Vol 16 No 02 March-April 2019 141 UROLOGICAL ONCOLOGY Association Study of Retinoic Acid Related Orphan Receptor A (RORA) Gene and Risk of Prostate Disorders Mohammad Taheri1, Rezvan Noroozi2, Arash Dehghan3, Golnaz Atri Roozbahani4, Mehrnoosh Musavi5, Mir Davood Omrani1*, Soudeh Ghafouri-Fard6* Purpose: Prostate cancer (PCa) and benign prostate hyperplasia (BPH) are two prevalent disorders among men with considerable mortality and morbidity. Several association studies have been conducted in different popula- tions to find genetic loci linked with these disorders. Retinoic acid-receptor-related orphan receptor alpha (RORA) codes for a transcription factor which regulates expression of several cancer-related genes. Besides, RORA has been shown to be down-regulated in PCa tissues and cell lines. Materials and Methods: In the present study we evaluated genotype and allele frequencies of rs11639084 and rs4774388 variants within RORA gene in PCa and BPH patients compared with healthy subjects. Result: The rs11639084 and rs4774388 alleles were not different between PCa and normal groups 95% CI: 0.52- 1.24, OR = 1.04, P = .34; 95% CI: 0.48-1.33, OR = .79, P = .39 respectively. Moreover, we did not detect any significant difference in allele, genotype or haplotype frequencies of these SNPs between the other study groups. Conclusion: The mentioned RORA variants are possibly not involved in the pathogenesis of PCa and BPH. Fu- ture studies are needed to assess the associations between other variant within this gene and PCa risk to suggest a putative mechanism for involvement of RORA in PCa. Keywords: benign prostatic hyperplasia; prostate cancer; retinoid-related orphan receptor alpha; RORA; single nucleotide polymorphism INTRODUCTION Prostate cancer (PCa) and benign prostate hyperpla-sia (BPH) are two prevalent disorders among men with considerable mortality and morbidity(1). Several researches have focused on evaluation of risk factors of these disorders at genomic (2,3) and transcriptomic levels (4). Among candidate genes whose involvement in PCa pathogenesis has been demonstrated is retinoic acid-receptor-related orphan receptor alpha (RORA). This gene encodes a transcription factor which is cate- gorized as one of the orphan nuclear receptors. The ex- istence of response elements for RORA in the promoter region of cell cycle-related genes implies its involve- ment in the regulation of cell cycle. Moreover, expres- sion of RORA in androgen-independent PCa cells has suppressed cell growth as demonstrated by in vitro and in vivo studies (5). More evidences for tumor suppressor role of RORA have been provided by observation of its down-regulation in various cancer tissues (6). Certain single nucleotide polymorphisms (SNPs) within this gene have been associated with risk of breast cancer in different ethnic groups (7,8). Despite the prominent role of RORA in PCa pathogenesis, there is no study for assessment of the association between RORA variants and risk of PCa or BPH. Consequently, in the present 1 Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 2 Phytochemistry Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. 3 Department of pathology, Hamadan University of Medical Sciences, Hamadan, Iran. 4 Faculty of Life science and Technology, Shahid Beheshti University, Tehran, Iran. 5 Student Research Committee, Hamadan University of Medical Sciences, Hamadan, Iran. 6 Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran. *Correspondence: Urogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Tel & Fax: +982123872572, E-mails: davood_omrani@yahoo.co.uk. Received January 2018 &Accepted April 2018 study we evaluated genotype and allele frequencies of rs11639084 and rs4774388 variants in PCa and BPH patients compared with healthy subjects to find if these variants are involved in the pathogenesis of these dis- orders or can be used as genetic risk factors for PCa or BPH. MATERIALS AND METHODS Study participants Subjects In the present case-control study a total of 144 PCa cas- es, 177 BPH cases and 112 normal males participated. PCa and BPH patients were selected from newly diag- nosed patients in whom the histological examination of samples obtained from transrectal needle biopsy or transurethral resection of the prostate confirmed the di- agnosis of disorder. Control subjects were normal age- matched males selected from a routine hospital-based health survey during 2016. The thee study groups have also been matched in age and body mass index (BMI). The study protocol has been approved by ethi- cal committee of Shahid Beheshti University of Medi- cal Sciences. All study participants were selected from hospitals affiliated with Shahid Beheshti University of Medical Sciences after assessment of their compli- Urological Oncology 142 ance with the study inclusion criteria and signing the informed consent. Control subjects had no history of lower urinary tract symptoms, prostate enlargement or family history of prostate cancer. PCa or BPH was diag- nosed through examination of clinical prostate biopsies by an expert pathologist especially in BPH patients with high PSA levels (4.0 ng/ml or more) in whom transrec- tal biopsies ruled out the presence of PCa. Those with insufficient pathologic sample for evaluation, patients with the history of previous malignancies in other or- gans and any former chemo-radiotherapy were ex- cluded from the study. Blood samples were collected from patients before initiation of any treatments such as surgery, radiotherapy, and chemotherapy. We also collected clinicopathologic characteristics of study participants including prostate weight, PSA levels and Gleason score through filling questionnaires and evalu- ation of patients' clinical reports. Genotyping of rs11639084 and rs4774388 We genotyped the rs11639084 (C/T) and rs4774388 (C/T) variants lcated in the intronic regions of the RORA gene by tetra primer-amplification refractory mutation system-PCR (4P-ARMS-PCR) method using primers designed by PRIMER1 online tool as reported in our previuos study(9). Briefly, for each reaction we used 100 ng of genomic DNA, 5 pmol/l of outer prim- ers, 10 pmol/l of inner primers and 12.5 μl Taq DNA Polymerase 2 × Master Mix Red (Ampliqon, Denmark). All reactions were performed in a FlexCycler (Analytik Jena, Germany) with a PCR program consisted of an initial denaturation at 95 °C for 5 min and subsequent 35 cycles of 95 °C for 45 seconds, 55 °C for 45 sec- onds and 72 °C for 45 seconds. The results obtained by 4P-ARMS-PCR method were verified by Sanger se- quencing of 10% of total samples in ABI 3730xl DNA analyzer (Macrogen, Korea). Statistical analysis SNPStats was used for assessment of allele, genotype and haplotype frequencies in distinct study groups and their accordance with Hardy-Weinberg equilibrium us- ing Chi-square test (two-sided)(10). The association of rs11639084 and rs4774388 polymorphisms with PCa and BPH risk was evaluated in all assumed inheritance models and described using odds ratios (ORs) and 95% confidence intervals (CIs). Haplotype frequencies for RORA were computed using the SNPAnalyzer pro- gram (Istech Ltd, Goyang-si, Korea) based on the ex- pectation-maximization algorithm. The pairwise link- age disequilibrium (LD) between mentioned SNPs was assessed through measurement of D' and r2 values. D' was described as the ratio of the unstandardized coef- ficient to its maximal/minimal value. P value less than 0.05 were considered statistically significant. RESULTS General data of study participants Clinical and demographic data of study participants in- cluding the 144 PCa cases, 177 BPH cases and 112 nor- mal males which were obtained through questionnaires and assessment of clinical reports are summarized in Table 1. Results of SNPs genotyping The assessed SNPs in the current study are located in intronic regions of RORA gene. Detailed information of these two SNPs is demonstrated in Table 2. We as- sessed the compliance of these SNPs with Hardy-Wein- berg equilibrium. As demonstrated in Table 3, geno- type frequencies of these SNPs in all three study groups are in Hardy-Weinberg equilibrium. No significant difference has been found in allele and genotype frequencies of rs11639084 and rs4774388 SNPs between three study groups (PCa, BPH and nor- mal controls). Table 4 shows the allele and genotypes frequencies of these SNPs in distinct study groups. We further assessed the frequencies of four supposed haplotypes of these two SNPs in distinct study groups but did not detect any difference between PCa, BPH and healthy subjects (Table 5). RORA and prostate disorders-Taheri et al. Table 1. Demographic and clinical data of study participants. Variables Prostate cancer group BPH group Controls Age (mean ± SD) 68.94 ± 9.89 66.96 ± 10.97 64 ± 9.1 BMI (mean ± SD) 25.07 ± 3.67 24.97 ± 3.47 24.17 ± 4.25 Prostate weight (gr) (mean ± SD) 52.01 ±25.32 62.17 ± 23.63 - PSA (ng/mL) (mean ± SD) 9.26 ± 9.53 8.54 ± 6.1 <4 < 4 27 (18.75%) 32 (18%) 112 (100%) 4 -10 75 (52.0%) 89 (50.2%) 0 > =10 42 (28.25%) 56 (31.8%) 0 Smoking Never smoker (%) 74 (51.3%) 118 (66.6%) 75 (67%) Current or former smoker (%) 70 (48.7%) 59 (33.4%) 37 (33%) Gleason score < =6 74 (51.3%) - - > 6 7 0 (49.7%) - - Abbreviations: BMI, Body Mass Index, BPH, Benign Prosttae Hyperplasia, PSA, prostate specific antigen, SD, standard deviation. SNP Position Minor Allele MAF MAC Type rs11639084 Chr15:60774317 T .24 1197 Intron rs4774388 Chr15:61174799 C .30 1507 Intron Abbreviations: MAF, Minor Allele Frequency, MAC, Minor Allele Content (the average amount of minor alleles per subject). Table 2. Descriptive information of rs11639084 and rs4774388 of RORA gene. Vol 16 No 02 March-April 2019 143 DISCUSSION In the present study we assessed allele, genotype and haplotype frequencies of two SNPs within RORA gene in Iranian patients with PCa and BPH compared with healthy subjects and demonstrated no significant dif- ference between three study groups. RORA has been shown to participate in the pathogenesis of a wide range of human disorders including attention-deficit hyperac- tivity disorder (11), bipolar disorder(12), major depression (13), autism(9), multiple sclerosis(14) and breast cancer (7). Previous studies have provided evidences for RORA contribution in suppression of PCa cell growth. RORA has been shown to down-regulate the expression of 5-lipoxygenase and restrict the mitogenic function of fatty acids on PCa cells. Consequently, RORA has been suggested as a therapeutic target in PCa (15). In addition, down-regulation of RORA in cancerous tissues might change the expression of its downstream target genes such as CDK inhibitor p21 and N-myc which partic- ipate in carcinogenesis process(16). We have recently demonstrated the association of the rs4774388 with- in this gene with autism spectrum disorder (9) as well as breast cancer risk(7). The rs4774388 is supposed to change the affinity of the encoded protein to bind with POU5F1 as predicted by HaploReg v4.1 (17). The ret- rogene POU5F1B encodes a homolog of the principal embryonic stem cell transcription factor Oct4. Breyer et al. have demonstrated the correlation between 8q24 risk alleles and decreased expression of POU5F1B gene in prostate tissues. In addition, they suggested the asso- ciation between harmful POU5F1B missense variants and PCa. Their experiments showed the involvement of POU5F1 in self-renewal capability of embryonic stem cells as well as the pathogenesis of cancer(18). Be- sides, participation of RORA in regulation of circadian rhythm(19) and dysregulation of circadian rhythm and melatonin pathways in PCa(20) increase the possibility of contribution of RORA variants in PCa risk. Taken together, we hypothesized that RORA variants might be regarded as risk factor for PCa. The absence of asso- ciations between these variants and PCa or BPH risk in Iranian population does not exclude the participation of RORA in the pathogenesis of these disorders. Our study has some limitations. First, due to the relative small sample size we could not analyze genotype and allele frequencies in subgroups of PCa patients includ- ing those with small tumor size, low Gleason grade or low stage separately. In addition, we could not assess the associations between mentioned SNPs and patients' survival or response to treatments. Finally, we did not assess associations between other functional RORA variants and risk of PCa or BPH. CONCLUSIONS Although we could not find any association between two variants within RORA gene and prostate disorders, based on the proposed function of this gene, other var- iants within this gene might alter risk of PCa or BPH. So, future studies are needed to assess the associations between other variant within this gene and PCa risk to suggest a putative mechanism for involvement of RORA in PCa. Table 3. Exact test for Hardy-Weinberg equilibrium. SNP rs11639084 P-value rs12826786 P-value All CC CT TT TT CT CC Cancer 144 85 (59%) 54 (37.5%) 5 (3.4%) .47 84 (58.3%) 49 (34%) 11 (7.6%) .37 BPH 177 119 (67.2%) 52 (29.3%) 6 (3.3%) 1 96 (54.2%) 63 (35.6%) 18 (10.1%) .13 Control 112 76 (67.8%) 30 (26.7%) 6 (5.3%) .21 58 (51.7%) 44 (39.2%) 10 (8.9%) .65 SNP Model Sample size (%) Cancer vs. Control BPH vs. Control Cancer vs. BPH Cancer(%) BPH(%) Control(%) OR P-value OR P-value OR P-value Allele T vs. 64 (22) 64 (18) 42 (19) 1.04 (.52-1.24) .34 1.04 (.68-1.61) .84 .77 (.52-1.14) .19 224 (78) 290 (82) 182 (81) Co-dominant TT vs. CC 5 (3.5) 6 (3.4) 6 (5.4) 1.34 (.39-4.58) .64 1.56 (.49-5.03) .45 .86 (.25-2.90) .80 CT vs. CC 54 (37.5) 52 (29.4) 30 (26.8) .62 (.36-1.07) .08 .90 (.53-1.54) .71 .69 (.43-1.10) .12 Dominant TT+CT vs. 59 (41) 58 (32.8) 36 (32.1) .68 (.41-1.14) .15 .97 (.59-1.61) .91 .70 (.44-1.12) .13 CC 85 (59) 119 (67.2) 76 (67.9) rs Recessive TT vs. 5 (3.5) 6 (3.4) 6 (5.4) 1.57 (.47-5.30) .46 1.61 (.51-5.13) .42 .97 (.29-3.26) .97 1163 CT+CC 139 (96.5) 171 (96.6) 106 (94.6) 9084 Over TT+CC vs. 90 (62.5) 125 (70.6) 82 (73.2) 1.64 (.96-2.81) .07 1.14 (.67-1.92) .63 1.44 (.90-2.30) .12 dominant CT 54 (37.5) 52 (29.4) 30 (26.8) Allele C vs. 71 (25) 99 (28) 64 (29) 1.22 (.82-1.81) .32 .91 (.62-1.32) .62 1.34 (.94-1.92) 0.10 T 217 (75) 255 (72) 160 (71) Co-dominant CC vs. TT 11 (7.6) 18 (10.2) 10 (8.9) 1.32 (.52-3.30) .56 .92 (.39-2.13) .84 1.43 (.64-3.20) .38 CT vs. TT 49 (34) 63 (35.6) 44 (39.3) 1.30 (.77-2.20) .33 1.16 (.69-1.91) .57 1.12 (.70-1.80) .62 Dominant CC+CT vs. 60 (41.7) 81 (45.8) 54 (48.2) 1.30 (.79-2.14) .3 1.10 (.69-1.77) .68 1.18 (.76-1.84) .64 TT 84 (58.3) 96 (54.2) 58 (51.8) Recessive CC vs. 11 (7.6) 18 (10.2) 10 (8.9) 1.18 (.48-2.90) .71 .87 (.38-1.95) .73 1.37 (.62-2.99) .43 rs TT+CT 133 (92.4) 159 (89.8) 102 (91.1) 477 Over TT+CC vs. 95 (66) 114 (64.4) 68 (60.7) .79 (.48-1.33) .39 .85 (.52-1.39) .53 .93 (.59-1.48) .77 4388 dominant CT 49 (34) 63 (35.6) 44 (39.3) Abbreviations: BPH, Benign Prostate Hyperplasia, OR, Odd Ratio Table 4. Allele and genotype frequencies of rs11639084 and rs4774388 SNPs in three study groups (PCa, BPH and normal controls). RORA and prostate disorders-Taheri et al. Urological Oncology 144 ACKNOWLEDGEMENT The current study was supported by a grant from Shahid Beheshti University of Medical Sciences. CONFLICT ON INTEREST The authors declare no conflicts of interest. REFERENCES 1. Ghafouri-Fard S, Ousati Ashtiani Z, Sabah Golian B, Hasheminasab SM, Modarressi MH. Expression of two testis-specific genes, SPATA19 and LEMD1, in prostate cancer. Arch Med Res. 2010;41:195-200. 2. Taheri M, Pouresmaeili F, Omrani MD, et al. Association of ANRIL gene polymorphisms with prostate cancer and benign prostatic hyperplasia in an Iranian population. Biomark Med. 2017;11:413-22. 3. Taheri M, Habibi M, Noroozi R, et al. HOTAIR genetic variants are associated with prostate cancer and benign prostate hyperplasia in an Iranian population. Gene. 2017;613:20-4. 4. Faramarzi S, Ghafouri-Fard S. Expression analysis of cancer-testis genes in prostate cancer reveals candidates for immunotherapy. Immunotherapy. 2017;9:1019-34. 5. Moretti RM, Marelli MM, Motta M, et al. Activation of the orphan nuclear receptor RORalpha induces growth arrest in androgen- independent DU 145 prostate cancer cells. Prostate. 2001;46:327-35. 6. Roshan-Moniri M, Hsing M, Butler MS, Cherkasov A, Rennie PS. Orphan nuclear receptors as drug targets for the treatment of prostate and breast cancers. Cancer Treatment Reviews. 2014;40:1137-52. 7. Taheri M, Omrani MD, Noroozi R, Ghafouri- Fard S, Sayad A. Retinoic acid-related orphan receptor alpha (RORA) variants and risk of breast cancer. Breast disease. 2017;37:21-5. 8. Truong T, Liquet B, Menegaux F, et al. Breast cancer risk, nightwork, and circadian clock gene polymorphisms. Endocr Relat Cancer. 2014;21:629-38. 9. Sayad A, Noroozi R, Omrani MD, Taheri M, Ghafouri-Fard S. Retinoic acid-related orphan receptor alpha (RORA) variants are associated with autism spectrum disorder. Metab Brain Dis. 2017;32:1595-601. 10. Solé X, Guinó E, Valls J, Iniesta R, Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics. 2006;22:1928-9. 11. Neale BM, Lasky-Su J, Anney R, et al. Genome- Wide Association Scan of Attention Deficit Hyperactivity Disorder. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics. 2008;147B:1337-44. 12. Le-Niculescu H, Patel SD, Bhat M, et al. Convergent Functional Genomics of Genome-Wide Association Data for Bipolar Disorder: Comprehensive Identification of Candidate Genes, Pathways and Mechanisms. American Journal of Medical Genetics Part B-Neuropsychiatric Genetics. 2009;150B:155- 81. 13. Garriock HA, Kraft JB, Shyn SI, et al. A genomewide association study of citalopram response in major depressive disorder. Biol Psychiatry. 2010;67:133-8. 14. Eftekharian MM, Noroozi R, Sayad A, et al. RAR-related orphan receptor A (RORA): A new susceptibility gene for multiple sclerosis. J Neurol Sci. 2016;369:259-62. 15. Moretti RM, Montagnani Marelli M, Sala A, Motta M, Limonta P. Activation of the orphan nuclear receptor RORalpha counteracts the proliferative effect of fatty acids on prostate cancer cells: crucial role of 5-lipoxygenase. Int J Cancer. 2004;112:87-93. 16. Zhu Y, McAvoy S, Kuhn R, Smith DI. RORA, a large common fragile site gene, is involved in cellular stress response. Oncogene. 2006;25:2901-8. 17. Ward LD, Kellis M. HaploReg: a resource for exploring chromatin states, conservation, and regulatory motif alterations within sets of genetically linked variants. Nucleic Acids Research. 2012;40:D930-D4. 18. Breyer JP, Dorset DC, Clark TA, et al. An expressed retrogene of the master embryonic stem cell gene POU5F1 is associated with prostate cancer susceptibility. Am J Hum Genet. 2014;94:395-404. 19. Solt LA, Kojetin DJ, Burris TP. The REV- ERBs and RORs: molecular links between circadian rhythms and lipid homeostasis. Future Medicinal Chemistry. 2011;3:623-38. 20. Gu F, Zhang H, Hyland PL, et al. Inherited variation in circadian rhythm genes and risks of prostate cancer and three other cancer sites in combined cancer consortia. Int J Cancer. 2017;141:1794-802. Table 5. Haplotype frequencies in PCa, BPH and healthy subjects. rs11639084 rs4774388 PCa BPH Control PCa vs. Control BPH vs. Control PCa vs. BPH OR (95% CI) P-value OR (95% CI) P-value OR (95% CI) P-value C T .58 .60 .57 1.00 --- 1.00 --- 1.00 --- C C .20 .22 .24 1.20 (.76 - 1.90) .44 1.12 (.73 - 1.72) .61 1.04 (.68 - 1.59) .85 T T .17 .12 .14 .81 (.46 - 1.43) .47 1.21 (.67 - 2.20) .53 .64 (.38 - 1.10) .11 T C .05 .06 .05 .98 (.32 - 3.04) .98 .82 (.30 - 2.22) .69 1.21 (.52 - 2.83) .66 Abbreviations: BPH, BPH, Benign Prostate Hyperplasia, OR, Odd Ratio, PCa, Prostate Cancer RORA and prostate disorders-Taheri et al.