RU2467330C1 - Method for prediction of risk of developing early occupational allergic dermatoses - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, and concerns a method for prediction of a risk of developing early occupational allergic dermatoses. The method involves venous blood sampling, genetic material recovery to be examined for polymorphy version A4889G of cytochrome P-450 1A1 by polymerase chain reaction followed by real-time high throughput sequencing. Conducting PCR requires specific primers C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T and C1A1-R Cag gAT AgC Cag gAA gAg AAA to be used; while high throughput sequencing involves specific primer C1A1-S ACC TCC Cag Cgg gCA A. If the derived pyrogram in position 4889 shows no adenine for guanine substitution and body exposure to an occupational hazard, the risk of developing early occupational allergic dermatoses less than 40% is predicted. If observing heterozygous version Cyp1A1*2C caused by adenine for guanine substitution in a nucleotide sequence in position 4889, and body exposure to said occupational hazard, the risk of 40%÷80% of developing early occupational allergic dermatoses is predicted.

EFFECT: method provides higher reproducibility and rate of the prediction analysis of the risk of occupational allergic dermatoses in case of body exposure to irritant and sensitising hazards.

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Description

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

A known method for predicting the risk of developing professional allergic dermatoses, 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 polymorphism variant of A4889G cytochrome P-450 1A1 (see N. Lazarashvili A.A. , The role of the oxidant-antioxidant system and genetic biochemical polymorphism in the pathogenesis of occupational allergic dermatoses. the scientific degree of a candidate of medical sciences, M., 2006, p. 48-87).

However, this method has a low speed of examination of patients due to the complexity and complexity of the used laboratory technologies, which are harmful to the medical staff conducting them. This makes it difficult to predict the risk of developing professional allergic dermatoses, for example, during medical examinations or periodic medical examinations of a large number of workers 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 risk of early development (up to 5 years of work with harmful production factors) of professional allergic dermatoses 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 increase the reproducibility and speed of predicting the risk of occupational allergic dermatoses when exposed to irritating and sensitizing factors on the body.

This task is achieved by the fact that in the known method for predicting the risk of early development of occupational allergic dermatoses, 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 polymorphism variant A4889G cytochrome P-450 1A1 when carrying out a polymerase chain reaction using specific primers

C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T

C1A1-R Cag gAT AgC Cag gAA gAg AAA,

and after carrying out the polymerase chain reaction, a real-time pyrosequencing reaction is carried out using a specific primer C1A1-S ACC TCC Cag Cgg gCA A and the nucleotide sequence is detected using a chemiluminescent signal, then the obtained nucleotide sequence at position 4889 is compared with reference sequences, if any on the resulting pyrogram, the absence of the replacement of adenine with guanine and when the organism is exposed to a harmful production factor irritating and sensitizing with a lysing effect, the risk of early development of occupational allergic dermatoses is predicted to be less than 40%, if a heterozygous variant of Sur1A1 * 2C is detected on the resulting pyrogram due to the replacement of adenine by guanine in the nucleotide sequence at position 4889, and when exposed to an harmful production factor of irritating and sensitizing effect, they predict 40 -80% risk of early development of occupational allergic dermatoses.

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 reproducibility and speed of predicting the risk of early development of professional allergic dermatoses, which, in turn, allows to increase the reliability of predicting the risk of early development of professional allergic dermatoses when exposed to harmful production factors of irritating and sensitizing effects, for example, during medical examinations or periodic medical examinations, pain th number of working in harmful and / or dangerous conditions for short (short) periods of time.

The proposed method is based on the study of a set of diagnostic tests.

Table 1 presents the sequence of primers used (see the end of the description).

The proposed method is illustrated in the drawing.

Figure 1 shows the principle of operation of the device for pyrosequencing. Figure 2 shows a schematic diagram of the proposed method for identifying a heterozygous variant of Sur1A1 * 2C in the nucleotide sequence. Figure 3 shows the light signal recorded during the enzyme-mediated conversion of the released pyrophosphate. Figure 4 shows a pyrogram with a variant of the homozygous genotype according to the determined allelic variant, which does not have the replacement of adenine with guanine at position 4889 (A / A) in the nucleotide sequence - corresponds to the norm (example 1). Figure 5 shows a pyrogram with a variant of the heterozygous genotype for a defined allelic variant having substitutions of adenine with guanine at position 4889 (A / G) (example 2).

The proposed method is as follows.

The patient performs a venous blood sampling. Blood is examined in the claimed way: from 100 μl of whole blood, the DNA sorbent is isolated by 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.

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

The volume of the polymer chain reaction is 25 μl, using reagents manufactured by the Central Research Institute of Epidemiology: “PCR-mixture-2-blue”, a mixture of desoxynucleotide triphosphates “dNTPs (T)”, “Wax for PCR”, “Mineral oil for PCR”. For amplification of the studied genetic locus, specific oligonucleotide primers C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T and C1A1-R Cag gAT AgC Cag gAA gAg AAA are used (the sequence of primers used is presented in table 1). The number of primers C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T and C1A1-R Cag gAT AgC Cag gAA gAg AAA in the polymerase chain reaction of 10 pmol.

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), the tubes are placed in the cells of the amplifier and kept at a temperature of 95 ° C for 5 minutes. Further, the tubes withstand 45 cycles: at 95 ° C - 10 s, at 65 ° C - 10 s, at 72 ° C - 10 s, and at the end at 72 ° C - 2 min.

When using amplifiers “Gradient Palm Cycler” (“MAXYGEN”), “GeneAmp PCR System 2700”, “PalmCycler”, when the temperature reaches 95 ° C (pause mode), put tubes in the cells of the amplifier and continue the program at a temperature of 95 ° C - 5 min After 45 cycles 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, samples are prepared for sequencing. After amplification, the PCR fragment is coupled to streptavidin coated Sepharose particles due to the formation of a biotin-streptavidin complex. Then it is purified from the free components of the reaction mixture using a series of washes and the alkaline denaturation stage is carried out. As a result, the amplified single-chain PCR fragment remains, which is used as a matrix in the sequencing synthesis reaction.

The sequencing primer C1A1-S ACC TCC Cag Cgg gCA A at a concentration of 0.3 pM diluted in an annealing buffer is hybridized to a single-chain PCR amplicon acting as a template.

After the polymerase chain reaction, a real-time pyrosequencing reaction is carried out using a specific primer C1A1-S ACC TCC Cag Cgg gCA A and nucleotide sequence detection using a chemiluminescent signal.

Using a pyrosequencer device, for example, PyroMark Q96 MD or PyroMark Q24 (Germany), pyrosequencing is performed (Fig. 1).

At this stage, during sequencing of the chain complementary to the matrix fragment, the released pyrophosphate undergoes a series of enzymatic transformations, as a result of which a chemiluminescent signal is recorded (Fig. 3). The immobilized DNA fragment under study was incubated in the presence of four enzymes (sulfurylase, luciferase, apyrase, Taq polymerase) and substrates (adenosine 5-phosphosulfate (APS) and luciferin) to convert the released pyrophosphate to a chemiluminescent signal.

The pyrosequencing reaction includes the following steps:

- Add one of four deoxynucleotide-3-phosphates (dNTP) to the reaction mixture. In the event that the added nucleotide is complementary to the DNA template strand, insertion into the growing strand catalyzed by the DNA polymerase enzyme takes place. Each act of incorporation of a nucleotide into a DNA strand is accompanied by the release of pyrophosphate (PPi) in an equimolar amount.

- The enzyme sulfurylase catalyzes the process of the formation of the ATP-adenosine 3 phosphate molecule from the released pyrophosphate and adenosine 5-phosphosulfate (APS) present in the reaction mixture.

- The created molecule ATP-adenosine 3 phosphate (ATP) starts the process of converting luciferin to oxyluciferin catalyzed by the enzyme luciferase, resulting in a chemiluminescent signal that is proportional to the amount of ATP.

- The chemiluminescent signal is recorded by a CCD camera, it is displayed as a peak on the pyrogram. Throughout the reaction, the apyrase enzyme degrades non-integrating nucleotides and excess ATP so that a new nucleotide is added to the DNA chain only with complete degradation of non-integrating deoxynucleotide-3-phosphates. The sequential addition of dNTP ensures the appearance of peaks in the pyrogram constituting the nucleotide sequence of the studied genetic locus (figure 3).

Next, a comparison is made of the obtained nucleotide sequence with reference sequences and the resulting pyrogram determines which variant of the mutation takes place in the nucleotide sequence.

If there is a lack of replacement of adenine with guanine on the resulting pyrogram, and when the harmful production factor of irritating and sensitizing effects is exposed to the body, the risk of early development of occupational allergic dermatoses is predicted to be less than 40%, if the heterozygous Sur1A1 * 2C variant due to the replacement of adenine with guanine in guanine is detected the nucleotide sequence at position 4889, and when exposed to an organism of a harmful production factor, it is irritating and sensitizing th action predict a 40-80% risk of early development of occupational allergic dermatoses.

Example 1. Patient M., 53 years old

When analyzing the patient’s medical history and professional route, it was revealed that patient M. worked from 1992 to the present time as a polisher of optical parts in contact with rosin. Considers herself a patient with occupational allergic dermatosis since 2005, when eruptions on the palmar surfaces, spreading to the back surfaces and accompanied by itching, first appeared. In 2006, rashes appeared on the skin of the lower extremities. The patient consulted a dermatologist at the place of residence, but received treatment with a temporary effect. On weekends, during the holidays notes improvement.

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

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.

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 a tube for polymerase chain reaction. The volume of the polymeric chain reaction is 25 μl; reagents manufactured by the Central Research Institute of Epidemiology: “PCR-mixture-2-blue”, a mixture of desoxynucleotide triphosphates “dNTPs (T)”, “Wax for PCR”, “Mineral oil for PCR” were used. For amplification of the studied genetic locus, specific oligonucleotide primers C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T and C1A1-R Cag gAT AgC Cag gAA gAg AAA were used, one of which is labeled with biotin. The number of primers C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T and C1A1 - R Cag gAT AgC Cag gAA gAg AAA in the polymerase chain reaction - 10 pmol

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 ° C - 10 s, at 65 ° C - 10 s, at 72 ° C - 10 s, and at the end at 72 ° C - 2 min.

After amplification, samples were prepared for sequencing. For pyrosequencing, we used instruments and a sample preparation station from Qiagen, Germany.

Next, the sequencing primer was annealed. The obtained samples were subjected to pyrosequencing on a PyroMark Q96 MD instrument (Germany).

When analyzing the results, a homozygous variant of A4889G was identified that does not have adenine to guanine substitution at position 4889 — a variant of the genotype corresponding to the normal activity of cytochrome P-450 1A1.

Conclusion: Patient M. revealed a homozygous variant of Sur1A1 * 2C polymorphism A4889G, which does not have the replacement of adenine with guanine, indicating normal enzyme activity. There is no early development of occupational allergic dermatosis with exposure to harmful production factors of an irritating and sensitizing effect; the disease developed 13 years after the start of work with irritating and sensitizing factors.

Example 2. Patient C., 58 years old

When analyzing the patient’s history and professional route, it was revealed that patient V. worked for 21 years at the AZLK Moscow plant as a fitter, in contact with allergen metals: chromium, nickel, cobalt and gasoline. A year after the start of work, the diagnosis was made: Occupational eczema of the upper extremities. When examined at the Research Institute of MT RAMS, the diagnosis was confirmed and specified: Occupational eczema of the upper limbs.

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.

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.

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. The volume of the polymeric chain reaction is 25 μl; reagents manufactured by the Central Research Institute of Epidemiology: “PCR-mixture-2-blue”, a mixture of desoxynucleotide triphosphates “dNTPs (T)”, “Wax for PCR”, “Mineral oil for PCR” were used. For amplification of the studied genetic locus, the specific oligonucleotide primers “C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T” and “C1A1-R Cag gAT AgC Cag gAA gAg AAA” were used, one of which is labeled with biotin. The number of primers "C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T" and "C1A1-R Cag gAT AgC Cag gAA gAg AAA" in the polymerase chain reaction - 10 pmol.

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. The tubes were then kept for 45 cycles: at 95 ° C for 10 s, at 65 ° C for 10 s, at 72 ° C for 10 s, and at the end at 72 ° C for 2 minutes.

After amplification, samples were prepared for sequencing. For pyrosequencing, we used instruments and a sample preparation station from Qiagen, Germany.

The sequencing primer was annealed, then the samples were pyrosequenced using a PyroMark Q96 MD instrument.

An analysis of the results revealed a heterozygous variant of Sur1A1 * 2C polymorphism A4889G, which has the replacement of adenine with guanine.

Conclusion: in patient B., a heterozygous variant of Sur1A1 * 2C polymorphism A4889G was detected, which has the replacement of adenine with guanine, indicating an increased activity of the enzyme. Therefore, there is an increased formation of intermediate toxic metabolites. This was the reason for the early development of professional allergic dermatosis a year after the start of work with harmful production factors of irritating and sensitizing effects.

The proposed method for predicting the risk of early development of professional allergic dermatoses allows to increase the accuracy of the results by minimizing errors in interpreting the results by eliminating the "human factor" and the analysis speed by 25% compared to the prototype through the use of pyrosequencing, which allows a unit of time to conduct a larger number of tests, for example, during medical examinations or periodic medical examinations ters a large number of working in harmful and / or dangerous conditions, in short (short) periods of time.

The proposed method for predicting the risk of early development of professional allergic dermatoses can be applied in clinical, biochemical, molecular genetic laboratories equipped with equipment manufactured by domestic or foreign industry.

Claims (1)

A method for predicting the risk of early development of occupational allergic dermatoses, 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 polymorphism variant A4889G of cytochrome P-450 1A1, characterized in that during polymerase chain reactions use specific primers
C1A1-F Biotin - ggT gTT Aag TgA gAA ggT gAT T
C1A1-R Cag gAT AgC Cag gAA gAg AAA,
and after the polymerase chain reaction, a real-time pyrosequencing reaction is carried out using a specific primer C1A1-S ACC TCC Cag Cgg gCA A and the nucleotide sequence is detected using a chemiluminescent signal, then the obtained nucleotide sequence at position 4889 is compared with reference sequences, if any on the resulting pyrogram, the absence of the replacement of adenine with guanine and when the organism is exposed to a harmful production factor irritating and of a sibilizing effect, the risk of early development of occupational allergic dermatoses is predicted to be less than 40%, if a heterozygous variant of Sur1A1 * 2C is detected on the resulting pyrogram due to substitution of adenine for guanine in the nucleotide sequence at position 4889, and 40 is predicted when the harmful production factor is irritating and sensitizing, ÷ 80% risk of early development of occupational allergic dermatoses.

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