RU2557977C1 - Method for prediction of risk of hyperplastic processes of endometrium - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: DNA is recovered from peripheral venous blood. Genetic polymorphisms of tumour necrosis factor α (-308 G/A TNFα), tumour necrosis factor 1 receptor (+36 A/G TNFR1), interferon-inducible T-cell chemoattractant (A/G I-TAC), interleukin 1A (-889 C/T IL-1A), lymphotoxin α (+250 A/G Ltα) are typed by polymerase chain reaction. A high risk of developing hyperplastic processes of endometrium is predicted if detecting a combination of alleles -308 G TNFα, +36 A TNFR1, A I-TAC, -889 T IL-1A and/or a combination of alleles +36 A TNFR1, A I-TAC, -889 T IL-1A and/or a combination of alleles -308 G TNFα,+250 G Ltα, -889 T IL-1A.

EFFECT: higher accuracy.

2 dwg, 1 tbl, 1 ex

Description

The invention relates to the field of medical diagnostics, namely to gynecology, and can be used to predict the risk of developing endometrial hyperplastic processes (GGE).

The endometrium is the mucous membrane lining the uterine cavity. When this shell begins to grow excessively in violation of hormonal processes in the body of a woman, they speak of endometrial hyperplasia. There are three types of endometrial hyperplastic processes:

- glandular cystic endometrial hyperplasia (proliferation of glandular tissue and the appearance of cysts in it);

- glandular and glandular-cystic polyps (endometrial polyps - benign tumor formations growing from the wall of the uterus into its lumen);

-atypical hyperplasia - perverse, uncharacteristic growth of endometrial tissue (adenomatosis, adenomatous hyperplasia) [Kipich N.V. The importance of molecular genetic and immune factors in the pathogenesis and management of patients with endometrial hyperplastic processes. // Abstract. Ph.D. - SPb., 2011. - 20 S.].

Hyperplastic processes of the endometrium are possible at any age, occur in almost every fourth woman with infertility and have a different degree of manifestation, but their frequency increases significantly by the period of perimenopause. The prevalence of endometrial hyperplasia is 10-30%, endometrial polyps - 0.5-35.7% [O. Lysenko Hyperplastic processes of the endometrium at different age periods study of the cytokine status and content of SFAS ligand // Obstetrics and Gynecology. - 2011. - No. 4.- S.12-15].

Hyperplastic processes of the endometrium as a possible basis for the formation of malignant tumors for many decades represent an important medical and social problem. The frequency of malignancy of endometrial hyperplastic processes varies widely (0.25-50%) and is determined by the morphological characteristics of the disease, the duration of its recurrence, and also the age of the patients. Simple endometrial hyperplasia without atypia passes into cancer in 1% of cases, the polypoid form without atypia is 3 times more likely.

The clinical picture of endometrial hyperplasia is characterized by anovulatory uterine bleeding, which occurs, as a rule, after a delay in menstruation. Bleeding is usually prolonged, with moderate hemorrhage or profuse, profuse. With hyperplastic processes of the endometrium (more often with endometrial polyps), intermenstrual bleeding sometimes appears [Maksimov S.Ya. Risk factors for the occurrence of malignant neoplasms of the organs of the reproductive system of women // Questions of Oncology.-2003. - T. 49, No. 3, - S. 496 - 501].

The final confirmation of the diagnosis of HPE is possible only after hysteroscopy with subsequent histological examination of the endometrium. Unfortunately, we have to admit that ultrasound diagnostics are not always effective. In a number of cases, in patients with unexplained even after diagnostic laparoscopy infertility GGE detected during a hysteroscopic examination may be the only likely cause.

The pathological transformation of the endometrium is a complex biological process that affects all parts of the neurohumoral regulation of a woman's body. It is known that ovulation defects, hyperestrogenism, progesterone deficiency, disruption of proliferation processes and suppression of apoptosis, as well as a change in the endometrial receptor phenotype can be the cause of the development of HPE. HPE often occurs with endometriosis, myoma, and chronic inflammatory processes in the endometrium. In recent years, a complex system of factors involved in cell regulation has been identified, and ideas about intercellular interaction and intracellular processes in hormone-dependent tissues have been expanded. Thus, it was found that a number of biologically active compounds (interleukins, tumor necrosis factors, chemokines) participate in the regulation of the proliferative activity of endometrial cells along with estrogens. In the regulation of tissue homeostasis and the pathogenesis of proliferative diseases, an important role is played not only by increased cell proliferation, but also by impaired cell death regulation (apoptosis). The resistance of endometrial cells to programmed cell death (apoptosis) leads to the accumulation of altered and excessively proliferating cells, which is characteristic of neoplastic changes in the endometrium [Lysenko OV Hyperplastic processes of the endometrium at different age periods, the study of the cytokine status and content of the SFAS ligand // Obstetrics and Gynecology. - 2011. - No. 4.- S.12-15].

The known method according to the patent of the Russian Federation No. 2466390, published on November 10, 2012, according to the application for invention No. 2011105301/15 of February 15, 2011. “A method for predicting the development of cancer of the uterine body in pathological processes of the endometrium in women of reproductive age” (I. Sidorova. Unanyan A.L. (RU), Vlasov R.S. (RU) and others), where a method is proposed for predicting the development of cancer of the uterine body, including the determination of clinical signs, curettage of the uterus, histological examination, determination of methylation of suppressor genes MLH1, RASSF1 , GSTP1, p16, RAR-b, CDX1 tumor growth method methyl-h comprehensible polymerase chain reaction (MCH-PCR), calculation of the coefficient of probability of developing cancer of the uterus. With a value of p greater than 0.6, a high probability of developing cancer of the uterine body is predicted, with p equal to 0.3-0.59, a moderate probability of developing cancer, with p below 0.29, a low probability of developing cancer.

The disadvantage is that it increases the accuracy of predicting the development of cancer of the uterus in case of already existing pathological processes of the endometrium in women of reproductive age, and does not make it possible to predict the tendency of patients to form isolated endometrial hyperplastic processes.

A known method of quantitative morphological assessment of the severity of chronic endometritis according to the patent of the Russian Federation No. 2475742, published on 02.20.2013. The essence of the method is that they examine the scraping of the endometrium. On a histological specimen, the presence of morphological signs of chronic inflammation in the predominant area and in individual areas is assessed in points, depending on their presence and uniformity. The total score, which is in the range from 1 to 10 points, is a quantitative assessment of the severity of chronic endometritis. The sum from 1 to 4 points allows you to establish a mild degree of severity of chronic endometritis. From 5 to 7 points - medium, from 8 to 10 - heavy. The disadvantage is that the method can only be used by examining a histomorphological study of scrapings from the uterine cavity.

0,00793+K₂

0,013 - K₃

0,043+3,09, где K₁ - показатель IgM, K₂ - показатель ЦИК, K₃ - показатель T-клеток (-CD3). Если F>0, то у пациента прогнозируют риск развития эндометриоза. Если F<0, то возникновение эндометриоза маловероятно. Предлагаемый способ дает вероятность правильного прогноза в 76% случаев.A known method for predicting the development of endometriosis, leading to impaired reproductive function in girls with algomenorrhea according to the patent of the Russian Federation No. 2161311, published 27.12. 2000. The method is based on the study of a number of indicators of the immune system and includes determining the number of T-lymphocytes by flow cytometry (using the FACS Calibur cytometric system from Beckton Dickinson), determining the concentration of immunoglobulins M (IgM) using the Mancini radial immunodiffusion test, determining the level of circulating immune complexes (CEC) in blood serum - by the method of isolation in polyethylene glycol as evaluated on a Multiscan spectrograph from Labsystems (wavelength 450 nm). The determination of the value of the diagnostic coefficient F by the formula F = - K ₁

0.00793 + K ₂

0.013 - K ₃

0.043 + 3.09, where K ₁ is the IgM indicator, K ₂ is the CEC indicator, K ₃ is the T-cell indicator (-CD3). If F> 0, then the patient predicts the risk of endometriosis. If F <0, then the occurrence of endometriosis is unlikely. The proposed method gives the probability of a correct forecast in 76% of cases.

The method allows, taking into account the main links in the pathogenesis of endometriosis, to predict the development of this disease in adolescent girls with algomenorrhea and chronic pelvic pain, to diagnose the disease in a timely manner and recommend pathogenetically based treatment that can affect the frequency of severe, advanced forms of endometriosis and concomitant infertility.

The disadvantage is that it can only be used to predict the development of endometriosis in adolescent girls with algomenorrhea and chronic pelvic pain, but it does not allow predicting the formation of isolated endometrial hyperplastic processes.

The objective of this study is to expand the arsenal of diagnostic methods, namely, to create a method for predicting the formation of isolated endometrial hyperplastic processes in women of Russian nationality, natives of the Central Black Earth Region of Russia, by combinations of genetic polymorphisms: -308 G TNFα, +36 A TNFR1, A I-TAC, - 889 T IL-1A and / or +36 A TNFR1, A I-TAC, -889 T IL-1A and / or -308 G TNFα, +250 G Ltα, -889 T IL-1A.

The technical result of using the invention is to obtain criteria for assessing the risk of developing isolated endometrial hyperplastic processes.

In accordance with the task, a method was developed for predicting the formation of isolated endometrial hyperplastic processes, including blood sampling, DNA extraction from peripheral venous blood, typing of genetic polymorphisms by the polymerase chain reaction, and genetic polymorphisms of the tumor necrosis factor gene α are typed (-308 G / A TNFα), tumor necrosis factor 1 receptor (+36 A / G TNFR1), interferon-inducing chemoattractant T-cells (A / G I-TAC), interleukin 1A (-889 C / T IL-1A), lymph syn α (+250 A / G Ltα) and analysis of combinations of polymorphisms of the tumor necrosis factor α gene (-308 G TNFα), tumor necrosis factor 1 receptor (+36 A TNFR1), interferon T-cell chemoattractant (A I-TAC) interleukin

1A (-889 T IL-1A), lymphotaxin α (+250 G Ltα), predicting an increased risk of developing isolated forms of endometrial hyperplastic processes in patients if a combination of the -308 G TNFα 36 A TNFR1, A I-TAC, -889 alleles is detected T IL-1A and / or combinations of alleles + 36 A TNFR1, A I-TAC, -889 T IL-1A and / or combinations of alleles -308 G TNFα, + 250 G Ltα, -889 T IL-1A.

The novelty and inventive step is that the prior art does not know the possibility of predicting the development of isolated endometrial hyperplastic processes by the presence of combinations of the -308 G allele of the tumor necrosis factor α gene (polymorphism -308 G TNFα), the + 36 A tumor necrosis factor receptor allele ( polymorphism + 36 A TNFR1), allele A of the interferon inducible chemoattractant T-cells (polymorphism A I-TAC), allele -889 T of interleukin 1A (polymorphism -889 T IL-1A), allele + 250 G lymphotaxin α (polymorphism + 250 G Ltα).

The invention is characterized by the following graphic images:

- гомозиготы GG,

- гомозиготы AA,

- гетерозиготы AG,

- отрицательный контроль).Figure 1. Allele discrimination at the G / A I-TAC locus (rs4512021) (where

- homozygotes GG,

- homozygotes AA,

- heterozygotes AG,

- negative control).

Figure 2. Electrophoretic separation of the restriction products of the gene -889 C / T IL-1A, where 2,3,4 - homozygotes -889 TT; 5,6,10 - homozygotes -889 SS; 1.7-9.11-14-heterozygotes -889 ST.

The method is as follows:

DNA is isolated from samples of peripheral venous blood of patients with isolated endometrial hyperplastic processes by phenol-chloroform extraction in 2 stages. At the first stage, 25 ml of lysis buffer containing 320 mM sucrose, 1% Triton X-100, 5 mM MgCl2, 10 mM Tris-HCl (pH = 7.6) is added to 4 ml of blood. The resulting mixture was stirred and centrifuged at 4 ° C, 4000 rpm for 20 minutes. After centrifugation, the supernatant is decanted, 4 ml of a solution containing 25 mM EDTA (pH = 8.0) and 75 mM NaCl are added to the precipitate, and they are resuspended. Then add 0.4 ml of 10% SDS, 35 μl of proteinase K (10 mg / ml) and incubate the sample at 37 ° C for 16 hours.

At the second stage, DNA is sequentially extracted from the obtained lysate in equal volumes of phenol, phenol-chloroform (1: 1) and chloroform with centrifugation at 4000 rpm for 10 minutes. After each centrifugation, the aqueous phase is selected. DNA is precipitated from solution in two volumes of chilled 96% ethanol. The formed DNA is dissolved in bidistilled, deionized water and stored at -200 ° C.

The isolated DNA is then subjected to polymerase chain reaction using standard oligonucleotide primers (table 1).

The structure of primers and probes used for genotyping of the studied DNA markers

Table 1

Gene and its polymorphism PCR conditions The structure of primers and probes Restriction enzyme Literature -308 G / A
TNFα
(rs 1800629) 95.0 ° C: 4.00m
54.0 ° C: 1.00m
95.0 ° C: 0.15m F: 5'-GAAATGGAGGCAATAGGTTTTTGAG-3 '
R: 5'-GGCCACTGACTGATTTGTGTGTAG-3 '
5'-FAM-CCGTCCTCATGCC- RTQ1-3 '
5'-ROX-CCGTCCCCATGCC - RTQ1-3 ' ____ [Hulkkonen J., 2002] +36 A / G TNFR1
(rs 767455) 95.0 ° C: 4.00m
59.0 ° C: 1.00m
95.0 ° C: 0.15m F: 5'- AGCCCACTCTTCCCTTTGTC-3 '
R: 5'-CCACCGTGCCTGACCTG-3 '
5'- FAM: CTGCTGCCACTGGT-RTQ1-3 '
5'- ROX: CTGCTGCCGCTGGT-BHQ2-3 ' ____ [Chae SJ
et al., 2008] A / g
I-tac
(rs 4512021) 95.0 ° C: 4.00m
52.5 ° C: 1.00m
95.0 ° C: 0.15m F: 5 '- CAAAGACCTAAGGGAACTAGGTGATAG - 3'
R: 5 '- GTGTCTTCCCAATGTGTGTTCCT - 3'
5 '- FAM - ATGACTCTGGCTAGTC - RTQ1-3'
5 '- ROX - AGCATGACTCCGGCTA-BHQ2-3' ____ [Derichs C., 2001] -889 C / T
IL-1A
(rs 1800587) 95.0 ° C: 4.00m
94.0 ° C: 0.40m
60.0 ° C: 0.40m
72.0 ° C: 1.00m 5'-aagcttgttctaccacctgaactaggc-3 '
5'-ttacatatgagccttccatg-3 ' Bsp 19I [Trevilatto
et al., 2003] +250 A / G
Lt α
(rs 909253) 95.0 ° C: 5.00m
56.3 ° C: 1.00m
95.0 ° C: 0.15m F: 5'-CAG TCTCATTGTCTCTGTCACACATT-3 '
R: 5'-ACAGAGAGAGACAGG AAGGGAACA-3 '
5'-FAM: CCATGGTTCCTCTC-RTQ1-3 '
5'-ROX: CTGCCATGATTCC-RTQ1-3 ' ____ [Sugiura S., 2002]

Molecular genetic analysis is carried out by polymerase chain reaction of DNA synthesis. PCR is carried out on amplifiers IQ5 (BioRad) and TP4-PCR-01- "TERTSIK".

IQ5 (Bio-Rad) amplifiers analyze gene polymorphisms of -308 G / A TNFα, + 36 A / G TNFR1, A / G I-TAC, + 250 A / G Ltα by the method of polymerase chain reaction of DNA synthesis using standard oligonucleotide primers and specific probes, followed by analysis of polymorphism by allele discrimination. The reaction mixture with a volume of 25 μl includes: 6.7 mm Tris-HCl (pH = 8.8), 2.5 mm MgCl2, 0.1 μg of genomic DNA, 10 pM of each primer, 5 pmol of each probe, 200 μM dATP, dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation, 40 amplification cycles are performed according to the scheme: primer annealing and denaturation.

The TP4-PCR-01- “TERTSIK” amplifier analyzes the polymorphism of the -889 C / T IL-1A gene by the polymerase chain reaction of DNA synthesis using standard oligonucleotide primers according to the method described in [Hulkkonen, J. Inflammotory cytokines and cytokine gene polymorphisms in chronic lymphocytic leukaemia, in primary Sjögren's Syndrome and Haelthy Subjects: [acad. diss.] / J. Hulkkonen; University of Tampere, Medical School Tampere University Hospital. - Tampere, 2002. - 81 p.].

The reaction is carried out in 12.5 μl of the total volume of the mixture containing 33.5 mm Tris-HCl (pH = 8.8), 1.25 mm MgCl2, 0.5 μg of genomic DNA, 5 pM of each primer, 100 μM dATP , dGTP, dCTP, dTTP and 1 unit of active Taq polymerase. After denaturation, 32 amplification cycles are performed according to the scheme: denaturation, annealing of primers and elongation. Then the samples are kept for 5 min at 72 ° C and cooled. Amplification products are analyzed on a 2% agarose gel stained with ethidium bromide for 30 minutes at 160V. As electrophoresis buffer use 1xTAE (Tris-acetate buffer). Samples are then identified in transmitted UV light.

Genotyping is carried out by allele discrimination using Tag Man probes. When performing PCR in a thermocycler with fluorescence detection (IQ5 amplifier), genotyping is carried out by the Tag Man method of probes according to the OEF values (relative fluorescence unit) of each probe.

Two bands, vertical and horizontal, divide the graph into four sections: one for each homozygous state, one for the heterozygous state and a section without reaction. Assigning genotypes to unknown samples is determined by plotting the OEF for one fluorophore on the x axis relative to the OEF for another fluorophore on the y axis in the allele discrimination diagram.

• If the OEF values of an unknown sample are above the horizontal strip and to the right of the vertical strip, the genotype is heterozygous (GA).

• If the OEF values of the unknown sample are above the horizontal strip and to the left of the vertical strip, the genotype is homozygous for allele 2 (the OEF of allele 2 is plotted along the y axis).

• If the OEF values of the unknown sample are below the horizontal strip and to the right of the vertical, the genotype is homozygous for allele 1 (RFU of allele 1 is plotted along the x axis).

• If the OEF values of the unknown sample are below the horizontal strip and to the left of the vertical, then the determination of the genotype is not possible, in this case an undefined sample is a negative control.

Genotyping of DNA markers (locus -889 C / T IL-1A) is performed by the analysis of restriction fragment length polymorphism (RFLP) of PCR amplification products of specific genome sections using appropriate restriction enzymes manufactured by Sibenzim (Novosibirsk).

After incubation of the restriction mixture for 16 hours at a temperature of 37 ° C, DNA fragments are separated by horizontal electrophoresis on electrophoretic cells manufactured by Helikon (Russia). Depending on the size of the separated DNA fragments, an agarose gel of 2-3% concentration is used, prepared on the basis of a TBE buffer, stained with ethidium bromide solution (0.01%). Visualization of phoreograms is carried out in a dark box with a transilluminator from UVP (Sweden).

The formation of the database and statistical calculations is carried out using the program "STATISTICA 6.0" [Borovikov, V. Statistica: the art of data analysis on a computer / V. Borovikov. - 2nd ed. - SPb .: Peter, 2003 .-- 688 p.: Ill. - (For professionals)].

The role of combinations of genetic variants of polymorphic markers of interleukin genes in the formation of isolated uterine fibroids was studied using the APSampler software [http: sources.redhat.com/cygwin/], using the Monte Carlo method of Markov chains and Bayesian nonparametric statistics [Favorov AV A Gibbs sampler for identification of symmetrically structured, spaced DNA motifs with improved estimation of the signal length / A. V. Favorov, M. S. Gelfand, A. V. Gerasimova [et al.] // Bioinformatics. - 2005. - Vol.21, No. 10. - R. 2240-2245].

The level of statistical significance of the obtained results is estimated using the Bonferroni correction (p _cor ), i.e. amendments minimizing the probability of obtaining false positive results [Rebrova O.Yu. Statistical analysis of medical data. https://sites.google.com/site/oyurebrova/book "\ t" _blank /O.Yu. Rebrova // M., Media Sphere. - 2006 - 312 p.].

The possibility of using the proposed method for assessing the risk of developing isolated endometrial hyperplastic processes is confirmed by an analysis of the observation results of 713 patients with various proliferative processes of the reproductive system, including uterine fibroids, endometriosis, endometrial hyperplastic processes and population control groups of 500 people. Among 713 examined, 150 women had endometrial hyperplastic processes. Patients were included in the appropriate group of patients only after a diagnosis of the disease was confirmed, confirmed by clinical, laboratory and instrumental methods of examination and the confirmed result of histological examination.

The studied groups included individuals of Russian nationality, who are natives of the Central Black Earth Region of Russia and have no kinship with each other.

It was found that the combination of molecular genetic markers -308 G TNFα, + 36 A TNFR1, A I-TAC, -889 T IL-1A occurs in 41.86% of patients, which is 2 times more often than in the control group (20, 92%, p = 0.000005, p _cor = 0.01, OR = 2.72 95% CI 1.76 - 4.18). Also, in patients with isolated endometrial hyperplastic processes, a combination of genetic variants +36 A TNFR1, A I-TAC, -889 T IL-1A (42.64%) is 1.9 times more often compared with the control (22.25%, p = 0.0000008, p _cor = 0.02, OR = 2.59, 95% CI 1.70 - 3.95). The combination of three genetic markers -308 G TNFα, + 250 G Ltα, -889 T IL-1A (14.81%) is significantly more (4.5 times) in this group than in the control group (3.28%, p = 0.000001, p _cor = 0.02, OR = 5.12, 95% CI 2.47 - 10.61).

Specific example.

A patient Kh. Of Russian nationality, a native of the Central Black Earth Region of Russia, underwent peripheral venous blood sampling, DNA extraction from peripheral venous blood; polymerase chain reaction typing of genetic polymorphisms of the gene for tumor necrosis factor α gene (-308 G / A TNFα), tumor necrosis factor 1 receptor (+36 A / G TNFR1), interferon T-cell inducible chemoattractant (A / G I-TAC), interleukin 1A (-889 C / T IL-1A), lymphotaxin α (+250 A / G Ltα) and analysis of combinations of tumor necrosis factor α gene polymorphisms (-308 G TNFα), tumor necrosis factor 1 receptor (+36 A TNFR1) interferon inducible chemoattractant T-cells (A I-TAC), interleukin 1A (-889 T IL-1A), lymphotaxin α (+250 G Ltα). The results are presented in figures 1 and 2.

It was revealed that due to the presence of the combination of the -308 G TNFα alleles, +36 A TNFR1, A I-TAC, -889 T IL-1A and the combination of the + 36 A alleles TNFR1, A I-TAC, -889 T IL-1A, a woman can be attributed to the risk group for the likelihood of isolated endometrial hyperplastic processes in order to prevent the development of clinical manifestations, as well as for the purpose of their early diagnosis to reduce the frequency of surgical intervention and improve a woman's quality of life.

Thus, the data obtained indicate that the combination of the alleles -308 G TNFα, + 36 A TNFR1, A I-TAC, -889 T IL-1A, alleles + 36 A TNFR1, A I-TAC, -889 T IL- 1A, as well as the alleles -308 G TNFα, + 250 G Ltα, -889 T IL-1A are risk factors for the development of endometrial hyperplastic processes (OR = 2.72, OR = 2.59 and OR = 5.12, respectively) among women of Russian nationality, natives of the Central Black Earth Region of Russia.

The application of this method will allow to form among women of Russian nationality, natives of the Central Black Earth Region of Russia, a group of patients with an increased risk of developing endometrial hyperplastic processes for mandatory dynamic ultrasound examination to monitor the endometrium, conduct a histomorphological study of the contents of the uterine cavity, followed by selection of preventive measures, observation and medication with a specialist doctor. With early detection and the correct differentiated approach to the management of patients with endometrial hyperplastic processes, it will be possible to prevent the malignancy of endometrial hyperplastic processes.

Claims (1)

A method for predicting the risk of developing endometrial hyperplastic processes in women of Russian nationality, natives of the Central Black Earth Region of Russia, including the extraction of DNA from peripheral venous blood, typing by polymerase chain reaction of genetic polymorphisms of the gene for tumor necrosis factor α (-308 G / A TNFα), tumor necrosis factor receptor 1 (+36 A / G TNFR1), interferon T-cell inducible chemoattractant (A / G I-TAC), interleukin 1A (-889 C / T IL-1A), lymphotaxin α (+250 A / G Ltα), and predicting an increased risk of developing hype rplastic processes of the endometrium when detecting a combination of -308 G TNFα alleles, +36 A TNFR1, A I-TAC, -889 T IL-1A and / or a combination of + 36 A alleles TNFR1, A I-TAC, -889 T IL-1A and / or combinations of the -308 G TNFα alleles, + 250 G Ltα, -889 T IL-1A.

Images