RU2466404C1 - Method for prediction of time of developing occupational allergic dermatoses after exposure to irritant and sensitising factors - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: genetic material is recovered from patient's venous blood, a PCR is conducted for detecting polymorphous versions of GSTM1 gene. If simultaneously observing deletions and GSTM1-del, and GSTT1-del in GSTM1 and GSTT1 genes respectively, the early development of occupational allergic dermatoses is predicted for the first 3 years from the beginning of contact with irritant and sensitising factors with the severe clinical course in the form of extensive forms of occupational allergic dermatoses and frequent recurrences. If observing one of GSTM1-del or GSTT1-del deletions in GSTM1 and GSTT1 genes respectively, the development of occupational allergic dermatoses is predicted for 3 to 5 years following the beginning of contact with irritant and sensitising factors, while the absence of deletions and GSTM1-del, and GSTT1-del in GSTM1 and GSTT1 genes respectively enables predicting the development of occupational allergic dermatoses at least in 5 years of contact with irritant and sensitising factors.

EFFECT: method provides higher accuracy of prediction of time of developing occupational allergic dermatoses.

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Description

The invention relates to medicine and can be used to predict the timing of the development of professional allergic dermatoses when exposed to factors of an irritating and sensitizing effect.

A known method for predicting the timing of the development of professional allergic dermatoses when exposed to factors of irritating and sensitizing effects, including venous blood sampling, isolation of genetic material, polymerase chain reaction with specific primers 5'-ggt-cat-tct-gaa-ggc-caa-gg and 5 '-ttt-gtg-gac-tgc-tga-gga-cg, determination of the nucleotide sequence and, on the basis of this, the determination of polymorphic variants of the GSTM1 gene (see N. Lazarashvili, Role of the “oxidant-antioxidant” system and genetic biochemical olymorphism in the pathogenesis of occupational allergic dermatoses. The dissertation for the degree of candidate of medical sciences, Moscow, 2006, p. 48-86).

However, the known method for predicting the timing of the development of occupational allergic dermatoses has low information content and, as a consequence of the low information content of the method, they can only predict that if there is a deletion in the GSTM1 gene, the disease can develop in the first 5 years from the start of work with harmful production factors. This method has low accuracy in predicting the timing of the development of professional allergic dermatoses, 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 improve the method for predicting the timing of the development of occupational allergic dermatoses by increasing its information content, 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 technical result of the invention is to improve the accuracy of predicting the timing of development of professional allergic dermatoses when exposed to factors of an irritating and sensitizing effect due to increased information content.

This task is achieved by the fact that in the known method for predicting the timing of the development of professional allergic dermatoses when exposed to factors of irritating and sensitizing effects, including venous blood sampling, isolation of genetic material, polymerase chain reaction with specific primers 5'-tgc-ttc-acg-tgt- tat-gga-ggt-tc and 5'-gtt-ggg-ctc-aaa-tat-acg-gtg-g, the determination of the nucleotide sequence and based on this the determination of the polymorphic variants of the GSTM1 gene, additionally determine the polymorphic variant of the gene GSTT1 during the polymerase chain reaction with specific primers 5'-ggt-cat-tct-gaa-ggc-caa-gg and 5'-ttt-gtg-gac-tgc-tga-gga-cg, and when detecting both deletions and GSTM1 -del and GSTT1-del, respectively, in the GSTM1 and GSTT1 genes predict early development of occupational allergic dermatoses when exposed to irritating and sensitizing factors during the first 3 years from the onset of contact with severe irritating and sensitizing factors in the form of common forms occupational allergic dermato call and frequent relapses, in the presence of one of the deletions of GSTM1-del or GSTT1-del in the GSTM1 and GSTT1 genes, respectively, they predict the development of occupational allergic dermatoses within 3 to 5 years after the onset of contact with irritating and sensitizing factors, with In the absence of deletions, both GSTM1-del and GSTT1-del in the GSTM1 and GSTT1 genes, respectively, predict the development of occupational allergic dermatoses after at least 5 years of work in contact with irritating and sensitizing factors.

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 that allows to increase the information content of the results, which, in turn, will improve the accuracy of predicting the timing of the development of professional allergic dermatoses when exposed to factors of irritating and sensitizing effects, for example, during medical examinations or periodic medical examinations of large the number of workers in harmful and / or hazardous working conditions for short (tight) time periods.

The proposed method is based on the study of a set of diagnostic tests, while the presence or absence of deletions of GSTM1-del and GSTT1-del in the GSTM1 and GSTT1 genes, respectively, plays a major role in predicting the timing of development of occupational allergic dermatoses when exposed to irritating and sensitizing factors.

Table 1 presents the sequence of primers used for the isolation of genetic material during the polymerase chain reaction.

The proposed method is illustrated in the drawing.

Figure 1 presents the electrophoregram of the amplification products GST-τ1 and GST-μ1 in a 3% agarose gel, where M is a marker, 1 is GST-μ1 0/0, GST-τ1 + / +, 2 is GST-μ1 + / + , GST-τ1 + / +, 3 - GST-µ1 + / +, GST-τ1 + / +, 4 - GST-µ1 + / +, GST-τ1 + / +, 5 - GST-µ1 + / +, GST -τ1 0/0, 6 - GST-µ1 0/0, GST-τ1 0/0.

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 the 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 is 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 are added to the tube for polymerase chain reaction with specific primers 5'-tgc-ttc-acg -tgt-tat-gga-ggt-tc and 5'-gtt-ggg-ctc-aaa-tat-acg-gtg-g, 5'-ggt-cat-tct-gaa-ggc-caa-gg and 5'- ttt-gtg-gac-tgc-tga-gga-cg.

The volume of the polymer chain reaction is 25 μl, using reagents manufactured by the Central Research Institute of Epidemiology: PCR-2-blue PCR, dNTPs (T) desoxynucleotide triphosphates, PCR Wax, and PCR Mineral Oil.

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

When using the Tercik amplifier (DNA-Technology company), when the temperature reaches 95 ° C (pause mode), put the tubes in the cells of the amplifier and maintain them at a temperature of 95 ° C for 5 minutes. The tubes are then kept for 45 cycles: at 95 ° С - 10 s, at 65 ° С - 10 s, at 72 ° С - 10 s, and at the end at 72 ° С - 2 min.

When using amplifiers “Gradient Palm Cycler” (“MAXYGEN”), “GeneAmp PCR System 2700”, “PalmCycler”, when the temperature reaches 95 ° C (pause mode), the tubes are placed in the cells of the amplifier and continue the program at a temperature of 95 ° C - 5 minutes. Further, the tubes withstand 45 cycles: at 95 ° C - 15 s, at 65 ° C - 15 s, at 72 ° C - 20 s, and at the end at 95 ° C - 2 min.

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). Homozygotes or heterozygotes by the normal “+” allele are determined by the presence of an amplification product on electrophoregrams of 271 bp in size. for GST-µ1 and fragment 315 n.p. for GST-τ1. The absence of an appropriate fragment indicates homozygosity of the individual for gene deletion.

When deletions of both GSTM1-del and GSTT1-del are detected in the GSTM1 and GSTT1 genes, early development of occupational allergic dermatoses is predicted when exposed to irritating and sensitizing factors during the first 3 years from the onset of contact with irritating and sensitizing factors with severe course in the form of common forms of occupational allergic dermatoses and frequent relapses. In the presence of one deletion of GSTM1-del or GSTT1-del in the GSTM1 and GSTT1 genes, the development of occupational allergic dermatoses is predicted within 3 to 5 years after the onset of contact with irritating and sensitizing factors. In the absence of deletion, both GSTM1-del and GSTT1-del in the GSTM1 and GSTT1 genes, respectively, predict the development of occupational allergic dermatoses after at least 5 years of work in contact with irritating and sensitizing factors.

Example 1. Patient F., 77 years old.

When analyzing the patient’s medical history and professional route, it was revealed that patient F. worked as a presser at the Moscow factory for the production of children's toys, being exposed to polystyrene. A year after the start of work, a professional eczema was diagnosed based on the results of the examination.

According to biochemical studies: a significant increase in the content of lipid peroxidation products (diene conjugates, ketodienes and carbonyls) in blood serum and inhibition of antioxidant protection - a significant decrease in the thiol disulfide ratio.

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 the 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 polymerase chain reaction with specific primers 5'-tgc-ttc-acg -tgt-tat-gga-ggt-tc and 5'-gtt-ggg-ctc-aaa-tat-acg-gtg-g, 5'-ggt-cat-tct-gaa-ggc-caa-gg and 5'- ttt-gtg-gac-tgc-tga-gga-cg.

The volume of the polymeric chain reaction was 25 μl, using reagents manufactured by the Central Research Institute of Epidemiology: PCR-2-blue PCB, dNTPs (T) desoxynucleotide triphosphates, PCR Wax, and PCR Mineral Oil.

The amplification reaction was carried out using a Tercik amplifier (DNA technology company). When it was heated to a temperature of 95 ° C (pause mode), the tubes were placed in the cells of the amplifier and kept at a temperature of 95 ° C for 5 minutes. Then the tubes were kept for 45 cycles: at 95 ° С - 10 s, at 65 ° С - 10 s, at 72 ° С - 10 s, and at the end at 72 ° С - 2 min.

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, deletions of GSTM1-del and GSTT1-del were revealed for the GSTM1 and GSTT1 genes, respectively.

Conclusion: in patient F., deletions of GSTM1-del and GSTT1-del were revealed according to the genes GSTM1 and GSTT1, i.e. Glutathione S-transferase M1 and T1 enzymes are not synthesized; therefore, antioxidant defense processes are not carried out. The consequence of this was the development of occupational eczema with frequent relapses in the first year of work with harmful production factors of irritating and sensitizing effects.

Example 2. Patient B., 60 years old.

When analyzing the patient’s history and professional route, it was revealed that patient B. worked as a hairdresser in a hairdresser, being exposed to the chemical composition “Lockon”. 4 years after the start of work, the results of the examination were diagnosed with occupational eczema of the extremities.

According to biochemical studies: an increase in the content of lipid peroxidation products (diene conjugates, ketodienes and carbonyls) in blood serum and inhibition of antioxidant protection - a decrease in the thiol disulfide ratio.

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 the 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 polymerase chain reaction with specific primers 5'-tgc-ttc-acg -tgt-tat-gga-ggt-tc and 5'-gtt-ggg-ctc-aaa-tat-acg-gtg-g, 5'-ggt-cat-tct-gaa-ggc-caa-gg and 5'- ttt-gtg-gac-tgc-tga-gga-cg.

The volume of the polymeric chain reaction was 25 μl, using reagents manufactured by the Central Research Institute of Epidemiology: PCR-2-blue PCB, dNTPs (T) desoxynucleotide triphosphates, PCR Wax, and PCR Mineral Oil.

The amplification reaction was carried out using a Tercik amplifier (DNA technology company). When it was heated to a temperature of 95 ° C (pause mode), the tubes were placed in the cells of the amplifier and kept at a temperature of 95 ° C for 5 minutes. Further, the tubes were kept for 45 cycles: at 95 ° С - 10 s, at 65 ° С - 10 s, at 72 ° С - 10 s, and at the end at 72 ° С - 2 min.

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 deletion of GSTM1-del by the GSTM1 gene and the absence of deletion of the GSTT1 gene were revealed.

Conclusion: patient B. revealed a deletion of GSTT1-del by the GSTT1 gene and the absence of deletion by the GSTM1 gene, i.e. the glutathione-S-transferase M1 enzyme is not synthesized, and the glutathione-S-transferase T1 enzyme is synthesized. Therefore, the processes of antioxidant protection are not fully implemented. The consequence of this was the development of occupational eczema of the extremities after 4 years of work with a harmful production factor of irritating and sensitizing effects.

Example 3. Patient R., 51 years old

When analyzing the patient’s history and professional route, it was revealed that patient R. had been working as a chip collector at the Komponent factory for 34 years, in contact with epoxy resin, POS 61 solder (tin-lead), which contains nickel. 34 years after the start of work according to the results of a survey at the Scientific Research Institute of Occupational Medicine RAMP, a diagnosis of occupational eczema of the upper extremities was diagnosed.

According to biochemical studies: a slight increase in the content of lipid peroxidation products (diene conjugates, ketodienes and carbonyls) in the blood serum and a slight inhibition of antioxidant protection - a decrease in the thiol disulfide ratio.

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 the 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 polymerase chain reaction with specific primers 5'-tgc-ttc-acg -tgt-tat-gga-ggt-tc and 5'-gtt-ggg-ctc-aaa-tat-acg-gtg-g, 5'-ggt-cat-tct-gaa-ggc-caa-gg and 5'- ttt-gtg-gac-tgc-tga-gga-cg.

The volume of the polymeric chain reaction was 25 μl, using reagents manufactured by the Central Research Institute of Epidemiology: PCR-2-blue PCB, dNTPs (T) desoxynucleotide triphosphates, PCR Wax, and PCR Mineral Oil.

The amplification reaction was carried out using a Tercik amplifier (DNA technology company). When it was heated to a temperature of 95 ° C (pause mode), the tubes were placed in the cells of the amplifier and kept at a temperature of 95 ° C for 5 minutes. Then the tubes were kept for 45 cycles: at 95 ° С - 10 s, at 65 ° С - 10 s, at 72 ° С - 10 s, and at the end at 72 ° С - 2 min.

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

An analysis of the results revealed the absence of deletions for the GSTM1 and GSTT1 genes.

Conclusion: patient R. revealed the absence of deletions for the GSTM1 and GSTT1 genes, i.e. enzymes glutathione-S-transferase M1 and T1 are fully synthesized, therefore, antioxidant defense processes are carried out satisfactorily. However, they are slightly reduced under the influence of production factors. The consequence of this was the development of occupational eczema of the upper extremities 34 years after the start of work with a harmful production factor of irritating and sensitizing effects.

The proposed method for predicting the timing of the development of occupational allergic dermatoses can improve the accuracy of predicting the timing of the development of occupational allergic dermatoses when the body is exposed to factors of irritating and sensitizing effects by 50% by increasing the information content of the studies, for example, during medical examinations or periodic medical examinations of a large number of workers harmful and / or dangerous working conditions for short (short) periods of time change, and also increase the reliability of predicting the timing of the development of professional allergic dermatoses when exposed to factors of an irritating and sensitizing effect,

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

Table Gene variant Primer used Gstm1 5'-tgc-ttc-acg-tgt-tat-gga-ggt-tc 5'-gtt-ggg-ctc-aaa-tat-acg-gtg-g GSTT1 5'-ggt-cat-tct-gaa-ggc-caa-gg 5'-ttt-gtg-gac-tgc-tga-gga-cg

Claims (1)

A method for predicting the timing of the development of occupational allergic dermatoses when exposed to irritating and sensitizing factors, including venous blood sampling, isolation of genetic material, polymerase chain reaction with specific primers 5'-tgc-ttc-acg-tgt-tat-gga-ggt-tc and 5'-gtt-ggg-ctc-aaa-tat-acg-gtg-g, determination of the nucleotide sequence and based on this the determination of polymorphic variants of the GSTM1 gene, characterized in that the polymorphic variant of the GSTT1 gene is additionally determined during polymerization a basic chain reaction with specific primers 5'-ggt-cat-tct-gaa-ggc-caa-gg and 5'-ttt-gtg-gac-tgc-tga-gga-cg, and when deletions and GSTM1-del are detected simultaneously, and GSTT1-del in the GSTM1 and GSTT1 genes, respectively, predict early development of occupational allergic dermatoses when exposed to irritating and sensitizing factors during the first 3 years from the onset of contact with severe irritating and sensitizing factors in the form of common forms of occupational allergic dermatoses and frequent relapses, etc. and the presence of one of the deletions of GSTM1-del or GSTT1-del in the GSTM1 and GSTT1 genes respectively, the development of occupational allergic dermatoses is predicted within 3 to 5 years after the onset of contact with irritating and sensitizing factors, in the absence of deletions and GSTM1-del and GSTT1- del, respectively, in the GSTM1 and GSTT1 genes predict the development of occupational allergic dermatoses in at least 5 years of work in contact with irritating and sensitizing factors.

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