title: Detailed investigations of proximal tubular function in Imerslund-Gräsbeck syndrome.
abstract: 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.
authors.forename: Tina
authors.initials: T
authors.lastname: Storm
authors.forename: Christina
authors.initials: C
authors.lastname: Zeitz
authors.forename: Olivier
authors.initials: O
authors.lastname: Cases
authors.forename: Sabine
authors.initials: S
authors.lastname: Amsellem
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Verroust
authors.forename: Mette
authors.initials: M
authors.lastname: Madsen
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Benoist
authors.forename: Sandrine
authors.initials: S
authors.lastname: Passemard
authors.forename: Sophie
authors.initials: S
authors.lastname: Lebon
authors.forename: Iben Møller
authors.initials: IM
authors.lastname: Jønsson
authors.forename: Francesco
authors.initials: F
authors.lastname: Emma
authors.forename: Heidi
authors.initials: H
authors.lastname: Koldsø
authors.forename: Jens Michael
authors.initials: JM
authors.lastname: Hertz
authors.forename: Rikke
authors.initials: R
authors.lastname: Nielsen
authors.affiliation: Department of Biomedicine, Aarhus University, Aarhus, Denmark. eic@ana.au.dk.
authors.forename: Erik I
authors.initials: EI
authors.lastname: Christensen
authors.forename: Renata
authors.initials: R
authors.lastname: Kozyraki
mesh_terms: D000419:Albuminuria / Q000175:diagnosis; D000749:Anemia, Megaloblastic; D000818:Animals; D016632:Apolipoprotein A-I / Q000652:urine; D001665:Binding Sites; D016466:CHO Cells; D016022:Case-Control Studies; D003412:Cricetulus; D005260:Female; D016368:Frameshift Mutation; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000378:metabolism / Q000503:physiopathology*; D008286:Malabsorption Syndromes / Q000235:genetics* / Q000503:physiopathology*; D008297:Male; D008565:Membrane Proteins; D008970:Molecular Weight; D020125:Mutation, Missense; D010375:Pedigree; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D011507:Proteinuria / Q000175:diagnosis / Q000235:genetics* / Q000503:physiopathology*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014168:Transferrin / Q000652:urine; D014806:Vitamin B 12 Deficiency / Q000235:genetics* / Q000503:physiopathology*; D014809:Vitamin D-Binding Protein / Q000652:urine
chemical_list: C484714:AMN protein, human; D016632:Apolipoprotein A-I; D008565:Membrane Proteins; D011506:Proteins; D011956:Receptors, Cell Surface; D014168:Transferrin; D014809:Vitamin D-Binding Protein; C029486:intrinsic factor-cobalamin receptor