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. Conclusion: 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.
Bibliographical noteFunding Information:
This work was supported by the National Institutes of Health Grants PO1 DK11794 , Project IV (to H.J.), and 1K08 DK078361 ; Young Investigator Awards by the National Kidney Foundation and the American Association for Clinical Investigation (to C.B.).