UROLOGICAL ONCOLOGY The Association of MYNN and TERC Gene Polymorphisms and Bladder Cancer in a Turkish Population Fikriye Polat 1, Meral Yilmaz 2*, Songul Budak Diler3 Purpose: Researchers reported that, MYNN rs10936599 polymorphism is in strong or moderate linkage disequilib- rium with SNPs within the 3q26.2 chromosomal regions that also include the TERC gene. In addition, it has been reported that MYNN rs10936599 had a strong cumulative association with bladder cancer risk, and TERC gene suppresses cell growth in bladder cancer cell lines. Therefore, we aimed to determine whether polymorphisms of MYNN rs10936599 and TERC rs2293607 play any roles for bladder cancer in the Turkish population in this study. Materials and Methods: In this case-control study, 70 patients and 150 controls were investigated. Genotyping analysis was performed by polymerase chain reaction, restriction fragment length polymorphism and DNA se- quencing techniques. Results: Genotype distribution between study groups for MYNN rs10936599 SNP was significantly different (P = .001); although there was no difference in genotype distribution for TERC rs2293607 SNP. In addition, pa- tients with CT genotype and CT+TT genotype combination of MYNN SNP have a decreased risk for bladder can- cer. Two times increased risk ratio on development of bladder cancer was obtained for CC genotype of the SNP (P = .001). Besides, it was found that genotype combination of GG+AG/CC versus AA/CC genotypes (TERC/MYNN) showed stronger correlation. We observed that statistically significant relationship between the C-G haplotypes of two polymorphisms and bladder cancer risk (P = .0001). Conclusion: At the end of the study, we suggested that there may exist an association between a combination of MYNN rs10936599 and TERC rs2293607 polymorphisms and development of bladder cancer in Turkish popula- tion. Keywords: Bladder cancer; MYNN gene; polymorphism; TERC gen INTRODUCTION Bladder cancer (BC) is a major problem and, 11th most common cancer in the world(1). BC is higher in men than women, and its incidence increases with age for both gender, peaking at the seventh decade(2). Genetic and potential environmental factors play role in the etiology of the disease and, familial cancer history increasing risk of BC. In addition, there is important heterogeneity in terms of its clinical and genetic back- grounds of urothelial carcinomas and, the heterogenei- ty partly originates from different changes in different genes that affect various mechanisms associated with cell proliferation and cancer(3, 4). A genome-wide associ- ation studies (GWAS) have informed that new locus on chromosome (chr) 3q may be correlated with BC risk. In addition, it has been reported that the locus on chro- mosome 3q is included in MYNN gene and TERC gene and, these genes are strong candidates for the associa- tion with bladder cancer(4). Myoneurin (MYNN) gene locates on 3q26.2 and en- codes a member of the BTB/POZ and zinc finger (ZF) domain-containing protein family that is involved in 1Department of Mathematics and Science Education, Faculty of Education, Kocaeli University, Kocaeli 41380, Turkey. 2Department of Research Centre, Faculty of Medicine, Cumhuriyet University, Sivas 58140, Turkey. 3Department of Biotechnology, Faculty of Science and Letters, Nigde Ömer Halisdemir University, Nigde 51240, Turkey. *Correspondence: Department of Research Centre, Faculty of Medicine, Cumhuriyet University, 58140-Sivas, Turkey. Phone: +90 346 2191156, Fax: +90 346 2191155, e-mail: meralylmz@cumhuriyet.edu.tr. Received August 2017 & Accepted May 2018 the control of gene expression(5). Certain polymor- phic regions were discovered on the MYNN gene. The rs10936599 polymorphism is one of these regions. The function of rs10936599 polymorphism on the MYNN gene is not known. Some research concludes that this synonymous variation is associated with both longer telomeres and the colorectal cancer or patients with adenomas(6-8). Additionally, it has been suggested that the MYNN polymorphism may be associated with ova- ry and bladder cancers(5,9). Furthermore, the variation which is close to Telomerase RNA component (TERC) gene participates at least partly in tumorigenesis at early stages(7,10). Telomerase is a specialized ribonucleoprotein poly- merase that adds TTAGGG repeats to telomere ends in soma human cells, including stem cells. The holo- enzyme consists of a protein component with reverse transcriptase (TERT) activity, and a RNA component (TERC). The TERT utilizes the TERC as a template to add repeats to the existing telomeres (7, 11). The TERC is a 451 base pairs (bp) long gene, located on chromo- some 3q26.2. TERC is a template for telomeric DNA Urological Oncology 50 Vol 16 No 01 January-February 2019 51 synthesis. Besides, it has the same important roles in- cluding catalysis, accumulation, localization and holo- enzyme assembly (12). Telomerase is up-regulated in the majority of human cancers. The increased telomerase activity is thought to be required to allow tumor cells to divide after genetic rearrangements enabled by tel- omere dysfunction(13). Consistent the data, Soder et al. (1997) reported that the level of TERC expression in- creases with tumor progression. It has been known that gene polymorphisms may cause significant changes in enzyme activity(14). According to remarkable data in the study of Figueroa and co-workers (2014), it has been re- ported that the rs10936599 SNP is in complete Linkage Disequilibrium with SNP rs2293607. TERC has been appeared to affect telomere length and mRNA fold- ing in several functional studies. Based on the litera- ture, no study has investigated the association of TERC rs2293607 polymorphism and BC risk. Until now, there has been no study investigating the effects of MYNN rs10936599 and TERC rs2293607 polymorphisms on BC risk. Therefore, we aimed to determine whether MYNN gene rs10936599 and TERC rs2293607 poly- morphisms that were closely located on 3q26.2 play any roles in the bladder cancer in a Turkish population. MATERIALS AND METHODS Study Population: The patients and controls were se- lected among the ones from urology clinic of Lulebur- gaz and Niğde State Hospital, Turkey. We investigat- ed the MYNN rs10936599 and TERC rs2293607 SNPs (Single Nucleotide Polymorphisms) in 70 bladder can- cer patients and 150 healthy controls. Protocol of the present study was reviewed and approved by the insti- tutional review board of the Local Human Ethics Com- mittee (Decision number: KAEK 2014/144). Informed consent was submitted by all subjects when they were enrolled. Inclusion and exclusion criteria: Patient group was gen- erated with individuals who have been diagnosed blad- der cancer by histopathological examination. Bladder cancer type of all patients is transitional cell carcinoma. Patients, who have received any chemotherapy or radi- otherapy, were not accepted in the study. The control group matched with age and gender distributions of patients was selected from healthy volunteers without bladder cancer history. DNA Isolation and Genotyping: Genomic DNA was extracted from the whole blood treated with EDTA using the QIAamp DNA Blood Mini Kit (Maryland, USA), according to the manufacturer’s guidelines. PCR amplifications of both the polymorphic regions in MYNN and TERC genes were using specific primer sets (for MYNN rs10936599 polymorphism F:5’- TCAA- GGGTAAAATTCCATTCTG-3’ and R:5’- TCACA- GAGAAAACCTGCTTCC-3’; for TERC rs2293607 polymorphism F:5’-AGTTCGCTTTCCTGTTGG-3’ and R:5’- ATTCATTTTGGCCGACTT-3’). The PCR was performed in a final reaction volume of 20 µl containing 10 ng of genomic DNA, 10 pmol of each primer, 5x FIREPol Master Mix (Solis BioDyne). PCR reaction was made on these conditions: after ini- tial denaturation at 95°C for 5 minutes, then followed by 38 cycles including of denaturation at 95°C for 40 seconds for denaturation, 58°C in the rs10936599 pol- ymorphism, 59°C in the rs2293607 polymorphism for 30 seconds for annealing, and 72°C for 30 seconds for extension. The reaction was completed by a final exten- sion of 5 min at 72°C. Evaluations: SNP rs10936599 is a point mutation oc- curring with C → T substitution at nucleotide 18 in MYNN gene and, this mutation causes coding of a syn- onymous variant (His6His). SNP rs2293607 is a point mutation occurring with A → G substitution at nucle- otide 514 in TERC gene (15). PCR products of MYNN and TERC polymorphic regions were digested with HpyCH4III at 37°C for over-night and BsrDI at 65°C for 30 min, respectively. PCR products were separated by electrophoresis on 2% agarose gels, and visualized under ultraviolet (UV) illumination after nucleic acid staining solution (ECO Safe). The PCR product size for MYNN rs10936599 SNP was 104 base pair (bp) and the wild-type allele (C) contains two fragments of 58 and 46 bp. The polymorphic variant (T) was seen a frag- ment of 104 bp. PCR product size for TERC rs2293607 SNP, was 159 bp. After enzymatic digestion of these products, the fragment sizes were 94 bp and 65 bp for the wild type (A). A fragment of 159 bp was seen for the variant allele (G). Statistical analysis: The genotype and allele frequen- cies of two SNPs were tested for Hardy-Weinberg Equilibrium using a chi-square (χ2) test. Deviations from Hardy-Weinberg equilibrium (HWE) were ana- lyzed by using Michael H. Court’s (2005-2008) online calculator. On the result of power analysis which was performed for detecting an association between BC and the studied polymorphisms, sample sizes were found to Table 1. Demographic characteristics of bladder cancer patients and controls Parameters Patients Controls p value OR (95% CI) n = 70 (%) n = 150 (%) aAge year ± SD 61.95 ± 10.63 59.41 ± 12.92 .15 (age range) (25-81) (22-94) bSex .09 Male 61 (87.1) 118 (78.7) Female 9 (12.9) 32 (21.3) bSmoking status .002* 2.55 (1.42-4.58) Smoker 36 (51.4) 44 (29.3) Non-smoker 34 (48.6) 106 (70.7) Abbreviation: ±SD, Standard derivation; *p < .05; significantly different from control group; aContinuous variables were compared by independent samples t-test; bCategorical variables including sex and smoking status were compared by chi square test. MYNN and TERC gene polymorphisms in bladder cancer-Polat et al. be sufficient for case and control groups consisting of 70 and 150 individuals (α: 0.05, β: 0.20 and test pow- er; 0.80). Statistically analysis of demographic feature was performed via student’s t-test and chi-square tests by using SPSS version 18. The frequencies of genotype and allele for these two SNPs in patients and controls were compared using Cochrans’s and Mantel-Haenszel statistics test. For each polymorphism, unconditional logistic regression was used to calculate adjusted (with smoking habit and gender) odds ratios (OR) in 95% confidence intervals (95% CI) for BC. P < .05 value was considered as statistically significant. EH program was used for analysis of haplotype frequencies. RESULTS The demographic characteristics of patients and con- trols were demonstrated on Table 1. When frequen- cies of mean age and gender were compared in both groups, the control group was found to be compatible with patients (for mean age P = .051, for gender P = .09). Smoker count in patient group (51.4%) compared with those in controls was found significantly higher (P = .002). Frequencies of genotype and allele for two polymor- phisms in patient and control groups were shown in Ta- ble 2. Genotype distribution of MYNN rs10936599 SNP among both groups was different and, the value was statistically significant (P = .001). In addition, patients with CT genotype and CT+TT genotype combination versus CC genotype and T allele versus C allele of the MYNN polymorphism compared those with controls have a decreased odds ratio for BC. Similar odds ratio was also obtained when the heterozygous genotype to- gether with other risk factors such as gender and smok- ing habit were evaluated. All these data are illustrated in Table 2. Interestingly, the frequency of CC genotype (wild type for rs10936599) in patients was higher than one in controls. When CC genotype was compared to other genotypes of the MYNN polymorphism among case-control groups, approximately 2 times increased odds ratio between BC development and this genotype was found [P = .001, OR = 2.64 (1.46-4.77)]. Frequencies of genotypes (AA, AG, GG) and alleles (A and G) for TERC rs2293607 polymorphism were ob- served as 38.6, 55.7, 5.7% and 66.4, 33.6% in patients, respectively and 45.3, 51.3, 3.3% and 71, 29% in con- trols, respectively. For the TERC SNP, we detected that GG genotype versus AA genotype resulted in odds ratio of two fold, but the ratio was not statistically significant (P = .44). In addition, gene-gene interaction and haplotype anal- ysis among cases and controls were made in the study. It was observed in gene-gene interaction analysis that genotype combination of GG+AG/CC (TERC gene/ MYNN gene) versus wild type genotypes of two pol- ymorphisms (AA/CC) revealed stronger correlation (Table 3). Besides, four possible haplotypes of MYNN (rs10936599) and TERC (rs2293607) SNPs were iden- tified in our study. C-A haplotype was accepted as a reference haplotype because it was more common in two groups. For the two variants, a linkage was found in both patients and controls (χ2 = 24.09, P = .0001 for patients; χ2 = 178.77, P = .0001 for controls). We ob- tained statistically significant relationship between pa- tients and controls for the C-G haplotypes (P = .0001) (Table 3). There is no data about histological types and stage of patients with bladder cancer, so these parameters were not evaluated in the study. DISCUSSION MYNN rs10936599 and TERC rs2293607 SNPs in 70 bladder cancer patients and 150 healthy controls were analyzed in this current study. In our study, genotype distributions in controls for two polymorphisms were not compatible with the principle of HWE. The cause of the drift from HWE is selection from hospital-based in- dividuals of control group in this study. In addition, we evaluated as reference allele, so C allele for the MYNN gene polymorphism is wild type in the current study. Table 2. The Genotypes and allele frequencies of MYNN (rs10936599) C/T and TERC (rs2293607) A/G genes SNPs in bladder cancer patients and control Individuals in Turkish population dCrude values eAdjusted values Gene Patients Controls P OR P OR cGenotypes n = 70 (%) n = 150(%) value CI (95%) value CI (95%) MYNN (rs10936599) CC 46 (66) 63 (42) 1 CT 19 (27) 78 (52) .001* 0.33 (0.17-0.62) .15 2.45 (0.71-8.41) TT 5 (7) 9 (6) .77 0.76 (0.23-2.42) .78 0.84 (0.25-2.77) CT+TT 24 (34) 87 (58) .001* 0.37 (0.20-0.68) .003* 2.53 (1.38-4.63) Alleles C 111 (79) 204 (68) 1 T 29 (21) 96 (32) .01* 0.55 (0.34-0.89) TERC (rs2293607) AA 27 (39) 68 (45) 1 AG 39 (55) 77 (51) .45 1.27 (0.70-2.29) GG 4 (6) 5 (4) .44 2.01 (0.50-8.07) .27 0.71 (0.38-1.30) AG+GG 43 (60) 82 (55) .38 1.32 (0.74-2.35) .35 0.50 (0.12-2.11) Alleles A 93 (66) 213 (71) 1 G 47 (34) 87 (29) .37 1.23 (0.80-1.90) cDistributions of genotypes in groups were compared by chi square test; dCrude values of odds ratios were calculated by Fisher exact test; eIndependent variables were compared by logistic regression. Adjusted with smoking habit and gender Adjusted values of odds ratios were calculated by using the statistic method. *P < .05 indicates statistically significant. MYNN and TERC gene polymorphisms in bladder cancer-Polat et al. Urological Oncology 52 Vol 16 No 01 January-February 2019 53 However, for rs10936599 SNP, more of the individu- als with C allele were obtained in patients compared to controls in our study. Therefore, all genotypes with- out pointing out a reference allele were analyzed again. We obtained significant odds ratio for CC genotype for bladder cancer among patients (these data were not shown on table). We suggest that T allele may have a protective effect in spite of C allele for bladder cancer. As consisted with our data, in a genome wide associ- ation studies (GWAS), Wang et al., (2014) reported that C allele of MYNN rs10936599 polymorphism may entail a risk for bladder cancer and the SNP together with other polymorphic side may be used, collective- ly, to effectively measure inherited risk for bladder cancer (16). Besides, it has been reported that T allele for rs10936599 polymorphism at 3q26.2 shows a pro- tective effect on bladder cancer in another GWAS by Figueroa et al., (2013) (ORadj per T allele = 0.85, 95% CI 0.81-0.90 and p = 4.53x10-9)(4). When the effect of the polymorphism in other cancer species has been investigated, similar results were de- tected. Carvajal-Carmona et al. (2013) examined some SNPs in colorectal cancers. They found that rs10936599 SNP of the MYNN gene was associated with adenoma risk(8). At the same time, in a GWAS studies conducted by Huolston et al., (2010), Lubbe et al., (2012), and Real et al., (2014), rs10936599 polymorphism was found risky in colorectal cancers(9,17,18). Furthermore, Speedy et al., (2014) identified new susceptibility loci mapping to 3q26.2 (rs10936599) for chronic lymphocytic leu- kemia (CLL) in a genome-wide association study(19). It has been reported in some studies by Houlston et al., (2010), Lubbe et al., (2012), and Kantor et al., (2014) that, C allele for rs10936599 SNP was major and risk allele in colorectal cancer(9,17, 20). Furthermore, as similar to our finding, T allele was shown as effective allele when the association of rs10936599 SNP and telomere length for coronary heart disease (CHD) was investigated in Han Chinese popu- lation(21). In addition, it has been found that the T allele may have a protective effect in the study OR = .907 (0.825–0.995). Telomere length was not analyzed in current study but we thought that bladder cancer risk of the polymorphism may be affected by alteration of tel- omere length. However, Broberg et al., (2005) reported that telomere length was significantly shorter in buccal cells from patients with bladder cancer than in control subjects. Telomere shortening increases the cancer risk rather than preventing it. It has been identified as the reason of the discrepancy in the study that short tel- omeres may increase the risk of developing cancer, particularly epithelial cancers via non-reciprocal trans- locations(22). The reason of this discrepancy may be the effects of other genes and risk factors. Wang et al., 2014 found that seven significant variants including MYNN rs10936599 had a strong cumulative association with bladder cancer risk. These loci showed the potential to predict the risk of bladder cancer in combination with the smoking risk factor in the Chinese population(16). In another study, it has been reported that this susceptibil- ity locus rs10936599 at 3q26.2 is in linkage disequi- librium with SNPs in TERC. In addition, it has been suggested that TERC gene suppresses cell growth in bladder cancer cell lines(23). On the other hand, it has been indicated that rs10936599 could change the regu- latory elements of MYNN or nearby genes to discuss the bladder cancer risk(16). Moreover, with regard to the results from a study by Jones et al. on MYNN and TERC, rs10936599 al- leles were associated with both longer telomeres and colorectal cancer risk. They reported that this variation close to TERC probably acts at an early stage in tum- origenesis. TERC rs2293607 is estimated by RNA fold to change the transcript’s secondary mRNA structure. Furthermore, Jones et al. stated that data from the EN- CODE Project had indicated H3K4Me1 and H3K4Me3 histones in the immediate vicinity of rs2293607. Ad- ditionally, the same region is considerably sensitive to DNaseI and estimated binding site of multiple transcrip- tion factors as like NFKB, PU.1, POU3F2 and MYC(7). Figueroa et al. (2014) found significantly higher TERC mRNA expression in muscle-invasive bladder tumors than adjacent normal bladder tissues(4). According to their report, TERC gene may have functional relevance for predisposition to bladder cancer but the possible functional effect of the MYNN gene in the associated LD block cannot be excluded as a molecular cause of this association. From some GWAS studies, some re- searchers found that rs10936599 SNP is in strong or moderate LD with SNP within the region 3q26.2 that includes the TERC gene(10). In our study, we also inves- tigated whether there is an association between TERC rs2293607 (A/G) polymorphism and bladder cancer risk. No significant association was found for the poly- Table 3. Analysis of gene- gene interaction and haplotype for MYNN (rs10936599) and TERC(rs2293607) polymorphisms Gene Patients Controls P OR Genotypes n (%) n (%) value CI (95%) MYNN gene (rs10936599)/TERC gene (rs2293607) CC-AA 24 (52.2) 62 (98,4) 1 CT/AA 3 (15.8) 6 (7.7) .71 1.29 (0.29-5.58) CT/AG 16 (84.2) 72 (92.3) .15 0.57 (0.28-1.17) TT/AG 1 (20) 4 (44.4) 1 0.64 (0.06-6.07) TT/GG 4 (80) 5 (55.6) .44 2.06 (0.51-8.35) CT+TT/AA 3 (11.1) 6 (8.8) .71 1.29 (0.29-5.58) GG+AG/CC 22 (47.8) 1 (1.6) .0001* 56.8 (7.25-445.3) CT+TT/GG+AG 21 (46.7) 81 (56.6) .30 0.67 (0.34-1.31) MYNN gene (C/T)/TERC gene (A/G) Haplotypes C A 45 100 1 C G 11 20 .0001* 12.22 (2.60-57.42) T A 2 6 1 0.74 (0.14-3.81) T G 12 42 .29 0.63 (0.30-1.32) *P < .05 indicates statistically significant. MYNN and TERC gene polymorphisms in bladder cancer-Polat et al. morphism. We did not find any data related to the TERC polymorphisms and bladder cancer risk in literature. So, our findings associated with TERC (rs2293607) SNP were not compared. When the data related to locali- zation of MYNN rs10936599 which is close to TERC genes(7) was considered, together with these MYNN and TERC SNPs were analyzed for bladder cancer risk in the present study. After gene-gene interaction analysis, a stronger correlation was obtained between GG+AG genotype combination for TERC (rs2293607) and CC genotype for MYNN (rs10936599) polymorphisms. It has been predicted that GG+AG combination of the TERC gene may cause telomere shortening. The re- markable finding was found consistent with those from Broberg et al. (2005)(22). In addition, it has been shown in our study that C and G haplotypes (for MYNN and TERC SNPs, respectively) had odds ratio value of ap- proximately 12 fold in development of bladder cancer. CONCLUSIONS We think that MYNN and TERC genes together may be associated with development of bladder cancer in the current study. In addition, establishing larger numbers of study groups, increasing the number of SNPs in the studied genes, and measuring the telomere lengths and evaluating them together will conclude more effective results. ACKNOWLEDGEMENT The authors confirm that all data underlying the find- ings are fully available without restriction. All relevant data are contained within the paper. The laboratory facilities of Medicine Faculty both Kocaeli and Sivas were used. A part of the manuscript has been presented in 5th International Molecular Biology and Biotechnol- ogy Congress in Tetova, Macedonia (orally-) at 25-29 August 2016. We would like to thank to Scientific Research Pro- ject Unit of Kocaeli University (Project No: 2014- 045) CONFLICT OF INTEREST STATEMENT All authors have no potential conflicts of interest to dis- close. REFERENCES 1. 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