Introduction
Focal segmental glomerulosclerosis (FSGS) is a kidney histological lesion with diverse etiologies and varying clinical manifestations. Clinically and pathologically, FSGS is characterized by proteinuria, glomerulosclerosis, and the fusion or effacement of podocyte foot processes [1]. Therapeutically, FSGS presents numerous treatment challenges. Corticosteroids, the primary treatment, are associated with numerous adverse events. A substantial proportion of patients exhibit steroid dependency or resistance, ultimately progressing to poor kidney outcomes [2]. However, the prognosis of FSGS patients is not only influenced by the histological lesion pattern, the degree of proteinuria, and the response to corticosteroids, but also by genetic factors. FSGS is broadly categorized into primary, secondary, and genetic forms. Accurate identification of the underlying causes of FSGS is critical for effective treatment but remains challenging [3]. As our understanding of genetic diseases in nephrology deepens and gene sequencing becomes more widespread, the role of genetic FSGS is becoming increasingly prominent. Despite the growing body of research, the role of genetic mutations in FSGS remains poorly understood.
Mutations in genes related to podocytes and the glomerular basement membrane (GBM) are the two major causes of genetic FSGS. Podocyte injury is a hallmark feature of FSGS. Mutations in podocyte-related genes, such as NPHS1, ACTN4, WT1, and TRPC6, are well-documented causes of structural and functional podocyte abnormalities that can lead to FSGS [4]. Type IV collagen is a component of the reticular matrix. Six α chains (α1–6), encoded by COL4A1–6 genes, form three trimeric structures: α1α1α2, α3α4α5, and α5α5α6. In mature kidneys, the α3α4α5 structure constitutes type IV collagen, a key component of the GBM [5]. In addition to the GBM, α3α4α consists of the basement membrane of distal kidney tubules and collecting ducts, the cochlea, and the eyes. Abnormalities in COL4A3–5 were initially believed to cause Alport syndrome. However, as genetics research deepens, the link between type IV collagen and the spectrum of kidney diseases has broadened. Recent studies have confirmed that mutations in COL4A3–5 can cause FSGS [6]. However, the relationship between gene mutation types and patient prognosis remains unclear.
Due to financial constraints and other limitations, gene sequencing is not routinely performed on all FSGS patients. Consequently, for adult FSGS patients, physicians typically prioritize those with steroid resistance (SR) and/or a family history of kidney disease (FH) for gene sequencing [7]. However, the diagnostic yield of gene sequencing in FSGS patients with SR and/or FH is unclear, and the significance of different genetic mutations in genetic FSGS patients is not well understood. This study aims to investigate the prevalence of genetic mutations in FSGS patients with SR and/or a FH. The clinicopathological characteristics and prognosis of these patients were further analyzed. Furthermore, additional analysis was conducted to explore the role of genetic mutation types in genetic FSGS, as confirmed by next-generation sequencing (NGS).
Methods
Patients
The study was divided into two parts based on the objectives. Part 1 aims to investigate the diagnostic yield of genetic FSGS in adult patients with SR and/or FH. Part 2 aims to explore the impact of different types of genetic mutations on the prognosis of genetic FSGS. Part 1 included FSGS patients with SR and/or a FH. Part 2 included patients with genetic FSGS proven by kidney biopsy and gene sequencing. A retrospective analysis was conducted on patients diagnosed with FSGS, confirmed by biopsy, at the National Clinical Research Center of Kidney Diseases, Jinling Hospital, between March 2017 and March 2022.
For part 1, eligible patients were required to meet the following inclusion criteria: 1) FSGS confirmed by biopsy; 2) having undergone gene sequencing due to SR and/or FH; 3) age at onset ≥16 years; 4) exclusion of secondary FSGS; 5) availability of complete clinical and laboratory data; and 6) no other kidney conditions.
For part 2, eligible patients were required to meet the following inclusion criteria: 1) FSGS confirmed by biopsy; 2) having undergone gene sequencing; 3) pathogenic mutations in the 54 genes listed Table 1; 4) age at onset ≥16 years; 5) exclusion of secondary FSGS; 6) availability of complete clinical and laboratory data; and 7) no other kidney conditions.
Data collection
Clinical data
Sex, age at onset, age at kidney biopsy, disease course at biopsy, family history, occurrence of acute kidney injury (AKI), hypertension, and drug use were collected.
Laboratory data
Urinary protein quantification, urinary red blood cell count, serum albumin, serum creatinine, and estimated glomerular filtration rate (eGFR, calculated by Chronic Kidney Disease Epidemiology Collaboration) were collected at baseline (onset) and the end of follow-up (100 months after onset). Data on peak proteinuria during the disease course, gene sequencing results, and time to event endpoint were also collected. Urinary red blood cell counts were graded based on microscopic examination as follows: grade 1, <30/µL; grade 2, 30–300/µL; and grade 3, >300/µL.
Gene sequencing and result interpretation
Gene sequencing was performed on peripheral blood obtained from each patient. Sequencing was conducted by Beijing Mygenostics Company using NGS technology.
For details, Genomic DNA was extracted from peripheral blood using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The DNA was quantified with Nanodrop 2000 (Thermo Fisher Scientific). Genomic DNA of 1–3 μg was fragmented to an average size of 150 bp using a S220 Focused-ultrasonicator (Covaris). A DNA Sample Prep Reagent Set (MyGenostics) was used for the preparation of standard libraries, including end repair, adapter ligation, and polymerase chain reaction (PCR) amplification, which would be further sequenced by DNBSEQ (DNBSEQ-T7).
The amplified DNA of targeted genes was captured using GenCap capture kit (MyGenostics). The biotinylated 100 bp capture probes were designed to tile along the coding exons plus 50 bp flanking regions of all the genes. The capture experiment was conducted according to the manufacturer’s protocol. The PCR product was purified using SPRI beads (Beckman Coulter) according to the manufacturer’s protocol. The enrichment libraries were sequenced on the DNBSEQ-T7 platform using paired-end 150 bp reads.
After sequencing, the raw data were saved in FASTQ format. Both sequencing adapters (MGI Tech Co., Ltd.) and low-quality reads were filtered using the Cutadapt software (http://code.google.com/p/cutadapt/). The clean reads were mapped to the UCSC hg19 human reference genome using the parameter BWA of Sentieon software (https://www.sentieon.com/). The duplicated reads were removed using the parameter driver of the Sentieon software, and the parameter driver is used to correct the base, so that the quality value of the base in the reads of the final output BAM (binary alignment/map) file can be closer to the real probability of mismatch with the reference genome and the mapped reads were used for the detection of variation. The variants of single nucleotide polymorphism and insertion-deletions were detected by the parameter driver of the Sentieon software. Then, the data would be transformed to VCF (Variant Call Format). Variants were further annotated by ANNOVAR software (http://annovar.openbioinformatics.org/en/latest/), and associated with multiple databases, such as 1000 Genomes, ESP6500, dbSNP, ExAC, Inhouse (MyGenostics), Human Gene Mutation Database, and also predicted by SIFT, PolyPhen-2, MutationTaster, and GERP++.
Fifty-four genes known to be associated with FSGS were selected according to the previous literature and reports [8–12] (Table 1). Mutations in any of these 54 genes that meet the following criteria were classified as disease-causing mutations [11,13,14]: 1) The mutation pattern corresponds to the pathogenic pattern. 2) According to variant interpretations published by the American College of Medical Genetics and Genomics, gene mutations were classified as “pathogenic” or “likely pathogenic.” 3) Mutations classified as “uncertain clinical significance” were predicted to be harmful by at least three of the five protein function prediction software tools, including SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.mutationtaster.org/), GERP++ (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html), and REVEL (Rare Exome Variant Ensemble Learner).
Definitions
The endpoint of this study was defined as a 40% decrease in eGFR from baseline. Simple massive proteinuria was defined as urinary protein >3.5 g/24 hr and serum albumin >30 g/L. Complete remission was defined as urinary protein <0.5 g/24 hr, serum albumin ≥35 g/L, and stable kidney function, with a serum creatinine increase of no more than 15% from baseline. Partial remission was defined as a reduction in urinary protein to 0.5–3.5 g/24 hr, less than 50% of baseline, with stable kidney function, regardless of serum albumin level. Remission included both complete and partial remission, while treatment failure was defined as the inability to achieve partial remission.
SR was defined as the failure of treatment with adequate doses of steroids (oral prednisone 1 mg/kg daily or 2 mg/kg every other day) for 16 weeks. Immunosuppressive resistance was defined as the failure of immunosuppressants to show efficacy after 4 to 6 months; the immunosuppressants included cyclosporine, tacrolimus, mycophenolate, cyclophosphamide, leflunomide, and tripterygium glycosides. The blood concentrations of cyclosporine, tacrolimus, and mycophenolate were maintained within the therapeutic range.
An FH was defined as having first- and/or second-degree relatives diagnosed with kidney disease. First-degree relatives share 50% of the proband’s DNA, while second-degree relatives share 25%. Kidney disease was defined according to the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines as abnormalities of kidney structure or function, present for more than 3 months. AKI was assessed according to the 2012 KDIGO guidelines and was defined as an increase or decrease in serum creatinine by 50% during hospitalization (expanded criteria).
Statistics analysis
Analysis was performed using IBM SPSS version 26.0 (IBM Corp.) and SAS version 9.4 (SAS Institute). The Shapiro-Wilk test was used to assess the normality of measurement data. Quantitative variables with a normal distribution are expressed as the mean ± standard deviation, while non-normally distributed variables are expressed as the median and interquartile range. Categorical variables are summarized as absolute frequencies and percentages. For continuous variables, the independent t tests were applied for normally distributed variables, and the Mann-Whitney U test was applied for non-normally distributed variables. For categorical variables, the Fisher exact test was used. Logistic regression and Fisher logistic regression were used to identify factors associated with genetic FSGS. Kaplan-Meier curves, the log-rank test, and the stratified log-rank test were used for survival analysis. All tests were two-tailed, and p < 0.05 was considered statistically significant.
Results
Baseline characteristics
We initially included 206 patients who underwent gene sequencing from the cohort at the FSGS outpatient department of the National Clinical Research Center of Kidney Diseases, Jinling Hospital, between March 2017 and March 2022. Sixty-four patients without a kidney biopsy and 95 diagnosed with other kidney diseases confirmed by biopsy were excluded. Of the remaining 47 patients, two underwent gene sequencing of a kidney tubule disease-related gene panel that did not include the 54 genes associated with FSGS and were therefore excluded. Seven patients with secondary causes of FSGS and two patients without complete clinical data were excluded. After the preliminary screening, 36 patients were eligible for inclusion. For part 1 of this study, four patients without SR and/or an FH were excluded, leaving 32 patients enrolled (Fig. 1). Seventeen of the 36 patients without genetic mutations were excluded, leaving 19 eligible patients for part 2 of the study (Fig. 1).
A total of 32 FSGS patients meeting the inclusion criteria were included in part 1, of whom 21 had an FH, and 15 exhibited SR (four patients had both SR and a FH). Eighteen patients (56.3%) with genetic mutations were assigned to the mutation group; 14 patients without gene mutations were assigned to the non-mutation group. The mutation group had a significantly higher rate of FH (83.3% vs. 42.9%, p = 0.03) and a lower incidence of AKI compared to the non-mutation group (5.6% vs. 50%, p = 0.01). The proportion of patients presenting with nephrotic syndrome in the mutation group was lower than that in the non-mutation group (16.7% vs. 50%, p = 0.06), as was the peak level of proteinuria (4.17 g/24 hr vs. 6.17 g/24 hr, p = 0.17), although these differences were not statistically significant (Table 2).
Nineteen patients who met the criteria for part 2 were included to explore the relationship between gene mutation types and the clinicopathological and prognostic features of genetic FSGS. Except for a female patient with a heterozygous COL4A3 mutation who had no FH and no SR, the remaining 18 cases were included in the mutation group of part 1. The 19 patients with genetic FSGS were categorized into the podocyte-related gene mutation group (PO group, n = 8) and the type IV collagen-related gene mutation group (IV-col group, n = 11). The median age at onset for the 19 genetic FSGS patients was 30 years. Of the 19 patients, 14 (73.7%) were female, and five were male. Compared to the PO group, the patients in the IV-col group were older at onset (38.0 years vs. 25.0 years, p = 0.03), had higher serum creatinine levels (0.90 mg/dL vs. 0.66 mg/dL, p = 0.01), and lower GFR (100 mL/min/1.73 m2 vs. 124 mL/min/1.73 m2, p = 0.003) at baseline. Patients with type IV collagen-related gene mutations had more urinary red blood cells (p = 0.04) and lower peak proteinuria levels (3.23 g/24 hr vs. 6.13 g/24 hr, p = 0.02). Among the 19 patients, one female patient with a COL4A5 heterozygous mutation exhibited high-frequency hearing loss, while the other patients showed no abnormalities in eye or ear examinations (Table 3).
Mutant genes
For part 1, 18 out of 32 patients were determined to have a pathogenetic mutation associated with FSGS. For part 2, there were three cases with an INF2 mutation, two cases with a TRPC6 mutation, and two cases with a WT1 mutation in the PO group. The IV-col group included five cases with a COL4A5 mutation (two males and three females), four cases with a COL4A4 mutation, and two cases with a COL4A3 mutation. In the IV-col group, all 11 cases had heterozygous mutations, except for the two male patients with a COL4A5 mutation (Fig. 2). Detailed information on mutant genes is listed in Supplementary Table 1 (available online).
Pathological characteristics
For part 1, the mutation group exhibited milder acute tubulointerstitial injury (p = 0.03) and a higher proportion of foam cells (33.3% vs. 0%, p = 0.02) compared to the non-mutation group (Table 2). In the mutation group, six patients had foam cells in the kidney interstitium, including one with clustered distribution (WT1 mutation), one with multifocal distribution (COL4A4 heterozygous mutation), two with focal distribution (both female, COL4A5 heterozygous mutation), and two with occasional foam cells in the interstitium (TRPC6 heterozygous mutation, COL4A4 heterozygous mutation). There were no significant differences between the two groups regarding the proportions of global glomerular sclerosis, segmental sclerosis, or the extent of chronic tubulointerstitial lesions (Table 4). The representative images of the histological staining of the kidney tissue are shown in Fig. 3.
Treatment and curative effect
In part 1, 28 of the 32 patients were treated with angiotensin-converting enzyme inhibitors (ACEIs) and/or angiotensin II receptor blockers (ARBs), 23 were treated with immunosuppressive agents, including cyclophosphamide, tacrolimus, cyclosporine A (CSA), mycophenolate mofetil, and tripterygium glycosides, and 18 received corticosteroids. Of the 18 patients who received steroids, one was treated with low-dose steroids, while 17 received adequate doses. Among the 17 patients with adequate steroid therapy, 15 exhibited SR, one patient with a WT1 mutation achieved partial remission (patient 14 in Supplementary Table 1, available online), and one patient in the non-mutation group achieved complete remission. Of the 15 SR patients, 14 were treated with immunosuppressants, and 12 showed resistance to immunosuppressants. In total, treatment was ineffective for 87.5% of the patients, and there was no statistically significant difference in overall efficacy between the two groups (Table 6).
In part 2, 17 of the 19 patients (89.5%) received ACEIs and/or ARBs, 14 patients (77.8%) received immunosuppressants, and eight patients received steroids. All eight patients who received steroids were started on adequate doses. One patient with a WT1 mutation achieved partial remission (patient 14), while seven patients exhibited SR. These seven SR patients received immunosuppressants, and all demonstrated resistance to immunosuppressive therapy. Among the 19 patients, one patient with a WT1 mutation achieved partial remission (patient 14), and one patient with a COL4A3 heterozygous mutation achieved complete remission (patient 19 in Supplementary Table 1, available online; Table 7). The patient 19 had no FH and presented with mild proteinuria, microscopic hematuria, and an initial episode of AKI. Following supportive care combined with renin-angiotensin system (RAS) blockade, complete kidney remission was achieved.
Regression analysis
For part 1 of the study, single-factor regression analysis revealed that an FH (odds ratio [OR], 6.667; 95% confidence interval [CI], 1.206–34.027; p = 0.02) and the presence of foam cells (OR, 8.470; 95% CI, 1.451–∞; p = 0.02) were positive predictive factors for a genetic diagnosis in these FSGS patients. Conversely, the occurrence of AKI during the disease was a negative predictive factor for genetic diagnosis (OR, 0.059; 95% CI, 0.006–0.571; p = 0.02) (Table 8).
Prognosis
Using a 40% decline in eGFR as the endpoint, Kaplan-Meier survival analysis revealed no significant difference in the cumulative kidney survival rate between the mutation and non-mutation groups after 100 months of follow-up (95% CI, 57.387–66.613; p = 0.32) (Fig. 5). Similarly, there was no significant difference in the kidney survival rate between the PO group and the IV-col group (95% CI, 50.816–75.184; p = 0.70) (Fig. 6). However, in Fig. 5, we observed that early in the follow-up, the survival curves of the mutation and non-mutation groups diverged, but over time, they gradually converged. The two curves were farthest apart at 24 months and then gradually overlapped. To further investigate, we performed a stratified log-rank test [15] to assess kidney prognosis in different event periods. The results showed that between 0 and 24 months, the non-mutation group had significantly better kidney prognosis than the mutation group (p = 0.03), whereas after 24 months, no significant difference in kidney prognosis was observed between the two groups (p = 0.57).
Discussion
This is the first study to investigate the rate of pathogenic gene mutations in FSGS patients with SR and/or FH in a real-world clinical setting. Concurrently, we performed a comparative analysis of clinicopathological and prognostic characteristics between patients with and without pathogenic variants. Furthermore, we evaluated the impact of distinct genetic mutation subtypes on clinical phenotypes and kidney outcomes in genetic FSGS.
Precision treatment is the goal in kidney disease management. And understanding the genetic underpinnings of FSGS is crucial. A confirmed genetic diagnosis allows clinicians to avoid corticosteroid and immunosuppressant regimens, reducing treatment-related complications. It further enables the timely initiation of protective agents such as RAS inhibitors or sodium-dependent glucose transporter 2 inhibitors, along with targeted therapies like coenzyme Q10 supplementation for patients with COQ mutations. Meanwhile, this genetic insight can provide essential guidance for reproductive planning and future kidney transplant strategies. Previous studies on genetic FSGS have mainly focused on pediatric populations [12,16,17]. However, pediatric and adult FSGS exhibit distinct etiological profiles, clinicopathological characteristics, and kidney outcomes [18]. Consequently, there remains a significant evidence gap regarding genetic FSGS in adults, with current studies providing limited guidance for clinical management. It has been reported that the diagnostic rate of genetic testing for children with steroid-resistant nephrotic syndrome (SRNS) is 23.6% to 30% [11,19–21]. The incidence of genetic FSGS in adolescents and adults has been reported to range between 10% and 43% [7,10,22,23]. The variations depend on the study population and genetic testing methods used. These data suggest the prevalence of genetic FSGS in adults may be substantially underestimated. The actual proportion of genetic FSGS in adult populations could approach or even exceed that observed in pediatric cohorts. The gene sequencing is not a routine test for adult FSGS patients. Thus, selecting high-risk patients for gene sequencing can increase the rate of genetic diagnosis. In clinical practice, adult FSGS patients presenting with SR and/or an FH are strongly suspected to have genetic FSGS, constituting the most frequently referred cohort for genetic sequencing in adult FSGS patients. However, in the real world, the actual diagnostic rate of genetic sequencing and clinicopathological and prognostic features among the patients remains unclear.
In this study, the patients included in part 1were all adults or adolescents (onset age ≥16 years) with SR and/or an FH. Our data show that the incidence of pathogenic gene mutations in this cohort was 56.3%, which is higher than previously reported (10%–43%). This suggests that selectively conducting genetic testing for FSGS patients with a SR and/or FH enhances genetic diagnosis. However, 40% of the patients in the non-mutation group had an FH, and most of whom had ineffective treatments. On the one hand, this phenomenon might be attributed to coincidental occurrence or environmental exposures shared among the patient and their immediate relatives that potentially contributed to FSGS pathogenesis. On the other hand, it may also indicate that other unknown genes may contribute to FSGS, either through monogenic or potentially polygenic mechanisms. Previous case reports have shown mutations in genes associated with tubulointerstitial and systemic diseases, such as UMOD, GLA, and FN1, that can lead to FSGS [24–26]. Future genetic testing protocols for FSGS should incorporate either expanded gene panels or, where resources permit, whole-exome sequencing (WES) to comprehensively assess emerging pathogenic variants. And future studies are needed to explore the unknown pathogenic genes and understand their mechanisms, which would support more precise diagnoses and personalized treatments.
Previous studies have highlighted a higher prevalence of FSGS among young males, with a male-to-female ratio of approximately 1.5:1. In contrast, our study found a higher proportion of females in this cohort, with a male-to-female ratio of approximately 1:2. This discrepancy may reflect the specific subpopulation characteristics that notably enrichment of X-linked variants manifesting later in females in our study or potentially stem from sampling bias due to limited cohort size. Further investigation in larger sample sizes to explore potential sex disparities in this specific FSGS population.
Regression analysis of part 1 in our study indicated that an FH is a significant predictor for genetic FSGS, consistent with earlier research [7]. Additionally, we find that the presence of foam cells can be an indicator of genetic FSGS. The presence of foam cells in the kidney tubulointerstitium is known to indicate macrophage activity and is linked to kidney inflammation and disease progression [27,28]. Foam cells are commonly seen in Alport syndrome and FSGS. In our study of part 1, foam cells were present in 18.8% of the patients, all of whom were in the mutation group. In part 2, although foam cells appeared more frequently in the IV-col group than in the PO group, this difference did not reach statistical significance. Regression analysis suggested that the presence of foam cells in FSGS patients may be a useful indicator for genetic FSGS and can prompt further genetic testing for diagnosis clarification. Further large-scale clinical studies and in-depth basic research are required to elucidate the role of foam cells in FSGS, as well as their indicative value for the diagnosis of FSGS subtyping and prognosis assessment.
AKI is a common complication in FSGS, with an incidence of 48.6% in primary FSGS [29]. It is often associated with acute tubulointerstitial injury. Our study found that the incidence of AKI in the non-mutation group was 50%, which is consistent with previous reports. However, in the mutation group, the incidence of AKI was significantly lower than in the non-mutation group. Additionally, compared to the non-mutation group, the degree of acute tubulointerstitial injury was milder in the mutation group. This may be due to differences in the underlying pathophysiology between genetic FSGS and primary FSGS. We hypothesize that the pathogenesis of primary FSGS, potentially driven by circulating soluble factors, may cause more extensive cellular damage compared to genetic FSGS. This could explain the more severe tubulointerstitial lesions, higher risk of AKI, and worse short-term prognosis observed in the non-mutation group.
This study found that genetic FSGS patients had better short-term kidney outcomes than those without genetic mutations, a result that contrasts with previous studies that have reported poor outcomes in genetic FSGS. The better short-term prognosis in our genetic FSGS cohort could be attributed to the fact that the non-mutation group exhibited more severe tubulointerstitial lesions and was more prone to develop AKI, which may have contributed to their worse short-term prognosis. However, when the observation period was extended, no statistically significant difference emerged in the long-term kidney outcomes. We hypothesize that the slower decline in kidney function observed in the later stages among patients in the mutation group may be related to recovery from AKI and better control of proteinuria. This suggests that severe kidney histology, AKI, and persistent proteinuria remain important risk factors for poor kidney outcomes, regardless of the presence of gene mutations. Further large-scale studies are needed to explore the relationship between gene mutations and clinical phenotypes, as well as the long-term prognosis of FSGS.
In genetic FSGS caused by podocyte-related gene mutations, the genetic pattern in childhood is typically autosomal recessive (AR), with common pathogenic genes being NPHS1, NPHS2, and PLCE1 [30]. In older children, adolescents, and adults, autosomal dominant (AD) inheritance is more common, with the main pathogenic genes being INF2, ACTN4, TRPC6, WT1, and LMX1B [23]. In our study, all podocyte-related gene mutations were AD heterozygous mutations, with INF2 being the most common mutant gene, consistent with previous reports. Although it is generally thought that immunosuppressive therapy is ineffective in genetic FSGS, some studies have reported that patients with genetic mutations can achieve remission with steroid and/or immunosuppressant therapy. Gellermann et al. [31] report that three children with WT1 mutations respond to CSA therapy given in combination with steroids and RAS inhibitors. Malina et al. [32] reported a partial remission of proteinuria in a child with a NPHS2 mutation following treatment with CSA. Callís et al. [33,34] and Charbit et al. [35] reported that the CSA can reduce proteinuria in pediatric patients with Alport syndrome. Hinkes et al. [36] described two children with truncating mutations of the PLCE1 who responded to treatment with steroids or CSA. Nagasaka et al. [37] reported a case of an adult FSGS patient with a TRPC6 mutation who responded to tacrolimus treatment, achieving partial remission. In our study, one patient with a WT1 mutation achieved partial remission under the therapy of steroids and RAS inhibition (RASi). Oo et al. [38] reported that genetic FSGS can present with clinical manifestations and pathological features highly resembling those of primary FSGS, while also occurring in sporadic cases. However, compared to primary FSGS patients, these genetic FSGS patients can experience spontaneous remission when supported only by RASi therapy [38]. Therefore, in patients with genetic FSGS who experience remission following immunotherapy, it is important to consider both the possibility of spontaneous remission and the potential protective effect of steroids or immunosuppressants, such as calcineurin inhibitors, in alleviating podocyte damage. At the same time, it cannot be ruled out that, in addition to the genetic mutation, other pathogenic factors of primary glomerulopathy may contribute to the disease's onset and progression. Although no kidney lesions other than FSGS were identified in any enrolled patients based on kidney biopsy findings, we cannot rule out the possibility that pathogenic factors associated with primary FSGS may have contributed to kidney injury. Future research should aim to more precisely define the genetic diagnosis of FSGS, while also investigating the immunological mechanisms underlying primary glomerulopathy, which will aid in the accurate diagnosis and treatment of FSGS.
This study also highlighted the importance of mutations in type IV collagen-related genes in genetic FSGS. Type IV collagen is an essential component of GBM, as well as the basement membranes of the eyes and ears. Mutations in the genes encoding type IV collagen (COL4A3–5) result in the inability of the corresponding protein products to form normal trimers, leading to a loose reticular structure in the basement membrane, which in turn affects the structure and function of the associated organs. Traditionally, mutations in the genes related to type IV collagen were thought to cause Alport syndrome. However, with a deeper understanding of genetic diseases, the notion that one pathogenic gene corresponds to a specific clinical phenotype has been challenged. Mutations in the type IV collagen-related genes have been associated with multiple kidney disease phenotypes, such as FSGS, immunoglobulin A nephropathy, Pierson syndrome, and polycystic kidney disease. Based on this, diseases caused by mutations in the COL4A3–5 genes have expanded from a singular Alport syndrome to a broader Alport disease spectrum. In the kidney, type IV collagen is produced by podocytes, and its abnormalities can affect adjacent podocytes, leading to damage to the glomerular filtration barrier. The genetic pattern of Alport syndrome is primarily X-linked dominant, AR, and, in rare cases, AD, with the GBM showing typical tearing-like changes, often accompanied by extra-kidney damage, such as eye and ear involvement. In contrast, FSGS caused by COL4A3–5 mutations is predominantly inherited in an AD heterozygous or X-linked dominant pattern, with no significant layering changes in the GBM and typically without extra-renal manifestations. In other words, kidney damage in Alport syndrome is primarily due to primary GBM damage caused by type IV collagen-related gene mutations, while FSGS due to COL4A3–5 mutations is characterized by podocyte damage and sclerotic lesions secondary to GBM abnormalities. In our study, all patients with COL4A3–5 gene mutations were heterozygous, except for the two male patients with a COL4A5 mutation. And all of the patients did not exhibit severe GBM damage or extra-renal involvement. It is recommended to RAS blockade early in patients with kidney damage caused by COL4A3–5 mutations to slow down the progression of kidney function decline. Recent studies have shown that tauroursodeoxycholic acid can improve podocyte dysfunction caused by COL4A3 mutations through alleviating endoplasmic reticulum stress, offering new hope for the precise treatment of FSGS [39].
Mutations in type IV collagen-related genes accounted for 59.7% of the genetic mutations in our cohort, with COL4A5 being the most common (45.5%). Gast et al. [40] included 81 FSGS patients from 76 families and screened 39 genes associated with FSGS or SRNS, including COL4A3–5, using NGS. They found that type IV collagen-related gene mutations accounted for 56% of all identified pathogenic genes, with COL4A5 mutations making up approximately half of these. Yao et al. [13] included 193 FSGS patients from Canada and screened 109 genes related to basement membrane and podocyte abnormalities, congenital urinary tract anomalies (CAKUT), and nephronophthisis. They found that COL4A3–5 mutations accounted for 55% of all identified pathogenic mutations, while mutations in podocyte-related genes accounted for 40%, and 5% were CAKUT-related. The latest study from Mayo Clinic showed that in adult hereditary FSGS, podocyte-related gene mutations accounted for 38.1%, while IV collagen mutations accounted for 52.4% [7]. Therefore, it is important to prioritize the sequencing of type IV collagen-related genes when diagnosing FSGS patients. Additionally, when gene sequencing is not available, clinicians should consider the possibility of other system involvement, such as ocular or auditory abnormalities, in patients with FSGS, especially when secondary causes have been excluded.
Our data also suggest that patients with type IV collagen-related gene mutations have a more insidious onset and more severe disease. It has been reported that patients with FSGS caused by type IV collagen-related gene mutations had a median age of onset of 36 years, compared to 26 years in patients with mutations in other pathogenic genes, although the difference was not statistically significant. The other pathogenic genes identified in that study included those associated with CAKUT and nephronophthisis [13]. Similarly, Gast et al. [40] found that the age of onset in patients with type IV collagen-related mutations was later than in those with podocyte-related gene mutations (36 years vs. 26 years, p = 0.15), but the difference also did not reach statistical significance. The median age of onset in our study was 38 years for the type IV collagen-related gene mutation group, significantly older than the 20.5 years in the podocyte-related gene mutation group (p = 0.03). Patients with type IV collagen-related gene mutations also had less severe proteinuria at baseline but presented with more advanced kidney damage. This may be due to the more gradual progression of kidney dysfunction in these patients, which makes early detection and administration more difficult. Although previous studies have suggested that the progression to end-stage kidney disease is slower in patients with type IV collagen mutations, our study did not observe significant differences in kidney outcomes between the two groups. Further long-term follow-up is necessary to explore the long-term prognosis of patients with different gene mutations.
This study has certain limitations. It is a single-center, small-sample retrospective study, which may introduce some bias in the results. The patients included in this study underwent gene panel sequencing or WES, which may have missed copy number variations in some genes. Conservative variants of uncertain significance exclusion may overlook variants with cryptic pathogenicity. Future studies with functional assays are warranted. Furthermore, the follow-up period in this study was relatively short, and thus, long-term kidney outcomes for these patients could not be observed.
Our study focuses on FSGS patients who exhibit SR and/or have an FH, as these individuals are more likely to have genetic mutations. Investigating the clinical and pathological characteristics of these patients is crucial for understanding their condition. We found that the genetic diagnosis rate in this cohort was 56.3%. Patients without genetic mutations were more prone to developing AKI, experienced more severe tubulointerstitial damage, and had worse short-term kidney outcomes compared to those with genetic mutations. Our findings highlight that foam cells, in addition to a FH, serve as important predictive factors for genetic FSGS.
Furthermore, we identified that type IV collagen-related gene mutations play a significant role in genetic FSGS, emphasizing the importance of routinely screening these genes during genetic testing. In comparison to podocyte-related gene mutations, patients with type IV collagen-related mutations have a more insidious onset, warranting increased attention in clinical practice.
All in all, our study suggests that genetic screening in FSGS patients with SR and/or an FH can provide valuable insights into their genetic diagnosis and clinical management.
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