|Year : 2019 | Volume
| Issue : 1 | Page : 16-24
Next-generation sequencing-based genetic diagnosis of steroid-resistant nephrotic syndrome: Benefits and challenges
Varsha Chottusing Pardeshi1, Ambily Narikot1, Anil Vasudevan2
1 Division of Molecular Medicine, St. John's Research Institute, Bengaluru, Karnataka, India
2 Division of Molecular Medicine; Department of Pediatric Nephrology, St. John's Medical College Hospital, Bengaluru, Karnataka, India
|Date of Web Publication||17-May-2019|
Department of Pediatric Nephrology, St. John's Medical College Hospital, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Steroid-resistant nephrotic syndrome (SRNS) is the second-most common cause of chronic kidney disease in children and in those requiring kidney transplantation. The disease shows significant heterogeneity in its age at onset and clinical course. The discovery of mutations in NPHS1, the gene encoding nephrin that is a key component of the podocyte slit diaphragm, in a subset of children with congenital NS, led to identification of a distinct subgroup of patients of SRNS that has an underlying genetic etiology. Subsequently, mutations in over 53 podocyte genes have been implicated in monogenic forms of SRNS with no clear genotype-phenotype correlations. The large number of genes implicated in SRNS, phenotypic variability, and lack of information about frequency of mutations in these genes, makes the use of genetic testing in the management of children with SRNS challenging in terms of decisions on who to test, which genes to screen, and how to use the information obtained from testing in the clinical setting. Given the genetic heterogeneity and phenotypic variability, Sanger sequencing is not a feasible approach for routine testing. Next-generation sequencing (NGS) technology is emerging as the preferred method to screen multiple genes in genetically heterogeneous diseases like SRNS. Such high-throughput sequencing method permits rapid and cost-effective simultaneous screening of large number of individuals and genes. However, the high throughput combined with significant phenotypic and genetic variability of monogenic SRNS poses unique challenges for clinicians in the interpretation of genetic result. This review provides an overview of utility of genetic testing with focus on NGS-based genetic testing and the challenges in the interpretation of genetic results in clinical settings.
Keywords: Genetic diagnosis, next-generation sequencing, steroid-resistant nephrotic syndrome
|How to cite this article:|
Pardeshi VC, Narikot A, Vasudevan A. Next-generation sequencing-based genetic diagnosis of steroid-resistant nephrotic syndrome: Benefits and challenges. Asian J Pediatr Nephrol 2019;2:16-24
|How to cite this URL:|
Pardeshi VC, Narikot A, Vasudevan A. Next-generation sequencing-based genetic diagnosis of steroid-resistant nephrotic syndrome: Benefits and challenges. Asian J Pediatr Nephrol [serial online] 2019 [cited 2020 May 30];2:16-24. Available from: http://www.ajpn-online.org/text.asp?2019/2/1/16/258566
| Introduction|| |
Nephrotic syndrome (NS) is the most common glomerular disease of childhood with the incidence of approximately 1–2/100,000 children., NS comprises a heterogeneous group of disorders and classification is based on response to steroids therapy into steroid sensitive NS (SSNS) and steroid-resistant NS (SRNS), representing 80% and 20% of NS cases, respectively. SRNS remains one of the most intractable causes of end-stage renal disease in children with 50%–70% of these children developing end-stage renal disease within 5–10 years of diagnosis.,,
The discovery of mutations in NPHS1 that encodes nephrin, a protein which is an important component of the podocyte slit diaphragm, in a subset of children with congenital NS, lead to identification of a distinct subgroup of SRNS who have an underlying genetic etiology., Subsequently, 70 podocyte gene mutation have been implicated in monogenic forms of SRNS and accounts for ~30% of pediatric cases of SRNS, inherited as autosomal recessive or autosomal dominant.,, The advent of next-generation sequencing (NGS) techniques has greatly facilitated the identification of numerous novel causative genes.
| Genes Implicated in Steroid-Resistant Nephrotic Syndrome and Utility of Genetic Testing|| |
Majority of the SRNS causing gene mutations have been identified in the podocyte, highly specialized epithelial cells that form an integral part of the glomerular filtration barrier. Genes associated with SRNS codes for podocyte structural protein complexes and signaling pathway proteins which are expressed at varied locations within podocyte such as cell membrane, nucleus, cytoskeleton, lysosomes, and mitochondria [Table 1]. A SRNS child with a genetic mutation can present as an isolated kidney disease or as a syndromic disorder with extrarenal manifestations that are often striking and diagnostic [Table 2].
|Table 1: Genes associated with steroid resistant nephrotic syndrome in relation to location of corresponding podocyte proteins|
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|Table 2: Genes associated with syndromic steroid-resistant nephrotic syndrome|
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While discovery of genetic mutations has contributed to a better understanding of the disease pathophysiology, it has also led to a paradigm shift in the genetic diagnosis and management of this disease. Identification of the causal genetic variant enables more precise diagnosis. Genetic testing facilitates family counseling regarding future pregnancies and aids in timely antenatal diagnosis. Identifying an underlying mutation in a child with SRNS predicts poor therapeutic responsiveness to immunosuppressants as only 0%–3% with genetic SRNS experience complete remission while 17% experience partial remission., Mutation detection also helps discover potential therapy and allows for a more personalized treatment approach. For example, it is reported that patients carrying the mutation in coenzyme Q10 biosynthesis pathway (COQ2, COQ6, ADCK4, or PDSS2) may be benefited by treatment with coenzyme Q10.,,,, Similarly, two patients with PLCE1 mutation responded fully to prednisolone treatment, whereas patients with CUBN mutation show response to Vitamin B12 treatment., Individuals with pathogenic variants in ARHGDIA gene may respond to therapy with eplerenone, a mineralocorticoid-receptor antagonist, whereas patients with TRPC6 mutations may potentially respond to treatment with calcineurin inhibitors., A genetic diagnosis helps in more specific prognostication as SRNS patients with mutation are more likely to progress quickly to end-stage renal disease as compared to most patients without a mutation. More than 75% of cases with a genetic etiology of SRNS progress to end-stage renal disease within 15 years, as compared to only 4% of SRNS patients with sporadic disease and responsiveness to immunosuppressive medications.
Genetic testing helps in screening and monitoring for extrarenal complications. For example, in patients with WT1 gene mutations, investigation of the karyotype of females to exclude XY with pseudohermaphroditism, and development of Wilms' tumor or gonadoblastoma, should be performed. Patients without extrarenal features may be carrying genetic variants typically identified in other syndromic kidney diseases, such as Alport syndrome (COL4A3 or COL4A4), nail–patella syndrome (LMX1B),,,, and juvenile nephronophthisis (TTC21B and NPHP4).,, Hence, screening of these genes should be performed in patients with focal segmental glomerulosclerosis (FSGS) even in the absence of extrarenal features. Several studies show low risk of posttransplant disease recurrence in patients with a genetic etiology.,,,,, Genetic screening in unaffected family members is important, especially in the case of autosomal dominant inheritance, when planning a living-related donor renal transplant.
| Genetic Testing Method in Steroid-Resistant Nephrotic Syndrome: Sanger Versus Next-Generation Sequencing|| |
The purpose of diagnostic genetic testing is to identify the mutation that is causal for disease in an individual patient. The common methods for diagnostic genetic testing include Sanger sequencing, NGS based approaches, and chromosomal microarrays. Sanger sequencing remains the gold standard for molecular diagnosis when a single-gene disorder is suspected [e.g., syndromic SRNS; [Table 2] and for confirmation of NGS findings as well sequencing regions that are not covered by NGS based testing. In diseases associated with significant genetic and phenotypic heterogeneity like SRNS, Sanger sequencing has limited utility as it is expensive and time-consuming. The advent of NGS technologies has enabled clinical genetic testing to move beyond single gene analysis to the simultaneous investigation of many genes. The variants identified using NGS is classified as pathogenic, likely pathogenic, uncertain significance, likely benign, or benign according to the stringent criteria of the American College of Medical Genetics and Genomics (ACMG). Many NGS-based approaches are available which could be used in clinical setting such as targeted panel sequencing of known genes, whole-exome sequencing (WES), and whole-genome sequencing (WGS) [Table 3]. Targeted NGS gene panels in which a set of genes are screened is recommended as a first-line test for the molecular diagnosis of SRNS. Targeted NGS is cost effective, has a short turnaround time and a low rate of incidental findings. This method tests only selected number of genes and to test the newly discovered genes with a clinical phenotype requires redesigning and revalidation of the panel. The European Society of Human Genetics guidelines, targeted diagnostic testing should be carried out with analysis focusing on reducing the likelihood of detecting incidental findings, focusing only on genes clinically actionable. WES and WGS are unbiased approaches with increased sensitivity for genetic discovery as unknown genes are also screened and in situ ations where precise clinical diagnosis is not possible. However, per-base coverage is generally lower with WES and WGS than with targeted panels, with clinically relevant segments of the genome at risk of being missed when using WES alone. For example, the regions corresponding to ~50% of reported pathogenic variants in the WT1 gene were poorly covered across three leading WES capture kits. WES is more expensive when compared to targeted panel-based testing. With improving technology, increasing access to NGS-based services and plummeting cost of NGS-based testing, NGS is establishing itself as a cost effective and useful method for routine genetic testing of genetically heterogeneous conditions such as SRNS. NGS-based testing not only facilitates rapid analysis of known disease genes but it also helps in discovery of previously unrecognized genes. In a small study done at our center, NGS-based testing using a panel of 17 genes identified variants in 25% of the children tested whereas screening only NPHS2 and WT1, the two most common genes associated with SRNS identified only 5% of patients with an underlying mutation. In this study, NGS based testing helped identify causal variants which would not have been considered in the conventional genetic testing algorithms for SRNS using Sanger sequencing.
|Table 3: Comparison of common assays for next-generation sequencing in the diagnosis of steroid-resistant nephrotic syndrome|
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| Key Findings from Studies of Next-Generation Sequencing-Based Genetic Testing in Steroid-Resistant Nephrotic Syndrome|| |
Due to low cost and improving turnaround time, several NGS-based studies using either the targeted or whole-exome (WES) sequencing were performed to determine the genetic defects in large NS cohorts. The important findings of these studies, [Table 4],,,,,,,,,, are provided below:
|Table 4: Summary of findings of next-generation sequencing-based genetic testing in pediatric steroid.resistant nephrotic syndrome|
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- A monogenic cause was identified in 24%–30% of patients with SRNS and none of those with steroid-sensitive NS. This suggests that 70% of cases of SRNS are caused by circulating (permeability) factor disease or as yet undiscovered genes
- The rate of detecting monogenic cause is inversely correlated with the age of onset. Patients with <3 months age of onset have the highest probability of carrying genetic mutations. In the largest cohort, the proportions of genetic etiology were 69.4%, 49.7%, 25.3%, 17.8%, and 10.8% in patients with disease manifesting during the first 3 months of life, 4–12 months, 1–6, 7–12, and 13–18 years, respectively,,,,,,
- The most common mutated genes vary with the age of onset. For example, NPHS1, NPHS2, WT1, and LAMB2 defects are common in children with disease manifesting within the first 3 months of life, whereas NPHS2 mutations are seen in children with onset of SRNS between 1 and 16 years
- Causative genes differ significantly by ethnic background. For example, in Chinese pediatric SRNS patients, ADCK4 was the most commonly mutated gene as compared to that of NPHS2. NPHS2 mutations were less prevalent in the Asian populations such as Chinese, Japanese, and Indians when compared to the European and American populations
- The genotype-phenotype correlations were poor except in cases of children with diffuse mesangial sclerosis who had mutations in WT1, PLCE1, or laminin gene. Long-term renal outcomes are similar in children with different genetic disease entities
- These studies could not determine the true prevalence rates of gene abnormalities in NS chiefly because most studies, except for the UK national study, were performed in a selected cohort of primary SRNS
- In genetic CNS, the most commonly mutated gene was NPHS1, and in genetic SRNS, it was NPHS2 gene. More than one-third of all SRNS cases, worldwide, that manifest before 25 years of age, are caused by a single-gene mutation in one of 21 SRNS genes. Recessive gene mutations were more frequently observed in patients with early-onset disease, whereas dominant genes mutations were frequent in adult-onset disease
- NGS application helped to identify risk alleles in SRNS patients and discovery of new disease-causing genes (COL4A3- 5, CLCN5)
- The renal outcome of patients with SRNS and genetic mutation is poor compared to that in patients without genetic mutations
- Various research groups have used different statistical and functional criteria for assessing the causality of mutations, changing the sensitivity for genes covered by the test and resulting in possible discrepancies between interpretations of identified variants in different studies.
[Table 4] highlights the salient features and limitations of studies that have used NGS-based testing in children with SRNS.
| Challenges With Next-Generation Sequencing-Based Genetic Testing|| |
Poor genotype-phenotype correlation
Patients with identical causative mutations show significant clinical heterogeneity rendering genetic testing and counseling a more complex task, especially when the variant detected is likely pathogenic or is of unknown significance. In these situations, clinicians face difficulties in conveying the results to patients or parents when results of NGS show poor genotype-phenotype correlation. In addition, the panel needs to be expanded as and when new genes implicated in causing SRNS are identified.
Challenges in the interpretation and uncertainty of results
NGS testing often results in the discovery of a large number of variants in genes of interest many of which are classified as variants of unknown significance (VUS). It is difficult to make sense of large quantities of sequence data in clinically meaningful way and explain to the patients. It is also difficult to differentiate pathogenic mutations from VUS and establish whether a VUS is contributing to disease development or is a polymorphic variant. There are no criteria defined to determine the clinically relevant and potentially disease-causing variant among multiple VUS., One of the main barriers to determine the pathogenicity of a variant is the absence or limited functional testing of variants discovered to identify specific variants that result in dysfunction of the protein product. For example, in the study, a novel homozygous variant R752X in PLCe1 gene was identified in a patient and was classified as likely pathogenic instead of pathogenic. Based on the clinical findings and histopathology of the patient, it is evident that PLCe1 gene variant can potentially be attributed to the disease development in this patient. However, due to the lack of segregation data, which would help with the segregation of alleles in cases and the ethnically matched reference population, and the absence of functional data, the variant could not be classified as pathogenic. Another challenge in NGS-based testing is the lack of clear guidelines to annotate heterozygous variants in dominant genes. For example, in our study, the novel LMX1B gene variant V145M with low allele frequency was predicted to be pathogenic in nature as per the Karbassi scoring algorithm but was classified only as VUS as per the ACMG criteria.,
In addition, NGS-based sequencing projects also revealed that healthy individuals may carry rare functional variants. This suggests that mutations published as pathogenic due to the lack of frequency information available at the time of publications may, in fact, be benign variants. Several such pathogenic variants were later annotated as benign.,,, Conversely, polymorphisms can sometimes be pathogenic or have a modifier effect on an underlying disorder. For example, the Arg229Gln variant in NPHS2 (c. 686G>A; rs61747728), which is found in 1%–2% of the Caucasian population, can be disease-causing when present “in trans” with specific other mutations. Thus, rarity alone is insufficient evidence for pathogenicity and indicate the need to sequence both in asymptomatic controls and the tested patient population of diverse ethnicity.
Careful collection and annotation revisions of sequencing data of people from all ethnic backgrounds and with various diseases should be carried out at regular intervals to better understand the significance of specific variants and correct the misclassified variants.,, Recent guidelines of ACMG are useful to increase the accuracy of variant annotations. However, such careful comparison gave rise to a large number of variants called “VUS”.
Genetic and medical counseling can be complex and challenging when VUS is identified. There is no consensus on optimal strategies to report such findings and for the clinician to communicate them with parents. Counseling parents with an affected child with a VUS is even more challenging in a prenatal setting if the variant is prospectively detected in the unborn fetus as quantifying the attributable risk of developing the disease is not possible.
Identification of multiple variants and incidental variants
Genetic testing using NGS-based approach not only identifies the disease-causing genes and variants but also potential modifying variants in other-related genes. In these cases, it is difficult to interpret the role of multiple variants in disease. For example, in a study of 50 children with SRNS, wherein targeted panel sequencing of 26 genes was performed, 22% of the patients with monogenic NS had a likely pathogenic variant in an additional gene. In other studies, a child with an early-onset disease had pathogenic WT1 variant along with the NPHS1 variant and the effect of the NPHS1 variant on the phenotype was unclear, whereas two NPHS1 variants and an NPHS2 variant were identified in five individuals suggesting possible phenotypic modifier effects.,, Similarly, an SRNS child who progressed to end-stage renal disease by 8 years of age carried two heterozygous variants in NPSH2 and CD2AP while a child with Pierson syndrome had heterozygous variants in LAMB2 and NPHS1.,
WES may result in obtaining variants in unrelated gene which has been reported to be causal for another disease. The clinical value of reporting such incidental findings has been highly debated.,,,,, It is difficult for clinician to interpret such ambiguous genomic information and decide how such information may affect clinical practices. Hence before genetic testing, it is mandatory to ensure that families are informed regarding such data, including privacy, legal, and social implications of identifying multiple variants in genes of interest or an incidental variant in unrelated genes.
Lack of reference database representing all ethnic backgrounds
A key step in variant interpretation is comparison of the frequency of the variant in the healthy population reported in public databases. Hence, it is essential that these databases should represent large, ethnically diverse population. Yet many of the world's populations are not well-represented in control databases, such as the Asian and African population. This leads to the potential for underestimations of allele frequencies and has contributed to the misclassification of benign variants as pathogenic. In addition, recent studies have showed that the frequency of specific variants varies with ethnicity. Therefore, it is critical to compare the identified variants in a patient against an ethnically matched dataset.
| Conclusions|| |
In order to provide optimal clinical management for genetic and clinical heterogeneous diseases such as SRNS, identifying the underlying genetic cause plays an important role. As multiple genes are implicated in SRNS, NGS-based methods are preferred for genetic testing. Various studies have demonstrated the feasibility of genetic screening using NGS based approach, especially using a targeted gene panel in a clinical setting. Diagnostic laboratories are expected to follow the standard guidelines for clinical variant interpretation, but they have not yet been implemented, hindering reproducibility in genetic interpretation. NGS-based testing results in the generation of a large amount of data making the interpretation a difficult task. Improving bioinformatics-based filtering strategy will help in differentiating pathogenic variants from those that are benign among VUS. In addition, integrating the data from various studies with large publicly accessible phenotype and genotype data may help in ascertaining the role of novel variants in disease development and determine the role of multiple variants on the phenotypic variability.
Parents need to be counseled for the benefits and potential risks of NGS-based testing including insurance eligibility. In view of challenges in interpretation and classification of variants, developing appropriate and effective clinical approaches to this challenge including additional training to clinicians in pretest counseling and consenting, interpretation of results, and communication of results to the parents is essential.
We would like to acknowledge the support of grant from the Department of Biotechnology, Division of Science and Technology, Government of India (BT/PR11030/MED/30/1644/2016).
Financial support and sponsorship
AV recipient of grant from the Department of Biotechnology, Division of Science and Technology, Government of India (BT/PR11030/MED/30/1644/2016).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]