Pathogenic Gene Mutations And Methylation Analysis Of Collagen Type Ⅰ Genes In Chinese Patients With Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a group of rare genetic connective tissue diseases characterized by frequently fragility and low bone mass, the other manifestations include deformities caused by severe fracture, scoliosis, blue sclera, dentinogenesis imperfecta, decreased skin elasticity, hyperlaxity of ligaments and joint. In additional to wide-ranging clinical spectrum, there is genetic heterogeneity with both autosomal-dominant and-autosomal recessive forms.01 has been classified into 15 subtypes according to clinical and pathogenic genes. About 90% of individuals with OI have dominantly inherited mutations in type I collagen genes, COL1A1 and COL1A2. Recessively inherited types of OI include type VI to XV, Till now, twelve genes including SERPINF1, CRTAP, LEPRE1, PPIB, SERPINH1, FKBP10, OSX, BMP1, PLOD2, TMEM38B, WNT1 and CREB3L1 have been found in autosomal recessive transmission forms of OI. PLOD2 is the pathogenic gene for OI-Bruck syndrome. ObjectivesThe study aims to identify gene mutations for Chinese osteogenesis imperfecta patients. The second aim is to explore the relationship between mutation and clinical phenotypes. MethodsUnder the approval of Shandong Academy of Medical Sciences Review Board, peripheral bloods and their clinical phenotypes of 110 enrolled OI patients were collected.. Genomic DNA was extracted from collected peripheral bloods. The coding region and exon-intron boundaries of pathogenic genes were amplified by touch-down PCR program according to our laboratory’s protocol. Initially, COL1A1 and COL1A2 genes were detected for their high prevalence of mutations. For these cases failed to identify mutations in these genes, all twleve of autosomal-recessive pathogenic genes and osteoporisis-related gene PLS3 were detected for futher analysis. PCR-based tranditonal Sanger sequencing was performed. Sequencing results were prelimnary anlyzed by Mutation Survery software to get candidate variations. Then polyPhen, Mutation Taster, Align GVGD and SIFT softwares were performed to predict the pathogeneic effects. All the vaitaions were screened using type I collagen mutation database, dbSNP and human gene mutation database. The relationship between genetype and clinical phenotypes were studied preliminary. ResultsA total of 77 OI patients were found to have mutations in all detected pathogenic genes and the left 33 cases were failed to identify any mutations. For these 77 cases,63 of them was found to have COL1A1/COL1A2 heterozygous mutations,7 cases with IFITM5 mutations,2 cases with SERPINF1 and 5 cases with WNT1.A total of 79 COL1A1/COL1A2 heterozygous mutations were identified in this study and most of them locate at the triple helix region of type I collagen. Thirty-six variations were identified for the first time and they need to further confirm by comparing with large healthy population. Missense mutation was the most common mutations of type I collagen genes and it’s predominated by glycine substitution, which was up to 58% of all mutations. Exon-preference is obvious and high-risk mutations were scattered at exon 8 of COL1A1, exon 17,19 and 38 of COL1A2. In additional, five mutations were found in the leathal regions of COL1A1 (p.869-1001) and six mutations in the lethal regions of COL1A2(p.409-454, p.637-670, p.712-727, p.847-901, p.949-997). Universal c.-14O>CT heterozygous mutation of IF1TM5 was found in 7 OI patients, who presented with severe clinical phenotypes and hyperplastic callus.Two complex heterozygous mutations were found in two cases with SERPINF1 gene mutations. One type III case was identify to have c.167C>CG and c.215c>CT mutations and the other relative mild patient have c.215C>CT and c.907C>CT heterozygous mutations. For 5 cases with WNT1, two of them were homozygous deletion mutations and the others were all heterozygous mutations. Patients with WNT1 mutations were found to have blepharoptosis phenotype and one cases showed excessive excitement. ConclusionA total of 110 clinical OI patients were diagnosed by gene mutations. Pathogeni cgene mutations were identified in seventy-seven cases and the left thrity-three cases were failed to find mutations. COL1A1/COL1A2 gene mutations were the most comm on form of 01 mutations and WNT1, SERPINF1 were the main form of autosomal-r ecessive gene muations in 01 patients.