Whole Exome Sequencing in Chinese Pediatric Patients With Nephrolithiasis

Abstract

Introduction

The incidence of pediatric nephrolithiasis has been increasing, and the role of genetic factors has garnered attention in recent years. This study aimed to explore the genetic basis underlying pediatric nephrolithiasis in Chinese population.

Methods

Whole exome sequencing (WES) was conducted in a consecutive cohort of 456 children over a 11-year period. Clinical and genetic data were systematically collected, analyzed, and comprehensively compared.

Results

Average age was 4.2 years with a male-to-female ratio of 2.2. A total of 260 causative variants in 16 genes were identified in 141 children, resulting in a positive molecular diagnosis rate of 31%. Of the causative variants, 43% were novel. The most prevalent diagnoses were primary hyperoxaluria (PH) (AGXT: 20%, GRHPR: 11%, and HOGA1: 24%) and cystinuria (SLC3A1: 18% and SLC7A9: 14%). Children with positive molecular diagnoses were more likely to have stone episodes, bilateral stones, multiple stones, or nephrocalcinosis (all P < 0.05). Children with AGXT defects were more prone to have severe clinical manifestations, and those with HOGA1 defects and males with SLC3A1 and SLC7A9 defects tended to be diagnosed at a younger age. The concordance rate between suspected clinical diagnoses and molecular diagnoses was 81%. At least 29% of children could benefit from additional clinical advice based on a molecular diagnosis.

Conclusion

A genetic etiology was identified in 141 of 456 of pediatric patients (31%) with nephrolithiasis in a Chinese cohort. A positive molecular diagnosis is a risk factor for severe clinical presentation of pediatric nephrolithiasis. WES has the potential to be used to confirm or even modify clinical diagnoses, thereby facilitating individualized therapeutic and preventive interventions.

Graphical abstract

Nephrolithiasis is a prevalent condition in the field of pediatric urology. Over the past few decades, there has been a significant increase in the incidence of pediatric nephrolithiasis, particularly among adolescents and females.¹ Children frequently experience recurrent stones, require multiple surgical interventions, and suffer from progressive impairment of kidney function; these factors not only compromise their physical and mental well-being but also impose significant financial burdens on their families.² In order to provide optimal care for patients and prevent recurrent stones, it is crucial to understand the etiology and underlying risk factors that cause nephrolithiasis. Etiology is commonly believed to be multifactorial, resulting from a complex interplay between environmental and genetic factors.

Over the past 10 years, studies have emphasized the clinical significance of genetic testing in patients with nephrolithiasis and demonstrated that 11.0% to 73.7% of pediatric stone formers have a monogenic molecular diagnosis.3, 4, 5, 6, 7, 8, 9, 10, 11, 12 A positive family history is observed in 35% to 65% of individuals with nephrolithiasis, and twin studies have reported the heritability of nephrolithiasis and urinary calcium excretion to be > 45%.¹³ Up to 83.0% of the variants detected were found to be novel.3, 4, 5, 6, 7, 8, 9, 10, 11, 12 Therefore, the etiology and genetic basis of nephrolithiasis remain poorly understood, necessitating further investigation and expanded genetic testing.

There is a paucity of studies on the prevalence and putative hotspots of monogenic nephrolithiasis in the Chinese population.⁸ We therefore performed high throughput WES analysis in a cohort of 456 children with nephrolithiasis recruited from 5 different tertiary care hospitals in China. We speculated that a significant proportion of Chinese individuals with nephrolithiasis possess causative variants, some of which might be identified as novel spots. Based on a definitive molecular diagnosis, individualized therapeutic and preventive measures can be provided for each patient.

Methods

Study Cohort

We carried out a case-control study based on Chinese population, which has received approval from the Institutional Review Board and Bioethics Committee of 3 participating centers (2021-P2-375-02; XHEC-D-2020-134; 2024-581). In this study, we selected children (aged 0–18 years) who were diagnosed with nephrolithiasis and with or without nephrocalcinosis by ultrasound or computed tomography from a prospectively maintained pediatric nephrolithiasis database spanning from June 2014 to June 2023. These children were consecutively recruited in typical nephrolithiasis clinics from each center, and the collection of blood and urine samples, as well as other clinical data, were conducted with informed consent obtained from all children’s parents. Children with congenital urinary tract anomaly or severe infection that may lead to secondary nephrolithiasis (n = 28) were excluded in our study. We finally enrolled 456 children with nephrolithiasis, all of whom except 2 brothers were from unrelated families.

Clinical Evaluation and Definition

Uribe samples were used for urine culture as well as metabolic analysis to identify the level of urine creatinine, hyperoxaluria, hypocitraturia, hypercalciuria, and hyperuricosuria. Blood samples were used for the calculation of the estimated glomerular filtration rate (serum creatinine), and DNA isolation. Stone composition was analyzed using an infrared spectrum analysis system. All children were evaluated with low-dose noncontrast computed tomography to determine stone features, such as number, burden, and location. Other clinical data such as demographic data, imaging findings, and medical records were collected from the electronic database. Detailed items of clinical data and their definitions were shown in the footnote of Table 1.

Table 1.

Genetic characteristics of each molecular diagnosis

Molecular diagnosis	Gene	Inheritance (patient n, %)	Truncating variants/ total variants, n (%)	Novel variants/ total variants, n (%)
Primary hyperoxaluria
Type 1	AGXT	AR (28, 20%)	12/27 (44%)	12/27 (26%)
Type 2	GRHPR	AR (16, 11%)	4/12 (33%)	4/12 (33%)
Type 3	HOGA1	AR (34, 24%)	9/22 (41%)	2/22 (9%)
Cystinuria
Type 1	SLC3A1	AD (4, 3%); AR (22, 16%)	16/33 (49%)	24/33 (73%)
Type 2	SLC7A9	AD (9, 6%); AR (10, 7%)	9/22 (41%)	13/22 (59%)
Calcium oxalate nephrolithiasis
Type 1	SLC26A1	AR (4, 3%)	2/7 (29%)	1/7 (14%)
Type 2 with NC	OXGR1	AD (1, 1%)	0/1 (0%)	1/1 (100%)
RHUC, type 2	SLC2A9	AD (3, 2%)	0/3 (0%)	2/3 (67%)
dRTA, type 1	SLC4A1	AD (3, 2%)	0/3 (0%)	1/3 (33%)
APRT	APRT	AR (1, 1%)	1/1 (100%)	1/1 (100%)
ADHH	CASR	AD (1, 1%)	0/1 (0%)	0/1 (0%)
FHHNC	CLDN16	AR (1, 1%)	1/2 (50%)	1/2 (50%)
Bartter syndrome, type 2	KCNJ1	AR (1, 1%)	0/2 (0%)	2/2 (100%)
HCINF, type 2	SLC34A1	AR (1, 1%)	1/1 (100%)	1/1 (100%)
Xanthinuria, type 1	XDH	AR (1, 1%)	0/2 (0%)	0/2 (0%)
HHRH	SLC34A3	AR (1, 1%)	0/2 (0%)	1/2 (50%)
Total	-	AD (21, 15%); AR (120, 85%)	55/141 (39%)	61/141 (43%)

AD, autosomal dominant; ADHH, autosomal-dominant hypocalcemia with hypercalciuria; APRT, adenine phosphoribosyl transferase deficiency; AR, autosomal recessive; DR, digenic recessive; dRTA, distal renal tubular acidosis; FHHNC, familial hypomagnesemia with hypercalciuria and nephrocalcinosis; HCINF, infantile hypercalcemia; HHRH, hereditary hypophosphatemic rickets with hypercalciuria; OMIM, Online Mendelian Inheritance in Man; RHUC, renal hypouricemia.

All children underwent endourological procedures according to European Association of Urology guidelines and achieved stone-free status (no residual fragments or fragments < 2.0 mm in diameter). Stone burden was estimated by adding the maximum diameter of each stone. Estimated glomerular filtration rate was calculated according to the Schwartz formula.¹⁴ Chronic renal disease was classified according to Kidney Disease: Improving Global Outcomes guidelines.¹⁵ Spot urine samples were used to evaluate hyperoxaluria, hypocitraturia, hypercalciuria, and hyperuricosuria; creatinine ratios (solute/creatinine) were calculated and normal values were determined based on European Association of Urology guidelines.¹⁶

WES and Variant Calling

Genomic DNA was isolated from the peripheral blood of all children and their parents, and the quality of the DNA was assessed using Qubit 3.0 Flurometer (Life Technologies, CA). The qualified DNA underwent whole-exome capture using Agilent SureSelect Target Enrichment System (Santa Clara, CA) according to standard protocols (Supplementary Method; Supplementary References). The pooled libraries were then sequenced on the Illumina HighSeq sequencing platform (San Diego, CA). The sequence encompasses all coding exons and splice sites (20 base pairs of intronic sequences on either side of the exons). After filtration, clean reads were mapped to the human reference genome assembly (UCSC GRCh37/hg19) and annotated with ANNOVAR (Annotate Variation) using different mutation databases and in silico analysis tools. The detailed process of variant calling is shown in Supplementary Method (Supplementary References). Sanger sequencing was conducted for single-variant confirmation. Parental DNA was used for segregation analysis. Variants were classified according to the 2015 guideline of American College of Medical Genetics and Genomics.¹⁴ Causative variants are defined as variants that are classified as pathogenic or likely pathogenic, and are either heterozygous in dominant genes, or homozygous or compound heterozygous in recessive genes. Monoallelic pathogenic or likely pathogenic variants in genes with reported autosomal recessive inheritance where evidence for these variants are consistent with the clinical picture are considered the ambiguous variants for nephrolithiasis. All causative variants had been submitted to ClinVar of National Center for Biotechnology Information (NCBI) and were identified as 141 submitted records in ClinVar (SCVs) (SUB14686847).

Statistical Analysis

All data were input in a designed spreadsheet and double-checked for correction. The normality of numerical variables was assessed through the Kolmogorov-Smirnov tests. Student t tests, Mann-Whitney U tests, and chi-square tests were employed to determine the statistical significance of variables. Logistic regression analyses were used to adjust for potential confounding variables (age, sex, body mass index, geographic distribution, ethnicity, family history, estimated glomerular filtration rate, and urine culture status). All P values were 2-sided, and P < 0.05 was the threshold of significance. SPSS (IBM, Armonk, NY; Version 24.0) were used for the statistical tests.

Results

Cohort Characteristics

A total of 456 children were recruited from 3 clinical cohorts from 31 provinces, municipalities, and autonomous regions across China, with the majority hailing from Eastern China (Figure 1). The population consisted of 312 males (68%) and 144 females (32%), resulting in a male-to-female ratio of 2.2:1. The average age of all children was 4.2 years. There was no difference in the distribution of sex and age among the 3 cohorts. More details were shown in Table 2.

Figure 1.

Geographical distribution of Chinese children clinically diagnosed with nephrolithiasis and screened with WES, along with the proportion of positive molecular diagnoses in provinces, municipalities, and autonomous regions with > 10 recruited children is shown. The geographical distribution of children with defected AGXT, GRHPR, HOGA1, SLC3A1, or SLC7A9 is plotted below respectively. WES, whole exome sequencing.

Table 2.

Characteristics of patients in the cohort

Characteristic	Cohort 1 n = 241	Cohort 2 n = 194	Cohort 3 n = 21	Total N = 456
Visiting age (yr), median (IQR)	2.1 (1.5–7.1)	2.0 (1.0–5.0)	2.2 (1.1–3.0)	2.1 (1.2–6.1)
Minority individual, n (%)	22 (9%)	5 (3%)	2 (10%)	29 (6%)
Sex, male, n (%)	170 (71%)	127 (65%)	15 (71%)	312 (68%)
BMI (kg/m2), median (IQR)	16.2 (14.9–18.4)	15.9 (14.8–17.4)	18.0 (15.7–19.4)	15.8 (14.9–18.0)
eGFR (ml/min per 1.73 m2), median (IQR)	124.8 (118.4–165.0)	156.0 (136.0–182.3)	140.0 (101.8–158.4)	130.4 (122.6–174.2)
Family history, n (%)	70 (29%)	30 (15%)	4 (19%)	104 (23%)
Symptomatic episode history, n (%)	88 (37%)	82 (42%)	3 (14%)	173 (38%)
Nephrocalcinosis, n (%)	12 (5%)	15 (8%)	0 (0%)	27 (6%)
Recurrent stone, n (%)	35 (15%)	17 (9%)	3 (14%)	55 (12%)
Bilateral stone, n (%)	76 (32%)	61 (31%)	7 (33%)	144 (32%)
Multiple stone, n (%)	122 (51%)	80 (41%)	11 (52%)	213 (47%)
Stone burden in diameter (cm), median (IQR)	2.0 (1.0–2.8)	1.5 (0.9–1.9)	1.5 (1.0–2.0)	1.5 (1.0–2.1)
Serum uric acid (μmol/l), median (IQR)	287 (247–331)	252 (205–316)	279 (266–308)	266 (223–324)
Serum calcium (mmol/l), median (IQR)	2.5 (2.4–2.5)	2.4 (2.4–2.5)	2.5 (2.4–2.6)	2.5 (2.4–2.5)
Serum phosphate (mmol/l), median (IQR)	1.7 (1.6–1.8)	1.7 (1.5–1.8)	1.7 (1.6–1.8)	1.7 (1.5–1.8)
Serum magnesium (mmol/l), median (IQR)	0.9 (0.8–0.9)	0.9 (0.9–1.0)	0.9 (0.9–1.0)	0.9 (0.9–1.0)
Stone composition, n (%)
Calcium oxalate	142 (70%)	72 (60%)	16 (80%)	230 (67%)
Cystine	22 (11%)	18 (15%)	1 (5%)	41 (12%)
Carbonate apatite	17 (8%)	17 (14%)	1 (5%)	35 (10%)
Brushite	11 (5%)	6 (5%)	1 (5%)	18 (5%)
Octacalcium phosphate	0 (0%)	1 (1%)	0 (0%)	1 (1%)
Xanthine	0 (0%)	1 (1%)	0 (0%)	1 (1%)
2,8-dihydroxyadenine	1 (1%)	0 (0%)	0 (0%)	1 (1%)
Magnesium ammonium phosphate	4 (2%)	1 (1%)	1 (5%)	6 (2%)
Uric acid	6 (3%)	3 (3%)	0 (0%)	9 (3%)
Ammonium urate	1 (1%)	1 (1%)	0 (0%)	2 (1%)

BMI, body mass index; eGFR, estimated glomerular filtration rate; IQR, interquartile range.

Genetic Information of Children With Positive Molecular Diagnosis

We finally identified a total of 141 children (31%) with a positive molecular diagnosis for 16 causative genes (Table 1). The most prevalent molecular diagnoses were PH (AGXT, GRHPR, and HOGA1) and cystinuria (SLC3A1 and SLC7A9), accounting for 87% (n = 123). A total of 141 unique variants were identified for the 16 causative genes, of which 71 variants (51%) were pathogenic, and 70 variants (50%) were likely pathogenic based on the American College of Medical Genetics and Genomics validation. Among these variants, 120 (85%) were found in 12 recessive genes and 21 (15%) were found in 6 dominant genes. Thirteen patients whose phenotypes were fitting for cystinuria had ambiguous variants in SLC3A1 or SLC7A9 genes, which exhibit autosomal dominant inheritance. Most of the causative variants identified in this study (43%, 61/141) were found to be novel, indicating that they have not been previously reported. Although nonsense and frameshift variants were less frequently observed (39%, 55/141), they exhibited a higher prevalence in specific genes such as AGXT, HOGA1, SLC3A1, and SLC7A9. More genetic information for the children with a positive molecular diagnosis are presented in Supplementary Table S2.

For provinces, municipalities, and autonomous regions with > 10 recruited children, the positive molecular diagnosis rates were comparable. Henan Province had the highest positive molecular diagnosis rate (63%, 10/16), whereas Hebei Province (22%, 16/71) and the Inner Mongolia Autonomous Region (24%, 4/17) had comparatively low rates. There were notable geographical differences in the number of people among the 5 major defected genes (AGXT, GRHPR, HOGA1, SLC3A1, and SLC7A9). More details were shown in Figure 1.

Clinical Influencing Factors for Positive Molecular Diagnosis

As shown in Table 3, the presence of a positive molecular diagnosis in all children was found to be associated with the episode history (adjusted odds ratio [OR]: 4.04, 95% confidence interval [CI]: 2.02–8.05, P < 0.001), bilateral stones (adjusted OR: 3.49, 95% CI: 1.58–7.70, P = 0.002), multiple stones (adjusted OR: 8.46, 95% CI: 3.27–21.88, P < 0.001), and nephrocalcinosis (adjusted OR: 5.05, 95% CI: 1.16–22.07, P = 0.031).

Table 3.

Multivariate logistic regression analyses for positive molecular diagnoses

Clinical variables	Positive group	Negative group	Crude OR (95% CI)	P value	Adjusted OR (95%CI)a	P value
Overall: positive (n = 141) vs. negative (n = 315)
Bilateral stone	64%	17%	8.67 (5.51–13.64)	<0.001	3.49 (1.58–7.70)	0.002
Multiple stone	86%	29%	14.60 (8.58–24.85)	<0.001	8.46 (3.27–21.88)	<0.001
Nephrocalcinosis	14%	2%	7.50 (3.09–18.20)	0.015	5.05 (1.16–22.07)	0.031
Episode history	57%	29%	3.59 (2.36–5.47)	<0.001	4.04 (2.02–8.05)	<0.001
Subgroup 1: AGXT (n = 28) vs. negative (n = 315)
Bilateral stone	89%	17%	40.12 (11.69–137.67)	<0.001	43.66 (8.81–216.47)	<0.001
Nephrocalcinosis	32%	2%	20.77 (6.98–61.85)	0.009	16.52 (1.47–186.12)	0.023
Episode history	64%	29%	4.39 (1.95–9.88)	0.017	3.70 (1.14–11.95)	0.029
Subgroup 2: GRHPR (n = 16) vs. negative (n = 315)
Stone burden (continuous), cm	3.4 ± 2.0	1.6 ± 1.0	2.03 (1.50–2.74)	0.005	1.66 (1.06–2.58)	0.025
Episode history	69%	29%	8.95 (2.44–32.81)	0.012	7.09 (1.25–40.35)	0.027
Subgroup 3: HOGA1 (n = 34) vs. negative (n = 315)
Stone burden (continuous), cm	2.5 ± 1.5	1.6 ± 1.0	1.70 (1.32–2.18)	0.008	1.74 (1.08–2.80)	0.022
Subgroup 4: SLC3A1 (n = 26) vs. negative (n = 315)
Multiple stone	81%	29%	10.14 (3.71–27.69)	<0.001	13.02 (2.79–60.76)	0.001
Episode history	69%	29%	5.49 (2.31–13.07)	<0.001	7.47 (2.19–25.45)	0.001
Subgroup 5: SLC7A9 (n = 19) vs. negative (n = 315)
Bilateral stone	63%	17%	8.25 (3.11–21.9)	<0.001	9.28 (2.13–40.40)	0.003
Stone burden (continuous)–cm	3.0 ± 2.0	1.6 ± 1.0	1.97 (1.47–2.63)	0.028	1.49 (1.01–2.21)	0.046

BMI, body mass index; CI, confidence interval; eGFR, estimated glomerular filtration rate; OR, odds ratio.

^aAdjusted for age, sex, BMI, geographic distribution, ethnicity, family history, eGFR, and urine culture status.

We conducted subgroup comparative analyses for children with AGXT, GRHPR, HOGA1, SLC3A1, and SLC7A9 defects against those with a negative molecular diagnosis. We found that children with AGXT and SLC7A9 defects were more likely to have bilateral stones, children with SLC3A1 defects were more likely to have multiple stones, and children with AGXT defects were more likely to have nephrocalcinosis (all P < 0.05). Besides, we found a marked difference in the level of stone burden between children with GRHPR and SLC7A9 defects and those with a negative one. Children with AGXT defects were at a higher risk of presenting with bilateral stones (adjusted OR: 43.66, 95% CI: 8.81–216.47). Children with HOGA1 and GRHPR defects were more likely to have large stone burden (adjusted OR: 1.66–1.74). SLC3A1 defects were associated with multiple stones (adjusted OR: 13.02, 95% CI: 2.79–60.76).

In the subgroup analysis, other distinct clinical phenotypes were observed among these genes (Table 4 and Figure 2). The AGXT subgroup exhibited the highest male-to-female ratio, with males accounting for 79% (22/28). Even if most children with defects in these genes were diagnosed at their young ages, those with HOGA1, SLC2A9, and SLC34A1 defects were diagnosed at an even younger age (average age: < 2 years old). Children with GRHPR and SLC2A9 defects seem to have higher body mass index. The highest proportion of positive family history was found in children with GRHPR defects (38%, 6/16). Children with AGXT defects were found to be more susceptible to nephrocalcinosis and defected estimated glomerular filtration rate. More clinical information for the children with a positive molecular diagnosis is presented in Supplementary Table S2.

Table 4.

Clinical characteristics of patients with defects in each gene

Gene (n)	Male, n (%)	Age of onset (yr)a	BMI, (kg/m2)a	eGFR, ml/min per 1.73m2)a,b	Family history, n (%)	Stone burden (cm)a,c	Nephrocalcinosis, n (%)	Recurrent stone, n (%)	Bilateral stones, n (%)	Multiple stones, n (%)
AGXT (28)	22 (79%)	4.5 ± 3.9	16.4 ± 3.1	100.8 ± 47.0	6 (21%)	3.6 ± 3.1	9 (32%)	4 (14%)	17 (61%)	18 (64%)
GRHPR (16)	8 (50%)	3.5 ± 3.1	18.5 ± 4.5	126.3 ± 51.2	6 (38%)	3.2 ± 2.0	2 (13%)	5 (31%)	8 (50%)	9 (56%)
HOGA1 (33)	20 (59%)	1.6 ± 1.8	16.5 ± 2.1	142.7 ± 34.3	6 (21%)	2.5 ± 1.5	2 (6%)	1 (3%)	9 (27%)	11 (32%)
SLC3A1 (27)	14 (54%)	3.8 ± 3.2	16.7 ± 4.1	148.1 ± 28.1	5 (19%)	2.7 ± 1.2	1 (4%)	5 (19%)	7 (26%)	15 (56%)
SLC7A9 (20)	9 (47%)	3.6 ± 3.7	16.7 ± 3.9	137.8 ± 48.2	2 (11%)	3.1 ± 1.9	1 (5%)	3 (14%)	8 (38%)	9 (43%)
SLC26A1 (4)	2 (50%)	3.5 ± 1.7	14.1 ± 1.7	195.3 ± 22.7	1 (25%)	2.0 ± 1.1	0 (0%)	0 (0%)	2 (50%)	3 (75%)
SLC2A9 (3)	3 (100%)	1.1 ± 0.5	19.0 ± 3.3	146.8 ± 15.3	0 (0%)	1.4 ± 0.8	0 (0%)	0 (0%)	0 (0%)	3 (100%)
SLC4A1 (3)	1 (33%)	7.0 ± 2.6	15.2 ± 1.2	163.7 ± 44.2	1 (33%)	1.8 ± 1.1	2 (67%)	1 (33%)	3 (100%)	3 (100%)
OXGR1 (1)	1	10.8	13.8	76.4	0	15.0	0	1	1	1
APRT (1)	0	1.8	19.0	159.4	1	3.0	0	1	0	1
CASR (1)	0	7.6	15.4	158.8	1	1.5	0	0	0	1
CLDN16 (1)	1	16.0	-	-	0	2.0	1	0	1	1
KCNJ1 (1)	1	4.0	15.8	205.1	1	1.0	0	1	1	1
SLC34A1 (1)	1	1.0	15.0	200.0	1	0.7	0	0	0	0
XADE (1)	1	2.0	17.0	241.9	1	4.2	0	0	0	1
SLC34A3 (1)	1	3.0	-	-	0	1.5	0	0	0	1

BMI, body mass index; eGFR, estimated glomerular filtration rate.

^aShown as mean ± SD.

^bCalculated according to the Schwartz formula.

^cEstimated by adding the maximum diameter of each stone.

Figure 2.

The distribution of onset age, stratified by gene and sex, is visualized using violin plots combined with scattered plots. The median, 25th percentile, and 75th percentile are indicated as dotted lines on the violin plots. In addition, the mean age of all children is depicted as a solid line in the background.

Conformity Between Suspected Clinical Diagnoses and Molecular Diagnoses

We conducted consistency analyses to identify the conformity between suspected clinical diagnoses and molecular diagnoses. Overall, the concordance rate was 81% (193/239). Stones composed of cystine, xanthine, or 2,8-dihydroxyadenine could help urologists to make an accurate diagnosis, even an accurate molecular diagnosis. Suspected clinical diagnoses of hyperoxaluria, cystinuria, and renal tubular acidosis could be diagnosed based on stone analysis and stone-specific metabolic evaluation; however, the identification of molecular subtyping needs further genetic analyses. At least 29% of children (70/239) could benefit from additional clinical advice based on a molecular diagnosis. Approximately 14% of children with idiopathic metabolic abnormalities (12/87) could gain an accurate diagnosis. In patients with negative urine metabolic screening, 94% (59/63) showed no gene defects in subsequent genetic testing (Table 5).

Table 5.

Conformity between suspected clinical diagnoses and molecular diagnoses and additional clinical advice for modified diagnoses

Stone type (N)	Suspected Clinical diagnosisa (N)	Molecular diagnosis (N)	Accurate diagnosis	Accuracy rate	Additional clinical adviceb
Calcium oxalate (CaOx) (160)	Primary hyperoxaluria (91)	AGXT (24)	Yes	67%	Pyridoxine therapy
		GRHPR (12)			-
		HOGA1 (35)			-
		Negative (30)	No		Monitoring urine oxalate
	Idiopathic hypercalciuria (9)	GRHPR (1)	No	89%	Alkaline citrate and hydrochlorothiazide therapy; Monitoring kidney function, and urine oxalate
		SLC34A3 (1)	Yes		-
		Negative (7)			-
	Idiopathic hypocitraturia (15)	AGXT (1)	No	73%	Pyridoxine and alkaline citrate therapy; Monitoring kidney function, and urine oxalate
		GRHPR (1)			Alkaline citrate therapy; Monitoring kidney function, and urine oxalate
		OXGR1 (1)			Alkaline citrate therapy; Monitoring kidney function, and urine oxalate
		SLC7A9 (1)			Alkaline citrate therapy; Possible treatment with tiopronin
		Negative (11)	Yes		-
	Idiopathic CaOx (45)	SLC2A9 (2)	No	93%	Monitoring urine uric acid; Purine reduced diet
		HOGA1 (1)			Monitoring kidney function, and urine oxalate
		Negative (42)	Yes		-
Cystine (44)	Cystinuria (44)	SLC3A1 (25)	Yes	98%	-
		SLC7A9 (18)			-
		Negative (1)	No		-
Carbonate apatite (CA) (24)	Primary hyperoxaluria (6)	HOGA1 (2)	Yes	33%	-
		SLC26A1 (1)	No		-
		Negative (3)			Monitoring urine oxalate
	Idiopathic hypocitraturia (2)	AGXT (1)	No	0	Pyridoxine and alkaline citrate therapy; Monitoring kidney function, and urine oxalate
		SLC34A1 (1)			Alkaline citrate therapy; Monitoring urine calcium
	Idiopathic hypercalciuria (6)	Negative (6)	Yes	100%	-
	Infection (2)	Negative (2)	Yes	100%	-
	Idiopathic CA (8)	SLC3A1 (1)	No	88%	Alkaline citrate therapy; Possible treatment with tiopronin
		Negative (7)	Yes		-
Brushite (CHPD) (1)	Renal tubular acidosis (1)	SLC4A1 (1)	Yes	100%	-
Xanthine (1)	Xanthinuria (1)	XDH (1)	Yes	100%	Purine reduced diet
2,8-dihydroxyadenine (1)	Adenine phosphoribosyltransferase deficiency (1)	APRT (1)	Yes	100%	Purine reduced diet
OCP, UA, AU, MAP (8)	Idiopathic and infection (8)	Negative (8)	Yes	100%	-
Total	239	-	-	81%	-

AU, ammonium urate; CHPD, calcium hydrogen phosphate dihydrate; MAP, magnesium ammonium phosphate ; OCP, octacalcium phosphate; UA, uric acid.

^aSuspected clinical diagnoses were based on stone analysis and stone-specific metabolic evaluation.

^bAll stone formers followed our general prevention advice, such as maintaining fluid intake of 2.5 to 3.0 L/d, a balanced diet rich in vegetables and limited in protein and salt; and normal body mass index with exercise, and monitoring stone recurrence by a regular follow-up.

Discussion

Among various kidney diseases, clinical exome sequencing can achieve a relatively high diagnostic rate (46%) in nephrolithiasis, with most of the identified variants being pathogenic or likely pathogenic.¹¹ Previous studies demonstrated that numerous causative genes have been identified in pediatric patients3, 4, 5, 6, 7, 8, 9, 10, 11, 12 (Supplementary Table S1). These findings suggest that WES represents a powerful tool for confirming the clinical diagnosis of nephrolithiasis, as well as elucidating distinct genetic backgrounds underlying identical phenotypes, ultimately impacting patient management and prognosis. However, a significant proportion of children with nephrolithiasis still lack a definitive etiology, and they may harbor multiple causative genes, which remain poorly elucidated. Therefore, it is imperative to conduct further studies using WES in a large cohort of children with nephrolithiasis. Our study may contribute to bridging the knowledge gaps in the genetic background of pediatric nephrolithiasis, particularly within the Chinese population.

To the best of our knowledge, this study represents the most extensive genetic analysis conducted to date in a Chinese cohort of children affected with nephrolithiasis. In 31% of children (n = 141), we identified causative variants in 16 of 37 genes (43%). The variants in other genes, particularly ATP6V0A4, ATP6V1B1, CLCN5, CYP24A1, SLC9A3R1, and SLC12A1, which have been previously identified in other studies, were not detected within our Chinese cohort. In addition, the detection rates vary across studies conducted on different cohort baselines. These findings provide substantial evidence for significant heterogeneity in causative variants of nephrolithiasis-causing genes among diverse populations.

In our Chinese cohort, the most prevalent monogenic disease-causing nephrolithiasis is PH (55%, n = 78), with AGXT (20%, n = 28), and HOGA1 (24%, n = 34) being the most prevalent disease-causing genes. The duplications, c.33dupC (21%, n = 9) and c.823_824dupAG (14%, n = 7) were the most frequent variants of AGXT in our Chinese cohort, whereas p.Gly170Arg was more common in a Western population (28%–30%).¹⁷ In our children with GRHPR defects, a frameshift deletion variant (c.866_867delTG) was the most common with an allelic frequency of 63% (n = 14). The most common frameshift deletion variant observed in this study was distinct from the one identified in a Caucasian cohort (c.103delG) and other children of Asian ancestry (c.404+3_404+6del).^18,19 Our study found that, within the cohort of children with PH, PH3 is the most prevalent type. This finding is confirmed in another study⁸ based on a Chinese cohort. In Huang’s study, 3 PH children were identified using genetic screening, 2 of whom had PH3, 1 had PH1, and no patients with PH2 were detected. In contrast, in European cohorts, PH3 is considered a relatively rare subtype, a point also noted by another review.²⁰ The majority of children with HOGA1 defects harbored variants at splicing sites with c.834G>A and c.834_834+1delinsTT, accounting for 22% (n = 14) and 21% (n = 14), respectively. However, the splice variant c.700+5G>T exhibited the highest prevalence (46%) in a European cohort.²¹ Therefore, we attribute the differences in the prevalence of PH types to different ethnicities.

Patients with PH1 exhibit more severe clinical manifestations and are more likely to progress to end-stage renal disease at an early stage.¹⁷ These findings are consistent with those in our study cohort, where patients with PH1 more frequently presented with bilateral stones (61%, n = 17), multiple stones (64%, n = 18), and nephrocalcinosis (32%, n = 9). In contrast, patients with PH2 (50%, n = 8; 56%, n = 9; 13%, n = 2) and PH3 (26%, n = 9; 32%, n = 11; 59%, n = 20) exhibited milder manifestations, resulting in a lower stone burden (Supplementary Table S2 for details). However, our study found that the age of diagnosis for patients with PH3 is significantly lower than that for patients with the other 2 types. This finding appears to contradict the relatively milder clinical manifestations of PH3. Therefore, we propose that an earlier age of onset does not necessarily indicate a worse prognosis in affected children. Moreover, the earlier age of onset in children with PH3 may represent one of the characteristics of the PH population in Chinese children, which is potentially associated with the distinct mutation profiles observed in Chinese patients with PH3 compared with other populations.

Cystinuria was the second most prevalent nephrolithiasis-causing disease (32%, n = 45) with SLC3A1 defects being more commonly detected (18%, n = 26); however, in other studies, the genes related to cystinuria (SLC3A1 and SLC7A9) were found to be less predominant.^3,4 The identified causative variants of SLC3A1 and SLC7A9 in this study were found to be distributed across various locations. We have observed a phenomenon: in children with cystinuria, the mean age of male patients is lower than that of female patients, regardless of the cystinuria type. In a study²² based on 224 patients with cystinuria, researchers found that males are indeed more prevalent, a finding consistent across different types of cystinuria. Moreover, they discovered that in the patient group aged 0 to 3 years, the proportion of males is the highest, far exceeding that of females. These findings are consistent with our study. In some, but not all, studies,²³ clinical symptoms of cystinuria are more severe in males, suggesting that sex is an important factor influencing cystinuria. In our study, no significant differences were observed in stone characteristics (including laterality and size) between male and female children with cystine stones. However, a higher recurrence rate was noted in male pediatric patients (Supplementary Table S2). No definitive mechanism has yet been identified, though. We speculate that this may be related to the differences in sex hormone levels between males and females. Evidence from some studies indicates that individuals with high levels of androgens and low levels of estrogens have a higher risk of developing stones.^24,25 Although the differences in sex hormone levels are limited in younger children, these differences lay the foundation for sexual development and may similarly affect the risk of cystine stone formation. In addition, differences in gene expression and penetrance also exist between different sex.

Given that approximately 1 in 3 children with nephrolithiasis harbor monogenic variants, a comprehensive genetic analysis or screening is imperative because of its practical implications in elucidating the etiology, personalizing treatment, and implementing preventive measures. According to our findings, a molecular genetic diagnosis may have significant implications in monitoring nephrocalcinosis and kidney function for affected children, especially those with an early onset age, female sex, a positive family history, recurrent stones, bilateral stones, multiple stones, and staghorn stones. WES could also help to identify the molecular type of each disorder for further treatment strategy. Although some treatment in children was conducted before WES, a molecular diagnosis could provide more precise treatment options and preventive measures. For children with PH, genetic analyses could confirm PH and its typing, which are pivotal to the management of these patients. In addition, it is important to note that relying solely on biochemical parameters for assessment can be unreliable.¹⁷ Invasive and potentially harmful procedures, such as a diagnostic liver biopsy in children with suspected PH, can be circumvented.³ The average outcome of children with AGXT defects (PH1) was found to be worse than that of PH2 or PH3, which is consistent with previous findings.¹⁷ A molecular diagnosis of children with PH1 could serve as a prompt for clinicians to adopt more vigorous treatment and monitoring. Moreover, certain PH1 genotypes exhibit a strong correlation with therapeutic response to pyridoxine,²⁶ and new RNAi therapies (lumasiran) have demonstrated efficacy exclusively in children diagnosed with PH1.²⁷ To confirm the diagnosis, classify the disease, and provide counseling to other family members, genetic testing was recommended for children with cystinuria; however, it was not mandatory for diagnosis.²⁸ Molecular diagnoses were also helpful to distinguish the enteric hyperoxaluria from PH. In our study, we have identified several monoallelic variants in SLC3A1 or SLC7A9 predisposing for nephrolithiasis. The interpretation of these causative variants underwent meticulous assessment, and the genetic findings were consistent with the clinical and biochemical observations. Children who have these predisposing monoallelic variants show no significant clinical differences from those with normally inherited cystinuria (Supplementary Table S3). The administration of phosphorus supplements and the avoidance of vitamin D are likely to be beneficial for children with SLC34A1 and SLC34A3 defects, which should only be considered following a definitive molecular diagnosis.^29,30 For children with abnormality of purine metabolism and reabsorption such as APRT, SLC2A9, and XDH variants, the administration of xanthine oxidase inhibitors and restriction of high purine diet may offer certain protection.³¹ In patients with negative urine metabolic screening, > 90% had negative molecular diagnoses; this indicates that urine metabolic screening is still a sensitive screening for genetic nephrolithiasis and suggests that combining urine metabolic screening with genetic testing can enhance diagnostic accuracy, improve the pertinence of genetic testing, and lower economic costs.

With the screening approach employed in this study, the copy number variations were detected with low sensitivity, and deleterious intronic variants were undetectable. The environmental effects were of great importance, some of which remained unknown and contributed to stone formation; thus, the rate of molecular diagnosis may be misestimated. Children with possible secondary causes of nephrolithiasis were excluded, which could potentially bias the results and affect prevalence data. These limitations as well as population genetic factors may have led to a selection bias in regard to the distribution of molecular diagnoses in this study cohort. Thus, we should give a cautious overall interpretation of these results.

In conclusion, our study has demonstrated that the molecular diagnosis is identified in 31% of children with nephrolithiasis, and the genetic findings of a Chinese population differ from those of other populations. Children with a positive molecular diagnosis have an early age of onset and are more likely to have severe manifestations. Specific molecular diagnoses in such cases held crucial clinical implications in personalized treatment plans and preventive care strategies.

Disclosure

All the authors declared no competing interests.

Supplementary Material

Supplementary File (PDF)

Supplementary Methods. Supplementary References. Table S1. Studies of genetic testing in pediatric patients with nephrolithiasis. Table S2. Detailed information of genetic and clinical data of each child with a positive molecular diagnosis. Table S3. Comparison of clinical characteristics between children with SLC3A1/SLC7A9 defects who have autosomal dominant inheritance and autosomal recessive inheritance. STROBE Checklist.