Vol 13 No 01 January-February 2016 2471Vol 13 No 01 January-February 2016 2509 ENDOUROLOGY AND STONE DISEASES Association of the BsmI, ApaI, TaqI, Tru9I and FokI Polymorphisms of the Vitamin D Receptor Gene with Nephrolithiasis in the Turkish Popula- tion Omer Onur Cakir,1 Akin Yilmaz,2 Emre Demir,3 Kutluhan Incekara,4 Mustafa Omur Kose,4 Nagehan Ersoy Tunali4* Purpose: To analyze the relationship between nephrolithiasis and vitamin D receptor (VDR) gene BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236), Tru9I (rs757343) and FokI (rs2228570) polymorphisms in a study group from the Turkish population. Materials and Methods: Ninety-eight patients with calcium oxalate kidney stones and 70 controls were enrolled in this study. Five polymorphisms of the VDR gene were studied using the Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method. Results: For all polymorphisms, genotype frequencies were in line with Hardy-Weinberg equilibrium in the pa- tients and controls. For the BsmI polymorphism, allele frequency distribution was found to differ significantly between the patients and the controls (P < .05). The “B” allele was found to increase the risk of nephrolithiasis by approximately 1.5-fold (odds ratio = 1.55, 95% confidence interval: 1.00-2.40; P = .048). However, we did not find any statistically significant differences in the allele and genotype frequencies for the ApaI, TaqI, Tru9I and FokI polymorphisms. Proportionally, the “BAt” and “baT” haplotypes were more common than other haplotypes in the cases and controls, respectively. For the haplotypes of the BsmI and TaqI polymorphisms, the “bT” haplo- type frequency was found to be common in both the patients and the controls. However, we did not find statisti- cally significant differences between the cases and the controls for either the BsmI / ApaI / TaqI or the BsmI/TaqI haplotypes. Moreover, no relationship was identified between family history and development of stone disease. Conclusion: The “B” allele of the BsmI polymorphism of the VDR gene may increase stone development risk. Further investigations are needed to improve our knowledge regarding the genetic factors affecting urinary stone development. Keywords: nephrolithiasis; genetics; mutation; humans; vitamin d3 24-hydroxylase/genetics; metabolism; hyper- calcemia; hypercalciuria; nephrocalcinosis. INTRODUCTION Nephrolithiasis is a common multifactorial disease that is influenced by both environmental and ge- netic factors.(1) Several studies have reported an increase in its morbidity rate in recent years.(2) Moreover, it has a recurrence rate of approximately 50% within 10 years. Most stones (up to 75%) are composed of calcium ox- alate (CaOx) crystals, and their formation occurs in a complex manner.(3) With regard to understanding the genetic basis of the dis- ease, several single nucleotide polymorphisms (SNPs) have been analyzed to determine the risk of stone for- mation in different populations. To date, SNPs found in osteopontin (SPP1),(4) calcium sensing receptor (CaSR), matrix-gla protein (MGP), urokinase (PLAU) (recently reviewed by Arcidiacono and colleagues),(5) E-cadher- in (CHD1),(6) calcitonin receptor (CALCR),(7) transient receptor potential vanilloid member 5 (TRPV5),(8) Fe- tuin-A(9) and vitamin D receptor (VDR)(10-23) genes have been analyzed, and some of them are related to nephro- lithiasis development. The VDR gene encodes a nuclear receptor for the ac- tive form of vitamin D, 1,25-dihydroxy vitamin D3 [1,25(OH) 2 D 3 ]. After it binds to its response element on DNA, it regulates hundreds of genes with different functions, including calcium homeostasis.(24) The VDR gene has several polymorphisms, and some of them have been associated with urinary stone for- mation in some populations.(10-23) Whereas the FokI (rs2228570, c.2T > C, p.Met1Arg) polymorphism 1 Department of Urology, Istanbul Bagcilar Research and Training Hospital, 34800, Istanbul, Turkey. 2 Department of Medical Biology, Faculty of Medicine, Hitit University, 19030, Corum, Turkey. 3 Department of Biostatistics, Faculty of Medicine, Hitit University, 19030, Corum, Turkey. 4 Halic University, Department of Molecular Biology and Genetics, Istanbul Halic University, 34060, Istanbul, Turkey. *Correspondence: Halic University, Department of Molecular Biology and Genetics, Istanbul Halic University, 34060, Is- tanbul, Turkey. Tel:+90 532 6850526. Fax:+90 212 3470042. E-mail: nagehanersoy@halic.edu.tr. Received January 2016 & Accepted February 2016 is located in the second exon of the VDR gene, the BsmI (rs1544410, 1024+283G > A), ApaI (rs7975232, c.1025-49G > T), TaqI (rs731236, c.1056T > C, p.Il- e352Ile) and Tru9I (rs757343, c.1024+443G > A) pol- ymorphisms are found at the 3′ end of the gene.(25) The FokI (rs2228570, c.2T > C, p.Met1Arg) polymorphism changes the translation start codon and causes the pro- duction of two different VDR protein variants. The first one is a long variant and is coded by the T allele or the “f” allele. The second short variant is shortened by three amino acids and is coded by the C-allele or the “F” allele.(25) Compared with the long VDR form, the short form has greater transcriptional activation capa- bility.(26) The BsmI (rs1544410, 1024+283G > A), ApaI (rs7975232, c.1025-49G>T) and Tru9I (rs757343, c.1024+443G > A) polymorphisms are located in the 8th intron of the gene,(25) whereas the TaqI (rs731236, c.1056T > C, p.Ile352Ile) polymorphism is a synony- mous variation at codon 352 in exon 9 of the gene, and this T > C alteration does not change the amino acid sequence (p.Ile352Ile).(27) The BsmI, ApaI, Tru9I and TaqI polymorphisms are located at the 3′ end of the gene and are near the regulatory 3’ untranslated region (3’-UTR) of mRNA. Thus, when the BsmI, ApaI, and TaqI polymorphisms are found in specific haplotypes, they have been shown to affect VDR mRNA stability and the rate of transcription.(19) For example, in green monkey kidney cells, the "BAt" haplotype of the Bsm / Apa / Taq polymorphisms was shown to increase VDR expression compared with the "baT" haplotype.(27) A relationship between the bb genotype of the BsmI pol- ymorphism and higher urinary calcium extraction and a link between the T allele of the TaqI polymorphism and hypocitraturia were shown in the literature. However, there are conflicting data regarding the relationship be- tween the FokI and ApaI polymorphisms and urinary stone formation mechanisms.(28) There is a lack of knowledge in the literature regard- ing the association of the BsmI, ApaI, TaqI, Tru9I and FokI polymorphisms of the VDR gene with nephrolith- iasis in the Turkish population. Thus, in this study, we aimed to investigate the possible relationship between development of nephrolithiasis and five common poly- morphisms [BsmI (rs1544410), ApaI (rs7975232), TaqI (rs731236), Tru9I (rs757343) and FokI (rs2228570)] of the VDR gene in patients and control subjects from the Turkish population. Moreover, a haplotype analysis was also performed for the BsmI, ApaI and TaqI pol- ymorphisms. MATERIALS AND METHODS Study Population Between March 2006 and March 2008, patients with urinary calcium oxalate stones who were treated at the Istanbul Sisli Etfal Research and Training Hospital and Erzurum Numune Hospital were enrolled in the study. The study population was composed of 98 patients with CaOx nephrolithiasis (65 male and 33 female) who were initially diagnosed by computerized tomography (CT) scan. Fifty-two (53%) cases out of 98 had familial stone history (maternal and/or paternal urolithiasis his- tory), and 33 of them were male. For the control group, seventy individuals (52 male and 18 female) without family history or clinical signs of urinary stone disease (assessed via urine testing, plain abdominal radiogra- phy, or ultrasonography) were included in the study. All cases and controls were matched according to age and gender. Eleven of 98 patients had recurrent stone disease. After treatment with extracorporeal shock wave lithotripsy (SWL) or surgery, stone samples were collected, and stone analysis was performed using an X-ray diffraction (XRD) machine. XRD analysis con- firmed that they contained calcium oxalate crystals. Pa- tients with Ca oxalate stones were included in the study. Patients with abnormal levels of serum creatinine, cal- cium, phosphate, uric acid, liver enzymes, sodium, po- tassium, chloride and 1,25(OH) 2 D 3 , thyroid-stimulating hormone, free thyroxin, free triiodothyronine, and par- athyroid hormone were excluded from the study. Fur- thermore, patients diagnosed with hypercalcemia, renal tubular acidosis, hyperuricemia, gout, hyperparathy- roidism, urinary tract infection, renal failure and hyper- thyroidism were also excluded from the study. The last group of exclusion criteria included patients who take drugs that effect calcium and hormone metabolism, such as diuretics, calcium and vitamin D supplements, and anti-diabetic and anti-hypertensive agents. Informed consent was obtained from all patients and healthy subjects in accordance with the Helsinki Dec- laration (revised 2001), and the study protocol was ap- proved by the local ethics committee (approval number: IRB 2007 / 88-685) Analysis of VDR Gene Polymorphisms Blood samples were collected in ethylenediamine- tetraacetic acid (EDTA) containing tubes, and the genomic DNA of the study subjects was isolated with a High Pure polymerase chain reaction (PCR) Template Preparation Kit (Roche, Mannheim, Germany) accord- ing to the manufacturer’s protocol. The concentrations and purity of the samples were checked spectrophoto- metrically. The detection of the 5 polymorphisms [BsmI VDR gene Polymorphisms in Nephrolithiasis-Cakir et al. Endourology and Stone Diseases 2510 Vol 13 No 01 January-February 2016 2511 (rs1544410), ApaI (rs7975232), TaqI (rs731236), Tru9I (rs757343) and FokI (rs2228570)] of the VDR gene was carried out using the conventional Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) method.(29-31) Each amplification reaction was performed in a 25 µL final volume consisting of 1 U/µL unit Taq DNA polymerase, each primer at a con- centration of 10 pmol/µL, a 100 ng DNA sample, 100 µM dNTP, and 2 mM MgCl2. Primer sequences, an- nealing temperatures, restriction endonucleases and al- lele sizes are provided in Table 1. The PCR and restric- tion enzyme products were electrophoresed in a 2.0% agarose gel and then stained with ethidium bromide. For all studied polymorphisms, agarose gel electropho- resis pictures demonstrating alleles and genotypes are provided in Figure 1 A-E. DNA Sequencing For all polymorphisms, homozygote and heterozygote samples identified via PCR-RFLP analysis were con- firmed by DNA sequencing in the laboratories of Ion- tek (Istanbul, Turkey). The sequences obtained were analyzed using Clustal W (version 1.83) alignment software and compared with the RFLP results. All se- quenced samples were in agreement with the RFLP data. Statistical Analysis Descriptive statistics with a normal distribution were presented as the mean ± standard deviation, and nom- inal variables were presented as numbers of cases and percentages (%). Distributions of the groups were evaluated with the Kolmogorov–Smirnov and Shap- iro-Wilk normality tests. The significances of the dif- ferences between the two groups were evaluated with Student’s t-test for the means. Categorical variables were evaluated using Pearson’s Chi-square or Fisher’s exact test. The distributions of the genotype and allele frequencies between the groups and their relationship to CaOx nephrolithiasis were compared with a Chi-square (χ2) test. Hardy–Weinberg equilibrium (HWE) was also tested using the χ2 test based on observed and expected VDR gene Polymorphisms in Nephrolithiasis-Cakir et al. Polymorphism Chr 12: Methodical Primer Annealing Product Restriction Alleles position Nomenclature Sequences Temperature (°C) Size (bp) Enzyme BsmI(29) 47846052 Intronic F: 5′-CAACCAAGACTACAAGTACCGCGTCAGTGA-3′ 63 825 BsmI G (b): 650+175 (rs1544410) (forward strand) (1024+283 G>A) R: 5′-AACCAGCGGGAAGAGGTCAAGGG-3′ A (B): 850 ApaI(29) 47871419 Intronic F: 5′-CAGAGCATGGACAGGGAGCAA-3′ 60 722 ApaI T (A): 722 (rs7975232) (forward strand) (c.1025-49 G > T) R: 5′-GCAACTCCTCATGGCTGAGGTCTC-3′ 60 G (a): 509+213 TaqI(29) 47844974 Synonymous F: 5′-CAACCAAGACTACAAGTACCGCGTCAGTGA-3′ 63 2000 TaqI T (T): 2000 (rs731236) (forward strand) (p.Ile352Ile) R: 5′-CACTTCGAGCACAAGGGGCGTTAGC-3′ C (t): 1800+200 c.1056 T > C Tru9I(28) 47845892 Intronic F: 5′-TGTATTGGTCCAGCTTGCTCT-3′ 63 252 Tru9I A (u): 193+59 (rs757343) (forward strand) c.1024+443G > A R: 5′-CAGGGTTTCTCCATGTTGGT-3′ G (U): 252 FokI(30) 47879112 Start lost F: 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ 63 265 FokI T (f): 196+69 (rs2228570) (forward strand) c.2T > C R: 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′ (p.Met1Arg) Table 1. Primers, annealing temperatures, product sizes, restriction enzymes, and allele sizes used for vitamin D receptor gene genotyping. Variables Patients (n = 98) Controls (n = 70) P Value Age, years, mean ± SD 47.2 ± 16.3 42.6 ± 13.5 .536 Gender, no (%) Male 65 (66.3) 52 (74.3) .349 Female 33 (33.7) 18 (25.7) .349 BMI, kg/m2, mean ± SD 24.9 ± 3.7 24.1 ± 3.1 .552 Smoking, no (%) 40 (40.8) 33 (47.1) .511 Family history, no (%) 52 (53) ----- ----- Abbreviations: BMI, body mass index; SD, standard deviation. Table 2. Characteristics of the study groups. genotype numbers with the ‘Hardy Weinberg’ package Table 3. Genotype and allele frequency of the five vitamin D receptor gene polymorphisms in the patients and controls and the results of the Har- dy-Weinberg Equilibrium tests.* Variables Patients (n = 98) HWE P Value Adjusted HWE**P Value Controls (n = 70) HWE P Value Adjusted HWE** P Value BsmI .568 .673 .781 .911 BB 29 (29.6) 13 (18.6) Bb 46 (46.9) 33 (47.1) bb 23 (23.5) 24 (34.3) BB+ Bb 75 (76.5) 46 (65.7) Allele frequency B 104 (53.1) 59 (42.1) b 92 (46.9) 81 (57.9) ApaI .271 .351 .834 .948 AA 43 (43.9) 26 (37.1) Aa 40 (40.8) 34 (48.6) aa 15 (15.3) 10 (14.3) Aa+aa 55 (56.1) 44 (62.9) Allele frequency A 126 (64.3) 86 (61.4) a 70 (35.7) 54 (38.6) TaqI .443 .541 .454 .582 TT 35 (35.7) 31 (44.3) Tt 44 (44.9) 29 (41.4) tt 19 (19.4) 10 (14.3) Tt+tt 63 (64.3) 39 (55.7) Allele frequency T 114 (58.2) 91 (65.0) t 82 (41.8) 49 (35.0) Tru9I .223 .290 .176 .249 UU 18 (18.4) 6 (8.6) Uu 41 (41.8) 37 (52.9) uu 39 (39.8) 27 (38.6) Uu+uu 80 (81.6) 64 (91.4) Allele frequency U 77 (39.3) 49 (35.0) u 119 (60.7) 91 (65.0) FokI .305 .399 .494 .658 FF 48 (49) 39 (55.7) Ff 38 (38.8) 25 (35.7) ff 12 (12.2) 6 (8.6) Ff+ff 50 (51.0) 31 (44.3) Allele frequency F 134 (68.4) 103 (73.6) f 62 (31.6) 37 (26.4) VDR gene Polymorphisms in Nephrolithiasis-Cakir et al. Endourology and Stone Diseases 2512 Abbreviation: HWE, Hardy-Weinberg Equilibrium. * Data are presented as no (%) ** Chi-Square P value with continuty correction. Vol 13 No 01 January-February 2016 2513 Table 4. Genotype and allele frequency of the five vitamin D receptor gene polymorphisms in the patients and controls.* Variables Patients (n = 98) Controls (n = 70) P Value OR (95% CI) P Value BsmI BB 29 (29.6) 13 (18.6) .158 2.33 (0.98-5.55) .055 Bb 46 (46.9) 33 (47.1) 1.45 (0.70-3.01) .311 bb 23 (23.5) 24 (34.3) 1.00** BB+ Bb 75 (76.5) 46 (65.7) 1.70 (0.86-3.36) .124 Allele frequency B 104 (53.1) 59 (42.1) .048 1.55 (1.00-2.40) .048 b 92 (46.9) 81 (57.9) 1.00** ApaI AA 43 (43.9) 26 (37.1) .595 1.00** Aa 40 (40.8) 34 (48.6) 0.71 (0.36-1.39) .317 aa 15 (15.3) 10 (14.3) 0.91 (0.35-2.31) .838 Aa+aa 55 (56.1) 44 (62.9) 0.77 (0.40-1.41) .382 Allele frequency A 126 (64.3) 86 (61.4) 1.00** a 70 (35.7) 54 (38.6) 0.88 (0.56-1.39) .593 TaqI TT 35 (35.7) 31 (44.3) .474 1.00** Tt 44 (44.9) 29 (41.4) 1.34 (0.68-2.63) .389 tt 19 (19.4) 10 (14.3) 1.68 (0.68-4.16) .258 Tt+tt 63 (64.3) 39 (55.7) 1.43 (0.76-2.68) .262 Allele frequency T 114 (58.2) 91 (65.0) 1.00** t 82 (41.8) 49 (35.0) 1.34 (0.85-2.09) .205 Tru9I UU 18 (18.4) 6 (8.6) .148 2.08 (0.73-5.91) .166 Uu 41 (41.8) 37 (52.9) 0.77 (0.40-1.49) .432 uu 39 (39.8) 27 (38.6) 1.00** Uu+uu 80 (81.6) 64 (91.4) Allele frequency U 77 (39.3) 49 (35.0) 1.20 (0.77-1.88) .424 u 119 (60.7) 91 (65.0) 1.00** FokI FF 48 (49) 39 (55.7) .614 1.00** Ff 38 (38.8) 25 (35.7) 1.23 (0.64-2.38) .529 ff 12 (12.2) 6 (8.6) 1.62 (0.56-4.72) .370 Ff+ff 50 (51.0) 31 (44.3) Allele frequency F 134 (68.4) 103 (73.6) 1.00** f 62 (31.6) 37 (26.4) 1.29 (0.80-2.08) .302 Abbreviations: OR, odds ratio; CI, confidence interval. * Data are presented as no (%). ** Chi-square P value with continuity correction. VDR gene Polymorphisms in Nephrolithiasis-Cakir et al. in R software. Possible haplotypes for the BsmI / ApaI / TaqI (rs1544410 / rs7975232 / rs731236) and BsmI/ TaqI (rs1544410 / rs731236) polymorphisms were de- termined using the HAPSTAT analysis tool, and their relationship to nephrolithiasis was analyzed using the χ2 test. Odds ratios (ORs) were presented with 95% confidence intervals (CIs), and P values less than 0.05 were considered statistically significant. All statistical procedures were performed using the Statistical Pack- age for the Social Science (SPSS Inc, Chicago, Illinois, USA) version 20.0. Statistical power was calculated us- ing QUANTO 1.2.4 software (Website: http://biostats. usc.edu/software).(32) QUANTO is specifically written to calculate either the power or the required sample size for association studies of genes, environmental factors, gene-environment interactions, or gene-gene interac- tions. In our study, for the less frequent alleles (42.1% for BsmI, 38.6% for ApaI, 35% for TaqI, 35% for Tru9I and 26.4% for FokI) with P = .05, the study had a power > 85% for the BsmI, ApaI, TaqI, and Tru9I polymor- phisms and > 80% for the FokI polymorphism (OR = 2.0; mode of inheritance: log-additive, population risk: 14.8%). RESULTS The characteristics of the subjects are provided in Ta- ble 2. The mean ages of the patients and normal con- trols were 47.2 ± 16.3 and 42.6 ± 13.5 years, respective- ly. The mean body mass index (BMI) (kg/m2) values of the patients and controls were 24.9 ± 3.7 and 24.1 ± 3.1, respectively. The genotype and allele distributions of the 5 VDR pol- ymorphisms (BsmI, ApaI, TaqI, Tru9I and FokI) in the nephrolithiasis patients and controls are provided in Ta- ble 3. None of the genotype frequencies were found to deviate from Hardy-Weinberg equilibrium in either the patients or the controls for the analyzed polymorphisms (P > .05). For the BsmI polymorphism, allele frequency distribu- tion was found to differ significantly between the pa- tients and the controls (P < .05). The “B” allele was found to increase the risk of nephrolithiasis by approx- imately 1.5-fold (OR = 1.55, 95% CI: 1.00-2.40; P = Abbreviations: OR, odds ratio; CI, confidence interval. * Referent haplotype. Haplotypes Patients (2n = 196) (%) Controls (2n = 140) (%) OR (95% CI) P Value BsmI / ApaI / TaqI baT 64 (0.3253) 53 (0.3768) 1.00* Bat 81 (0.412) 48 (0.3448) 1.40 (0.84-2.33) .198 BaT 7 (0.03575) 2 (0.01199) 2.90 (0.58-14.54) .297 BAT 17 (0.08861) 9 (0.06802) 1.56 (0.64-3.79) .320 bAT 27 (0.1383) 28 (0.1984) 0.80 (0.42-1.51) .492 BsmI / TaqI bT 91 (0.4639) 81 (0.5755) 1.00* Bt 81 (0.4124) 48 (0.3453) 1.50 (0.94-2.39) .086 BT 24 (0.1237) 11 (0.07914) 1.94 (0.90-4.21) .089 Table 5. BsmI / ApaI / TaqI and BsmI / TaqI haplotypes of the patients and controls. Figure 1. Gel electrophoresis images of the PCR products after digestion with restriction enzymes. A: BsmI polymorphism, B: ApaI polymorphism, C: TaqI polymorphism, D: Tru9I polymorphism, and E: FokI polymor- phism. Sizes of the alleles specifying genotypes are given in Table 1. VDR gene Polymorphisms in Nephrolithiasis-Cakir et al. Endourology and Stone Diseases 2514 Vol 13 No 01 January-February 2016 2471Vol 13 No 01 January-February 2016 2515 Table 6. Genotype and allele frequency of the five vitamin D receptor gene polymorphisms in patients with or without family history.* Variables Positive (n = 52) Negative (n = 46) P Value OR (95 CI) P Value BsmI BB 16 (30.8) 13 (28.3) .846 1.00** Bb 23 (44.2) 23 (50.0) 0.81 (0.32-2.06) .662 bb 13 (25.0) 10 (21.7) 1.07 (0.35-3.18) .922 Bb+bb 36 (69.2) 33 (71.7) 0.89 (0.37-2.12) .786 Allele frequency B 55 (52.9) 49 (53.3) 1.00** b 49 (47.1) 43 (46.7) 1.01 (0.58-1.78) .958 ApaI AA 23 (44.2) 20 (43.5) .801 1.00** Aa 20 (38.5) 20 (43.5) 0.87 (0.37-2.06) .751 aa 9 (17.3) 6 (13.0) 1.30 (0.39-4.31) .662 Aa+aa 29 (55.8) 26 (56.5) 0.97 (0.44-2.16) .94 Allele frequency A 66 (63.5) 60 (65.2) 1.00** a 38 (36.5) 32 (34.8) 1.08 (0.60-1.94) .798 TaqI TT 21 (40.4.) 14 (30.4) .391 1.00** Tt 20 (38.5) 24 (52.2) 0.56 (0.27-1.37) .199 tt 11 (21.1) 8 (17.4) 0.92 (0.29-2.85) .880 Tt+tt 31 (59.6) 32 (69.6) 0.65 (0.28-1.49) .305 Allele frequency T 62 (59.6) 52 (56.5) 1.00** t 42 (40.4) 40 (43.5) 0.88 (0.50-1.55) .661 Tru9I UU 8 (15.4) 10 (21.7) .583 0.76 (0.25-2.33) .631 Uu 24 (46.1) 17 (37.0) 1.34 (0.55-3.24) .514 uu 20 (38.5) 19 (41.3) 1.00** Uu+uu 44 (84.6) 36 (78.3) 1.13 (0.50-2.53) .774 UU+Uu 32 (61.5) 27 (58.7) 0.59 (0.24-1.48) .261 Allele frequency U 40 (38.5) 37 (40.2) 0.93 (0.52-1.65) .802 u 64 (61.5) 55 (59.8) 1.00** FokI FF 28 (53.8) 20 (43.5) .584 1.00** Ff 18 (34.6) 20 (43.5) 0.64 (0.27-1.51) .311 ff 6 (11.6) 6 (13.0) 0.71 (0.20-2.54) .602 Ff+ff 34 (46.2) 26 (56.5) 0.93 (0.43-2.01) .862 Allele frequency F 74 (71.2) 60 (65.2) 1.00** f 30 (28.8) 32 (34.8) 0.76 (0.42-1.39) .372 Abbreviations: OR, odds ratio; CI, confidence interval. * Data are presented as no (%). ** Referent genotype/allele. VDR gene Polymorphisms in Nephrolithiasis-Cakir et al. .048). Likewise, the BB genotype was more frequently observed in the patients than in the controls. Howev- er, this difference was not statistically significant (P = .055). Moreover, the frequency of individuals carrying BB+Bb genotypes was more frequent in the patients than in the controls (76.5% vs. 65.7%) (Table 4). The proportional frequencies of the “a” allele of the ApaI polymorphism, the “t” allele of the TaqI polymor- phism, the “U” allele of the Tru9I polymorphism, and the “f” allele of the FokI polymorphism were shown to be less than those of their second alleles in the patients and control subjects. However, we did not find any sig- nificant differences in the allele and genotype frequen- cies of the ApaI, TaqI, Tru9I and FokI polymorphisms (Table 4). We also analyzed the haplotypes of the BsmI / ApaI / TaqI polymorphisms and BsmI / TaqI haplotypes. Haplotype analysis revealed that there were 5 common haplotype blocks for the BsmI, ApaI and TaqI polymor- phisms, as shown in Table 5. Proportionally, the “BAt” and “baT” haplotypes were found to be more common than other haplotypes in the cases and controls, respec- tively. With regard to the haplotypes of the BsmI and TaqI polymorphisms, the “bT” haplotype frequency was found to be common in both patients and controls. For the haplotype distribution, we did not show statisti- cally significant differences between the cases and the controls for either the BsmI / ApaI / TaqI or the BsmI/ TaqI haplotypes. Of the 98 patients, 52 of them (17 females and 35 males) had a positive family history of nephrolithiasis, and the remaining patients (15 females and 31 males) were neg- ative. We did not find any differences between family history and VDR gene polymorphisms (Table 6). DISCUSSION The involvement of the VDR gene locus and its pol- ymorphisms in nephrolithiasis development has been shown in several studies. Initially, a strong linkage was shown between calcium stone formation and the D12S339 marker, which is located near the VDR gene locus on chromosome 12, by Scott and colleagues. using a candidate gene approach.(28) Then, genetic as- sociation studies were performed to analyze the rela- tionship between stone formation and VDR gene pol- ymorphisms in different populations,(10-23) and as seen in the literature, contradictory results were obtained for commonly analyzed VDR SNPs. In one of the analy- ses performed by Bid and colleagues,(10) a significant difference in genotype frequencies for the VDR FokI polymorphism between subjects with calcium oxalate nephrolithiasis and control subjects in the Indian popu- lation was shown. In another study, Bid and coworkers analyzed VDR gene FokI and calcitonin receptor (CTR) gene polymorphisms in pediatric stone patients. With regard to the FokI variation, an apparent difference was demonstrated between the pediatric cases and the con- trol group.(11) In a study performed in Taiwan by Liu and colleagues,(12) the FokI variation of the VDR gene was demonstrated to be a meaningful parameter in the clinical appearance of calcium stone formers. The FF genotype of the FokI polymorphism was found to be associated with recurrence of calcium stones and stone development age. However, no significant difference was shown between patients and healthy controls in terms of FokI genotype frequency. Similarly, we found no significant differences in the allele and genotype fre- quencies for the FokI polymorphism in the study group from Turkish population (Table 4). In the Chinese Han population, Wang and colleagues(13) studied five polymorphisms (FokI, BsmI, DdeI, ApaI and TaqI) of the VDR gene, and they showed an as- sociation for only the ApaI polymorphism. The ApaI genotypes were found to differ between groups, and the “a” allele was shown to be related to an increased risk of stone recurrence. In Japanese stone patients, the TaqI “t” allele was shown to correlate with an approxi- mately 5-fold increased risk of stone development and increased urinary calcium levels. However, they were not able to show differences between ApaI polymor- phism distribution and severe and recurrent stones.(22) In Korean stone patients, Seo and colleagues did not show any relationship between the AlwI, FokI, ApaI, and TaqI polymorphisms of the VDR gene and urinary stone development.(23) The BsmI, ApaI, and TaqI genotypes were analyzed in hypercalciuric children from Turkey, and the AA gen- otype of the ApaI SNP was shown to be related to a 3.5-fold increased risk of idiopathic hypercalciuria.(14) Moreover, in pediatric stone patients and matched con- trols, Ozkaya and colleagues(15) analyzed VDR ApaI, BsmI and TaqI SNPs and found that ApaI and TaqI variations were related to nephrolithiasis and family history of the disease, respectively. In another study from Turkey, Gunes and colleagues(16) found a relation- ship between ApaI polymorphisms and family history of stone development. However, in our study, such an association was not found for the ApaI polymorphism for either stone development or family history. How- ever, we found that the bb genotype was present less frequently in stone patients, as shown in Table 3. Gunes and colleagues(16) did not find any significant VDR gene Polymorphisms in Nephrolithiasis-Cakir et al. Endourology and Stone Diseases 2516 Vol 13 No 01 January-February 2016 2517 differences between urolithiasis patients and controls in the Black Sea Coastal Region of Turkey with respect to genotype frequencies of the BsmI, ApaI and TaqI poly- morphisms. With regard to this study, we found a more similar genotype and allele distribution in the healthy control group. Likewise, similar frequencies were also observed in the patients, except for the BsmI polymor- phism (B allele 43%; b allele 57%). An approximately 10% difference between the BsmI allele frequencies of the two studies may be attributed to various factors, such as stone type or patient characteristics. In another study, the bb genotype of the BsmI poly- morphism was found to be significantly higher in hy- percalciuric stone patients than in normacalciuric stone patients in the Brazilian population.(16) In the Taiwan- ese population, the BsmI polymorphism was not found to be associated with calcium oxalate stone disease.(20) Heilberg and colleagues showed that the BsmI poly- morphism was not related to bone loss in hypercalciuric calcium-stone-forming Brazilian patients.(21) As seen in the literature, conflicting results have been noted regarding the relationship between VDR gene polymorphisms and nephrolithiasis development. These differences among studies may result from the complex etiology of stone formation, ethnicity, patient characteristics, sample size, environmental effects and other genetic factors. Recently, a meta-analysis aiming to reveal a possible relationship between VDR gene polymorphisms and nephrolithiasis was performed by Zhou and colleagues. (18) Six studies were included in their analysis, and they showed no association between the VDR BsmI, FokI, TaqI or ApaI polymorphisms and stone formation in either the overall population or Caucasians. However, FokI, TaqI and ApaI polymorphisms were related to the development of nephrolithiasis in the Asian population. (18) Some alleles of VDR gene polymorphisms also tend to show linkage disequilibrium with each other.(25) There- fore, a haplotype analysis of BsmI, ApaI and TaqI polymorphisms has been performed in some studies. Mossetti and colleagues(19) analyzed the BsmI/TaqI haplotypes and did not show any differences between stone patients and controls with respect to haplotype frequencies. However, they showed that the bT hap- lotype was associated with an earlier age of onset and elevated stone rate, as well as diminished urinary citrate excretion. In our study, the BAt, baT and bAT haplotypes of the BsmI / ApaI / TaqI polymorphisms and Bt and bT hap- lotypes of the BsmI/TaqI polymorphisms were found to have higher frequencies in both patients and controls. However, we did not show statistically significant dif- ferences between the groups (Table 5). The limitations of current study include a low number of patients (98), which seems to be insufficient to rep- resent the entire Turkish population. However, when looking at our results, the BsmI polymorphism of the VDR gene is related to nephrolithiasis, and this associa- tion may be stronger in a study including a higher num- ber of cases and controls. However, this specific cohort consisted of patients with urinary CaOx stone and for all polymorphisms, the power was > 80%, which was acceptable. Another limitation was the need for more detailed serum and 24 hour urine tests that could be per- formed for all patients. This would have resulted in a stronger discussion. CONCLUSIONS More studies should be performed to reveal the possible association between VDR gene polymorphisms and the risk of nephrolithiasis in a larger cohort. Understanding the genetic tendency toward kidney stone development and recurrence may provide an opportunity for early diagnosis and may also be helpful for the clinical fol- low-up of urinary stone sufferers. CONFLICT OF INTEREST None declared. REFERENCES 1. Mittal RD, Bid HK, Manchanda PK, Kapoor R. Predisposition of genetic polymorphism with the risk of urolithiasis. Indian J Clin Biochem. 2008;23:106–16. 2. Shoag J, Tasian GE, Goldfarb DS, Eisner BH. The New Epidemiology of Nephrolithiasis. Adv Chronic Kidney Dis. 2015;22:273–8. 3. Aggarwal KP, Narula S, Kakkar M, Tandon C. 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