Genomes and Genes
Exon capture and large-scale sequencing for disease-cause identification, early d
Principal Investigator: Friedhelm Hildebrandt
Abstract: DESCRIPTION (provided by applicant): This application addresses: Broad Challenge Area: (15) Translational Science Topic: 15-OD (ORDR)-101* Pilot projects for prevention, early detection and treatment of rare diseases. Exon capture and large-scale sequencing for disease-cause identification, early detection and drug discovery in nephrotic syndrome. Background: Chronic kidney diseases (CKD) take one of the highest tolls on human health. They insidiously lead to end- stage kidney disease (ESKD) requiring dialysis or kidney transplantation for survival. 20 million individuals in the U.S. suffer from CKD with a treatment cost of >$20 billion/yr. Steroid-resistant nephrotic syndrome (SRNS) is a rare disease that constitutes the second most frequent cause of ESKD in children and young adults. No curative treatment is available. We demonstrated that many pediatric cases with SRNS are rare recessive single-gene disorders. 25% of all SRNS cases are caused by recessive NPHS2/podocin mutations and many additional single-gene causes of SRNS exist. Identification of rare single-gene causes for SRNS has provided fundamental insights into disease mechanisms of nephrotic syndrome in children and adults. It has allowed to perform unequivocal molecular genetic diagnostics for early detection of SRNS (www.renalgenes.org) and to stratify patient cohorts for therapeutic trials. The study of recessive disease mechanisms is particularly powerful, as they can be recapitulated in animal models by gene knockdown/knockout in zebrafish or mice. These animal models allow for high throughput drug screening to define new molecular targets for treatment. Knowledge Gap and New Technology: The current knowledge gap in disease causes of SRNS has hampered diagnostics, early detection and target- oriented drug treatment for this incurable disease. The heterogeneity of recessive SRNS-causing genes, and their rarity, pose severe limitations to gene identification. Recently, we introduced a new technology into gene discovery of rare recessive single-gene causes by developing a combined approach of homozygosity mapping (HM) with consecutive exon capture and large-scale sequencing. Feasibility and Existing Resources: We have ascertained DNA samples and clinical data from over 2,000 families with SRNS world-wide and have clarified the molecular cause of SRNS in 15% of cases. We applied HM successfully to the identification of 11 novel CKD genes. Recently, we demonstrated that HM is broadly applicable even to single cases with rare diseases. In 30-80% of cases it yielded homozygous candidate regions, which contained the disease causing homozygous mutation in 93% of cases. When identifying by HM recessive PLCE1 mutations as a rare cause of SRNS (~1%), we recapitulated the disease in zebrafish, thereby generating a model for drug screening. By total genome HM in 250 of the 2,000 families with SRNS (using 250k and 1Mill SNP arrays) we yielded new homozygous candidate regions in 100/250 families (40%), and demonstrated that more than 20 additional unknown recessive single-gene causes of SRNS must exist. Thus, we will address the following specific aims: Specific Aims SA1. Identify novel causes of the rare disease steroid-resistant nephrotic syndrome (SRNS) by total genome homozygosity mapping, with consecutive targeted exon capture and large-scale sequencing. SA2. Establish zebrafish models of novel SRNS genes and test new treatment approaches. SA3. Rapidly expand the new strategy of gene identification to other autosomal recessive conditions with a urogenital phenotype. Significance: The new approach that we developed will have the following impact for SRNS and other rare diseases: i) Gene identification will generate novel insights into the etiology and disease mechanisms of SRNS. ii) It will allow screening and early detection for this rare disease and etiologic stratification for clinical trials. iii) Gene identification will generate novel molecular targets to treat SRNS, which currently is incurable. iv) Further development of disease models in zebrafish for SRNS-causing genes will enable high throughput screening assays for drug discovery of novel compounds to treat SRNS. We already demonstrated the feasibility of this approach. The project will expand our successful preliminary results of HM with consecutive targeted exon capture and large-scale sequencing to a large number of families with rare diseases. Following this ARRA, we will enlarge the project (with existing resources) to study all recessive pediatric diseases at the U of M Mott Children's Hospital and beyond. These studies, for which all resources and technologies are in place, would greatly benefit from an influx of funds to quickly and significantly advance disease-cause identification in SRNS and other rare diseases by rapidly generating data using highly efficient, but costly, new technology of exon capture and large-scale sequencing, which we have established. Chronic kidney diseases take one of the highest tolls on human health. Steroid-resistant nephrotic syndrome (SRNS) is a rare disease that constitutes the second most frequent cause of ESKD in children and young adults. No curative treatment is available. 25% of all SRNS cases are caused by recessive NPHS2/podocin mutations, and we recently demonstrated, by genetic mapping, that many additional single-gene causes of SRNS must exist. Identification of rare single-gene causes for SRNS has provided fundamental insights into disease mechanisms of nephrotic syndrome in children and adults. Recently, we introduced a new technology into gene discovery of rare recessive single-gene causes by developing a combined approach of homozygosity mapping with consecutive exon capture and large-scale sequencing. We have ascertained DNA samples and clinical data from over 2,000 families with SRNS world- wide and have clarified the molecular cause of SRNS in 15% of cases. We here propose to 1) Identify novel causes of the rare disease SRNS by total genome homozygosity mapping, with consecutive targeted exon capture and large-scale sequencing;2) Establish zebrafish models of novel SRNS genes and test novel treatment approaches;and 3) Rapidly expand the new strategy of gene identification to other autosomal recessive conditions with a urogenital phenotype.
Funding Period: ----------------2009 - ---------------2011-
more information: NIH RePORT
- ADCK4 mutations promote steroid-resistant nephrotic syndrome through CoQ10 biosynthesis disruptionShazia Ashraf
J Clin Invest 123:5179-89. 2013..These data indicate that individuals with SRNS with mutations in ADCK4 or other genes that participate in CoQ10 biosynthesis may be treatable with CoQ10. ..
- ARHGDIA mutations cause nephrotic syndrome via defective RHO GTPase signalingHeon Yung Gee
Division of Nephrology, Department of Medicine, Boston Children s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
J Clin Invest 123:3243-53. 2013..RAC1 inhibitors were partially effective in ameliorating arhgdia-associated defects. These findings identify a single-gene cause of NS and reveal that RHO GTPase signaling is a pathogenic mediator of SRNS. ..
- Mutation analysis of NPHS1 in a worldwide cohort of congenital nephrotic syndrome patientsBugsu Ovunc
Department of Pediatrics and Department of Human Genetics, University of Michigan, Ann Arbor, MI, USA
Nephron Clin Pract 120:c139-46. 2012..Mutations in the NPHS1 gene encoding nephrin, are a major cause for CNS. Currently, more than 173 different mutations of NPHS1 have been published as causing CNS, affecting most exons...
- Integrin α3 mutations with kidney, lung, and skin diseaseCristina Has
Department of Dermatology, University Freiburg Medical Center, Freiburg, Germany
N Engl J Med 366:1508-14. 2012..The renal and respiratory features predominated, and the lung involvement accounted for the lethal course of the disease. Although skin fragility was mild, it provided clues to the diagnosis...
- Inducible podocyte injury and proteinuria in transgenic zebrafishWeibin Zhou
University of Michigan Medical School, 8240 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109 5646, USA
J Am Soc Nephrol 23:1039-47. 2012..These data support the use of these transgenic zebrafish as a model system for studies of glomerular pathogenesis and podocyte regeneration...
- Exome sequencing reveals cubilin mutation as a single-gene cause of proteinuriaBugsu Ovunc
Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109, USA
J Am Soc Nephrol 22:1815-20. 2011..In summary, we report successful use of exome capture and massively parallel re-sequencing to identify a rare, single-gene cause of nephropathy...
- MYO1E mutations and childhood familial focal segmental glomerulosclerosisCaterina Mele
Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases, Aldo e Cele Daccò Ranica, Bergamo, Italy
N Engl J Med 365:295-306. 2011..However, the molecular cause in over half the cases of primary focal segmental glomerulosclerosis is unknown, and effective treatments have been elusive...
- COQ6 mutations in human patients produce nephrotic syndrome with sensorineural deafnessSaskia F Heeringa
Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, USA
J Clin Invest 121:2013-24. 2011..These data suggest that coenzyme Q10-related forms of SRNS and hearing loss can be molecularly identified and potentially treated...
- Exome capture and massively parallel sequencing identifies a novel HPSE2 mutation in a Saudi Arabian child with Ochoa (urofacial) syndromeWisam Al Badr
Division of Nephrology, King Fahad Specialist Hospital Dammam, Dammam, Saudi Arabia
J Pediatr Urol 7:569-73. 2011..Early intervention and recognition of Ochoa syndrome with control of risk factors and close surveillance will decrease complications and renal failure...
- Respiratory-chain deficiency presenting as diffuse mesangial sclerosis with NPHS3 mutationEsra Baskin
Department of Pediatric Nephrology, Baskent University, Ankara, Turkey
Pediatr Nephrol 26:1157-61. 2011..Another, more likely, possibility is that the mitochondriopathy phenotype is caused by another mutation homozygous by descent in a yet unidentified recessive gene...
- Immunosuppression and renal outcome in congenital and pediatric steroid-resistant nephrotic syndromeAnja K Büscher
Pediatric Nephrology, Pediatrics II, University of Duisburg Essen, Essen, Germany
Clin J Am Soc Nephrol 5:2075-84. 2010..The aim of this study was to evaluate the influence of podocyte gene defects in congenital nephrotic syndrome (CNS) and pediatric SRNS on the efficacy of CsA therapy and preservation of renal function...
- Genetic kidney diseasesFriedhelm Hildebrandt
Howard Hughes Medical Institute and Department of Pediatrics, University of Michigan, Ann Arbor, MI, USA
Lancet 375:1287-95. 2010....
- Nineteen novel NPHS1 mutations in a worldwide cohort of patients with congenital nephrotic syndrome (CNS)Dominik S Schoeb
Department of Pediatrics, University of Michigan, 1150 W Medical Center Drive Drive, Ann Arbor, MI, USA
Nephrol Dial Transplant 25:2970-6. 2010..CNS is defined as steroid-resistant nephrotic syndrome (SRNS) within the first 90 days of life. Currently, more than 119 different mutations of NPHS1 have been published affecting most exons...