Iben Møller Jønsson, Sophie Lebon, Erik Ilsø Christensen, Heidi Koldsø, Mette Madsen, Jens Michael Hertz, Rikke Nielsen, Francesco Emma, Sabine Amsellem, Jean-François Benoist, Sandrine Passemard, Pierre J. Verroust, Olivier Cases, Christina Zeitz, Renata Kozyraki, Tina Storm, Department of Biomedicine, Aarhus University [Aarhus], Institut de la Vision, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biochimie, Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Robert Debré-Université Paris Diderot - Paris 7 (UPD7), Service de Génétique, Physiopathologie et neuroprotection des atteintes du cerveau en développement, Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Pediatrics, Aarhus University Hospital, Department of Nephrology and Urology, IRCCS Ospedale Pediatrico Bambino Gesù [Roma]-IRCCS, Centre for Insoluble Protein Structures (inSPIN) and Interdisciplinary Nanoscience Center (iNANO), Department of Clinical Genetics, Odense University Hospital, CHU Saint-Antoine [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), University of Aarhus, the Danish Medical Research Council, the NOVO-Nordisk Foundation, The Lundbeck Foundation, The Danish Kidney Association, Region Viborg, Fondation Voir et Entendre, and the program of the European Community, EUNEFRON (FP7, GA#201590)., European Project: 201590,EC:FP7:HEALTH,FP7-HEALTH-2007-A,EUNEFRON(2008), BMC, Ed., and European Network for the Study of Orphan Nephropathies - EUNEFRON - - EC:FP7:HEALTH2008-05-01 - 2012-04-30 - 201590 - VALID
Background Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B12 receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. Methods Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. Results Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B12 binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. Conclusions In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria.