Pathogenesis Of Novel Splicing Mutations In Osteogenesis Imperfecta And Aniridia

Part Ⅰ A novel splicing mutation c.3814 + 1G> T of COL1A1 gene in an family with Osteogenesis Imperfecta type ⅠObjectives:This section studies the pathogenicity of a novel splicing mutation in COL1A1 gene in a family with osteogenesis imperfect type Ⅰ,thereby revealing the gene and genetic model of this family and providing a basis for future prenatal diagnosis and treatment of this family.Methods:This study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University,we collected peripheral blood samples from eight members in the family with osteogenesis imperfect type Ⅰ including three patients and four normal persons.Patients of this pedigree are commonly characterized by blue or light blue sclera,normal height or slightly short.Using whole-exome sequencing technology,bioinformatics analysis,polymerase chain reaction(PCR)technology and Sanger sequencing,fluorescent quantitative PCR technology(PCR),Minigene technology,reverse transcription PCR technology(RT-PCR)and Western-blot method to detect pathogenicity of the novel mutations.Results:1.Whole-exome sequencing was performed on the peripheral blood sample of the proband,and a novel splicing mutation of COL1A1 gene c.3814 + 1G> T was screened by biochemical analysis.2.Genomic samples of the proband and other seven members of the family were extracted,and Sanger sequencing was used to verify the carrying status of COL1A1 c.3814 + 1G> T in the family.The results showed that three patients carried the mutation and four normal members didn’t carried this mutation.3.Use qRT-PCR technology to detect the expression level of COL1A1 mRNA in patients’ blood.Compared with normal family members,the expression of COL1A1 gene in patients was down-regulated.4.Using MaxEntScan(MES),Human Splicing Finder,and Spliceman software to perform biometric analysis to predict the pathogenicity of the splice site,and the results show that the site is pathogenic.5.Minigene results showed that the mutation resulted from the insertion of a 132 bp Intron 48 between Exon 48 and Exon 49.6.Western blot results showed that the mutant cells produced a truncated protein,which was due to the premature translation termination caused by a stop codon in the inserted intron,which led to the truncated protein.7.Sequence homology alignment found that this mutation site is highly conserved in different species;homology modeling analysis of the tertiary structure of the protein found that COL1A1 c.3814 + 1G> T(p.Gly1272 Valfs * 6)mutation caused its encoding The carboxy terminus of the pre-α chain of type Ⅰ collagen is lost,thereby affecting the type Ⅰ collagen synthesis.Conclusion:Using whole-exome sequencing technology and a series of cell molecular biology techniques,it was confirmed that the new splicing mutation COL1A1 c.3814 + 1G> T is the pathogenic gene of this family with osteogenesis imperfecta type Ⅰ.Part Ⅱ A novel splicing mutation c.682 + 2T> C of PAX6 gene in an family with AniridiaObiective: This section studies the pathogenic mechanism of a novel splicing mutation in PAX6 in an family with Aniridia.Methods: This study was approved by the Ethics Committee of the Second Hospital of Shanxi Medical University,and peripheral blood samples were collected from five members of the family,including two patients and three normal persons.Using whole-exome sequencing technology,bioinformatics analysis,polymerase chain reaction(PCR)and Sanger sequencing,fluorescent quantitative PCR technology(q PCR),Minigene technology,reverse transcription PCR technology(RT-PCR)and Western-blot,immunofluorescence technology for pathogenicity of the novel mutation.Results: 1.Whole-exome sequencing technology was used to detect peripheral blood sample of the proband.After bioinformatics analysis and screening,a novel splicing mutation of PAX6 gene c.682 + 2T> C was obtained.2.Genomic samples of probands and other five members of this family were extracted and PAX6 c.682 + 2T> C was detected by Sanger sequencing.The results showed that two patients carried the mutation and four normal members did not carry the mutation.3.Quantitative real-time PCR technology is used to detect m RNA expression in the body after gene mutation.Compared with the wild type,the novel mutation was not affect the normal expression of m RNA in the patient.4.Using RT-PCR technology to detect abnormal bands in the patient’s body in addition to normal transcripts.After Sanger sequencing analysis,the abnormal bands were 39 bp intron 8 inserted between Exon 8 and Exon 9.;5.Minigene technology was used to simulate the transcription of patients in vitro.It was proved that the c.682 + 2T> C mutation results to abnormal splicing of m RNA precursors.6.Conservation analysis of homologous sequences showed that the novel mutation of PAX6 gene was located at the conserved region where evolution of multiple species is important.7.Immunofluorescence results showed that the protein localization and particle changes were not obvious.8.The downstream target gene ALDH1A1 gene expression decreased due to PAX6 gene mutation.Conclusion: Novel splicing mutation c.682 + 2T> C of PAX6 gene was confirmed to be the pathogenic gene of this family by using whole exome sequencing technology and a series of cell molecular biology techniques.