Next-Generation Sequencing for New Gene Identification and for Diagnosis in Steroid-Resistant Nephrotic Syndrome

Nephrotic syndrome is clinically characterized by massive proteinuria, and hypoalbuminemia. It represents a heterogeneous group of glomerular disorders characterized by distinct causes and histopathologic lesions. The present thesis evaluates the contribution of genetics in conditions commonly associated with NS: the immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN), the C3 glomerulopathy (C3G) and the podocytopathies, usually characterized by steroid-resistant nephrotic syndrome (SRNS). The first part of the thesis focused on IC-MPGN and C3G. We found likely pathogenic (LP) variants in complement alternative pathway genes in 18% of patients and describe, for the first time, thrombomodulin rare pathogenic variants in C3G patients. Interestingly, mutations alone did not increase the risk of developing IC-MPGN or C3G, but they did so when combined with common susceptibility variants. The prevalence of alternative pathway abnormalities (mutations and/or C3 Nephritic Factors, C3NeFs) was similar in IC-MPGN (53-56%) and C3G (64-65%). To investigate for more homogeneous subgroups within IC-MPGN/C3G, an operator-independent approach was applied using genetic, biochemical, histological and clinical data. Four different groups emerged showing distinct pathogenesis, and histological and clinical features. In the second part, the genetic causes of podocytopathies were investigated. LP variants in podocytopathy-associated genes are found in 23% of SRNS patients, prevalently in COL4A3-5 genes. Moreover, 8% of patients carried LP variants in Congenital Anomalies of the Kidney and the Urinary Tract (CAKUT)-associated genes, which were not previously associated with podocytopathies. LP variants in CAKUT-associated and podocytopathy-associated genes frequently combined together. Finally, possibly pathogenic variants in EPB41L45, a candidate gene for podocytopathies, were identified in 3 unrelated patients. In conclusion, this thesis contributes to understand the complex genetic basis of both IC-MPGN/C3G and podocytopathies. It introduces new players in the pathogenesis of these diseases. Finally, it provides evidence of the presence of subgroups within IC-MPGN/C3G with distinct underlying mechanisms, and clinical and histopathologic features

Genetic basis of steroid resistant nephrotic syndrome in Indian and Black South African children.

Master of Medicine in Paediatrics & Child Health. University of KwaZulu-Natal. Durban, 2017.Abstract available in PDF file

An Update on Glomerulopathies

An Update on Glomerulopathies - Clinical and Treatment Aspects is a systemic overview of recent advances in clinical aspects and therapeutic options in major syndromes of glomerular pathology. The book contains twenty four chapters divided conveniently into five sections. The first section deals with primary glomerulopathies, and the second section is devoted to glomerulopathies complicating infectious conditions. The third section deals with systemic autoimmune disorders and vasculitides which constitute major causes of glomerular disease and often renal failure. The fourth section includes chapters discussing the glomerular involvement in some major metabolic and systemic conditions. The final section has chapters which relate to some general aspects of glomerular diseases. This book will form an excellent reference tool for practicing and academic nephrology community

Genomic profiling in rare kidney disease

Rare diseases (RD), generally defined by an incidence of less than 1:2000, affect about 3-6% of the population. To date, over 600 different genetic kidney diseases have been identified. Most of them with the exception of autosomal dominant polycystic kidney disease (ADPKD) are rare to ultrarare disorders. Due to their rarity and often genetic heterogeneity, analysis is difficult and diagnosis is frequently delayed. Clinically, (rare) kidney diseases (RKD) are mainly divided into the following categories: Congenital or developmental kidney and urogenital tract malformations, electrolytes or metabolic disorder, glomerular disease, secondary renal, hereditary renal cancer syndromes, or tubulointerstitial kidney disease. Steroid-resistant nephrotic syndrome (SRNS) and focal segmental glomerulosclerosis (FSGS), are leading causes of end-stage renal disease (ESRD) in children, adolescents, and adults. Although several SRNS genes could be identified mostly in younger children, the genetic basis of SRNS/FSGS in adolescents and adults is far from being completely understood. Reliable discrimination of genetic versus non-genetic forms is an imperative as the identification of monogenic rare kidney disease has numerous implications in a precision medicine setting. Currently, the predominant application of short-read based sequencing techniques results in preferential detection of point mutations and small-sized deletions/insertions while larger structural aberrations (large deletions/insertions), gene rearrangements, and mutations in homologous or repetitive regions frequently escape detection. This project combines short-read based high throughput next-generation sequencing (GPS/WES/WGS) with unparalleled structural variant analyses to overcome previous limitations in genetic analyses. Clinically relevant examples, that such structural variants (SV) are important in rare kidney diseases, are complement activation gene cluster (RCA; chromosome1q32) in atypical haemolytic uremic syndrome (aHUS) as well as the deletion of the chloride channel ClC-Kb associated with Bartter syndrome type 3. The major goal of this project is to unravel the genetic basis of genetic forms of RKD like SRNS/FSGS, aHUS, and tubulopathies and to define a pipeline for the molecular genetic analyses of rare kidney diseases as a best-practice clinical routine at the University Hospital of Cologne. Specifically, my aim was to perform profound genome analyses using biosamples from a cohort of paediatric and adult SRNS/FSGS patients, that have been collected over the last years and integrate this with already existing genetic data. By a comprehensive genomic approach, we aim to improve the diagnostics of chronic kidney disease, enhance our IV understanding of the underlying pathomechanisms, and contribute to practice-changing discoveries by precision diagnostics, that allow individualized therapies

Biomarkers Predicting Treatment-Response in Nephrotic Syndrome of Children: A Systematic Review

Purpose Nephrotic syndrome (NS) is the most common form of glomerulopathy in children. Most pediatric patients respond to glucocorticosteroid treatment (steroid-sensitive NS, SSNS), while approximately 10–15% will remain unresponsive or later become steroid-resistant. There has been a long-standing effort to find biomarkers that may predict steroid responsiveness. Methods We systematically reviewed current studies which investigated clinically relevant biomarkers for predicting steroid responsiveness in pediatric NS. We performed a PubMed and EMBASE search to identify eligible articles. We collected data on urinary markers, blood/serum markers (including cellular phenotypes and mRNA expression), genotypes and HLA allele frequency. Results A total of 659 articles were identified following electronic and manual searches. After reviewing the titles, abstracts, and full texts, 72 eligible articles were finally included. Vitamin D-binding protein (VDBP) seemed to be significantly elevated in SRNS than in SSNS, in both serum and urine specimen, although further validation is required. Conclusions The present paper narratively illustrates current understandings of potential biomarkers that may help predict steroid responsiveness. Further investigation and collaboration involving a larger number of patients are necessary

Loss of cytoskeletal and polarity regulation as key pathogenic principles in glomerular disease

Injury to glomerular podocytes constitutes the predominant cause of glomerular diseases and therefore loss of kidney function. Podocytes are specialized epithelial cells within glomeruli indispensable for the filtration process. Their cell bodies extrude numerous large primary and smaller foot processes that completely enwrap glomerular capillaries. The only cell-cell contact established between adjacent foot processes of neighbouring cells is the slit diaphragm. In addition to contributing to the filtration barrier and preventing the loss of blood proteins into the urine, the slit diaphragm is a vital signaling platform that integrates external signals with the intracellular signaling machinery. The sophisticated architecture of podocytes and their foot processes requires a dynamic actin cytoskeleton and its tight regulation through pathways such as polarity signalling. Upon injury, podocytes start to retract their foot processes, leading to loss of the slit diaphragm and ultimately disruption of the filter apparatus. A common finding upon this so-called effacement are actin cytoskeleton rearrangements. Understanding pathways and key players of actin regulation in podocytes that could prevent the progression of foot process effacement is therefore desirable. This thesis investigated the loss of actin and polarity regulation as key principles of podocyte injury. A first focus was thereby put on utilizing the fruit fly Drosophila melanogaster and in particular nephrocytes as in vivo model for glomerular diseases and to establish solid and reproducible experimental read-outs. Expression of human transgenes in nephrocytes was then used to characterize a so far unknown patient mutation in alpha-Actinin 4, an actin-crosslinking protein. In addition to demonstrating that the mutation alters actin localization, the Drosophila model was utilized to assess the pathogenic potential of the mutant protein. In light of the fact that expression of the mutant variant resulted in severe morphological and functional phenotypes in nephrocytes, it was determined that the novel variant is indeed a pathogenic ACTN4 variant. By this, the power of Drosophila as in vivo tool to complement clinical and genetic diagnostics was emphasized. Lastly, a possible link between podocyte polarity signaling and actin regulation was further investigated by using both, murine and Drosophila models. Here, single and double knockout of Par3A and Par3B in murine podocytes revealed, that the proteins share redundant functions, as only double knockout of Par3A and -B led to a glomerular phenotype. Further analyses in Drosophila nephrocytes focussed on knockdown of the Drosophila Par3 homologue bazooka. Depletion of Bazooka in nephrocytes led to differential expression of various actin-binding and -regulating proteins, including increased activity of the small GTPase Rho1 (the Drosophila RhoA homologue). Interestingly, Rho1 activation could be reverted upon transgenic expression of murine Par3A but not Par3B, indicating that the two proteins also exhibit distinct but unknown functions. These findings strengthen the link between perturbed polarity signalling and dysregulated actin dynamics, making polarity proteins a strong target to possibly counteract the progression of podocyte disease

Rare Kidney Diseases

Rare kidney diseases comprise a large group of different life-threatening or chronically debilitating disorders that affect very small numbers of people (<1 in 2000 individuals in Europe and <200,000 in USA) with local or systemic manifestations. For several years, the research and development of treatments in this field have been neglected in favor of more common diseases. The main reasons for the lack of interest in rare kidney diseases seem to be the small numbers of patients and limited epidemiological data on the natural history of many of these diseases. Rare diseases can affect people differently. Even patients with the same condition can exhibit very different signs and symptoms, or there may be many subtypes of the same condition. This diversity constitutes a significant challenge to healthcare practitioners and scientists alike, in terms of being able to acquire sufficient experience for the most appropriate and timely definition, diagnosis, and management. Fortunately, in the last ten years, concerted efforts have led to a marked improvement in the understanding of these disorders. In particular, an important step forward has been taken with the employment of innovative technologies (including next-generation sequencing), in order to replace obsolete phenotypic classifications and to discover new useful diagnostic biomarkers. These new tools are, in fact, becoming part of routine clinical practice, increasing diagnostic accuracy and facilitating genetic counseling. Moreover, biomedical research, providing insights into the pathologies of these rare diseases and elucidating their underlying mechanisms, is revealing new therapeutic avenues and driving the industry to develop safer and more effective orphan drugs. Finally, in this field, it is desirable that, in the future, the crosstalk between basic scientists and clinicians could achieve a great clinical benefit by improving the quality of life of these patients as well. This Special Issue welcomes scientific contributions and critical reviews describing new pathogenetic insights, reporting novel and specific disease biomarkers, and underlying new pharmacological targets or therapies for rare diseases of the kidney and urinary tract

Genetics of atypical haemolytic uraemic syndrome

PhD ThesisAtypical haemolytic uraemic syndrome (aHUS) is a life threatening renal disease, caused by deregulation of the alternative complement pathway. Several genes within this pathway are associated with aHUS. At the outset of this project, the genetic cause had been identified in 45% of familial cases in the Newcastle aHUS cohort. The aim of this project was to identify the genetic cause of disease in the remaining 55%. Complement Factor H (CFH) and Complement Factor H-related (CFHRs) are found on chromosome 1. This area contains several low copy repeats, the result of genomic duplications that occurred early in evolution. This causes genomic instability, which can lead to gene conversions or rearrangements. Sanger sequencing will not always detect these abnormalities, therefore patients were also screened using multiplex ligation-dependent probe amplification and western blotting. A novel hybrid CFH/CFHR3 gene was described, which arose by microhomology-mediated end joining. Functional analysis demonstrated that it was defective at regulating complement at the cell surface, which was predicted to predispose this patient to disease. Review of all patients in the Newcastle aHUS cohort with CFH abnormalities, identified a third of patients had a genomic rearrangement between CFH and CFHRs. The relative frequency of genomic rearrangements emphasised the importance of undertaking copy number analysis in aHUS diagnostic testing, because often they are not detected by Sanger sequencing. Whole exome sequencing was then undertaken in Newcastle familial cohort with an unknown genetic aetiology. Pathogenic sequence variants were identified in genes, known to be associated with thrombotic microangiopathies. Sequence variants that were predicted to be pathogenic, were found in three genes not previously associated with disease. Two of these genes were located outside of the complement system, indicating that complement-directed therapies may be contraindicated. In this project, a genetic cause of disease was found in 54% of familial cases tested