Gregory Papagregoriou

A miR-1207-5p binding site polymorphism abolishes regulation of HBEGF and is associated with disease severity in CFHR5 nephropathy.

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 3′UTR 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 3′UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p 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 hsa-miR-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.

Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies.

Cystic diseases of the kidney: molecular biology and genetics.

CONTEXT Cystic diseases of the kidney are a very heterogeneous group of renal inherited conditions, with more than 33 genes involved and encompassing X-linked, autosomal dominant, and autosomal recessive inheritance. Although mostly monogenic with mendelian inheritance, there are clearly examples of oligogenic inheritance, such as 3 mutations in 2 genes, while the existence of genetic modifiers is perhaps the norm, based on the extent of variable expressivity and the broad spectrum of symptoms. OBJECTIVES To present in the form of a mini review the major known cystic diseases of the kidney for which genes have been mapped or cloned and characterized, with some information on their cellular and molecular biology and genetics, and to pay special attention to commenting on the issues of molecular diagnostics, in view of the genetic and allelic heterogeneity. Data Sources.-We used major reviews that make excellent detailed presentation of the various diseases, as well as original publications. CONCLUSIONS There is already extensive genetic heterogeneity in the group of cystic diseases of the kidney; however, there are still many more genes awaiting to be discovered that are implicated or mutated in these diseases. In addition, the synergism and interaction among this repertoire of gene products is largely unknown, while a common unifying aspect is the expression of nearly all of them at the primary cilium or the basal body. A major interplay of functions is anticipated, while mutations in all converge in the unifying phenotype of cyst formation.

New miRNA Profiles Accurately Distinguish Renal Cell Carcinomas and Upper Tract Urothelial Carcinomas from the Normal Kidney

Background Upper tract urothelial carcinomas (UT-UC) can invade the pelvicalyceal system making differential diagnosis of the various histologically distinct renal cell carcinoma (RCC) subtypes and UT-UC, difficult. Correct diagnosis is critical for determining appropriate surgery and post-surgical treatments. We aimed to identify microRNA (miRNA) signatures that can accurately distinguish the most prevalent RCC subtypes and UT-UC form the normal kidney. Methods and Findings miRNA profiling was performed on FFPE tissue sections from RCC and UT-UC and normal kidney and 434 miRNAs were significantly deregulated in cancerous vs. the normal tissue. Hierarchical clustering distinguished UT-UCs from RCCs and classified the various RCC subtypes among them. qRT-PCR validated the deregulated expression profile for the majority of the miRNAs and ROC analysis revealed their capability to discriminate between tumour and normal kidney. An independent cohort of freshly frozen RCC and UT-UC samples was used to validate the deregulated miRNAs with the best discriminatory ability (AUC>0.8, p<0.001). Many of them were located within cytogenetic regions that were previously reported to be significantly aberrated. miRNA targets were predicted using the miRWalk algorithm and ingenuity pathway analysis identified the canonical pathways and curated networks of the deregulated miRNAs. Using the miRWalk algorithm, we further identified the top anti-correlated mRNA/miRNA pairs, between the deregulated miRNAs from our study and the top co-deregulated mRNAs among 5 independent ccRCC GEO datasets. The AB8/13 undifferentiated podocyte cells were used for functional assays using luciferase reporter constructs and the developmental transcription factor TFCP2L1 was proved to be a true target of miR-489, which was the second most upregulated miRNA in ccRCC. Conclusions We identified novel miRNAs specific for each RCC subtype and UT-UC, we investigated their putative targets, the networks and pathways in which they participate and we functionally verified the true targets of the top deregulated miRNAs.

NPHS2 screening with SURVEYOR in Hellenic children with steroid-resistant nephrotic syndrome

Sirs,The idiopathic nephrotic syndrome is a common clinico-pathological entity characterized by massive proteinuria,hypoalbuminaemia, hyperlipidaemia, oedema, and variousglomerular changes, occurring mainly in children in 15–20% of whom the condition is steroid-resistant. About 85%of patients with steroid-resistant nephrotic syndrome(SRNS) exhibit renal histology of focal segmental glomer-ulosclerosis (FSGS), and the rest exhibit mesangial prolif-erative glomerulonephritis (MsPGN) or other rarerhistological phenotypes [1]. Mutations in the NPHS2 gene,encoding podocin, which is one of the important proteins ofthe slit diaphragm, are a frequent cause of sporadic SRNSin children, occurring in 2.8–28% of the cases [2–4].Mutations in exons 8 and 9 of the WT1 gene have also beenreported (more frequently in girls) with isolated SRNS [5].Other genes that are responsible have been recentlyreported, accounting for rare cases of SRNS.Idiopathic nephrotic syndrome is a frequent glomerulardisease in Cyprus and Greece, where 15–20% of cases aresteroid resistant, in accordance with the literature. In thiswork we studied for the first time in Greece and Cyprus acohort of 24 children (ten boys, 14 girls) with SRNS. Renalhistology, based on 1–3 biopsies, showed changes in FSGSin 21 children and MsPGN in three children. We investi-gated these children at the molecular level by searching formutations in the NPHS2 and WT1 (exons 8 and 9) genes. Indoing so, we used, for the first time to our knowledge,SURVEYOR endonuclease (Transgenomic, UK), an en-zyme that cleaves double-stranded DNA at positions ofheteroduplex mismatches, as a method for identifyingmutations and/or polymorphic variants. For this, genomicsequences, amplified by polymerase chain reaction (PCR)and encompassing the exons, the consensus exon

Frequent COL4 mutations in familial microhematuria accompanied by later-onset Alport nephropathy due to focal segmental glomerulosclerosis

Familial microscopic hematuria (FMH) is associated with a genetically heterogeneous group of conditions including the collagen‐IV nephropathies, the heritable C3/CFHR5 nephropathy and the glomerulopathy with fibronectin deposits. The clinical course varies widely, ranging from isolated benign familial hematuria to end‐stage renal disease (ESRD) later in life. We investigated 24 families using next generation sequencing (NGS) for 5 genes: COL4A3, COL4A4, COL4A5, CFHR5 and FN1. In 17 families (71%), we found 15 pathogenic mutations in COL4A3/A4/A5, 9 of them novel. In 5 families patients inherited classical AS with hemizygous X‐linked COL4A5 mutations. Even more patients developed later‐onset Alport‐related nephropathy having inherited heterozygous COL4A3/A4 mutations that cause thin basement membranes. Amongst 62 heterozygous or hemizygous patients, 8 (13%) reached ESRD, while 25% of patients with heterozygous COL4A3/A4 mutations, aged >50‐years, reached ESRD. In conclusion, COL4A mutations comprise a frequent cause of FMH. Heterozygous COL4A3/A4 mutations predispose to renal function impairment, supporting that thin basement membrane nephropathy is not always benign. The molecular diagnosis is essential for differentiating the X‐linked from the autosomal recessive and dominant inheritance. Finally, NGS technology is established as the gold standard for the diagnosis of FMH and associated collagen‐IV glomerulopathies, frequently averting the need for invasive renal biopsies.

Therapeutic potential of omega-3 fatty acids supplementation in a mouse model of dry macular degeneration

Purpose To evaluate the therapeutic effects of omega-3 (ω-3) and omega-6 (ω-6) fatty acids in the CCL2−/− model of dry age-related macular degeneration (AMD). The blood level of eicosapentaenoic acid (EPA) and arachidonic acid (AA) served to adjust the treatment dosage (AA/EPA=1–1.5). Methods Nine-month-old animals were allocated to different groups: (A) C57BL/6 untreated , (B) CCL2−/− untreated, (C) CCL2−/− treated with ω-3+ω-6, and (D) CCL2−/− treated with ω-3. Treatment was daily administered by gavage for 3 months. Fatty acids analysis was performed and retinas were histologically examined. Three-month-old wild type mice were used for comparison purposes. Real-time PCR and Western blot were performed for retinal inflammatory mediators. Results Increased EPA and decreased AA levels were observed in both blood and retinas in the treatment groups. The outer nuclear layer thickness was increased in groups C (45.0±3.9 µm) and D (62.8±4.9 µm), compared with groups B (65.6±3.0 µm) and A (71.1±4.2 µm), and in younger mice, it was 98.0±3.9 µm. A decrease in NF-κB expression was noted in the treatment groups. Interleukin (IL) 18 protein levels demonstrated a significant reduction in the ω-3-treated group only. Conclusion Supplementation with ω-3+ω-6 or ω-3 alone (AA/EPA=1–1.5) suggests a protective mechanism in the CCL2−/− animal model of dry AMD, with a more beneficial effect when ω-3 are used alone. Our findings indicated that inflammation is not the only determining factor; perhaps a regenerative process might be involved following administration of ω-3 fatty acids.

A novel splice-site mutation in ATP6V0A4 gene in two brothers with distal renal tubular acidosis from a consanguineous Tunisian family

1LR11IPT05, Laboratoire de Genomique Biomedicale et Oncogenetique, Institut Pasteur de Tunis, 1002, Universite de Tunis El Manar, 1068 Tunis, Tunisia 2Molecular Medicine Research Center and Laboratory of Molecular and Medical Genetics, Department of Biological Sciences, University of Cyprus, Kallipoleos 75, 1678 Nicosia, Cyprus 3Department of Pediatrics B, Children’s Hospital of Tunis, Boulevard 9 avril, Bab Saadoun, Jabbary, 1007 Tunis, Tunisia

Renal graft outcome in autosomal dominant medullary cystic kidney disease type 1.

BACKGROUND Medullary cystic kidney disease (MCKD) is an inherited interstitial nephritis, leading to end-stage renal disease (ESRD) between the fourth and seventh decade of life. MCKD shares clinical and morphological features with nephronophthisis, although advances in molecular genetics have distinguished these 2 entities. Data regarding graft survival after kidney transplantation in MCKD patients are extremely limited. The aim of this study was to compare renal graft survival in transplanted MCKD1 and non-MCKD1 patients, to discover whether renal transplantation can be considered as an acceptable treatment for MCKD. METHODS Thirty-three transplanted patients with MCKD1 and 33 controls (transplanted due to other causes) were included in the study. Graft losses were considered censored for death. Graft survival was evaluated with the Kaplan-Meier method, and comparisons between groups were made by log-rank test. Cox regression analysis was used to estimate the effect of several variables on graft survival, and the chi-square test was used to compare groups of categorical data. RESULTS The 1-year cumulative graft survival rate for the MCKD1 group was 97%, while at 5 and 10 years it was 94% and 86%, respectively. For the control group, the respective values at years 1, 5 and 10 were 97%, 97% and 90%. Comparisons of graft survival rates between the 2 groups revealed no significant differences. CONCLUSIONS Renal graft survival of transplanted MCKD1 patients was not shown to be inferior in comparison with that for patients undergoing transplants due to other causes. Therefore, it may represent a treatment of choice in MCKD1 patients with ESRD.

Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease

BACKGROUND Autosomal dominant tubulointerstitial kidney disease caused by mucin-1 gene (MUC1) mutations (ADTKD-MUC1) is characterized by progressive kidney failure. Genetic evaluation for ADTKD-MUC1 specifically tests for a cytosine duplication that creates a unique frameshift protein (MUC1fs). Our goal was to develop immunohistochemical methods to detect the MUC1fs created by the cytosine duplication and, possibly, by other similar frameshift mutations and to identify novel MUC1 mutations in individuals with positive immunohistochemical staining for the MUC1fs protein. METHODS We performed MUC1fs immunostaining on urinary cell smears and various tissues from ADTKD-MUC1-positive and -negative controls as well as in individuals from 37 ADTKD families that were negative for mutations in known ADTKD genes. We used novel analytic methods to identify MUC1 frameshift mutations. RESULTS After technique refinement, the sensitivity and specificity for MUC1fs immunostaining of urinary cell smears were 94.2% and 88.6%, respectively. Further genetic testing on 17 families with positive MUC1fs immunostaining revealed six families with five novel MUC1 frameshift mutations that all predict production of the identical MUC1fs protein. CONCLUSIONS We developed a noninvasive immunohistochemical method to detect MUC1fs that, after further validation, may be useful in the future for diagnostic testing. Production of the MUC1fs protein may be central to the pathogenesis of ADTKD-MUC1.