Konstantinos Voskarides

Identification of a mutation in complement factor H-related protein 5 in patients of Cypriot origin with glomerulonephritis.

Summary Background Complement is a key component of the innate immune system, and variation in genes that regulate its activation is associated with renal and other disease. We aimed to establish the genetic basis for a familial disorder of complement regulation associated with persistent microscopic haematuria, recurrent macroscopic haematuria, glomerulonephritis, and progressive renal failure. Methods We sought patients from the West London Renal and Transplant Centre (London, UK) with unusual renal disease and affected family members as a method of identification of new genetic causes of kidney disease. Two families of Cypriot origin were identified in which renal disease was consistent with autosomal dominant transmission and renal biopsy of at least one individual showed C3 glomerulonephritis. A mutation was identified via a genome-wide linkage study and candidate gene analysis. A PCR-based diagnostic test was then developed and used to screen for the mutation in population-based samples and in individuals and families with renal disease. Findings Occurrence of familial renal disease cosegregated with the same mutation in the complement factor H-related protein 5 gene (CFHR5). In a cohort of 84 Cypriots with unexplained renal disease, four had mutation in CFHR5. Overall, we identified 26 individuals with the mutation and evidence of renal disease from 11 ostensibly unrelated kindreds, including the original two families. A mutant CFHR5 protein present in patient serum had reduced affinity for surface-bound complement. We term this renal disease CFHR5 nephropathy. Interpretation CFHR5 nephropathy accounts for a substantial burden of renal disease in patients of Cypriot origin and can be diagnosed with a specific molecular test. The high risk of progressive renal disease in carriers of the CFHR5 mutation implies that isolated microscopic haematuria or recurrent macroscopic haematuria should not be regarded as a benign finding in individuals of Cypriot descent. Funding UK Medical Research Council and Wellcome Trust.

COL4A3/COL4A4 Mutations Producing Focal Segmental Glomerulosclerosis and Renal Failure in Thin Basement Membrane Nephropathy

Mutations in the COL4A3/COL4A4 genes of type IV collagen have been found in approximately 40% of cases of thin basement membrane nephropathy, which is characterized by microscopic hematuria and is classically thought to cause proteinuria and chronic renal failure rarely. Here we report our observations of 116 subjects from 13 Cypriot families clinically affected with thin basement membrane nephropathy. These families first came to our attention because they segregated microscopic hematuria, mild proteinuria, and variable degrees of renal impairment, but a dual diagnosis of focal segmental glomerulosclerosis (FSGS) and thin basement membrane nephropathy was made in 20 biopsied cases. Molecular studies identified founder mutations in both COL4A3 and COL4A4 genes in 10 families. None of 82 heterozygous patients had any extrarenal manifestations, supporting the diagnosis of thin basement membrane nephropathy. During follow-up of up to three decades, 31 of these 82 patients (37.8%) developed chronic renal failure and 16 (19.5%) reached end-stage renal disease. Mutations G1334E and G871C were detected in seven and three families, respectively, and were probably introduced by founders. We conclude that these particular COL4A3/COL4A4 mutations either predispose some patients to FSGS and chronic renal failure, or that thin basement membrane nephropathy sometimes coexists with another genetic modifier that is responsible for FSGS and progressive renal failure. The findings presented here do not justify the labelling of thin basement membrane nephropathy as a benign condition with excellent prognosis.

Familial C3 Glomerulopathy Associated with CFHR5 Mutations: Clinical Characteristics of 91 Patients in 16 Pedigrees

BACKGROUND AND OBJECTIVES Complement factor H and related proteins (CFHR) are key regulators of the alternative complement pathway, where loss of function mutations lead to a glomerulopathy with isolated mesangial C3 deposits without immunoglobulins. Gale et al. (12) reported on 26 patients with the first familial, hematuric glomerulopathy caused by a founder mutation in the CFHR5 gene in patients of Cypriot descent living in the United Kingdom. CFHR5 nephropathy is clinically characterized by continuous microscopic hematuria whereas some patients present with additional episodes of synpharyngitic macrohematuria, associated with infection and pyrexia. A subgroup of patients, particularly men, develop additional proteinuria, hypertension, and chronic renal disease or ESRD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS We herewith expand significantly on the study by Gale et al., reporting on histologic, molecular, and clinical findings in 91 patients from 16 families with the same founder mutation. RESULTS Eighty-two patients (90%) exhibited microscopic hematuria; 51 (62%), exhibited only microscopic hematuria, whereas the remaining 31 additionally had proteinuria (38%); 28 proteinuric patients developed chronic renal failure (CRF). Among carriers of CFHR5 mutation aged >50 years, 80% of the men and 21% of the women developed CRF; 18 developed ESRD (14 men [78%], 4 women [22%]). CONCLUSIONS The diagnosis of CFHR5-related, isolated C3 glomerulopathy was established in 2009 using newly described mutation analysis after decades of follow-up with unclear diagnoses, occasionally confused with IgA nephropathy. This larger patient cohort establishes the clinical course, significant variable expressivity, and marked gender difference regarding the development of CRF and ESRD.

Molecular genetics of familial hematuric diseases

The familial hematuric diseases are a genetically heterogeneous group of monogenic conditions, caused by mutations in one of several genes. The major genes involved are the following: (i) the collagen IV genes COL4A3/A4/A5 that are expressed in the glomerular basement membranes (GBM) and are responsible for the most frequent forms of microscopic hematuria, namely Alport syndrome (X-linked or autosomal recessive) and thin basement membrane nephropathy (TBMN). (ii) The FN1 gene, expressed in the glomerulus and responsible for a rare form of glomerulopathy with fibronectin deposits (GFND). (iii) CFHR5 gene, a recently recognized regulator of the complement alternative pathway and mutated in a recently revisited form of inherited C3 glomerulonephritis (C3GN), characterized by isolated C3 deposits in the absence of immune complexes. A hallmark feature of all conditions is the age-dependent penetrance and a broad phenotypic heterogeneity in the sense that subsets of patients progress to added proteinuria or proteinuria and chronic renal failure that may or may not lead to end-stage kidney disease (ESKD) anywhere between the second and seventh decade of life. In addition to other excellent laboratory tools that assist the clinician in reaching the correct diagnosis, the molecular analysis emerges as the gold standard in establishing the diagnosis in many cases of doubt due to equivocal findings that complicate the differential diagnosis. Recent work led to the description of candidate genetic modifiers which confer a variable risk for progressing to chronic renal failure when co-inherited on the background of a primary glomerulopathy. Finally, more families are still waiting to be studied and more genes to be mapped and cloned that are responsible for other forms of heritable hematuric diseases. The study of such genes and their protein products will likely shed more light on the structure and function of the glomerular filtration barrier and other important glomerular components.

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.

Evidence for Activation of the Unfolded Protein Response in Collagen IV Nephropathies

Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)-related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.

Evidence that NPHS2-R229Q predisposes to proteinuria and renal failure in familial hematuria

BackgroundFamilial hematuria (FH) is associated with at least two pathological entities: thin basement membrane nephropathy (TBMN), caused by heterozygous COL4A3/COL4A4 mutations, and C3 nephropathy caused by CFHR5 mutations. It is now known that TBMN patients develop proteinuria and changes of focal segmental glomerulosclerosis when biopsied. End-stage kidney disease (ESKD) is observed in 20% of carriers, at ages 50–70. A similar progression is observed in CFHR5 nephropathy. Recent evidence suggests that NPHS2-R229Q, a podocin polymorphism, may contribute to proteinuria in TBMN and to micro-albuminuria in the general population.Case-Diagnosis/TreatmentNPHS2-R229Q was screened in a Cypriot FH cohort. 102 TBMN patients with three known COL4 mutations and 45 CFHR5 male patients with a single mutation were categorized as “Mild” or “Severe”, based on the presence of microhematuria only, or proteinuria and chronic kidney disease. Nine R229Q carriers were found in the “Severe” category and none in the “Mild” (p=0.010 for genotypic association; p=0.043 for allelic association, adjusted for patients’ relatedness), thus supporting the possible contribution of 229Q allele in disease progress.ConclusionsOur results offer more evidence that in patients with FH, NPHS2-R229Q predisposes to proteinuria and ESKD. R229Q may be a good prognostic marker for young hematuric patients.

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.

A European spectrum of pharmacogenomic biomarkers: Implications for clinical pharmacogenomics

Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant inter-population pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective.

Increased number of microRNA target sites in genes encoded in CNV regions. Evidence for an evolutionary genomic interaction

MicroRNAs (miRNAs) and copy number variations (CNVs) are two newly discovered genetic elements that have revolutionized the field of molecular biology and genetics. By performing in silico whole genome analysis, we demonstrate that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA-binding sites are significantly higher than those of genes found in non-CNV regions. This suggests that miRNAs may have acted as equilibrators of gene expression during evolution in an attempt to regulate aberrant gene expression and to increase the tolerance to genome plasticity.