Exploratory analysis of CaSR gene polymorphisms and haplotypes in relation to urolithiasis in the Qiongnan population of Hainan, China: a small-sample case–control study

Abstract

Background

Calcium urolithiasis arises from complex interactions between environmental and genetic factors regulating calcium metabolism. The calcium-sensing receptor (CaSR) is essential for maintaining calcium homeostasis, yet population-specific genetic effects remain insufficiently characterized.

Methods

A case–control study was conducted in the Qiongnan region of Hainan Province, including 50 patients with urolithiasis and 54 matched controls. Five CaSR single-nucleotide polymorphisms (rs7652589, rs6776158, rs1501899, rs1801725, and rs1042636) were genotyped by Sanger sequencing. Genotype–phenotype associations were assessed under multiple inheritance models, and haplotype analyses were performed to evaluate combined variant effects.

Results

None of the five CaSR SNPs showed a significant association with urolithiasis after multivariable adjustment. Four major haplotypes were identified, and the AGAGA haplotype was nominally associated with a markedly reduced risk of urolithiasis (adjusted OR = 0.217, 95% CI = 0.054–0.872, P = 0.031). No significant relationship was found between CaSR variants and stone composition.

Conclusions

The CaSR AGAGA haplotype showed a nominal association with reduced susceptibility; however, given the limited sample size and low post-hoc power, these findings should be interpreted as exploratory and require validation in larger, independent cohorts.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12894-026-02094-5.

Summary

In this exploratory case–control study, the CaSR AGAGA haplotype was nominally associated with reduced susceptibility to urolithiasis; however, given the limited sample size and low post-hoc power, this finding should be interpreted cautiously and requires validation in larger, independent cohorts.

Introduction

Urolithiasis is a significant global public health challenge, with both incidence and disease burden increasing over the past decades. The Global Burden of Disease (GBD) 2021 report shows increasing urolithiasis incidence worldwide, highlighting the need for improved prevention and risk stratification strategies [1]. Epidemiological data indicate that the global prevalence of urolithiasis among adults ranges from 2 to 3% [2], while in China, the incidence reaches approximately 6%, with higher rates observed in males, in rural populations, and in southern regions compared with their counterparts [3, 4]. Without preventive measures, about 50% of patients experience recurrence within five years [5].

The etiology of urolithiasis is multifactorial, involving interplay among environmental exposures, metabolic derangements, and genetic predisposition [6]. Genetic association studies have increasingly implicated genes involved in calcium and phosphorus handling, oxalate metabolism, citrate homeostasis, and renal tubular function. Among these, the calcium-sensing receptor (CaSR) plays a pivotal role in systemic calcium homeostasis, modulating parathyroid hormone (PTH) secretion and renal tubular calcium reabsorption [7]. Genetic variants in CaSR that alter receptor sensitivity or signaling may therefore influence urinary calcium excretion and, by extension, stone formation risk.

Common polymorphisms in CaSR—including A986S (rs1801725), R990G (rs1042636), and Q1011E (rs1801726)—have been studied in various populations, and meta-analyses suggest modest associations with calcium stone risk, though effect sizes and directionality are inconsistent across ethnic groups and environmental contexts [8, 9]. Functional studies have also linked some CaSR variants (e.g. A986S) to alterations in serum ionized calcium and PTH levels, supporting a mechanistic role in calcium handling [10, 11].

Hainan’s southern region (often termed “Qiongnan”) is characterized by a tropical monsoon climate with high temperature and humidity. Such environmental conditions may promote increased sweating and fluid loss, thereby reducing effective urine volume and raising supersaturation risk of lithogenic salts. Moreover, the local population’s genetic background, dietary habits, water mineral content, and occupational heat exposure patterns may differ significantly from those in mainland China, suggesting potential region-specific gene–environment interactions for stone risk.

Thus, a population-based investigation of CaSR polymorphisms in relation to urolithiasis prevalence and characteristics in the Qiongnan region is warranted. Such a study can help elucidate how CaSR genetic variation interacts with the distinctive climatic and lifestyle milieu of this tropical region, identify possible region-specific risk alleles, and ultimately inform targeted prevention strategies or personalized risk stratification in this endemic setting.

Material and methods

Study design and population

A total of 104 subjects were enrolled in this study, including 50 patients with urolithiasis (case group) and 54 non-stone controls (control group), all recruited from the Department of Urology, Sanya Central Hospital, between January 2024 and October 2025. All cases were diagnosed by computed tomography (CT), intravenous pyelography (IVP), and ultrasonography. Routine laboratory tests, including blood and urine analyses, liver and renal function tests, and serum electrolytes, were performed. Routine urinalysis included leukocyte esterase, nitrite, urinary protein, glucose, ketone bodies, urine pH, and urine specific gravity. Stone samples were collected after surgery, and the composition analysis by Fourier transform infrared (FTIR) spectroscopy was available for 39 of the 50 urolithiasis patients. Genotype–phenotype association analyses for stone composition were therefore conducted only in this subset of patients. Control subjects were age- and sex-matched inpatients from the same period, with no personal or family history of urolithiasis. Ultrasonography was performed to exclude urinary tract stones, nephrocalcinosis, or other abnormalities. The same routine laboratory tests were conducted, and individuals with disorders affecting calcium or phosphorus metabolism (such as rickets, thyroid or parathyroid dysfunction) or with active urinary tract infection were excluded. All subjects were native residents of Hainan Province. The study protocol was approved by the Ethical Committee of Sanya Central Hospital (approval number: LLKY2507079) and informed consent was obtained from all patients and control subjects after a full explanation of the study.

SNP selection

Five single nucleotide polymorphisms (SNPs) of CaSR gene (rs7652589, rs1501899, rs6776158, rs1801725, and rs1042636) were selected for genotyping based on their reported functional relevance and minor allele frequency (MAF). The tagging SNPs were identified using Haploview 4.2 software (Broad Institute of MIT and Harvard, Cambridge, MA, USA), based on the Han Chinese database loaded from 1000 Genomes Brower. The selection criteria were set at a linkage disequilibrium (LD) threshold of r² > 0.8 and a MAF > 0.10, ensuring adequate genomic coverage and representativeness of common variants within the CaSR gene region.

Because the tagging SNPs were initially selected based on the Han Chinese reference panel, the present study included participants of both Han and Li ethnic groups from Hainan Province. Therefore, these loci were also examined to evaluate their applicability and allelic distribution within a mixed ethnic background representative of the local population.

Genotyping for CaSR polymorphisms

Genomic DNA was extracted from peripheral blood leukocytes using the standard phenol–chloroform method. The concentration and purity of DNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and samples were stored at − 80 °C until analysis.

Genotyping of the five SNPs was performed using Sanger sequencing. Primers were designed with online Primer 3 software. Polymerase chain reaction (PCR) amplification was performed using standard reaction mixtures, and the PCR products were purified prior to sequencing. Sequencing reactions were carried out in a total volume of 5 µL, containing 2 µL of sequencing primer, 1–3 µL of purified PCR product, and 1 µL of BigDye v3.1 Cycle Sequencing Kit (ABI, Foster City, CA, USA). The sequencing reaction was performed under the following thermal cycling conditions: 94 °C for 1 min, followed by 28 cycles of 94 °C for 20 s, 50 °C for 10 s, and 60 °C for 4 min, with a final hold at 4 °C for 30 min. Sequencing was conducted on a 3730XL Genetic Analyzer (ABI, Foster City, CA, USA). Raw chromatograms were processed using DNA Sequencing Analysis Software v5.2.0 (Applied Biosystems), and genotype calling and alignment were performed with Sequencher v5.1 software (Gene Codes Corporation, Ann Arbor, MI, USA). All genotypes were verified manually to ensure accuracy and reproducibility.

Statistical analysis

Continuous variables were expressed as mean ± SD, and categorical variables as frequencies and percentages, N (%). Differences in demographic and clinical characteristics between groups were analyzed using the Student’s t-test for continuous variables and the chi-square test or Fisher’s exact test for categorical variables. Genotype and allele frequencies were calculated by direct counting, and deviations from Hardy–Weinberg equilibrium (HWE) were tested in the control group using the exact test. Associations between individual CaSR polymorphisms and urolithiasis risk were evaluated under codominant, dominant, and recessive genetic models using binary logistic regression, with results presented as odds ratios (ORs) and 95% confidence intervals (CIs). Multivariate logistic regression was subsequently performed to adjust for potential confounders, including age, sex, BMI, ethnicity. Haplotype blocks and linkage disequilibrium (LD) coefficients (D′ and r²) among the five CaSR loci were estimated using the ‘haplo.stats’, ‘SNPassoc’ and ‘genetics’ packages in R. Haplotypes with frequencies greater than 5% were included in the analysis. The associations between common haplotypes and urolithiasis susceptibility were determined using logistic regression models adjusted for the same covariates. P < 0.05 was considered statistically significant. All statistical analyses were performed using R software (version 4.5.1; R Foundation for Statistical Computing, Vienna, Austria).

Result

Baseline demographic characteristics of the study population

The baseline demographic characteristics of the two groups are summarized in Table 1. The mean age of the urolithiasis group was 63.08 ± 14.27 years, which was not significantly different from that of the control group (64.46 ± 15.30 years, P = 0.635). Likewise, there were no significant differences in height (P = 0.117) or weight (P = 0.060) between the two groups. Males accounted for 64.0% of the urolithiasis group and 61.1% of the control group, with no significant difference in sex distribution (P = 0.919). The ethnic composition was also comparable between groups (P = 0.563), with Han individuals comprising the majority in both. However, the mean body mass index (BMI) was significantly higher in the urolithiasis group than in the control group (24.37 ± 3.86 kg/m² vs. 21.94 ± 4.52 kg/m², P = 0.004).

Table 1.

Demographic and characteristics of the urolithiasis patients and controls

	Control group	Urolithiasis group	P
	n = 54	n = 50
Age (years)	64.46 ± 15.30	63.08 ± 14.27	0.635
Height (m)	1.66 ± 0.07	1.64 ± 0.09	0.117
Weight (kg)	60.57 ± 13.49	65.61 ± 13.47	0.06
BMI (kg/m2)	21.94 ± 4.52	24.37 ± 3.86	0.004
Gender, n (%)			0.919
Male	33 (61.1)	32 (64.0)
Female	21 (38.9)	18 (36.0)
Ethnicity, n (%)			0.563
Han	44 (81.5)	39 (78.0)
Hui	0 (0.0)	1 (2.0)
Li	10 (18.5)	10 (20.0)

Distributions of alleles and genotype frequencies

The genotype distributions of five CaSR polymorphisms were compared between urolithiasis patients and healthy controls (Table 2). No significant deviation from Hardy–Weinberg equilibrium was observed in the control group (P > 0.05). As shown in Table 2, logistic regression analysis adjusted for age, sex, BMI, and ethnicity revealed no statistically significant associations between any of the studied polymorphisms and the risk of urolithiasis. Specifically, the dominant models for rs7652589 (adjusted OR = 1.14, 95% CI: 0.46—2.87, P = 0.776) and rs1042636 (adjusted OR = 0.71, 95% CI: 0.26—1.91, P = 0.495) demonstrated no increased susceptibility. Although the variant G/T genotype of rs1801725 was detected only in the patient group (6.0%), this difference did not reach statistical significance (P = 0.990). Similarly, no significant differences were found under codominant or recessive models for all loci.

Table 2.

Genotype distributions and allele frequencies of CaSR polymorphisms in urolithiasis patients and controls

	Control group, n (%)	Urolithiasis group, n (%)	OR (95%CI)	p	OR—adjust (95%CI)	p-adjust
rs7652589
Codominant
A/A	21 (38.9)	20 (40)	ref		ref
A/G	27 (50)	24 (48)	0.93 (0.41—2.13)	0.869	1.06 (0.41—2.78)	0.910
G/G	6 (11.1)	6 (12)	1.05 (0.29—3.8)	0.941	1.51 (0.36—6.53)	0.568
Dominant
A/A	21 (38.9)	20 (40)	ref		ref
A/G-G/G	33 (61.1)	30 (60)	0.95 (0.43—2.10)	0.908	1.14 (0.46—2.87)	0.776
Recessive
A/A-A/G	48 (88.9)	44 (88)	ref		ref
G/G	6 (11.1)	6 (12)	1.09 (0.32—3.73)	0.887	1.47 (0.39—5.75)	0.569
rs6776158
Codominant
G/G	21 (38.9)	20 (40)	ref		ref
G/A	27 (50)	24 (48)	0.93 (0.41—2.13)	0.869	1.06 (0.41—2.78)	0.910
A/A	6 (11.1)	6 (12)	1.05 (0.28—3.89)	0.941	1.51 (0.36—6.53)	0.568
Dominant
G/G	21 (38.9)	20 (40)	ref		ref
G/A-A/A	33 (61.1)	30 (60)	0.95 (0.43—2.10)	0.908	1.14 (0.46—2.87)	0.776
Recessive
G/G-G/A	48 (88.9)	44 (88)	ref		ref
A/A	6 (11.1)	6 (12)	1.09 (0.32—3.73)	0.887	1.47 (0.39—5.75)	0.569
rs1501899
Codominant
A/A	21 (38.9)	20 (40)	ref		ref
A/G	27 (50)	24 (48)	0.93 (0.41—2.13)	0.869	1.06 (0.41—2.78)	0.910
G/G	6 (11.1)	6 (12)	1.05 (0.28—3.89)	0.941	1.51 (0.36—6.53)	0.568
Dominant
A/A	21 (38.9)	20 (40)	ref		ref
A/G-G/G	33 (61.1)	30 (60)	0.95 (0.43—2.10)	0.908	1.14 (0.46—2.87)	0.776
Recessive
A/A-A/G	48 (88.9)	44 (88)	ref		ref
G/G	6 (11.1)	6 (12)	1.09 (0.32—3.73)	0.887	1.47 (0.39—5.75)	0.569
rs1801725
Codominant
G/G	54 (100)	47 (94)	ref		ref
G/T	0 (0)	3 (6)	inf (0—inf)	0.990	inf (0—inf)	0.990
rs1042636
Codominant
G/G	14 (25.9)	14 (28)	ref		ref
A/G	24 (44.4)	28 (56)	1.17 (0.46—2.95)	0.743	0.81 (0.28—2.32)	0.702
A/A	16 (29.6)	8 (16)	0.50 (0.16—1.52)	0.228	0.52 (0.14—1.84)	0.314
Dominant
G/G	14 (25.9)	14 (28)	ref		ref
A/G-A/A	40 (74.1)	36 (72)	0.90 (0.38—2.15)	0.812	0.71 (0.26—1.91)	0.495
Recessive
G/G-A/G	38 (70.4)	42 (84)	ref		ref
A/A	16 (29.6)	8 (16)	0.45 (0.17—1.15)	0.104	0.59 (0.19—1.73)	0.343

Odds ratios adjusted for age, sex, BMI and ethnicity

Association between CaSR genotypes and stone composition

Stone composition data were available for 39 of the 50 patients with urolithiasis. The associations between CaSR genotypes and urinary stone composition were analyzed within this subgroup. No significant associations were observed between the genotypes of rs7652589/rs1501899, rs6776158, rs1801725, or rs1042636 and specific stone compositions, including calcium oxalate, carbonate apatite, or anhydrous uric acid (all P > 0.05, Table 3). In both the calcium oxalate and carbonate apatite subgroups, the distribution of genotypes was similar across all loci. Likewise, no genotype differences were detected among patients with anhydrous uric acid stones.

Table 3.

Association between CaSR genotypes and urinary stone composition among patients with available stone analysis (n = 39)

	rs7652589/rs1501899				rs6776158				rs1801725			rs1042636
	AA	AG	GG	p	AA	GA	GG	p	GG	GT	p	AA	AG	GG	p
	16	20	3		3	20	16		37	2		5	20	14
Calcium oxalate (n = 5)
yes	3 (18.8)	2 (10.0)	0 (0.0)	0.581	0 (0.0)	2 (10.0)	3 (18.8)	0.581	5 (13.5)	0 (0.0)	0.998	0 (0.0)	4 (20.0)	1 (7.1)	0.357
no	13 (81.2)	18 (90.0)	3 (100.0)		3 (100.0)	18 (90.0)	13 (81.2)		32 (86.5)	2 (100.0)		5 (100.0)	16 (80.0)	13 (92.9)
Carbonate apatite (n = 27)
yes	9 (56.2)	16 (80.0)	2 (66.7)	0.307	2 (66.7)	16 (80.0)	9 (56.2)	0.307	26 (70.3)	1 (50.0)	0.997	3 (60.0)	13 (65.0)	11 (78.6)	0.625
no	7 (43.8)	4 (20.0)	1 (33.3)		1 (33.3)	4 (20.0)	7 (43.8)		11 (29.7)	1 (50.0)		2 (40.0)	7 (35.0)	3 (21.4)
Anhydrous uric acid (n = 36)
yes	14 (87.5)	19 (95.0)	3 (100.0)	0.614	3 (100.0)	19 (95.0)	14 (87.5)	0.614	34 (91.9)	2 (100.0)	0.998	5 (100.0)	19 (95.0)	12 (85.7)	0.478
no	2 (12.5)	1 (5.0)	0 (0.0)		0 (0.0)	1 (5.0)	2 (12.5)		3 (8.1)	0 (0.0)		0 (0.0)	1 (5.0)	2 (14.3)

Stone composition analysis was available for 39 patients; sample numbers differ across stone types. Stone components are mixed composition allowed, counts represent presence of each component

Linkage disequilibrium analysis of CaSR polymorphisms

The pairwise linkage disequilibrium (LD) coefficients (D′) among the five CaSR polymorphisms are presented in Table 4. A strong LD was observed among rs7652589, rs6776158, rs1501899, and rs1801725, with D′ values close to 1.0, indicating that these loci are in high linkage disequilibrium. In contrast, rs1042636 showed relatively weaker LD with the other three SNPs (D′ = 0.493), Fig. 1.

Table 4.

Pairwise linkage disequilibrium coefficients (D′) among CaSR polymorphisms

	rs7652589	rs6776158	rs1501899	rs1801725	rs1042636
rs7652589	-	1.000	1.000	0.986	0.493
rs6776158	-	-	1.000	0.986	0.493
rs1501899	-	-	-	0.986	0.493
rs1801725	-	-	-	-	0.990
rs1042636	-	-	-	-	-

Fig. 1.

Linkage disequilibrium (LD) plot of the five CaSR gene polymorphisms

Haplotype analysis of CaSR polymorphisms and urolithiasis risk

Haplotype analysis based on the five CaSR SNPs (rs7652589, rs6776158, rs1501899, rs1801725, and rs1042636) was performed to evaluate the combined effects of genetic variants on urolithiasis susceptibility. Four common haplotypes with frequencies greater than 0.05 were identified, including AGAGG, AGAGA, GAGGA, and GAGGG. The AGAGG haplotype was the most prevalent in both groups and was used as the reference. After adjustment for age, sex, BMI, and ethnicity, the AGAGA haplotype was nominally associated with a reduced risk of urolithiasis (adjusted OR = 0.217, 95% CI: 0.054–0.872, P = 0.031). No significant associations were observed for the GAGGA or GAGGG haplotypes (all P > 0.05, Table 5).

Table 5.

Haplotype analysis of CaSR polymorphisms and risk of urolithiasis

Haplotype	Frequency	Controls n (%	Urolithiasis n (%)	OR	95%CI	P	OR-adjust	95%CI—adjust	P-adjust
AGAGG	0.42	21 (0.39)	23 (0.45)	ref		0.3832	ref		0.432
AGAGA	0.20	13 (0.24)	8 (0.16)	0.473	0.189—0.695	0.0242	0.217	0.054—0.872	0.031
GAGGA	0.26	15 (0.28)	12 (0.24)	0.757	0.374—1.529	0.6202	0.671	0.283—1.588	0.366
GAGGG	0.10	5 (0.09)	6 (0.12)	0.903	0.235—3.470	0.4409	0.651	0.112—3.778	0.633

Odds ratios adjusted for age, sex, BMI and ethnicity

Discussion

In this case–control study of the Qiongnan population in Hainan Province, we investigated five common CaSR polymorphisms and their haplotypic combinations in relation to urolithiasis susceptibility. The principal finding is that no single CaSR SNP showed a statistically significant association with urolithiasis after multivariable adjustment, whereas a specific five-locus haplotype (AGAGA) was significantly associated with reduced risk. This pattern suggests that, in this regional cohort, the detectable genetic contribution of CaSR to stone susceptibility is better captured at the multi-locus (haplotype) level than at individual markers.

An additional noteworthy feature is that the four tagging SNPs located in the 5′/intronic region (rs7652589, rs6776158, rs1501899) were all in high linkage disequilibrium (D′ close to 1.0) but none of them showed a disease-causing signal in any of the genetic models. This "LD-no-association" pattern has also been reported in cohorts from the Middle East (e.g., Iran) and Indonesia, where exon-7 missense variants (A986S, R990G, Q1011E) or their haplotypic combinations explained the association, while promoter-region SNPs did not [12, 13]. Our finding suggests that the coding region of exon 7 may represent a key susceptibility segment of CaSR for calcium stone disease in this population.

CaSR is a G-protein-coupled receptor that senses extracellular Ca²⁺ and modulates PTH secretion and renal tubular calcium reabsorption; alterations of this receptor therefore have direct effects on urinary calcium excretion and stone risk [14–16]. Classically, gain-of-function CaSR variants have been linked to lower Ca²⁺ set-points, increased calciuria and a higher frequency of nephrolithiasis or nephrocalcinosis [17–19]. However, in our Qiongnan cohort, single-marker analyses did not identify statistically significant associations between any of the five genotyped CaSR loci and urolithiasis after multivariable adjustment. This pattern suggests that, within this regional population, any CaSR-related genetic contribution is likely modest and/or context-dependent, and may be better captured by multi-locus genetic background rather than by individual common variants alone.

Across populations, reported associations between CaSR polymorphisms and nephrolithiasis have been heterogeneous. For example, a case–control study in a high-altitude Han Chinese cohort from Kunming reported locus-specific associations and biochemical correlates (including differences in 24-h urinary calcium), whereas our cohort did not show robust single-variant signals after adjustment [20]. Such discrepancies are plausible given differences in genetic background, environmental exposures (e.g., altitude, climate, hydration, salt and calcium intake), phenotype definition, and statistical power. Moreover, regional variation in allele frequencies and linkage disequilibrium structure can influence which markers tag the underlying functional architecture in candidate gene studies. In this context, our observation of a significant CaSR haplotype association, despite null single-locus results, supports the view that haplotype-based approaches may provide additional resolution for detecting population-specific susceptibility patterns.

Haplotype analysis provided further support for this interpretation. We identified one haplotype, AGAGA, that was under-represented in patients and remained significant after adjustment (adjusted OR = 0.217, 95% CI 0.054–0.872, P = 0.031), indicating a protective allelic configuration across the CaSR locus. Protective haplotypes of CaSR or of calcium-handling genes (e.g. CLDN14, TRPV5) have also been reported in pediatric and adult stone-forming cohorts and are thought to reflect small compensatory changes in receptor sensitivity or expression that counterbalance calciuric tendencies [21–23]. The coexistence in our data of a protective haplotype suggests that both single-locus and multi-allelic effects within CaSR contribute to the genetic architecture of urolithiasis in this region.

We further examined whether CaSR variants predisposed individuals to specific stone types and found no genotype differences between calcium oxalate, carbonate apatite and mixed/uric acid stones (all P > 0.05). This finding is consistent with the hypothesis that CaSR-related lithogenicity acts upstream, by influencing urinary calcium load, while the final crystal phase is more strongly driven by urinary pH, citrate, inhibitors and matrix proteins, as described in recent reviews on stone pathobiology [24, 25]. In other words, CaSR appears to create a “lithogenic milieu”, but does not determine the mineralogy of the stone. In line with this interpretation, urine pH was descriptively summarized in the present study and did not differ significantly between patients and controls (Table S1). However, comprehensive biochemical profiling, including serum electrolyte measurements, was not available for all participants, which limits further mechanistic interpretation of SNP–phenotype associations.

Mechanistically, several pathways support a role of CaSR-related variation in stone susceptibility and may help explain why associations can be detectable at the haplotype level despite null single-marker results. Functional studies have shown that coding changes within CaSR—particularly exon-7 variants—can alter receptor signaling and shift the Ca²⁺ EC₅₀, thereby influencing renal tubular calcium handling and downstream pathways such as CLDN14-regulated paracellular transport in the thick ascending limb, with potential consequences for calciuria [18, 26]. At the population level, however, the phenotypic expression of calcium-handling genotypes is likely to be strongly modified by environmental exposures. Gene–environment frameworks suggest that genetic effects may become clinically overt in settings characterized by high salt intake, suboptimal hydration, or high ambient temperature, whereas modest regulatory effects may remain difficult to detect when exposures are unmeasured or heterogeneous [22, 27]. This perspective is relevant to our study’s geographic context; Northern and Northeastern Thailand were referenced only as climatically comparable settings rather than as direct epidemiological counterparts, based on reports of ion-transport–related renal disorders in Southeast Asia [28]. Nonetheless, because we did not identify region-specific epidemiological or genetic reports documenting channelopathies in the Qiongnan area, and such conditions were not systematically assessed in our cohort, mechanistic interpretation should be cautious. Overall, these considerations align with evidence that genetic susceptibility scores explain only a limited proportion of risk unless diet and fluid intake are incorporated, and may partly account for inconsistent replication of CaSR signals across cohorts, particularly when analyses rely on single non-coding markers rather than multi-locus configurations [27, 29].

Several limitations should be acknowledged. First, the modest sample size (n = 104) limited statistical power, particularly for recessive models and low-frequency haplotypes, and effect estimates may therefore be unstable. The findings should be considered exploratory and require validation in larger cohorts. Second, urinary calcium and other biochemical parameters reflecting CaSR activity were not available, precluding functional interpretation of the studied variants. Third, participants were recruited from a single regional population, which may limit generalizability. Finally, environmental and lifestyle factors were not comprehensively assessed, and residual confounding cannot be excluded.

In conclusion, our findings suggest that a CaSR haplotype (AGAGA), rather than any individual common SNP, may be associated with reduced susceptibility to urolithiasis in the Qiongnan population. Given the modest sample size and limited statistical power, these results should be interpreted as exploratory and require validation in larger, independent cohorts. Importantly, urolithiasis is a multifactorial disease influenced by both genetic susceptibility and metabolic/environmental factors, as reflected by the observed association with BMI in our cohort. Future studies integrating broader genomic coverage, detailed physiological phenotyping (including urinary calcium measurements), and environmental exposure data will be essential to clarify the population-specific determinants of stone risk. Extending analyses to other calcium-handling genes (e.g., TRPV5, CLDN14, and VDR) may further refine our understanding of the complex genetic architecture underlying urolithiasis.

Abbreviations

ABI

Applied Biosystems, Inc

BMI

Body mass index

CaSR

Calcium-sensing receptor

CI

Confidence interval

CT

Computed tomography

DNA

Deoxyribonucleic acid

FDR

False discovery rate

FTIR

Fourier transform infrared

GBD

Global Burden of Disease

HWE

Hardy–Weinberg equilibrium

IVP

Intravenous pyelography

LD

Linkage disequilibrium

MAF

Minor allele frequency

OR

Odds ratio

PCR

Polymerase chain reaction

PTH

Parathyroid hormone

SD

Standard deviation

SNP

Single-nucleotide polymorphism

Authors’ contributions

JW, GL, and GC conceived, designed, and supervised the study. XZ conducted the clinical work, sample collection, and laboratory analysis. JW, GL, XZ, BL, and LL performed data curation, statistical analysis, and interpretation. JW, GL, BL, GC, and LL wrote, reviewed, and revised the manuscript drafts. All authors read and approved the final version of the manuscript.

Funding

This study was supported by the Sanya Science and Technology Special Fund (Grant No. 2024KJFX063), the Sanya Central Hospital(The Third People's Hospital of Hainan Province) Natural Science Foundation (Grant No. SYZXYY202409), and the Joint Program on Health Science and Technology Innovation of Hainan Province (Grant No. WSJK2025MS165).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Ethical Committee of Sanya Central Hospital (approval number: LLKY2507079). The study was conducted in accordance with the principles of the Declaration of Helsinki. Written informed consent was obtained from all participants after a full explanation of the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.