Human Molecular Genetics Advance Access originally published online on May 22, 2009
Human Molecular Genetics 2009 18(16):2963-2974; doi:10.1093/hmg/ddp235
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Uromodulin mutations causing familial juvenile hyperuricaemic nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum
1 Academic Endocrine Unit 2 Diabetes Research Laboratories, Churchill Hospital, Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Headington, Oxford OX3 7LJ, UK 3 AP-HP, Department of Genetics, Hopital Necker-Enfants Malades, 75015 Paris, France 4 INSERM U574, Hôpital Necker-Enfants Malades, 75015 Paris, France 5 Universite Paris Descartes, Paris, France 6 Institute of Human Genetics, Newcastle University, Newcastle-upon-Tyne NE1 3BZ, UK 7 Cambridge Institute for Medical Research, Addenbrookes Hospital, Cambridge CB2 0XY, UK 8 Renal Research Institute, New York, NY 10128, USA 9 Department of Nephrology and Dialysis, Academic Teaching Hospital Feldkirch, AT-6800 Feldkirch, Austria 10 Department of Nephrology, Ipswich Hospital, Heath Road, Ipswich IP4 5PD, UK 11 Starship Children's Health, Park Road, Auckland 1023, New Zealand
* To whom correspondence should be addressed. Tel: +44 1865857501; Fax: +44 1865857502; Email: rajesh.thakker{at}ndm.ox.ac.uk
Received March 18, 2009; Revised May 2, 2009; Accepted May 13, 2009
Familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol-anchored protein that is expressed in the thick ascending limb of the loop of Henle and excreted in the urine. Uromodulin contains three epidermal growth factor (EGF)-like domains, a cysteine-rich region which includes a domain of eight cysteines and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and by the use of western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.
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