Genetic Identification And Phenotypic Analysis Of Rare Types Of Osteogenesis Imperfecta

Introduction:Osteogenesis imperfecta(OI)is a group of single gene inherited fragile bone disease characterized by recurrent fractures and skeletal dysplasia.The pathogenic variants are responsible for damage of processing and synthesis of collagen type I,osteoblast differentiation and bone mineralization anomalies.Majority of OI cases were caused by pathogenic variants in COL1A1 or COL1A2.At present,21 genes related to OI have been found.Due to different mutant sites and gene,phenotypes of OI are complex.In recent years,OI caused by non-COL1A1 or COL1A2 gene mutations have attracted more attention in academic circles,because it may contribute to elucidate new pathogenesis mechanism of osteoporosis and open up new fields of bone metabolism research.In this study,23 patients without COL1A1 or COL1A2 gene mutations were examined to determine the etiology and further explore the relationship between genotype and phenotype.Methods:A total of 23 patients were diagnosed with OI by clinical phenotypes.The clinical manifestations,blood tests and image findings of the 23 patients were collected,and genomic DNA were extracted from peripheral blood of the patients and their relatives.Firstly,we screened COL1A1 and COL1A2 in the patients by Sanger sequencing,and found no mutation.Secondly,genomic DNA of patients was analyzed by next-generation sequencing based panel for targeted and the filtered damaging mutations were verified using Sanger sequencing for the patients and their parents.Besides,the sequencing results were compared in 250 cases of normal control.Finally,bioinformatics,homologous analysis and spatial structure construction of mutant proteins were used to predict the function of mutant sites.Meanwhile,the efficacy of long-term bisphosphonate therapy was evaluated in patients with WNT1 mutation.Results:Four cases of rare gene type OI were found in this study.Proband 1,male,10 years old,carrys compound heterozygous variants in the WNT1: c.506G>A(p.Gly169Asp)and c.500dupG(p.Cys170Leufs).Proband 2,female,17 years old,were found compound heterozygous variants in TMEM38B: c.150C>G(p.Ser50Arg)and c.507G>A(p.Trp169X),The unaffected parents of the probands above were heterozygous carriers of each of the variants,respectively.Proband 3,female,45 years old,was identified a heterozygous mutation in the P4 HB gene: c.1198T> C(p.Cys400Arg);Proband 4,male,11 years old,had a heterozygous mutation of PLS3(c.892-1G>A),which resulted in an abnormal splicing site,and his unaffected mother carried the same heterozygous mutation.Proband 1 experienced 5 fractures involving the left femur,the left ulnar and radial and the manubrium sternum.Multiple compression fractures of the vertebrae were found on X-ray,and mitral regurgitation was shown on cardiac ultrasound.Since the age of 1,proband 2 has suffered 2 to 3 fractures every year,involving bilateral upper arms and both lower limbs.After the age of 18,the number of fractures was reduced,and the patient’s spine and lower limbs are severely deformed.The proband 3 has suffered 1 to 2 fractures every year since the age of 2,involving both lower limbs.The visual acuity began to decline around the age of 8.Currently,the visions acuity of both eyes is 0.4.Mild ocular herniation,flat forehead,and flat nose were observed in Proband 3 and her lower limbs are short and malformed.She also suffered severe scoliosis and thoracic deformity.Proband 4 had a mild phenotype,characterized by multiple fractures of the distal radius caused by wrestling,without any skeletal deformity.All the patients had normal sclera,intelligence,dentin and hearing,and their parents without abnormal symptoms.In addition,bisphosphonates exerted potentially beneficial effects on P1,who carried the WNT1 mutations,by increasing BMD Z-score,reshaping the compressed vertebrae and decreasing the fracture risk.Conclusion:We reported novel mutations in WNT1,TMEM38 B,P4HB and PLS3 of Chinese cohort,which expanded the spectrum of phenotype and genotype of extremely rare OI.It lays a foundation for the research on the pathogenesis of subsequent genes and its relationship with phenotype.For sporadic OI cases without COL1A1 or COL1A2 mutations,targeted next-generation sequencing of rare OI-related genes is the economical and effective way to clear etiology.