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

A proposed hypothetical model for the mechanism of disease development when a causative heterozygous mutation is inherited on the <i>COL4A3/A4</i> gene or when a heterozygous mutation is co-inherited with a variant on a genetic modifier.

<p>This general model implies that hypomorphic mutations such as <i>NEPH3</i>-p.V353M are benign in heterozygosity on their own but may confer malfunction and impair the integrity of the glomerular filtration barrier when co-inherited with a collagen IV pathogenic mutation, or in homozygosity.</p

Frequencies and statistical analysis of variant <i>NEPH3</i>-p.V353M in the various hematuric sub-cohorts, based on disease severity.

<p>Frequencies and statistical analysis of variant <i>NEPH3</i>-p.V353M in the various hematuric sub-cohorts, based on disease severity.</p

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

Results of the densitometry analysis of western blots where we assayed the elevation of markers of the unfolded protein response pathway, in the presence of the two <i>NEPH3</i> alleles (normal “V” vs mutant “M”).

<p>Note that in the presence of tunicamycin (TM, a potent factor adding additional cellular stress), three markers (BiP, IRE1a, p-elF2a) are rising significantly (asterisks mark the significance) for the “M” allele.</p

Alignment of orthologs and paralogs sequences of the Neph3 protein (filtrin) around the 353V residue position.

<p>Note that there is absolute conservation of the relevant amino-acid residue, across a very broad evolutionary range.</p

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

Demographic data of the three general population cohorts genotyped for <i>NEPH3</i>-p.V353M.

<p>Demographic data of the three general population cohorts genotyped for <i>NEPH3</i>-p.V353M.</p

Characteristics of the pooled HEMATURIA cohort.

<p>Characteristics of the pooled HEMATURIA cohort.</p