Haplotype of CaSR gene is associated with risk of renal stone disease in West Indian population

Calcium is the most abundant metabolite involved in the stone matrix. The CaSR gene controls calcium homeostasis, and genetic variation in the CaSR gene could lead to the development of renal stone disease. Therefore, the current study has been designed to assess the association of genetic variants of CaSR gene polymorphisms with renal stone disease. A single-centric prospective study has been carried out on a total of 300 participants (150 cases and 150 controls). Serum levels of calcium, creatinine, parathyroid hormone, and 24 h urine metabolites were measured. Two polymorphisms, rs1801725 and rs1042636, of the CaSR gene, have been genotyped for each participant. T test, binary logistic regression, and Chi-square analysis were used for statistical analysis. Renal stone patients had significantly higher levels of serum parathyroid hormone, creatinine, and 24-h urine metabolites in comparison to the controls. CaSR gene variants, rs1801725 (GG) and rs1042636 (AA), both have shown significant association with renal stone disease. In addition, individuals having specific genotypes along with metabolic abnormalities such as hypercalcemia and hyperparathyroidism are found to be at a higher significant risk of developing the renal stone disease. In the present study, the haplotype of the CaSR gene has shown an association with renal stone disease. Individuals with hyperparathyroidism and hypercalcemia and risk genotype have a higher susceptibility to developing renal stone disease.


Introduction
Renal stone disease is among the most prevalent renal disease.Humanity has suffered kidney stones for centuries dating back to 4000 B.C [1].Preventing the recurrence of kidney stones is still a major issue in human health [2].Chronic kidney disease [3], end-stage renal disease [3], cardiovascular disease [4], diabetes, and hypertension [5] have all been linked to renal stone disease.As a result, stone recurrence avoidance has become a major responsibility, and doing so necessitates greater knowledge of the mechanisms underlying stone formation.The renal stone disease affects 12% of the world's population and causes a tremendous economic burden that developed countries like America spend 2-3% of their health budget on renal stone disease [5].
The prevalence of the renal stone disease is the highest among all renal diseases and affects almost 20 million new cases that are reported in the Indian population every year.In the absence of metaphylaxis, the recurrence rate of renal stone formation is greater, accounting for 10-23 percent per year, 50 percent in 5-10 years, and more than 75 percent in 20 years of the first episode [6].
Since the beginning, it has been believed that nutrition and environment are the most prominent cause of urinary stone formation.Indeed, with the advancement in medical science and technology, different possibilities and areas have been explored; genetic polymorphisms and mutation could be the potential factor that may be involved in the pathology of renal stones.
Renal stone disease is a multifactorial idiopathic disease that includes very complex mechanisms which could be linked to metabolic abnormalities.Calcium is the major component of 80% of renal stones.So, any change in any mechanism or pathways of calcium ion homeostasis may lead to renal stone formation directly or indirectly.
The calcium-sensing receptor (CaSR) gene encodes the calcium-sensing receptor, which regulates parathyroid hormone (PTH) secretion and calcium reabsorption, making it the primary controller of extracellular calcium homeostasis.CaSR receptor activation would decrease the PTH secretion and reabsorption of calcium from the thick ascending limb (TAL).CaSR receptor activation thereby enhances urine calcium excretion via a direct and PTH-mediated Pi transport action on renal tubular cells.[7] While CaSR activation, particularly in the TAL, causes hypercalciuria, which is the primary risk factor for renal stone formation.[8] So, any mutation or polymorphism in the CaSR gene can cause major changes in this series of events and lead to the formation of stone.
Genetic variants of CaSR, "rs1801725" and "rs1042636", both SNPs are being studied in various populations for many of severe diseases, including cancer [9,10], hyperparathyroidism [11][12][13], and renal stone disease [14][15][16][17], which has shown a potential association with disease pathophysiology.Also, Guha et al. studied these two SNP in the East Indian population (from Kolkata) and found them to be associated with the risk of development of renal stone disease [18]; indeed, no study has been carried out on the West Indian population, so considering the facts we have included only these two SNPs for the study.
Although a study has been conducted in the eastern Indian population to evaluate the association between CaSR gene polymorphism and renal stone disease [18], they did not evaluate the role of metabolic abnormalities and their susceptibility toward the CaSR gene polymorphisms.To the best of our knowledge, no study has been conducted in the western part of India, and therefore present study was carried out to determine the association between CaSR gene polymorphism and renal stone disease and its influence on the major metabolic abnormalities in the West Indian population.

Participants
A total of 150 patients (male and female both) aged between 18 and 65 having a current or history of renal stone(s) supported by conforming renal ultrasound or CT scan and admitted to the department of urology at Muljibhai Patel Urological Hospital (MPUH), Nadiad, Gujarat, India were enrolled into the case group.Patients with a known history of renal disorders other than renal stone, known metabolic disorder, pregnancy, or on drugs like diuretics and calcium supplements, which may affect the stone formation, have been excluded from the study.Age-and sex-matched 150 healthy individuals were enrolled in the control group who belonged to the same geographical region and had no known history of renal stones.Renal ultrasound sonography was performed for control participants to avoid asymptomatic renal stones and calcification; individuals diagnosed with renal stone(s) or calcification were excluded from the control group.Detailed socio-demographic information about age, gender, drinking water, food habit (vegetarian or nonvegetarian), salt intake, and family history for renal stones were collected with the help of paper case record forms from the participants of both groups.

Blood and urine analysis
Once the written consent was taken from the participant, a total of 6 ml of peripheral blood was withdrawn, from which 2 ml of blood was withdrawn into the ethylene diamine tetraacetic acid (EDTA) vacutainer stored at -20 °C for further genetic analysis and remaining 4 ml of blood sample was used to perform biochemistry parameters like serum creatinine, serum calcium, and parathyroid hormone levels.The 24-h urine sample was collected to perform 24-h metabolite levels, including creatinine, calcium, magnesium, phosphorus, oxalate, citrate, and uric acid.All the biochemistry parameters from blood and urine samples were performed on the fully automated analyzer of Beckman AU480.Parathyroid hormone level was performed by radioimmunoassay (RIA) on Maglumi 800 CLIA analyzer.A colorimetry method developed by MPUH was used to perform 24-h citrate levels.

Genomic DNA extraction
Genomic DNA was extracted by GeneJET mini Whole Blood Genomic DNA Purification Kit (K0781), Thermo Scientific, USA, as per the manufacturer's instruction.Isolated DNA was checked for quality using QIAxpert, QIAGEN, Germany.

Statistical analysis
A binary logistic regression test was used to investigate the association of various genotypes of CaSR gene variants, rs1801725 and rs1042636, with a renal stone disease by applying various genetic models (odds ratios were adjusted for the age and gender distribution among case and control groups).Student t test was applied to calculate statistically significant differences in continuous independent variables like age, gender, food habit, drinking water, family history, serum creatinine, serum calcium, parathyroid hormone level, and 24-h urine metabolites between the case and control groups.All the biochemical parameters and genotyping data were statistically analyzed using SPSS 29.0.0,IBM software.We have calculated linkage disequilibrium (LD) [19] for our studied population and done haplotype selection using the genotype distribution of both the SNPs to carry out the association study of haplotypes.

Results
Socio-demographic characteristics of stone patients and controls are presented in Table 1.The mean age of stone patients was 44.15 ± 11.71 years; out of 150 stone patients, the renal stone disease was significantly observed in males accounting for 74% incidence, while females were less affected (26%) compared to the males.Family history of stone has been significantly observed in 58.66% of stone patients.Though there is no significant role of drinking water observed in stone formation, non-vegetarian food has remarkably impacted stone formation.
Biochemical determinants of stone patients and healthy controls are manifested in Table 2. Serum creatinine, parathyroid hormone levels, and all the 24-h urine metabolites showed a significant difference between stone patients and healthy controls, though no significant difference was found in serum calcium levels.In addition, some metabolic abnormalities, such as hyperparathyroidism (HPT), hypercalciuria, and hyperoxaluria, are diagnosed in 51.33, 52, and 67.33% of stone patients, respectively.
In the current study, CaSR gene variants, rs1801725 and rs1042636, have been analyzed for the frequency distribution among the West Indian population, and frequencies have been found to differ from the various populations around the world.(rs1801725; G allele-63%, T allele-37% and rs1042636; A allele-58%, G allele-42%).(Fig. 1.)

CaSR gene rs1801725 polymorphism and renal stone disease
Homozygous genotype GG is present in a higher percentage of stone patients as compared to healthy controls (31.33 vs. 20.66).Heterozygous co-dominant (GG vs. GT) and dominant (GG vs. GT/TT) models showed significant association (OR 1.785, p = 0.0300 95% CI 1.033-3.083and OR 1.785, p = 0.0360 95% CI 1.039-3.067,respectively) (Table 3).Hypercalcemia has been found to be significantly associated with rs1801725 polymorphism.The heterozygous co-dominant (GG vs. GT) model has shown a significantly increased risk of hypercalcemia (OR 2.13, p = 0.0105, 95% CI 1.21-3.88) in renal stone patients.While there was no significant association found between hyperparathyroidism and hypercalciuria and rs1801725 polymorphism.(Table 4)

CaSR gene rs1042636 polymorphism and renal stone disease
Homozygous genotype AA is present in a higher percentage of stone patients compared to healthy controls (32.66 vs.

Combined effect of rs1801725 and rs1042636 polymorphisms on renal stone disease
We analyzed the combined effect of all the genotypes of rs1801725 and rs1042636 as we expected that haplotypes could show a significant additive or synergistic effect on renal stone disease.Homozygous GG/AA dominant genotype is present in 32% of stone patients compared to 20.66% in healthy controls, suggesting a significant association with renal stone disease.(Table 7)

Discussion
Renal stone disease is among the most prevalent renal disorders, affecting a large population of the world and putting 50% of patients at risk of developing other severe renal diseases.Renal stone disease is an idiopathic multifactorial disease, and environmental factors such as temperature, dietary habits, and drinking water play a critical role in stone formation.[20,21] The considerable regional and ethnic heterogeneity in renal stone incidence implies that it is influenced by genetic and environmental factors.So, we hypothesized that genetic abnormality could influence the occurrence of renal stone disease and investigated the role of CaSR genetic variants in renal stone disease.
In the present study, we have analyzed socio-demographic parameters, blood parameters, 24-h urine metabolite profiles, and the CaSR gene polymorphisms rs1801725 and rs1042636, their genotype distribution in case and control groups, and their association with renal stone disease and other metabolic disorders that can play a significant role in stone formation.Most patients were from the 31 years to 58 years age group accounting for 70%.Males have a higher incidence rate (77%) with a male-to-female ratio of 3:1, similar to other studies conducted on different populations [22,23].
Parathyroid hormone, calcium, and urine metabolites like calcium, oxalate, and uric acid have a significant presence in the stone matrix or promote stone formation.The present study found that parathyroid hormone, calcium, urine calcium, oxalate, and uric acid showed a remarkable difference between stone patients and healthy controls.However, citrate and magnesium are associated with the inhibition of stone formation.In our study, we observed significant protection with a high level of citrate in urine samples of healthy individuals.An increased level of magnesium in urine suggests an increase in filter load and reduced reabsorption from the loop of Henle.[24] This reabsorption mechanism can be affected by renal stone disease.Altered reabsorption of urine mechanism may explain the high level of urine magnesium in our study.
Although the presence of calcium in the stone matrix among 80% of all stones signifies its importance in studying the complex mechanism of calcium homeostasis of calcium ions, PTH and CaSR gene has a central role.
Studies have reported that polymorphism in CaSR gene variants, rs1801725 and rs1042636, are associated with various diseases, i.e., renal stone disease [11,18,25], prostate cancer [26], colorectal cancer [27], and primary HPT [28].In our study, we have observed that individuals carrying GG genotype (rs1801725) and AA genotype (rs1042636) are associated with a high risk of developing the renal stone disease.Shakhssalim Et al. carried out a similar study on the Iranian population and have reached on same conclusion pointing to a strong association between GG (rs1801725) allele and renal stone disease.[16] Moreover, Ding et al. found the GG (rs1801725) and AA (rs10426363) genotypes to be associated with an increased risk of renal stone disease in the Chinese population [15].On the contrary, a study from East India conducted by Guha et al. has observed a significant association between the GT genotype (rs1801725) and AG genotype (rs1042636) of the CaSR gene with renal stone disease [18].Ali et al. also reported that T allele and TT genotype (rs1801725) are associated with the increased risk of renal stone disease in the Egyptian population [17].European ancestry population has shown a strong association of CaSR gene polymorphism of GG genotype (rs1801725) with a high level of serum phosphate, and AA genotype (rs1042636) has been shown to be associated with a higher level of serum PTH [29].
Both SNPs of the CaSR gene were in linkage disequilibrium; therefore, we performed the haplotype analysis.We have observed that GG/AA haplotype increases the risk of renal stone disease by twofold.
In the present study, we found that allele frequencies of CaSR gene variants, rs1801725 and rs1042636, differ from the previously reported allele frequencies among different populations around the world, including South Asian, European, African, and East Indian populations (Fig. 1), which suggest that the West Indian population has a different genotype distribution compared with other population.It has been shown that genetic variants occur in all ethnic groups but with variable frequency.For example, genetic variants of NAT2-associated phenotypes are variable due to population frequency variability-East Asians (14%), Black Americans (34%), and Whites (54%) [30,31].
Studies have reported that HPT, hypercalciuria, and hypercalcemia are the primary metabolic disorders and significantly increase the risk of the development of renal stone disease [32][33][34][35].In the present study, we have observed these metabolic abnormalities in stone patients with a high percentage of HPT (51.53%) and hypercalciuria (52%).In comparison, the incidence of hypercalcemia has been observed in less number (18.66%) of stone patients.CaSR gene controls ionized calcium in the blood through PTH upregulation and downregulation and reabsorption of calcium ions from urine.In addition, many reports have suggested a positive association of gene polymorphisms with hypercalciuria and HPT [36][37][38].
In the present study, we have examined the association of CaSR gene variants, rs1801725 and rs1042636, with these significant metabolic abnormalities.Individuals with GG genotype of rs1801725 and hypercalcemia were found to be associated with a high risk of developing the renal stone disease.In addition, the individuals with HPT and AA genotype of rs1042636 were associated with a high risk of developing the renal stone disease.Results suggest the remarkable association of CaSR gene polymorphisms rs1801725 and rs1042636 with significant metabolic abnormalities that contribute to the development of renal stone disease.
In summary, the present results indicate that the GG genotype of the rs1801725 variant and AA genotypes of the rs1042636 variant of the CaSR gene could be associated with a significantly increased risk of development of renal stone disease.The renal stone disease results from convoluted reciprocity between environmental and genetic variables.Gene polymorphisms in CaSR gene variants could be partly responsible for an individual's susceptibility in the presence of an appropriate environmental condition.The current study only examined a small number of populations; therefore, large-scale genetic epidemiology investigations are needed to validate the study.

Table 1
a Values for a variable are represented in mean ± SD

Table 2
Fig. 1 Frequency distribution of CaSR gene variants rs1801725 (A) and rs1042636 (B) among various populations *We have recorded the alleles frequency in healthy individuals of the Indian population for CaSR gene variants rs1801725 (A) and rs1042636 (B) from IndiG-enomes data (https:// cling en.igib.res.in/ indig en/).The allele frequencies for European, South African, and South Asian populations were recorded from National Center Biotechnology Information (https:// www.ncbi.nlm.nih.gov)

Table 3
Genotype distribution and association of CaSR gene variant rs1801725 with renal stone disease risk according to the genetic association models a Adjusted for age and gender

Table 5
Genotype distribution and association of CaSR gene variant rs1042636 with renal stone disease risk according to the genetic association models a Adjusted for age and gender

Table 6
Association of CaSR