Introduction

Chronic kidney disease (CKD) is a set of conditions characterized by gradual loss of kidney function. Kidney failure is the last stage of CKD, where treatment with dialysis or kidney transplant is needed to sustain life [1]. A multitude of factors, including genetic influences, contribute to the occurrence of CKD in children. Without knowledge of the etiologic causes, a full understanding of the pathogenesis, guiding clinical classification, predicting prognosis, and providing a suitable medical approach for patients with CKD cannot be achieved [2, 3]. About 30% of children with CKD suffer from a monogenic condition, which rises to a higher percentage when considering children with kidney failure [4,5,6]. Genetic testing is instrumental in assessing pediatric CKD, offering a reduction in the clinical ambiguity associated with the disease [7].

Currently, more than 600 genes have been reported to be involved in the occurrence of CKD. A total of 53 monogenic causes have been identified for isolated cases of congenital anomalies of the kidneys and urinary tract (CAKUT) and 131 for cases of syndromic CAKUT. Monogenic causation has been reported in 14 to 20% of cases in pediatric cohorts with CAKUT [8]. A genetic etiology accounts for about 30% of patients with childhood-onset steroid-resistant nephrotic syndrome (SRNS) involving one of the roughly 60 genes associated with genetic SRNS. Mutations in approximately 100 genes have been described as single-gene causes in renal cystic ciliopathies, some of which have high genetic diagnostic rates: for example, 100% for Bardet–Biedl syndrome and nearly 30% for nephronophthisis (NPHP). Roughly 30 genes are known to cause nephrolithiasis (NL) or nephrocalcinosis (NC), and the monogenic contribution rate of those genes in NL/NC is 29%. Furthermore, more than 50 monogenic disease-causing genes have been identified in children with renal tubulopathies, with a genetic diagnostic rate of about 64% [2, 8,9,10].

Our earlier data from the Chinese Children Genetic Kidney Disease Database (CCGKDD) revealed that the most common primary diseases leading to CKD in children are CAKUT, chronic glomerulonephritis, SRNS, and ciliopathy. A molecular genetic diagnosis was confirmed in 42.1% of the total patients [11] and in almost 40% of children with kidney failure. A diagnosis rate of 100% was observed in cases of nephrolithiasis, followed by ciliopathies (54.1%), SRNS (48.1%), chronic glomerular nephritis (29.4%), and CAKUT (23.8%) [12].

Sanger sequencing is a technique that examines a single or a small number of genes sequentially, and which previously constituted the primary method for the diagnosis of kidney diseases. Nowadays, next-generation sequencing (NGS) is increasingly being applied in diagnostics. An exome-based approach, such as targeted gene sequencing (TGS) and exome sequencing (ES), provides dynamic gene updates with minimal design and validation, making it more attractive to diagnostic laboratories. The primary scope of TGS and ES is to identify small variants such as single-nucleotide variants (SNVs) or small insertions/deletions (INDELs) within the coding region of the genome. Nonetheless, large copy number variants (CNVs), which are also important causes of kidney diseases are not easily picked up by TGS or ES. Genome sequencing (GS) can pick up large CNVs and detect pathogenic variants in complex genomic regions, addressing the limitations of TGS and ES. However, as the mass of data output in GS is essentially 100-fold higher than in ES, leading to difficulties in classification and interpretation of intronic and other noncoding variants, GS is not yet commonly used in clinical practice [13, 14].

The role of genetic testing for kidney disease is vital. Nevertheless, there are two main obstacles in clinical genetics for CKD. First, the increasingly diverse landscape of genetic testing options may present a formidable challenge to clinicians who lack an extensive background in genetics. Without the guidance of a geneticist, clinicians often feel confused about choosing a suitable genetic testing approach for their patients with complex penetrance. Without the comprehensive phenotypic assessment from clinicians, a geneticist may omit or overlook a candidate positive mutation during analysis of the sequencing data. Multidisciplinary discussions between clinicians and geneticists are important. Second, as the genetic testing technology improves and the costs and turnaround times for sequencing decrease, genetic testing is suitable for daily clinical practice in many regions around the world. However, as the first-line approaches of whole exome sequencing (WES) and whole genome sequencing (WGS) may fail to identify a genetic cause in up to 60–70% of pediatric patients with CKD, the development of new genetic testing technologies is urgently needed. For instance, using machine learning, additional genetic causes may be identified and diagnostic yields may be improved through reanalysis of unresolved cases of CKD [15].

Results of Caliment et al.’s study

Previous studies have confirmed the importance of clinical genetics in pediatric nephrology including the different emerging sequencing technologies in recent years. However, the approach to selection of genetic testing in pediatric cases is still not unified. This issue of Pediatric Nephrology features a retrospective study by Caliment et al. of 126 pediatric patients with CKD [16]. The cohort were included for genetic testing based on the current international guidelines [13, 17], with the aim of clarifying the indications for genetic testing in pediatric patients. Disease groups enrolled among these patients included glomerulopathies, tubulopathies, and ciliopathies because of the evidence of high diagnostic yields of monogenic etiology [18]. CAKUT cases were only enrolled if patients presented with visual or hearing phenotypes. Caliment et al. propose a step-by-step, multidisciplinary strategy for the diagnostic evaluation of pediatric patients with CKD. Patients firstly attended a pediatric nephrology consult to decide the necessity of genetic testing, and cases were then discussed during a multidisciplinary meeting involving at least one pediatric nephrologist and one geneticist. Patients with more complex presentations were offered a multidisciplinary consultation before an oriented genetic testing was proposed. Types of genetic testing included Sanger sequencing, SNaPshot mini-sequencing, and targeted phenotype-associated gene panel sequencing, while WES was employed as a second line of testing. Human phenotype ontology (HPO) terms chosen during the multidisciplinary meeting or consultation were used to encode clinical and/or paraclinical findings to evaluate the diagnostic relevance of HPO entries.

Ninety-three patients (74%) were proposed for genetic testing according to the step-by-step, multidisciplinary strategy. Among them, definitive genetic results were available for 44 patients (47%), 93% (41/44) of which benefited from NGS-targeted phenotype-associated gene panel sequencing. The authors found that overall genetic yield reached 63%, and the distribution of renal diseases among patients was balanced and matched previously described pediatric cohorts in terms of glomerulopathies, tubulopathies, and ciliopathies [19]. These results support application of their step-by-step approach in clinical practice. The most frequent causal mutations identified were in PKD1 and PKD2, HNF1B, COL4A3/5, SLC34A3, and SLC5A2 genes. This distribution is different from reported cases, which could be explained by the selection of the disease group [12]. Importantly, genetic testing resulted in a modification of pharmacologic treatments in 10 patients, which indicates the importance of applying genetic technology in patients with kidney disease [20, 21].

The authors did not mention any variants in mitochondrial genes or pathogenic CNVs because of the limitations of the sequencing technology. However, previous studies have indicated pathogenic variants and CNVs play important roles in ciliopathies and tubular diseases, especially those with syndromic features [22]. Furthermore, they reported a relatively poor performance of HPO, which could be explained by a lack of precision in the definition of the HPO terms and in the association of terms with potentially causal mutations. This reminds both nephrologists and geneticists when using HPO to interpret the sequencing result.

Generalizability and future application

Introducing precision medicine to CKD patients offers significant potential for improving their quality of life. Genetic assessment of children with CKD has already made advances in the accuracy of diagnosis, prognosis, and treatment. Lots of different approaches to genetic testing are now widely available to find new disease-causing variants in previously unrecognized biological pathways, and to highlight the mechanisms involved in the etiology, morbidity, and mortality of CKD. Comprehending the merits of genetic testing and the associations between genotype and phenotype is helpful for guiding nephrologists in the diagnosis and treatment of patients with CKD. There is an urgent need to bridge the gap in awareness of the indications of clinical genetics across the range of pediatric CKD.

Indications for genetic testing in CKD

There are some patients with CKD who do not require genetic testing [14]. As mentioned previously, patients proposed for genetic testing were selected in accordance with the current international guidelines [23], even though family history and parental consanguinity have been shown to not be related to a positive genetic diagnosis in CKD [12]. Clinical genetics is recommended when the etiology of CKD is uncertain, and the genetic component is clinically considered (positive family history, early onset, extra-renal manifestations, unusual disease course, complicated treatment process) [14]. There is no doubt that additional reference predictors should be further studied and added to better determine the application of clinical genetics in CKD [12].

Approaches to identify disease-causing genes in CKD

To date, there are lots of different approaches to genetic testing, in nephrology. Sanger sequencing is a convenient way of reading the sequence of small targeted regions of the genome, which is applied in diseases with minimal locus heterogeneity, such as Fabry disease and Alport syndrome. TGS is used in disorders with locus heterogeneity, overlapping phenotypes, or disorders associated with genes from a common pathway, such as SRNS and other complement-related disorders. In cases where the phenotype is not clear and the underlying cause is unknown, such as in unexplained kidney failure, WES (which captures almost all coding sequences) is recommended in order to find new disease-causing genes when TGS fails to find specific pathogenic variants. WGS covers almost the entire genome and is well suited to identify SNVs and INDELs, large CNVs, and to detect pathogenic variants in complex genomic regions. WGS is proposed to discover disease-causing variants that cannot be resolved by WES [13].

Strategies for genetic testing in CKD

As the cost of high-throughput sequencing declines, genetic testing has become a commonly available practice in CKD. Caliment et al.’s study proposes that before an oriented genetic testing, children with CKD should receive a step-by-step, multidisciplinary strategy: patients attend a pediatric nephrology consult to decide the necessity of genetic testing first, cases were then discussed during a multidisciplinary meeting involving at least one pediatric nephrologist and one geneticist. Genetic testing was employed as a second line of testing in children with CKD.

Multiple kidney diseases caused by single genes have been identified and functionally characterized by Dr. Hildebrandt’s group. They have recommended the application of WES to the diagnostic approach in CAKUT, to provide an accurate etiology-based diagnosis and to improve clinical management [24]. Simone et al. found that the burden of rare CNVs is high in CAKUT, using a multidisciplinary approach (about 5% of CAKUT cases are explained by CNVs) [25]. Non-coding variants can also cause CKD, suggesting that using WGS to elucidate genetic causes should be considered [26].

Prior to 2020, we used a three-pronged approach (TGS, proband-based WES, and trio-WES) to identify disease-causing variants. TGS was utilized to sequence protein-coding regions of the targeted genes. WES was utilized in children with CKD without a genetic diagnosis. In our cohort, 40% of pediatric patients with kidney disease were molecularly diagnosed with the application of TGS or WES. The precise definition of a phenotype could assist in making a genetic diagnosis and a comprehensive understanding of the phenotypic variability in kidney diseases may benefit from genetic findings. A strategy combining analysis of the phenotype and genotype in clinical genetics in CKD has been proposed [6].

Future application of clinical genetics in CKD

The optimal application of clinical genetics in CKD requires a preferred strategy for practicing nephrologists and geneticists across the healthcare spectrum [14]. RNA-sequencing (RNA-Seq) is an efficacious complementary tool to WES or WGS for improving genetic diagnosis. A recent study revealed that up to 31% of splicing variants of unknown significance (VUSs) could be classified as either likely pathogenic or likely benign by RNA-seq analysis [27, 28]. Since the second half of 2020, the innovative sequential WGS with dual-omics (DNA-mRNA) approach (combined RNA-seq analysis with WGS) has been applied in pediatric patients with CKD in our center and has identified a molecular diagnosis in nearly 50% of this cohort.

For all the patients with CKD of unknown origin, after multiple assessment by clinicians, we firstly use TGS; for example, we use COL4A3/COL4A4/COL4A5 exome panels in patients with hematuria, and when causative mutations are found, and the patient has a molecular diagnosis, other genetic tests will not be used. For cases still genetically unsolved after TGS, trio-WES, CNV seq and mitochondrial genome (mtDNA) seq are applied: if causative genetic factors are found, WGS will not be applied. In patients where causative mutations are not found by trio-WES, CNV seq and mtDNA seq sequential WGS with dual-omics approach is applied. All sequencing data are analyzed by geneticists, and with an in-depth discussion with clinicians to identify the pathogenicity of genetic variants and to receive a certain molecular diagnosis (Fig. 1).

Fig. 1
figure 1

Sequential WGS strategy in pediatric patients with CKD. For all CKD patients with unknown origin, after multiple assessment by clinicians, peripheral blood is collected once. Firstly, TGS is used; for unsolved cases, trio-WES, CNV seq and mitochondrial genome (mtDNA) seq are applied secondly; in patients where causative mutation is not found, sequential WGS with dual-omics approach is applied finally. All the sequencing data should be analyzed by geneticists, and with an in-depth discussion with clinicians to identify the pathogenicity of genetic variants and to receive a certain molecular diagnosis

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In our view, first, a combined phenotype-genotype analysis is vital in clinical genetics. Following-up clinical characterization of genetically unsolved patients has enabled us to revise and detect relevant new clinical features in a more appropriate pathogenic context—an accurate definition of a phenotype will be helpful for improving the rate of the genetic diagnosis. Second, for patients with CKD unsolved by routine genetic testing, the sequential WGS with dual-omics approach should be applied: new causative gene discovery would help the production of new TGS (large exome panels) and provide new targets for precision treatment. The sequential WGS strategy has not only improved the genetic diagnosis rates but also reduces economic costs.

Conclusion

In conclusion, the multifaceted significance of clinical genetics in the realm of CKD cannot be overstated. To begin with, it is important to note that in the age of precision medicine, genetic testing can provide accurate diagnosis, thereby facilitating personalized treatment and prognosis. Moreover, molecular diagnostics can frequently obviate the requirement for invasive kidney biopsies, particularly when such biopsies yield ambiguous results. In addition, in instances of hereditary kidney diseases, molecular genetic analysis can facilitate genetic counseling and the assessment of family members at risk, which is crucial for making knowledgeable decisions, especially when considering living related kidney transplants. Finally, in addition to providing insights into disease pathogenesis, the discovery of a novel disease-causing gene also opens the possibility of developing gene therapy strategies for specific disease foci [2].

The study by Caliment et al. represents a step-by-step, multidisciplinary strategy to maximize the yield of genetic testing in pediatric patients with CKD. This approach optimizes patient care while avoiding unnecessary treatments or procedures. Future studies with a larger patient cohort, based on a comprehensive solution that integrates genomics, proteomics, transcriptomics, epigenetics, and machine learning techniques, will be instrumental in elucidating the underlying mechanisms, as well as the onset and progression of CKD, and thus in finding new therapeutic options, in addition to genetic analyses.