Assay Development for the Detection of FGF23 Mutations That Cause Familial Tumoral Calcinosis by Amplification Refractory Mutation System (ARMS)

Abstract

Hyperphosphatemic Familial tumoral calcinosis (HFTC) is an autosomal recessive disorder characterized by hyperphosphatemia and the presence of ectopic or other periarticular calcifications around major joints.The disease is caused mainly by Fibroblast growth factor-23 (FGF-23) loss-of-function. Seven mutations throughout the FGF23 gene (H41Q, Q54K, S71G, M96T, G123W, S129F, and S129P) have been identified as pathogenic mutations in HFTC patients.So far, DNA sequence analysis is the only method for detection of these mutations. For this purpose, we report a rapid and efficient method using amplification refractory mutation system (ARMS) that can be used for the primary genetic diagnosis of HFTC. Human FGF23 exons were cloned into plasmid to perform in vitro site-directed mutagenesis. We generated the different mutants and confirmed each one of them by DNA sequencing. We optimized the conditions for ARMS-PCR and tested each mutant. Finally, we validated S129F mutant by testing 10 patient’s from a clan with HFTC. Seven different mutations in FGF-23 known to causes HFTC have been successfully generated using In vitro site-directed mutagenesis. And the mutated sequences were confirmed by DNA sequence analysis. We designed ARMS specific primers to identify each of these mutations and we optimized the conditions needed to run ARMS-PCR. And To validate our designed method, we tested ten patients from two different family clinically diagnosed with HFTC. Our results indicate the successful use of this method in the detection of FGF23 mutation. The development of rapid and simplified methods of diagnosis of HFTC is important to improve the prognosis of the disease. Early identification of the carriers and the affected of the disorder can provided a proper genetic counseling and therapy. Here, we reported rapid, simple, and inexpensive method for diagnosis HFTC.