Application Of High Resolution Melting Analysis In Screening COL1A1/COL1A2 Mutations For Osteogenesis Imperfecta

Objective:We aimed to put forward application of PCR-HRM as a reliable way for screening COL1A1/COL1A2 mutations prior to Sanger sequencing from OI patients.The relevance of genotypes and phenotypes of OI patients in mainland China were summarized.Methods:1.We proposed to explore the optimum reaction conditions of PCR-HRM,including concentration of template(DNA),primers design and primers concentration,annealing temperature,cycle numbers,etc.2.PCR-HRM analysis combined with Sanger sequencing were applied to detected COL1A1/COL1A2 mutations from 87 OI individuals.The relevance of the genotype and phenotype of OI patients were also summarized.3.Three cases were selected randomly among the OI patients who have aberrant mutations on COL1A1/COL1A2 that detected by PCR-HRM.Whole genome sequencing analysis was further processed to identify the causes of the OI patients without abnormal mutations.Results:1.COL1A1/ COL1A2 mutations in 87 cases were detected by PCR-HRM.Eventually,we found 44 mutations,including 22 novel mutations.Total detection rate of PCR-HRM was 52.87%.The results showed 24 patients with COL1A1 mutations and 22 with COL1A2.There was no significant difference in the mutation detection rate between the two genes(P>0.05).The incidence was higher only for COL1A1/COL1A2 mutations in familial patients than that in sporadic patients(P<0.01).2.The mutation pattern of COL1A1/COL1A2 was predominantly single-base substitutions.The incidence of missense mutations was 65.22%,which held the highest ratio among COL1A1/COL1A2 mutations.Among all 44 mutations,mutations that lead to amino acid substitutions were found in 30 cases,28(93.33%)were caused by the Gly substitution,Gly being replaced by Ser accounted for 46.43% of the 28 cases.Compared with COL1A1 mutations,COL1A2 mutations which resulted in Gly substitutions were more prone to helical mutations(P<0.01).3.In addition to the fracture,blue sclera showed the highest incidence among the 46 patients with COL1A1/COL1A2 mutations(95.7%),followed by limb deformities(91.3%),tooth dysplasia(71.7%).Hearing loss was not found in those 46 patients.Compared with COL1A1,patients with COL1A2 mutations were more prone to have severe phenotypes such as tooth dysplasia,fractures before the age of one year,and a total number of fractures(P<0.05).As the amino acid position changed,the height of the patients with COL1A1 and COL1A2 missense mutations appeared with no obvious regularity.4.After detection,a special case was observed which two different mutations were shown on the same exon.Three function prediction softwares(PolyPhen,SIFT and Align GVGD)showed that the two mutations are both likely to cause OI.5.After screening of genome-wide areas,two COL1A2 mutations and one causative pathogenic gene PAPSS2 mutation were found which PCR-HRM did not identify.Conclusions:1.COL1A1/COL1A2 mutations of OI patients in mainland China were screened by optimal PCR-HRM firstly with effectiveness and little-cost.2.22 novel mutations detected in our research were integrated to the Human Collagen Mutation Database.3.It confirmed that the PCR-HRM can be used with genes with heavy workload testing,muti-exons and no hotspot regions.4.A special case was discovered by PCR-HRM and Sanger sequencing,where two different mutations were inherited from his parents one from each.Both of two mutations may be pathogenic after function prediction.5.WGS can make up for the undetected abnormity of PCR-HRM to a certain extent.