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Original Clinical Science—General

Targeted Next-Generation Sequencing in Brazilian Children With Nephrotic Syndrome Submitted to Renal Transplant

Feltran, Luciana S. MD, PhD1; Varela, Patricia PhD2; Silva, Elton Dias MSc2; Veronez, Camila Lopes PhD2; Franco, Maria Carmo PhD1; Filho, Alvaro Pacheco MD, PhD1; Camargo, Maria Fernanda MD1; Koch Nogueira, Paulo Cesar MD, PhD3; Pesquero, Joao Bosco PhD2

Author Information
doi: 10.1097/TP.0000000000001846

Nephrotic syndrome (NS) is one of the most common and challenging diseases in pediatric nephrology. NS patients present a dysfunction of the glomerular filtration barrier (GFB) and according to recent discovery of mutations in genes related to steroid-resistant NS (SRNS) the GFB dysfunction may have 2 distinct causes1: (1) a systemic origin in which GFB is the target of a yet unknown circulating permeability factor, (2) a hereditary dysfunction in which the anomaly lies in the GFB itself.

Generally, steroid-sensitive NS (SSNS) patients, as well a subgroup of SRNS patients, show an immunologic flaw. On the other hand, SRNS of genetic origin do not respond to immunosuppressive therapy,2 almost invariably progress to ESRD and rarely recurs after transplantation.3-8 To date, the number of genes potentially involved in SRNS is 53.2,8

The hereditary form of NS is more frequent in children who develop early symptoms. The literature describes 66% of hereditary NS in children whose disease starts before 3 months of age (congenital NS) and 15% to 16% in schoolchildren and adolescents.7

Molecular diagnosis is not indispensable for the treatment of this disease, but identifying pathogenic mutations in SRNS is important for the following therapeutic reasons: (a) avoiding adverse effects of steroids, (b) modifying the intensity and duration of immunosuppressive therapies, (c) aiding donor selection, and (d) delaying the progression of the disease in rare cases.9 It also provides genotype-phenotype correlations and prognoses, enabling genetic counseling and to identify mutations which would rarely be considered.10

Currently, mutations in NS-related genes are being studied to explain cases with atypical renal phenotypes or with high clinical intrafamilial variability.11 High cost of genetic analysis makes the diagnosis of hereditary NS unfeasible in many countries. The extent of genetic screening in Latin America varies among countries, including 91% of cases in Chile and 26% in Colombia.7 In Brazil, genetic research in SRNS is available only in some specific protocol studies. NPHS2, NPHS1, and exons 8 and 9 of WT1 were surveyed in a recent Brazilian study on 27 nephrotic children resulting in 14.8% mutated for NPHS2.12

Genetic analysis with next-generation sequencing (NGS) is a cost-efficient method for simultaneously sequencing multiple genes with high sensitivity and specificity for diagnosis of SRNS.9-11,13,14

Children who underwent kidney transplantation (KT) due to SRNS are a group in which disease has evolved unfavorably, without response to therapy and with loss of renal function. The long-term follow-up shows the evolution of the disease, making this group interesting for genotype-phenotype correlation research. To the best of our knowledge, there are no studies evaluating genetic mutations in multiple genes in these patients. Therefore, the aim of our study was to identify the most frequent genetic mutations in 24 genes related to SRNS and to contribute to the understanding of the genotype-phenotype correlations in this disease.

MATERIALS AND METHODS

Children with NS who underwent KT younger than 19 years and were followed up for a minimum of 6 months at Universidade Federal de São Paulo or at Hospital Samaritano de São Paulo were selected. These were patients who had been referred to transplant centers because of NS, with a history of edema and/or proteinuria and had been treated at least with oral steroids, with or without other immunosuppressive drugs. familial/syndromic NS or patients with acquired disease, with or without kidney biopsy were included. Familial cases were defined as patients with affected second-degree or closer relatives. Congenital cases, defined as NS beginning in the first 3 months of life, were excluded.

Patient information (demographics and clinical details) were collected from medical files and checked through parent interviews (in-person or by phone calls) to carefully select patients with clinical data indicative of NS.

The following data were collected: (a) age at NS onset; (b) time to ESRD defined as time from disease onset to the beginning of dialysis or KT; (c) occurrence of associated extra-renal abnormalities; (d) family history of NS; (e) primitive kidney biopsy report, if any; (f) date of transplantation; (g) donor type; (h) NS recurrence, defined as nephrotic proteinuria after transplantation in patients who were treated either with plasmapheresis and/or Rituximab, regardless of the graft biopsy result; and (i) graft loss or failure to attend follow up.

After signing the informed consent, the patients underwent collection of peripheral blood for genetic sequencing using Ion Torrent NGS platform. Because there are no previous local studies assessing the prevalence of genetic mutations in our country, we chose to analyze the 24 genes published by McCarthy et al.10

The inheritance pattern of the studied genes is described in Table 1.

TABLE 1
TABLE 1:
Inheritance pattern of studied genes

Genetic Analysis

DNA samples were extracted from blood using the QIAmp DNA Blood Mini Kit (Qiagen) per the manufacturer's instructions. The DNA content was determined in a Qubit 2.0 Fluorometer (Invitrogen Life Technologies).

An Ion Torrent barcoded library was set up following the manufacturer's protocol for the Ion AmpliSeq Library Kit 2.0V (Life Technologies, Carlsbad, CA). Specific primers designed through the AmpliSeq Designer software (Life Technologies), targeting coding regions and splicing donor and acceptor sites in the genes are listed in Table 1.

Amplified libraries were submitted to emulsion PCR carried out on the Ion OneTouch (Life Technologies). Sequencing was performed in the Ion 316 Chip v2 at Ion PGM Sequencer as described by Veronez et al.15

Bioinformatic Analyses

Data analysis was carried out using Torrent Suite v3.2.1. The readings were mapped with the human genome (hg19/GRCh37) and variant interpretation performed using software Ion Reporter 4.0 (Life Technologies), whereby variants were reviewed and annotated using dbSNP (single-nucleotide polymorphism database (ncbi.nlm.nih.gov/projects/SNP/) and Human Genome Mutation Database (HGMD) professional, biobase-international.com/product/hgmd). Exome Aggregation Consortium (ExAC) (exac.broadinstitute.org) and 1000 Genomes (1000genomes.org) databases were used to achieve known population frequency. We considered minimum allele frequency less than 1% for any of the 7 populations presented.

A combination of prediction programs: Sift (sift.jcvi.org), Polyphen2 (genetics.bwh.harvard.edu/pph2), and Mutation Taster (mutationtaster.org) were analyzed to predict the impact of amino acid alteration on the structure and function of proteins. Missense variants were evaluated by at least 2 programs to be predicted as potentially deleterious. Comparison of the amino acid sequence variant with those of phylogenetically distant species was performed using Mutation Taster database.

The standards and guidelines for the interpretation of sequence variants of American College of Medical Genetics and Genomics and the Association for Molecular Pathology were used to describe and identify pathogenic variants.16

Validation

We used Integrative Genomics Viewer (http://www.broadinstitute.org/igv/) to confirm all alterations. Sanger sequencing method validated the mutations of hereditary NS group and APOL1 variations.

The amplification of the genomic DNA was achieved using PCR with the TopTaq Master Mix Kit (Qiagen, Melbourne, Victoria, Australia) and sequenced with BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) according to manufacturer’s conditions in an ABI Prism 3500xl Genetic Analyzer sequencer (Life Technologies) as described by Turaça et al.17 Sequences were compared with the references sequences (http://www.ncbi.nih.gov) and confirmed by reverse strand.

Statistical Analyses

Median and interquartile ranges (IQR) were used to describe the data in the case of quantitative variables and frequency tables were employed to represent qualitative values. Proportions between groups were compared with the χ2 or Fisher exact test, whereas quantitative variables were compared through the Mann-Whitney U Test. All tests were bicaudal, conducted in Stata (Version 14.1, Stata Corp, College Station, TX) and assuming the limit of 5% (α < 0.05) to reject the null hypothesis.

RESULTS

Clinical Data

One hundred thirty-nine patients were selected for this study, 110 from Universidade Federal de São Paulo (1989-2012) and 29 from Hospital Samaritano (2009-2014). Based on inclusion and exclusion criteria, the genetic study was performed on 95 patients (Figure 1), 61 were men (64%). The median age at NS onset was 4 years (IQR, 2-9 years). Eighteen (19%) patients had their first symptoms before 2 years old and 38 (40%) before 3 years. Eight patients had associated extrarenal anomalies, 9 others had family history, and parents were consanguineous in 3 cases.

FIGURE 1
FIGURE 1:
Flow diagram showing patients inclusion criteria.

Edema was the main symptom of the disease onset in 86 (91%) patients. Other immunosuppressive therapy apart from steroids was used in at least 64 (67%). Eighty-seven (92%) patients had histological diagnosis: 52 (55%) FSGS, 10 (11%) mesangial proliferation, 10 (11%) global sclerosis, 6 (6%) minimal change disease, and 9 (10%) insufficient material for diagnosis.

Median time from disease onset to ESRD was 3.5 years (IQR, 1.9-7.0 years) and median age at KT was 12.5 years (IQR, 8.5-15 years). At blood collection, 85 patients had a functioning graft with a median follow-up of 48 months (IQR, 18-97 months). Ten patients were undergoing dialysis.

Twenty-three patients presented recurrence of NS after their first KT (24%). Among these 23, 9 lost their graft (39%), whereas of 72 patients who did not have recurrence, only 9 (12.5%) lost their graft (P = 0.005).

From the total 95 patients, 9 underwent retransplant, 5 of whom lost their first graft due to NS recurrence. These 5 children also had recurring NS post-second transplant.

Table 2 summarizes clinical and demographic data of selected patients.

TABLE 2
TABLE 2:
Summary of clinical and demographic data of selected patients

Demographic and clinical information of NS before transplantation do not correlate with chance of recurrence of NS after transplantation.

Table S1 SDC (https://links.lww.com/TP/B460) describes clinical data of all 95 children individually.

Variant Analysis

A total of 149 unique variants were identified in 22 of 24 sequenced genes. No variants were found in PDSS2 and LMX1B genes.

Variants were classified according to the chance of causing disease into 4 groups: pathogenic, likely pathogenic, likely benign and benign using the American College of Medical Genetics and Genomics guideline.16 A list of all detected variants in 24 studied genes and their classification is shown in Table S2 SDC (https://links.lww.com/TP/B460). Five variants in 3 genes (MYO1E, NPHS2, and SMARCAL1) were classified as pathogenic and 20 other variants in 16 genes as likely pathogenic. Finally, 44 variants were grouped as benign and 80 as likely benign.

Considering the pathogenicity of variants and Mendelian inheritance pattern of the studied genes, we found 8 patients with hereditary NS. Table 3 correlates genotype and phenotype of patients with hereditary NS and possibly genetically caused NS. We briefly describe below the hereditary NS patients individually. NPHS2 (codes for podocin) was the most common mutated gene. Patients 12 and 101, even though both had the Gln215Ter (Q215X) variant in homozygosis, presented different age of disease onset and histology. Patients 73 and 128 are compound heterozygous, presenting both the variant Glu310Lis (E310K), previously described as pathogenic,18 and the variant Arg229Gln (R229Q), classified as a polymorphism. Patient 60 has the 2 mutations Val260Glu (V260E) and Arg138Ter (R138X) in heterozygosis, both already described as pathogenic.18 The DNA from the parents was analyzed because of the prior knowledge of pathogenicity in cases of compound heterozygosis in this gene, and the presence of the mutations was confirmed in different alleles (Figure 2).COL4A3 and COL4A5 (coding for alpha3 and alpha5 chain of type IV collagen, a major component of basement membranes): patient 132 has the mutation Gly681Asp in COL4A5 gene in hemizygosis, associated with the variant Gly566Ala in COL4A3 gene in heterozygosis, presenting hearing loss associated with ESRD. His diagnosis was defined as Alport’s Syndrome although it was clinically indistinguishable to NS. It is described that individuals carrying COL4A3 mutations in heterozygosis could present a broad range of symptoms, from familial benign hematuria to the complete features of Alport syndrome nephropathy.19

TABLE 3
TABLE 3:
Genotype and phenotype correlation of variants identified as pathogenic and likely pathogenic by analysis of 24 genes related to NS in Brazilian kidney transplanted children
FIGURE 2
FIGURE 2:
Compound heterozygosity in NPHS2 gene.

WT1 (codes for the transcription factor WT1): patient 116 has the p.Cys453Arg variant (C453R, c.1357T > C, exon 9) in heterozygosis and presents short penis, hypospadia, and cryptorchidism.

MYO1E (codes for nonmuscle membrane-associated class I myosin): patients 46 and 49 have the variant Arg169X in homozygosis, a variant never described before. Both patients had comparable onset and evolution of the disease. Patient 46's mother has NS as well.

COQ2 (codes for coenzyme Q2): patient 95, son of first-degree cousins, has the variant Phe383Leu, predicted to be pathogenic, in homozygosis. He is currently in the second KT, also presenting retinitis and hypertrophic cardiomyopathy.

CD2AP (encodes a scaffolding molecule that regulates the actin cytoskeleton): patient 10 presents the variant Arg74Met and the patient 78 has the variant Lys369Asn, both in heterozygosis. The variant Lys369Asn has never been described before and the variant Arg74Met has already been implicated in NS.20

APOL1 Gene

Per some authors, patients with high-risk alleles of APOL1 (G1 = rs73885319 and rs60910145 and G2 = rs71785313) should be classified as having genetic disease. Considering the importance of these variations in the progression of NS to ESRD,14 we analyzed separately these variations because APOL1 is not traditionally considered a genetic cause of NS. We found 8 patients having high-risk alleles of APOL1 (6 G1G1 and 2 G1G2), but none of them presented hereditary NS. The age at NS onset in APOL group was 11.1 ± 3.4 years and time to ESRD was 2.5 ± 2.6 years. Patients from high-risk APOL1 group did not present NS recurrence, and the rate of first graft loss was 12% in the first 2 groups compared with 20% in nonhereditary NS patients.

Age at disease onset showed a tendency to be earlier in hereditary NS patients (2.3 ± 1.4 years vs 5.5 ± 4.2 years; P = 0.06), and time to ESRD was significantly longer in hereditary NS group when compared with patients without known genetic background (8.3 ± 5.0 vs 4.4 ± 3.4; P = 0.03). No patient from hereditary NS group presented recurrence of NS, whereas 26% of other patients had NS recurrence (P = 0.19). Family history of NS was present in 25% of hereditary NS group and in 8% in other groups (P = 0.17) and no patient from hereditary NS group had parental consanguinity or extra-renal manifestation.

DISCUSSION

KT patients constitute an interesting group to study SRNS because the complete history and the incidence of disease recurrence are known. The severe outcomes in these patients increase the clinical relevance of studying genetic markers that could better characterize them.

Although genetic diagnosis is not indispensable for clinical treatment, it has a strong significance for the patient and families when the etiology of the disease is confirmed. Moreover, the genotype-phenotype correlation can improve the understanding of the GFB.

Per the stringency of criteria adopted to classify the variants, results can be considerably different. It is easy to separate 1 group of mutations that must be the cause of NS (pathogenic variants) from another group with no involvement (benign variants). However, there are numerous variants (likely pathogenic) that should be studied apart. Using the proposed strategy in our study, we could identify 8 patients with hereditary NS (8.4%). This result is in accordance to the literature, considering that sporadic cases were the majority and congenital NS patients were excluded.7,20,21

Younger age at NS onset was a known signal of hereditary NS and was confirmed in this study. However, the time to ESRD was strangely longer in our hereditary NS group. This finding could probably be related to the presence of 1 patient with Alport syndrome in this group and to the presence of patients with NPHS2 mutations in compound heterozygosis with the variant R229Q.22,23 Patients with confirmed hereditary NS, as expected, had no recurrence of this disease after transplantation.8

In this work, we briefly described the patients with mutations causing NS; however, the possible interactions among genes were not considered.

NPHS2 was the most frequent gene associated with NS (5 patients), and this result is in consonance with the literature.7,24-26 Interestingly, we found the variant Q215X in homozygosis in 2 unrelated patients from the same region of the country. This variant had previously been described as a cause of disease only in compound heterozygosis with the R138Q variant.27-29 Because we did not evaluate the parents of these patients, it is also possible that they present a gross deletion in 1 allele. We also described 1 patient with V260E/R138X and another 2 patients presenting E310K/R229Q in compound heterozygosis. It is known that the R229Q variant is a polymorphism with a nonneutral feature and when inherited with a specific NPHS2 pathogenic mutation (in trans) the individuals develop SRNS.18,22,23 None of NPHS2 patients presented recurrence of the disease after transplantation, in accordance with Jungraithmayr et al.30

Remarkably we found many variants in COL4A3-5 genes in our sample and these findings have been repeatedly reported.31-33 The question whether these cases should be considered isolated FSGS or simply a misdiagnosed type of the Alport spectrum is open to debate. Alport or Thin Basement Membrane Nephropathy could both potentially lead to secondary FSGS, meaning that the COL4A3-5 genes should be considered in a sequencing-based approach to define pathogenesis in FSGS.34 Furthermore, secondary variants in other genes in patients carrying mutations in a gene causing NS might have additive effects on the disease manifestation. In this sense, variants in COL4A3 gene have been associated with increased disease severity when they coexist with a disease-causing mutation in another gene.11,32

We also described in this article the new mutation Arg169X in MYO1E in 2 patients with similar clinical symptoms and evolution of NS.

WT1 and NPHS1, which are frequently reported as causes of SRNS, were not relevant in this study probably because we excluded congenital NS. We had just 1 patient showing a variant in WT1, which was already described as a cause of NS.35,36

Clinical diagnosis of NS caused by mutations in COQ2 is very difficult. Molecular diagnosis is important in these cases especially because early recognition and ubiquinone supplementation can improve symptoms and prevent neurologic complications.37,38 We observed this result in 1 patient who is now under coenzyme Q10 replacement therapy, although after his second KT.

High-risk APOL1 patients presented later NS onset but faster evolution to ESRD when compared with other groups in this study. This result could mean that the presence of a high-risk APOL1 does not necessarily represent a higher risk to NS recurrence or graft loss.

This study highlights the difficulties in identifying phenotypic differences between hereditary and acquired NS. Clinically the age at disease onset and time to ESRD were the principal parameters that distinguished patients with confirmed genetic NS from others. We emphasize that 40% of children submitted to pediatric KT had the onset of NS before 3 years of age. Our data also suggest that distinguishing NS from other diseases is sometimes a complex task, given that 1 patient from our sample had the diagnosis of Alport Syndrome after genetic tests.

Recurrence of NS after KT was 24% in this study, which is a slightly lower frequency than the 30-50% described in the literature.39,40 This is possibly because we used strict criteria to accept the diagnosis of recurrence.

Based on our results we considered NGS a useful tool in the study of diseases with genetic heterogeneity as NS. We could quickly carry out the sequencing of several genes simultaneously and to identify rare and unknown variants. However, it also brings a sizeable amount of information that generates a real “puzzle” to be understood. Each patient has many variations, many with unknown significance, and their interactions are poorly understood. Collaboration between geneticists and clinicians is necessary to translate the results into benefits for the patients. In addition, bioinformatic tools should be used with caution. The information contained in HGMD is useful but any variant should be individually studied in the literature. Moreover, the available predictors are not always able to reproduce the effects of the variant in vivo. In addition, many populations have never been studied before, which makes the use of population frequency doubtful.

In conclusion, this is the first study in children with SRNS who underwent KT using NGS. Considering the results showing that 8.4% of our patients had hereditary NS, we believe this study has shed some light on the uncertainties of genotype-phenotype correlation in NS, where several genes cooperate to produce or even modify the course of the disease. This complex scenario should foster collaborative international studies aiming to enlarge patients number and to find more candidate genes to provide robust evidence to solve the existing puzzles in genotype-phenotype correlation of this important disease.

ACKNOWLEDGMENTS

The authors thank Dr. Suzana M. Oliveira for the editing assistance.

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