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.

FGF23-S129F mutant bypasses ER/Golgi to the circulation of hyperphosphatemic familial tumoral calcinosis patients.

FGF23 is essential for the homeostasis of phosphate, and vitamin D. Loss-of-function mutations in this hormone cause hyperphosphatemic familial tumoral calcinosis (HFTC). Earlier reports suggested that intact FGF23 from loss of function mutants such as FGF23/S129F (iFGF23/S129F) is retained intracellularly while the carboxy-terminal fragment is secreted. We sought to investigate the fate of iFGF23/S129F mutant hormone in vivo and in vitro. Five patients clinically diagnosed with HFTC and confirmed by DNA sequencing to carry the c.386 C>T; p.S129F mutation in the homozygous state were studied. Healthy and heterozygous individuals were used as controls in the study. Using ELISA assays, we showed that iFGF23/S129F was 2-5 folds higher in patients' plasma, compared to heterozygous or healthy controls. Importantly, the mutant hormone could not be detected in the patients' sera. However, using proteinase inhibition profiling, we found that a serum metalloproteinase degraded the iFGF23/S129F explaining our failure to detect it in sera. The serum metalloproteinase degrades the WT and the mutant at different rates. Also, confocal microscopy imaging using wild-type (WT) FGF23 or FGF23/S129F mutant in transiently transfected HEK293 and HeLa cells showed weak staining of the Golgi complex with some vesicular staining resembling the ER. Additionally, FGF23 variants (FGF23/WT, FGF23/S129F, FGF23/S71G, and FGF23/R176Q) from stably transfected HEK293 cells secreted high levels into a serum-free medium that can be detected by ELISA and Western blot. Our results suggest that iFGF23/S129F mutant bypasses the ER/Golgi quality control system to the circulation of HFTC patients by an unknown pathway. Finally, we hypothesize that either the mutant hormone is unable to bind α-Klotho-FGFR1c, or it binds the dyad receptor with low affinity and, therefore, incapable of initiating maximal intracellular signaling. Our findings raise the potential use of the WT hormone in therapies of some HFTC patients.

Hypercholesterolemia among apparently healthy university students.

BACKGROUND: Hypercholesterolemia (HC) is a major risk factor in the development of coronary heart disease (CHD). Serum cholesterol is directly related to complications and mortalities associated with heart diseases. There are a few studies that describe HC among youths in the Arab Gulf countries. We sought to evaluate HC among young healthy university students to assess their risk of developing CHD. METHODS: Lipid profile of 166 students between the ages of 16-30 years (Mean: 20.49±2.96) were examined and blood glucose, total protein, albumin, thyroid stimulating hormone (TSH) and the inflammation marker high sensitivity CRP (hsCRP) were determined. Each volunteer filled a questionnaire about her/his lifestyle and personal and family medical histories and height and weight were measured to determine body mass index (BMI). The data were analyzed using SPSS version 17. Chi-Square was used to determine the relation between categorical variables. A p-value <0.05 was considered statistically significant. RESULTS: According to the American Heart Association criteria, 44 (26.5%) students were identified with primary hypercholesterolemia (PHC) in the first testing round. After proper health counseling, the same tests were repeated after 2-3 weeks in all 44 hypercholesterolemic students. We found only 26 (15.6%) of them to be hypercholesterolemic. There was a significant relation between high total cholesterol (TC) and high TC/HDLC, as well as high or very high hsCRP and high TC/HDLC (both, p<0.001). Males tend to have higher TC/HDLC and hsCRP than females (both p0.002 and 0.005, respectively). Family history of CHD was found in 8 students and obesity was recorded in 5 volunteers. CONCLUSION: The results necessitate further studies in determining the cause of PHC. We predict a genetic element contributing to the high percentage of PHC in the current study.

The Arabic allele: a single base pair substitution activates a 10-base downstream cryptic splice acceptor site in exon 12 of LDLR and severely decreases LDLR expression in two unrelated Arab families with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is a monogenic autosomal dominant disorder caused by defects in LDLR. Few reports describe FH mutations among Arabs. We describe a mutation in LDLR of two unrelated Arab families. We investigated 19 patients using DNA sequencing, RFLP, and real-time (RT) PCR. DNA sequencing showed a base pair substitution (c.1706-2 A>T) in the splice acceptor site of LDLR intron 11. Our results were confirmed by RFLP on 2% agarose gel. In silico analysis predicted a new cryptic splice site downstream of the original position generating a 10-base deletion from the beginning of exon 12; (c.1706-1715del.ATCTCCTCAG). cDNA sequencing of exon 12 confirmed the computational analysis. The deletion was visualized on 4% agarose gel. The deletion generates a frameshift and a premature termination codon (c.1991-1993; p.(Asp569Valfs*93). RT-PCR revealed that LDLR mRNA is 9.3%±6.5 and 17.9%±8.0 for FH homozygote and heterozygote individuals respectively, compared to a healthy family control. We predict a class II LDLR mutation that leads to a truncated receptor missing exons 14-18. We called this mutation "the Arabic allele". We expect a significant contribution of this mutation to the prevalence of FH among Arabs. Also, we propose that the severe down regulation of LDLR mRNA expression is due to nonsense-mediated-decay.