EURenOmics Project Summary Year 5

Summary description of project context and objectives

Rare kidney diseases impact markedly on the life expectancy and quality of life of affected individuals. The current diagnostic and therapeutic management of rare kidney diseases is highly unsatisfactory. We are typically unable to explain the genetic or molecular abnormality underlying the disease phenotype, to predict the individual risk and rate of disease progression, or to quantitate the risk of relatives to develop the same disorder. Even in patients with known genetic causes, individual risk prediction is limited by considerable phenotypic variability. Molecular disease and progression risk markers are lacking. Effective therapies are largely unavailable; the lack of suitable disease models is a major barrier to therapeutics development.

Kidney diseases are well suited to high-throughput research due to the opportunity to examine molecular events in the end organ that is manifesting the disease: kidney biopsy, a standard diagnostic procedure, provides a unique opportunity to study intrarenal biological processes ex vivo using transcriptomic, proteomic and morphological approaches. Urine is a readily available non-invasive bioresource to study molecular readouts directly derived from the organ of interest, the kidney. Amniotic fluid, the fetal urine, allows prenatal epigenetic, proteomic and metabolomic profiling in the context of renal maldevelopment. Recent technological progress in exosome isolation from urine and amniotic fluid even has created the opportunity to study non-invasively cellular biomaterials originating from the diseased tissues.

EURenOmics has prioritized five disease groups based on their urgent need and significant potential for diagnostic and therapeutic progress: Steroid-resistant nephrotic syndrome (SRNS), membranous nephropathy (MN), tubulopathies, complement disorders and malformations of the kidney and urinary tract (CAKUT). The consortium has access to a unique assembly of large existing patient cohorts and biorepositories, encompassing more than 15,000 patients with >10,000 DNA, >3,000 serum, 2,000 urine, 500 amniotic fluid and 3,000 kidney biopsy specimens. Some 30 academic and industry partners have joined EURenOmics to apply a wide range of high-throughput technologies, innovative systems biology approaches and a plethora of in vitro, ex vivo and in vivo models to study disease mechanisms and explore novel therapeutic approaches.

Initial research efforts focus on methodological standardization, involving procedures for acquisition and processing of biospecimens but also the construction of an integrated phenome database for uniform clinical phenotyping. The next objective is to search for new genes causing, modifying or predisposing to individual disease phenotypes. Screening strategies encompass next-generation sequencing of known genes, regulatory regions, exomes and whole genomes, application of cutting-edge bioinformatic tools for data mining, filtering and gene network analysis, and extensive genotype-phenotype analyses utilizing standardized phenotypic information. Novel epitopes/antigens and antibodies are searched systematically in those disorders in which an auto-immune pathology is suspected. The protein products of the newly identified gene variants, as well as the mechanisms of auto-antibody formation, are functionally characterized by an array of in vitro, ex vivo and in vivo technologies. Multi-level -omics profiling (mRNA, miRNA, peptidome/proteome, metabolome) in body fluids and renal tissues is performed to identify unique molecular disease signatures (deep phenotyping), develop a prognostically indicative new ontology of rare kidney diseases, discover molecular markers and pathways associated with disease activity and progression, and develop diagnostic tools and biomarkers. Finally, in vitro and in vivo models will be developed that allow high-throughput screening of compound libraries for novel therapeutic agents reversing or attenuating disease phenotypes.

Work performed since the beginning of the project and the main results achieved so far

Numerous novel methodologies including groundbreaking diagnostic and disease activity assays and in vivo disease models, a central ‘renal phenome’ database integrating clinical information from local and regional patient registries as well as a unique kidney-focused bioinformatic analysis pipeline have been established.

Exome sequencing has been performed in 315 families (WP2: 103; WP4: 87; WP5: 35; WP6: 190). These efforts contributed to the discovery of 11 novel SRNS, 5 tubulopathy, 3 complement disease and 5 CAKUT genes, and 13 new genomic rearrangements in complement disorders. Moreover, causative mutations in known genes were found in 23 SRNS, 11 tubulopathy, 10 complement disease and 4 CAKUT families. Several candidate disease genes are undergoing functional characterization.

In parallel, targeted NG sequencing assays capturing all known genes within a disease group have been developed for glomerular (34 genes), tubular (44 genes), complement diseases (13 genes) and CAKUT (two panels, 208/388 genes). So far, panel sequencing has been applied in >1700 SRNS, >600 tubulopathy, >750 atypical hemolytic uremic syndrome (aHUS) and >1000 CAKUT cases. Disease causing mutations were identified in 21% of the SRNS, 49% of the tubulopathy, 40% of the aHUS and 6% of the CAKUT patients.

The integration of patient databases and advances in gene diagnostics has allowed deep phenotyping and genotype-phenotype association studies in the largest cohorts of genetically classified patients assembled to date (e.g., WT1, ADCK4, DGKE glomerulopathies, Schimke syndrome, Dent disease, Bartter syndrome, familial hypercalcemic hypocalciuria, renal Fanconi syndrome, AD tubulointerstitial kidney diseases, Bardet-Biedl syndrome, distal renal tubular acidosis, aHUS and C3 glomerulopathy).

The compilation of large biorepositories fostered unbiased searches for novel diagnostic and prognostic biomarkers. Integrative bioinformatic analysis based on tissue transcriptome information unsurfaced urinary EGF as a novel biomarker of renal failure progression in adult and pediatric patients with chronic kidney disease. Peptidomic, metabolomic and miRNA profiling of body fluids has yielded promising molecular signatures discriminating different disease entities in SRNS and complement diseases, as well as favorable from poor prognosis in MN and prenatal CAKUT.

Fundamental progress in the understanding of genetic disease penetrance was achieved. Mutation type dependent interallelic complementation of mutations in compound heterozygous NPHS2 nephropathy and common gene variants associated with disease risk were identified in MN, aHUS, and hypomagnesemia were identified as novel mechanisms explaining variable phenotype expression.

Major progress was also made concerning molecular mechanisms of auto-immune disease. The B cell epitope in PLA2R, target of autoantibodies in MN, and the HLA peptidome have been identified, the latter paving the way for unbiased identification of immune response targets in autoimmune diseases. In CFH-antibody mediated aHUS, autoantibody formation was found to emerge from deficient B-cell regulation by genetic CFHR3 deficiency, demonstrating a mechanistic link between genetic disease and autoimmunity.

For the search for new therapies, drug re-purposing has allowed rapid progress in several areas such as rituximab for MN, eculizumab for secondary aHUS, and CoQ10 supplementation for hereditary mitochondrial podocytopathies. Promising new molecular therapies are tested experimentally, including monoclonal C3 and C5a antibodies for complement diseases, and flavaglines for TRPM6-associated hypomagnesemia. High-throughput compound screening in novel cell and animal models has identified several re-purposable drugs reversing the pathogenic effects of mutations in podocytopathies and ciliopathies, and the development of models suitable for compound screening have been completed for several tubulopathies, metabolic nephropathies and complement disorders.

Expected final results and their potential impact and use

The research results expected from EURenOmics will have substantial impact on diagnostic management, risk assignment and preventative and therapeutic strategies in patients with rare kidney diseases, with potential implications also for patients with common kidney and other diseases and the society at large.

Comprehensive exome sequencing of large patient cohorts is profoundly advancing the state of knowledge by unraveling the as yet unknown monogenic and complex genetic causes underlying the diseases of interest. The newly discovered genes are continuously included in the targeted next-generation sequencing (NGS) gene arrays developed by the partners. The advent of these NGS panels is considered a major breakthrough as it will render the current sequencing technology with its high cost and time demands obsolete and will make rapid and complete genetic diagnostics a routine clinical practice. For those patients in whom a genetic diagnosis cannot be established, we seek to develop disease-specific peptidomic, metabolomic and miRNA signatures and novel auto-antibody assays.

The availability of such diagnostic tests should constitute a major step towards precision medicine. The need for invasive kidney biopsies will be greatly reduced or eliminated. Exposure to ineffective, potentially toxic and costly medications can be avoided and replaced by rational, risk-adapted therapies tailored to the molecular basis of mechanistically defined disease entities. The recent development of a sensitive and specific bioassay that allows to monitor therapeutic complement blockade and prevent both hazardous under- and costly overtreatment with the C5 inhibitor eculizumab in complement disorders exemplifies the remarkable health and socio-economic impact of the Consortium’s activities.

In addition to the expected diagnostic progress, the anticipated identification of genetic disease causes and risk factors as well as prognostic biomarkers should greatly facilitate individual risk assignment regarding disease occurrence, progression, complications and post-transplant recurrence compared to the currently used clinical, histopathological or imaging criteria. The efficiency of genetic counselling to families with inherited disorders will be much improved, alleviating their psychological burden and distress. Also, accurate risk assignment will form a basis for the development of early intervention and secondary prevention protocols.

Moreover, the partners are developing innovative in vitro and in vivo disease models aimed to discover novel therapeutics including alternatives to existing superexpensive drugs, and to allow a more rational, calculated use of already available agents. Our preliminary results and ongoing research efforts with respect to in vitro and in vivo model development, the repurposed use of approved pharmaceutical agents to attenuate hereditary and immune-mediated glomerulopathies, the development of novel complement blocking monoclonal antibodies and the early work on activators of mutated magnesium channels look promising and will contribute to the IRDiRC goal of establishing new therapies for a significant number of rare diseases by 2020.

Finally, EURenOmics seeks to generate clinical and socioeconomic impact beyond the field of rare kidney disease. The study of the genetic abnormalities and molecular mechanisms underlying the selected disorders is hoped to produce insights into general mechanisms of kidney disease progression and even universal risk factors relevant to the general population. Our identification of a mechanism by which common genetic variation of a kidney-specific protein involved in a rare kidney disorder (uromodulin) predisposes to chronic kidney disease and hypertension impressively exemplifies the far-reaching potential of rare kidney disease research.