title: Recessive osteogenesis imperfecta caused by LEPRE1 mutations: clinical documentation and identification of the splice form responsible for prolyl 3-hydroxylation.
abstract: BACKGROUND
  Recessive forms of osteogenesis imperfecta (OI) may be caused by mutations in LEPRE1, encoding prolyl 3-hydroxylase-1 (P3H1) or in CRTAP, encoding cartilage associated protein. These proteins constitute together with cyclophilin B (CyPB) the prolyl 3-hydroxylation complex that hydroxylates the Pro986 residue in both the type I and type II collagen alpha1-chains.
  
  
  METHODS
  We screened LEPRE1, CRTAP and PPIB (encoding CyPB) in a European/Middle Eastern cohort of 20 lethal/severe OI patients without a type I collagen mutation.
  
  
  RESULTS
  Four novel homozygous and compound heterozygous mutations were identified in LEPRE1 in four probands. Two probands survived the neonatal period, including one patient who is the eldest reported patient (17 7/12 years) so far with P3H1 deficiency. At birth, clinical and radiologic features were hardly distinguishable from those in patients with autosomal dominant (AD) severe/lethal OI. Follow-up data reveal that the longer lived patients develop a severe osteochondrodysplasia that overlaps with, but has some distinctive features from, AD OI. A new splice site mutation was identified in two of the four probands, affecting only one of three LEPRE1 mRNA splice forms, detected in this study. The affected splice form encodes a 736 amino acid (AA) protein with a "KDEL" endoplasmic reticulum retention signal. While western blotting and immunocytochemical analysis of fibroblast cultures revealed absence of this P3H1 protein, mass spectrometry and SDS-urea-PAGE data showed severe reduction of alpha1(I)Pro986 3-hydroxylation and overmodification of type I (pro)collagen chains in skin fibroblasts of the patients.
  
  
  CONCLUSIONS
  These findings suggest that the 3-hydroxylation function of P3H1 is restricted to the 736AA splice form.
authors.affiliation: Department of Medical Genetics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Willaert
authors.forename: F
authors.initials: F
authors.lastname: Malfait
authors.forename: S
authors.initials: S
authors.lastname: Symoens
authors.forename: K
authors.initials: K
authors.lastname: Gevaert
authors.forename: H
authors.initials: H
authors.lastname: Kayserili
authors.forename: A
authors.initials: A
authors.lastname: Megarbane
authors.forename: G
authors.initials: G
authors.lastname: Mortier
authors.forename: J G
authors.initials: JG
authors.lastname: Leroy
authors.forename: P J
authors.initials: PJ
authors.lastname: Coucke
authors.forename: A
authors.initials: A
authors.lastname: De Paepe
mesh_terms: D017398:Alternative Splicing; D015153:Blotting, Western; D002478:Cells, Cultured; D015331:Cohort Studies; D024042:Collagen Type I / Q000378:metabolism; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D016326:Extracellular Matrix Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D005808:Genes, Recessive; D005820:Genetic Testing; D006801:Humans; D006900:Hydroxylation; D007150:Immunohistochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D018832:Molecular Chaperones; D009154:Mutation; D010013:Osteogenesis Imperfecta / Q000175:diagnosis / Q000235:genetics*; D064828:Prolyl Hydroxylases; D011509:Proteoglycans / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053719:Tandem Mass Spectrometry
chemical_list: C108053:CRTAP protein, human; D024042:Collagen Type I; D016326:Extracellular Matrix Proteins; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D011509:Proteoglycans; C081786:cyclophilin B; D064828:Prolyl Hydroxylases; C485657:P3H1 protein, human; D021983:Cyclophilins