Targeted Gene Sequencing For Hypophosphatemic Rickets And Osteomalacia

PART ONE Targeted Resequencing of Phosphorus Metabolism Related Genes in 86 Patients with Hypophosphatemic Rickets/OsteomalasiaBackgrounds: Hypophosphatemic rickets/osteomalacia is characterized by abnormal bone mineralization and defective renal phosphate reabsorption.Hypophosphatemic rickets/osteomalacia consists of acquired and inherited forms,many of which have unknown etiologies.Using next-generation sequencing-based resequencing,we selected Chinese subjects with hypophosphatemic rickets/osteomalacia and aimed to detect the pathogenic genes in our patients.Methods: 86 hypophosphatemic rickets/osteomalacia patients(ranging from 3 to 70 years old) were recruited.Patients with tumor-induced osteomalacia(TIO),renal osteodystrophy,renal tubular acidosis,adefovir-induced Fanconi syndrome were excluded.Targeted massively parallel resequencing of 196 candidate genes for hypophosphatemic rickets/osteomalacia were performed in the 86 affected unrelated patients and in 100 unrelated healthy controls to identify new mutations in known genes and new genes that cause hypophosphatemic rickets/osteomalacia.Results: Among the 86 patients with hypophosphatemic rickets/osteomalacia,seven had a family history of the disease.Twelve of the hypophosphatemic rickets patients were ?18 years old,with an average age of 10.4±5.6 years.Seventy-four of the osteomalacia patients were >18 years of age,with an average age of 42.1±15.5 years.The clinical manifestations of these hypophosphatemic rickets/osteomalacia patients included short stature,bone pain,lower extremity deformities,calcification of the ligaments,and dental abnormalities.The serum phosphate levels of the majority of patients were <0.7 mmol/L(normal range is 0.8-1.6 mmol/L for adults and 1.29–1.94 mmol/L for patients aged 2-18 years),and serum alkaline phosphatase levels exhibited by the majority of patients were >160 u/L(normal range is 15-112 u/l for adults and 58-400 u/l for patients aged from 2-18 years).We identified seven PHEX gene mutations(two were novel)and one novel DMP1 mutation in eight patients respectively.After targeted sequencing data analysis,we selected 14 candidate disease-related gene loci,two of which were of most concern regarding disease severity.These results must be validated by further sequencing and functional studies.Conclusions: According to the available literature report,this study is the largest cohort of cases with hypophosphatemic rickets/osteomalacia to undergo targeted resequencing.Through the second generation sequencing of the designated target region,accurate and rapid molecular diagnosis can be made for patients with hypophosphatemic rickets/osteomalacia,which is of great clinical diagnostic value for expanding the pathogenic gene spectrum of hypophosphatic rickets/osteomalacia.PART TWO Relationship between Vitamin D Pathway Gene Polymorphisms and Changes of Bone Turnover Markers in Postmenopausal Women Supplemented with Calcium and Low-Dose CalcitriolBackgrounds:The aim of the study was to explore the association between the Vitamin D pathway gene variations and the bone biomarkers response to low dose calcitriol and calcium supplementation in postmenopausal Chinese women.Methods: In our study,a total of 110 healthy postmenopausal Chinese women(61.51±6.93 years)were enrolled.The participants were supplemented with Calcitriol D(calcium 600 mg,Vitamin D3 125IU) and Calcitriol Soft Capsules(calcitriol 0.25?g) pey day for one year.Four biomarkers,serum levels of amino-terminal propeptide of type I collagen(P1NP),beta C-terminal cross-linked telopeptides of type I collagen(?-CTX),parathyroid hormone(PTH) and 25-hydroxyvitamin D[25(OH)D] were measured at baseline and 1-year follow-up.Multivariate regression models were established to explore the statistical association between 96 single nucleotide polymorphisms(SNP) of 15 key genes within the vitamin D metabolic pathway and the change rate of the four biomarkers.Results: The complete data of 95 SNPs and 98 subjects in the vitamin D metabolism pathway were analyzed.Serum 25(OH)D level had no significant change(P>0.05).And serum levels of P1 NP,?-CTX and PTH were significantly decreased at the 12-month follow-up(all P<0.05),P1 NP decreased by 21%,?-CTX decreased by 31.8% and PTH decreased by 25%.No association was found between the bone biomarkers response to calcium and low dose calcitriol supplementation and vitamin D pathway gene polymorphisms.Conclusion: Genetic background of postmenopausal Chinese women might not influence supplemental response of the biomarkers to low dose calcitriol and calcium.Studies with larger sample size are needed to confirm these results.