A comprehensive analysis of NPHS1 gene mutations in patients with sporadic focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a syndrome with unique clinical and pathological manifestations. Although new medications continue to emerge, FSGS is still one of the most common causes which contributes to refractory nephrotic syndrome [1‐3]. The previous studies suggested that FSGS could be idiopathic or secondary to a process originating outside the kidneys as well as to a specific genetic mutation [1, 3, 4], including INF2, ACTN4, TRPC6, WT1, NPHS1, NPHS2, etc. [5, 6]. The efficacy of cyclosporin A (CsA) is much better in nonhereditary steroid-resistant nephrotic syndrome (SRNS) than in genetic SRNS [7]. It suggests that genetic mutations not only contribute to the pathogenesis of FSGS but also influence the outcome of clinical treatment. Therefore, it is of great clinical significance to elucidate the characteristics of genetic variation for FSGS patients.

About 8–14% of patients with FSGS could be explained by podocyte-related gene mutations. NPHS1, that relates to congenital nephrotic syndrome (CNS) and SRNS, is one of the most frequently reported genes. Initially, Kestilä described NPHS1 as the pathogenic gene of the congenital nephrotic syndrome of the Finnish type (CNF) [8]. The NPHS1 gene (OMIM *602716) locates in the chromosome 19q13.1 and consists of 29 exons that span a 150 kb region. Nephrin, a transmembrane protein encoded by NPHS1 gene, is one of the important components of slit diaphragm (SD) [9]. As a signaling scaffold via interactions at its short intracellular region, nephrin also serves as the core component of the glomerular filtration barrier [10]. The mutations of NPHS1 gene could lead to the occurrence of different degrees of kidney diseases, however, studies which investigated the relationship between NPHS1 gene mutations and FSGS were limited, especially in China [8, 11‐13].

Santin et al selected SRNS patients to conduct a series of research of podocyte-associated genes [6]. The patients showed familiar heredity and 57% of them developed FSGS at extremely early ages [6]. NPHS1 gene was the most common mutant gene in these subjects, and patients with NPHS1 mutations were more likely to progress to ESRD than those with other podocyte genes [6]. NPHS1 mutations have exhibited increasing prevalence across the world over recent decades, while most of the reports have only focused on one or several mutations of NPHS1 in relatively small FSGS samples. In this study, we performed an analysis of NPHS1 gene mutations in Chinese patients with sporadic FSGS by direct sequencing of all exons. While comparing these variants with the data from 1000 genome project as well as the mutations reported previously, we hoped to further elucidate the characteristics of NPHS1 gene mutations in sporadic FSGS patients and to provide a profile for future precision medicine and pathogenetic studies of FSGS.

Although NPHS1 was first demonstrated to be the causal gene for congenital nephrotic syndrome of Finnish type [20], subsequent studies have confirmed that it was also a causative gene or susceptibility gene for a variety of kidney diseases, such as SRNS, FSGS, minimal change disease with nephrotic syndrome (MCNS), IgA nephropathy, et al. [9, 11, 12, 21‐24]. Among them FSGS was one of the most common causes of SRNS and a common type of genetically related kidney disease [18, 24, 25]. Most studies about genetic mutations in FSGS were based mainly on familial FSGS patients and lots of FSGS-causing genes have been identified [1, 5]. Especially in individuals presenting with FSGS or nephrotic syndrome before or at the age of 18 years old, the most common genes in which a mutation was found continues to be limited to only a few genes, including NPHS1 and NPHS2 [2, 23, 26‐29].

Nephrin was located only in glomerular podocytes, which participated in intercellular junctions of mature podocyte and formations of the slit diaphragms [30]. It was demonstrated by lowering nephrin expression in an inducible model of nephrin deletion that normal nephrin expression was necessary for podocyte intercellular junction in the glomerulus [31]. A low level of nephrin expression could result in progressive proteinuria with glomerular hypertrophy and FSGS of glomeruli [31, 32]. Nephrin-knockdown mice developed more podocyte apoptosis and depletion after doxorubicin challenge [32]. NPHS1 knockout mice subjected to podocyte injury failed to recover from foot process effacement as well as the persistence of proteinuria [31]. Ephrin-B1, a membrane-bound protein, bound to and interacted with nephrin by immunoprecipitation assay [33]. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1-promoted cell motility [33]. The interaction of nephrin and ephrin-B1 maintains the structure and barrier function of the slit diaphragm [33]. It suggested that nephrin was required to maintain slit diaphragm integrity and slit diaphragm-mediated signaling, and it played an important role for the maintenance of podocyte function, production of proteinuria and pathogenesis of FSGS.

So far, more than 220 different mutations have been described affecting most exons in NPHS1 [11]. These mutations have been found in various kidney diseases, however, the relationship between NPHS1 mutation and FSGS was relatively less explored [34‐37]. Santin, et al. found that the mutations of NPHS1 gene were detected in patients with CNF, congenital FSGS, childhood FSGS, as well as adulthood FSGS [37]. A previous study reported that no pathogenic NPHS1 mutations were found in 33 FSGS patients with SRNS [36], but the study only explored the previously reported pathogenic mutations in a small FSGS sample. Another study indicated that gene interactions between NPHS1 and TRPC6 variants have important implications on post-transplantation FSGS [35]. In this study, we focused on sporadic FSGS patients to analyze the NPHS1 mutations. We identified 32 mutations in NPHS1 in a relatively large cohort of FSGS patients in China, including two novel mutations.

Twelve synonymous mutations, 17 missense mutations, one splicing mutation and two intron mutations were found in our study. The MAFs of four synonymous mutations (c.C294T [p.I98I], c.C2223T [p.T741 T], c.C2289T [p.V763 V], c.G3315A [p.S1105S]) and four missense mutations (c.G349A [p.E117K], c.G1339A [p.E447K], c.G1802C [p.G601A], c.C2398T [p.R800C]) were much higher in FSGS patients than the data in controls. The MAFs of a missense mutation (c.A3230G [p.N1077S]) and two intron mutations (c.C3286 + 36 T and c. C3481 + 45 T) were lower in FSGS patients than the data in controls. Interestingly, of the four synonymous and five missense mutations mentioned above, eight other mutations except synonymous mutation c.C294T (p.I98I) have been reported to be associated with SRNS, MCNS or FSGS (Tables 1 and 2). Five missense mutations, c.C616A (p.P206T), c.G1802C (p.G601A), c.C2309T (p.P770L), c.G2869C (p.V957 L), and c.C3274T (p.R1092C), were predicted to be disease-causing variants by three different software.

As listed in Table 1, several synonymous mutations were previously reported to be associated with SRNS, MCNS, or FSGS. But the pathogenesis was still unknown. For the missense mutations, although five mutations in our study were reported previously, the exact pathogenesis of these mutations remained unclear, and no appropriate animal model of these mutations has been established for further study. Nephrin contained eight Ig-like domains, a fibronectin type III-like module (Fn), a transmembrane domain and an intracellular domain [13, 38]. A previous study revealed that most mutations of NPHS1 were observed in Ig-2, Ig-4, and Ig-7 domains [39]. In our study, the differentially distributed mutations in FSGS patients mainly located in Ig-1, Ig-6, Ig-7, and C-terminal cytoplasmic regions. It was also consistent with the concept that Ig-like part of the intracellular domain remained to be crucial for nephrin function [29]. All of the results would be helpful for future pathogenic mechanism research.

It was demonstrated in our study that the mutations of NPHS1 gene are common in Chinese sporadic FSGS patients. A comprehensive evaluation of NPHS1 mutations would be helpful for the management of FSGS. The mutations of NPHS1 gene could play an important role in the pathogenesis of FSGS, and we still needed more researches to clarify the pathogenesis of these mutations.