Polycystic ovary syndrome (PCOS) is a multifactorial, heterogeneous, complex genetic, endocrine and metabolic disorder, diagnostically characterized by chronic anovulation. The autonomic nervous system plays an important role in the regulation of ovarian physiology [1,2]. It is well known that one of the major neurotransmitters that control LH secretion is noradrenaline (NA). As women with PCOS have significantly higher sympathetic nerve activity than their matched controls, the increased sympathetic outflow may be related to hormonal and metabolic features that may be relevant to the pathophysiology of the syndrome [3]. Rodent models of polycystic ovaries have shown that ovarian sympathetic outflow may be increased [4,5] transection of the superior ovarian nerve in the EV-induced rat PCO model reduces the steroid response, increases b2-adrenoceptor expression to more normal levels, and restores estrus cyclicity and ovulation [6]. Consequently, the identification of genes related to PCOS is complicated by the heterogeneity of its etiology. Studies identifying familial clustering of cases have established a genetic basis of PCOS [7]. Evidence suggests it to be a complex heterogeneous syndrome in which both genetic and environmental influences play an important role in its manifestation. Beta2-adrenergic receptor may represent a functional candidate gene.

Beta2 adrenergic receptor

The gene encoding ADRB2 displays a moderate degree of heterogeneity in the human population and the distributions of singlenucleotide polymorphisms (SNPs) at amino acid positions 16, 27, and 164 are changed in asthma, obesity, and hypertension and in the autoimmune disease myasthenia gravis [8,9]. An involvement of the beta2-AR has also been suggested in human rheumatoid arthritis (RA) and its animal model. The ADRB2 gene has 9 different single nucleotide polymorphisms (SNPs); in which, four of these polymorphisms involve the change of amino acids at residues 16, 27, 34 and 164 [10]. Numerous reports have suggested possible associations between SNPs in the coding region of the ADRB2, mostly for Arg16Gly, Gln27Glu and Threonine 16 Isoleucine (Thr164Ile), but have produced conflicting results [11,12]. This polymorphism has been studied in a transfected cell system and has been shown to alter the agonist-binding properties of the receptor (Figure 1).

Figure 1: The structure of the human β2-receptor and the sites of polymorphic mutations of the receptor are shown in image.

Adrb2 variants and functional studies

Individual variations in physiological responses, expression and function of Adrb2, as well as individual differences in response to drugs that act on these receptors may relate to polymorphic variants of the receptor [13]. The Adrb2 regulates a number of metabolic and physiologic processes in various organs such as heart, lung and kidney [14]. While variation of Valine to Methionine at codon 34 (Val34Met) and Threonine to Isoleucine at codon 164 (Thr164Ile) are rare mutations [10,15]. A number of reports revealed that both the Arg16Gly and Gln27Glu variants may play a role in receptor down regulation. In studies among asthmatic patients, Arg16Gly substitution exaggerates agonist-mediated receptor down-regulation in response to β₂ agonists [16]. In another experiments, the Isoleucine substitution of the Threonine in residue 164 causes that receptor to have decreased basal and agonist-stimulated adenylyl cyclase activities and therefore, decreased affinity for β₂ agonists [17].

Adrb2 Variants and PCOS

Waterworth et al., first reported a strong linkage and association between the III/III genotype of INS VNTR and anovulatory PCOS [18]. Several studies have since looked for association of these VNTRs with PCOS and related phenotypes in different ethnic populations. Vanková et al., found no association with PCOS or related traits like BMI, insulin, glucose, or c-peptide levels [19]. Ferk et al., reported a significant association of class III INS VNTR alleles with PCOS and obese women with III/III INS VNTR genotype showed elevated insulin levels [20]. Similar associations with PCOS were not observed in Finnish [21], Croatian [22], Korean [23], and Han Chinese [24] populations. Recent meta-analysis confirmed a strong association of INS VNTR polymorphism with PCOS risk only in anovulatory women but not with the overall women with PCOS which may explain the contradictory results mentioned above [25]. Sheikh et al., reviewed Genetic Markers of PCOS on insulin resistance. They summarized the role of putative genetic variants contributing to the insulin resistance state frequently observed in PCO women [26]. These studies help to understand complex phenotypes of PCOS and are useful for in designing of therapies approaches. The aim of this study was to investigate polymorphism codon 16, 27,113 and 164 of beta2 adrenergic receptor in polycystic ovary syndrome (PCOS). The reason for choosing of these codons is the role of them in diseases associated with hyperactivity of the SAS, which has been reported till now. In this study, we investigated them in PCOS.

Subjects

This Case/control study was carried out at May 2014-2015. Totally 103 PCO patients from Reproductive Health Research Center of Imam Khomeini Hospital, Tehran, Iran participated in this study. In this study inclusion criteria were aged 20-40 years and the body mass index (BMI) fewer than 28 and exclusion criteria were no specific disease and no drugs for all women. The diagnosis of PCOS was made according to the joint criteria of the European Society of Human Reproduction and Embryology and the American Society of Reproductive Medicine (ESHRE/ASRM) [27]. Two out of three of the following criteria were met for the diagnosis: oligo-ovulation and/or anovulation (irregular menstrual cycle), clinical and/or biochemical signs of hyperandrogenism, polycystic ovaries (PCO) by ultrasound (≥ 12 or more follicles in each ovary measuring 2-8 mm in diameter). This study was approved by the Ethics Committee of Tehran University of Medical Sciences. The study objectives were explained to the patients before they entered the study, and an informed consent was obtained from all.

Sampling

Preparation of peripheral blood samples of PCO and control groups. Blood samples were collected in EDTA treated tubes and maintained at -20°C.

DNA extraction

Total Genomic DNA was extracted from these samples by using Blood & cell culture kit, Qiagen (Iran) according to manufacturer's instructions. Purity and concentration of genomic DNA was evaluated using Nanodrop and prepared a concentration 50 μg/ml as working tubes. Primer had been designed for PCR-based gene bank.

Genotyping of ADRB2gene: amplification of sample for preparation of PCR

ADRB2 Information (NC_000005.10:148826593...14882863 4) was extracted from NCBI Site. ADRB2 functional SNPs was selected for genotyping in this study is rs180088 (Thr164Ile). The SNP genotypes were determined by polymerase chain reaction (PCR). PCR reactions were performed using specific primers was designed by Primer3 online software. PCR reactions were carried out in a volume of 25 μl using 1 μl of genomic DNA, 7 μl Master Mix(Taq DNA Polymerase 2X Master Mix Red, 1.5 mm MgCL₂ ) , 15 μl ddH₂O, 1 μl Forward primer and 1 μl Reverse primer was used. The primer pairs were: 5' AAGCGGCTTCTTCAGAGCA 3' (forward) and 5'-GATGGCTTCCTGGTGGGTG-3' (reverse). The generated PCR product size using these primers is 759 bp. Reaction buffers were those included with these polymerases from the manufacturers. Temperature cycling was 95°C for 5 min, 95°C for 3 s, 61°C for 30 s, 72°C for 20 s and 72°C for 5 min for 30 cycles. 5 μl of the PCR reactions were then electrophoresed on 1.5% agarose gels and visualized with ethidium bromide staining and ultraviolet illumination. The allele-specific PCR technique was verified by direct dideoxy sequencing of PCR products generated using sequencing primers different from those used in the PCR (Figure 2).

Figure 2: Jells electrophorese .

Data was presented as frequency and percentage also odds ratio and 95% confidence interval. Comparison of mutation and zygote between PCO and control groups were done with chi-square test. Because unbalance and low sample size in control group a bootstrap estimation was used to check the hypothesis. Statistical analysis was done in SPSS 19 (SPSS Inc, Chicago, ILL). Statistical power of analysis was calculated with PASS (NCSS PASS program, NCSS, Kaysville, UT).

Sequencing results

Mutation in codon 27 was higher than other codon 70% but it was not significant difference with control group (Figure 3). All calculation approves this hypothesis that codon 27 mutation there is no significant difference between PCOs and normal women.

Figure 3: Sequencing of Codon27, CAA, mutant.

Mutation in codon 16 was 69.7%. It was not significant difference with control group. According to calculation we could not had any powerful inference about hypothesis, that codon 16 mutation was different between PCOs and normal women (Figure 4).

Figure 4: Sequencing of Codon16, CAA, mutant.

Mutation in codon 113 was 7% in PCOs women while it was not seen in control group because mutation in control group was not seen, calculation of power, odds ratio and bootstrapping method was not possible. If one woman with mutant gene added to control group, mutation rate in control group increase to 7.9%. Mutation rate in control comparison with 7% in PCOs group have not significant difference with good power. In other way if one woman without mutation added to control group, mutation rate in control comparison to PCOs group have not significant difference. We can show if 13 women without mutation add to control group mutation rate are not statistically significant differences. Finally we approve this hypothesis that mutation in codon 13 there is no significant difference between PCOs and normal women (Figure 5).

Figure 5: Sequencing of Codon113, GAT, normal.

Mutation rate in codon 164 in PCOs women was 44.4%. It was significantly higher than control group. If one woman without mutant gene added to control group, difference between two groups become deeply so it still be significant. In other way if one woman with mutation added to control group, mutation rate in control comparison to PCOs group have still be significant differences. Also if increase control groups to 16 women without mutation that significance in mutation rate will be remain. So we can reject this hypothesis that mutation in codon 164 there is no significant difference between PCOs and normal women (Figure 6). Comparison of mutation between PCO and normal women has shown in (Table 1).

Figure 6: Sequencing of Codon164, ATC, mutant.

Table 1: Comparison of mutation at different codon in Beta2 adrenergic gene between PCO and normal women.

Zygote in codon 27 and 16 were significant difference with control group (Table 2).

Table 2: Comparison of zygote in Beta2 adrenergic gen between PCO and normal women.

Our results show that women with PCOS had polymorphism only in codon 164 (P<0.002). Zygote in both codon 27 and 16 were significant difference in PCO and control group.

Studies on the β₂-adrenoceptor identified a total of 9 different polymorphisms. All of these differed from the accepted wild-type sequence by a single base change at different positions in the coding sequence of the gene. Because of redundancy in the amino acid code, a number of these polymorphisms are clinically silent. However, 4 polymorphisms, resulting from single base changes, altered the amino acid sequence of the receptor protein [28].

The relationship between neuronal activity of b-adrenergic receptors and the stimulatory effect of noradrenaline (NA) on steroid secretion found in the ovary [29] gives further support for a regulatory and complementary role between gonadotropins and sympathetic nerves. Lara in 1993 found that PCO induced by the administration of a single dose of EV to rats results in profound changes in ovarian catecholamine homeostasis, which were initiated before the development of cysts and persist after the cysts were formed [30,31]. These changes include an increased ovarian NA content, enhanced NA release from ovarian nerve terminals and down-regulation of b-adrenergic receptors in theca-interstitial cells and in granulosa cells [32]. The β₂-receptor is in the activated form when it is associated with the α-subunit of the Gs protein, together with a molecule of guanosine triphosphate (GTP). The replacement of the GTP by guanosine diphosphate dramatically reduces the affinity of the α-subunit for the receptor, causing dissociation and inducing the receptor to return to its low-energy inactivated form. It is probable that β₂-agonists have their effects not through inducing a conformational change in the receptor but rather by binding to and temporarily stabilizing receptors in their activated state (ie, bound to Gs-GTP) [33]. Associated with b2-adrenoceptor activation is the autoregulatory process of receptor desensitization. This process operates as a safety device to prevent overstimulation of receptors in the face of excessive b2-agonist exposure. Desensitization occurs in response to the association of the receptor with the agonist molecule. The mechanisms by which desensitization can occur consist of 3 main processes: (1) uncoupling of the receptors from adenylate cyclase, (2) internalization of uncoupled receptors, and (3) phosphorylation of internalized receptors. The extent of desensitization depends on the degree and duration of the b2-adrenoceptor/b2- agonist response. The principal mechanism of homologous, shortterm b2-agonist–promoted desensitization of the b2-adrenoceptor is phosphorylation of the receptor by PKA, beta adrenoceptor kinase, or other closely related G protein–coupled receptor kinases [34]. Under experimental conditions, increased ovarian sympathetic tone in rat with EV-induced PCO has been evidenced by elevations in tyrosine hydroxylase activity and NA concentration, down-regulation of the b2- AR, and increased production of ovarian nerve growth factor, a target-derived neurotrophin [35]. Desensitization or down-regulation of beta2 adenoceptors can be accrue after more prolonged agonist exposure, an internalization of receptors occurs that results in some loss from the cell surface. This process, termed sequestration, might play a major role in short-term regulation of the receptor, because while sequestered, dephosphorylation of the receptor occurs (Figure 7) [36]. Internalization takes longer to reverse than uncoupling, but full reversal normally occurs within hours. Receptor trafficking as part of the overall process of receptor desensitization has now been investigated in the form of kinetic analysis of internalization and recycling of the human b2-receptor. Under resting conditions, recycling of the receptor reflects 1-phase exponential kinetics with a half-life of 7.5 minutes [28].

Figure 7: Beta2-Adrenoceptor trafficking. After b2-agonist binding, PKA, b-adrenoceptor kinase, or both phosphorylate the b2-receptor, leading to desensitization. b- Arrestin binding is stimulated, and the resulting b2-receptor/ b-arrestin complex is sequestered from the cell surface and internalized. The receptor is then dephosphorylated, b-arrestin dissociates, and the b2-receptor is recycled back to the membrane. Beta2-AR, b2-adrenergic receptor; Pi, phosphorylation.

In this study, we tried to investigate the link between this desensitization and probability of polymorphism in Beta 2-Adrenergic receptor.

The Arg16/Gln27 receptor underwent 26 ± 3% down-regulation. In contrast, the Gly16/Gln27 receptor displayed 41 ± 3% downregulations. The Gly16/Glu27 receptor displayed a similar level of down-regulation (39 ± 4%) compared to the Gly16/Gln27 receptor. The data suggest that position 16 is the major polymorphic locus that affects agonist-promoted down-regulation [36]. The polymorphism of Beta 2-Adrenergic receptor is at amino acid 164, which can either be threonine (Thr) or isoleucine (Ile). This polymorphism is much rarer than that at amino acid 16 or 27, with an allelic frequency of about 1%, but is potentially interesting in that amino acid 164 is situated in the fourth transmembrane-spanning domain of the receptor and is adjacent to serine 165, which has been predicted to interact with the OH groups of adrenergic ligands [37]. This polymorphism has been studied in a transfected cell system and has been shown to alter the agonist-binding properties of the receptor. Cells expressing Ile 164 were found to have approximately 4 times less ligand affinity. This alteration in binding affinity was reflected in a reduced capacity for the receptor to activate adenylate cyclase, relative to the wild-type (Thr 164) form of the receptor [38]. Association studies have been hampered by the low prevalence of the Thr164Ile polymorphism, which necessitates large patient groups to reach robust conclusions. Piscione et al. now report that Ile164 is much more prevalent in a population of coronary artery disease patients than in a control population (12% vs. 3%), and that within the patient group, Ile164 carriers exhibit a more severe pathology than those with Thr164 genotype. Similarly, a group of patients with peripheral artery disease also exhibited a high prevalence of the Ile164 genotype (7%) and a more severe clinical phenotype than those with Thr164 [39]. However, the Thr164Ile polymorphism may not only have pathophysiological but also therapeutic relevance. Reduced B2AR responses of Ile164 carriers were also reported for lipolysis in isolated adipocytes [40]. Results of this study indicated that in review of gen polymorphism of codon 16, 27, 113 and 164 from beta2 adrenergic receptor, only codon 164 (44.4%) has accompanied with Polycystic Ovary Syndrome. However, the Thr164Ile polymorphism may not only have pathophysiological but also therapeutic relevance [41]. Identification of the candidate genes and understanding of their function holds the promise for the establishment of the specific molecular basis for PCOS. In the future more studies on different populations with a larger sample size are needed for proving of the functional candidate genes that play important role in the etiology of PCOS. We had two limitations in this study: financial obligation and small sample size in control group. The pharmacogenetics studies can help to etiology and drug therapy in PCOS.