1. Background
ULM is one of the most common type of tumor; it originates from smooth muscle cells, is considered as a gynecological benign tumor, and affects 20% - 50% of women at fertility ages. ULM prevalence rates in black women are higher than in white women (1, 2). Although the molecular pathogenesis of ULM remained unknown, it has reported that the genetic and environmental factors contributed to ULM (3). Previous investigations have reported that several candidate gene polymorphisms, such as estrogen receptor alpha (ERα), cytochromes P450 (CYP), COMT, androgen receptor, and interleukin (IL) could have an impact on ULM susceptibility (4-10). COMT enzymes play roles in degradation of catecholamines, including dopamine, epinephrine, and norepinephrine. In humans, COMT enzyme was encoded by the COMT gene (11), which plays as a transfer from the methyl group to the carboxyl catechol estrogen group (10). COMT enzyme contributed as an inactivator of catecholamine neurotransmitters (dopamine, epinephrine, and norepinephrine). Levodopa is a precursor of catecholamines, which is an important substrate of COMT (12). A functional single-nucleotide polymorphism (rs4680 G>A) of the COMT gene leads to changing a valine to methionine amino acid (13). The enzyme with valine amino acid is notably active compared to the enzyme-containing methionine (14). However, the methionine variant upregulated in the brain leads to a 40% decrease in the enzyme activity (15). In regards to the preferential role of COMT in the prefrontal dopamine degradation, the Val158Met variant may have an impact on cognition by modulating the dopamine signaling pathway (16, 17). The COMT regulation activity may play a pathogenesis role in estrogen-induced tumorigenesis such as ULM.
2. Objectives
Due to discrepancies on the results reported to the significant roles of COMT (rs4680) gene polymorphism with the risk of ULM, we aimed to survey the frequency of COMT (rs4680 G>A) gene polymorphism with the risk of ULM.
3. Methods
3.1. Sample Preparation
Our study was conducted in a case-control investigation, which comprised of 100 peripheral blood samples of female patients with ULM and 100 peripheral blood samples of healthy women who were referred to the department of infertility and sterility of Tabriz Al-Zahra hospital during 2015 - 2017. The inclusion criteria for the patients in the ULM group were those who underwent elective surgery such as laparoscopic myomectomy, open myomectomy, transcervical resection of the myoma, or hysterectomy. The inclusion criteria for the control group were lack of uterine fibroids after clinical and ultrasonographic evaluation as well as no family history of uterine leiomyoma. The control subjects matched for age, race (Turk), and geographic area with the ULM patients. The survey protocol was confirmed by the Ethics Committee of Tabriz Al-Zahra hospital and written informed consent was collected from all participants.
Genomic DNA was obtained from 2 mL peripheral blood samples, with EDTA as an anti-coagulant, by the salting-out method (18).
The primers and restriction enzyme were used for the gene sequence (accession number: NC_000022.11) listed in Table 1 (19).
| COMT (rs4680) Gene Polymorphism | Primer Sequence (5' - 3') | Product Size (bp) | Restriction Enzyme |
|---|---|---|---|
| COMT -F | 5'-TACTGTGGCTACTCAGCTGTGC-3' | 236 | NIaIII |
| COMT -R | 5'-GTGAACGTGGTGTGAACACC-3' |
Primer Sequences for Amplifying the Region of COMT (rs4680) Gene Polymorphism (19)
To evaluate the quantity and quality of extracted genomic DNA, we used NanoDrop (Thermo Scientific™. UAS) and 1% agarose gel electrophoresis, respectively. To determined genotype frequencies of COMT (rs4680) polymorphism, we used the PCR-RFLP method. The following conditions applied to amplify the region of involved SNP: 2 μL (100 ng) genomic DNA, 1 μL (10 picomoles) forward and reverse primers, 15 μL of Master Mix Red 2x (Ampliqon, Denmark), and 7 μL of distilled water. Amplification steps included a first denaturation at 95°C for 10 minutes, then at 35 rounds with a temperature of 95°C for 30 seconds, an annealing temperature of 59°C for 50 seconds, and a temperature of 72°C for 60 seconds. Final amplification was performed at 72°C for five minutes. Before the enzyme digestion, the amplified products run on a 1% agarose gel electrophoresis and are stained with Safe Stain (Sina genes, Iran) to obtain amplification assurance. Digest was performed using restriction enzymes NIaIII (Fermentas, Germany), according to the manufacturer's protocol, which consisted of 10 μL of PCR-amplified products, 2 μL of Buffer 10x, 1 μL (1 unit/μL) enzyme, and 17 μL of distilled water, which were then mixed and incubated at 37°C for 12 hours. The digested PCR product was incubated for five minutes at 80°C for enzyme inactivation. Then, in order to evaluate the enzymatic digestion process, the amplified products electrophoresis on a 4% agarose gel and illuminated by the safe stain. The digested fragments were obtained for GG genotype (114 bp, 54 bp, 41 bp, 27 bp), for GA genotype (114 bp, 96 bp, 54 bp, 41 bp, 18bp, 27 bp), and AA genotype (96 bp, 54 bp, 41 bp, 18 bp, 27 bp) (Figure 1).
PCR-RFLP results for COMT (rs4680) gene polymorphism in 4% agarose gel. M: 50 bp molecular weight standard (50bp), line 1: Heterozygote (GA), line 2: Homozygote (AA), line 3: Homozygote (GG)
3.2. Statistical Analysis
To determine the different genotype and allele frequencies between ULM cases and control groups, the chi-square test was used. Odds ratios (OR) and 95% confidence intervals (CI) were done to evaluate the strength of correlation between genotypes and ULM. Two-tailed P values < 0.05 were considered statistically significant. The results were analyzed by SPSS software version 25.
4. Results
The case group comprised of 100 females with ULM (mean age 37.92 ± 4/722 years) and 100 healthy females without leiomyoma (mean age 37.27 ± 5.82 years). No significant difference in the mean age of groups was observed (P = 0.695). The genotype frequencies of COMT showed that GG, GA, and AA was 46, 35, and 19 in patients with ULM and 55, 18, and 27 in healthy women, respectively (Table 2). Our findings revealed a statistically significant difference between both groups (P = 0.022) (Table 2). In addition, we assessed a significant difference of genotype frequencies in different genetic models. The AA + GG vs. GA genetic model showed a statistically significant difference between groups (OR = 2.453; CI = 1.4 - 274/723, P = 0.006). Our results revealed that the COMT (rs4680) variation was associated with the risk of ULM. However, in dominant and recessive models, a statistically significant difference between two groups was not shown (Table 3).
| COMT Gene Polymorphism | Case Group (n = 100) | Control Group (n = 100) | P Value |
|---|---|---|---|
| COMT (rs4680 G>A) | 0.022 | ||
| GG | 46 (46) | 55 (55) | |
| GA | 35 (35) | 18 (18) | |
| AA | 19 (19) | 27 (27) |
The Genotype Frequencies of COMT (rs4680) Gene Polymorphism Between Groupsa
| COMT (rs4680) Gene Polymorphism | Case Group (n = 100) | Control Group (n = 100) | P Valuec | ORd | 95 % Confidence Intervals (CI)e | |
|---|---|---|---|---|---|---|
| High | Low | |||||
| Dominant | ||||||
| GG | 46 (46) | 55 (55) | 0.203 | 1.435 | 2.503 | 0.822 |
| GA + AA | 54 (54) | 45 (45) | ||||
| Recessive | ||||||
| GG + GA | 81 (81) | 73 (73) | 0.179 | 1.577 | 3.071 | 0.810 |
| AA | 19 (19) | 27 (27) | ||||
| Over-dominant | ||||||
| AA + GG | 65 (65) | 82 (82) | 0.006 | 2.453 | 4.723 | 1.274 |
| GA | 35 (35) | 18 (18) | ||||
| Frequency of A allele | 73 (36.5) | 72 (36) | 0.917 | 1.022 | 1.536 | 0.680 |
| Frequency of G allele | 127 (63.5) | 128 (64) | ||||
5. Discussion
The study was aimed to find out if COMT (rs4680) gene polymorphism was associated with the risk of ULM in the women of Tabriz. Our results showed that the COMT (rs4680) gene polymorphism increased the risk of ULM. The ULM may develop from the cervix and form a lump or swelling in the stomach. Its precision molecular pathogenesis of ULM remained unknown, however, several surveys have found that it arises from smooth muscle cells. Of note, ULM has a low potential to the malignancy (20). Previous investigations showed that regulation of COMT activity may be modulating the biologic impact of estrogen and plays a critical role in leiomyoma formation (21, 22). In addition, the preliminary investigations have revealed that whole polymorphisms are not associated with a certain disease, however, they are a useful marker for predicting the susceptibility to multifactorial disorders, including leiomyoma of the uterus. The polymorphisms, which occurred in the regulatory or structural regions, may influence the genes expression levels and is also associated with the immunological reactions (23, 24).
Previous investigations revealed that both genetic and environmental factors are associated with the development of ULM. The associations between COMT (rs4680) gene polymorphism, with the risk of ULM, have been considered in different ethnic communities with conflicting results. Morikawa et al. (25) showed that COMT (rs4680) gene polymorphism does not refer to be associated with ULM in the Japanese population (25). Denschlag et al. (26) did not reveal statistically significant differences in allele and genotype frequencies in Caucasian women (26). Gooden et al. (27) reported that the COMT (rs4680) variant was not associated with the risk of fibroids between African-American or white women (27).
Similar to previous findings, de Oliveira et al. (28) reported no significant association between COMT (rs4680) genotypes/ alleles and ULM in Brazilian white/non-white women (28). According to the above surveys and the study of Ates et al. (12) regarding Turkish women with ULM, COMT (rs4680) polymorphism was not associated with an increased risk of ULM (12). These findings are contradictory to our investigation. Al-Hendy and Salama indicated that COMT Val/ Val genotype was associated with the risk of ULM (29). These discrepancies may be influenced by several parameters including differences between the patient's groups studied and the fibroid size of selected patients in different studies. Different geographic regions, race, ethnicity, and genetics or error analysis may lead to controversial results. In addition, the samples selection criteria may lead to controversy results. Low activity of COMT has been considered a risk factor for breast cancer (30), endometrial cancer (31), endometriosis, and adenomyosis (32). COMT change catechol estrogens, 2-hydroxyestradiol, and 4-hydroxyestradiol into inactive metabolites, which leads to stimulating uterine growth (33). Increased function of 4-hydroxycstradiol was demonstrated in the human ULM compared with the uterine myometrium (34). 2-methoxy estradiol inhibits DNA and collagen synthesis as well as apoptotic cell death. During decreasing activity of COMT enzyme, the 4-hydroxycstradiol levels was up-regulated and down-regulated 2-methoxyestradiol, induced cell propagation, reduced DNA synthesis, and inhibited apoptotic cell death in leiomyomas (25). It has been hypothesized that the COMT 158 Met allele may be a risk factor for the development of large fibroids (28). Alternatively, this controversial result could be due to heterogeneity between populations and the small number of samples used in the studies. In addition, other environmental factors should be considered in different geographic areas due to the interaction between gene and environmental factors (35).
However, to confirm our findings more investigations in the future are needed. In conclusion, the genotypes frequency of COMT (rs4680) gene polymorphism AA + GG vs. GA revealed a significant association with the risk of ULM. The findings suggested that COMT (rs4680) gene polymorphism may be contributed as a predisposing risk factor to ULM.
