title: Defective O-glycosylation due to a novel homozygous S129P mutation is associated with lack of fibroblast growth factor 23 secretion and tumoral calcinosis.
abstract: BACKGROUND
  Homozygous mutations in fibroblast growth factor (FGF23) have recently been described as the genetic cause of one form of hyperphosphatemic tumoral calcinosis (HFTC). However, it remained unclear to date how these mutations lead to loss of biologically active FGF23 in the circulation.
  
  
  METHODS
  We here report a novel homozygous mutation, c.385T>C in FGF23 exon 2, which changes codon 129 from serine to proline (S129P) in a previously described individual affected by HFTC. The S129P mutation as well as two known FGF23 mutations, S71G and S129F, were introduced into an expression vector encoding wild-type (wt) human (h) FGF23 to yield [P129]hFGF23, [F129]hFGF23, and [G71]hFGF23; whole lysates, glycoprotein fractions, and conditioned media from HEK293 and COS-7 cells expressing these constructs were subjected to Western blot analysis using affinity-purified goat anti-hFGF23(51-69) and anti-hFGF23(206-222) antibodies.
  
  
  RESULTS
  We detected 25- and 32-kDa protein species in total lysates of HEK293 cells expressing wt-hFGF23. The 32-kDa band, representing O-glycosylated hFGF23, was not detectable in the glycoprotein fraction of lysates from HEK293 cells expressing [P129]hFGF23, and in comparison with wt-FGF23 only small amounts of [P129]hFGF23 were secreted into the medium. Similar results were obtained for cells expressing [G71]hFGF23 and [F129]hFGF23.
  
  
  CONCLUSIONS
  Our data for the first time directly show that FGF23 mutations associated with HFTC impair O-glycosylation in vitro resulting in poor secretion of the mutant hormone thereby explaining the characteristic hyperphosphatemic phenotype of homozygous carriers in vivo.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. cbergwitz@partners.org
authors.forename: Clemens
authors.initials: C
authors.lastname: Bergwitz
authors.forename: Santanu
authors.initials: S
authors.lastname: Banerjee
authors.forename: Hilal
authors.initials: H
authors.lastname: Abu-Zahra
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kaji
authors.forename: Akimitsu
authors.initials: A
authors.lastname: Miyauchi
authors.forename: Toshitsugu
authors.initials: T
authors.lastname: Sugimoto
authors.forename: Harald
authors.initials: H
authors.lastname: Jüppner
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002114:Calcinosis / Q000235:genetics*; D002353:Carrier State; D002522:Chlorocebus aethiops; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005091:Exons / Q000235:genetics; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006720:Homozygote; D006801:Humans; D007015:Hypophosphatemia, Familial / Q000150:complications / Q000235:genetics*; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000150:complications / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D011392:Proline; D012694:Serine
chemical_list: D003062:Codon; D017931:DNA Primers; C000717427:FGF23 protein, human; D012694:Serine; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23; D011392:Proline