title: Diagnostic value of immunostaining in cultured skin fibroblasts from patients with oxidative phosphorylation defects.
abstract: In the last decades, a large variety of oxidative phosphorylation (OXPHOS) defects have been reported, expressed as an increasing variety of clinical phenotypes. With the expanding number of genes and proteins involved, new screening techniques leading to more effective diagnostic routes are in ever-increasing demand. Cultured skin fibroblasts from a cohort of patients with various OXPHOS defects, previously recognized by enzyme activity studies and blue native PAGE, were investigated with an immunocytochemical technique. Cytospins of cultured fibroblasts were air dried, fixed, and stained with antibodies specifically directed against subunits of each OXPHOS complex. Control cells stained homogeneously and strongly. In fibroblasts from five out of seven patients with a severe deficiency of one of the OXPHOS complexes, a homogeneous reduction of cytoimmunoreactivity of the affected complex was observed. In five out of seven fibroblast strains harboring a mitochondrial tRNA mutation, a mosaic pattern of staining was observed for both complexes I and IV, reflecting the heteroplasmic nature of the defect. The proportion of deficient fibroblasts varied considerably between cell strains from different subjects. The method described offers a convenient and rapid approach to first-line screening of OXPHOS defects. In association with routine assays of enzyme activity, the technique is helpful in orienting molecular investigation further.
authors.affiliation: Department of Pediatrics, Ghent University Hospital, Belgium.
authors.forename: Boel
authors.initials: B
authors.lastname: de Paepe
authors.forename: Joél
authors.initials: J
authors.lastname: Smet
authors.forename: Jules G
authors.initials: JG
authors.lastname: Leroy
authors.forename: Sara
authors.initials: S
authors.lastname: Seneca
authors.forename: Edith
authors.initials: E
authors.lastname: George
authors.forename: Dirk
authors.initials: D
authors.lastname: Matthys
authors.forename: Lionel
authors.initials: L
authors.lastname: van Maldergem
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Scalais
authors.forename: Willy
authors.initials: W
authors.lastname: Lissens
authors.forename: Linda
authors.initials: L
authors.lastname: de Meirleir
authors.forename: Ann
authors.initials: A
authors.lastname: Meulemans
authors.forename: Rudy
authors.initials: R
authors.lastname: van Coster
mesh_terms: D000293:Adolescent; D000328:Adult; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D045222:Electron Transport Chain Complex Proteins / Q000032:analysis*; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D007223:Infant; D008297:Male; D008875:Middle Aged; D028361:Mitochondrial Diseases / Q000175:diagnosis*; D010085:Oxidative Phosphorylation; D012867:Skin / Q000201:enzymology* / Q000473:pathology
chemical_list: D045222:Electron Transport Chain Complex Proteins