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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 4  |  Page : 884-887

Q192R variant in paraoxonase 1 gene confers susceptibility to leiomyoma


1 Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2 Department of Obstetrics and Gynecology, Iran University of Medical Sciences, Tehran, Iran
3 Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

Date of Submission23-Aug-2016
Date of Acceptance24-Feb-2018
Date of Web Publication24-Oct-2018

Correspondence Address:
Shirin Shahbazi
Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Al-E-Ahmad and Chamran Cross, POB 14115-111, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_923_16

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 > Abstract 


Objective: Paraoxonase 1 (PON1) plays a defensive role against oxidative stress by destroying oxidized lipids. Q192R single nucleotide polymorphism of PON1 gene alters the enzyme's activity. Several investigations reported a link between Q192R and an increased risk of developing tumors including uterine leiomyomas. We assessed the antioxidant effects of Q192R on myoma which fluctuate in frequency between populations.
Study Design: The cohort consisted of 68 unrelated uterine leiomyoma patients and 93 healthy controls that were randomly selected from women with no ultrasonographic evidence of myoma.
Materials and Methods: Genotyping was performed using tetra-primer amplification refractory mutation system-polymerase chain reaction. Chi-square test was selected to evaluate differences between the groups.
Results: To analyze the correlation between PON1 Q192R and leiomyoma risk, the AA genotype was given as a reference genotype then the two other genotypes were compared with the reference. A significantly (P < 0.05) increased risk of myoma was observed with both Q192R homozygote GG and heterozygote AG genotypes. The odds ratio (OR) of AG genotype was calculated 1.8 (confidence interval [CI]: 0.94–3.62). A higher OR was seen with GG genotype (OR: 2.8; 95% CI: 0.98–8.18).
Conclusion: Oxidative stress has been suspected of having a link with tumor development, and the role of endogenous-free radical scavenger is taken into consideration. Increased protein oxidative stress status and reduced antioxidant capacity have been observed in leiomyomas patients. Our study indicates that the low-antioxidant PON1 R192 allele correlates to leiomyoma development.

Keywords: Amplification refractory mutation system-polymerase chain reaction, leiomyoma, oxidative stress, paraoxonase 1 gene, single nucleotide polymorphisms


How to cite this article:
Shahbazi S, Zarei S, Torfeh M, Fatahi N. Q192R variant in paraoxonase 1 gene confers susceptibility to leiomyoma. J Can Res Ther 2020;16:884-7

How to cite this URL:
Shahbazi S, Zarei S, Torfeh M, Fatahi N. Q192R variant in paraoxonase 1 gene confers susceptibility to leiomyoma. J Can Res Ther [serial online] 2020 [cited 2020 Sep 29];16:884-7. Available from: http://www.cancerjournal.net/text.asp?2020/16/4/884/243503




 > Introduction Top


Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme which is secreted mainly by the liver. PON1 plays a defensive role against oxidative stress by destroying oxidized lipids. Oxidized lipids are responsible for the initiation of inflammation and the formation of several cytotoxic and mutagenic substances that may contribute to the occurrence of certain diseases such as cancer. Furthermore, PON1 acts as an endogenous-free radical scavenging molecule in the human body by decreasing the level of systemic oxidative stress.[1]

PON1 enzyme is a glycoprotein composed of 354 amino acids that encoded by the PON1 gene located at chromosome 7q21.3. Genetic polymorphisms of PON1 alters the enzyme's activity, varying by over 40-fold between individuals.[2] The exonic Q192R (rs662) is a well-studied PON1 single nucleotide polymorphism (SNP). The Q and R alleles are in the majority of European and Asian/African populations, respectively.[3] It seems that the Q allele presents a most effective at protecting low-density lipoprotein oxidation, whereas the R allele is least efficient and is related to risk for coronary heart disease.[4]

Several investigations reported a link between PON1 Q192R polymorphism and an increased risk of cancers including ovarian, lung, breast, and prostate.[5] However, to date, published results have been conflicting. In an effort to dissect the role of PON1, Attar et al. have described a connection between Q192R with an increased risk of uterine leiomyomas.[6] Leiomyomas are common benign uterine tumors in women of reproductive age. Although the pathogenesis of the disease remains poorly understood, researchers suggest that changes in the ovarian hormones contribute to myoma development.[7] In addition to hormonal factors, genetic and environmental exposures are effective on the prevalence of myoma.[8] Genetic variations that reduce the activity of detoxifying carcinogen substances could possibly exert an influence on leiomyomas. It has been shown that, 8-OH-dG, one of the biomarkers of oxidative stress, is significantly higher in uterine myoma than their respective tumor-free tissues.[9] To assess the anti-inflammatory and antioxidant effects on myoma development, we explored PON1 polymorphism in Iranian females. We hypothesized that PON1 Q192R polymorphism, which fluctuates in frequency between populations, contributes in pathogenesis of myoma.


 > Materials and Methods Top


Participants

The cohort consisted of 68 unrelated uterine leiomyoma patients and 93 healthy controls that were randomly selected from women with no ultrasonographic evidence of myoma.

All the contributors were visited by an expert gynecologist and subjected to imaging and laboratory tests. Diagnostic criteria were based on the standard international guidelines. Each subject contributed to the study signed a written consent. Using a designed questionnaire, women were interviewed to obtain the characteristics of their gynecological complications. This study was approved by Ethics Committee of Iran University of Medical Sciences.

DNA extraction and genotyping

Five milliliters peripheral blood samples were procured in ethylenediaminetetraacetic acid anticoagulant containing tubes from each individual. Genomic DNA extraction was performed according to the standard salting-out protocol. The concentration and quality (Thermo Fisher Scientific) of the DNA were measured using NanoDrop® ND-1000 spectrophotometer. DNA sample aliquots were stored at −20°C and fresh working solutions (30–50 ng/μL) were prepared immediately before each polymerase chain reaction (PCR) experiment. Genotypes were tested based on the developed tetra-primer amplification refractory mutation system (ARMS) PCR described before[10] which is an efficient procedure for genotyping of SNP.[11]

PCR and genotyping were performed in a total volume of 20 μl containing a set of four primers (two allele-specific and two common primers) according to the following program: an initial denaturation step for 5 min at 94°C then 40 amplification cycles of denaturation at 95°C for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 60 s. Final extension was allowed to proceed for 7 min at 72°C. PCR product size of common primers that provides an internal control was 354 bp. Amplification of reverse inner and forward outer primers results in a 239 bp fragment that indicates the existence of A allele. In the same way, amplification of forward inner and reverse outer occurs in the presence of G allele and results in a 168 bp product.

Randomly selected PCR products were subjected to DNA sequencing to verify the ARMS results.

Statistical analysis

The collected data were loaded on statistical analysis software SPSS V.16 (IBM Corporation). Chi-square test was selected to weigh up the null hypothesis (H0) among the groups. The P < 0.05 was considered to be statistically significant. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated by unconditional logistic regression.


 > Results Top


Subject's data

Patients and controls' mean age was 39.06 ± 7.22 and 43.81 ± 10.57 years, respectively. Ultrasound examination revealed that most of the tumors were intramural leiomyomas which situated anteriorly. They vary in size from minimal lesions to 3–4 cm nodules. The most common symptoms of the patients included uterine abnormal bleeding and low back pain. Despite the lack of statistical significance, oral contraceptive usage was linked to increased risk of the disease. However, parity and BMI yield no correlation with the risk of myoma development.

Genotyping and statistics

As it is shown in [Figure 1], the desired tetra-primer ARMS PCR fragments were revealed by gel electrophoresis. The accuracy of the ARMS assay was confirmed by DNA sequencing of randomly selected representative samples [Figure 2].
Figure 1: Tetra-primer amplification refractory mutation system-polymerase chain reaction fragments on gel electrophoresis. lane 1, 100 bp ladder, lanes 1, 4, and 5 present AG genotype, lanes 3 and 7 indicate GG genotype, and lane 6 shows AA genotype

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Figure 2: DNA sequencing of randomly selected samples

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The allele frequencies of PON1r s662 did not show a significant difference from the expected Hardy-Weinberg equilibrium. Our data revealed the minor allele frequency of 0.25 in healthy controls as are summarized in [Table 1]. The AA genotype was detected in 39.7% of the patients versus 57% of the controls. To analyze the correlation between PON1 Q192R and leiomyoma risk, the AA genotype was given as a reference genotype; then, the two other genotypes were compared with the reference. As a result, a significantly (P < 0.05) increased risk of myoma was observed with both Q192R homozygote GG and heterozygote AG genotypes. The OR of AG genotype was calculated 1.8 (CI, 0.94–3.62). A higher OR was seen with GG genotype (OR, 2.8; 95% CI: 0.98–8.18). The Chi-square was estimated 3.18 and 3.73, with a degree of freedom of 1 for AG and GG genotype, respectively.
Table 1: The allele frequencies determined for PON1rs662 among the study cohort

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This data suggest that the G allele may involve in susceptibility to myoma lesion development, either in heterozygote or homozygote state. The association of PON1 rs662 and tumor characteristic was assessed. Any interaction of AG genotypes with size and number of fibroids or menstrual status was not observed. Our results showed a slight increase in size of the lesions among GG genotypes, although it was not statistically significant. There was no significant association between GG genotype and menstrual status.


 > Discussion Top


Regardless of the benign neoplasm features, uterine fibrum is responsible for the mortality and morbidity of a significant portion of the female and is the most common cause of hysterectomy. Evidence is emerging that oxidative stress has a pivotal role in the development of uterine leiomyomas. In addition to myometrial tissues, increased protein oxidative stress status and reduced antioxidant capacity have been observed in the serum of patients with uterine leiomyomas.[12] Since PON1 decreases the level of systemic oxidative stress, it may protect myometrial tissue from free radical accumulation and induced proliferation. Considerable evidence now supports the concept that isozyme consisting of Q allele underlies a higher antioxidant effect than the R allele.[13]

In this study, we assessed the correlation between PON1 gene rs662 SNP and susceptibility to the development of leiomyomas. To establish the possible difference in the allelic distribution of the rs662, we conducted a case–control study. Our results revealed that this variant is significantly associated with increased risk of myoma. The obtained data present that the QQ genotype has lowest risk of developing fibroids followed by the QR genotype.

Reflected by several publications, oxidative stress and PON1 activity are involved in pathogenesis of disorders of the female genital tract. It has been shown that plasma PON1 levels were significantly lower in endometriosis patients.[14] Younis et al. showed that after ovarian stimulation forin vitro fertilization or intrauterine insemination, women with high serum concentrations of PON1 were found have a higher chance of pregnancy compared to others.[15] In a subsequent study by the same investigators, it has been shown that gonadotropin stimulation in women with mild endometriosis, polycystic ovary syndrome (PCOS), or unexplained infertility increases the serum levels of PON1. They reported no relationship between serum PON1 concentration and PCOS, endometriosis or unexplained infertility.[16] Oxidative stress through the process of ovulation has been suspected of having a link with DNA damage. As a result, the potential role of endogenous-free radical scavenger is taken into consideration in ovarian diseases. It has been shown that women with PCOS have reduced PON1 activity. Furthermore, a study showed that lean women with Q192R polymorphism were significantly at reduced PCOS risk.[17] A decreased risk of ovarian cancer has been shown in association with rs662 A allele.[18] Although these results do not agree with the results of Arpaci et al., who found the AA genotype significantly higher in the Turkish patients with ovarian cancer.[19] A possibility that may explain the conflicting results are due to ethnic difference. The study of correlation between PON1 rs662 and myoma showed again a significant low occurrence of the R allele in Turkish patients.[6]


 > Conclusion Top


The significance of PON1 as a biomarker in clinical diagnosis and treatment of oxidative stress has been a growing interest. Our study indicates that the low-antioxidant PON1 R192 allele correlates to leiomyoma development. Considering the impact of uterine leiomyoma on patient's lives, our results emphasized the importance of the genome variation and ethnic differences. This finding needs to be confirmed in further studies with a larger number of ethnically diverse individuals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Mackness M, Mackness B. Human paraoxonase-1 (PON1): Gene structure and expression, promiscuous activities and multiple physiological roles. Gene 2015;567:12-21.  Back to cited text no. 1
    
2.
Mackness B, Durrington PN, Mackness MI. Human serum paraoxonase. Gen Pharmacol 1998;31:329-36.  Back to cited text no. 2
    
3.
Adkins S, Gan KN, Mody M, La Du BN. Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: Glutamine or arginine at position 191, for the respective A or B allozymes. Am J Hum Genet 1993;52:598-608.  Back to cited text no. 3
    
4.
Mackness B, Mackness MI, Arrol S, Turkie W, Durrington PN. Effect of the human serum paraoxonase 55 and 192 genetic polymorphisms on the protection by high density lipoprotein against low density lipoprotein oxidative modification. FEBS Lett 1998;423:57-60.  Back to cited text no. 4
    
5.
Fang DH, Fan CH, Ji Q, Qi BX, Li J, Wang L, et al. Differential effects of paraoxonase 1 (PON1) polymorphisms on cancer risk: Evidence from 25 published studies. Mol Biol Rep 2012;39:6801-9.  Back to cited text no. 5
    
6.
Attar R, Atasoy H, İnal-Gültekin G, Timirci-Kahraman Ö, Güleç-Yilmaz S, Dalan AB, et al. The effects of PON1 gene Q192R variant on the development of uterine leiomyoma in Turkish patients. In Vivo 2015;29:243-6.  Back to cited text no. 6
    
7.
Shahbazi S, Fatahi N, Amini-Moghaddam S. Somatic mutational analysis of MED12 exon 2 in uterine leiomyomas of Iranian women. Am J Cancer Res 2015;5:2441-6.  Back to cited text no. 7
    
8.
Yang Q, Mas A, Diamond MP, Al-Hendy A. The mechanism and function of epigenetics in uterine leiomyoma development. Reprod Sci 2016;23:163-75.  Back to cited text no. 8
    
9.
Foksinski M, Kotzbach R, Szymanski W, Olinski R. The level of typical biomarker of oxidative stress 8-hydroxy-2'-deoxyguanosine is higher in uterine myomas than in control tissues and correlates with the size of the tumor. Free Radic Biol Med 2000;29:597-601.  Back to cited text no. 9
    
10.
Masud R, Qureshi IZ. Tetra primer ARMS-PCR relates folate/homocysteine pathway genes and ACE gene polymorphism with coronary artery disease. Mol Cell Biochem 2011;355:289-97.  Back to cited text no. 10
    
11.
Shahbazi S, Mashayekhi A, Fatahi N, Mahdavi MR. Association of ABO and colton blood group gene polymorphisms with hematological traits variation. Medicine (Baltimore) 2015;94:e2144.  Back to cited text no. 11
    
12.
Santulli P, Borghese B, Lemaréchal H, Leconte M, Millischer AE, Batteux F, et al. Increased serum oxidative stress markers in women with uterine leiomyoma. PLoS One 2013;8:e72069.  Back to cited text no. 12
    
13.
Aviram M, Hardak E, Vaya J, Mahmood S, Milo S, Hoffman A, et al. Human serum paraoxonases (PON1) Q and R selectively decrease lipid peroxides in human coronary and carotid atherosclerotic lesions: PON1 esterase and peroxidase-like activities. Circulation 2000;101:2510-7.  Back to cited text no. 13
    
14.
Verit FF, Erel O, Celik N. Serum paraoxonase-1 activity in women with endometriosis and its relationship with the stage of the disease. Hum Reprod 2008;23:100-4.  Back to cited text no. 14
    
15.
Younis A, Clower C, Nelsen D, Butler W, Carvalho A, Hok E, et al. The relationship between pregnancy and oxidative stress markers on patients undergoing ovarian stimulations. J Assist Reprod Genet 2012;29:1083-9.  Back to cited text no. 15
    
16.
Younis A, Hawkins K, Mahini H, Butler W, Garelnabi M. Serum tumor necrosis factor-α, interleukin-6, monocyte chemotactic protein-1 and paraoxonase-1 profiles in women with endometriosis, PCOS, or unexplained infertility. J Assist Reprod Genet 2014;31:1445-51.  Back to cited text no. 16
    
17.
Dadachanji R, Shaikh N, Khavale S, Patil A, Shah N, Mukherjee S, et al. PON1 polymorphisms are associated with polycystic ovary syndrome susceptibility, related traits, and PON1 activity in Indian women with the syndrome. Fertil Steril 2015;104:207-16.  Back to cited text no. 17
    
18.
Lurie G, Wilkens LR, Thompson PJ, McDuffie KE, Carney ME, Terada KY, et al. Genetic polymorphisms in the paraoxonase 1 gene and risk of ovarian epithelial carcinoma. Cancer Epidemiol Biomarkers Prev 2008;17:2070-7.  Back to cited text no. 18
    
19.
Arpaci A, Görmüs U, Dalan B, Berkman S, Isbir T. Investigation of PON1 192 and PON1 55 polymorphisms in ovarian cancer patients in Turkish population. In Vivo 2009;23:421-4.  Back to cited text no. 19
    


    Figures

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    Tables

  [Table 1]



 

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