Clinical And Molecular Genetic Mechanism Of FGF23 Mutation-related Diseases. Study On The Metabolic Regulation Mechanism Of ENPP1 On FGF23

Part 1 Clinical and molecular genetic mechanism of FGF23 mutation related Diseases[Background]Fibroblast growth factor 23(FGF23)is an important phosphate regulator.Secreted by bone cells,FGF23 act through increasing the excretion of urine phosphate in kidney.Meanwhile,FGF23 can reduce the intestinal absorption of phosphate by decreasing the production of 1,25(OH)2D3.Activating mutation of FGF23 lead to autosomal dominant hyperphosphatemic rickets(ADHR),while inactivating mutation of FGF23 lead to hyperphosphatemic familial tumoral calcinosis/hyperostosis-hyperphosphatemia syndrome(HFTC/HHS).The experience of dealing with the two diseases is very insufficient due to the clinical heterogeneity and rarity.Studying the clinical and molecular genetic mechanisms of the two diseases can provide experience for diagnosis and treatment of the diseases.Besides,it is helpful to clarify the pathophysiology role of FGF23 and provide clues for the metabolic regulatory mechanism of FGF23.[Objective]1.Provide diagnosis and treatment experience for ADHR and HFTC/HHS by summarizing the clinical manifestations,biochemical characteristics,bone microstructure and treatment of the diseases.2.Explore the genotype-phenotype correlation of ADHR.3.Explore the molecular mechanism of HFTC/HHS.[Methods]Eight ADHR families and one HFTC/HHS family were enrolled.Genetic diagnosis was based on Sanger sequencing.The clinical manifestations,laboratory examination and radiographic imaging of patients were collected.The bone microarchitectures of patients were detected by HR-pQCT.Multiple regression analysis and Pearson correlation test were used to explore the correlation between serum phosphates level and other laboratory indexes of ADHR patients.The glycosylation level of HFTC/HHS mutant and wild-type FGF23 protein were detected by wheat germ agglutinin affinity chromatography and Western blot.The localization of mutant FGF23 protein in endoplasmic reticulum and Golgi were detected by immunofluorescence colocalization.[Results]1.Among 8 ADHR kindreds,there were 23 patients carrying heterozygous FGF23 mutations(p.R1 76 W,p.R1 76Q,p.R1 79W or p.Rl 79Q).14 patients presented with overt symptoms.Patients with childhood onset presented with bone pain,lower extremity deformity,growth retardation and periodontal abscess,while patients with adulthood onset revealed bone pain,weakness,mobility limitation and fractures.The laboratory examinations revealed hypophosphatemia,elevated ALP and PTH and normal/low 1,25(OH)aD3.The intact FGF23 level of i-FGF23 was elevated in 6/11 patients,with average level of 86.8±91.9 pg/ml.2.Patients with p.R1 79 mutation have earlier onset of ADHR than those with p.R176 mutation[R179 v.s R176:1.25(1,37)years v.s 29(19,47)years,p<0.01].Meanwhile,patients with p.R179 mutation presented with more history of rickets and lower extremity deformity.The same phenomenon also exists in previously reported ADHR patients.3.10/14 ADHR patients with overt symptoms had iron deficiency anemia at the onset of disease,which correlated with pregnancy,abortion,menarche,etc.Correction of the iron deficiency relieved patients' symptoms significantly.The serum phosphate level of ADHR patients positively correlated with iron metabolism indexes and hemoglobin related indexes.4.In distal radius,the diameter and total area were increased in ADHR patients,while in distal tibia,the trabecular bone microstructure was significantly damaged.Compared with XLH patients,the bone microarchitecture of ADHR patients was better except for the trabecular thickness in distal tibia.5.HFTC/HHS patients presented with subcutaneous and periarticular ectopic calcified nodules,with bone pain and hypoplasia of dental root.One patient presented with generalized calcification of cardiovascular system.His X-ray revealed hyperostosis of long bones.The laboratory examinations revealed severe hyperphosphatemia,low renal phosphate excretion,and elevated C-terminal FGF23(c-FGF23).Their i-FGF23 was normal or mildly elevated.Besides,the ?-CTX and serum RANKL were elevated in both patients.The hsCRP was elevated in one patient.6.Patients carried unreported p.Ll 38R and p.I164N mutations in FGF23.The FGF23 protein with two mutations revealed defective O-glycosylation and secretion compared to wild type FGF23.7.Etidronate relieved the bone pain and ectopic calcification of one HFTC/HHS patient.Meanwhile,it significantly improved the bone microarchitecture of the patient.[Conclusion]1.We reported the largest cohort of ADHR patients around the world so far.Patients with ADHR presented with various onset ages.Patients with childhood onset revealed bone pain,lower extremity deformity,growth retardation and periodontal abscess,while patients with adulthood onset revealed bone pain,weakness,mobility limitation and fractures.2.We found the genotype-phenotype correlation in ADHR patients for the first time.Patients with p.R179 mutation have earlier onset than those with p.R176 mutation.Meanwhile,patients with p.R179 mutation presented with more history of rickets and lower extremity deformity.3.Iron deficiency is an important trigger of ADHR,which is related to pregnancy,abortion and menarche.Prevention and correction of iron deficiency play an important role in prevention,treatment and prognosis of ADHR.4.We illustrated the bone microarchitecture of ADHR patients for the first time.FGF23 mainly influenced the development and mineralization of trabecular bone in weight-bearing site.5.Our study reported the first FGF23 mutation caused HFTC/HHS family in China.The patient presented with ectopic calcification of subcutaneous,periarticular and cardiovascular system.The laboratory examinations of HFTC/HHS patients revealed hyperphosphatemia and elevated c-FGF23,hsCRP was elevated in some patients.Etidronate can relieve ectopic calcification and improve bone microstructure parameters in HFTC/HHS patients.6.The mechanism of HFTC/HHS may be related to the defective O-glycosylation of FGF23 protein.Part 2 The regulatory mechanism of ENPP1 on FGF23[Background]FGF23 plays an important role in maintaining phosphate homeostasis.Its elevation is related to many diseases,including hypophosphatemic rickets,left ventricular hypertrophy,coronary heart disease and adverse events of chronic kidney disease.Autosomal recessive hypophosphatemic rickets type 2(ARHR2)is a rare disease caused by mutation of ENPP1 gene.Patients presented with bone pain,growth retardation,lower limb deformity,fractures and ectopic calcification,with elevated FGF23 level.ENPP1 encodes exonucleotide pyrophosphatase/phosphodiesterase.Its main function is to hydrolyze extracellular triphosphate nucleotides(mainly ATP)to monophosphate(AMP)and pyrophosphate(PPi).PPi is an important inhibitor of mineralization.At present,the regulatory mechanism of ENPP1 on FGF23 remain unclear.Exploring the regulatory mechanism of ENPP1 on FGF23 is helpful to clarify the pathogenic mechanism of ARHR2,and can provide therapeutic target for ARHR2 and other FGF23 related diseases.[Objective]1.Explore the effect of ENPP1 on bone mineralization.2.Explore the regulatory mechanism of ENPP1 on FGF23 and clarify the pathogenic mechanism of ARHR2.[Methods]ENPP1 floxp mice was established by CRISPR/Cas9.Osteocyte specific ENPP1 knockout mice was established by mating ENPP1 floxp mice and DMP1 cre mice.Bone microstructure,serum phosphate and urinary phosphorus excretion indexes of 12-week-old male mice were detected.Ectopic calcification of aorta and kidney was detected by alizarin red staining and von Kossa staining.The enzyme activities of wild type and ENPP1 mutant protein were detected by colorimetry.ENPP1 in rat osteoblast-like cell line UMR106 cells was inhibited by siRNA and its inhibitor.The expression of FGF23 was detected by quantitative PCR.[Results]1.12-week-old male cKO mice revealed increased trabecular BV/TV?trabecular thickness and trabecular number,the trabecular pattern molecules and SMI were decreased.The cortical open pore volume and bone mineral density were decreased.There was no ectopic calcification in kidney and aorta of the mice.i-FGF23 in ENPP1 cKO mice was significantly increased(496.9±108.7 pg/ml v.s 321.8±57.0 pg/ml,p=0.001).Accordingly,the renal phosphate excretion index was increased(30.8±6.9%v.s 22.4 ±7.9%,p=0.04).The serum phosphate revealed decreasing trend,but did not reach statistically significance.2.ENPP1 protein with ARHR2 causing mutations revealed decreased enzyme activity compared to wild-type protein.Inhibition of ENPP1 led to reduced degradation of extracellular ATP and increased FGF23 production in UMR106 cells.ATP-?S,a non-hydrolytic analogue of ATP,significantly upregulated the expression of FGF23,while pyrophosphate had no effect on FGF23 expression.3.PPADS is a non-selective purinergic P2 receptor inhibitor.100?M PPADS significantly inhibited the FGF23 expression in UMR106 cells,while P1 receptor inhibitor MRS 1745 upregulated the synthesis of FGF23 in UMR106 cells.PPADS also inhibited the increase of FGF23 induced by ENPP1 inactivation.[Conclusion]1.ENPP1 regulate bone mineralization and ectopic calcification by regulate the ratio of phosphate and pyrophosphate.2.Loss of catalytic activity in ENPP1 lead to elevated FGF23 in bone.ENPP1 regulate FGF23 through ATP-P2 purinergic receptor signaling.P2 purinergic receptor might be a therapeutic target for ADHR2 and FGF23 related diseases.3.Regulation of ATP on FGF23 reveals role of FGF23 in energy metabolism.