Massive parallel sequencing in steroid-resistant nephrotic syndrome (SRNS)

<p><strong>ABSTRACT</strong></p> <p><strong>Introduction</strong>. To date abnormalities in more than 20 genes have been associated with SRNS. Sequencing of all SRNS genes requires ~ 600 PCR amplicons, rendering conventional mutation testing unfeasible due to financial and time constraints. Hence, current screening algorithms usually include only the most common disease genes and/or use preselection according to additional phenotypic criteria. This practice typically allows for mutation detection in ~15% of patients. We have evaluated targeted NGS screening of SRNS patients enrolled in the PodoNet registry who were found negative for mutations in the first-line SRNS-associated genes.</p> <p><strong>Material and Methods</strong>. Molecular analysis of 31 known or plausible SDNS disease genes was performed by NGS using a custom-designed multiplex PCR kit (MASTR FSGS, Multiplicom). The pilot group consisted of 22 patients with 4.7 years median age at disease onset (range 0.5-20 years), positive family history in 68%, parental consanguinity in 18% and chronic renal insufficiency at last observation in 59%.</p> <p><strong>Results.</strong> Mean coverage was 1269x (median 1286, range 929-1826). 18/22 runs had at least 15x read depth covering 99% of the target sequences. One patient was diagnosed with hereditary SRNS due to a previousy described homozygous pathogenic mutation in SMARCAL1 gene. In addition, three novel sequence variants in the genes PLCE1 (homozygous), LAMB2 (homozygous) and WT1 (heterozygous) were detected. In silico studies support their classification as pathogenic, even though the patients do not present the characteristic clinical and/or histopathological features typically reported for patients with mutations in these genes.</p> <p><strong>Conclusions</strong>. Our detection of pathogenic mutations in 4 out of 22 SRNS patients screened negative by conventional selective screening approaches support targeted NGS testing in all SRNS patients, regardless of age at diagnosis, absence of extrarenal manifestations or histological subtype. We anticipate that systematic NGS screening of the SRNS cohorts collected in EURenOmics will allow re-evaluation of mutation incidence rates in SRNS and become the new standard of genetic diagnostics in this condition.</p

Podocyte foot process effacement in <i>Nphs2</i>^{<i>R140Q/-</i>} mice.

<p>(A) Ultrastructural studies showed regular foot processes (FP) in healthy control animals on the opposite side of endothelial cells (En) lining the capillary lumen (L). (B) Irregularly shaped or fused FPs in <i>Nphs2</i>^{<i>R140Q/-</i>} mice one week after induction (arrow). (C and D) Progression of focal changes to global fusion of FPs in <i>Nphs2</i>^{<i>R140Q/-</i>} animals over time (Magnification, X10000). 3D modelling of glomerular structure showed no GBM denudation in <i>Nphs2</i>^{<i>R140Q/-</i>} animal (F) compared to controls (E). Blue colour represents GBM, pink and green represent FPs of adjacent podocytes. Severely affected FP number and organization in <i>Nphs2</i>^{<i>R140Q/-</i>} animals (G). GBM thickening in <i>Nphs2</i>^{<i>R140Q/-</i>} animals (H) (G and H: analysis is based on 3 animals per group). ** p<0.01, **** p<0.0001.</p

Podocin loss leads to renal damage.

<p>(A) control, (B) week 2, (C) week 4, (D) week 12. (E) Percentage of total kidney area affected by fibrosis in <i>Nphs2</i>^{<i>R140Q/-</i>} mice increased with observation time (columns represent 4–10 animals per group and time point). (F) Proteinuria 2 weeks after induction is correlated with the tubulointerstitial fibrosis score at week 4 (n = 17; p = 0.01). SR staining; Magnification, X150. * p<0.05, ** p<0.001.</p

Progressive loss of glomerular podocin abundance in the course of disease.

<p>Podocin (green), nidogen (red) and nucleus (blue) staining of glomeruli of healthy and <i>Nphs2</i>^{<i>R140Q/-</i>} animals. (A) Normally expressed podocin in glomerulus of a healthy animal. (B) Partial podocin loss one week after the induction. (C) Immense podocin loss in <i>Nphs2</i>^{<i>R140Q/-</i>} animals four weeks after the induction. (D) Subtotal to total podocin loss at the end stage disease. Magnification, X640.</p

Whereas mRNA expression of mutant <i>Nphs2</i> is elevated, podocin protein abundance is diminished.

<p>(A) <i>Nphs2</i>^{<i>R140Q/-</i>} animals showed an elevated expression level of mutated podocin mRNA during the first four weeks following induction (analysis is based on 4–6 animals per group and time point) WT: wild type. (B) Western blot analysis of total kidney extracts showing partial podocin protein loss during first two weeks, subtotal loss after 4–6 weeks and complete loss at attainment of end-stage renal disease (week 12–16) (analysis is based on 4–6 animals per group and time point; p<0.05).</p

Podocyte loss in <i>Nphs2</i>^{<i>R140Q/-</i>} mice.

<p>(A) Number of podocytes reduced in induced mice with the course of the disease (columns represent 4–6 animals per group and time point). (B) Wt1 labelled podocytes in glomerulus in a healthy animal. Decreased Wt1 signal in <i>Nphs2</i>^{<i>R140Q/-</i>} animals 1 week (C), 2 weeks (D), and 4 weeks after induction (E). Wt1: green, nidogen: red, nucleus: blue. Magnification, X640.</p

Time course of clinical phenotype in different hereditary podocytopathy mouse models.

<p>Time course of clinical phenotype in different hereditary podocytopathy mouse models.</p

<i>Wt1</i> regulation in induced animals.

<p><i>Wt1</i> expression is significantly reduced in <i>Nphs2</i>^{<i>R140Q/-</i>} animals 4 weeks after disease induction (columns represent 5–7 animals per group and time point). **p = 0.0002.</p

<i>Nphs2</i>^{<i>R140Q/-</i>} mice develop focal-segmental glomerulosclerosis (FSGS).

<p>(A) Glomerular sclerosis index (GSI) in healthy and <i>Nphs2</i>^{<i>R140Q/-</i>} mice. (B) Percentage of glomeruli affected by sclerosis in <i>Nphs2</i>^{<i>R140Q/-</i>} mice increased drastically over time (columns represent 4–10 animals per group and time point). (C-F) Evolution of glomerular lesions in induced animals. PAS staining; Magnification, X200.</p

Novel <i>NEK8</i> Mutations Cause Severe Syndromic Renal Cystic Dysplasia through YAP Dysregulation

<div><p>Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. <i>NEK8/NPHP9</i> mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, <i>situs inversus</i>, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of <i>NEK8</i> mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to <i>NEK8</i> loss of function, highlighting their likely gain-of-function effect. We also showed that <i>NEK8</i> missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of <i>Nek8</i>-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of <i>Jck</i> mice. Moreover, co-injection of <i>nek8</i> MO with WT or mutated <i>NEK8-GFP</i> RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human <i>YAP</i> RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of <i>Nek8</i>-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that <i>NEK8</i> human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.</p></div