphic variant of 2282del4 filaggrin, characterized in that when carrying out the polymerase chain reaction, specific primers are used: "FLG-2 F1" 5'Biotin-TgA-CCA-gCC-TgT-CCA-Tgg-C3 '; "FLG-2 R1" 5'CAA-gTg-CAg-gAg-AAA-gAC-ATg-gAT3 ", the amplicon size is 205 base pairs, and after the polymerase chain reaction, the pyrosequencing reaction is carried out in real time using a specific primer" FLG-2 S1 "5'gAC-ATT-CAg-AAg-ACT-CAg-AC3 'and detection of the nucleotide sequence using a chemiluminescent signal, then compare the obtained nucleotide sequence at position 2282 with reference sequences, after which, if detected on the resulting pyrogram no deletion at position 2282 of the phi gene aggrin when exposed to an organism of a harmful production factor of an irritating and sensitizing effect determines a low hereditary predisposition to a violation of the barrier function of the skin; when a heterozygous ins / del variant at position 2282 of the filaggrin gene is detected on the resulting pyrogram, it determines a high production factor of an irritating and sensitizing effect on the body hereditary predisposition to impaired barrier function of the skin.

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