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

Description of cohorts and patients under study.

<p>Please note that cohort B1 is the male only patients with CFHR5 nephropathy.</p><p>MH: Microscopic Hematuria, ESKD: End Stage Kidney Disease, XLAS: X-linked Alport syndrome.</p>1<p>“Mild” patients born before 01/1963. Gender difference (Mild vs Severe) is not significant (p = 0.141).</p>2<p>“Mild” patients born before 01/1975. Gender difference (Mild vs Severe) is significant (p = 0.001).</p>3<p>“Severe” patients: ESKD≤40 yo.</p>4<p>“Mild” patients born before 01/1979. Gender difference (Mild vs Severe) is significant (p = 0.001).</p

<i>MYH9</i> rs11089788 statistical analysis for a replicate cohort (D) and for the sum of cohorts B and D, that gave statistical significance.

<p>Patients in cohort D belong to families that segregate microscopic hematuria but no known mutation has been found in any of the genes <i>COL4A3</i>, <i>COL4A4</i>, or <i>CFHR5</i>, so far.</p

Genotype associations for the three <i>MYH9</i> variants genotyped in this study.

<p>P-values were calculated by Pearson Chi-Square test. The whole CFHR5 cohort (B) was genotyped only for <i>MYH9</i> rs11089788, the only SNP that gave p-value close to 0.1 for the male CFHR5 patients (B1).</p>*<p>P-values calculated by Fisher's Exact Test (2-sided) due to existence of genotypes values less than 10.</p>**<p>Odds ratio (OR) cannot be estimated due to zero genotypic values in the “Severe” category.</p

Genotype distribution of the studied <i>MYH9</i> variants, by cohort and by severity.

<p>Genotype distribution of the studied <i>MYH9</i> variants, by cohort and by severity.</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

Characteristics of the pooled HEMATURIA cohort.

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