RU2373862C1 - Method of determining person's predisposaition to development of aggressive non-hodgkin's lymphomas - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, in particular to medical genetics, oncology and oncohematology. Method is technically simpler and less consumptive in terms of time and means. Genotype of gene XRCC1 399-th codon is studied. If in examined person genotype Gln/Gln is detected, conclusion about their predisposition to development of aggressive non-Hodgkin's lymphomas, in particular, of diffuse B- large cell lymphoma is made, if in examined person genotype Arg/Arg is detected, conclusion about lower in comparison with population risk of development of aggressive versions of NHL in said examined patient is made.

EFFECT: method is technically simple and less consumptive in terms of time and means, and provides possibility to determine person's predisposition to development of aggressive NHL in the cases when other methods do not allow to realise said determination.

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Description

The invention relates to medicine, more specifically to medical genetics, oncology and oncohematology.

The authors did not find in the prior art information about methods for determining a person’s predisposition to develop precisely aggressive variants of non-Hodgkin’s lymphomas, therefore, information relating to methods for determining a person’s predisposition to develop lymphomas was analyzed.

Several genetic mutations have been identified that can determine a person’s predisposition to the development of a number of solid neoplasms and contribute to the development of lymphomas: MTS, MTS2, ATM.

The invention is known "MTS gene, mutations of this gene and methods for diagnosing malignant tumors using the MTS gene sequence" (RF patent No. 2164419, prototype). According to this method, the detection in a germline of a human cell line of a mutation in the gene of a multifunctional tumor suppressor (MTS) indicates a person’s predisposition to such forms of cancer as melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin’s lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiosarcoma, squamous cell carcinoma, chronic lymphoid lymphocytic leukemia (CLL), as well as tumors of the pancreas, breast, brain, prostate, bladder, thyroid glands, ovaries, uterus, testes, kidneys, stomach, colon and rectum.

Mutation events at the MTS locus can include deletions, insertions, and point mutations in both coding and non-coding sequences. Therefore, when diagnosing a patient's predisposition to these types of tumor diseases, all loci of a given gene should be investigated and compared with similar wild-type loci of this gene.

Implementation of the prototype method requires expensive, high-tech techniques (single-chain conformational analysis ("SSCA"); denaturing gradient gel electrophoresis ("DGGE"); RNase protection test; allele-specific oligonucleotide ("ASOs") method; use of proteins that recognize nucleotide mismatches, such as E. coli mutS protein; cloning, sequencing, etc.). Since the identification of mutations in the MTS gene requires a comparison with the “healthy control” (wild type of the gene), it is necessary to develop or acquire the standard of the wild type of the gene.

In addition, genetic mutations are rare. People who do not have a mutation in the MTS gene also have oncological diseases, in particular aggressive variants of non-Hodgkin lymphomas. Other methods can help to detect a predisposition to them, in particular, the method we offer.

The present invention is based on the study of the relationship between the presence in humans of Arg399Gln polymorphism of the XRCC1 gene (X-ray cross-complementing group I) and the occurrence of aggressive non-Hodgkin lymphomas. No intrinsic enzymatic activity was found in the XRCC1 protein, however, it forms complexes of enzymes involved in DNA repair and is part of a family of proteins involved in controlling the cell cycle and genome stability.

Three oligonucleotide polymorphisms were detected in the protein coding regions of the XRCC1 gene, one of which is located in codon 399 and consists in replacing arginine with glycine (Arg> Gln).

The codon 399 is located within the 10 exon of the XRCC1 gene, and with Arg> Gln polymorphism, replacement occurs in the region that encodes the biologically important region of BRCT-I. Polymorphism is recognized by the absence of restriction by endonuclease Msp I in the minor gene allele. Replacing arginine with glycine in the primary structure leads to conformational changes in the protein and a decrease in its activity.

Regarding the Arg399Gln polymorphism of the XRCC1 gene, the following information is available in the prior art:

- the presence of the 399C1n allele of the XRCC1 gene in humans is a factor contributing to the development of non-small cell lung cancer, especially when exposed to carcinogenic substances of tobacco (The XRCC1 codon 399 Gln allele is associated with adenine to guanine p53 mutations in non-small cell lung cancer. // Casse C., Hu YC, Ahrendt SA // Mutat Res., - 2003, Jul 25. - 528 (1-2). - P.19-27);

- Gln homozygosity shows a significant association with lung adenocarcinoma in smokers (The XRCC1 399 glutamine allele is a risk factor for adenocarcinoma of the lung. / Divine KK, Gilliland FD, Crowell RE, Stidley CA, Bocklage TJ, Cook DL, Belinsky SA / / Mutat Res., - 2001, Jan 5. - 461 (4). - P.273-8);

- a positive relationship was found between Arg / Gln and the Gln / Gln genotype at 399 codon XRCC1 and breast cancer in black American women. In white patients, it is absent (Polymorphisms in the DNA repair gene XRCC1 and breast cancer. / Duell EJ, Millikan RC, Pittman GS, Winkel S., Lunn RM, Tse CK, Eaton A., Mohrenweiser HW, Newman B., Bell DA // Cancer Epidemiol Biomarkers Prev. - 2001, Mar. - 10 (3). - P.217-22);

- studies by Hu et. al. The Gln allele has been found to increase sensitivity to radiation, alkylating agents and oxygen free radicals, factors predisposing to the development of breast cancer (Genetic regulation of ionizing radiation sensitivity and breast cancer risk. / Hu JJ, Smith TR, Miller MS, Lohman K ., Case LD // Environ Mol Mutagen. - 2002. - 39 (2-3). - P.208-15);

- studies by Abdel-Rahman et. al. in Egypt, it was shown that the Gln allele, both in the homozygous and heterozygous state, increases the risk of colorectal cancer (Inheritance of the 194 Trp and the 399 Gln variant alleles of the DNA repair gene XRCC1 are associated with increased risk of early-onset colorectal carcinoma in Egypt. / Abdel-Rahman SZ, Soliman AS, Bondy ML, Omar S., El-Badawy SA, Khaled HM, Seifeldin IA, Levin B. // Cancer Lett. - 2000, Oct. - 159 (1) .- P.79-86). A similar study was performed on a group of patients with colorectal cancer in Korea. The analysis showed that the amount of alcohol in terms of alcohol ≥80 g / week is a significant risk factor for colorectal cancer in combination with the 399Gln genotype (Polymorphisms of XRCC1 gene, alcohol consumption and colorectal cancer. / Hong YC, Lee KH, Kim WC, Choi SK , Woo ZH, Shin SK, Kim H. // Int J Cancer. - 2005, Sep. - 116 (3). - P.428-32);

- Chinese scientists Yu et. al. revealed that the frequency of the XRCC1 399 Gln / Gln genotype in squamous cell carcinoma of the esophagus was significantly higher than in the group of healthy individuals. Smoking significantly increased the risk of people with the genotype XRCC1 399 Gln / Gln getting sick with squamous cell cancer of the esophagus (DNA repair gene XRCC1 polymorphisms, smoking, and esophageal cancer risk. / Yu HP, Zhang XY, Wang XL, Shi LY, Li YY, Li F., Su YH, Wang YJ, Lu B., Sun X., Lu WH, Xu SQ // Cancer Detect Prev. - 2004 .-- 28 (3). - P.194-9);

- Sturgis et. al. revealed a relationship between the 399Gln allele and the risk of developing squamous cell carcinoma of the head and neck. / Sturgis EM, Castillo EJ, Li L., Zheng R., Eicher SA, Clayman GL, Strom SS, Spitz MR, Wei Q. // Carcinogenesis. - 1999, Nov. - 20 (11). - P.2125-9).

Thus, there is information in the prior art that shows that the presence of the 399Cln allele of the XRCC1 gene in a heterozygous or homozygous state in humans is a risk factor for the development of certain types of tumors.

At the same time, there are a number of studies that show that:

- The 399Gln allele of the XRCC1 gene may not have a pro-oncogenic, but protective effect against certain types of tumors. For example, in a study by Skjelbred et. al., a decrease in the risk of developing colorectal adenocarcinomas in carriers of the Gln allele 399 of the codon of the XRCC1 gene (Polymorphisms of the XRCC1, XRCC3 and XPD genes and risk of colorectal adenoma and carcinoma, in a Norwegian cohort: a case control study. / Skjelbred CF, Saebo M., Wallin H., Nexo BA, Hagen PC, Lothe IM, Aase S., Johnson E., Hansteen IL, Vogel U., Kure EH // BMC Cancer. - 2006, Mar. - 6. - P. 67). Two studies noted the protective effect of the Gln / Gln genotype in smokers (DNA repair gene XRCC1 polymorphisms, smoking, and bladder cancer risk. / Stem MC, Umbach DM, van Gils CH, Lunn RM, Taylor JA // Cancer Epidemiol Biomarkers Prev. - 2001, Feb. - 10 (2) .- P.125-31; Polymorphisms of the DNA repair genes XRCC1, XRCC3, XPD, interaction with environmental exposures, and bladder cancer risk in a case-control study in northern Italy. / Shen M., Hung RJ, Brennan P., Malaveille C., Donato F., Placidi D., Carta A., Hautefeuille A., Boffetta P., Porru S. // Cancer Epidemiol Biomarkers Prev. - 2003, Nov. - 12 (11 Pt 1). - P.1234-40);

- The 399Gln allele of the XRCC1 gene may not affect the risk of a tumor. No association of this polymorphism with oral cancer was found (Polymorphism in DNA repair genes and oral squamous cell carcinoma in Thailand. / Kietthubthew S., Sriplung H., Au WW, Ishida T. // Int J Hyg Environ Health. - 2006, Jan . - 209 (1). - P.21-9). In a study by Zhang et. al. it was shown that the Arg399Gln polymorphism of the XRCC1 gene does not have an independent modifying effect on the risk of developing stomach cancer (Correlation of genetic polymorphisms in DNA repair genes ADPRT and XRCC1 to risk of gastric cancer. / Zhang Z., Miao XP, Tan W., Guo YL, Zhang XM, Lin DX // Ai Zheng. - 2006, Jan. - 25 (1). - P.7-10). In one study of bladder cancer, no association of this polymorphism with bladder cancer was found (DNA repair gene polymorphisms, bulky DNA adducts in white blood cells and bladder cancer in a case-control study. / Matullo G., Guarrera S., Carturan S., Peluso M., Malaveille C., Davico L., Piazza A., Vineis P. // Int J Cancer. - 2001, May. - 92 (4). - P.562-7);

- the risk of developing certain tumors is associated with the normal (wild) allele of the XRCC1 gene. Lee G. M. et al. showed a statistically significant increase in the risk of esophageal cancer in Arg / Arg of alcohol abusers compared to Arg / Gln and Gln / Gln patients (Genetic polymorphisms of XRCC1 and risk of the esophageal cancer. / Lee JM, Lee YC, Yang SY, Yang PW, Luh SP, Lee CJ, Chen CJ, Wu MT // Int J Cancer. - 2001, Jul. - 95 (4). - P.240-6).

Thus, it is known from the prior art that the Arg399Gln polymorphism of the XRCC1 gene predisposes its carrier to certain oncological diseases, protects against other oncological diseases and is indifferent to the third types of oncological diseases.

Data on the role of the Arg399Gln polymorphism of the XRCC1 gene in lymphomas, in particular aggressive non-Hodgkin lymphomas, are not available in the prior art.

Disclosure of invention

One of the most common forms of hemoblastosis is non-Hodgkin’s lymphoma (NHL), a tumor of the blood system characterized by neoplastic proliferation of lymphoid cells.

The essence of the proposed method for determining a person’s predisposition to the development of aggressive non-Hodgkin lymphomas is as follows. The genotype of the 399th codon of the XRCC1 gene is examined. If a Gln / Gln genotype is found in a person being examined, a conclusion is made about the predisposition of the subject to the development of aggressive non-Hodgkin lymphomas, in particular to the development of diffuse B-large cell lymphoma, while when the Arg / Arg genotype is found in the examined person, they conclude that the development risk is reduced in relation to the population aggressive NHL options in this subject. Upon detection of the Arg / Gln genotype, it is concluded that the patient’s risk of developing aggressive lymphomas corresponds to the population one.

Comparison of the proposed method with the prototype shows the following:

- while in the prototype all loci of the MTS gene should be investigated and compared with similar wild-type loci of this gene, which requires the development or acquisition of a wild-type standard of the MTS gene, in the proposed method it is necessary to analyze the only change, namely, the replacement of Arg by Gln in the 399th codon of the XRCC1 gene, which does not require comparison with the wild type of the XRCC1 gene, and therefore, the development or acquisition of a wild-type gene standard;

- the proposed method uses PCR-RFLP analysis (PCR - polymerase chain reaction; RFLP - polymorphism of the lengths of restriction fragments), which is technically much simpler and less costly in time and money in comparison with the prototype;

- the proposed method can reveal a person’s predisposition to the development of aggressive non-Hodgkin lymphomas in those cases when the prototype does not work, i.e. in humans, there are no mutations in the MTS gene.

When implementing the proposed method at the first stage in the PCR reaction, a fragment of the XRCC1 gene of the examined patient is obtained with a length of 615 nucleotides (bp). This fragment may contain Gln (mutant allele) or Arg (wild allele) in the 399th codon. Then, the amplicon obtained in the first stage is restricted using the restriction endonuclease MspI, which recognizes only the wild variant of the allele (containing Arg in the 399th codon) and cuts it into two fragments 376 bp long. and 239 bp Next, electrophoresis of hydrolysis products is carried out. Three variants of electrophoresis patterns can be obtained. The presence on the electrophoresis of one band of 615 bp in length indicates the absence of amplicon hydrolysis, and therefore, the homozygous minor genotype of the patient Gln / Gln. The presence on the electrophoresis of two bands with a length of 376 bp and 239 bp indicates the hydrolysis of the entire amplicon, and therefore, the homozygous wild genotype of the patient Arg / Arg. The presence on electrophoresis of three bands with a length of 615 bp, 376 bp and 239 bp indicates partial hydrolysis of the amplicon, and therefore, the heterozygous genotype of the patient Arg / Gln.

List of figures illustrative material

Figure 1. Electrophoretic analysis of the results of PCR of a DNA fragment containing the 399th codon of the XRCC1 gene.

Figure 2. Results of electrophoretic analysis of the products of MspI hydrolysis of amplicons obtained in the previous step.

Figure 3. Frequency distribution of alleles and genotypes of Arg399Gln of the XRCC1 gene in patients with NHL.

Figure 4. Frequency distribution of alleles and genotypes of Arg399Gln of the XRCC1 gene in patients with aggressive and indolent lymphomas.

Figure 5. Frequency distribution of the alleles and genotypes of Arg399Gln of the XRCC1 gene in patients with DVCL and DVML.

The rationale for the method and its implementation

Clinical characteristics of patients

The examined group consisted of 98 patients with NHL from the Novosibirsk City Hematology Center. The patient genotype was examined in 399 codon of the XRCC1 gene.

The average age of patients was 52.2 ± 15.5 years (15-85 years). Patients were distributed by sex as follows: men - 43 (44%), women - 55 (56%).

In the examined group of patients with NHL, the vast majority of patients had advanced stages of the disease: 65 people (66%) - stage IV, 13 people (13%) - stage III, 17 people (18%) - stage II and 3 people (3%) - Stage I lymphoma.

In the examined group, 42 patients (43%) had lymphomas of a high degree of malignancy (SCD, aggressive), which included diffuse large-cell, burkitt-like, pleomorphic, lymphoblastic, centroblast, immunoblastic, plasmoblast, anaplastic, follicular 3rd cytological types, mantle cell. 56 people (57%) had lymphomas of a low degree of malignancy (NHA, indolent), among which were diffuse small cell, prolymphocytic, centrocytic, lymphoplasmacytic, follicular 1st cytological type, mushroom mycosis, marginal cell, MALT lymphoma.

All patients were examined before the start of active chemotherapy.

The control was the data on the prevalence of Arg399Gln polymorphism of the XRCC1 gene among a healthy population (n = 180) of Novosibirsk and the Novosibirsk Region (Belyavskaya VA, Smetannikova NA, Maksimov VN et al. Polymorphism in XRCC1 DNA basic exision repair gene and some functionale related genes: Assosiation with longelity and cancer risk among the population of the Novosibirsk region. // Materials of the international conference on chemical biology, June 2–7, Novosibirsk, 2005, p. 49).

The diagnosis of non-Hodgkin lymphoma was established on the basis of a histological examination of lymph node biopsies with immunohistochemical verification of a tumor variant using a wide panel of monoclonal antibodies to hematopoietic cell differentiation clusters.

Isolation of DNA from leukocytes of the whole venous blood of patients with lymphomas

Blood for the study of the patient's genotype was taken from a vein in a volume of 5 ml. The resulting material was stored until DNA isolation at a temperature of -20 ° C.

DNA extraction procedure

1st day. Before DNA extraction, the blood sample was thawed, homogenized, and 5 ml of freshly prepared buffer A was added to it (it contains: 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl _2. ) After thorough shaking and centrifugation of the sample in for 5 minutes at 4 thousand revolutions the supernatant was drained.

5 ml of buffer A was again added to the pellet. The contents of the tube were shaken, centrifuged and the supernatant was drained. This sequence of actions was repeated 2-3 more times.

1.9 ml of buffer B was added to the washed precipitate (it contains 10 mM EDTA, 100 mM NaCl, 50 mM Tris-HCl, pH 8.0). After thoroughly shaking the pellet with buffer, 20% SDS (sodium dodecyl sulfate) was added in an amount of 50 μl and proteinase K (Medigen, Novosibirsk) at a dilution of 20 mg / ml in an amount of 160 μl. The contents of the tube were mixed and left for protein treatment with proteinase K overnight in an incubator at 37 ° C.

2nd day. 400 μl of 5 M NaCl, 3 ml of phenol saturated with 0.1 M Tris-HCl buffer, pH 8.0, containing 0.2% 2-mercaptoethanol was added to the sample tube in which the protein was treated with proteinase K. The contents of the tube were shaken and centrifuged at 4 thousand revolutions for 5 minutes. After separation of the phases into aqueous and phenolic, the upper (aqueous) phase was carefully pipetted into a clean tube, 3 ml of a mixture of phenol with chloroform in a 1: 1 ratio (chloroform contained isoamyl alcohol in a 24: 1 ratio) was added, shaken and centrifuged again.

After separating the phases into aqueous and chloroform-phenolic, the upper (aqueous) phase was carefully pipetted into a clean tube, 3 ml of chloroform with isoamyl alcohol was added to it in a ratio of 24: 1, the contents of the tube were shaken and centrifuged again.

After separation of the phases into aqueous and chloroform, the upper (aqueous) phase was carefully pipetted into a clean tube, 3 ml of isopropyl alcohol was added to it, and the contents of the tube were gently mixed until a bundle of DNA strands appeared.

Test tubes with a DNA sample in isopropyl alcohol were closed and left overnight at -20 ° C to sediment small fragments of DNA.

3 day. Tubes with DNA and isopropyl alcohol were centrifuged at 4 thousand revolutions for 5 minutes to precipitate DNA, the alcohol was carefully drained.

3 ml of 75% ethanol was carefully added to the precipitate, centrifuged at 5,000 rpm for 10 minutes, the alcohol was carefully drained.

3 ml of 96% ethyl alcohol was added to the precipitate and centrifuged again, the alcohol was carefully drained, the residues were removed with filter paper and the DNA was dried at 37 ° C until the alcohol was completely removed.

The precipitate was dissolved in bi-distilled water to a DNA concentration of about 1 mg / ml and stored until PCR at -20 ° C.

Genotyping of Arg399Gln polymorphism of XRCC1 gene

The analysis of the patient's genotype by Arg399Gln polymorphism of the XRCC1 gene included several steps described below.

1) Statement (PCR). The composition of the reaction mixture per 1 DNA sample for PCR is shown in table 1. The note to table 1 indicates the primers used. These primers provide amplification of the XRCC1 gene fragment in the 19q13.2 chromosome from 23614 bp up to 24228 bp 615 bp containing 399 codon.

The program of temperature-time cycles of PCR (automatic thermal cycler "Eppendorf"):

- preliminary denaturation - 94 ° C, 3 min;

Table 1 The composition of the reaction mixture for 1 sample for PCR Reagent Volume μl Buffer × 10 (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 25 mM MgCl ₂ ) 1,5 A mixture of deoxynucleotide triphosphates (dNTPS) (each concentration in a mixture of 2 mm) 1,5 Direct primer (F, I) * (diluted to 0.25 μM) 1,5 Reverse primer (R, II) ** (diluted to 0.25 μM) 1,5 Tag-polymerase (in reaction 1 ЕА) “Sibenzym”, Novosibirsk 1,0 Water 7.0 1 mg / ml diluted DNA sample 1,0 Note: * Direct primer: 5'-TTGTGCTTTCTCTGTGTCCA. ** Reverse Primer: 5'-TCCTCCAGCCTTTTCTGATA

- then 30 cycles: denaturation - 94 ° C, 30 sec; primer annealing - 55 ° С, 60 sec; elongation - 72 ° C, 60 sec;

- post-elongation - 72 ° C for 3 minutes

2) Electrophoretic analysis of PCR results in a 1.5% agarose gel. 5 μl of the amplification sample of several patients is applied to the gel. A molecular weight marker of 100 bp is used. in the amount of 0.8 μl. A marker is a mixture of DNA fragments of successively increasing length, differing from each other by 100 bp. As can be seen from figure 1, as a result of PCR, fragments of the XRCC1 gene of 615 bp were obtained.

3) Hydrolysis of the resulting amplicons using restriction endonuclease MspI. The composition of the reaction mixture per 1 DNA sample for hydrolysis is given in table.2. The hydrolysis was carried out for 3 hours at 37 ° C.

4) Electrophoretic analysis of the results of MspI hydrolysis in a 1.5% agarose gel. On the gel, 10 μl of the restriction restriction of each patient and a DNA marker of molecular weights of 100 bp are applied. (in an amount of 1.5 μl). Variants of the distribution of bands on the electrophoregram are shown in figure 2. The absence of hydrolysis (the presence of one band of 615 bp in lanes number 1) indicates a homozygous minor genotype (Gln / Gln) of these patients. Complete hydrolysis (the presence of two bands of 376 bp and 239 bp in lane number 3) indicates the homozygous wild genotype (Arg / Arg) of this patient. Partial hydrolysis (the presence of three bands of 615 bp, 376 bp, and 239 bp in lanes number 2) indicates heterozygosity (Arg / Gln genotype) of patient data on the Arg399Gln polymorphism of the XRCC1 gene.

Statistical Research Methods

Analysis of genetic polymorphisms began with the identification of the frequency of occurrence of each of the alleles and genotypes. For the studied polymorphism in the sample of population control and patients with lymphomas, the distribution of genotypes corresponded to that expected at Hardy-Weinberg equilibrium, which can be written in the form of the formula: p ² AA + 2pqAa + q ² aa = 1.

When comparing the frequencies of alleles and genotypes, as well as to compare the observed and expected frequencies, the standard Pearson χ ² criterion was used. If the absolute frequencies of more than 20% of the characteristics in the group did not exceed 5, the exact Fisher test was used.

To assess the relationship between certain genotypes and the risk of developing the disease, the odds ratio OR (odd ratio) was calculated. The odds ratio is indicated with a 95% confidence interval (CI). If the upper and lower values of CI were more than unity, then the risk of developing the disease was considered significantly higher in the group with the studied change in genotype compared with the population (Rebrova O.U. Statistical analysis of medical data. Application of the STATISTICA application package. - M., Media Sphere. - 2003. - 312 p.).

Research results

As mentioned above, the polymorphism of the XRCC1 DNA repair gene was studied in 98 patients with NHL and 180 healthy individuals in the population (control) sample of Novosibirsk and the Novosibirsk Region (Belyavskaya VA, Smetannikova NA, Maksimov VN et al. Polymorphism in XRCC1 DNA basic exision repair gene and some functionale related genes: Assosiation with longelity and cancer risk among the population of the Novosibirsk region. // Materials of the international conference on chemical biology, June 2–7, Novosibirsk, 2005, p. 49).

Of the 196 XRCC1 genes belonging to 98 examined patients with NHL, 100 genes (51%) carried the Gln allele. In the control sample of 360 genes (180 people), the Gln allele carried 144 genes (40%) (Fig. 3). The frequency of occurrence of the Gln allele in the group of patients with NHL is significantly higher than the frequency of its occurrence in the control group (p = 0.012).

The frequency of the Arg allele in the group of patients with NHL and in the control group was 49% (96/196) and 60% (216/360), respectively. The frequency of occurrence of the Arg allele in the group of patients with NHL is significantly lower than the frequency of its occurrence in the control group (p = 0.012) (Fig. 3).

The homozygous Gln / Gln genotype was found in 25% of patients with NHL (in 25 out of 98 people), in the control group it was found in 13% of the examined (in 24 out of 180 people), i.e. the frequency of occurrence of the homozygous Gln / Gln genotype in the group of patients with lymphomas is significantly (p = 0.016) higher than the frequency of its occurrence in the control group (Table 3, Fig. 3).

The homozygous Arg / Arg genotype was found in 22% of patients (22/98) and in 34% of healthy subjects (60/180). The frequency of occurrence of the homozygous Arg / Arg genotype in the group of patients with lymphomas is significantly (p = 0.037) lower than the frequency of its occurrence in the control group (Table 3, Fig. 3).

The Arg / Gln genotype was found in 53% of cases in the control group and in 52% in the group of patients with NHL (96/180 and 51/98, respectively).

To assess the relationship between genotypes and the risk of developing NHL, the odds ratio of OR with 95% CI was calculated (Table 3). According to the data obtained, the risk of developing NHL in individuals with a homozygous Gln / Gln genotype significantly exceeded the population 1.9 times (OR = 1.9). At the same time, the maximum risk of developing NHL in individuals with the Gln / Gln genotype exceeded the population by 3.6 times and a minimum of 1.04 times (95% CI [1.04; 3.6]). The Arg / Arg and Arg / Gln genotypes did not significantly change the risk of developing NHL.

Non-Hodgkin lymphomas are a heterogeneous group of diseases, therefore, a comparative analysis of the frequency of the XRCC1 gene polymorphism was carried out in groups of patients with indolent, aggressive variants of the disease and in the control sample.

table 2 The composition of the reaction mixture per 1 DNA sample for hydrolysis Reagent Volume μl one 2 3 MspI B * restriction endonuclease buffer 2.0 2.0 2.0 Restriction endonucleosis MspI ** (to reaction 2 EA) 0.1 0.1 0.1 Water 15.9 12.9 7.9 Amplicon 2.0 5,0 10.0 Note: * 10 mM Tris HCl pH 7.6 at 25 ° С, 10 mM MgCl, 1 mM DTT are included. ** "Sibenzyme", Novosibirsk.

Table 3 The relationship between the Arg399Gln genotype of the XRCC1 gene and the risk of developing NHL Genotype Arg399Gln XRCC1 Control (n * = 180)
one Group of patients with NHL (n * = 98)
2 OR
[95% CI] Abs. qty % Abs. qty % Arg / Arg 60 34 22 22
P _1-2 <0.05 0.58
[0.33; 1.02] Arg / gln 96 53 51 52 0.95
[0.58; 1.55] Gln / gln 24 13 25 26
P _1-2 <0.05 1.9
[1.04; 3.6] Note: * the letter n indicates the number of people in the group.

The incidence rate of the Gln allele in the group of patients with aggressive lymphomas (42 patients) was 55% (47 alleles out of 84), in the control sample - 40% (144/360), in the group of patients with indolent lymphomas (56 people) - 47% ( 53/112). The frequency of occurrence of the Arg allele in the group of patients with aggressive lymphomas was 45% (37/84), in the control sample - 60% (216/360), and in the group of patients with indolent lymphomas - 53% (59/112).

Differences in the frequencies of occurrence of minor (Gln) and wild (Arg) alleles of the XRCC1 gene in the group of patients with indolent lymphomas in comparison with the group of patients with aggressive lymphomas and also in comparison with the control group are not significant (p> 0.05). At the same time, the frequency of occurrence of the minor allele in the group of patients with aggressive lymphomas is significantly (p = 0.005) higher than the frequency of its occurrence in the control group. The frequency of occurrence of wild allele in the group of patients with aggressive lymphomas was significantly (p = 0.005) lower than the frequency of its occurrence in the control group (Fig. 4).

The incidence of homozygous minor genotype (Gln / Gln) in the group of patients with aggressive lymphomas was 28% (12/42), in the group of patients with indolent lymphomas - 21% (12/56), and in the control group - 13% (in 24 of 180) (table 4, figure 4).

The frequency of occurrence of the homozygous minor genotype was significantly higher in the group of patients with aggressive lymphomas compared with the frequency of its occurrence in the control group (p = 0.021), but did not differ significantly from the frequency of occurrence of this genotype in the group of patients with indolent lymphomas.

The incidence rate of the normal homozygous genotype (Arg / Arg) in the group of patients with aggressive lymphomas was 17% (7 out of 42), in the group of patients with indolent lymphomas - 27% (15/56), and in the control group - 34% (60/180 ) (Table 4).

The frequency of occurrence of the normal homozygous genotype was significantly lower in the group of patients with aggressive lymphomas compared with the frequency of its occurrence in the control group (p = 0.019), but did not differ significantly from the frequency of occurrence of this genotype in the group of patients with indolent lymphomas (Fig. 4).

The frequency of occurrence of the heterozygous Arg / Gln genotype was 55% (23/42) in the group of patients with aggressive lymphomas, 52% (29/56) in the group of patients with indolent lymphomas, and 53% (96/180) in the control group (Table 4).

Differences in the frequency of occurrence of heterozygous genotypes in groups of patients with aggressive and indolent lymphomas and in the control group are not significant (p> 0.05).

The OR odds ratio with a 95% confidence interval (CI) was calculated (Table 4). In individuals with the Gln / Gln genotype, the risk of developing aggressive NHL exceeded the population average by 2.3 times [1.04; 5.0]. In individuals with the Arg / Arg genotype, the risk of developing aggressive NHL was significantly lower than the average population. There was no association between the Arg / Gln genotype and the risk of developing aggressive lymphomas, as well as the risk of developing indolent lymphomas.

Thus, previously discovered differences in the distribution of genotypes and alleles of the 399th codon of the XRCC1 DNA repair gene between diseased lymphomas and population control were mainly due to patients with aggressive NHL.

Due to the fact that both aggressive and indolent NHLs include many morphological forms, the frequencies of alleles and genotypes of the studied polymorphism of the XRCC1 gene were analyzed for the most common variants of the disease: diffuse B-large cell lymphoma (DVCL, n = 14) and diffuse B-small cell lymphoma (DVMKL, n = 19).

The frequency of occurrence of the Gln allele of the XRCC1 gene in the group of patients with diffuse large B-cell lymphoma was 68% (19/28) and was significantly higher than in the control sample - 40% (144/360) (p = 0.004) and the group of patients with diffuse B-small cell lymphoma - 37% (14/38) (p = 0.012).

The frequency of occurrence of the Arg allele in patients with DVBCL was 32% (9/28) and was significantly lower (p = 0.004) than in the control sample, where it was 60% (216/360) and significantly lower (p = 0.012) than in patients with DVMCL, where it was equal to 63% (24/38) (figure 5).

The frequency of occurrence of the Gln / Gln genotype in the group of patients with dvccl was 43% (6/14), which significantly exceeded (3.5 times) the frequency of its occurrence in the control group (p = 0.008) (Table 5), as well as in the group DVMKL, where it was 10.5% (2/19) (p = 0.032).

The frequency of occurrence of the wild Arg / Arg genotype among patients with dvccl was 7% (1/14) and was significantly (p = 0.033) lower (6 times) than in the control sample, where it was 34% (60/180) (Table 5) and significantly (p = 0.049) lower than in patients with DVMLC, where it was 37% (7/19).

The frequency of occurrence of the heterozygous genotype in the group of patients with dvccl was 50% (7/14), in the group of patients with dvmcl - 52% (10/19), in the control group 53% (96/180), i.e. there were no significant differences in the frequency of the heterozygous genotype between the groups of patients with DVMKL, DVMKL and healthy people (p> 0.05) (Fig. 5)

The distribution of the Arg399Gln alleles and genotypes of the XRCCl gene in patients with DVMLC was generally consistent with the population.

Thus, when assessing the relationship between the Arg399Gln XRCC1 polymorphism genotypes and the risk of developing DECL, it was found that individuals with the Gln / Gln genotype were 4.2 times [1.4: 14.9] more likely to develop DECCL compared with the control group (Table. 5). The Arg / Arg and Arg / Gln genotypes did not significantly change the risk of developing DECLC in relation to the population.

Table 4 The relationship between the Arg399Gln genotype of the XRCC1 gene and the risk of developing aggressive and indolent lymphomas Genotype Arg399Gln XRCC1 Control (n = * 180)
one Patients with aggressive lymphomas (n * = 42)
2 Patients with indolent lymphomas (n * = 56)
3 OR _1-2
[95% CI] Abs. % Abs. % Abs. % Arg / Arg 60 34 7 17
P _1-2 <0.05 fifteen 27 0.4
[0.17; 0.95] Arg / gln 96 53 23 55 29th 52 1.06
[0.54; 2.08] Gln / gln 24 13 12 28
P _1-2 <0.05 12 21 2 3
[1.04; 5.0] Note: * the letter n indicates the number of people in the group.

Table 5
The relationship between the Arg399Gln genotype of the XRCC1 gene and the risk of developing dvccl Genotype Arg399Gln XRCC1 Control (n * = 180)
one Patients DVKKL (n * = 14)
2 OR
[95% CI] Abs. % Abs. % Arg / Arg 60 34 one 7
P _1-2 <0.05 0.15
[0.02; 1,2] Arg / gin 96 53 7 fifty 0.88
[0.29; 2.6] Gln / gln 24 13 6 43
P _1-2 <0.01 4.2
[1.4; 12.9] Note: * the letter n indicates the number of people in the group.

Claims (1)

A method for determining a person’s predisposition to the development of lymphomas, including analyzing the patient’s genotype, characterized in that the predisposition to the development of aggressive non-Hodgkin lymphomas is determined, the genotype of the 399th codon of the XRCC1 gene is examined and, when the examined genotype Gln / Gln is detected, it is concluded that it is predisposed to develop aggressive non-Hodgkin’s lymphomas, in particular, to the development of diffuse B-large cell lymphoma, whereas when Arg / Arg is detected in the examined genotype, they conclude that the population the risk of developing aggressive NHL variants in this subject.

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