| Abstract|| |
Genetic screening paradigms for the nephrotic syndrome (NS) in the developed world are well established; however, screening in developing countries has received only minor attention. We retrospectively analyzed a cohort of all children who underwent genetic testing for challenging NS from our registry in the 10-year interval from 2000 to 2010 and based on 58 patients aged 0–12 years with at least one of the following clinical diagnosis: Nonsyndromic steroid-resistant nephrotic syndrome (SRNS), familial NS, and congenital NS. Of these, 23 patients (~40%) had a history of familial disease occurrence. All cases were screened for NPHS2 and WT1 mutations by direct sequencing of all exons of the genes. In addition, all patients who were diagnosed during the first three months of life were screened for NPHS1 mutations too. A genetic disease cause was identified in 12 patients (20.7%); of these, five novel mutations, all in NPHS2 accounting for 42% of all mutations and 9% of the cohort. Nine patients were found to have NPHS2 mutations. Only one case with SRNS had a mutation in WT1. Of the five congenital NS, two cases were found to have NPHS1 mutations and one case with NPHS2 mutation. Therefore, mutations in NPHS2 were the most commonly identified and explained in 15.5% of the screened patients and WT1 mutation in 1.7% of cases, whereas NPHS1 mutations were found in 40% of congenital NS cases. A genetic disease cause was identified in 20.7% of the screened patients. Among 12 identified mutations, abnormalities in NPHS2 (n = 9) were most commonly identified.
|How to cite this article:|
Saeed B. Genetic screening in children with challenging nephrotic syndrome. Saudi J Kidney Dis Transpl 2020;31:1189-97
| Introduction|| |
The diagnosis and management of steroid-resistant nephrotic syndrome (SRNS), congenital NS, and familial NS remain a challenge to pediatric nephrologists. SRNS is a rare kidney disease involving either immune-mediated or genetic alterations of podocyte structure and function with highly variable outcomes, and 50% of children with SRNS progress to end-stage renal disease within 15 years., Historically, diagnostic evaluation and prognostic classification relied largely on histo-pathologic assessment.
In recent years, abnormalities in a growing number of genes essential for podocyte development, structure, and function have been identified in patients with SRNS and congenital NS. NPHS2 and NPHS1 mutations are the most common genetic causes in children with SRNS and congenital NS, respectively. The ongoing discovery of genetic podocyto-pathies is about to redefine the physiopatho-logic understanding, diagnostic assessment, prognostic judgment, and therapeutic approaches in childhood-onset SRNS. However, the development of evidence-based management algorithms has been hampered by the low incidence of SRNS, which is estimated at 2–4 per million person-years. To overcome these limitations imposed by the rarity of these diseases, the PodoNet Consortium has created an international registry for congenital NS and childhood-onset SRNS. To date, 103 pediatric nephrology centers in 31 countries including our center in Damascus have registered with PodoNet. Currently, 2419 patients from 81 centers are followed in the PodoNet registry.
Genetic screening paradigms for NS in the developed world are well established; however, screening in developing countries has received only minor attention. To help rectify this, we retrospectively analyzed a cohort of all children who underwent genetic testing for challenging NS of likely genetic origin from our registry.
| Patients and Methods|| |
We retrospectively collected and analyzed clinical, biochemical, genetic, and histopathologic information from 58 patients aged 0 to 12 years at disease onset with challenging NS of likely genetic origin which includes all patients fulfilling one or more of the following: Non-syndromic childhood-onset SRNS, familial form of NS which is referred to nephrotic children with a history of at least one sibling with NS, and congenital NS from the pediatric nephrology registry at Kidney Hospital in Damascus from 2000 to 2010.
Prior to enrolment in our clinical registry, all families have been informed in detail about the goals and procedures of our study before asking them for informed consent which was subsequently obtained from the parents of all patients who were enrolled.
All cases were screened for NPHS2 and WT1 mutations by direct sequencing of all exons of the genes. In addition, all patients with congenital NS were screened for NPHS1 mutations too. We did not screen for NPHS1 mutations in patients who first manifested their NS after the age of three months because NPHS1 mutations occur in congenital NS only.
As our center was one of the PodoNet registered centers since May 2010, all genetic testing has been carried out at the laboratory of the Faculty of Medicine at Hacettepe University in Ankara which is one of the participating laboratories in the PodoNet Registry Consortium. The PodoNet project (www.podonet.org) encompasses clinical, genetic, and experimental research into hereditary podocyte disorders. The clinical activities encompass a web-based international clinical registry and a central biobank for SRNS.
Our participation in this international registry involves the collection of pseudonymized clinical data (by locally defined number code, allowing only the local investigator to assign data to an individual patient) every six months, of blood and urine samples every 12 months, and of blood for DNA analysis at one point in time.
Only children with primary NS who had genetic testing results available for NPHS2, NPHS1, and WT1 were included in the study. We excluded children with a secondary cause for NS (such as lupus nephritis or infections). The diagnosis of SRNS was made if the child did not respond to the standard steroid therapy with oral prednisone 60 mg/m2/day for four weeks. Secondary steroid resistance was defined as no response after four weeks of prednisone 60 mg/m2/d in a child previously known to have a steroid-sensitive course. All children received intravenous pulse methyl prednisolone 600 mg/m2 daily for three consecutive days after the failure of the four-week treatment with oral prednisolone. Ultrasound-guided kidney biopsy was performed in all patients. The biopsy specimens were examined by light and immunofluorescence microscopy. Electron microscopy facility was not available to our patients. An adequate biopsy was defined as the presence of at least five glomeruli in the specimen on light microscopy.
Data analysis was performed using the Statistical Package for the Social Sciences (SPSS) for Windows, version 19.0 (SPSS Inc., Chicago, IL, USA). When appropriate, data were presented using the mean standard deviation, and count numbers and percentages. Students’ t-test and one-way ANOVA were used to assess the difference in age among the different types of NS. When appropriate, Chi-square or Fisher’s exact test were used to assess the association between the categorical variables, for example, type of NS, type of mutation, and the pattern of incidence (sporadic vs. familial). A P <0.05 was considered statistically significant.
| Results|| |
Fifty-eight children with non-syndromic challenging NS from 51 families were recruited. Patients were distributed into several clinical types [Table 1] based on steroid response pattern, the pattern of incidence: familial or sporadic, and age at onset of the disease: congenital NS (during the first three months of life) or thereafter.
|Table 1: Distribution of patients according to steroid response, pattern of incidence, and age at onset.|
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Twenty-three patients out of 58 (39.7%) had a history of familial disease occurrence. The distribution of these familial cases according to steroid responsiveness and age at onset is as follows: 12 patients (20.7% of the cohort) with familial SRNS, eight patients (13.8%) with familial nephrotic syndrome and unknown steroid response, two patients (3.4%) with familial and congenital nephrotic syndrome, and only one patient (1.7%) with familial SSNS.
Steroid therapy had been given to 45 patients from this cohort, 44 were shown to be resistant and only one patient had shown steroid responsiveness but considered as challenging NS given its familial incidence regardless of steroid response. 13 patients did not receive steroid therapy including all patients with congenital lNS (5 patients) and eight patients with familial incident NS.
Renal biopsy revealed focal and segmental glomerulosclerosis (FSGS) in 24 patients (41%), minimal change disease (MCD) in 20 patients (34%), mesangioproliferative glomerulonephritis in eight patients (14%), diffuse mesangial sclerosis in two (3.5%), global glomerulosclerosis in one (~2%) patient, and nonspecific findings in three patients (5%).
Twelve out of 58 tested patients had confirmed pathogenic mutations which makes the overall diagnostic rate of genetic origin in challenging NS 20.7%. Of these 12 patients, nine had NPHS2 mutations, two had NPHS1 mutations, and one had WT1 mutation. Probably, pathogenic mutations were identified in an additional two patients which makes the combined diagnostic rate of genetic origin (definitely and probably pathogenic mutations) in challenging NS around 24% (14/58). The clinical and genetic details of these 12 patients with confirmed pathogenic mutations in NPHS2 (known and novel), NPHS1, and WT1 are shown in [Table 2].
|Table 2: Clinical and genetic details of the 12 patients with confirmed pathogenic mutations in NPHS2 (Known and novel), NPHS1, and WT1.|
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Five of these 12 confirmed pathogenic mutations were novel sequence variants in the NPHS2 gene, accounting for 55.6% of all NPHS2 mutations, and 9% of the cohort. To verify this, the five novel variants in NPHS2 were checked in a control sample from the same ethnicity consisting of 100 healthy Syrian children; later on, none of these variants was detected in any of the tested children and thus were considered as novel mutations in NPHS2.
Out of 44 patients with SRNS in this cohort, eight patients (18.2%) had confirmed pathogenic mutations: seven (15.9%) in NPHS2 and one (2.3%) in WT1. Hence, podocin (NPHS2) mutations were identified in 15.9% of children presenting with SRNS.
Four patients (17.4%) out of 23 familial NS cases had confirmed pathogenic mutations, all in NPHS2. Three of the five patients with congenital NS had confirmed pathogenic mutations: two in NPHS1, and one in NPHS2. Hence, nephrin (NPHS1) and podocin (NPHS2) mutations were identified in 40% and 20% of children with congenital NS, respectively.
Of the12 patients with identified mutations, eight had sporadic incidence of the disease and four had familial NS. Two patients with NPHS2 were siblings from one family.
Eight out of 35 patients with sporadic incident NS and four out of 23 patients with familial NS were found to have identified mutations (22.9% and 17.4% respectively).
Mutations in NPHS2 were found in nine patients from eight families, accounting for 75% (9/12) of all identified mutations; of these, seven patients with SRNS (5 sporadic and 2 familial), one patient with congenital NS of familial incidence, and one patient with familial NS and unknown steroid response. The inheritance pattern of NPHS2 mutations was autosomal recessive (AR) in eight cases and one single case with heterozygous state where in Exon 7 of the podocin gene, the following heterozygous mutation was found. (Variation: c.868G>A X, Consequence: p.V290M). Mutations in NPHS1 were found in two sporadic congenital NS cases accounting for 16.7 % (2/12) of all pathogenic mutations. One mutation in WT1 was found in one patient with sporadic SRNS; the parents of this patient were tested and found not to carry this mutation which means that the identified mutation was de novo. Ultimately, this child turned out to be a case of Frasier syndrome with male pseudo-hermaphroditism and female external genitalia. WT1 mutation accounted for 8.3% (1/12) of all pathogenic mutations.
There was no statistical association between the type of NS (SRNS and congenital NS) and the pattern of incidence (sporadic vs. familial). (Fisher exact test statistic value = 1, P >0.05).
Patients with podocin mutations were more likely to have a positive family history of NS since familial cases accounted for ~44% (4/9) of patients with NPHS2 versus 39% (18/46) of patients without mutation. However, this did not reach statistical significance as there was no statistical association between the familial incidence of NS and the likelihood of carrying NPHS2 mutations; (Fisher exact test statistic value = 0.718318, P >0.05). Familial SRNS cases were not significantly more likely to carry NPHS2 mutations than sporadic SRNS ones; (Fisher exact test statistic value = 1, P >0.05). However, one has to remember that statistically insignificant tests may still be clinically significant, and the reason for the insignificant figures may be the small samples.
The median age at onset of NS in this cohort was 4.7 years (range: 1 month–12 years). Patients with podocin (NPHS2) mutations were younger at presentation since the mean age at presentation of NPHS2 cases (9 patients) was three years (2 months–9.9 years) versus 5.7 years (1 month–12 years) in patients without detected mutation (46 patients).
The clinical variables of NS in children with mutations versus without mutation are given in [Table 3].
|Table 3: Clinical variables of nephrotic syndrome in children with mutations versus without mutation.|
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The mean age at presentation of all patients with any pathogenic mutation and those with NPHS2 mutations were 2.7 years and three years, respectively versus 5.7 years inpatients without mutation.
Among the 44 patients with SRNS in this cohort, 27 patients did receive further immuno-suppressive therapy consisting of cyclosporine (CsA), they were distributed as follows: five patients with a genetic cause of SRNS (4 with NPHS2 mutation, one with WT1 mutation) and 22 patients without mutation. None of the five patients with a genetic cause of SRNS showed a complete response to intensified immunosuppression other than corticosteroids and only one SRNS patient with NPHS2 mutation showed a partial response to CsA therapy, whereas 50% (11/22) of patients without mutation showed some response to CsA, either complete response in four patients (18%) or partial response in seven patients (32%).
In 16% of the patients, one or several extra-renal abnormalities were observed. Alterations of the morphology and/or function of the central nervous system were the most common including microcephaly and mental retardation, other features included urogenital abnormalities, short stature, minor thalassemia, vision disturbances, impaired hearing, poly-dactyly, eczema, and Frasier syndrome with sex reversal (WT1 disease).
| Discussion|| |
In our cohort, 60.3% of cases occurred sporadically and 39.7% showed familial disease occurrence. A genetic cause was identified in 17.4% of the familial cases and 22.9% of the sporadic cases. Taken together, the overall mutation detection rate was 20.7%.
The primary goal of this study was to determine the frequency of NPHS2, NPHS1, and WT1 mutations in children with challenging NS, particularly those with SRNS. Our results showed that 15.9% and 2.3% of children with primary SRNS had mutations in the NPHS2 and WT1 genes, respectively. Our results are comparable to those of Hinkes et al who found a frequency of 18.1% podocin mutations in 430 children with SRNS; however, this is higher than those reported by Kari et al from Saudi Arabia and from Abid et al from Pakistan where the frequencies of NPHS2 mutations in children with primary SRNS were 6.8% and 3.4%, respectively.,
The most common histopathologic diagnosis was FSGS followed by MCD. Another noteworthy aspect of this study is the observation that children with genetic NS tend to manifest their disease at younger age, three years for patients with NPHS2 mutation, 2.7 years for all patients with any mutation versus 5.7 years for patients without the mutation. Our observation is similar to what was reported by Tan et al from Boston Children’s Hospital in the US who found a median age at onset of 4.1 years in patients without a mutation and 3.2 years in those in whom the causative mutation was detected. Moreover, this is in agreement too with results of the PodoNet Registry as reported by Trautmann et al who found the detection rate of the genetic cause was lower with increasing age at first manifestation.
Almost 85% of our cohort presented between one and 12 years of age, whereas 8.6% were categorized as congenital, and 6.8% were categorized as early infantile (onset age of 4–12 months). Among the children who underwent mutation screening, the genetic disease detection rate was markedly higher (60%, 3/5) in patients with congenital NS than in children first presenting at older age (17%, 9/53). This figure is in keeping with previous screening studies in SRNS patients suggesting a steep inverse relationship of the rate of genetic diagnoses with age. Reported prevalence figures of genetic causes were 81%–100% for congenital NS,, and 44% for 4–12 months. Our rates are also similar to those of Trautmann et al from the PodoNet Registry Consortium who found a genetic disease detection rate of 66% in patients with congenital NS and in 16% of children first presenting at age six years old and older. Hinkes et al have also reported that as high as two-thirds of NS manifesting in the 1st year of life can be explained by mutations in four genes (NPHS1, NPHS2, WT1, or LAMB2). The chances of finding a mutation drop considerably in older children as shown in a Spanish national study with detection rates of 24% for toddlers (1–5 years), 36% for school children (6–12 years), 25% for adolescents (13–17 years), and 14% for adults. The only population study performed in an adult cohort reported a mutation rate of 8%.
The inheritance pattern of NPHS2 mutations was AR in eight cases and one single case with heterozygous state where in Exon 7 of the podocin gene, the following heterozygous mutation was found. (Variation: c.868G>A X, Consequence: p.V290M). This mutation has been defined previously. However, it does not explain the phenotype alone as it is in heterozygous state.
Among all the patients in this cohort, parental consanguinity and familial disease occurrence were observed in 60% and 40%, respectively. This is even higher than the average rates reported from several Middle Eastern countries including Syria (28.6% and 25.6%, respecttively) by Trautmann et al on behalf of the PodoNet Registry Consortium who concluded that consanguinity and familial disease occurrence were most common in the Middle Eastern countries.
The overall mutation frequency in our cohort of 20.7% (12/58) indicates that the availability of rapid genetic testing for NPHS1, NPHS2, and WT1 would influence the management of genetic NS patients who made up about one-fifth of all patients and 18.2% of patients with SRNS since we observed nonresponse to invasive immunosuppressive treatment in patients with a genetic cause of SRNS. Therefore, our study underlines the clinical usefulness of genetic testing in children with SRNS and by recognizing those patients early on we would spare them a renal biopsy and prolonged exposure to immunosuppressive drugs and unnecessary treatment attempts can be avoided., Moreover, studies have shown that the establishment of a genetic diagnosis is far superior to histopathologic disease classification not only in predicting intensified immunosuppressive therapy responsiveness but also post-transplant disease recurrence in patients with SRNS since they show no disease recurrence after renal transplantation. However, the genes identified to date, altogether explain less than half of the SRNS cases unresponsive to any immunosuppressive treatment.,
The diagnosis of WT1-associated nephro-pathy is of particular clinical relevance because of its involvement in sex determination and the associated risk of Wilms’ tumor and/or gonadoblastoma. Lipska et al evaluated 61 patients with WT1-related SRNS from the PodoNet Registry Consortium which included our single patient with WT1 mutation and found a wide range of expressivity, solid genotype-phenotype associations, and high risk and significance of extrarenal complications in WT1-associated nephropathy; that is exactly the scenario of our patient who turned out to be a case of Frasier syndrome with male pseudo-hermaphroditism (female phenotype and male genotype). At the end of this important study, the biggest ever done for patients with WT1 mutations, Dr. Lipska et al suggested that all children with SRNS undergo WT1 gene screening.
Nine patients in this cohort (16%, 9/58) had extra-renal abnormalities, including four (33%, 4/12) with pathogenic mutations versus five patients (11%, 5/46) without mutations. The frequency of extra-renal symptoms in our cohort (16%) is comparable to what was reported by Trautmann et al (17.3%).
In summary, the 20.7% overall prevalence of mutation causing challenging NS in our cohort is closer to figures found in other studies. However, the low mutation detection rate even in familial cases, 17.4% versus 22.9% in sporadic cases, points toward a large fraction of as yet undiscovered genetic forms. The advent of next-generation exome sequencing is expected to complete our knowledge of the genetic causes of SRNS in the near future and should remove the current limitations.,
| Conclusion|| |
A genetic disease cause was identified in 20.7% of the screened patients, 18.2% of SRNS, 17.4% of familial NS, and 60% of congenital NS. Mutations in NPHS2 were the most commonly identified (9 out of 12 mutations), five of them were novel NPHS2 mutations.
Children with genetic NS tend to manifest their disease at younger age. The most common histopathologic diagnosis in this cohort was FSGS followed by MCD. Our study confirms that genetic testing is indicated in pediatric patients with SRNS.
Conflict of interest: None declared.
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Farah Association for Child with Kidney Disease, P.O. Box: 8292, Damascus
[Table 1], [Table 2], [Table 3]