A functional variant in NEPH3 gene confers high risk of renal failure in primary hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in the general population.

BACKGROUND: Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms. METHODS: We looked in silico for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as "Severe" or "Mild", based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the NEPH3-V353M variant has any renal effect in the general population. RESULTS AND CONCLUSIONS: Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant NEPH3-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10-3 adjusting for patients' kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a "rare variant-strong effect" role for NEPH3-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria

Evidence for activation of the unfolded protein response in collagen IV nephropathies

Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)–related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies

Co-immunoprecipitation experiments, testing for the binding effectiveness of Neph3 protein with methionine (M) at the 353 position.

<p><b>(A)</b> Left panel: FLAG-Neph3[353<b>V</b>] and FLAG-Neph3[353<b>M</b>] were immuno-precipitated with anti-FLAG antibody and then they were analyzed by western blot using an anti-HA antibody (for HA-Neph3[353<b>V</b>] and HA-Neph3[353<b>M</b>]) in order to check all possible hetero- and homo-dimer interactions. Neph3[353<b>M</b>]-Neph3[353<b>M</b>] homodimers are strongly increased compared with the Neph3[353<b>V</b>]-Neph3[353<b>M</b>] and the Neph3[353<b>V</b>]-Neph3[353<b>V</b>] ones. Right panel: FLAG-Neph3[353<b>V</b>] and FLAG-Neph3[353<b>M</b>] were immuno-precipitated with anti-FLAG antibody and then they were analyzed by western blot using an anti-V5 antibody (for sV5-Nephrin]. Nephrin-Neph3[353<b>M</b>] heterodimers are slightly increased compared with the Nephrin -Neph3[353<b>V</b>] ones. Anti-V5 and anti-FLAG western blots from lysates (input) were used for loading normalization. V5-NPHP1 (nephrocystin) served as an experiment control. <b>(B)</b> Statistics of densitometry of the blots in Fig 3A, n = 3. Intensity (±SEM) is given as a percentage of wild-type intensity, which is set by definition at 1.0 (100%). There is statistical significance (unpaired t-test) for the homodimerization and heterodimerization comparisons described in Fig 3A. For more details see text.</p

Flowchart of the genotyping strategy followed to investigate the significance of the two SNPs in four genes that emerged to have functional significance based on the <i>in silico</i> assay (see material and methods).

<p>Black arrow symbolizes the <i>NEPH3</i>-V353M variant which initially derived indicative significant association and was investigated further (black not filled arrows symbolize the meta-analysis). Light grey arrows symbolize the three SNPs found not to be significantly associated in sub-cohort A and not tested further. Dot-lined arrows symbolize the two SNPs that were found to be non-polymorphic in this cohort and not tested further.</p

A functional variant in <i>NEPH3</i> gene confers high risk of renal failure in primary hematuric glomerulopathies. Evidence for predisposition to microalbuminuria in the general population

<div><p>Background</p><p>Recent data emphasize that thin basement membrane nephropathy (TBMN) should not be viewed as a form of benign familial hematuria since chronic renal failure (CRF) and even end-stage renal disease (ESRD), is a possible development for a subset of patients on long-term follow-up, through the onset of focal and segmental glomerulosclerosis (FSGS). We hypothesize that genetic modifiers may explain this variability of symptoms.</p><p>Methods</p><p>We looked <i>in silico</i> for potentially deleterious functional SNPs, using very strict criteria, in all the genes significantly expressed in the slit diaphragm (SD). Two variants were genotyped in a cohort of well-studied adult TBMN patients from 19 Greek-Cypriot families, with a homogeneous genetic background. Patients were categorized as “Severe” or “Mild”, based on the presence or not of proteinuria, CRF and ESRD. A larger pooled cohort (HEMATURIA) of 524 patients, including IgA nephropathy patients, was used for verification. Additionally, three large general population cohorts [Framingham Heart Study (FHS), KORAF4 and SAPHIR] were used to investigate if the <i>NEPH3</i>-V353M variant has any renal effect in the general population.</p><p>Results and conclusions</p><p>Genotyping for two high-scored variants in 103 TBMN adult patients with founder mutations who were classified as mildly or severely affected, pointed to an association with variant <i>NEPH3</i>-V353M (filtrin). This promising result prompted testing in the larger pooled cohort (HEMATURIA), indicating an association of the 353M variant with disease severity under the dominant model (p = 3.0x10^-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2x10^-3 adjusting for patients’ kinships). Subsequently, genotyping 6,531 subjects of the Framingham Heart Study (FHS) revealed an association of the homozygous 353M/M genotype with microalbuminuria (p = 1.0x10^-3). Two further general population cohorts, KORAF4 and SAPHIR confirmed the association, and a meta-analysis of all three cohorts (11,258 individuals) was highly significant (p = 1.3x10^-5, OR = 7.46). Functional studies showed that Neph3 homodimerization and Neph3-Nephrin heterodimerization are disturbed by variant 353M. Additionally, 353M was associated with differential activation of the unfolded protein response pathway, when overexpressed in stressed cultured undifferentiated podocyte cells, thus attesting to its functional significance. Genetics and functional studies support a “rare variant-strong effect” role for <i>NEPH3</i>-V353M, by exerting a negative modifier effect on primary glomerular hematuria. Additionally, genetics studies provide evidence for a role in predisposing homozygous subjects of the general population to micro-albuminuria.</p></div

Frequencies and statistics of <i>NEPH3</i>-p.V353M in three general population cohorts including a meta-analysis.

<p>Frequencies and statistics of <i>NEPH3</i>-p.V353M in three general population cohorts including a meta-analysis.</p