الفهرس 
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Abstract
Kidney stone disease (KSD) refers to a disease caused by calcifications that form stones in the human urinary system leading to intense colicky pain in the back, flank, lower abdomen, groin region along with hematuria and sometimes in bilateral cases, renal failure. Individuals with family history of stone disease have a high risk of developing kidney stones than those without a positive family history indicating that genetics play a classic role in stone formation and its recurrence. As a consequence, genetic markers of calcium stone development could be clinically valuable for recognizing subjects at risk of stone formation and for preventing its occurrence.
from the genome-wide association studies and candidate gene studies, several genes like transient receptor potential vanilloid member 5 (TRPV5), calcitonin receptor (CALCR), calcium sensing receptor (CaSR), have been found to be associated with increasing risk of urolithiasis in different populations of the world; however, the role of these genes needs to be evaluated in the pathogenesis of the disease in Egyptian population for better diagnosis and treatment of Egyptian urolithiasis patients.
Majority of kidney stones are calcium-rich. As a consequence, genetic markers of calcium stone development like TRPV5 rs4236480, CALCR rs1801197 and CaSR rs1801725 SNPs could be clinically valuable for recognizing subjects at risk of stone formation and for preventing its occurrence.
It is generally known that urolithiasis and vascular disease are related. In a prior study, it was discovered that the levels of many osteogenic factors, including BMP2, were much higher in the kidney tissue of genetically hypercalciuric rats than in control rats. Hence, kidney stone development may occur in the same way that bone does, and BMP2 may help with kidney stone formation prediction.
The aim of this study was to evaluate the association of these candidate genetic variants (TRPV5 rs4236480, CALCR rs1801197 and CaSR rs1801725) with the risk of developing KSD in an Egyptian population. Moreover, to determine the efficacy of bone morphogenetic protein-2 (BMP-2) in the prediction of kidney stone formation in Egyptian population.
This study includes 134 patients with calcium kidney stone disease together with 86 healthy control subjects. Patients selected from those attending the inpatient of Department of Urology, El-Demerdash Hospital, Ain Shams University, Cairo, Egypt.
Blood samples were collected from patients and healthy controls each blood sample was divided into 2 parts one of them was kept in clean glass tube without additives to clot at 37 ºC for 10 minutes, and then centrifuged at 3000 rpm for 10 minutes. The serum was then separated into aliquots and stored at -20 ºC to be thawed only once on demand for the determination of biochemical investigations. Another part of blood was collected on ethylene diamine tetra-acetic acid (EDTA) coated tubes for DNA extraction and genotyping studies of TRPV5, CALCR and CaSR.
All samples were analysed for genotyping of TRPV5 rs4236480, CALCR rs1801197 and CASR rs1801725 polymorphisms using allelic discrimination assay and Serum levels of Calcitonin and bone morphogenetic protein-2 (BMP-2) were measured using ELISA technique. The levels of blood urea nitrogen (BUN), creatinine, serum calcium, serum phosphorus, urinary calcium, urinary phosphorus were also analyzed.
The results of the present study were clearly indicated that:
Calcium KSD patients had highly significant elevation of BUN, urinary calcium, calcitonin, BMP-2 and significant increase of urinary phosphorus levels when compared to the control group.
In control group, a highly significant negative correlation was shown between urinary Ca and creatinine. In the meantime, a significant positive correlation was shown between urinary Ca and serum Ca also between calcitonin and serum Ca. Also there was a highly significant positive correlation was observed between urinary Ca and urinary phosphorus.
In Ca KSD group, a significant positive correlation was observed between creatinine and either BUN, urinary phosphorus or calcitonin. Also, a highly significant negative correlation was observed between urinary phosphorus and BUN. Moreover, a significant negative correlation was observed between calcitonin and serum phosphorus.
The genotypic distribution was in consistent with Hardy-Weinberg equilibrium in both KSD and control groups in the 3 SNPs.
KSD patients showed a significantly higher frequency of homozygous mutant (TT) genotype of TRPV5 with a decrease in the homozygous wild (CC) genotype compared with controls. Correspondingly, KSD patients had a higher frequency of the minor T allele than normal subjects.
KSD subjects showed a significantly higher frequency of GA and AA genotypes of CALCR with a decrease in the homozygous wild (GG) genotype compared to controls. Accordingly, KSD subjects had higher A minor allele frequency than normal subjects.
KSD subjects showed a significantly higher frequency of TT genotype of CaSR compared with controls. KSD patients had higher CaSR 2956T allele frequency than normal subjects.
Considering TRPV5 rs4236480, urinary calcium was significantly increased in the sera of TT carriers compared to CT carriers in control group. Moreover, urinary calcium showed a highly significant elevation in TT and CT carriers compared to CC carriers in Ca KSD group.
Regarding CALCR rs1801197, serum and urinary calcium were significantly increased in the sera of AA and GA carriers, compared to GG carriers in control group. Additionally, a highly significant decrease of serum calcium was observed in the sera of GA carriers compared to GG carriers and a highly significant elevation of serum calcium was observed in the sera of AA carriers compared to GA carriers in Ca KSD group.
Referring to CaSR rs1801725, calcitonin level was significantly increased in the sera of TT carriers compared to GG and GT carriers in control group. As well as, serum and urinary calcium were significantly increased in the sera of TT carriers compared to GT carriers. Also, a highly significant increase of BMP-2 was observed in the sera of TT carriers compared to GG carriers in Ca KSD group.
The TRPV5 rs4236480 SNP was associated with increased susceptibility to develop KSD under all the tested genetic models; (homozygous codominant, heterozygous codominant, dominant, recessive and allelic models).
The CALCR G1340A polymorphism was associated with an increased risk of calcium KSD under all the tested genetic models except the recessive model.
The CaSR G2956T polymorphism was associated with an increased risk of calcium KSD under all the tested genetic models.
The results of the exhaustive MDR analysis revealed that among the three loci, the best statistically significant interaction model predicting a potential KSD risk was that of the three-locus interaction consisting of TRPV5 rs4236480, CALCR rs1801197 and CASR rs1801725 polymorphisms.
The results of dendrogram and Fruchterman-Reingold graph describe the interactions between the three SNPs showed that the strongest interaction effect was found between TRPV5 rs4236480 and CALCR rs1801197 with the information gain (IG) value of 1.46%.
In conclusion, in the Egyptian population, the polymorphisms of TRPV5 rs4236480, CALCR rs1801197 and CASR rs1801725 showed a significant association with the risk of KSD and play an important role in the pathogenesis of calcium urolithiasis. Additionally, their complex interactions might have an impact on the genetic susceptibility to develop KSD. As a result, these genes seem to be suitable candidate markers to explain the individual propensity to KSD and may aid in a more accurate diagnosis of that complex disease. Moreover, elevated BMP-2 level in calcium KSD patients compared to controls could explain the role of vascular calcification in the pathogenesis of calcium urolithiasis.