EA024837B1 - Method for predicting risk of early development of professional allergodermatosis affected by chrome, nickel and cobalt - Google Patents

Abstract

The invention relates to medicine and can be used for predicting a risk of early development of professional allergodermatosis affected by chrome, nickel and cobalt. The present invention is aimed at predicting a risk of development of professional allergodermatosis during 3 years from the initiation of continuous work with chrome, nickel and cobalt - factors of sensibilizing action in the process of preliminary or periodic medical examination for short undertime of a great number of working or newly-hired staff to be engaged in harmful and/or dangerous working conditions. The assigned task is solved by that the method of predicting an early development of professional allergodermatosis affected by chrome, nickel and cobalt comprises taking venous blood, isolating genetic material, performing a quasi-uniform chain reaction with specific primers, determining nucleotide sequence, further performing amplification of the sequence in the promoter region of the IL-10 gene in the polymorphic position - 1082, the nucleotide sequence is determined and polymorphic variant of the IL-10 gene is determined based thereon, in case of detecting homozygous variant of the IL-10 G-1082A-AA gene the early development of professional allergodermatosis affected by chrome, nickel and cobalt during first 3 years from the initiation of the professional continuous contact therewith is correspondingly predicted, wherein if a heterozygous variant of the IL-10 G-1082A-GA gene or homozygous variant of the IL-10 G-1082A-GG gene are resent, a possibility of developing professional allergodermatosis after 3 and more years of work in contact with chrome, nickel and cobalt is predicted. The proposed method provides to predict early development of professional allergodermatosis during 3 years from the initiation of professional continuous work with chrome, nickel and cobalt - factors of sensibilizing action in the process of preliminary or periodic medical examination for short undertime of a great number of working or newly-hired staff to be engaged in harmful and/or dangerous working conditions. The proposed method can be used in clinical, biochemical, molecular-genetic laboratories, equipped with national or foreign-made facilities.

Description

The invention relates to medicine and can be used to predict the risk of early development of occupational allergic dermatoses when exposed to chromium, nickel and cobalt.

A known method for predicting the risk of early development of professional allergic dermatoses when exposed to sensitizing factors, including chromium, nickel and cobalt, including venous blood sampling, isolation of genetic material, polymerase chain reaction with specific primers, determination of the nucleotide sequence and based on this determination of the polymorphic Variants L4889C of cytochrome P-450 1A1 (Method for predicting the risk of early development of occupational allergic dermatitis call. № RF Patent 2467330, cl .. C 01 N 33/50, 2011).

However, the known method for predicting the timing of the development of occupational allergic dermatoses has low specificity for chromium, nickel and cobalt - factors of a sensitizing effect, because cytochrome P-450 1A1 is involved mainly in the activation to the carcinogenic, mutagenic and toxic effects of a wide variety of xenobiotics. Using the known method, they predict the risk of developing professional allergodermatosis in a worker during continuous work under conditions of exposure to the body of chromium, nickel and cobalt - sensitizing factors 4-5 years from the start of work, which does not allow predicting the risk of earlier development (with continuous work up to 3 years from the start of work) of professional allergic dermatoses.

The objective of the present invention is to predict the risk of developing occupational allergic dermatoses within 3 years from the beginning of continuous work with chromium, nickel and cobalt - sensitizing factors during preliminary or periodic medical examinations of a large number of workers who work or go to work in harmful and / or dangerous conditions labor for short (short) periods of time.

The technical result of the invention is the ability to individually forecast the risk of developing occupational allergic dermatoses up to 3 years from the beginning of continuous work with chromium, nickel and cobalt - sensitizing factors, which will allow timely identification of persons predisposed to the development of occupational allergic dermatoses to prevent the development of occupational pathology through rational employment out of contact with chromium, nickel and cobalt - factors of sensitizing action, timely correction and increase of adaptive abilities of the body in various non-specific ways.

This task is achieved by the fact that in the known method for predicting the early development of occupational allergic dermatoses when exposed to chromium, nickel and cobalt, including venous blood sampling, isolation of genetic material, polymerase chain reaction with specific primers, determination of the nucleotide sequence, the sequence is also amplified in the promoter regions of the 1b-10 gene in the polymorphic position 1082 determine the nucleotide sequence and based on this they determine the polymorphic variant of the 1L-10 gene, when a homozygous variant of the 1L-10 gene C-1082A-AA is detected, the early development of professional allergic dermatoses under the influence of chromium, nickel and cobalt is accordingly predicted during the first 3 years from the start of professional continuous contact with them, and in the presence of a heterozygous variant of the 1b-10 C-1082A-CA gene or a homozygous variant of the 1b-10 C-1082A-SS gene, the possibility of developing professional allergic dermatoses after 3 or more years of work in contact with ohm, nickel and cobalt.

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 and drugs manufactured by domestic or foreign industry.

Therefore, the claimed method is affordable and practically applicable.

The proposed method is based on the study of a set of diagnostic tests, while the presence or absence of a polymorphic variant AA, CA, or CC of the 1L-10 C-1082A gene plays a major role in predicting the early onset and course of occupational allergic dermatoses when exposed to chromium, nickel and cobalt - sensitizing factors actions.

The homozygous variant of the 1L-10 C-1082A-AA gene determines the hypersecretion of interleukin-10, which is fundamental in the pathogenetic mechanisms of professional allergic dermatoses, as it inhibits the production of pro-inflammatory cytokines and stimulates the secretion of immunoglobulins, especially immunoglobulin E, B-lymphocytes. The discovery of the aforementioned variant of the 1L-10 C-1082A-AA gene indicates a risk of early development of occupational allergic dermatoses during the first 3 years from the onset of continuous occupational exposure to chromium, nickel and cobalt sensitizing factors. In the presence of a heterozygous variant of the 1L-10 C-1082A-CA gene or a homozygous variant of the 1b-10 C-1082A-SS gene, the development of professional allergic dermatoses is predicted after 3 or more years of continuous operation in contact with chromium, nickel and cobalt - sensitizing factors.

The proposed method is illustrated in the drawing.

The drawing shows an electrophoregram of amplification products of gene 1B-10 (O-1082A) in a 3% agarose gel, where K- is a negative control sample, K + is a positive control sample, 1 is homozygote 00 (normal), 2 is homozygote 00 ( norm), 3 - OA heterozygote, 4 - OA heterozygote, 5 - AA homozygote (pathology).

The proposed method is as follows.

Venous blood sampling is performed in patients. Blood is examined in accordance with the proposed method. DNA sorbent is isolated from 100 μl of whole blood using a single-tube method using a DNA sorb-B isolation kit (TU9398-071-01897593-2008) in accordance with the instructions for the kit used.

From the obtained DNA preparation (50 μl), a series of dilutions in TE buffer is prepared to a final concentration of 1-3 ng / μl, 10 mg of the final DNA solution is added to the tube for genotyping of Ш-10 using Litech kits, Russia. In this case, reagents are used, for example, manufactured by Litech, Russia: diluent, reaction mixture (normal - Ν, pathology P), Tad polymerase. The volume of the amplification mixture is 25 μl.

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

When using a thermal cycler Т 100 Тегта1 Сс1eg (company KAO, USA), when the temperature reaches 94 ° С - the Raike mode, put the tubes in the cells of the thermal cycler and maintain them at a temperature of 93 ° С - 1 min. The test tubes are then kept for 35 cycles: at 93 ° C for 10 s, at 64 ° C for 10 s, at 72 ° C for 20 s, and at the end at 72 ° C for 1 min, then 10 ° §!

After amplification, the reaction products are analyzed in a 3% polyacrylamide gel, followed by staining with ethidium bromide and visualization in transmitted UV light (Fig. 1). Interpretation of the result + - homozygote according to Alleli 1, ++ - heterozygote, - + - homozygote according to Alleli 2.

When a homozygous variant of the Sh-10 O-1082A-AA gene is detected, early development of professional allergic dermatoses when exposed to chromium, nickel and cobalt, respectively, sensitizing factors during the first 3 years from the start of professional continuous contact with chromium, nickel and cobalt is accordingly predicted - factors of a sensitizing effect, in the presence of a heterozygous variant of the Sh-10 O-1082A gene - OA or a homozygous variant of the Sh-10 O-1082A gene - O predict the possibility of developing professional allergies dermatoses of 3 or more years of continuous work in contact with chromium, nickel and cobalt - factors sensitizing effect.

Example 1

Patient R., 45 years old. Upon admission to work in contact with cement containing chromium, nickel and cobalt, the proposed method, the blood of a patient R. was examined as follows.

The total DNA was isolated from 100 μl of whole blood using a single-tube method using a DNA-sorb-B isolation kit (TU9398-071-01897593-2008) in accordance with the instructions for the kit used.

From the obtained DNA preparation (50 μl), a series of dilutions was prepared in TE buffer to a final concentration of 1-3 ng / μl, 10 mg of the final DNA solution was added to the tube for genotyping of Ш-10 using Litech kits, Russia. In this case, reagents manufactured by Litech, Russia were used: diluent, reaction mixture (norm - Ν, pathology - P), Tad polymerase. The volume of the amplification mixture is 25 μl.

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

When using a thermal cycler Т 100 Тегта1 Сс1eg (KAI firm, USA), when the temperature reaches 94 ° С - the Raike mode, put tubes in the thermal cycler cells and maintain them at a temperature of 93 ° С - 1 min. The test tubes are then kept for 35 cycles: at 93 ° C for 10 s, at 64 ° C for 10 s, at 72 ° C for 20 s, and at the end at 72 ° C for 1 min, then 10 ° §!

After amplification, the reaction products were analyzed in a 3% polyacrylamide gel, followed by staining with ethidium bromide and visualizing in transmitted UV light.

When analyzing the results, a homozygous variant of AA was identified, which has the substitution of guanine for adenine at position 1082, a variant of the genotype corresponding to reduced activity of interleukin 10.

According to biochemical studies: the normal content of the inflammatory protein - the eosinophilic cationic protein and antibodies responsible for the allergic reaction - immunoglobulin E in the blood serum. When undergoing a mandatory periodic medical examination 2 years after starting work with chromium, nickel and cobalt - the sensitizing factors that make up the cement, the patient R. revealed clinical and functional signs of allergic dermatosis.

According to biochemical studies: an increased content of the inflammatory protein - the eosinophilic cationic protein and antibodies responsible for the allergic reaction - immunoglobulin E in the blood serum.

Patient R. was sent to the center of Occupational Pathology for examination of the connection of the disease with the profession. By

- 2 024837 the results of the examination were diagnosed with occupational eczema of the upper extremities 2 years after the beginning of continuous work with chromium, nickel and cobalt - sensitizing factors that make up the cement.

Conclusion: in patient R., a homozygous variant of AA gene 1b-10 (O-1082A) was detected, i.e. the synthesis of interleukin-10 is reduced, which led to a violation of the ratio of inflammatory and anti-inflammatory reactions with a predominance of inflammatory, which resulted in the early development of occupational eczema of the upper extremities after 2 years of continuous work with chromium, nickel and cobalt with the sensitizing factors that make up the cement.

Example 2

Patient P., 51 years old. When analyzing the patient’s medical history and professional route, it was revealed that patient P. worked as an operator at a concrete factory, being exposed to compounds of nickel, chromium and cobalt, which are part of cement. A year after the start of work, the results of the examination were diagnosed with occupational eczema of the upper extremities.

According to biochemical studies: a significant increase in the content of the inflammatory protein - eosinophilic cationic protein and antibodies responsible for the allergic reaction - immunoglobulin E in blood serum.

According to the proposed method, the blood was examined as follows.

The total DNA was isolated from 100 μl of whole blood using a single-tube method using a DNA-sorb-B isolation kit (TU9398-071-01897593-2008) 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 the tube for genotyping 1B-10 using kits from Litech, Russia.

The volume of the polymer chain reaction was 25 μl, using reagents manufactured by Litech, Russia: diluent, reaction mixture (normal - Ν, pathology - P), Tatz polymerase.

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

When using the thermal cycler Т 100 ТНсгта1 Сс1ег (company ΒΙΟ ΚΑΌ, USA), when the temperature reaches 94 ° С - the Raike mode, put the tubes in the cells of the thermal cycler and maintain them at a temperature of 93 ° С - 1 min. Next, the tubes are kept for 35 cycles: at 93 ° С - 10 s, at 64 ° С - 10 s, at 72 ° С 20 s, and at the end at 72 ° С - 1 min, then 10 ° §1 place.

After amplification, the reaction products were analyzed in a 3% polyacrylamide gel, followed by staining with ethidium bromide and visualizing in transmitted UV light.

When analyzing the results, a homozygous variant of AA was identified, which has the substitution of guanine for adenine at position 1082, a variant of the genotype corresponding to reduced activity of interleukin 10.

Conclusion: in patient F., a homozygous variant of AA of the Sh-10 O-1082A gene was detected, i.e. the synthesis of interleukin-10 is reduced, this leads to a more pronounced inflammatory reaction, which increases the risk of early development in the first year of work, the consequence of this was the development of occupational eczema in the first year of continuous work when exposed to the compounds of nickel, chromium and cobalt, which are part of the cement.

Example 3

Patient G., 50 years old. When analyzing the patient’s medical history and professional route, it was revealed that patient G. worked as an annealer in a metal processing plant, being exposed to chromium. 9 years after the start of work, the results of the examination were diagnosed with occupational eczema of the upper extremities.

According to biochemical studies: an increase in the content of the inflammatory protein - the eosinophilic cationic protein and antibodies responsible for the allergic reaction - immunoglobulin E in the blood serum.

According to the proposed method, the blood of patient G. was investigated as follows.

The total DNA was isolated from 100 μl of whole blood using a single-tube method using a DNA-sorb-B isolation kit (TU9398-071-01897593-2008) 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 the tube for genotyping of Ш-10 using Litech kits, Russia.

The volume of the amplification mixture is 25 μl, using reagents manufactured by Litech, Russia: diluent, reaction mixture (normal - Ν, pathology - P), Tatz polymerase.

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

When using a thermal cycler Т 100 ТНегта1 Сс1ег (company ΒΙΟ ΚΑΌ, USA), when the temperature reaches 94 ° С - the Raike mode, put the tubes in the cells of the thermal cycler and maintain them at a temperature of 93 ° С - 1 min. Next, the tubes are kept for 35 cycles: at 93 ° С - 10 s, at 64 ° С - 10 s, at 72 ° С 20 s, and at the end at 72 ° С - 1 min, then 10 ° §1 place.

After amplification, the reaction products were analyzed in 3% polyacryl-3,048,837 amide gel, followed by staining with ethidium bromide and visualizing in transmitted UV light.

An analysis of the results revealed a heterozygous variant of CA, which has the substitution of guanine for adenine at position 1082, a variant of the genotype corresponding to a slightly reduced activity of interleukin 10.

Conclusion: in patient B., a heterozygous variant of CA was detected, i.e. the synthesis of interleukin-10 is slightly reduced, this leads to an inflammatory reaction that increases the risk of development after 3 or more years of work, the consequence of this is the development of occupational eczema after 9 years of work in contact with chromium, a sensitizing substance.

Example 4

Patient B., 60 years old. In the analysis of history and prof. it was revealed that patient B. worked as a model designer at AMO ZIL, for 26 years had contact with chromium salts, putty, PVA glue, and hardener. 26 after the start of work, according to the results of the examination at the Scientific Research Institute of Occupational Medicine, the diagnosis was made - occupational eczema of the upper extremities.

According to biochemical studies: a slight increase in the content of the inflammatory protein - the eosinophilic cationic protein and the normal content of antibodies responsible for the allergic reaction of immunoglobulin E in the blood serum.

According to the proposed method, the blood of patient B. was examined as follows.

The total DNA was isolated from 100 μl of whole blood using a single-tube method using a DNA-sorb-B isolation kit (TU9398-071-01897593-2008) 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 the tube for genotyping 1B-10 using kits from Litech, Russia.

The volume of the amplification mixture is 25 μl, using reagents manufactured by Litech, Russia: diluent, reaction mixture (normal - Ν, pathology - P), Tatz polymerase.

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

When using a thermal cycler Т 100 ТНегта1 Сс1ег (KAI firm, USA), when the temperature reaches 94 ° С - Raise mode, put tubes in the thermal cycler cells and maintain them at a temperature of 93 ° С - 1 min. Next, the tubes are kept for 35 cycles: at 93 ° С - 10 s, at 64 ° С - 10 s, at 72 ° С 20 s, and at the end at 72 ° С - 1 min, then 10 ° §1 place.

After amplification, the reaction products were analyzed in a 3% polyacrylamide gel, followed by staining with ethidium bromide and visualizing in transmitted UV light.

When analyzing the results, a homozygous SS variant was identified that does not have the replacement of guanine with adenine at position 1082, a variant of the genotype corresponding to the normal activity of interleukin 10.

Conclusion: in patient B., the absence of homo- and heterozygotes IL 10 С1082А was revealed, i.e. interleukin-10 is synthesized in normal amounts. The anti-inflammatory function of interleukin-10 has been preserved, but the prolonged exposure to chromium salts, a sensitizing substance, helps to weaken the immune status, which resulted in the later development of occupational eczema of the upper extremities, only 26 years after the start of work with a harmful production sensitizing factor, chromium salts.

Thus, the proposed set of techniques allows predicting the early development of professional allergic dermatoses within 3 years from the onset of professional continuous contact with chromium, nickel and cobalt - sensitizing factors during preliminary or periodic medical examinations of a large number of working or arriving at work in harmful and / or hazardous working conditions for short (short) periods of time.

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

Claims (1)

CLAIM A method for predicting the risk of early development of occupational allergic dermatoses under the influence of chromium, nickel and cobalt, including venous blood sampling, isolation of genetic material, polymerase chain reaction with specific primers, determination of the nucleotide sequence, characterized in that the sequence is further amplified in the promoter region of the 1b- gene 10 in the polymorphic position - 1082, determine the nucleotide sequence and on the basis of this determine the polymorphic 1B-10 Ianthe gene. if a homozygous variant of the 1L-10 C-1082A-AA gene is detected, the early development of professional allergic dermatoses under the influence of chromium, nickel and cobalt is predicted accordingly during the first 3 years from the beginning of professional contact with them, and in the presence of heterozygous variant of the 1L-10 C-1082L gene - CA or the homozygous variant of the 1L-10 C-1082A - OS gene predict the possibility of developing professional allergic dermatoses after 3 or more years of continuous work in contact with chromium, nickel and cobalt.