Steroid Resistant Nephrotic Syndrome (SRNS) in children and young adults has differing etiologies with monogenic disease accounting for 2.9–30% in selected series. Using whole exome sequencing we ...sought to stratify a national population of children with SRNS into monogenic and non-monogenic forms, and further define those groups by detailed phenotypic analysis. Pediatric patients with SRNS were identified via a national United Kingdom Renal Registry. Whole exome sequencing was performed on 187 patients, of which 12% have a positive family history with a focus on the 53 genes currently known to be associated with nephrotic syndrome. Genetic findings were correlated with individual case disease characteristics. Disease causing variants were detected in 26.2% of patients. Most often this occurred in the three most common SRNS-associated genes: NPHS1, NPHS2, and WT1 but also in 14 other genes. The genotype did not always correlate with expected phenotype since mutations in OCRL, COL4A3, and DGKE associated with specific syndromes were detected in patients with isolated renal disease. Analysis by primary/presumed compared with secondary steroid resistance found 30.8% monogenic disease in primary compared with none in secondary SRNS permitting further mechanistic stratification. Genetic SRNS progressed faster to end stage renal failure, with no documented disease recurrence post-transplantation within this cohort. Primary steroid resistance in which no gene mutation was identified had a 47.8% risk of recurrence. In this unbiased pediatric population, whole exome sequencing allowed screening of all current candidate genes. Thus, deep phenotyping combined with whole exome sequencing is an effective tool for early identification of SRNS etiology, yielding an evidence-based algorithm for clinical management.
MAGI2 Mutations Cause Congenital Nephrotic Syndrome Bierzynska, Agnieszka; Soderquest, Katrina; Dean, Philip ...
Journal of the American Society of Nephrology,
05/2017, Letnik:
28, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Steroid-resistant nephrotic syndrome (SRNS), a heterogeneous disorder of the renal glomerular filtration barrier, results in impairment of glomerular permselectivity. Inheritance of genetic SRNS may ...be autosomal dominant or recessive, with a subset of autosomal recessive SRNS presenting as congenital nephrotic syndrome (CNS). Mutations in 53 genes are associated with human SRNS, but these mutations explain ≤30% of patients with hereditary cases and only 20% of patients with sporadic cases. The proteins encoded by these genes are expressed in podocytes, and malfunction of these proteins leads to a universal end point of podocyte injury, glomerular filtration barrier disruption, and SRNS. Here, we identified novel disease-causing mutations in
(
) through whole-exome sequencing of a deeply phenotyped cohort of patients with congenital, childhood-onset SRNS. Although
has been shown to interact with nephrin and regulate podocyte cytoskeleton and slit diaphragm dynamics,
mutations have not been described in human SRNS. We detected two unique frameshift mutations and one duplication in three patients (two families); two siblings shared the same homozygous frameshift mutation, whereas one individual with sporadic SRNS exhibited compound heterozygosity. Two mutations were predicted to introduce premature stop codons, and one was predicted to result in read through of the normal translational termination codon. Immunohistochemistry in kidney sections from these patients revealed that mutations resulted in lack of or diminished podocyte MAGI2 expression. Our data support the finding that mutations in the
gene are causal for congenital SRNS.
Intensified immunosuppression in steroid-resistant nephrotic syndrome is broadly applied, with disparate outcomes. This review of patients from the United Kingdom National Study of Nephrotic Syndrome ...cohort aimed to improve disease stratification by determining, in comprehensively genetically screened patients with steroid-resistant nephrotic syndrome, if there is an association between response to initial intensified immunosuppression and disease progression and/or post-transplant recurrence.
Pediatric patients with steroid-resistant nephrotic syndrome were recruited
the UK National Registry of Rare Kidney Diseases. All patients were whole-genome sequenced, whole-exome sequenced, or steroid-resistant nephrotic syndrome gene-panel sequenced. Complete response or partial response within 6 months of starting intensified immunosuppression was ascertained using laboratory data. Response to intensified immunosuppression and outcomes were analyzed according to genetic testing results, pattern of steroid resistance, and first biopsy findings.
Of 271 patients, 178 (92 males, median onset age 4.7 years) received intensified immunosuppression with response available. A total of 4% of patients with monogenic disease showed complete response, compared with 25% of genetic-testing-negative patients (
=0.02). None of the former recurred post-transplantation. In genetic-testing-negative patients, 97% with complete response to first intensified immunosuppression did not progress, whereas 44% of nonresponders developed kidney failure with 73% recurrence post-transplant. Secondary steroid resistance had a higher complete response rate than primary/presumed resistance (43% versus 23%;
=0.001). The highest complete response rate in secondary steroid resistance was to rituximab (64%). Biopsy results showed no correlation with intensified immunosuppression response or outcome.
Patients with monogenic steroid-resistant nephrotic syndrome had a poor therapeutic response and no post-transplant recurrence. In genetic-testing-negative patients, there was an association between response to first intensified immunosuppression and long-term outcome. Patients with complete response rarely progressed to kidney failure, whereas nonresponders had poor kidney survival and a high post-transplant recurrence rate. Patients with secondary steroid resistance were more likely to respond, particularly to rituximab.
The recent Chandos House meeting of the Alport Variant Collaborative extended the indications for screening for pathogenic variants in the COL4A5, COL4A3 and COL4A4 genes beyond the classical Alport ...phenotype (haematuria, renal failure; family history of haematuria or renal failure) to include persistent proteinuria, steroid-resistant nephrotic syndrome, focal and segmental glomerulosclerosis (FSGS), familial IgA glomerulonephritis and end-stage kidney failure without an obvious cause. The meeting refined the ACMG criteria for variant assessment for the Alport genes (COL4A3-5). It identified 'mutational hotspots' (PM1) in the collagen IV α5, α3 and α4 chains including position 1 Glycine residues in the Gly-X-Y repeats in the intermediate collagenous domains; and Cysteine residues in the carboxy non-collagenous domain (PP3). It considered that 'well-established' functional assays (PS3, BS3) were still mainly research tools but sequencing and minigene assays were commonly used to confirm splicing variants. It was not possible to define the Minor Allele Frequency (MAF) threshold above which variants were considered Benign (BA1, BS1), because of the different modes of inheritances of Alport syndrome, and the occurrence of hypomorphic variants (often Glycine adjacent to a non-collagenous interruption) and local founder effects. Heterozygous COL4A3 and COL4A4 variants were common 'incidental' findings also present in normal reference databases. The recognition and interpretation of hypomorphic variants in the COL4A3-COL4A5 genes remains a challenge.
Background
Focal segmental glomerulosclerosis (FSGS) in pediatric patients is typically difficult to treat and will progress to end-stage renal disease (ESRD) in about 10% of cases. Following kidney ...transplantation, FSGS can recur in up to 56% of renal allografts—with a near 100% recurrence in subsequent transplants.
Methods
Four different pediatric centers across the USA and the UK employed a protocol using LDL-apheresis (LDL-A) and pulse solumedrol to treat recurrent FSGS after transplantation in seven patients. All the patients included in this series demonstrated immediate, or early, recurrence of FSGS, which clinically presented as nephrotic-range proteinuria within hours to days after implantation of the kidney.
Results
All patients experienced reductions in urinary protein to creatinine ratios resulting in partial or complete remission. All patients demonstrated improvements in their estimated GFRs at their most recent follow-up since LDL-A discontinuation.
Conclusions
This case series describes the successful treatment, across four different pediatric centers, of seven pediatric patients with recurrent post-transplant FSGS using the Liposorber® LA-15 in combination with pulse solumedrol.
Steroid-resistant nephrotic syndrome (SRNS) is characterized by high-range proteinuria and most often focal and segmental glomerulosclerosis (FSGS). Identification of mutations in genes causing SRNS ...has improved our understanding of disease mechanisms and highlighted defects in the podocyte, a highly specialized glomerular epithelial cell, as major factors in disease pathogenesis. By exome sequencing, we identified missense mutations in TBC1D8B in two families with an X-linked early-onset SRNS with FSGS. TBC1D8B is an uncharacterized Rab-GTPase-activating protein likely involved in endocytic and recycling pathways. Immunofluorescence studies revealed TBC1D8B presence in human glomeruli, and affected individual podocytes displayed architectural changes associated with migration defects commonly found in FSGS. In zebrafish we demonstrated that both knockdown and knockout of the unique TBC1D8B ortholog-induced proteinuria and that this phenotype was rescued by human TBC1D8B mRNA injection, but not by either of the two mutated mRNAs. We also showed an interaction between TBC1D8B and Rab11b, a key protein in vesicular recycling in cells. Interestingly, both internalization and recycling processes were dramatically decreased in affected individuals’ podocytes and fibroblasts, confirming the crucial role of TBC1D8B in the cellular recycling processes, probably as a Rab11b GTPase-activating protein. Altogether, these results confirmed that pathogenic variations in TBC1D8B are involved in X-linked podocytopathy and points to alterations in recycling processes as a mechanism of SRNS.
Nephrotic syndrome (NS), the association of gross proteinuria, hypoalbuminaemia, edema, and hyperlipidemia, can be clinically divided into steroid-sensitive (SSNS) and steroid-resistant (SRNS) forms. ...SRNS regularly progresses to end-stage renal failure. By homozygosity mapping and whole exome sequencing, we here identify recessive mutations in Crumbs homolog 2 (CRB2) in four different families affected by SRNS. Previously, we established a requirement for zebrafish crb2b, a conserved regulator of epithelial polarity, in podocyte morphogenesis. By characterization of a loss-of-function mutation in zebrafish crb2b, we now show that zebrafish crb2b is required for podocyte foot process arborization, slit diaphragm formation, and proper nephrin trafficking. Furthermore, by complementation experiments in zebrafish, we demonstrate that CRB2 mutations result in loss of function and therefore constitute causative mutations leading to NS in humans. These results implicate defects in podocyte apico-basal polarity in the pathogenesis of NS.
Background
Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing
NUP93
...variants suggest these could be a significant cause of paediatric onset SRNS. We report
NUP93
cases in the UK and demonstrate in vivo functional effects of
Nup93
depletion in a fly (
Drosophila melanogaster)
nephrocyte model.
Methods
Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS).
D. melanogaster Nup93
knockdown was achieved by RNA interference using nephrocyte-restricted drivers.
Results
Six novel homozygous and compound heterozygous
NUP93
variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis.
Silencing of the two orthologs of human
NUP93
expressed in
D. melanogaster
,
Nup93-1
, and
Nup93-2
resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in
Nup93-1
and
Nup93-2
silenced flies surviving to adulthood.
Conclusion
We expand the spectrum of
NUP93
variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either
D. melanogaster Nup93
ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology.
Graphical Abstract
A higher resolution version of the Graphical abstract is available as Supplementary information
Up to 95% of children presenting with steroid-resistant nephrotic syndrome in early life will have a pathogenic single-gene mutation in 1 of 24 genes currently associated with this disease. Others ...may be affected by polymorphic variants. There is currently no accepted diagnostic algorithm for clinical genetic testing. The hypothesis was that the increasing reliability of next generation sequencing allows comprehensive one-step genetic investigation of this group and similar patient groups.
This study used next generation sequencing to screen 446 genes, including the 24 genes known to be associated with hereditary steroid-resistant nephrotic syndrome. The first 36 pediatric patients collected through a national United Kingdom Renal Registry were chosen with comprehensive phenotypic detail. Significant variants detected by next generation sequencing were confirmed by conventional Sanger sequencing.
Analysis revealed known and novel disease-associated variations in expected genes such as NPHS1, NPHS2, and PLCe1 in 19% of patients. Phenotypically unexpected mutations were also detected in COQ2 and COL4A4 in two patients with isolated nephropathy and associated sensorineural deafness, respectively. The presence of an additional heterozygous polymorphism in WT1 in a patient with NPHS1 mutation was associated with earlier-onset disease, supporting modification of phenotype through genetic epistasis.
This study shows that next generation sequencing analysis of pediatric steroid-resistant nephrotic syndrome patients is accurate and revealing. This analysis should be considered part of the routine genetic workup of diseases such as childhood steroid-resistant nephrotic syndrome, where the chance of genetic mutation is high but requires sequencing of multiple genes.
Background
Idiop athic nephrotic syndrome (INS) is classified in children according to response to initial corticosteroid therapy into steroid-sensitive (SSNS) and steroid-resistant nephrotic ...syndrome (SRNS), and in adults according to histology into minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). However, there is well-recognised phenotypic overlap between these entities. Genome-wide association studies (GWAS) have shown a strong association between SSNS and variation at HLA, suggesting an underlying immunological basis. We sought to determine whether a risk score generated from genetic variants associated with SSNS could be used to gain insight into the pathophysiology of INS presenting in other ways.
Methods
We developed an SSNS genetic risk score (SSNS-GRS) from the five variants independently associated with childhood SSNS in a previous European GWAS. We quantified SSNS-GRS in independent cohorts of European individuals with childhood SSNS, non-monogenic SRNS, MCD, and FSGS, and contrasted them with SSNS-GRS quantified in individuals with monogenic SRNS, membranous nephropathy (a different immune-mediated disease-causing nephrotic syndrome), and healthy controls.
Results
The SSNS-GRS was significantly elevated in cohorts with SSNS, non-monogenic SRNS, MCD, and FSGS compared to healthy participants and those with membranous nephropathy. The SSNS-GRS in all cohorts with non-monogenic INS were also significantly elevated compared to those with monogenic SRNS.
Conclusions
The shared genetic risk factors among patients with different presentations of INS strongly suggests a shared autoimmune pathogenesis when monogenic causes are excluded. Use of the SSNS-GRS, in addition to testing for monogenic causes, may help to classify patients presenting with INS.
Graphical abstract
A higher resolution version of the Graphical abstract is available as
Supplementary information
PDF
