Exploring The Gene Mutation Profile Of Large Osteogenesis Imperfecta In China And The Clinical Characteristics And Bisphosphonate Efficacy Analysis Of Adult Patients With Osteogenesis Imperfecta

Part 1.DNA sequence analysis and gene mutation spectrum in a large Chinese cohort with osteogensis imperfectaObjective:Osteogenesis imperfecta(OI)is a group of hereditary skeletal disorders characterized by low bone mass,increased bone fragility and recurrent fractures.Molecular diagnosis of OI is challenging due to the clinical and genetic heterogeneity,and few such large-scale studies about OI have been conducted among Chinese population.The present study aims to reveal the gene mutation spectrum,autosomal dominant and recessive genotype-phenotype correlations in a large Chinese cohort with OI by targeted next-generation sequencing(NGS).Methods:We developed a novel NGS panel for the targeted sequencing of all 19 genes related to OI,and performed diagnostic DNA sequencing for 152 Chinese OI patients admitted in PUMCH(2013-2016).Variants identified by NGS were futher confirmed by Sanger sequencing and co-segregation analysis in each OI family.We analyzed the OI gene mutation spectrum in a total of 261 OI patients with sequcening results in our research group histherto.Besides,multiple clinical phenotypes were also collected,such as height Z-scores,fracture rates,bone mineral density Z-scores at lumbar spine and femoral neck(LS and FN BMD),serum levels of alkaline phosphatase(ALP)and cross-linked C-telopeptide of type ? collagen(P-CTX),bone morphologies and some extra-skeletal features.The genotype and phenotype relationships were then analyzed by comparison of the above phenotypes among patients with different gene mutations.Results:Using NGS and Sanger sequencing,a total of 115 different variants in 10 OI-genes were identified,including 50 novel mutations among 140 OI patients.The sequencing platform showed a high OI diagnostic yield(92%)and accuracy rate(100%).Mutations in genes encoding type ? collagen(COL1A1/COL1A2)accounted for 76%(n= 198)of all the molecularly diagnosed patients in our research group,followed by the autosomal dominant IFITM5 gene(n=15,6%),autosomal recessive SERPINF1 and WNT1 genes(n=12,4.6%).In mild OI type ?,we observed COL1A1 and COL1A2 mutations were responsible for 01 in 84%of patients;whereas in moderate-severe 01(type ?-?),mutations in COL1A1 and COL1A2 were observed in only 68%of patients,but mutations in recessive genes were in 21%of patients.Patients with type I collagen quantitative defects were taller(-0.4 � 1.3),with higher BMD Z-scores(LS,-1.7 � 1.3 and FN,-2.7� 2.2)and less skeletal deformities(11%,all P<0.05)compared with patients with type I collagen qualitative defects.The position of qualitative defects in al chain was found a correlation to the presence of blue sclera(BS)and absence of dentinogenesis imperfecta(DI);and the position in a2 chain was found a negative correlation with FN-BMD.We found no relations between the amino acid substitution types in ?1 or ?2 chains and any clinical phenotypes.Moreover,compared with collagen type ? mutant patients,autosomal recessive inherited patients exhibited higher P-CTX levels(1.09 � 0.48 ng/ml),lower LS-BMD Z-scores(-3.5 � 2.4),more severe deformities and mobility restrictions(80%),but less presense of BS(40%)and DI(7%).Conclusion:We revealed the comprehensive gene mutation spectrum in Chinese OI patients,and the novel mutations identified here expanded the mutation catalogue of OI.Mutations in autosomal dominant COL1A1 and COL1A2 genes were the most common,followed by IFITM5 gene,autosomal recessive SERPINF1 and WNT1 gene.However,the mutation spectrum showed dramatical differences between mild and moderate-severe OI.Patients with type I collagen quantitative defects were less severe than those with qualitative defects.And patients with autosomal recessive gene mutations were all belong to moderate-severe OI.We demonstrated our new NGS panel as an effective method for molecular diagnosis of OI,and may contribute to the progress of future research on prenatal diagnosis and molecular targeted therapy in OI.Part 2.The Clinical Characteristics and Efficacy of Bisphosphonates in Adult Patients with Osteogenesis ImperfectaObjective:Osteogenesis imperfecta(OI)is characterized by bone fragility and recurrent fractures.Patients with 01 have significant variable expressivity ranging from mild to neonatal lethality(type I-IV).Adults with 01 are often treated with oral or intravenous bisphosphonates(BPs).We investigated the clinical phenotypes of adult 01 patients and prospectively compared the efficacy of oral alendronate(ALN)with intravenous zoledronic acid(ZOL)in 01 patients.Methods:The clinical data of 102 01 patients admitted before the age of 18 years were collected and analyzed retrospectively(2007-2016).These included bone mineral density(BMD)at lumbar spine and proximal hip,serum levels of total alkaline phosphatase(ALP),cross-linked C-telopeptide of type I collagen(P-CTX),25-hydroxyvitamin D(250HD)and parathyroid hormone(PTH),and also clinical fractures,X-ray and extra-skeletal features.We compared the differences in BMD and serum biomarkers between OI adults and healthy subjects.Besides,we conducted a 24-month,observational clinical study in 60 adult patients recruited between the year 2011 and 2015.They were randomized at a 2:1 ratio to receive either weekly oral ALN 70 mg or once-yearly infusion of ZOL 5 mg.The efficacy outcomes were changes in BMD,bone turnover biomarkers,and fracture incidence.Results:Three-fourths of the 102 OI patients were classified with mild or moderate OI.Adult OI patients had significantly higher ALP(type ?/?/?)and P-CTX(type ?)levels than age/sex/BMI-matched healthy subjects.Vitamin D deficiency and insufficiency accounted for 73.4 and 89.0%patients among OI adults,with the mean levels of 16.2 � 8.1 ng/ml.BMD at lumbar spine and proximal hip were extremely low in OI type ?/?/? patients,with two-thirds having at least one BMD Z-score of less than or equal to-2.0.For OI type ?,this percentage was as high as 90%,and skeletal deformity,scoliosis and dental problems were also common.As for the BPs treatment,52 patients completed the 24-month clinical study.BMD at lumbar spine,femoral neck,and total hip were equivalently elevated in the ALN(10.5,13.2,and 14.7%,respectively)and ZOL(11.3,13.7,and 11.7%,respectively;all P>0.05)groups.Serum ALP and P-CTX levels decreased dramatically in ALN and ZOL group,with no significant differences between two groups(P=0.12 and 0.48).Compared to the prior fracture rates,clinical fracture incidences were decreased in the ALN and ZOL groups.Conclusion:OI adults present significantly higher bone resorption biomarkers,lower vitamin D and bone mass than healthy populations.Oral ALN and intravenous ZOL are equally effective in increasing BMD and inhibiting bone turnover in adults with OI.The treatment may reduce fracture riskes in patients of this study,but further efforts are still needed to demonstrate the anti-fracture efficacy of BPs.