title: Identification of the molecular defect in patients with peroxisomal mosaicism using a novel method involving culturing of cells at 40 degrees C: implications for other inborn errors of metabolism.
abstract: The peroxisome biogenesis disorders (PBDs), which comprise Zellweger syndrome (ZS), neonatal adrenoleukodystrophy, and infantile Refsum disease (IRD), represent a spectrum of disease severity, with ZS being the most severe, and IRD the least severe disorder. The PBDs are caused by mutations in one of the at least 12 different PEX genes encoding proteins involved in the biogenesis of peroxisomes. We report the biochemical characteristics and molecular basis of a subset of atypical PBD patients. These patients were characterized by abnormal peroxisomal plasma metabolites, but otherwise normal to very mildly abnormal peroxisomal parameters in cultured skin fibroblasts, including a mosaic catalase immunofluorescence pattern in fibroblasts. Since this latter feature made standard complementation analysis impossible, we developed a novel complementation technique in which fibroblasts were cultured at 40 degrees C, which exacerbates the defect in peroxisome biogenesis. Using this method, we were able to assign eight patients to complementation group 3 (CG3), followed by the identification of a single homozygous c.959C>T (p.S320F) mutation in their PEX12 gene. We also investigated various peroxisomal biochemical parameters in fibroblasts at 30 degrees C, 37 degrees C, and 40 degrees C, and found that all parameters showed a temperature-dependent behavior. The principle of culturing cells at elevated temperatures to exacerbate the defect in peroxisome biogenesis, and thereby preventing certain mutations from being missed, may well have a much wider applicability for a range of different inborn errors of metabolism.
authors.affiliation: Department of Clinical Chemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
authors.forename: Jeannette
authors.initials: J
authors.lastname: Gootjes
authors.forename: Frank
authors.initials: F
authors.lastname: Schmohl
authors.forename: Petra A W
authors.initials: PA
authors.lastname: Mooijer
authors.forename: Conny
authors.initials: C
authors.lastname: Dekker
authors.forename: Hanna
authors.initials: H
authors.lastname: Mandel
authors.forename: Meral
authors.initials: M
authors.lastname: Topcu
authors.forename: Martina
authors.initials: M
authors.lastname: Huemer
authors.forename: M
authors.initials: M
authors.lastname: Von Schütz
authors.forename: Thorsten
authors.initials: T
authors.lastname: Marquardt
authors.forename: Jan A
authors.initials: JA
authors.lastname: Smeitink
authors.forename: Hans R
authors.initials: HR
authors.lastname: Waterham
authors.forename: Ronald J A
authors.initials: RJ
authors.lastname: Wanders
mesh_terms: D002374:Catalase / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D003080:Cold Temperature; D003241:Consanguinity; D004252:DNA Mutational Analysis / Q000379:methods; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D005455:Fluorescent Antibody Technique / Q000379:methods; D005816:Genetic Complementation Test / Q000379:methods; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D009030:Mosaicism / Q000235:genetics* / Q000473:pathology; D018901:Peroxisomal Disorders / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D012867:Skin / Q000473:pathology
chemical_list: D008565:Membrane Proteins; C105251:PEX12 protein, human; D002374:Catalase