Genotype-phenotype Relationship Analysis And A Murine Model Phenotype Identification Of Osteogenesis Imperfecta

Context and ObjectiveOsteogenesis imperfecta(OI)is a rare genetic bone disease that mainly characterized by bone fragility and skeletal deformities.Other extraskeletal manifestations have also been reported,such as blue sclerae,hearing loss,skin fragility,muscle weakness,and dentinogenesis imperfecta(DI).Most OIs are caused by autosomal dominant mutations in the collagen I genes(COL1A1 and COL1A2),which result in the quantity or structure defects of type I collagen.In the last decade,many non-collagen I genes have been identified that are participated in the synthesis,processing,secretion and post-translational modification of type I collagen,as well as in the regulation of bone mineralization and osteoblast differentiation.The clinical diagnosis of OI is based on phenotypes and history of recurrent fractures as well as imaging features,but confirmation of the diagnosis of OI depends on genetic testing.The classification of OI has become very complicated due to the discovery of non-collagen I genes.In recent years,the genetic classification was confirmed.Sillence types Ⅰ-Ⅳ are used for patients with COL1A1 or COL1A2 mutations and additional type numbers to new genes.The diversity of OI causative genes also complicates the relationship between its genotype and phenotype.Recently,investigators around the world have performed on the genotype-phenotype relationship of OI.However,these previous studies have limitations.In addition,there are few investigations analyzing the correlation between treatment effect and different genotypes in OI patients.Bisphosphonates are widely used to treat OI.Currently,lack of a cure for OI and the treatment of OI patients is primarily focus on reducing fractures and improving mobility.Research on OI treatments has relied on a variety of animal models,and in recent years several new approaches to treat OI have been discovered by the classic mouse model of OI,with gene therapy and stem cell therapy promising to cure OI.we enrolled 276 hospitalized OI patients and 116 patients were diagnosed as OI by genetic testing.We analyzed the correlation between genotype and phenotype based on the different types,pathogenic mechanisms,and gene inheritance patterns.Additionally,we assessed the effect of treatment on different pathogenic genes.We have performed phenotypic identification of the classical murine model of OIaying the foundation for the study of new approaches to OI treatment.Part 1 Genotype-phenotype analysis of osteogenesis imperfectaMethods1.SubjectsFrom 2010 to 2020,a total of 276 OI patients were hospitalized and treated in the Shandong Provincial Hospital affiliated with Shandong University.Finally,116 patients were diagnosed as OI by genetic testing in this study.A total of 116 patients were followed up for an average of 6 years,and subsequent analysis was performed on these patients.2.Clinical and treatment data collectionThe clinical data include height,weight,age,and location of the first fracture,the total number of fractures,frequency of fracture,blue sclera,DI,independent walking,long bone deformity,scoliosis,and chest deformity.The treatment data included therapeutic methods and therapeutic effects.3.Genetic testingGenetic testing of OI patients by Whole-exome sequencing(WES).4.Statistical analysisNormality was examined by the Kolmogorov-Smirnov test,and all continuous variables in our study followed a skewed distribution.Continuous variables were summarized by the median(first quartile,third quartile).Categorical variables are presented as counts.The MannWhitney test was used to evaluate two group differences in continuous variables,and differences in more than three groups were evaluated using the Kruskal-Wallis test and pairwise multiple comparison tests.We used Pearson’s chi-squared test or Fisher exact tests to analyze differences among the categorical variables,and multiple testing correction was done using the Bonferroni method.Calculations were performed using IBM SPSS Statistics 25 software.P<0.05 was used to indicate statistic difference.Results1.Phenotype analysisThe Z score for height was-2.18(-4.15,-0.78),which showed that patients were generally short.The average age at the first fracture of OI patients was 0.96(0.09,2.00)years,however the clinical and genetic diagnoses were confirmed 2 and 10 years after the first fracture,respectively.On average,every person OI patients suffered 1.50 fractures annually.We observed that blue sclerae was the most common clinical manifestation in the patients with OI(86.2%),followed by long bone deformity(81.0%).2.Genotype analysisA total of 116 mutations were identified in the 6 genes(COL1A1,COL1A2,IFITM5,SERPINF1,FKBP10,and WNT1)including 96 mutations in collagen Ⅰ genes and 20 in noncollagen Ⅰ genes.A total of 61 helical mutations and 21 haploinsufficiency mutations were identified in this study.3.Genotype and phenotype correlation analysis3.1 For the collagen Ⅰ gene mutations,the mutations in two genes led to similar phenotypes of OI,except that the age at first fracture was younger in patients with COL1A2 mutations than those with COL1A1 mutations.3.2 The clinical manifestations of patients with helical mutations were more serious than those with haploinsufficiency mutations,such as shorter height,younger age of first fracture,and more frequent incidence of DI and long bone deformity.3.3 We performed genotype-phenotype correlation in helical,haploinsufficiency,and noncollagen Ⅰ gene mutations.Comparing these three groups,patients with helical mutations were the shortest and the most prone to DI.Patients with non-collagen Ⅰ gene mutations were least susceptible to blue sclerae and had the highest fracture frequency.Moreover,patients with haploinsufficiency mutations exhibited the oldest age at first fracture.3.4 There were some minor phenotypic differences between non-collagen Ⅰ genes.3.5 AR patients were more susceptible to fracture and had a poorer ability to walk independently but were less prone to blue sclerae and DI.3.6 After pamidronate treatment,the vast majority of the patients presented with significantly reduced fracture numbers,and the pamidronate effect seems independent of the genotypes.Conclusion1.Our findings help reveal the genotype-phenotype relationship of OI patients and contribute to the classification and diagnosis of OI.2.Pamidronate treatment effect appeared to be unrelated to genotypes of OI patients,which could provide a basis for guiding clinical treatment and predicting the prognosis of OI.Part 2 Phenotype identification of a mouse model of osteogenesis imperfectaMethods1.Osteogenesis imperfecta model mouse(oim)purchased from Jackson Labory,USA,which have a spontaneous single nucleotide deletion mutation in the COL1A2.The genotypes were identified by Polymerase Chain Reaction(PCR)and Sanger sequencing.2.Body weight was observed in Wild Type(WT)mice and oim at 4-8 weeks of age.3.X-rays were taken at 8 weeks of age in WT and oim mice.4.Femur and tibia bones of 8-week-old oim and WT mice were collected respectively,and the transcript levels of COL1A2 were detected by quantitative real-time PCR(qPCR);the protein levels of COL1A2 were detected by Western blot.5.Hematoxylin-Eosin staining(HE)was used to detect bone growth in the femur;masson staining was used to detect the distribution of type Ⅰ collagen;and goldner staining was used to detect the mineralization.6.Micro Computed Tomography(Micro-CT)was used to examine the microstructure of trabeculae and bone cortex in the femur.7.A three-point force bending test was used to examine the biomechanics of the femur.Results1.PCR and Sanger sequencing results showed no single nucleotide deletion mutations on the COL1A2 in WT mice and single nucleotide deletion mutations on both alleles in oim.2.Compared to WT mice,oim are smaller and lighter in weight.3.Compared to WT mice,oim with low bone density throughout the body.4.Compared with WT mice,both qPCR and western blot revealed a significant decrease in the expression of COL1A2 of oim.5.Compared with WT mice,HE staining showed that the number of trabeculae was significantly reduced and the bone cortex was significantly thinner in oim;masson staining showed that the type Ⅰ collagen content was significantly reduced;goldner staining showed that the bone mineralization was significantly abnormal.6.Compared with WT mice,Micro-CT showed that oim had thinner,less numerous and more separated bone trabeculae,thinner bone cortex and smaller cross-sectional area.7.Compared with WT mice,the maximum load and stiffness of the femur in oim were significantly reduced.Conclusion1.The oim showed a marked reduction in bone mass,decreased bone mechanics and a significant decrease in type Ⅰ collagen expression,consistent with the clinical features of patients with osteogenesis imperfecta.2.The oim can be served as model mouse for osteogenesis imperfecta and used in subsequent therapeutic studies.