Nephrotic syndrome (NS) is one of the most common glomerular diseases in children. It is generally divided into steroid-sensitive NS (SSNS) and steroid-resistant NS (SRNS), depending on the response of the patient to steroid therapy. SRNS is a challenging clinical disease, in which 50% of the patients progress to end-stage renal disease within 15 years (1, 2). However, in some patients, temporary or sustained remission may be achieved. Some patients exhibit multidrug-resistant phenotypes, even with enhanced immunosuppressive therapy (3). In children with SRNS, there are usually genetic mutations affecting either podocytes or the glomerular basement membrane (4). NPHS1 is one of the most common genetic SRNS causes, accounting for 13% of the genetic cases (3). The human NPHS1 gene is located on the long arm of chromosome 19 (19q13.1) and contains 29 exons, whose protein product “nephrin” is a member of the immunoglobulin-like superfamily. NPHS1 mutations are primarily responsible for CNS of the Finnish type. SRNS caused by mutations in the NPHS1 gene is manifested by NS but lacks its extrarenal manifestations, and virtually all patients are unresponsive to steroid and immunosuppressant therapy (5). Fortunately, the recurrence rate after transplantation is low. SRNS can be divided into three types according to age: congenital (presenting within the first 3 months of life, most of which are steroid-resistant), childhood, and adulthood (6). As the NPHS1 variants are not common, it may be difficult to identify them before genetic testing has been carried out in cases with late-onset NS. Furthermore, pediatric clinicians have a relatively insufficient understanding of this condition. To better understand NPHS1 variants in pediatric patients in China, we present the clinical and genetic data from a pediatric study involving 30 patients, derived from a multicenter registration system. In this research, the gene mutation spectrum and the resultant clinical manifestations in children were analyzed aiming to describe the general situation of NPHS1 variants in children with NS in China and raise awareness of this disease among clinical pediatricians.

The National Multicenter Registry (Chinese Children Genetic Kidney Disease Database [CCGKDD], www.ccgkdd.com.cn) has assembled the largest genetically screened cohort with pediatric renal disease in China (7). The data was submitted to the CCGKDD registry monthly by the participating centers in the nation. Eligibility criteria for the registration were complete information of phenotype and genotype for each family, and each medical center identified all the eligible patients post standardized training from the “Internet Plus” Nephrology Alliance of National Center for Children's Care. The phenotype data was identified by clinician experts in pediatric nephrology. The data-entry clerks who had only the ID number for the probands were trained to collect the phenotype information for the registration and they checked the birthdate and phenotype for duplication, and finally contacted clinicians to confirm the individual information before the final registry version. We retrospectively collected information regarding the genotype and phenotype of NPHS1-associated kidney disease from the registry and investigated the associations between clinical and genetic findings.

The current study was a nationwide, multicenter study, with the largest number of pediatric cases of NPHS1 gene variants in the country. This research yielded important information regarding the distribution of clinical phenotypes and genotypes in childhood SRNS. The 30 patients were from 30 unrelated families located in 11 provinces and autonomous regions in China. By analyzing the characteristics of clinical manifestations and genotypes, we aimed to enable clinicians to understand better disease-related NPHS1 variants and optimize decision making.

In this study, most of the NPHS1 variants were found in the CNS group, accounting for 80.0% (24/30) of cases. They were also found in early childhood-onset NS (age ≤ 3 years), accounting for 20.0% (6/30) of the cases. The results of the current study and previous findings confirm that NPHS1 variants can cause a broader variety of clinical phenotypes in nephrotic syndrome including childhood- and adult-onset focal segmental glomerulosclerosis than CNS (3, 6, 23, 27, 28). The variants of NPHS1 showed no obvious difference between patients with MCD and FSGS. Similarly, the previous study showed the spectrum of renal histologic findings atypical for CNS with NHPS1 variants ranging from MCD to FSGS (6). The histopathological manifestations may be related to podocyte injury and persistent proteinuria caused by NPHS1 variants. Extrarenal manifestations were present in 16 of the 24 patients with CNS in our study, mainly in congenital hypothyroidism, hernia, and congenital heart disease. From the results, there was no considerable difference in variants between patients with and without extrarenal manifestations. Combined with the literature review, we consider that the extrarenal manifestations in our study were possibly secondary to CNS or cooccurring complications along with CNS rather than related to genetic variation.

A previous consensus indicated that patients with NS caused by NPHS1 variants exhibit a weak response to steroid therapy (5, 24, 29). In our study, one patient (case 12), responded effectively to initial steroid therapy and maintained an effective response in subsequent nonfrequent relapses. The NPHS1 variants of this child exhibited three-compound heterozygous variants, namely c.928G>A in exon 8, c.2207T>C in exon 16, and c.3312-23C>T in intron 25. This finding from our study was inconsistent with previous reports and may indicate that steroid therapy can be usefully applied in CNS patients with these variants loci in NPHS1. However, it was just an individual observation result from clinical practice in our study. A recent genome-wide association study reported that there are some common risk variants in NPHS1 that are associated with childhood SSNS (30). However, the steroid-sensitive mechanism remains unclear. We speculate that perhaps the nephrin protein, resulting from these variants, maintains its partial function and shows the response to steroid or immunosuppressive therapy, which may help actin reorganization in the cytoskeleton of podocytes. Further study of its molecular mechanism and long-term follow-up is required. In this study, it was also indicated that some children with early childhood-onset NS caused by NPHS1 variants may be effectively treated with steroid and immunosuppressive agents. Studies involving greater numbers of patients will be needed to observe the effects of immunosuppressive therapy, along with further mechanism studies to determine the significance of these variants.

The results of NPHS1 variant analysis in our study revealed that there were mainly compound heterozygous variants, accounting for 93.3% (28/30) of cases, with one homozygous variant. This finding suggests that compound heterozygous variants are the major variation pattern in NPHS1 in Chinese patients. The most frequent variant was c.928G>A in exon 8 (eight patients), which was present in CNS patients, followed by c.616C>A in four, c.2207T>C in three, and c.1394G>A, c.2783C>A, and c.3478C>T in two, respectively. These findings were, to some extent, different from reports from other Chinese studies (15, 31–33). Our findings indicated that heterozygous variants, including c.928G>A and c.616C>A, are “pathogenic recurrent variants” of NPHS1 in China, whereas c.928G>A is the main genotype of CNS. It is generally believed that recurrent genetic variants occur via two mechanisms: one is a founder effect and the other is a mutational hot spot. In our study, the variant 928G>A occurred with high frequency and was rarely reported in other populations, and so we speculated whether it is a founder effect. However, there remains the little specific supporting basis for this speculation since there is little data about researches on NPHS1 in China, and data in our study was so limited that it was difficult to perform further analysis for the founder effect. The mutation results revealed that missense mutations were the main mutation types in the two groups. Splice mutations, nonsense mutations, and intronic mutations mainly occurred in patients with a phenotype of congenital nephrotic syndrome, similar to previous reports. In the present study, 13 variants were found to be of uncertain significance, accounting for 21.3% of cases, of which 11 variants have not been reported yet. Most of the patients who carried the novel variants, along with a pathogenic variant detected in trans, presented typical clinical features of CNS. Among the 11 novel variants of VUS, eight were located in exons with “damaging” in silico predictions. Although the pathogenicity of variants with VUS has not been identified, from the perspective of clinical practice based on factors involved, all of them in this study were thought to be disease-causing variants through fully integrated analyses of the phenotypical manifestations and genotypes. However, the introns and copies of these variants need to be further detected and analyzed to determine their pathogenicity.

In China, the prognosis for CNS was poor before the turn of the 21st century. Significant progress has been made in healthcare, as well as in pediatric nephrology during the past few decades, and the prognosis has improved significantly. In terms of treatment and prognosis for the 30 patients in this study, 12.5% (3/24) of children diagnosed with CNS received kidney transplantation. Up to 30.0% (9/30) of the patients did not receive appropriate treatment after genetic diagnosis because their parents declined treatment due to the high financial burden involved or concerns about poor prognosis. A cross-sectional nationwide survey of CNS and infantile NS in Japan reported that up to 78.8% (26/33) of patients underwent peritoneal dialysis and up to 51.5% (17/33) underwent subsequent renal transplantation (25). On the one hand, due to the very low frequency of renal disease resulting from NPHS1 variants, individuals and general clinicians are unfamiliar with the disorder as well as its prognosis. On the other hand, most physicians exhibit inadequate recognition and understanding of this illness and its multidisciplinary requirements. We, therefore, need to develop a greater understanding of CNS, both in medical care and patient care personnel, across the nation in the future.

Some potential limitations should be recognized in the current study. First, it was a retrospective analysis where the interventions could not be well controlled. Such as some patients underwent genetic testing by the clinical panel (targeted gene sequencing) rather than all by WES. Although the clinical panel used in our study included 249 genes which almost covered the main responsible genes in hereditary kidney disease, WES is recommended for these patients, if available. Second, it may be underpowered to perform the statistical analyses in comparison between CNS group and non-CNS group due to the overall sample size of the cohort. Significant differences could be made if a larger cohort size is available. Finally, it was a limited number involving only 30 patients collected from 2014 to 2020, although it represents the largest cohort with NPHS1 variants in China currently. In the next step, it is necessary to expand the cohort size nationwide and longer follow-up is needed.

In conclusion, variants of NPHS1 not only cause CNS but also NS in early childhood-onset disease. NPHS1 genetic testing for CNS with onset within 3 months after birth, and also NS with steroid resistance, is helpful for early diagnosis and prognosis evaluation. NPHS1 variants in Chinese individuals with NS were mainly compound heterozygous variants, and c.928G>A(p.Asp310Asn) in exon 8 may act as a recurrent variant in the Chinese population, followed by c.616C>A(p.Pro206Thr) in exon 6. Steroids and immunosuppressants may have a beneficial effect on selected patients. Kidney transplantation in children with NPHS1 variants is effective. Efforts are also needed to raise awareness of CNS among patient family members and to improve treatment coverage in China.

The data that support the findings of this study are available from the corresponding authors upon reasonable request. The datasets presented in this article are not readily available for public repository due to the regulation on the management of human genetic resources from the State Council, CHINA. Requests to access the datasets should be directed to the database for Chinese children renal disease which is publicly available datasets in Chinese language (https://www.ccgkdd.com.cn/).

The studies involving human participants were reviewed and approved by the Institutional Review Board (IRB) of the Children's Hospital of Fudan University (Shanghai, China) (IRB No. 2018286). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

HX and XJ designed the study, reviewed, and revised the manuscript. LR, LC, JR, and QS performed the search, performed the analysis and wrote the manuscript, and they have contributed equally to this work. JM, CF, XW, XK, WH, QM, XL, CL, RF, XG, GD, HY, ZH, MH, QL, QZ, and YL collected the data. GL and JL rechecked the data. All authors contributed to the article and approved the submitted version.

This work was supported by the Science and Technology Planning Project of Guangzhou, China (Grant No. 202103000001).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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