Date Published: March 23, 2017
Publisher: Public Library of Science
Author(s): Konstantinos Voskarides, Charalambos Stefanou, Myrtani Pieri, Panayiota Demosthenous, Kyriakos Felekkis, Maria Arsali, Yiannis Athanasiou, Dimitris Xydakis, Kostas Stylianou, Eugenios Daphnis, Giorgos Goulielmos, Petros Loizou, Judith Savige, Martin Höhne, Linus A. Völker, Thomas Benzing, Patrick H. Maxwell, Daniel P. Gale, Mathias Gorski, Carsten Böger, Barbara Kollerits, Florian Kronenberg, Bernhard Paulweber, Michalis Zavros, Alkis Pierides, Constantinos Deltas, Christos Chatziantoniou.
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.
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.
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 = 3.0×10-3, OR = 6.64 adjusting for gender/age; allelic association: p = 4.2×10-3 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 = 1.0×10-3). 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 = 1.3×10-5, 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.
Podocytes are terminally differentiated epithelial cells with multiple foot processes. A highly specialized cell junction, known as the slit diaphragm (SD), links adjacent foot processes of podocytes and is considered to be the most important selective barrier to protein leakage into the glomerular filtrate[1, 2].
Progression in primary hematuric glomerulopathies, inherited or not, is still an open question in nephrology. The phenotypic heterogeneity observed among patients with inherited monogenic disorders, including AS, TBMN and CFHR5 nephropathy, prompted us to hypothesize that the full spectrum of the phenotype behaves as a multifactorial condition, implicating primary genes, modifier genes and environmental factors [34, 37]. Here we considered that excellent candidates to act as genetic modifiers could be non-synonymous SNPs located in specific genes of the SD. Many of these genes have been linked with inherited nephrotic syndromes and corresponding encoded proteins are the main scaffold of the glomerular filtration barrier in the glomeruli. Under these assumptions we hypothesized that specific missense variants on these genes, perhaps acting as hypomorphic mutations, may contribute to progression in a subset of hematuric patients. Following a strategy that is detailed in Methods and Results sections, we focused on filtrin (Neph3), largely of unknown function, which has been shown to interact with various slit diaphragm proteins. After deriving a suggestive significance in a previously well-studied TBMN sub-cohort of 103 patients, we further evaluated this SNP in the pooled hematuric cohort, HEMATURIA, with 524 patients (Table 1), comprised mainly by TBMN and IgA nephropathy patients. Statistical analysis revealed a high risk for the carriers of this variant (genotypic association: p = 3.0×10-3, OR = 6.63 adjusting for gender/age; allelic association: p = 2.0×10-4) (Table 2). Pending replication by other researchers, our study suggests that variant NEPH3-V353M may have a prognostic value for an adverse outcome when it occurs in patients with a background of another primary hematuric glomerulopathy, such as TBMN or IgA nephropathy. This makes the variant qualify as a hypomorphic allele which on its own is not adequate to cause any perceptible symptom, when in heterozygosity. A similar inheritance pattern explained some rare severe and early onset cases of autosomal dominant polycystic kidney disease when hypomorphic PKD1 mutations were co-inherited with variants in the PKHD1 or the HNF-1β gene [58, 59].
We provided three lines of evidence based on evolutionary conservation, genetic association studies and functional assays, which support the role of variant NEPH3-p.V353M as a hypomorphic mutant with low or incomplete penetrance. Our findings may afford the opportunity for early detection of a subgroup of patients with glomerular hematuria who are at increased long-term risk of kidney function decline. With this in mind, this finding conforms to the low-frequency variant-large effect hypothesis in opposition to the common variant hypothesis. At the same time, homozygosity in the general population may also arise as a risk factor for micro-albuminuria of unknown significance to health. Admittedly, it is more than obvious that more genetic variants (perhaps hundreds or thousands), and environmental factors exist that contribute to this end-phenotype, and waiting to be identified. In our view, every family or patient could have their own additional variant that either predispose them to, or protect them from, adverse renal function developments, on the background of another primary glomerulopathy. At the same time, these same variants may predispose subjects of the general population, when inherited in homozygosity. If this is the case, molecular examinations of prognostic value could be massive parallel sequencing of panels of candidate genes, something that nowadays has become easier and cheaper with Next Generation Sequencing technologies.