| Part 1.Serum sclerostin levels in a large sample of Chinesechildren with osteogenesis imperfectaBackgroundOsteogenesis imperfecta(OI)is a group of clinically and genetically heterogeneous hereditary bone disorder characterized by decreased bone mass,recurrent bone fractures and bone deformities.At present,the diagnosis of OI is determined by either genetic testing or by clinical manifestation and radiography features.There are no specific biochemical markers available for the diagnosis of OI.The abnormity of bone matrix proteins and osteoblasts in OI patients might obviously impair the function of osteocytes and cytokines secreted by osteocytes.Sclerostin is an important regulator of bone remodeling,which is a glycoprotein almost exclusively produced by mature osteocytes.Studies with a small sample of patients showed that SOST levels in OI were inconsistent.Whether sclerosin is a specific biomarker in 01 patients should be assessed in a large sample of patients.The association between serum sclerostin and genotypes,phenotypes,and bisphosphonates(BPs)treatment in 01 patients is worthy to be investigated.Objective1.We evaluated serum sclerostin levels in children with OI,and compared with healthy children in order to assess whether sclerosin could act as a specific biomarker in OI patients.2.To determine the association between serum sclerostin and disease classification,and bisphosphonates treatment,we compared sclerostin levels in different types of OI on the basis of accurate molecular diagnosis and Sillence classification,and identify its associations with BPs treatment history.Methods1.This was a cross-sectional study included 139 children with 01 who had definite clinical and genetic diagnosis and 53 healthy age-and gender-matched controls who went to the hospital for regular physical examination.In OI patients,medical history,clinical manifestation,physical examination,biochemical examination and radiography were collected in detail.Serum levels of P-cross linked C-telopeptide of type I collagen(?-CTX,bone resorption marker),25-hydroxyvitamin D(250HD),and intact parathyroid hormone(PTH)were measured using automated electrochemiluminescence system.Total alkaline phosphatase(TALP,bone formation marker)was detected by enzyme-linked immunoassay.Bone mineral density at lumbar spine and femoral neck was measured by dual-energy X-ray absorptiometry(DXA,Lunar Prodigy,GE Healthcare,USA).The genomic DNA of the probands were sequenced using a targeted next-generation sequencing(NGS)panel(Illumina HiSeq2000 platform,USA)and confirmed by Sanger sequencing.2.Serum sclerostin levels were quantitatively detected by an established microtiter-plate-based enzyme-linked immunosorbent assay(ELISA,Biomedica Medizinprodukte GmbH,Vienna)in OI children and healthy controls.Then we compared the differences of serum sclerostin levels between these two groups.3.OI was then divided into types ?,?,and ? according to classical Sillence classification.To compare the differences between different phenotypes,patients were divided into two subgroups:mild(OI type ?,OI-?)and moderate-severe(OI type ? and?,OI-?-?).To compare the differences among various genotypes,patients were divided into four groups,including COL1A1,COL1A2,IFITM5 and the rare gene mutation group(gene mutations other than COL1A1,COL1A2 and IFITM5).To analyze the effects of different mutations in encoding genes of type I collagen,patients were divided into COL1A1 haploinsufficiency mutation,COL1A1 triple helical mutation and COL1A2 triple helical mutation group.Patients were divided into BPs-treated and BPs-untreated group.The differences of height Z-scores,fracture rates,bone turnover markers(BTMs),sclerostin,bone mineral density Z-scores were compared among patients with different phenotypes and genotypes.4.The associations between sclerostin levels and BTMs,250HD,PTH,BMD Z-score,fracture rates were analyzed.Results1.A total of 139 patients with OI(mean age 9.4±4.6 years)were included in this study,which consisted of 97 boys and 42 girls.61 patients had positive family history of fragility fractures.The average long bone fracture rate was 1.00±1.02 fractures/year.Blue or gray sclerae were found in 118(84.9%)patients,and dentinogenesis imperfecta existed in 30(21.6%)patients.Apparent hearing loss was identified in 3(2.2%)patients.The mean BMD Z-score at lumbar spine and femoral neck were-0.86±2.47 and-2.10±33.13,respectively.2.Serum sclerostin levels did not differ significantly between OI patients and healthy controls(25.3±9.8 vs.25.0±5.5 pmol/l,p=0.514).3.In different phenotypes of OI,patients with mild OI(type I,n=78)were taller,heavier,had higher BMD Z-score at lumbar spine and femoral neck and had fewer fractures before BPs treatment than that in moderate and severe OI patients(type ?-?,n=61)(p<0.05).Serum sclerostin levels were mildly lower in mild OI patients than those in moderate and severe OI patients(24.2±9.8 vs.26.5±9.8 pmol/L,p=0.035),which was not significant after adjusting for age(p=0.160).4.In different genotypes of OI,patients with rare gene mutations were the tallest,and patients with COLIA1 mutations were the shortest.Patients with rare gene mutations(n=18)had significantly higher serum sclerostin levels than healthy controls(31.9±12.9 and 25.0±5.5 pmol/1,p=0.018)and patients with mutations in COL1A1(n=69),COL1A2(n=42)and IFITM5(n=10)(all p<0.05)even after adjusting for age.IFITM5 mutation group had a trend to have higher serum sclerostin levels than control group(29.3±9.0 and 25.0±5.5 pmol/l,p=0.054).5.Patients with COL1A1 haploinsufficiency mutations(n=26)were taller,heavier than patients with COL1A1 triple helical mutations(n=17)and COL1A2 triple helical mutations(n=31)(p<0.05).Patients with COL1A1 haploinsufficiency mutations had lower serum sclerostin levels(21.6±6.0 vs.25.0±.5 pmol/l,p=0.013)than healthy controls after adjusting for age.However,there was no significant difference of sclerostin between patients with COL1A1 haploinsufficiency mutations and triple helical mutations.6.Serum ?-CTX and TALP levels were lower in patients with BPs treatment(n=61)than that in BPs-untreated groups(n=78)(p<0.001).BMD Z-score of lumbar spine and femoral neck were higher in patients with BPs treatment(all p<0.001).Serum sclerostin levels were similar in BPs-treated and BPs-untreated group(26.4±11.3 vs.24.3±8.4 pmol/l,p=0.596).7.Serum ?-CTX levels were positively correlated to sclerostin levels,after adjusting for age,sex and BPs treatment(r=0.24,p=0.006).No correlations were found between serum sclerostin levels and TALP,BMD and bone fracture frequency.ConclusionWe investigated the serum levels of sclerostin in a large sample of OI children and healthy children in China for the first time,and we also analyzed the differences of serum sclerostin levels in patients with different phenotypes and genotypes of OI.We found that serum sclerostin levels were similar between different phenotypes of OI patients and healthy controls,which indicated that sclerostin was not a reliable parameter for the discrimination of OI in children patients or reflecting the severity of OI.Patients with rare gene mutations had higher sclerostin levels,which suggested serum sclerostin levels were associated with molecular classification of 01.Elevated serum sclerostin levels in patients with rare gene mutations could partially explain the poor response to BPs treatment in these patients.Sclerostin antibody is considered in these OI patients in the future.Part 2.Bone damage and prevention in patients with myastheniagravisBackgroundBone and muscle are closely related.Muscles affect bones primarily through mechanical and chemical effects.In neuromuscular disorders,low muscle strength,vitamin D deficiency,decreased bone mineral density(BMD)and increased fracture incidence have been observed.Myasthenia gravis(MG)is a common autoimmune neuromuscular disorder,which is a disease model to study the interrelationship between muscle and bone.However,the influence of MG on bone metabolism,the relationship between muscle and bone in MG patients are unclear.Glucocorticoids(GCs)act as the first-line treatment in MG as long-term immunosuppressants,which could increase the risk of bone loss and bone fracture.However,the prevention and treatment of osteoporosis in MG patients is inadequate.The efficacy and safety of treatment with alendronate and alfacalcidol in MG patients are worthy to be studied.Objective1.To evaluate the muscle and bone metabolism,and their associations in Chinese patients with MG before GCs treatment.2.To assess the effects of oral alendronate and alfacalcidol,or alfacalcidol alone on bone in Chinese patients with MG who initiated GCs treatment.Methods1.This study included 86 MG patients who had ocular muscle weakness only or mild-to-moderate generalized weakness in the Neurology and Endocrinology Department of Peking Union Medical College Hospital between December 2014 and June 2016.86 healthy,age-and gender-matched normal subjects from the database of the Chinese Bone Turnover Marker Study were also included.Serum levels of P-cross linked C-telopeptide of type I collagen(?-CTX,bone resorption marker),25-hydroxyvitamin D(25OHD),and intact parathyroid hormone(PTH)were measured using automated electrochemiluminescence system.Total alkaline phosphatase(TALP,bone formation marker)was detected by enzyme-linked immunoassay.Serum level of osteocalcin(OC,bone formation marker)was measured by radioimmunoassay.Bone mineral density at lumbar spine and femoral neck was measured by dual-energy X-ray absorptiometry(DXA,Lunar Prodigy,GE Healthcare,USA).These markers were compared between the two groups.In MG patients,muscle strength was assessed by handgrip,which was measured using a digital hand dynamometer(CAMRY,EH101).One-leg-stand-time(OLST)was measured individually on each leg with open eyes.The relationships between these markers were analyzed.2.The patients who initialed glucocorticoid treatment were given anti-osteoporosis drugs.Patients with bone mineral density(BMD)T-score less than-1.0 at baseline were treated with alendronate 70 mg/vitamin D3 2800IU/week.Patients with BMD T-score more than-1.0 at baseline were included in alfacalcidol group.Patients in both groups were treated with alfacalcidol 0.25 ?g/2d and calcium 600 mg/d.Follow-up evaluations were scheduled at baseline,3,6,and 12 months of treatment to assess bone turnover markers,BMD,and side effects.Results1.Compared to healthy controls,MG patients had lower BMD at the femoral neck(P<0.001),lower 250HD levels(17.36±6.64 vs.22.11 ±7.28 ng/ml,p<0.001),higher?-CTX and PTH levels(p<0.001 and p=0.001,respectively).2.Low grip strength and short OLST were found in 11(12.8%)and 12(14.0%)MG patients,respectively.There were 3(3.5%)MG patients with low bone mass for chronological age.In patients with MG,grip strength was positively correlated with BMD.3.After 12 months of treatment,the mean BMD at lumbar spine and total hip increased by 3.4%(p=0.002)and 2.6%(p=0.020)in alendronate group.Serum OC,ALP and P-CTX levels decreased by 54.5%,31.2%,and 63.5%(all p<0.05 vs.baseline).4.In alfacalcidol group,mean BMD at lumbar spine,femoral neck,and total hip reduced by 6.1%,3.2%,and 3.3%(all p<0.001 vs.baseline).The OC,ALP levels reduced by 41.3%(p<0.001)and 18.3%(p<0.001)after 12 months of treatment,while ?-CTX was not changed(p=0.652).5.Tolerance of patients to alendronate and alfacalcidol was relatively well,but incidence of hypercalciuria(24UCa?7.5 mmol/24h)was high in both groups.There were 40.9%and 50.9%of the patients with one or more hypercalciuria in alendronate group and in alfacalcidol group,respectively(p=0.458).ConclusionOur study evaluated the relationship between muscle and bone,and the efficacy and safety of anti-osteoporosis drugs in Chinese MG patients for the first time.We identified that MG patients without glucocorticoids treatment tended to have low proximal hip BMD compared to healthy controls,which may partially be explained by reduced muscle strength,elevated bone resorption markers,vitamin D deficiency,and increased PTH levels in those with MG.Treatment with alendronate combined with alfacalcidol was demonstrated for the first time to significantly increase BMD,and decrease bone turnover biomarker levels in a relatively large cohort of Chinese MG patients who initiated glucocorticoids treatment.However,alfacalcidol alone failed to prevent bone loss in MG patients receiving glucocorticoid therapy.During the treatment,urinary calcium levels should be closely monitored to reduce drug-related side effects. |
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