Development and Validation of a Simple Diagnostic Method to Detect Gain and Loss of Function Defects in Fibroblast Growth Factor-23.

BACKGROUND: Fibroblast growth factor-23 (FGF23) is a bone-derived hormone that regulates the homeostasis of phosphate and vitamin D. Three substitutions in the hormone are reported to cause autosomal dominant hypophosphatemic rickets and seven substitutions to cause autosomal recessive hyperphosphatemic familial tumoral calcinosis (HFTC). Both disorders are rare in the general population and occur most often in the Eastern Mediterranean region and Africa. None of the mutations could be identified using standard restriction fragment length polymorphism. The only technique currently available to confirm the clinical diagnosis is DNA sequencing. METHODS: Using a tri-primer ARMS-PCR, in vitro site-directed mutagenesis and DNA sequencing, we developed, verified and validated a rapid and reliable diagnostic test for the ten mutations in FGF23. RESULTS: We generated a test for all ten mutations and confirmed each test by DNA sequencing. We increased the specificity of the test by introducing a mismatch at position -2 in the 3'-terminus of the reverse primer of the normal and the mutant sequences. Finally, using DNA sequencing, we validated the technique for FGF23/S129F substitution by testing samples from 80 individuals from two unrelated Arab families harboring HFTC. CONCLUSIONS: This inexpensive and specific method could be adopted where DNA sequencing is not available or affordable.