The Association between TNF-alpha Gene Polymorphisms
and Endometriosis in An Iranian Population
Tumor necrosis factor-alpha (
Materials and Methods
We recruited 150 women with endometriosis and 150 women without endometriosis in this case-control study and collected 4 ml of blood from all subjects. After DNA extraction, the polymorphisms were geno- typed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
The allele frequency of
For the first time the association of the four polymorphisms in the promoter region of the
Endometriosis is developed as a result of endometrial tissue exposing outside the uterine cavity. Studies have reported the pelvic and the peritoneum as the most common sites of replacement (1, 2). This highly prevalent disease can be really enervating (about 30% in infertile and 10% in fertile women) (3). Approximately, 25-50% of infertile women develop endometriosis while 30-50% of women with endometriosis are infertile (4).
This polygenic disease with its complex genetic background
(5, 6) occurs as a result of interactions between genetically
determined factors and environment. The genetic
component of endometriosis has been shown through studying
the kinship of patients (7, 8). To date, the most common
method for investigating genetic factors underlying
complex diseases is the hypothesis-based candidate gene
studies (8). One of the most important factors in endometriosis
is mutations in cytokine genes. The tumor necrosis
Studies on patients diagnosed with endometriosis have
Materials and Methods
This case-control study enrolled a total of 150 Iranian women with endometriosis who had referred to Avicenna Infertility Clinic and Tehran Clinic Hospital, Tehran, Iran. Diagnostic laparoscopy was performed in all patients. The severity of endometriosis was determined using the revised American Society for Reproductive Medicine (ASRM) classification (stages I-IV of disease). The control group consisted of 150 women without endometriosis. Only women who underwent laparoscopy for non-endometriosis infertility and showed absence of endometriosis were included as controls. Stages I and II of endometriosis are commonly found in asymptomatic women (21). The exclusion criteria in our study were the following: having a history of rheumatoid arthritis, diabetic retinopathy and Behcet’s disease. Approval from the Avicenna Research Institute Ethics and Human Rights Committee was obtained for using blood samples and the designed protocol. Written informed consent was obtained from all patients with inclusion criteria to take part in the study.
DNA extraction and genotyping
Blood was collected in tubes with 200 µl EDTA (0.5
M), as an anti-clotting factor, and stored at -20ºC until
DNA extraction. Genomic DNA was extracted by salting
out method from peripheral blood samples. Genotyping
of the -238G/A (rs361525), -308G/A (rs1800629), -857C/
T (rs1799724) and -863C/A (rs1800630) polymorphisms
in the 5'-untranslated region of
The PCR reactions carried out in final volume of 25 µl containing: 10X PCR Buffer (Roche, Germany), 1.5 mM MgCl2 (Roche, Germany), 0.4 µM of each dNTP (Fermentas, Germany), 5 pmol of each primer, 50 ng template DNA, 1 U Taq DNA polymerase (Roche, Germany) and sterile distilled water up to 25 µl. Amplification conditions start with an initial denaturation step of 5 minutes at 94ºC, followed by 30 cycles of 30 seconds denaturation (94ºC), 30 seconds annealing (63ºC) for -238G/A, -857C/T and -863C/A and 30 seconds annealing (66ºC) for -308G/A and 30 seconds extension (72ºC), ended by a final extension for 5 minutes (72ºC) and finally cooling to 4ºC.
Polymerase chain reaction products were electrophoresed on a 1.5% agarose gel in 1X TAE and stained with ethidium bromide and visualized by ultraviolet light. After reviewing the PCR products, they were treated with restriction enzymes (Hpa II and NcoI at 37ºC and TaiI at 65ºC) overnight. The digestion products were subjected to 10% polyacrylamide gel electrophoresis and stained with silver nitrate (Fig .1,).
Results were analyzed by SPSS 24.0 software (IBM SPSS Statistical Software, USA). The analysis of age and body mass index (BMI) in the study groups were performed using t test. The allele frequencies were compared using the Chi-squared test. Genotype distributions in the case and control groups were also analyzed. Age and BMI were considered as potential confounders. The analyses were performed and adjusted in terms of age and BMI using logistic regression. P<0.05 was considered statistically significant. The P value corrected using Bonferroni method for the multiple testing. Logistic regression was used to predict the odds of developing a given disease based on observed characteristics of the patients. In our study, the criterion variable was the logistic regression of disease and no-disease. To perform the statistical analysis using SPSS, we considered the two case and control groups as dependent variables. Age and BMI were considered as covariants and genotype selected as the basis of categorical covariant.
|Gene||Variation||Primers (5ˊ-3ˊ)||Size (bp)||Restriction enzyme||Allele||Cutting product (bp)|
|-238G/A||F: AGAAGACCCCCCTCGGAACC||165||Hpa II (New England BioLabs)||G||136|
|-308G/A||F: AGGCAATAGGTTTTGAGGGCCAT||107||NcoI (New England BioLabs)||G||87|
|-857C/T||F: GGCTCTGAGGAATGGGTTAC||128||TaiI (New England BioLabs)||C||107|
|-863C/A||F: GGCTCTGAGGAATGGGTTAC||125||TaiI (New England BioLabs)||A||101|
For interaction analysis, the STRING online server
According to the analysis of descriptive variables, the
age range was from 19 to 50 years (mean=31, SD=6.1)
in the patients, and from 19 to 44 years (mean=29.2,
SD=5.2) in the control group. The mean BMI (Kg/m2) in
the case and control groups were 25.2 (SD=3.7) and 26.2
(SD=4) respectively. Genotypes of the
|Polymorphisms*||Cases||Controls||OR||95% CI||P value||Corrected P value**|
|The -238 (rs361525)||Genotype||GG||137 (91.3%)||135 (90.6%)|
|GA||11 (7.3%)||14 (9.4%)||0.65||0.24-1.71||0.381||0.762|
|AA||2 (1.3%)||0 (0.0%)||NA||NA||NA||NA|
|Allele||G||285 (95.0%)||284 (95.3%)|
|A||15 (5.0%)||14 (4.7%)||1.07||0.51-2.25||0.862||1|
|The -308 (rs1800629)||Genotype||GG||131 (87.3%)||127 (84.7%)|
|GA||18 (12.0%)||21 (14.0%)||0.8||0.35-1.84||0.604||1|
|AA||1 (0.7%)||2 (1.3%)||0||NA||0.999||1|
|Allele||G||280 (93.3%)||275 (91.7%)|
|A||20 (6.67%)||25 (8.3%)||0.79||0.43-1.45||0.438||1|
|The -857 (rs1799724)||Genotype||CC||102 (68.9%)||102 (71.3%)|
|CT||43 (29.1%)||36 (25.2%)||1.62||0.86-3.04||0.137||0.274|
|TT||3 (2.0%)||5 (3.5%)||0.46||0.05-4.35||0.499||0.998|
|Allele||C||247 (83.5%)||240 (83.9%)|
|T||49 (16.5%)||46 (16.1%)||1.03||0.66-1.61||0.887||1|
|The -863 (rs1800630)||Genotype||CC||114 (76.0%)||99 (66.0%)|
|CA||32 (21.3%)||44 (29.3%)||0.66||0.35-1.27||0.215||0.43|
|AA||4 (2.7%)||7 (4.7%)||0.68||0.19-2.47||0.557||1|
|Allele||C||260 (86.7%)||242 (80.67%)|
|A||40 (13.3%)||58 (19.3%)||0.64||0.41-0.99||0.047||0.188|
OR; Odds ratios, CI; Confidence interval, BMI; Body mass index, NA; No answer, *; The effect of genotypes were adjusted by age and BMI, and **; The P value corrected using Bonferroni method for the multiple testing.
|SNP name||Association with susceptibility|
|Association Population/(number of cases and controls)||No-association Population/(number of cases and controls)|
|-1031T/C||Japanese (123, 165) (Teramoto et al.) (20)Japanese (130, 185) (Asghar et al.) (12)Iranian (135, 173) (Saliminejad et al.) (15)Iranian (65, 65) (Abutorabi et al.) (16)||Australian (958, 959) (Zhao et al.) (19)|
|-863C/A||Japanese (123, 165) (Teramoto et al.) (20)Iranian (150, 150) (This study)*, £||Japanese (130, 185) (Asghar et al.) (12)|
|-857C/T||Japanese (123, 165) (Teramoto et al.) (20)Iranian (148, 143) (This study)£||Japanese (130, 185) (Asghar et al.) (12)Australian (958, 959) (Zhao et al.) (19)Iranian (148, 143) (This study)**|
|-308G/A||Iranian (150, 150) (This study)£||Taiwanese (120, 106) (Hsieh et al.) (13)Korean (70, 202) (Lee et al.) (17)Austrian (92, 69) (Wieser et al.) (18)Japanese (130, 185) (Asghar et al.) (12)Australian (958,959) (Zhao et al.) (19)Chinese (76,87) (Lu et al.) (14)Iranian (65, 65) (Abutorabi et al.) (16)Iranian (150, 150) (This study)**|
|-238G/A||Iranian (150, 150) (This study)£||Korean (70, 202) (Lee et al.) (17)Austrian (92, 69) (Wieser et al.) (18)Japanese (130, 185) (Asghar et al.) (12)Australian (958, 959) (Zhao et al.) (19)Iranian (65, 65) (Abutorabi et al.) (16)Iranian (149, 150) (This study)**|
BMI; Body mass index, *; Allele frequencies, **; Genotype adjusted by age and BMI, and £; case and control and BMI adjusted by age.
Endometriosis is a multifactorial disease with both genetic
and environmental components (8). Studies have scanned
the genome and specific candidate genes to determine the
genetic aetiology of this disease (22), reporting on endometriosis
and related genes involved in "detoxification, galactose
metabolism, steroid hormone production and inflammation"
(15, 21, 22). Studies have also shown that any change
in function and number of immune cells as well as high
levels of inflammatory cytokines may lead to endometriosis
(23). The present study aimed at examining the association
The effects of polymorphisms on cytokines genes have
been examined by many studies (22) as well as the possible
All in all, endometriosis has been reported to be linked
with a number of polymorphisms of
According to the studies mentioned,
It has been shown that the presence of polymorphism
in promoter regions can affect gene expression (26).
STRING showed that
This study has some limitations in spite of its strengths. The limitations of our study on endometriosis were not only the difficulty in choosing the controls, but also in recruiting patients. This is because laparoscopy should be undertaken to confirm the disease and its steady state that was a matter of time to collect samples.
We investigated the association of four polymorphisms
in the promoter region of
We offer our special thanks to all women who participated in this study, the Avicenna Research Institute for financial supporting the study and Ms. Fatemeh Javadi for editing the manuscript. The authors declare that they have no financial or conflict of interest.
A.A., H.R.K.K., F.S.; Participated in the conception and design of the study. A.A.; Was responsible for overall supervision of the project, revised statistical analysis and data interpretation. B.B.; Collected the data, conducted molecular and statistical analyses, interpreted the data and wrote the first draft of the manuscript. H.B.-F., Undertook literature search and contributed to data analysis and statistical analysis. All authors performed editing and approved the final version of this manuscript prior to submission.