The high rate of steroid resistance in Black South African children compared to other racial groups prompted the need to further investigate the genetic basis for this racial disparity. Asharam et al. identified the NPHS2 p.V260E mutation as a frequent cause of AR SR-FSGS in Black children with NS, potentially sparing these children the use of steroids and other immunosuppressive drugs as well as the need to undergo kidney biopsy[18, 32]. As the NPHS2 p.V260E is not infrequent in the general South African population (MAF 0.2-1%) and explains approximately 30% of SR-FSGS in Black South African children; therefore, we first genotyped all cases and controls for the p.V260E mutation.
In this study, we detected NPHS2 p.V260E homozygosity in 21.43% of Black children with a general diagnosis of SRNS and 23.81% with a specific diagnosis of SR-FSGS. The detection rate of 23.81% for the homozygous p.V260E mutation-specific to SR-FSGS in our study was lower than the two previous reports from South Africa that reports rates of 33% (Asharam et al., 2018) and 50%. The NPHS2 gene, encoding podocin, is an integral structural protein of the podocyte. Although the NPHS2 p.V260E variant likely arose in Africans, it was first identified in several consanguineous families from the previous Omani empire[18, 41, 42]. Like the European founder variant p.R138Q, the p.V260E variant disrupts the transport of the modified podocin protein from the endoplasmic reticulum to the plasma membrane [43–45].
A variable and earlier age of onset (range 24 to 178 months) in children was observed in children homozygous for the NPHS2 p.V260E mutation. In an earlier report, Black SR-FSGS cases homozygous for p.V260E had an earlier age of onset compared to SRNS cases homozygous for the p.V260 reference allele (median onset of age, 34 months vs. 78 months, p= 0.01). The findings of our study in children carrying the p.V260E mutation highlight the variability of the phenotype seen in SR-FSGS. That said, it is important to consider NPHS2 p.V260E mutation as a cause for SRNS in all paediatric age groups. In contrast to the findings in SRNS, we did not detect any homozygous or compound heterozygous mutations for NPHS2 p.V260E among patients with SSNS, consistent with previously published reports showing that this mutation is specifically associated with SR-FSGS[18, 32, 46–48].
NPHS2-mediated disease is resistant to corticosteroids and other immunosuppressants[21, 42, 46, 49]. Additionally, several studies have highlighted a high risk of progression to ESKD in monogenic SRNS, with many patients requiring kidney transplantation and a low risk of disease reoccurrence post-transplant when a genetic aetiology has been confirmed[19, 42, 50–53]. In keeping with published reports, none of the children in our study homozygous for the NPHS2 p.V260E variant responded to any second-line treatment. We also found that individuals who did not have the NPHS2 p.V260E variant progressed more slowly to ESKD. Moreover, three of the five children that progressed to ESKD underwent a kidney transplant and no disease recurrence occurred. Screening for the NPHS2 p.V260E mutation has the potential to provide a precision diagnosis of SR-FSGS thereby informing the differential diagnosis, prognosis, and treatment in 20-30% of Black African children presenting with NS.
In those children lacking the NPHS2 p.V260E mutations, we observed 12 children with sporadic SR-FSGS carrying putative causal mutations in the following AD genes [INF2 (n=8), CD2AP (n=3) and TRPC6 (n=1)]. Autosomal dominant SRNS typically presents later in life, in adolescence or adulthood, and has significant phenotypic variability. In our study, a later age of onset in children carrying an AD mutation (range 62-220 months) was observed when compared to AR mutation (range 24-178 months). Two children carrying an AD mutation in CD2AP and TRPC6 were in partial remission following treatment with calcineurin inhibitors in contrast to all 15 children with an NPHS2 mutation who did not respond to any additional immunosuppressants.
Recently, mutations in INF2 were reported as the most common cause of adolescent-onset autosomal dominant SR-FSGS[54, 55]. Only three INF2 mutations have been previously reported in a total of 436 sporadic SR-FSGS cases (<1%), all of which occur in the N-terminus[54–57]. In this study, a putative causal variant INF2 p.P516L in the INF2 gene was present in the heterozygote state in eight Black SRNS cases with biopsy-proven FSGS with an age of onset ranging from 6 to 18 years. The clinical characteristics of the children carrying the p.P516L variant in this study are in corroboration with previous reports which show INF2 mutations as a common cause of FSGS with incomplete penetrance and variable age of onset ranging from 10-70 years[27, 54, 57]. The finding that 16% of Black children with SRNS undergoing WES carried the mutation compared to 3% of the South African population indicates that this predicted pathogenic variant merits further investigation to determine penetrance and causality.
Despite the clear association of CD2AP defects with a glomerular pathology suggestive of idiopathic FSGS in animal models, data is sparse in humans[58, 59]. In our study, we identified three Black children with SR-FSGS harbouring a heterozygous putative causal mutation (p.K633R) in the CD2AP gene. The first report by Kim et al. described one heterozygous nucleotide variant resulting in an aberrant CD2AP splicing in two patients with idiopathic FSGS. More recently, Asharam et al. reported on a heterozygote CD2AP p.K346N variant in an Indian child with SR-FSGS. Notably, the only child with CD2AP-associated nephropathy was in partial remission at last hospital visit after treatment with cyclosporin A. This treatment reduced the proteinuria by ∼50%, whilst the remaining two patients failed to respond. A case report by Tsvetkov et al. showed that ciclosporin A is a treatment option for CD2AP-associated nephropathy. However, further studies and clinical trials are required on the use of cyclosporin A as a therapeutic option for patients with CD2AP-associated nephropathy.
The TRPC6 gene encodes the transient receptor potential cation channel, which is located on the podocyte membrane where together with podocin, it regulates mechano-sensation at the slit diaphragm. autosomal dominant SR-FSGS, with variable age of onset from early childhood through adulthood[62–65]. In our study, we report a novel putative causal mutation, TRPC6 p.G162V in one SRNS Indian child with biopsy-proven FSGS with an age of onset of 18 years. The clinical phenotype of this patient improved after treatment with calcineurin inhibitors (tacrolimus). At last hospital visit, the patient was in partial remission. Our findings together with previous reports suggest that calcineurin inhibitors could open up new avenues on the treatment of TRPC6-associated FSGS[65, 66].
This study has several limitations. Family members were not available for segregation studies to determine the penetrance of the heterozygous variants. Due to the small number of controls typed for INF2 P.516L, and the limited number typed for the population frequency estimate of this variant the statistical significance of its association with disease is not clearly established. Causality of INF2 p.P516L, CD2AP p.K346N, and TRC6 p.G162V has not been demonstrated in animal models or in vitro cell studies.
This is the largest study in a paediatric South African population to perform sequence analysis of ten candidate genes (NPHS1, NPHS2, WT1, LAMB2, ACTN4, TRPC6, INF2, CD2AP, PLCE1, MYO1E) associated with SR-FSGS. We have confirmed that the NPHS2 p.V260E mutation is a prevalent cause of SR-FSGS among Black South African children occurring in 23.81% of children with SRNS. Given the large number of children with NPHS2 mutations, sufficiently powered clinical trials may be possible on testing different treatment therapies on these children and comparing outcomes. We also report putative causal missense variants in INF2, CD2AP and TRPC6. Screening all Black African children with NS for NPHS2 p.V260E and possibly INF2 p.P516L, if causality is shown, has the potential to be highly predictive of a diagnosis of SR-FSGS and multi-resistant forms of NS. Detection of pathogenic mutations will obviate the need for a kidney biopsy and the use of additional immunosuppressant drugs thus avoiding complications of an invasive procedure and the serious adverse effects of treatment. Precision genetic diagnosis will enable clinicians to provide genetic counselling, detect carriers, and propose prenatal diagnosis to couples at risk.