Mara Sanches Guaragna1*, Anna Cristina GB Lutaif2, Vera MS Belangero2 Andréa T. Maciel-Guerra3,4, Gil Guerra-Junior4,5 and Maricilda P. De Mello1
Nephrotic syndrome (NS) represents one of the most common kidney conditions that affect children. It manifests as proteinuria, edema, hypoalbuminemia and hyperlipidemia. According to the response to standardized corticosteroid therapy, 80 to 90% of children that respond well to the treatment are classified as steroid sensitive (SSNS), whereas the remaining 10 to 20% that are unresponsive to the treatment are classified as steroid resistant (SRNS)1. NS is a highly heterogeneous condition and many SRNS cases remain idiopathic, although a fraction of them have a clear genetic etiology. The most frequent renal histologic feature of SRNS is focal segmental glomerulosclerosis (FSGS). Almost 40% of SRNS/FSGS children develop end-stage renal disease (ESRD) before adulthood and may receive kidney transplantation with 10 to 50% risk of FSGS recurrence in the allograft kidney2–4. Conversely, the post-transplant risk of proteinuria recurrence in patients with inherited forms of SRNS is only 1-2%5.
expressed in the visceral epithelial podocytes, specialized cells of the outer surface of the glomerular filtration barrier6. Two recent studies in large SRNS cohorts report a monogenic cause of the disease in approximately 30% of cases in whom mutations were found in one of those 50 genes7,8. Despite the increasing number of genes associated with SRNS, there are three that are frequently mutated in patients with early onset of the disease: NPHS1, NPHS2 and WT17–9. NPHS1 (OMIM *602716) mutations are typically associated with congenital nephrotic syndrome, with proteinuria onset before 3 months of age, although there are reports on mutations identified in childhood onset cases of SRNS10–12. NPHS1 encodes nephrin, an essential protein of the glomerular slit diaphragm formed between adjacent podocytes13. NPHS2 (OMIM *604766) mutations are responsible for 6-17% and 40% of sporadic and familial forms of SRNS in childhood, respectively, and encodes podocin, a close interactor of nephrin14–16. The third main gene associated with early onset SRNS is WT1 (OMIM *607102), which encodes a transcription factor that plays a key role during kidney and genital development. Dominant de novo mutations in exons 8-9 have been associated to 2 to 7% of sporadic cases of SRNS in childhood17.
Our publication “NPHS2 mutations account for only 15 % of nephrotic syndrome cases” was the first cohort study focusing on genetics of SRNS in Brazilian children. In this study we included 27 SRNS patients that enrolled from 2008 to 2013 in the reference public hospital of clinics from the South East region of Brazil18. The patients comprised seven unrelated familial cases and 20 sporadic cases with onset of proteinuria between 3 months and 18 years of age. We performed sequence analysis of the entire NPHS2 and NPHS1 genes and of exons 8 and 9 of the WT1 gene in all the patients. A genetic cause of the NS was identified in only four cases (4/27, 14.8%), two sporadic (2/20, 10%) and two familial (2/7, 28.5%) (Table 1). All variants considered as the cause of the NS in these patients were identified in NPHS2 gene. Therefore, mutations in NPHS1 and WT1 genes have not been identified in those patients. The sporadic patients were compound heterozygous for [p.Arg229Gln];[p.Ala284Val] and [p.Arg229Gln];[p.Glu310Lys] NPHS2 mutations, as indicated by the segregation of the variants in the family. Those results confirmed the autossomal recessive inheritance in both cases. The association of those mutations have been already described as the cause of SRNS by other groups19–22. In the case of the two familial cases, the heterozygosis for NPHS2 mutations were, respectively, [p.Arg229Gln (;) p.Ala284Val] and [p.Lys239Argfs*13 (;) p.Val260Glu]. We also identified the same combinations in the affected siblings, but we could not evaluate the segregation in these two families although we considered the compound heterozygosis as the most probable genotype. We are not aware if other members of the family have renal disease. The age at onset of NS in the two patients with the p.Arg229Gln variant along with the p.Ala284Val mutation was 12 and 13 years old, confirming a later onset of the disease as the result of this association21. One of them progressed to CKD and received kidney transplant with no FSGS recurrence. On the other hand, the other two familial cases presented NS symptoms with 1.2 and 2.2 years old and with no progress to ESRD yet.
Table 1:Clinical data from SRNS patients with NPHS2 variants
NPHS2Two heterozygous variants
|P6||12||spo||FSGS||Yes||[p.Ala284Val] ; [p.Arg229Gln]|
|P67||2.2||spo||DMP||No||[p.Glu310Lys] ; [pArg229Gln]|
|P103||1.2||fam||FSGS||No||[p.Lys239Argfs*13 (;) p.Val260Glu]|
|P154||13||fam||FSGS||No||[p.Arg229Gln (;) p.Ala284Val]|
|One heterozygous variant|
aSpo: sporadic; Fam: familial;bFSGS:focal segmental glomerular sclerosis; DMP: diffuse mesangial proliferation; CO: complex = minimal change disease or diffuse mesangial proliferation or focal segmental glomerular sclerosis.
Our findings also included SRNS patients with only one heterozygous alteration in the NPHS2 gene: three sporadic cases presented promoter variants c.-164C>T, c.-196C>G and c.-537_-531delCTTTTTT and a familial case presented the p.Arg229Gln variant. Two of these patients progressed to ESRD and both received kidney transplant, although one of them presented FSGS recurrence post-transplant. Regarding promoter variants, Di Duca et al.23 studied regulatory elements in the NPHS2 promoter and described the c.-537_-531delCTTTTTT variant (rs146791300, MAF = 0.069; http://www.ncbi.nlm.nih.gov/) as a “functional polymorphism” that downregulated the gene expression of podocin by 85% when transfected in podocytes. Since there are no records of association of c.-164C>T and c.-196C>G variants with SRNS patients and our in-silico predictions indicated a possible creation of a new WT1 transcription binding site after c.-164C>T transition, we are investigating their role on podocin expression using reporter vector transfection assays in podocytes. For the familial case presenting the p.Arg229Gln, he and his father presented this variant and both had already received kidney transplant with no FSGS recurrence, clearly indicating a genetic background of their NS. Therefore, we performed whole exome sequencing (WES) for this family and the results will be published further.
Our paper’s main limitation was the sample small size. Approximately 200 patients with NS are routinely followed up in our service, one of the reference centers in Brazil; our 27 SRNS patients represent a frequency of 13.5% (27/200), which is in line with the reported worldwide frequency of 10-20% for this form of the condition. However, the sample is considered small for mutation frequency and epidemiological estimations16,24. An example of this limitation is the NPHS2 mutation rate – 28.5% - detected for our four out of seven familial cases. We cannot assure that the frequency described by us for the Brazilian population is lower then those described by others in different populations (~ 40%), since our cohort is not robust enough for making such comparisons. The Brazilian population is highly miscegenated and presents diverse regional features therefore our cohort from South East did not represent the entire country. The novelty of the work was the genetic study in Brazilian SRNS patients and the identification of a new frameshift mutation associated with SRNS.
Since we were able to identify the genetic origin of the NS only in 14.8% of our SRNS cases, our conclusion was that target-oriented next generation sequencing of glomerulopathy related genes is recommended to search for mutations in other genes related to SRNS in the remaining 74.2% patients. It is worth to mention that such studies are being carried out. Finally, the unequivocal molecular genetic diagnose and the correct establishment of genotype-phenotype correlation are essential because they can influence physicians’ decision on individual treatment, as patients carrying mutations can be spared the side effects of immunosuppressive therapy and ultimately can be considered for kidney transplantation from a living donor.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - 478444/08-7 to G.G-J and 141072/2010 to MSG) and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - 2012/51109-0 to MPdeM).