Urolithiasis (UL) is a highly prevalent health problem worldwide, affecting 4%–20% of all individuals (Quhal and Seitz, 2021). It is associated with significant morbidity because of frequent recurrence, the necessity for surgical intervention, and progression to chronic kidney disease, which heavily burdens the development of social healthcare (Rule et al., 2009). Patients with UL usually present with renal colic, hematuria, urinary obstruction, and urinary tract infections that can aggravate renal dysfunction and cause kidney loss. The specific risk of developing UL varies according to geography, dietary structure, socioeconomic status, and genetic factors (Monico and Milliner, 2011).

Significant increases in the incidence and prevalence of UL in adult populations have been observed for decades (Turney et al., 2012). Although UL is less prevalent in children and adolescents, the annual incidence of pediatric nephrolithiasis has increased by 4%–10% over the last two decades (Dwyer et al., 2012; Alfandary et al., 2018). The unique congenital deformities, anatomical structures, and metabolic patterns contribute to a higher UL recurrence rate in children than in adults. Among patients diagnosed with UL during childhood, there is a 50% probability of recurrence within 3 years (Tasian et al., 2017). Various factors are involved in the development of nephrolithiasis, including fluctuations in urinary pH, inadequate liquid intake, medications, metabolic abnormalities such as hypercalciuria and hyperoxaluria, and hereditary disorders (Miah and Kamat, 2017). However, the cause of UL has not yet been elucidated.

In pediatric patients, genetic and anatomical causes account for up to 75% of the risk factors for UL development (van’t Hoff, 2004). A positive family history of UL has been reported in 30%–80% of cases (Scoffone and Cracco, 2018). A cohort study estimated that the relative risk for UL was 2.57-fold in individuals with a positive family history compared to those with a negative family history (Curhan et al., 1997). Genetic or metabolic alterations usually lead to remarkable changes in serum or urine components, disrupting the balance between pro- and anti-lithogenic substances in body fluids (Hoppe and Martin-Higueras, 2020)^. According to the Online Mendelian Inheritance in Man (OMIM) catalog, monogenic mutations in 30 known nephrolithiasis (NL)-causing genes can contribute to the formation of NL by autosomal recessive, autosomal dominant, or X-linked transmission. These mutations were detected in 20.8% of the children with the onset of NL (Halbritter et al., 2015). Metabolic disorders involved in nephrolithiasis have been identified in more than 75% of cases. Hypercalciuria and hypocitraturia are the most common metabolic disorders in UL (Bowen and Tasian, 2018). Because pediatric patients with UL are more susceptible to monogenic alterations, genetic sequencing is particularly critical in patients with early disease onset because of its benefits in tailoring personal treatment regimens and follow-up. Misdiagnosis and missed diagnosis of inherited UL usually occur because of the lack of an effective genetic mutation detection approach.

Recently, exome sequencing (ES), as a breakthrough next-generation technique, has offered an in-depth and comprehensive way to detect potential pathogenic mutations at a relatively low cost and in a short time and is regarded as a cost-effective screening method (Aggarwal, 2021). Although a growing number of studies have outlined the prevalence of monogenic mutations and novel disease-causing loci (Halbritter et al., 2015; Daga et al., 2018; Wang et al., 2020), the contribution of monogenic causes of UL has not been extensively studied in Chinese pediatric patients. In the present study, we used ES to screen 82 children with UL for hereditary causes. Six novel mutations were identified in 6 patients. In addition, metabolic evolution and stone component analyses were performed to clarify the genotype–phenotype correlations in the pediatric UL group.

Pediatric UL often frustrates pediatricians and urologists because of its early onset, high recurrence rate, relatively poor prognosis, and treatment ambiguity. In the clinical setting, this condition is often overlooked or misdiagnosed. Recently, several genetic causes of UL have been identified. At least 30 genes have been shown to cause the monogenic forms of UL via autosomal-dominant, autosomal-recessive, or X-linked transmission (Halbritter et al., 2015). Previously, monogenic ULs were considered to have an incidence rate of less than 2%. However, growing evidence suggests that they might be more common than expected (Singh et al., 2022). Current molecular genetic diagnostic techniques make screening for hereditary diseases feasible. Next-generation sequencing combined with a designed multigene panel can be conducted for a comprehensive analysis of specific diseases or disease groups with a faster and more precise process (Ziyadov et al., 2021).

In this study, ES was performed on a cohort of 82 pediatric patients with UL. We sequenced the exon regions of 30 genes known to cause the monogenic form of UL and identified a causative mutation in 38 of 82 individuals (46.3%). This rate of identifying causative mutations in children with UL is higher than that reported by Braun et al., who found a positive mutation rate of 16.8% in a pediatric NL/nephrocalcinosis (NC) cohort (Braun et al., 2016). This was also higher than that in a previous report that found a monogenic cause in 29.4% of the families presenting with NL or NC at the age of onset less than 25 years (Daga et al., 2018). Halbritter et al. (2015) also observed a relatively low percentage of monogenic cases in the pediatric NL/NC subgroup (20.8%). In our study, the causative mutation detection rate plunged to 25.6% after excluding patients with uncertain variants, which is slightly higher than previous reports. All these individuals were recruited from a referral center for the management of pediatric UL in China, where early onset, severe, and stone-recurrent cases present more frequently than in other national or local hospitals. Moreover, only UL cases were enrolled in our research rather than including NL/NC together, and UL cases usually sustain higher genetic abnormality rates and more severe clinical symptoms than NC. Therefore, selective bias in our cohort and different inclusion criteria may have contributed to the differences in the genetic landscapes among the multiple cohort studies.

The most commonly mutated gene in our cohort was SLC3A1, which encodes a 685-amino acid glycoprotein rBAT that plays a role in the reabsorption of cystine, ornithine, lysine, and arginine as subunits of heterodimeric amino acid transporters and proximal tubular transporters (Yan et al., 2020). Mutations in SLC3A1 lead to high urinary cystine excretion, precipitation, and crystal formation, resulting in an increased risk of cystine stone formation (Hoppe and Martin-Higueras, 2020). Heterozygous carriers of the SLC3A1 mutation normally show an increased urinary cystine excretion pattern (Eggermann et al., 2012). We detected 15 genetic mutations in SLC3A1, including three pathogenic mutations and three novel variants. Only the novel mutation, c.1320G>T (p.Trp440Cys), was detected in the homozygous state. This mutation has never been reported in the literature, whereas other mutations were found in heterozygote carriers. Some undetected alleles or loci may underlie the heterozygous carriers of SLC3A1 mutations that cause urolithiasis. The Met467Thr mutation in SLC3A1 is the most frequent in European and North American populations (Shen et al., 2017). However, this mutation was not found in our results, indicating genetic variant heterogeneity among ethnicities.

Age distribution analysis of monogenetic mutations in the UL-causing gene panel was performed in our cohort. Individuals with autosomal recessive variants typically manifest earlier onset in life than dominant mutation carriers, such as those with hereditary polycystic kidney disease and previous inherited UL reports (Braun et al., 2016; Ghata and Cowley, 2017). Interestingly, we found that cases with the age of onset ranging from 6 to 15 years showed a high positive causative mutation detection rate, but the overall sample size was limited, and a larger number of cases needs to be included in further studies. The prevalence and heritability of urolithiasis vary according to sex. Traditionally, male cases predominated the sex distribution, with a ratio of 3:1 to female cases (Cicerello et al., 2021). Two twin studies reported that the heritability of kidney stones was at an estimated rate of 56%–57% in male twins, while the rate for female twins was only 46% (Halbritter, 2021). Notably, in pediatric UL, a higher prevalence was observed among boys in the first decade of life and among girls in the second decade of life, and girls represented 70% of the pediatric UL population in general (Van Batavia and Tasian, 2016). Fang et al. (2021) reported 39.9% of idiopathic stone patients were male in his hospitalized pediatric UL cohort. Our findings demonstrated a similar sex proportion to previous studies, with a ratio of 2.03/1 (male/female) in all subjects and 1.3/1 in positive individuals, suggesting that male patientss still accounted for most of the pediatric stone probands and needed more urgent early intervention. However, only six patients were older than 10 years in this study, and the inheritability of UL in terms of sex distribution during the second decade of life remains undetermined.

A pivotal step in ES analysis is the identification of potentially pathogenic mutations and benign variants. All mutations identified in our study were evaluated for pathogenicity and allele frequency by cross-checking them against public data banks, such as HGMD and ClinVar, and predicted using the ACMG criteria. The pathogenicity of 27 (50.0%) of the 54 detected variants was uncertain based on the ACMG classification, whereas 6 variants were novel and had not been previously identified. Two novel variants in SLC3A1, c.1320G>T(p.Trp440Cys), and c.1772_1773del p.(Arg591fs), were predicted to be likely pathogenic by ACMG classification. MutationTaster (http://www.mutationtaster.org/) yielded consistent results, implying that a frameshift mutant resulting in truncated proteins was likely to cause disease. c.2006G>C (p.Gly669Ala) (two individuals) in XDH was the most common novel mutation identified in our study. Although they were identified in an ACMG “uncertain” subgroup, multiple in silico analysis tools predicted it as potentially disease-causing. Notably, this variant and c.878T>C p.(Phe293Ser) in SLC7A9 were detected in a compound heterozygous state in patient P29, a cystic stone case who experienced recurrent nephrolithiasis three times. Therefore, these novel mutations broaden our knowledge of known pathogenic gene mutations implicated in UL. Novel mutational pathogenicity that is not yet recognized or has obscured clinical significance will be identified through genetic profiling mediated by high-throughput sequencing and complementary functional studies.

Normally, a genetic diagnosis must be consistent with the patient’s actual metabolic pattern abnormalities to confirm inherited UL. Thus, a comparative analysis between the metabolic evaluation and genetic results in positive cases was performed to confirm the consistency of our study. The hyperoxaluria-related variant was detected in eight of the nine (88.9%) patients in whom calcium oxalate was confirmed by stone component analysis. The AGXT and GRHPR defects showed 100% (3/3) conformity with the stone analysis results, whereas the HOGA1 defect sustained a conformity rate of 66.7%. In the cystine stone subgroup, a high level of conformity was also observed in the SLC3A1 (80.0%) variants. Our finding demonstrated ES yielded high diagnostic precision for genetic UL with metabolic abnormalities.

This study has some limitations. The relatively small number of patients, single ethnic background, and nature of the single-center study limit the universality and comprehensiveness of the interpretation and application of the results. In some studies, a female predominance was found in the pediatric UL population, especially in the second decade of life. The gender ratio in our study presents a male predominance. Therefore, a large, multicenter population-based study may be needed to determine this demographic factor in the Chinese Han population. The technical limitations of ES, which include uncovering untranslated regions and undetected complex deletion-insertion variants, compromise the reliability and credibility of high-throughput sequencing. Novel causative mutations may be missed because the gene set we selected to study may not cover some genes that cause urolithiasis/nephrocalcinosis phenocopies rather than urinary stones. Although the current results may be compromised by such selection bias, the actual prevalence of monogenic UL in children will be refined through further research. Additionally, obtaining sufficient stone samples for component analysis is difficult due to specific surgical interventions, such as extracorporeal shockwave lithotripsy or undersized calculus that result in incomplete metabolic evaluation. Finally, further functional studies are required to systematically verify the candidate pathogenic genes.

In this study, we analyzed the genotypes and phenotypes of pediatric urolithiasis in 82 patients and identified stone-causing genetic defects in 8 genes in 25.6% of the individuals. Six novel genetic mutations were identified, and a high degree of association between genotype and phenotype was observed. Chinese Han UL pediatric patients exhibited high genetic mutation frequency and early onset. ES is a cost-effective and reliable tool for the molecular diagnosis of monogenetic urolithiasis. Genetic screening should be applied in pediatric UL patients because individual genetic profiles may facilitate the personalization of treatment plans, monitoring treatment responses, and target drug development. This study identified the monogenic cause of pediatric UL, improved our understanding of the underlying molecular mechanisms, and provided novel genetic targets for further research.