Fall 2012 - 08.pdf 640 | 1Department of Pediatric Nephrology, Celal Bayar University, Manisa, Turkey 2Department of Public Health, Celal Bayar Univer- sity, Manisa, Turkey 3Department of Pediatrics, Molecular Medicine Laboratory, Ege University, Izmir, Turkey Gökhan Tekin,1 Pelin Ertan,1 Gönül Horasan,2 Afig Berdeli3 SPP1 Gene Polymorphisms Associated With Nephrolithiasis in Turkish Pediatric Patients Corresponding Author: Pelin Ertan, MD Mithatpaşa Cad. 900/15 Göztepe, İzmir, Turkey E-mail address: pelinertan@ hotmail.com Tel: +90 236 232 3133 Received October 2011 Accepted April 2012 Purpose: To investigate the association between SPP1 gene polymorphisms and nephrolithiasis. Materials and Methods: A total of 65 pediatric patients and 50 healthy controls were enrolled in this study. Two known polymorphisms of the SPP1 gene, c.240T > C and c.708C > T nucleotide substitutions, both of which were also known as synonymous aminoacid polymorphisms, D80D and A236A, respectively, at SPP1 gene cDNA level, were investigated. SPP1 gene polymorphism was evaluated using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism method. Results: - dence Interval (CI), 1.170 to 3.880; P = .013] and CC genotype distribution (OR, 2.946; 95% CI, 0.832 to 10.431; P = .094) and in c.708C > T polymorphism, T allele frequency (OR, 2.183; 95% CI, 1.197 to 3.980; P = .011) and TT genotype distribution (OR, 3.056; 95% CI, 0.861 to 10.839; P = .084) were found to be higher in the patient group. Conclusion: SPP1 polymorphisms were found to be associated with nephrolithiasis and it may be suggested that SPP1 gene polymorphism could be a useful marker for evaluation of the early genetic risk factor in childhood nephrolithiasis. Keywords: osteopontin, nephrolithiasis, SPP1 gene, genotype, case-control studies, polymorphism ENDOUROLOGY AND STONE DISEASE Endourology and Stone Disease 641Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L INTRODUCTION Kenvironmental as well as hereditary factors, and - crystals involves stages of nucleation, crystal growth, ag- gregation, and retention in sequence.(1) Urinary analyses of kidney stones show that they contain proteinous ingredi- ents, and several reports have emphasized the importance of proteins in preventing nephrolithiasis.(2,3) Many macro- molecules, organics, and inorganics are known to inhibit stone formation, including Tamm-Horsfall, glycosamino- glycans, Bikunin, Calgranulin, and Osteopontin.(1) Osteopontin (OPN) is a 44-kDa negatively-charged acidic hydrophilic, multifunctional protein encoded by the SPP1 gene that is located on chromosome 4q21-25 and consists (3-5) tissue, including osteoclasts as well as osteoblasts, and in other cell types, such as endothelial, smooth muscles, and epithelial cells.(4,6) Osteopontin has many crucial biologi- cal functions, including leucocyte function and recruitment, wound repair, cell survival as well as regulation of normal bone resorption and inhibition of urinary crystallization.(1,7) Urinary OPN may prevent the renal stone formation by de- tubular epithelial cells. Quantitative and structural investigation of OPN have been studied in renal stones previously by various researchers in order to determine its genetic heritance.(1,3,7) Our aim was to investigate the SPP1 gene polymorphism distribution among pediatric patients with nephrolithiasis and to deter- mine its association with nephrolithiasis. MATERIALS AND METHODS The study group consisted of 65 Turkish pediatric patients with nephrolithiasis who were followed-up in Department of Pediatric Nephrology of Celal Bayar University, Manisa, Turkey, and 50 age and gender-matched healthy subjects without a history of nephrolithiasis or a family history of urinary stone disease. Urinary ultrasonography was also performed for the con- trol group to demonstrate that they did not have any urinary calculi. The patients and healthy groups without any history of nephrolithiasis were selected from the same geographi- cal area as well as race. Blood samples were obtained from both patient and control groups. Written informed consents were obtained from parents of all participants. The study was approved by the local ethics committee. Molecular Analysis - tracted from ethylenediaminetetraacetic acid (EDTA) an- ticoagulated venous blood using QiAamp DNA Blood Mini kit (QIAGEN GmbH, Hilden, Germany) according to manufacturer’s. c.708C > T polymorphism in the 7th of SPP1 gene was genotyped by polymerase chain reaction (PCR) and endonuclease digestion.(5) Polymerase chain re- in PCR strip tubes containing 100 ng genomic DNA solu- tion. Platinum Taq Enhancer 2.0 mmol MgCl 2 , 50 mmol/L each of the dGTP, dATP, dTTP, and dCTP (Promega), 5 pmol each of forward and reverse primers, and 1.0 U Plati- num Taq polymerase (Invitrogen Co, Paisley, UK). The sequences of the forward and reverse primers were used; 5’-TACCCTGATGCTACAGACGAGG-3’ and 5’-CT- GACTATCAATCACATCGGAATG-3’, respectively. The cycling conditions comprised a denaturation step at 95 ºC SPP1 gene were analyzed on a 2.0% agarose gel prestained with ethidium bromide. Genotyping was performed using the AluI (New England Biolabs, Beverly, MA, USA) re- and incubated at 37 ºC during a 16-h period. The fragments of 147 + 61 + 44 bp for the TT genotype, 147 + 105 bp for the CT genotype, and four fragments of 105 + 61 + 44 + 42 bp for the CC genotype were separated on a 3% metaphor agarose gel (FMC BioProducts, BioConcept, Allschwill, Switzerland) stained with ethidium bromide, and visualized under ultraviolet light (Figure 1). SPP1 Gene Polymorphisms and Nephrolithiasis | Tekin et al 642 | DNA Sequencing Method of the SPP1 Gene oligonucleotide primers were synthesized and purchased from Invitrogen (Invitrogen, Paisley, UK) as the HPLC pu- Primer details were: 5’-reverse-TACGTTTCTTGCACCTCTCG 5’-reverse- ATGGCCTGAGTGTGGCTATC - GGG; 5’-reverse-TGCAAACTGTGGTTTCCTAGAC 5’-reverse-CAGACTCAAATAGATACACATTCAACC on Corbett Palm-Cycler gradient thermal cycler (Corbett CA) containing 1 μL genomic DNA solution, 1.0 U Plat- inium TAQ with Enhancer Buffer (Invitrogen Ltd. Paisley, UK), 50 μmol/L each of the dGTP, dATp, dTTP, and dCTP (Promega, Madison,WI), 5 pmol each forward and reverse primers. The cycling conditions comprised of a hot start at 95 ºC for 45 sec, 58 to 60 ºC (gradient program) for 45 sec, - - ing to the appropriate protocol. Cycle sequencing PCR was performed using BigDye Terminator v.3.1 kit as manufac- turer’s (PE Applied Biosystems, Foster City, CA). Cycle se- Terminator kit (PE Applied Biosystems, Foster City, CA) were analyzed by ABI 3130×l Genetic Analyzer. DNA se- quencing was performed in both directions, initiated from the forward, and the reverse primers was used in the initial PCR reaction. The primers design was similar to the one SeqScape 2.0 sequencing analysis software was employed used as reference to determine the nucleotide substitution - tion to protein structure could be achieved using NCBI pro- Statistical Analysis All statistical analyses were performed with SPSS software (the Statistical Package for the Social Sciences, Version 11.0, SPSS Inc, Chicago, Illinois, USA) using the Chi- different genotypes and their alleles were calculated using logistic regression models. The Hardy-Weinberg equilib- rium test was done for each polymorphism. RESULTS The study population consisted of 37 (56.9%) males and 28 (43.1%) females with the male-to-female ratio of 1.37. The mean age of the patient group was 84.4 months (range, 4 months to 17.5 years) and the mean age at diagnosis was 67.2 months (range, 3 to 210 months). Twenty-one (32.3%) chil- dren were younger than 12 months of age at diagnosis. Follow- up duration was 13 months (range, 1 to 45 months). Family history of nephrolithiasis was found in 46 (70.8%) patients. Seventeen (26.2%) patients were born from con- sanguineous marriages. In 15 (23.0%) patients, stones were bilateral, and 29 (44.0%) patients had multiple stones. Only common compound. During the follow-up period, 4 (6.1%) Figure 1. Agarose gel electrophoresis image of c.708C > T poly- morphism. Well 1 was 100 bp DNA ladder CT genotype was indicated in wells 2, 3, and 6. TT genotype was indicated in wells 4 and 8. CC genotype was indicated in wells 5 and 7. Endourology and Stone Disease 643Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L SPP1 Gene Polymorphisms and Nephrolithiasis | Tekin et al patients had recurrence. Genotype distribution and allele frequency of c.240T > C polymorphism was analyzed in each group. In patient group, C allele frequency was higher than in control group (OR, 2.13; 95% CI, 1.170 to 3.880; P = .013). Regarding genotype distribution, the TC genotype (OR, 2.448; 95% CI, 1.066 to 5.622; P = .035) and the CC genotype (OR, 2.946; 95% CI, 0.832 to 10.431; P = .094) were higher in the patient group. Genotype distribution and allele frequen- cies of c.240T > C polymorphism are shown in Table 1. Genotype distribution and allele frequency of c.708C > T polymorphism was analyzed in each group. In patient group, T allele frequency was higher than in control group (OR, 2.183; 95% CI, 1.197 to 3.980; P = .011). Regarding Figure 2. Electropherogram for c.240T > C homozygous nucleo- tide substitution in exon 6 (D80D) of SPP1 gene. Figure 3. Electropherogram for c.240T > C homozygous nucleo- tide substitution in exon 6 (D80D) of SPP1 gene. Figure 4. Electropherogram for c.708C > T homozygous nucleo- tide substitution in exon 7 (A236A) of SPP1 gene. Figure 5. Electropherogram for c.708C > T homozygous nucleo- tide substitution in exon 7 (A236A) of SPP1 gene. 644 | genotype distribution, the CT genotype (OR, 2.538; 95% CI, 1.102 to 5.848; P = .029) and the TT genotype (OR, 3.056; 95% CI, 0.861 to 10.839; P = .084) were higher in the patient group. Genotype distribution and allele frequen- cies of c.708C > T polymorphism are shown in Table 2. In case-only analysis of patients with nephrolithiasis, c.240T > C and c.708C > T polymorphisms were found not to be associated with urinary metabolic risk factors, gender, early age at diagnosis, positive family history, consanguin- ity, stone recurrence, and bilateral or multiple stones (Table 3). DISCUSSION Nephrolithiasis is a commonly known disease threatening the human kind for many years. There are not many studies investigating the genetic role of OPN in renal stones. Some researchers have been emphasized the importance of the re- lation between OPN and crystal formation. Limited number of studies have been conducted on OPN gene structure for familial and recurrent renal stones.(7) Single nucleotide polymorphisms (SNP) of the human OPN gene has been reported to be associated with many diseases. (5,8-11) Especially, Gao and colleagues have investigated 61 polymorphisms and evaluated four haplotypes among these polymorphisms. Two of these haplotypes have been identi- shed a light to understand the mechanism of how the OPN gene can change the structure of OPN molecule.(7) In this study, we have investigated the difference in SPP1 gene polymorphisms between patients with nephrolithiasis Endourology and Stone Disease Table 1. Genotype distribution and allele frequencies of c.240T > C polymorphism in patients and controls. Patient Control Genotype n (%) n (%) Odds Ratio 95% Confidence Interval P CC 10 (15.4) 4 (8) 2.946 0.832 to 10.431 .094 TC 27 (41.5) 13 (26) 2.448 1.066 to 5.622 .035* TT 28 (43.1) 33 (66) 1 (ref ) Total 55 (100.0) 50 (100.0) Allele C 47 (36.2) 21 (21.0) 2.130 1.170 to 3.880 .013* T 83 (63.8) 79 (79.0) 1 (ref ) Total 130 (100.0) 100 (100.0) *P < .05 Table 2. Genotype distribution and allele frequencies of c.708C > T polymorphism in patients and controls. Patient Control Genotype n (%) n (%) Odds Ratio 95% Confidence Interval P TT 10 (15.6) 4 (8) 3.056 0.861 to 10.839 .084 CT 27 (42.2) 13 (26) 2.538 1.102 to 5.848 .029* CC 27 (42.2) 33 (66) 1 (ref ) Total 55 (100.0) 50 (100.0) Allele T 47 (36.7) 21 (21.0) 2.183 1.197 to 3.980 .011* C 81 (63.3) 79 (79.0) 1 (ref ) Total 130 (100.0) 100 (100.0) *P < .05 645Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L SPP1 Gene Polymorphisms and Nephrolithiasis | Tekin et al and normal subjects. Two SNPs in SPP1 gene were found to be associated with nephrolithiasis. As was previously stated in literature, although both nucleotide substitutions take place in the SPPI gene coding region, this does not mutations. Synonymous substitutions between nucleotides in genes produce structurally identical OPN. Difference may be present in the sequence of transcription factor binding sites and synthesis of the transcription factors suggesting that a change in transcription step may generate differences in the amount of mRNA and protein.(12) was thought to be due to reduced synthesis of OPN or its incorporation into growing stones with normal amount of synthesis.(13) In a rat model, OPN and its mRNA were en- hanced in rats in which urinary stone formation was induced (14) Furthermore, in an urolithiasis model, OPN mRNA was observed to enhance in ethylene glycol-administered rats.(3) Yamate and colleagues investigated the difference in OPN DNA between normal subjects and patients with urolithi- asis. Nucleotide substitution of GCC to GCT, encoding synonymous Ala-250, was reported to be higher in patients with urolithiasis. Based on the difference in gene frequency suggested to be a diagnostic method for patients with uro- lithiasis and a predisposing hereditary factor.(12) Gao and coworkers reported that an SNP at position 9401 was de- termined to be seen more frequently in patients with renal stones. Hence, they have speculated on the relationship be- tween OPN sequence variants and the risk of nephrolithi- asis. Due to these results, they have stated that there is an association between OPN and calcium stones and it might be a candidate genetic marker for evaluating the genetic risk of renal stone disease(15) In our study, we have found no association between urinary metabolic risk factors and OPN polymorphisms. There are certain factors indicating that urolithiasis may be related to genetic predisposition, such as family history of urolithiasis, higher incidence of recurrences, and early onset of the disease in such patients, and drag the clinical Table 3. Genotype distribution and allele frequencies of c.240T > C and c.708C > T polymorphisms and urinary metabolic risk factors. Hypercalciuria Hyperoxaluria Hypocitraturia Hyperuricosuria Cystinuria Hypomagnesuria n (%) 15 (23.1) 14 (21.5) 23 (35.4) 16 (24.6) 9 (13.8) 10 (15.4) 240T > C TT 6 (40.0) 5 (35.7) 9 (39.1) 6 (37.5) 5 (55.6) 5 (50.0) TC 6 (40.0) 2 (14.3) 8 (34.8) 6 (37.5) 3 (33.3) 4 (40.0) CC 3 (20.0) 7 (50.0) 6 (26.1) 4 (25.0) 1 (11.1) 1 (10.0) P 0.8 0.7 0.2 0.4 0.7 0.8 T 18 (60.0) 17 (60.7) 26 (56.5) 18 (56.3) 13 (72.2) 14 (70.0) C 12 (40.0) 11 (39.3) 20 (43.5) 14 (43.8) 5 (27.8) 6 (30.0) P 0.6 0.6 0.1 0.3 0.4 0.5 708C > T CC 6 (40) 5 (35.7) 9 (39.1) 6 (37.5) 4 (50.0) 5 (50) CT 6 (40) 2 (14.3) 8 (34.8) 6 (37.5) 3 (37.5) 4 (40) TT 3 (20) 7 (50.0) 6 (26.1) 4 (25) 1 (12.5) 1 (10) P 0.8 0.7 0.2 0.4 0.7 0.8 C 18 (60.0) 17 (60.7) 26 (56.5) 18 (56.3) 13 (72.2) 14 (70.0) T 12 (40.0) 11 (39.3) 20 (43.5) 14 (43.8) 5 (27.8) 6 (30.0) P 0.6 0.7 0.2 0.3 0.6 0.4 646 | focus to genetic factors.(16,17) However, we have not found any association between family history and early presenta- tion of nephrolithiasis and OPN polymorphisms. In a study from Turkey performed on adult patients with nephrolithiasis, the association of A236A SNP in 7th as well as -593T > A polymorphism in the promoter region with nephrolithiasis was investigated. In this study, the im- portance of this polymorphism in the promoter region of the SPP1 gene is emphasized.(18) - tigated in the present study. We have determined that the SNP is located on this region. It was aimed to associate the synonymous aminoacid mutation distributions of the more common SNP’s D80D and A236A with phenotypic proper- gene (unpublished data). The study was performed on a low number of patients and and nephrolithiasis (Table 1) or between TT genotype and nephrolithiasis (Table 2). This may be due to the low num- ber of study sample. The power for the comparison of CC genotype between patients and controls was 21.4% and that was also found 22.3% for the comparison of TT genotype between patients and controls. Therefore, this should be studied in greater number of subjects.(19) CONCLUSION - ture to investigate the relationship between childhood neph- rolithiasis and OPN. SPP1 polymorphisms were found to be associated with nephrolithiasis and we suggest that OPN may be a useful marker evaluating the early genetic risk of childhood nephrolithiasis. Although SNPs were determined - sion of OPN mRNA in urine and blood to further clarify this association. We believe, in the future, further studies on different races and ethnic groups are required to verify nephrolithiasis and OPN. CONFLICT OF INTEREST None declared. REFERENCES 1. Wesson JA, Johnson RJ, Mazzali M, et al. Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules. J Am Soc Nephrol. 2003;14:139- 47. 2. Tawada T, Fujita K, Sakakura T, et al. Distribution of osteo- pontin and calprotectin as matrix protein in calcium-con- taining stone. Urol Res. 1999;27:238-42. 3. Yasui T, Fujita K, Sasaki S, et al. Expression of bone matrix proteins in urolithiasis model rats. Urol Res. 1999;27:255-61. 4. Mazzali M, Kipari T, Ophascharoensuk V, Wesson JA, John- son R, Hughes J. Osteopontin--a molecule for all seasons. QJM. 2002;95:3-13. 5. Forton AC, Petri MA, Goldman D, Sullivan KE. An osteopon- tin (SPP1) polymorphism is associated with systemic lupus erythematosus. Hum Mutat. 2002;19:459. 6. Xie Y, Sakatsume M, Nishi S, Narita I, Arakawa M, Gejyo F. Expression, roles, receptors, and regulation of osteopontin in the kidney. Kidney Int. 2001;60:1645-57. 7. Gao B, Yasui T, Itoh Y, et al. Association of osteopontin gene haplotypes with nephrolithiasis. Kidney Int. 2007;72:592-8. 8. Shin HD, Park BL, Cheong HS, Yoon JH, Kim YJ, Lee HS. SPP1 polymorphisms associated with HBV clearance and HCC occurrence. Int J Epidemiol. 2007;36:1001-8. 9. Erdogan H, Mir S, Berdeli A, Aksu N. Renal scarring and osteopontin gene C/T polymorphism in children with pri- mary vesicoureteral reflux. Indian Pediatr. 2012;49:311-3. 10. Niino M, Kikuchi S, Fukazawa T, Yabe I, Tashiro K. Ge- netic polymorphisms of osteopontin in association with multiple sclerosis in Japanese patients. J Neuroimmunol. 2003;136:125-9. 11. Mochida S, Hashimoto M, Matsui A, et al. Genetic polymor- phims in promoter region of osteopontin gene may be a marker reflecting hepatitis activity in chronic hepatitis C patients. Biochem Biophys Res Commun. 2004;313:1079- 85. 12. Yamate T, Tsuji H, Amasaki N, Iguchi M, Kurita T, Kohri K. Analysis of osteopontin DNA in patients with urolithiasis. Urol Res. 2000;28:159-66. Endourology and Stone Disease 647Vol. 9 | No. 4 | Fall 2012 |U R O LO G Y J O U R N A L SPP1 Gene Polymorphisms and Nephrolithiasis | Tekin et al 13. Yasui T, Fujita K, Hayashi Y, et al. Quantification of osteo- pontin in the urine of healthy and stone-forming men. Urol Res. 1999;27:225-30. 14. Jiang XJ, Feng T, Chang LS, et al. Expression of osteopontin mRNA in normal and stone-forming rat kidney. Urol Res. 1998;26:389-94. 15. Gao B, Yasui T, Okada A, Tozawa K, Hayashi Y, Kohri K. A polymorphism of the osteopontin gene is related to uri- nary calcium stones. J Urol. 2005;174:1472-6. 16. Ertan P, Tekin G, Oger N, Alkan S, Horasan GD. Metabolic and demographic characteristics of children with urolithi- asis in Western Turkey. Urol Res. 2011;39:105-10. 17. Koyuncu HH, Yencilek F, Eryildirim B, Sarica K. Family his- tory in stone disease: how important is it for the onset of the disease and the incidence of recurrence? Urol Res. 2010;38:105-9. 18. Gogebakan B, Igci YZ, Arslan A, et al. Association between the T-593A and C6982T polymorphisms of the osteopontin gene and risk of developing nephrolithiasis. Arch Med Res. 2010;41:442-8. 19. Dean AG, Sullivan KM, Soe MM. OpenEpi: Open Source Epi- demiologic Statistics for Public Health, Version 2.3.1. www. OpenEpi.com, updated 2011/23/06, accessed 2012/02/10.