GRHPR gene variations in Iraqi patients infected with calcium oxalate kidney stones

a slow complex


Introduction
Urolithiasis or nephrolithiasis widely known as kidney stone disease (KSD) 1 has increased prevalence and recurrence rates worldwide, particularly over the past 30 years 2 , 3 and these rates have varied greatly in different countries, reaching nearly 5-9% in Europe, 12% in Canada, and 13-15% in the USA and 20.% in Saudia Arabia 4,5 .It is related to high financial costs, as well as high recurrence rates 6 .The recurrence rates are very high 7 , leading to an increase in the number of patients who need adequate therapy and a proper prevention method for the recurrence 8 .
Published Online First: January, 2024 https://doi.org/10.21123/bsj.2024.9066P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal sizes, structures, and locations 10 .Kidney stones are classified into the following types, calcium oxalate (CaOx) is the most predominant type of all kidney stones around the world 11,12 , in Iraq; previous studies have reached the same results indicating that CaOx stones are the most common type 13 .It is followed by calcium phosphate, struvite, uric acid, and cysteine which are the least common 14,15 .Kidney stones diseases are characterized by the accumulation of oxalate crystals in the kidneys and eventually forming calcium oxalate stones, there are also several other genetic diseases that lead to the formation of other types of kidney stones, such as distal renal tubular acidosis forming cysteine stones and calcium phosphate stones respectively 16 .
The disease is quite complex and has several possible etiologies 17 including sex, age, race 18 , pregnancy 19 , diabetes and hypertension 20,21 , high temperature degrees 22 , nutrition regime 23 like depending on animal protein-rich diet and consuming foods that contain high amounts of oxalates, Vitamin C and D deficiency and salt intake with low potassium and low citrate content in the nutrition are main factors that lead to stones formation [23][24][25][26][27][28] and insufficient drinking water intake 29,30 Also, biofilm forming microorganisms have a role in stone forming; including Pseudomonas spp., Klebsiella spp., Proteus spp., Oxalobacter formigenes play a role in calcium oxalate formation and other certain bacterial species [30][31][32] .Some drugs have a potential role in promoting kidney stone formation like nephrocalcin, osteopontin, urinary prothrombin fragment-1, bikunin and glycosaminoglycans 33 .Moreover, genetic factors have an important role in stone formation 29 .Hereditary kidney stone disease could be associated with monogenic rare recessive, and X-linked transmission genes that inherited in mendelian manner with distinct phenotype with full penetrance and lead to severe congenital disorders in newborns and adolescents, such as CASR, SLC34A1, AGXT, GRHPR, and HOGA1 genes 34,35 , while dominant polygenic risk variants and single nucleotide polymorphism are more frequent in adults with less penetrance 36,37 .
One of KSD associated genes is the GRHPR, it's a protein coding gene, located in the pericentromeric region of chromosome 9, composed of 21280 base pairs with 9 exons and 8 introns spanning 9 Kbp, encodes for 328 amino acid, 36 kDa, this gene encodes for GRHPR enzyme and mutations in GRHPR gene cause its deficiency 38 .The enzyme catalyzes glyoxylate and hydroxylpyruvate reduction using NADPH coenzyme, glyoxylate is normally removed through the alteration to glycolate in the liver cytosol and mitochondria also hydroxyl-pyruvate is typically reduced to D-glycerate by this enzyme, the absence of this enzyme results in the oxidation of glyoxylate to oxalate and hydroxyl-pyruvate is reduced to Lglycerate by L-lactate dehydrogenase effect 39 .Numerous reported polymorphisms and mutations in the GRHPR gene have been linked to primary hyperoxaluria type2 [40][41][42] .The GRHPR gene has a potential protective effect as a tumor suppressor gene in association with a number of cancers, including breast cancers and kidney cancers 43 .Additionally, low GRHPR expression has been linked to a high degree of risk of hepatocellular carcinoma (HCC) 44 .A recent study found that its expression is regulated by miR-138-5p 45 , as well as, that it is expressed at low levels in the malignant tissues compared to neighboring normal tissues 46 Moreover, GRHPR expression is downregulated by methylation of CpG regions and chromatin remodeling in the human steatohepatitis leading to metabolic pathway disorders 46 .

Patients and controls:
The study extended from February 2022 until August 2022 and included 80 individuals; the patients' group included 50 subjects distributed as 25 males (50%) and 25 females (50%), and their ages ranged from 19 to 60 years.They were diagnosed as having calcium oxalate stones by specialists, in Al Karama Teaching Hospital, according to an abdomen ultrasound, stone analysis, biochemical tests, and the presence of oxalate in the urine.The healthy control group consisted of 30 individuals.They were 17 males (57%) and 13 https://doi.org/10.21123/bsj.2024.9066P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal females (43%), their ages ranged from 15 to 53 years.All participants in the two groups were not smokers, did not have hypertension, diabetes, or obesity.They filled out a prepared questionnaire, and provided oral consent.The ethical approval was registered in the Iraqi Ministry of Health.

Clinical samples and Laboratory investigations
The clinical samples were collected from both patients and controls at Al Karama Teaching Hospital they included urine samples for general urine examination (GUE).The urine was collected in sterile screw caped container and examined within two hours by centrifuging the samples in a bench centrifuge for 10 minutes at 1500 rpm then the supernatant was thrown and the sediment was put on the glass sild and covered by a coverslip then examined by the compound microscopic under the power of 40X, to conform the oxalate crystals presence in the patients' urine.Also, five milliners of peripheral whole blood samples were collected from both patients and controls by vein puncture in sterile plan tube (3ml) then left for 20 minutes to coagulate then centrifuged by bench centrifuge for 5 minutes to collect the sera.The renal functions were investigated by testing the sera for Urea, Creatinine and Uric acid, by using uric acid measuring kit (JTC Diagnostics/Germany), urea measuring kit (JTC Diagnostics/Germany) and creatinine measuring kit (Linear/ Spain) in the sera of both of the patients and controls.

Enzyme Linked Immunosorbent Assay (ELISA) for GRHPR
The concentration of human Glyoxylate Reductase/Hydroxy pyruvate Reductase (GRHPR) enzyme kit (Shanghai YL Biotec / China) was measured in the sera of both patients and controls by sandwich enzyme -linked immune-sorbent assay (Biotech, Greece) according to the manufacture instructions.

Genetic investigation
The genetic investigation included the extraction of genomic DNA from peripheral whole blood samples.The remaining blood (2 ml) was poured into EDTA tubes and kept at -20C˚ till it is used for DNA extraction for the genetic study by using a Reliaprep TM kit (Promega, USA) in accordance with the manufacturer's instructions.The efficacy of the extraction method was assessed directly by measuring the purity of the extracted DNA samples using a Nano-spectrophotometer (Thermo Science, USA).The purity of DNA samples ranged from 1.6 to 2, and the findings were computed using the Nanodrop method.The target DNA fragments were amplified by the Polymerase chain reaction (PCR) technique using a thermal cycler (Applied Biosystems, USA).For a single reaction, the PCR's components were master mix (12.5 μl) and forward primer (1μl), reverse primer (1 μl), DNA (2 l) the final volume was adjacent to 25 μl by adding nuclease-free water.The oligonucleotide primers were designed using primer 3 plus software.the oligonucleotide primers were designed using primer 3 plus software for the studied fragments for exon 4, 6 and 9 as shown in Table 1.final extension was performed at 72°C for 7 minutes.Agarose gel electrophoresis was used to separate PCR products, and then they were visualized using ethidium bromide staining under the UV-trans-illuminator apparatus (OPTEMA /Japan) to ensure the accurate size of the DNA bands.The DNA sequencing of the PCR products was performed by using the Sanger method (Macrogen/Korea), the PCR products sequences were analyzed by Geneious Prime software, and then the variations of the sequence were calculated and statistically analyzed by T-test and least significant difference -LSD (analysis of variance-ANOVA) were used to compare the means.Chisquare test was used to compare the significant percentage in this study according to the statistical analysis system -SAS (2018) program.

Results and Discussion
General Urine Examination: The microscope examination is a basic examination used to identify crystals' type and shape in the urine 41 .Calcium oxalate monohydrate (thermostable form) and dihydrate (soluble form) are the most common crystals found in the urine of urolithiasis infected patients, while anhydrous and trihydrates forms are rarely seen forms in the patients' urine 47 .The initial step for calcium oxalate crystallization is the supersaturation of urine, the attachment of the saturated urine to a sold surface such as red or white blood cells, bacterium or tubular epithelial cells induce the nucleation of the crystals spontaneously, then the crystals increase in size and number facilitate their aggregation, leading to hyperoxaluria and calcium oxalate kidney stone 48 .Basically, crystallization is started due to urine supersaturation with minerals, mainly calcium, and proteins which act as a glue to gather the crystals 49 .Supersaturated urine occurred by several factors include: the high consumption of animal protein which has high purines that are metabolized to oxalate and uric acid.Animal protein consumption and their metabolism lower urine pH, which is between 5.0 and 6.5 thus promoting the growth of calcium oxalate stones 17 .Also, less fluid intake decreases urine flow, hypercalciuria, high salt consumption, Vitamin D deficiency, hyperthyroidism, low calcium intake, hypocitraturia and hyperoxaluria 50,51 .Alternatively, it may be caused by the absence of the GRHPR enzyme, which results in calcium oxalate crystal formation and consequently kidney stone formation Fig. 1.

Kidney functions tests
The results of urea showed highly significant differences between the control (15.95 ±0.19) and the patients' group (31.44 ±2.17), the normal ranges are (7-37 mg\dl).The results of creatinine have also showed highly significant differences between the control (0.721 ±0.04) and the patients' group (1.29 ±0.11) as they were above the normal range, the standard normal ranges are (0.5-1.2 mg\dl).The  2.
Laboratory testing of serum urea and creatinine is rottenly used to diagnose kidney stone 46 51 .Waikar and his colleagues found comparable outcomes.The high levels of urea and creatinine products may be the result of a metabolic disorder in the kidneys caused by direct renal epithelia injury and reduced glomerular filtration rate (GFR), which is attributed to the precipitation of calcium oxalate crystals in the kidney tubules lumen that obstructed the nephrons.Renal dysfunction reduces waste excretion and leads to an accumulation of toxic compounds and waste products in the blood and urine 52,53 .In this study, the results of uric acid in the control were nonsignificant compared to the patients may be related to genetic factors.In Park et al study including the serum metabolites, they found that serum uric acid in was not significantly differing between families with stone history than those without stone family history 54 .The risk of urolithiasis had been found to increase in gout patients suffering from serum uric acid and calcium high levels as a consequence of thyroid hormone dysregulation 55 .In this study, the uric acid concentration was not significantly differed between the two groups may be related to the normal function of the thyroid hormones that maintain normal calcium levels in the blood.

Enzyme Linked Immunosorbent Assay (ELISA) for GRHPR:
Both patient and control groups had their GRHPR enzyme concentrations evaluated, by comparing the two groups the results revealed highly significant differences between the two groups Fig. 2. The results revealed that the concentration Mean ± SE of the controls was (4.78 ± 1.06) while the patients group lacked this enzyme's activity (0.411 ± 0.02) Fig. 2, for that reason the comparison between the two groups was highly significant which is consistent with the findings of Takayama and his collegues 35 .This significant difference between the two groups suggests that a deficiency in GRHPR may play a role in the formation of calcium oxalate kidney stones.Previous studies have reported similar associations between GRHPR deficiency and the formation of kidney stones 56 .This may be because the enzyme's primary function is to keep levels of glyoxylate and hydroxypyruvate relatively low in order to prevent the production of oxalate stones, and it also converts glyoxylate to glycolate, in the case of enzyme absence, glyoxylate is oxidized to oxalate through lactate dehydrogenase (LDH) effect 56 .Since the human body cannot hydrolyze oxalate; calcium oxalate crystals will precipitate and aggregate as calcium oxalate stones 57 .

The genetics investigation
The amplified PCR products of exon 4, exon 6 and exon 9 are shown in Fig. 3, 4 and 5 respectively.The amplified fragments were subjected to the Sanger sequencing technique to identify possible mutations and /or SNPs for both of patients and controls.
Table 3 shows the interpretation of SNPs detected in the amplified products of exon 4, 6 and 9 by sequencing technique.The rs2768659 (A>G) intronic single nucleotide variant with benign effect, is located upstream of the exon4 displays the genotype and allele frequency of both control and patient groups.The study found three genotypes (AA, AG, and GG) in both groups, with varying frequencies.The distribution of genotypes in both the control and patient groups was consistent with Hardy-Weinberg's law at a significance level of 0.05, indicating that the population was in genetic equilibrium.The Chi-Square test showed no significant differences in the number of genotype distribution and allele frequency between the control and patient groups for the AA and AG genotypes.However, the dominant AA genotype accounted for almost half of the genotype in the controls (40%) while in the patients' group it was (28%) therefore, the Chi-Square value 0.154 was non-significant.The GG genotype was significantly higher in the patient group compared to the control group, with a Chi-Square value of 7.758 and a pvalue of 0.0053.The odds ratio (OR) for the GG genotype was 1.47 (0.88-2.08), indicating a moderate association with kidney stone formation.

Table 3. The
The allele frequency of the A allele was higher in the control group (0.58) compared to the patient group (0.42), while the G allele frequency was higher in the patient group (0.58) compared to the control group (0.42).This suggests that the G allele may be associated with an increased risk of kidney stone formation in this population as shown in Table 4.This study may provide evidence for an association between this variant and kidney stone formation.The rs1294628807 is a single nucleotide variant, its located upstream exon 4 was identified in the patients and in the controls.The wild type AA was not found in any group the results were nonsignificant between the two groups, the results of the hetero type AG was highly significant (10.89), it is considered as the causative type, finally the mutant type GG was non-significant (0.580), as shown in Table 5.The Chi-Square test showed a significant difference in the number of genotype distribution and allele frequency between the control and patient groups for the AG and GG genotypes.The AG genotype was significantly higher in the patient group compared to the control group, with a Chi-Square value of 10.89 and a pvalue of 0.001.The odds ratio (OR) for the AG genotype was 1.74 (0.86-3.16), indicating a moderate association with kidney stone formation.
In contrast, the GG genotype was significantly higher in the control group compared to the patient group, with a Chi-Square value of 0.580 and a nonsignificant p-value of 0.446.The OR for the GG genotype was 0.492 (0.21-0.89), indicating a protective effect against kidney stone formation in this population.The allele frequency of the A allele was higher in the patient group (0.16) compared to the control group (0.03), while the G allele frequency was higher in the control group (0.97) compared to the patient group (0.84).This suggests that the A allele may be associated with an increased risk of kidney stone formation, while the G allele may be protective against kidney stone formation in this population.The Chi-Square test showed no significant difference in the number of genotype distribution and allele frequency between the control and patient groups for the TC genotype.The OR for the TC genotype was 0.071 (0.03-26), indicating no association with kidney stone formation.However, the CC genotype was significantly higher in the patient group compared to the control group, with a Chi-Square value of 5.261 and a p-value of 0.0218.
The OR for the CC genotype was 1.29 (0.78-1.84), indicating a moderate association with kidney stone formation.The allele frequency of the T allele was similar between the control group (0.03) and the patient group (0.02), while the C allele frequency was higher in the patient group (0.98) compared to the control group (0.97).This suggests that the C allele may be associated with an increased risk of kidney stone formation in this population as shown in Table 6.The results of genotype and allele frequency of the c.295C>T(Arg99ter) rs119490108 nonsense transition in exon 4 in both groups indicated that there was no significant difference in the genotype distribution of this variant between patients with kidney stones and control individuals.All individuals in both groups had the TT genotype, and no individuals had the CC or CT genotypes.The allele frequency of the T allele was 1 in both groups, indicating that the T allele is the dominant allele for the studied population, and the C allele was not detected in either group as shown in Table 7, and this study may provide some evidence that this variant may not play a significant role in kidney stone formation in the Iraqi population.This variant was found in a PH2 patient by Garrelfs et al 42 .Moreover, the result did not agree with the findings of Konkoľová, et al 35 and Takayama, et al that found it was common in Caucasian individuals of American and European origen 18 , because the patients in this study initially did not have PH2, so, it most likely they did not have the pathogenic mutations.The single nucleotide variants in exon 4 are shown in the fig.6.The c.579A>G (p.Ala193=) rs309458 is located within exon 6 53 , it's a benign single nucleotide variant.This variant was identified in both groups.The genotype frequencies in the control group were AA=0%, AG=7%, and GG=93%, while in the patient group, the genotype frequencies were AA=0%, AG=22%, and GG=78%.The allele frequencies of A and G were 0.03 and 0.97 in the control group, respectively, and 0.11 and 0.89 in the patient group, respectively.The chi-square test was used to evaluate the significance of the differences in genotype frequencies between the two groups.
The results showed a statistically significant difference between the control and patient groups in the distribution of genotypes for this SNP (χ2=6.231,p=0.0126).The odds ratio (OR) for the AG genotype compared to the AA genotype was 0.98 (95% CI: 0.43-2.18),indicating that there is no significant association with the disease.On the other hand, the OR for the GG genotype compared to the AA genotype was 0.335 (95% CI: 0.19-0.77),which suggests a protective effect against the disease.The results indicate that the GG genotype of this SNP in the gene is associated with a lower risk of the disease, while the AG genotype does not appear to be associated with the disease as shown in Table 8.Cregeen, et al. found that, the c.579A>G (A193A) rs309458 had no effect on the GRHPR enzyme activity as it does not form a splicing site in 68% of total in non PH2 patients 58 .he c.494-68A>G rs309459 intronic variant, is located upstream exon 6 (ncbi.nlm.nih.gov/SNP/).This benign mutation was identified in the patients and in controls, the genotype and allele frequencies showed three genotypes (AA, AG, and GG) in both groups, with varying frequencies.None of the individuals in the control or patient groups had the AA genotype.The Chi-Square test showed no significant difference in the number of genotype distribution and allele frequency between the control and patient groups for the GG genotype.The OR for the GG genotype was 0.521 (0.23-1.09), indicating no association with kidney stone formation.However, the AG genotype was significantly higher in the patient group compared to the control group, with a Chi-Square value of 4.571 and a p-value of 0.0325.The OR for the AG genotype was 1.06 (0.72-1.61), indicating a slight association with kidney stone formation, although the effect size is small.The allele frequency of the A allele was higher in the patient group (0.11) compared to the control group (0.05), while the G allele frequency was higher in the control group (0.95) compared to the patient group (0.89).This suggests that the A allele may be associated with an increased risk of kidney stone formation in this population as shown in Table 9. Cregeen, et al. found that, the c.494-68A>G rs309459 had an effect on the GR activity but less on the HPR enzyme activity 58 .The results of the genotype and allele frequency of the c.494G>A (p.Gly165Asp) rs180177314 (G>A) variant in exon 6 of the GRHPR gene in both control and patient groups indicated that there was no significant difference in the genotype distribution of this variant between patients with kidney stones and control individuals.All individuals in both groups had the AA genotype, and no individuals had the AG or GG genotypes.The allele frequency of the A allele was 1 in both groups, indicating that A allele is the major allele for this variant in the population, and the G allele was not detected in either group as shown in Table 10.In this study it had been found that three single nucleotide intronic variants at rs 309458 and rs309459 as well as the pathogenic variant at c.494G>A (p.Gly165Asp) rs180177314 which is restricted in the GG genotype in both of the patients and the controls, Fig. 7 shows the sequences of the nucleotides of all GRHPR SNPs that were found at exon 6.The nonpathogenic c.*146A>G rs1057507, intron variant was identified in both groups, the results of genotype frequencies in the control group were AA=87%, AG=13%, and GG=0%, while in the patient group, the genotype frequencies were AA=90%, AG=10%, and GG=0%.The allele frequencies of A and G were 0.975 and 0.025 in the control group, respectively, and 0.95 and 0.05 in the patient group, respectively.The chi-square test was used to evaluate the significance of the differences in genotype frequencies between the two groups.
The results showed a significant difference between the control and patient groups in the distribution of genotypes for this SNP (χ2=5.084,p=0.0241).The odds ratio (OR) for the AG genotype compared to the AA genotype was 0.317 (95% CI: 0.15-0.80),indicating a significant protective effect against the disease.The results suggest that the AG genotype of this SNP is associated with a lower risk of the disease.The absence of individuals with the GG genotype in both control and patient groups suggests that this genotype may be rare in the population studied or may be associated with severe phenotypes that are not included in the study as shown in Table 11.This variant was found to be dominant in several other populations like European, African American, African and others (ncbi.nlm.nih.gov/SNP/).The nucleotide sequence for the SNPs that had been found at exon 9 is shown in Fig. 8  The results of genotype and allele frequency of GRHPR c.904C>T (p.Arg302Cys) rs180177322 in exon 9 in the control and patient groups indicated that there were no significant differences in the genotype or allele frequency of this pathogenic variant between the control and patient groups.Both groups had a 100% frequency of the dominant CC genotype and a 0% frequency of the CT and TT https://doi.org/10.21123/bsj.2024.9066P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal genotypes as shown in Table 12.This suggests that this particular SNP may not be associated with an increased risk of kidney stone formation in the studied population.The pathogenic missense mutation at c.165 G>A (Gly165Asp) rs180177314, the c.295C>T (Arg99ter) rs119490108, and the c.904C>T p. (Arg302Cys) rs180177322 had been associated with reduced GRHPR enzyme activity and an increased risk of PH2, which lead to the formation of kidney stones 37,58 .These pathogenic mutations are rare in the studied population and they were not detected due to the presence of the wild genotype and the absence of the heterozygous genotypes, the patients did not have PH2 because of the protective alleles are the dominant alleles while the causative alleles were not shown in the studied population.
Table 13 elucidates the association of the GRHPR single nucleotide variants with enzyme concentration in control and patient.The results showed that all SNPs were significantly associated with enzyme concentration in both control and patient at (p<0.0001).In the control group, the mean enzyme concentrations for each genotype were significantly different for all SNPs.The highest mean enzyme concentrations were observed for the homozygous wild-type genotypes (AA, GG, TT and CC) while the lowest mean concentration was found in the heterozygous genotypes (AG, CT).
In the patient group, the mean enzyme concentrations for each genotype were also significantly different for all SNPs.However, the pattern of association was different compared to the control group, and the lowest mean enzyme concentrations were consistently observed in the homozygous mutant genotypes, while the highest mean concentrations were found in the heterozygous genotypes.These results suggest that the SNPs are associated with changes in enzyme concentration in both control and patient populations, and this association is likely to be mediated by the effect of these genetic variants on the activity or expression of the enzyme 59 .
The reasons that may have led to these results are the existence of mutations in another place that was not studied in this study 56 .On the other hand, the presence of microRNA (miR-138-5p) could attack GRHPR mRNA and inhibits its expression 45 .It may be led to DNA methylation of the CpG sequences in the promoter region or other CpG islands, which may affect the GRHPR gene expression 40 .However, it is important to note that the study has some limitations, such as the small sample size and the lack of information on other potential confounding factors that may influence enzyme concentration.Therefore, further studies with larger sample sizes and more comprehensive analyses are necessary to confirm these findings and elucidate the underlying mechanisms of these associations.However, it is important to note that the study has some limitations, such as the relatively small sample size, and further studies with larger sample sizes are needed to confirm these findings.

Conclusion
This study found that GRHPR enzyme concentration was lower in the sera of patients than in the sera of the control group.The pathogenic mutations at c.295C>T, c.165G>A and c.904C>T (p.Arg302Cys) rs180177322 were not found in the kidney stone patients in this study and the alleles were in the dominant not mutant type, therefore they did not have PH2.The wild genotypes were dominant in the studied groups and the SNP variants and the benign intron variants collectively may lead to reduce enzyme gene expression and eventually lead to stone formation.

Figure 3 .
Figure 3. PCR product gel electrophoreses at 2% Agarose using 100 bp DNA ladder in TBP for 40 minutes at 80 Volt for patient's exon 4, band size 879bp.

Figure 4 .
Figure 4. PCR product gel electrophoreses at 2% Agarose using 100 bp DNA ladder in TBP for 40 minutes at 80 Volt for patient's exon 4 and 6, bands sizes 876bp and 765 bp respectively.

Figure 5 .
Figure 5. PCR product gel electrophoreses at 2% Agarose using 100 bp DNA ladder in TBP for 40 minutes at 80 Volt for patient's exon 9, band size 376 bp.

Figure 6 .
Figure 6.The chromatograms for the nucleotide sequences of the GRHPR exon 4 showed the reference sequences of the rs2768659, rs1294628807, rs2736664, and rs119490108 respectively.The chromatograms also demonstrated the transitions from A to G in rs2768659, G to A in rs1294628807, C to T in rs2736664, and the dominant allele C in rs119490108 respectively.

PublishedFigure 7 .
Figure 7.The chromatograms of nucleotide sequences of GRHP exon 6, showing the reference sequences of c.579A>G (p.Ala193=) rs309458, rs180177314 and c.494-68A>G rs309459 intronic variant respectively.Also, chromatograms show dominant allele A transition to G at the c.579A>G (p.Ala193=) rs309458, the dominant allele G of rs180177314 and the transition from A to G the transition from A to G in the c.494-68A>G rs309459.

Figure 8 .
Figure 8.The chromatogram of nucleotide sequence of GRHPR exon 9.The chromatogram shows the intronic variant c.*146A>G rs1057507 nucleotide sequence and the transition from A to G. and the pathogenic variant at c.904C>T (p.Arg302Cys rs180177322, only the C dominant allele is appeared in both patients and controls.

Table 2 . concentration of Urea, Creatinine, and Uric acid by mg/dl in patients with renal stones compared to controls.
**

Table 5 . Genotype and allele frequency of GRHPR gene/SNP (rs1294628807) in control and patients' groups
* Significant at (P≤0.05), ** significant at (P≤0.01), NS: Non-Significant at (P≤0.05).The c.288-11 C>T (rs2736664) variant was found at exon 4 of the patients and controls.The study found three genotypes (TT, TC, and CC) in both groups, with varying frequencies.None of the individuals in the control or patient groups had the TT genotype.

Table 10 . Genotype and allele frequency of GRHPR c.494G>A (p. Gly165Asp) rs180177314 in control and patients' groups
*