- Email: [email protected]
Association of urokinase and Vitamin D receptor genes SNPs and urolithiasis in an Iraqi population
Journal Pre-proof Association of urokinase and Vitamin D receptor genes SNPs and urolithiasis in an Iraqi population
Zainab Nizar Jawad, Weaam Awad PII:
S2214-5400(20)30034-7
DOI:
https://doi.org/10.1016/j.mgene.2020.100679
Reference:
MGENE 100679
To appear in:
Meta Gene
Received date:
28 January 2020
Accepted date:
20 February 2020
Please cite this article as: Z.N. Jawad and W. Awad, Association of urokinase and Vitamin D receptor genes SNPs and urolithiasis in an Iraqi population, Meta Gene(2019), https://doi.org/10.1016/j.mgene.2020.100679
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
Association of Urokinase and Vitamin D receptor Genes SNPs and Urolithiasis in an Iraqi Population Zainab Nizar Jawad*1, and Weaam Awad2 1-Department of Biology, College of Education for pure sciences, University of kerbala, kerbala, Iraq, email: [email protected], [email protected] 2-College of Medicine, University of Kerbala, Kerbala, Iraq, email: [email protected]
*Corresponding
author:
Zainab
Nizar
Jawad,
email:
[email protected],
Jo u
rn
al
Pr
e-
pr
oo
f
[email protected]
Journal Pre-proof
Abstract Background Several genetic polymorphisms have been supposed to participate in urolithiasis progression. We investigated the association of the urokinase gene 3'-UTR and vitamin D receptor (VDR) polymorphisms with urolithiasis in an Iraqi population. Methods
oo
f
Totally, 104 first kidney stone formers (63 males and 41 females), 100 recurrent stone formers (56 males and 44 females) and 127 healthy subjects (71 males and 56 females) were enrolled. The DNA was extracted from the peripheral blood; next the urokinase 3'-UTR and vitamin D receptor (VDR) genes polymorphisms (rs4065 and rs731236, respectively) were evaluated using polymerase chain reaction (PCR) and PCR- restriction fragment length polymorphism (RFLP) using ApaL I and Taq1 restriction enzymes, respectively.
pr
Results
Conclusion
Jo u
rn
al
Pr
e-
There was a significant difference between those who had urolithiasis with familial history and those patients without history (179 vs 25, p<0.001). TT genotype (rs4065 polymorphism) was predominant among first-time stone formers (73/104, 70.2%) and recurrent stone formers (84/100, 84%) as compared to control group (42/127, 33.1%) (p=0.002 and p<0.001, respectively). CC genotype was significantly higher in recurrent stone formers (20/100, 20%) than first-time stone formers (8/104, 7.7%, p=0.022). Notably, total stone formers had significant more rs4065 SNP (157/204 or 76.96% vs. 33.1%, p<0.001) than control group (76.96 vs 33.1%, p<0.001). Furthermore, VDR CC genotype (rs731236 polymorphism) of stone formers (18%) was significantly higher than control (12/127, 9.44%) (p=0.030 and p=0.021, respectively). Control group had significantly higher rate of CG than those from other groups. Using multinomial logistic analysis, urokinase TT genotype and VDR CC genotype were associated with urolithiasis.
There was a significant association between urokinase gene 3'-UTR TT (rs4065) and VDR genes (rs731236) polymorphisms urolithiasis. In addition, VDR CC genotype was significantly associated with recurrent urolithiasis. However, more in-depth verifications will be helpful towards disclosure of genetic mutations predisposing first-time and recurrent urolithiasis. Keywords Urokinase polymorphism, Vitamin D receptor, Urolithiasis, PCR- RFLP
Journal Pre-proof
1. Introduction Urolithiasis is common among women and men (Li et al., 2013) caused by several genetic and epigenetic factors. The most common types of kidney stones include calcium oxalate and calcium phosphate (Lai et al., 2010). Idiopathic hypercalciuria is the most common metabolic risk factor for calcium kidney stone disease and other important risk factors include hyperoxaluria, hypocitraturia and low urine volume. Urokinase gene is located in the
f
chromosome 10q24 and encodes and enzyme converting plasminogen into
oo
plasmin, which in turn stimulates fibrinolysis and breaks down the basement
pr
membrane glycoproteins (Riccio et al., 1985; Tripputi et al., 1985). This gene has TT polymorphism predisposing to kidney stones (Jawad, 2014). Among
e-
them, a T/C polymorphism at the +4065 nucleotide in the 3'-untranslated region
Pr
(3'-UTR) has been mostly studied. Previous studies have demonstrated correlation between the incidence of kidney stone this polymorphism (Assari,
al
Angaji, & Morovvati, 2016; Li, et al., 2013; A. Ozturk, Minster, DeKosky, & Kamboh, 2007; Segarra et al., 2014; Wei et al., 2011; Zhang et al., 2003). Two
rn
studies found no association between 3'UTR polymorphism and nephrolithiasis.
Jo u
Another candidate gene is the vitamin D receptor (VDR) gene, which is involved in calcium metabolism, including calcium intestinal absorption and renal reabsorption (Haussler et al., 2013). The polymorphism at rs731236 locus predisposes individuals to urolithiasis according to previous findings. As these genes have not been studied in this area before, this study assessed their role in urolithiasis. The aim of this study was assessment the association of the urokinase gene polymorphisms at 3’-UTR and VDR with the risk of calcium kidney stones in Iraq . 2. Materials and Methods 2.1. Study Participants
Journal Pre-proof
A total of 104 first stone formers and 100 recurrent stone formers admitted to Al Hussein Educational Hospital in the holy of Karbala, Iraq, were selected randomly as well as medical clinics for urinary diseases compared to 127 matched healthy subjects. The duration of study was from January 2018 to February 2019. A patient was defined stone former (at least one calcium kidney stone) when a stone was observed at ultrasound or X-ray of the kidney or he reported a spontaneous urinary stone emission or a history of surgical stone removal. All ages were included in the study, but subjects from other cities or a
oo
f
foreign country or another race were excluded. Additionally, patients with drugs consumption (steroids, vitamin D, etc.), endocrine or other disorders,
pr
recurrent infections, abnormal serum creatinine or electrolytes and kidney
e-
cystic disorders were excluded. Notably, chemical or infrared spectrometric analyses were applied to confirm calcium oxalate or calcium phosphate stones.
Pr
We excluded patients having drug affecting electrolyte or citrate handling.
al
2.2. Genotyping
rn
Peripheral blood samples were collected for DNA extraction using the QIAamp Blood Kit (QIAGEN, Hilden, Germany) and the PCR technique was used to the
genetic
Jo u
amplify
region
using
CCAGGCGTCTACACGAGAGT-3'
primers
and
F:
R:
5'5'-
AAAGACAAGTTGCTGGTCAGTAACA-3' for urokinase SNP and F: 5'CAGAGCATGGACAGGGAGCAA-3' AAGGGGCGTTAGC-3′.
The
cycling
and
R:
conditions
5′-CACTTCGAGCAC were
as
following:
denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 56 °C (for both genes) for 30 s, 72 °C for 40 s, and one final extension cycle at 72°C for 7 min. The PCR products (210bp) were analyzed by use RFLP technique after products digested by 2 units of ApaL I and Taq1 restriction enzymes (New England Biolabs, Beverly, USA). The restriction buffer for ApaL I and Taq1 included at 37 °C for 16 h and 65° for 3 h, respectively. A 210 bp size fragment
Journal Pre-proof
represented the genotype CC, 185bp and 25bp sizes referred to the genotype TT, and the 210bp, 185bp and 25bp fragments referred to genotype C/T for urokinase. Furthermore, For Taq1 VDR SNP, C/C homozygotes yielded 208 and 293 bp fragments, T/T homozygotes yielded a 501 bp fragment, and T/C heterozygotes exhibited 501, 293, and 208 bp fragments. All samples were run and kept using a 2% agarose gel electrophoresis (70V/30mA). Data analysis
oo
f
The IBM SPSS 21 (IBM, Armonk, NY, USA) was employed. Comparison of SNPs differences between first-stone formers and recurrent stone formers and
pr
also control population was implemented using Chi-square and multinomial
e-
logistic regression tests.
Pr
3. Results
al
3.1. Demographic data
Of 104 first stone formers, the mean age was 47.56±6.21 year not being
rn
significant different from recurrent stone formers (50.32±9.14) and control
Jo u
(48.18±3.06) group. We also observed a correlation between familial history and incidence of urolithiasis (p=0.023). The composition of patients’ stones included calcium oxalate (80%) and calcium phosphate (12%) stones and 8% mixed.
3.2. PCR-RFLP It was revealed that CC genotype was significantly higher in recurrent stone formers than first-time stone formers and control groups (p=0.022 and p=0.003, respectively). Furthermore, CT genotype was not significantly different between first stone formers than recurrent stone formers, though being significantly different between control and recurrent stone formers. However
Journal Pre-proof
the TT genotype was significantly different between each first stone formers and recurrent stone formers and control group (table1). Furthermore, total stone formers had significant more rs4065 SNP than control group significant more (157/204 or 76.96% vs. 33.1%, p<0.001) using Chi-square test, table1). Noticeably, using multinomial logistic analysis, rs4065 TT genotype was significantly associated with urolithiasis (table1). Therefore, there were significant differences in genetic models of Urokinase
f
gene 3'-UTR T/C polymorphism, where the gene model TT recorded the
oo
highest incidence in patients compared with the control group and the other
pr
genotypes.
e-
Table1. Distribution of genotypes in Urokinase gene 3'-UTR T/C polymorphism in
Pr
patients and control group Population
CT
8
23
73
7.7%
22.1%
70.2%
2.4%
6.9%
22.05%
20
16
84
%within sample
20%
16%
84%
% of Total
6.0%
4.83%
25.37%
11.76%
9.80%
76.96%
45
40
42
%within sample
35.4%
31.5%
33.1%
% of Total
13.59%
12.08%
12.68%
-
-
33.1%
Count
rn
%within sample % of Total
rs4065 SNP Control
rs4065 SNP
Count
Jo u
Recurrent SF
Total ST Count
-
P value Total
Total
CC
al
First SF
Genotypes TT
104
100
127
P<0.001 Count %within sample
ST: stone formers
73 22.05%
79 23.86%
199 60.12%
331
Journal Pre-proof
In addition, VDR CC genotype was significantly higher in recurrent stone formers than first stone formers and control groups (p=0.016 and p=0.011, respectively). Moreover, neither CT nor TT genotype was significantly different between each of first stone formers or recurrent stone formers and control population. (table2). Hence, CC genotype was associated with recurrent urolithiasis as evaluated with Chi-square and multinomial logistic analysis. Table2. Distribution of VDR genotypes in patients and control group Genotypes
Total
oo
f
Population CC
43.26%
46.15%
% of Total
3.32%
6.9%
14.50%
19
33
48
19%
33%
48%
6.0%
9.96%
14.50%
12
60
55
%within sample % of Total
al
Count
9.44%
47.24%
43.30%
% of Total
3.62%
18.12%
16.61%
42
138
151
12.68%
41.69%
45.61%
Count
rn
%within sample
Jo u
Total
48
10.57%
Count
Control
45
%within sample
e-
Recurrent SF
11
TT
pr
Count
Pr
First SF
CT
%within sample
104
100
127
331
ST: stone formers
4. Discussion In this study, there was a significant correlation between urolithiasis and familial history of patients (179/204, p=0.023). Our results revealed that the TT genotype was predominant among first-time stone formers (73/104) and recurrent stone formers (84/100) as compared to control group (42/127) (p=0.002 and p<0.001, respectively). CC genotype was significantly higher in recurrent stone formers than first-time stone formers and control groups (p=0.022 and p=0.003, respectively). Therefore, this genotype can be
Journal Pre-proof
Pr
e-
pr
oo
f
considered a risk factor among recurrent stone formers. Notably, total stone formers had significant more rs4065 SNP than control group (76.96 vs 33.1%, p<0.001, table1). Furthermore, VDR TT genotype was significantly higher in recurrent stone formers than control group. Interestingly, first stone formers but not recurrent stone formers were significantly associated with both genes SNPS. Hence, TT and C/T genotypes are significant predisposing risk factor for urolithiasis. The mechanism of urolith formation is still not fully understood till date. However, urinary super- saturation, crystal nucleation, precipitation, growth, aggregation of crystals and their retention in renal tubules participate in the process (Aykan et al., 2016; Riedi, Nathues, Knubben‐Schweizer, Nuss, & Meylan, 2018). The plasminogen activator, urokinase enzyme causes fibrinolysis, but some conditions cause epithelial damage and growth of kidney stone. As a counter mechanism, urokinase increases to dissolve the plasminogen (Deng, Ding, Lavker, & Sun, 2001). Genetic factors play a crucial role in the occurrence of kidney stones, in which, single nucleotide polymorphisms (SNPs) play an important role in the disease occurrence (Lorder, 1999).
Jo u
rn
al
Studies (M. Ozturk et al., 2008), Zainab, 2018) have revealed that polymorphisms correlate with the risk of diseases such as cancer, kidney stone and other disorders. Type of urolith depends on diet, urine pH, anatomic variation and occurrence of infection (Radostits, Gay, Blood, & Hinchcliff, 2000). Results of the current study indicated the effect of urokinase SNPs on the urolithiasis, where the genotype TT had the highest incidence and association with the kidney stone formation compared to the genotypes CT and CC with lower effects. These results are consistent with previous findings (Kim et al.,2011). Other studies also indicated that the presence of T-allele in the gene increases the risk of diseases such as kidney stones (Chen et al., 2014), Alzheimer's (Segarra etal.,2014) and bladder cancer. Li et al. (Li et al.,2013) exhibited that T/C polymorphism in the 3'-UTR of urokinase gene increases the risk of kidney stones, especially in Asian countries. They also pointed out that dysfunction of the urokinase gene with the presence of calcium kidney stones is related to polymorphism in the 3'-UTR region of urokinase gene. This is owing to the crucial role of 3-'TTR sequence in the regulation of gene expression. Regulatory regions within the 3'-untranslated region can influence polyadenylation, translation efficiency, localization, and stability of the mRNA. Some previously published data (Ozturk etal.,2008) have outlined a significant association between 3'UTR polymorphism and the formation of kidney stones
Journal Pre-proof
(Hagikura et al., 2013; Morovvat, Assari, Angaji, & Daraii, 2016). However, some studies (Mittal (Kim et al.,2011)(Mittal, Bid, Kumar, & Bhandari, 2006) have not indicated this association between the polymorphism and disease. This difference in results can be explained by the difference in populations, selection and number of patient samples and control group. It is noteworthy that this association needs more in-depth verifications in various populations. Major limitations of this study included lack of entire evaluation of genetic variations in other genes behind the occurrence of urolithiasis.
f
5. Conclusion
oo
Conclusion
There was a significant association between urokinase gene 3'-UTR TT
pr
genotype and first time and recurrent urolithiasis. In addition, VDR CC
e-
genotype was significantly associated with recurrent urolithiasis. Significant
Pr
difference regarding the existence of these genes SNPs at rs4065 and rs731236 loci between patients and control populations was observed. However, more indepth verifications will be helpful towards disclosure of major genetic predisposing
first-time
al
mutations
and
recurrent
urolithiasis.
rn
Declaration of Competing Interest
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The authors declare no conflicts of interest for this article. Acknowledgements
This study was supported by College of Education for pure sciences, University of kerbala, kerbala, Iraq References Assari, N., Angaji, S., & Morovvati, S. (2016). Association of Polymorphism at 3 ׳-UTR of Urokinase Gene with Risk of Calcium Kidney Stones. Biomacromolecular Journal, 2(1), 21-27. Aykan, S., Tuken, M., Gunes, S., Akin, Y., Ozturk, M., Seyhan, S., et al. (2016). ApaL1 urokinase and Taq1 vitamin D receptor gene polymorphisms in first-stone formers, recurrent stone formers, and controls in a Caucasian population. Urolithiasis, 44(2), 109-115.
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pr
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Cambareri, G. M., Kovacevic, L., Bayne, A. P., Giel, D., Corbett, S., Schurtz, E., et al. (2015). National multi-institutional cooperative on urolithiasis in children: Age is a significant predictor of urine abnormalities. Journal of pediatric urology, 11(4), 218-223. Chen, C.-H., Chen, S.-Y., Shu, K.-H., Wen, M.-C., Cheng, C.-H., Wu, M.-J., et al. (2014). Urokinase gene 3′-UTR T/C polymorphism is associated with malignancy and ESRD in idiopathic membranous nephropathy. BioMed research international, 2014. Deng, F.-M., Ding, M., Lavker, R. M., & Sun, T.-T. (2001). Urothelial function reconsidered: a role in urinary protein secretion. Proceedings of the National Academy of Sciences, 98(1), 154-159. Hagikura, S., Wakai, K., Kawai, S., Goto, Y., Naito, M., Hagikura, M., et al. (2013). Association of calcium urolithiasis with urokinase P141L and 3′-UTR C> T polymorphisms in a Japanese population. Urolithiasis, 41(1), 47-52. Haussler, M. R., Whitfield, G. K., Kaneko, I., Haussler, C. A., Hsieh, D., Hsieh, J. -C., et al. (2013). Molecular mechanisms of vitamin D action. Calcified tissue international, 92(2), 77-98. Jawad, Z. N. THE ASSOCIATION OF UROKINASE GENE 3’-UTR T/C POLYMORPHISM WITH URINARY BLADDER CANCER. Lai, K.-C., Lin, W.-Y., Man, K.-M., Tsai, C.-H., Chen, H.-Y., Tsai, F.-J., et al. (2010). Association of interleukin-18 gene polymorphisms with calcium oxalate kidney stone disease. Scandinavian journal of urology and nephrology, 44(1), 20-26. Li, D., Liu, J., Ren, J., Yan, L., Liu, H., & Xu, Z. (2013). Meta‑ analysis of the urokinase gene 3'‑ UTR T/C polymorphism and susceptibility to urolithiasis. Biomedical reports, 1(3), 369-374. Lorder, N. (1999). Genetic variations can point the way to disease gene. Nature, 401, 734. Mittal, R. D., Bid, H. K., Kumar, A., & Bhandari, M. (2006). Association of urokinase gene 3′-UTR polymorphism with calcium oxalate nephrolithiasis. Journal of endourology, 20(2), 157-160. Morovvat, S., Assari, N., Angaji, S., & Daraii, S. (2016). Association between polymorphism at 3 ׳UTR of urokinase gene and risk of calcium kidney stones. The Journal of Qazvin University of Medical Sciences, 20(2), 4-9. Ozturk, A., Minster, R. L., DeKosky, S. T., & Kamboh, M. I. (2007). Association of tagSNPs in the urokinase‐plasminogen activator (PLAU) gene with Alzheimer's disease and associated quantitative traits. American Journal of Medical Genetics Part B: Neuropsychiatric Genetics, 144(1), 79-82. Ozturk, M., Kordan, Y., Cangul, H., Dogan, H. S., Kilicarslan, H., Vuruskan, H., et al. (2008). Association of urokinase gene 3′-UTR T/C polymorphism with calcium oxalate urolithiasis in children. International urology and nephrology, 40(3), 563-568. Perera, M., Papa, N., Kinnear, N., Wetherell, D., Lawrentschuk, N., Webb, D., et al. (2016). Urolithiasis treatment in Australia: The age of ureteroscopic intervention. Journal of endourology, 30(11), 1194-1199. Radostits, O., Gay, C., Blood, D., & Hinchcliff, K. (2000). A textbook of the diseases of cattle, sheep, pigs, goats and horses. Veterinary medicine, 1371-1441. Riccio, A., Grimaldi, G., Verde, P., Sebastio, G., Boast, S., & Blasi, F. (1985). The human urokinase plasminogen activator gene and its promoter. Nucleic Acids Research, 13(8), 2759-2771. Riedi, A. K., Nathues, C., Knubben‐Schweizer, G., Nuss, K., & Meylan, M. (2018). Variables of initial examination and clinical management associated with survival in small ruminants with obstructive urolithiasis. Journal of veterinary internal medicine, 32(6), 2105-2114. Segarra, A., Jatem, E., Quiles, M. T., Arbós, M. A., Ostos, E., Ostos, H., et al. (2014). Diagnostic value of soluble urokinase-type plasminogen activator receptor serum levels in adults with idiopathic nephrotic syndrome. Nefrología (English Edition), 34(1), 46-52. Takeuchi, M., Yasunaga, H., Matsui, H., & Fushimi, K. (2017). Pediatric urolithiasis associated with acute gastroenteritis: an inpatient database study in Japan. European journal of pediatrics, 176(4), 501-507.
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rn
al
Pr
e-
pr
oo
f
Tripputi, P., Blasi, F., Verde, P., Cannizzaro, L. A., Emanuel, B. S., & Croce, C. M. (1985). Human urokinase gene is located on the long arm of chromosome 10. Proceedings of the National Academy of Sciences, 82(13), 4448-4452. Wei, C., El Hindi, S., Li, J., Fornoni, A., Goes, N., Sageshima, J., et al. (2011). Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis. Nature medicine, 17(8), 952. Yu, D.-S., Yang, Y.-T., & Lai, C.-H. (2016). Epidemiology and treatment of inpatients urolithiasis in Taiwan. Formosan Journal of Surgery, 49(4), 136-141. Zhang, G., Kim, H., Cai, X., López-Guisa, J. M., Alpers, C. E., Liu, Y., et al. (2003). Urokinase receptor deficiency accelerates renal fibrosis in obstructive nephropathy. Journal of the American Society of Nephrology, 14(5), 1254-1271.
Zainab Nizar Jawad, Weaam Awad PII:
S2214-5400(20)30034-7
DOI:
https://doi.org/10.1016/j.mgene.2020.100679
Reference:
MGENE 100679
To appear in:
Meta Gene
Received date:
28 January 2020
Accepted date:
20 February 2020
Please cite this article as: Z.N. Jawad and W. Awad, Association of urokinase and Vitamin D receptor genes SNPs and urolithiasis in an Iraqi population, Meta Gene(2019), https://doi.org/10.1016/j.mgene.2020.100679
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
Journal Pre-proof
Association of Urokinase and Vitamin D receptor Genes SNPs and Urolithiasis in an Iraqi Population Zainab Nizar Jawad*1, and Weaam Awad2 1-Department of Biology, College of Education for pure sciences, University of kerbala, kerbala, Iraq, email: [email protected], [email protected] 2-College of Medicine, University of Kerbala, Kerbala, Iraq, email: [email protected]
*Corresponding
author:
Zainab
Nizar
Jawad,
email:
[email protected],
Jo u
rn
al
Pr
e-
pr
oo
f
[email protected]
Journal Pre-proof
Abstract Background Several genetic polymorphisms have been supposed to participate in urolithiasis progression. We investigated the association of the urokinase gene 3'-UTR and vitamin D receptor (VDR) polymorphisms with urolithiasis in an Iraqi population. Methods
oo
f
Totally, 104 first kidney stone formers (63 males and 41 females), 100 recurrent stone formers (56 males and 44 females) and 127 healthy subjects (71 males and 56 females) were enrolled. The DNA was extracted from the peripheral blood; next the urokinase 3'-UTR and vitamin D receptor (VDR) genes polymorphisms (rs4065 and rs731236, respectively) were evaluated using polymerase chain reaction (PCR) and PCR- restriction fragment length polymorphism (RFLP) using ApaL I and Taq1 restriction enzymes, respectively.
pr
Results
Conclusion
Jo u
rn
al
Pr
e-
There was a significant difference between those who had urolithiasis with familial history and those patients without history (179 vs 25, p<0.001). TT genotype (rs4065 polymorphism) was predominant among first-time stone formers (73/104, 70.2%) and recurrent stone formers (84/100, 84%) as compared to control group (42/127, 33.1%) (p=0.002 and p<0.001, respectively). CC genotype was significantly higher in recurrent stone formers (20/100, 20%) than first-time stone formers (8/104, 7.7%, p=0.022). Notably, total stone formers had significant more rs4065 SNP (157/204 or 76.96% vs. 33.1%, p<0.001) than control group (76.96 vs 33.1%, p<0.001). Furthermore, VDR CC genotype (rs731236 polymorphism) of stone formers (18%) was significantly higher than control (12/127, 9.44%) (p=0.030 and p=0.021, respectively). Control group had significantly higher rate of CG than those from other groups. Using multinomial logistic analysis, urokinase TT genotype and VDR CC genotype were associated with urolithiasis.
There was a significant association between urokinase gene 3'-UTR TT (rs4065) and VDR genes (rs731236) polymorphisms urolithiasis. In addition, VDR CC genotype was significantly associated with recurrent urolithiasis. However, more in-depth verifications will be helpful towards disclosure of genetic mutations predisposing first-time and recurrent urolithiasis. Keywords Urokinase polymorphism, Vitamin D receptor, Urolithiasis, PCR- RFLP
Journal Pre-proof
1. Introduction Urolithiasis is common among women and men (Li et al., 2013) caused by several genetic and epigenetic factors. The most common types of kidney stones include calcium oxalate and calcium phosphate (Lai et al., 2010). Idiopathic hypercalciuria is the most common metabolic risk factor for calcium kidney stone disease and other important risk factors include hyperoxaluria, hypocitraturia and low urine volume. Urokinase gene is located in the
f
chromosome 10q24 and encodes and enzyme converting plasminogen into
oo
plasmin, which in turn stimulates fibrinolysis and breaks down the basement
pr
membrane glycoproteins (Riccio et al., 1985; Tripputi et al., 1985). This gene has TT polymorphism predisposing to kidney stones (Jawad, 2014). Among
e-
them, a T/C polymorphism at the +4065 nucleotide in the 3'-untranslated region
Pr
(3'-UTR) has been mostly studied. Previous studies have demonstrated correlation between the incidence of kidney stone this polymorphism (Assari,
al
Angaji, & Morovvati, 2016; Li, et al., 2013; A. Ozturk, Minster, DeKosky, & Kamboh, 2007; Segarra et al., 2014; Wei et al., 2011; Zhang et al., 2003). Two
rn
studies found no association between 3'UTR polymorphism and nephrolithiasis.
Jo u
Another candidate gene is the vitamin D receptor (VDR) gene, which is involved in calcium metabolism, including calcium intestinal absorption and renal reabsorption (Haussler et al., 2013). The polymorphism at rs731236 locus predisposes individuals to urolithiasis according to previous findings. As these genes have not been studied in this area before, this study assessed their role in urolithiasis. The aim of this study was assessment the association of the urokinase gene polymorphisms at 3’-UTR and VDR with the risk of calcium kidney stones in Iraq . 2. Materials and Methods 2.1. Study Participants
Journal Pre-proof
A total of 104 first stone formers and 100 recurrent stone formers admitted to Al Hussein Educational Hospital in the holy of Karbala, Iraq, were selected randomly as well as medical clinics for urinary diseases compared to 127 matched healthy subjects. The duration of study was from January 2018 to February 2019. A patient was defined stone former (at least one calcium kidney stone) when a stone was observed at ultrasound or X-ray of the kidney or he reported a spontaneous urinary stone emission or a history of surgical stone removal. All ages were included in the study, but subjects from other cities or a
oo
f
foreign country or another race were excluded. Additionally, patients with drugs consumption (steroids, vitamin D, etc.), endocrine or other disorders,
pr
recurrent infections, abnormal serum creatinine or electrolytes and kidney
e-
cystic disorders were excluded. Notably, chemical or infrared spectrometric analyses were applied to confirm calcium oxalate or calcium phosphate stones.
Pr
We excluded patients having drug affecting electrolyte or citrate handling.
al
2.2. Genotyping
rn
Peripheral blood samples were collected for DNA extraction using the QIAamp Blood Kit (QIAGEN, Hilden, Germany) and the PCR technique was used to the
genetic
Jo u
amplify
region
using
CCAGGCGTCTACACGAGAGT-3'
primers
and
F:
R:
5'5'-
AAAGACAAGTTGCTGGTCAGTAACA-3' for urokinase SNP and F: 5'CAGAGCATGGACAGGGAGCAA-3' AAGGGGCGTTAGC-3′.
The
cycling
and
R:
conditions
5′-CACTTCGAGCAC were
as
following:
denaturation at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 56 °C (for both genes) for 30 s, 72 °C for 40 s, and one final extension cycle at 72°C for 7 min. The PCR products (210bp) were analyzed by use RFLP technique after products digested by 2 units of ApaL I and Taq1 restriction enzymes (New England Biolabs, Beverly, USA). The restriction buffer for ApaL I and Taq1 included at 37 °C for 16 h and 65° for 3 h, respectively. A 210 bp size fragment
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represented the genotype CC, 185bp and 25bp sizes referred to the genotype TT, and the 210bp, 185bp and 25bp fragments referred to genotype C/T for urokinase. Furthermore, For Taq1 VDR SNP, C/C homozygotes yielded 208 and 293 bp fragments, T/T homozygotes yielded a 501 bp fragment, and T/C heterozygotes exhibited 501, 293, and 208 bp fragments. All samples were run and kept using a 2% agarose gel electrophoresis (70V/30mA). Data analysis
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The IBM SPSS 21 (IBM, Armonk, NY, USA) was employed. Comparison of SNPs differences between first-stone formers and recurrent stone formers and
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also control population was implemented using Chi-square and multinomial
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logistic regression tests.
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3. Results
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3.1. Demographic data
Of 104 first stone formers, the mean age was 47.56±6.21 year not being
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significant different from recurrent stone formers (50.32±9.14) and control
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(48.18±3.06) group. We also observed a correlation between familial history and incidence of urolithiasis (p=0.023). The composition of patients’ stones included calcium oxalate (80%) and calcium phosphate (12%) stones and 8% mixed.
3.2. PCR-RFLP It was revealed that CC genotype was significantly higher in recurrent stone formers than first-time stone formers and control groups (p=0.022 and p=0.003, respectively). Furthermore, CT genotype was not significantly different between first stone formers than recurrent stone formers, though being significantly different between control and recurrent stone formers. However
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the TT genotype was significantly different between each first stone formers and recurrent stone formers and control group (table1). Furthermore, total stone formers had significant more rs4065 SNP than control group significant more (157/204 or 76.96% vs. 33.1%, p<0.001) using Chi-square test, table1). Noticeably, using multinomial logistic analysis, rs4065 TT genotype was significantly associated with urolithiasis (table1). Therefore, there were significant differences in genetic models of Urokinase
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gene 3'-UTR T/C polymorphism, where the gene model TT recorded the
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highest incidence in patients compared with the control group and the other
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genotypes.
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Table1. Distribution of genotypes in Urokinase gene 3'-UTR T/C polymorphism in
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patients and control group Population
CT
8
23
73
7.7%
22.1%
70.2%
2.4%
6.9%
22.05%
20
16
84
%within sample
20%
16%
84%
% of Total
6.0%
4.83%
25.37%
11.76%
9.80%
76.96%
45
40
42
%within sample
35.4%
31.5%
33.1%
% of Total
13.59%
12.08%
12.68%
-
-
33.1%
Count
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%within sample % of Total
rs4065 SNP Control
rs4065 SNP
Count
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Recurrent SF
Total ST Count
-
P value Total
Total
CC
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First SF
Genotypes TT
104
100
127
P<0.001 Count %within sample
ST: stone formers
73 22.05%
79 23.86%
199 60.12%
331
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In addition, VDR CC genotype was significantly higher in recurrent stone formers than first stone formers and control groups (p=0.016 and p=0.011, respectively). Moreover, neither CT nor TT genotype was significantly different between each of first stone formers or recurrent stone formers and control population. (table2). Hence, CC genotype was associated with recurrent urolithiasis as evaluated with Chi-square and multinomial logistic analysis. Table2. Distribution of VDR genotypes in patients and control group Genotypes
Total
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Population CC
43.26%
46.15%
% of Total
3.32%
6.9%
14.50%
19
33
48
19%
33%
48%
6.0%
9.96%
14.50%
12
60
55
%within sample % of Total
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Count
9.44%
47.24%
43.30%
% of Total
3.62%
18.12%
16.61%
42
138
151
12.68%
41.69%
45.61%
Count
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%within sample
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Total
48
10.57%
Count
Control
45
%within sample
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Recurrent SF
11
TT
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Count
Pr
First SF
CT
%within sample
104
100
127
331
ST: stone formers
4. Discussion In this study, there was a significant correlation between urolithiasis and familial history of patients (179/204, p=0.023). Our results revealed that the TT genotype was predominant among first-time stone formers (73/104) and recurrent stone formers (84/100) as compared to control group (42/127) (p=0.002 and p<0.001, respectively). CC genotype was significantly higher in recurrent stone formers than first-time stone formers and control groups (p=0.022 and p=0.003, respectively). Therefore, this genotype can be
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considered a risk factor among recurrent stone formers. Notably, total stone formers had significant more rs4065 SNP than control group (76.96 vs 33.1%, p<0.001, table1). Furthermore, VDR TT genotype was significantly higher in recurrent stone formers than control group. Interestingly, first stone formers but not recurrent stone formers were significantly associated with both genes SNPS. Hence, TT and C/T genotypes are significant predisposing risk factor for urolithiasis. The mechanism of urolith formation is still not fully understood till date. However, urinary super- saturation, crystal nucleation, precipitation, growth, aggregation of crystals and their retention in renal tubules participate in the process (Aykan et al., 2016; Riedi, Nathues, Knubben‐Schweizer, Nuss, & Meylan, 2018). The plasminogen activator, urokinase enzyme causes fibrinolysis, but some conditions cause epithelial damage and growth of kidney stone. As a counter mechanism, urokinase increases to dissolve the plasminogen (Deng, Ding, Lavker, & Sun, 2001). Genetic factors play a crucial role in the occurrence of kidney stones, in which, single nucleotide polymorphisms (SNPs) play an important role in the disease occurrence (Lorder, 1999).
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Studies (M. Ozturk et al., 2008), Zainab, 2018) have revealed that polymorphisms correlate with the risk of diseases such as cancer, kidney stone and other disorders. Type of urolith depends on diet, urine pH, anatomic variation and occurrence of infection (Radostits, Gay, Blood, & Hinchcliff, 2000). Results of the current study indicated the effect of urokinase SNPs on the urolithiasis, where the genotype TT had the highest incidence and association with the kidney stone formation compared to the genotypes CT and CC with lower effects. These results are consistent with previous findings (Kim et al.,2011). Other studies also indicated that the presence of T-allele in the gene increases the risk of diseases such as kidney stones (Chen et al., 2014), Alzheimer's (Segarra etal.,2014) and bladder cancer. Li et al. (Li et al.,2013) exhibited that T/C polymorphism in the 3'-UTR of urokinase gene increases the risk of kidney stones, especially in Asian countries. They also pointed out that dysfunction of the urokinase gene with the presence of calcium kidney stones is related to polymorphism in the 3'-UTR region of urokinase gene. This is owing to the crucial role of 3-'TTR sequence in the regulation of gene expression. Regulatory regions within the 3'-untranslated region can influence polyadenylation, translation efficiency, localization, and stability of the mRNA. Some previously published data (Ozturk etal.,2008) have outlined a significant association between 3'UTR polymorphism and the formation of kidney stones
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(Hagikura et al., 2013; Morovvat, Assari, Angaji, & Daraii, 2016). However, some studies (Mittal (Kim et al.,2011)(Mittal, Bid, Kumar, & Bhandari, 2006) have not indicated this association between the polymorphism and disease. This difference in results can be explained by the difference in populations, selection and number of patient samples and control group. It is noteworthy that this association needs more in-depth verifications in various populations. Major limitations of this study included lack of entire evaluation of genetic variations in other genes behind the occurrence of urolithiasis.
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5. Conclusion
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Conclusion
There was a significant association between urokinase gene 3'-UTR TT
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genotype and first time and recurrent urolithiasis. In addition, VDR CC
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genotype was significantly associated with recurrent urolithiasis. Significant
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difference regarding the existence of these genes SNPs at rs4065 and rs731236 loci between patients and control populations was observed. However, more indepth verifications will be helpful towards disclosure of major genetic predisposing
first-time
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mutations
and
recurrent
urolithiasis.
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Declaration of Competing Interest
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The authors declare no conflicts of interest for this article. Acknowledgements
This study was supported by College of Education for pure sciences, University of kerbala, kerbala, Iraq References Assari, N., Angaji, S., & Morovvati, S. (2016). Association of Polymorphism at 3 ׳-UTR of Urokinase Gene with Risk of Calcium Kidney Stones. Biomacromolecular Journal, 2(1), 21-27. Aykan, S., Tuken, M., Gunes, S., Akin, Y., Ozturk, M., Seyhan, S., et al. (2016). ApaL1 urokinase and Taq1 vitamin D receptor gene polymorphisms in first-stone formers, recurrent stone formers, and controls in a Caucasian population. Urolithiasis, 44(2), 109-115.
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