title: A new heterozygous mutation (R714C) of the osteopetrosis gene, pleckstrin homolog domain containing family M (with run domain) member 1 (PLEKHM1), impairs vesicular acidification and increases TRACP secretion in osteoclasts.
abstract: We studied phenotypic and cellular aspects in a patient with a heterozygous mutation of the PLEKHM1 gene and obtained some indications regarding the role of the protein in bone cell function. Plekhm1 is involved in osteoclast endosomal vesicle acidification and TRACP exocytosis, contributing to events involved in osteoclast-osteoblast cross-talk.
  
  
  BACKGROUND
  The gene PLEKHM1 encodes a nonsecretory adaptor protein that localizes to endosomal vesicles. A highly truncated Plekhm1 protein was previously found in a patient with intermediate autosomal recessive osteopetrosis.
  
  
  METHODS
  We describe a new heterozygous mutation in the PLEKHM1 gene in a patient presenting with low vertebral and femoral T-scores and areas of focal sclerosis. Clinical evaluation, mutational analysis, assessment of in vitro osteoclast morphology and activity, transfection studies, and evaluation of osteoclast-osteoblast cross-talk were carried out.
  
  
  RESULTS
  Direct DNA sequencing showed a heterozygous C to T substitution on cDNA position 2140 of the PLEKHM1 gene, predicted to lead to an R714C mutant protein. The mutation was not found in 104 control chromosomes. In vitro, patient's osteoclasts showed normal formation rate, morphology, number of nuclei, and actin rings but lower TRACP activity and higher endosomal pH than control osteoclasts. The patient had high serum PTH and TRACP, despite low TRACP activity in osteoclasts in vitro. HEK293 cells overexpressing either wildtype or Plekhm1-R714C showed no difference in localization of the variants, and co-transfection with a TRACP vector confirmed low TRACP activity in cells carrying the R714C mutation. RAW 264.7 osteoclast-like cells expressing the Plekhm1-R714C variant also showed low TRACP activity and reduced ability to acidify endosomal compartments compared with cells expressing the wildtype protein. Reduced intracellular TRACP was caused by increased protein secretion rather than reduced expression. TRACP-containing conditioned medium was able to increase osteoblast alkaline phosphatase, suggesting the focal osteosclerosis is a result of increased osteoclast-osteoblast coupling.
  
  
  CONCLUSIONS
  We provide further evidence for a role of Plekhm-1 in osteoclasts by showing that a novel mutation in PLEKHM1 is associated with a complex bone phenotype of generalized osteopenia combined with "focal osteosclerosis." Our data suggest that the mutation affects endosomal acidification/maturation and TRACP exocytosis, with implications for osteoclast-osteoblast cross-talk.
authors.affiliation: Department of Experimental Medicine, University of L'Aquila, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Del Fattore
authors.forename: Rachele
authors.initials: R
authors.lastname: Fornari
authors.forename: Liesbeth
authors.initials: L
authors.lastname: Van Wesenbeeck
authors.forename: Fenna
authors.initials: F
authors.lastname: de Freitas
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Timmermans
authors.forename: Barbara
authors.initials: B
authors.lastname: Peruzzi
authors.forename: Alfredo
authors.initials: A
authors.lastname: Cappariello
authors.forename: Nadia
authors.initials: N
authors.lastname: Rucci
authors.forename: Giovanni
authors.initials: G
authors.lastname: Spera
authors.forename: Miep H
authors.initials: MH
authors.lastname: Helfrich
authors.forename: Wim
authors.initials: W
authors.lastname: Van Hul
authors.forename: Silvia
authors.initials: S
authors.lastname: Migliaccio
authors.forename: Anna
authors.initials: A
authors.lastname: Teti
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000328:Adult; D019943:Amino Acid Substitution; D000071183:Autophagy-Related Proteins; D002450:Cell Communication / Q000235:genetics; D002460:Cell Line; D004252:DNA Mutational Analysis; D011992:Endosomes / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D005089:Exocytosis / Q000235:genetics; D005260:Female; D006579:Heterozygote; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism* / Q000473:pathology; D010022:Osteopetrosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D010281:Parathyroid Hormone / Q000378:metabolism; D017354:Point Mutation; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D007527:Isoenzymes; D008562:Membrane Glycoproteins; C476215:PLEKHM1 protein, human; D010281:Parathyroid Hormone; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase