A miR-1207-5p Binding Site Polymorphism Abolishes Regulation of HBEGF and Is Associated with Disease Severity in CFHR5 Nephropathy

et al. (2012) A miR-1207-5p Binding Site Polymorphism Abolishes Regulation of HBEGF and Is Associated with Disease Severity in CFHR5 Nephropathy. PLoS ONE 7(2): e31021. doi:10.1371/journal.pone.0031021 A miR-1207-5p Binding Site Polymorphism Abolishes Regulation of HBEGF and Is Associated with Disease Severity in CFHR5 Nephropathy Gregory Papagregoriou 0 Kamil Erguler 0 Harsh Dweep 0 Konstantinos Voskarides 0 Panayiota Koupepidou 0 Yiannis Athanasiou 0 Alkis Pierides 0 Norbert Gretz 0 Kyriacos N. Felekkis 0 Constantinos Deltas 0 Niels Olsen Saraiva Camara, Universidade de Sao Paulo, Brazil 0 1 Molecular Medicine Research Center and Laboratory of Molecular and Medical Genetics, Department of Biological Sciences, University of Cyprus , Nicosia, Cyprus, 2 Medical Research Center, University of Heidelberg , Mannheim, Germany , 3 Department of Nephrology, Nicosia General Hospital , Nicosia , Cyprus , 4 Department of Nephrology, Hippocrateon Hospital , Nicosia , Cyprus , 5 Department of Life and Health Sciences, University of Nicosia , Nicosia , Cyprus Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 39UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 39UTR of HBEGF that corresponds to the second base of the hsa-miR1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsamiR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder. - Funding: This work was supported mainly by the George & Maria Tyrimos endowment through a grant to CD by the Pancyprian Gymnasium, Nicosia, as a scholarship to support GP and by a grant NEW INFRASTRUCTURE/STRATEGIC/0308/24 by the Cyprus Research Promotion Foundation (www.research.org.cy) to CD. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The inherited monogenic glomerulopathies is a genetically and phenotypically highly heterogeneous group of conditions. Even in specific monogenic diseases, the exact molecular pathomechanism underlying the variable expressivity is rarely well understood. This heterogeneity is exemplified by the observation that not all patients who develop chronic kidney disease (CKD) due to a primary genetic cause will proceed to end-stage kidney disease (ESKD). In such diseases, glomerular defects that include but are not limited to the glomerular basement membrane, the glomerular endothelium and the podocytes can alter the kidneys filtration barrier integrity and lead to an adverse outcome in patients. A subset of glomerular defects emerging from germinal mutations in specific genes or are acquired are directly reflected on podocytes, which may lose their structural integrity and functional properties [1,2]. Microscopic hematuria (MH) of glomerular origin can be a benign condition persisting for life or can be the starting point of a progressive process that may lead many years later to proteinuria and decline of renal function resulting in CKD or ESKD [3]. A prime example is thin basement membrane nephropathy (TBMN), where patients in the same family who bear an identical heterozygous mutation in either the COL4A3 or COL4A4 gene that encodes for the a3 or a4 chain of collagen type IV respectively, may follow a quite diverse disease course. In recent studies on a large cohort of patients we showed that a small percentage of patients will remain for life with benign isolated MH; however a larger fraction of patients will proceed to proteinuria and CKD. Overall 1520% of patients will have an even worse course and reach ESKD at ages after 50 years of age. In fact, nearly 50% of patients after 50 years will require hemodialysis or a renal transplant [4]. Similarly, in another recently revisited C3 glomerulopathy that is caused by mutations in the CFHR5 gene which plays a role in the regulation of the alternative pathway of complement activation, nearly all patients present with MH since childhood while they may also develop macroscopic hematuria as a response to infections of the upper respiratory tract. A subset of patients will remain stable but about 15%, predominantly males will develop proteinuria and CKD or ESKD [5]. Female patients appear to have a milder disease progression and according to our recently published work, 14/18 patients who reached ESKD were males. This variable expressivity might be explained by a host of factors including genetic modifiers through yet unknown molecular mechanisms. MicroRNA (miRNA) regulation of gene expression could be one of these factors. The role of miRNAs in processes such as maturation of the mammalian kidney was recently established by the podocytespecific inactivation of Dicer, the RNAse III endonuclease responsible for miRNA maturation, in mice [6,7,8]. Podocyte foot processes were consequently depleted, while apoptosis commenced. The affected animals initially developed albuminuria followed by glomerular sclerosis and tubulo-interstitial fibrosis with acute renal disease progression and eventually death of mice by 6 8 weeks. The pathological phenotype was completed by proteinuria, glomerular basement membrane abnormalities and mesangial expansion, assimilating a congenital glomerulopathy. This proves that miRNAs have a fundamental role in regulating kidney physiological development; hence they must have a role in renal disease as well. miRNAs belong to the most abundant class of small RNAs in animals. It is a recently discover (...truncated)