Screening Of Candidate Pathogenic Genes In Congenital Malformations And MicroRNA Expression Profiles In Abnormal Ear Tissues

Part IObjective:(1) To screen the candidate genes of congenital microtia using Affymetrix SNP6.0and direct sequencing.(2) To determine the mutational features of TCOF1in3Chinese patients with TCS.Methods:(1) Clinical informations and Blood samples of112patients were collected, and we performed genome-wide scan in3of them using Affymetrix SNP6.0arry. Then DNA sequencing analysis was performed on all exons of the candidate genes in the rest patients.(2) DNA sequencing analysis was performed on all exons and the exon-intron borders of TCOF1in addition to the1200bp upstream in the three Chinese patients with TCS.Results:(1) Five candidate genes were screened following the analysis of bioinformation, which were MSX1, MSX2, GSC, HOXA2 和PACT.(2) We identified3SNPs in GSC, HOXA2和PACT and2novol alterations in the5’-UTR of HOXA2without apparent pathogenicity.(3) We identified12different variations in TCOF1. Among the12variations in TCOF1, four have not been previously reported as a TCOF1mutation or polymorphism and were not in the dbSNP. All four variations were also identified in healthy unaffected controls in the form of compound heterozygosity.Conclusions:(1) GSC, HOXA2和PACT are not the hot spots in microtia, the function of the alterations in the5’-UTR of HOXA2should be further investigated.(2) Our results suggest the possibility of genetic heterogeneity or different mechanisms leading to the disease. Further functional study of the silent mutation is necessary to obtain more definitive information. Part IIObjective:To investigate the globle expression profiling of microRNAs in human deformed external ear tissue by using microRNA array and explore the specific miRNAs’ clinical significance.Material and Methods:19deformed external ear tissue and5normal ear tissue were accumulated and their total RNAs were isolated routinely. Expressions of miRNAs in4deformed external ear tissue and2normal ear tissue were detected by using Affymetrix miRNA array. Furthermore, all the differentially expressed miRNAs were chosen to determine through Real-time PCR analysis. Last we use bioinformatics methods to predict the target genes of miRNA.Results:(1) From the microarray results we found that a set of aberrant miRNAs were detected in the human deformed external ear tissue. In particular, a significant increase of7miRNAs and decrease of5miRNAs were detected in comparison to normal tissues.Furthermore,7upregulated miRNAs included hsa-miR-16, hsa-miR-140-3p, hsa-miR-126, hsa-miR-185, hsa-miR-378, hsa-miR-451and hsa-miR-486-5p, and5downregulated miRNAs included hsa-miR-203, hsa-miR-205, hsa-miR-200c, hsa-miR-708and hsa-miR-1308.(2) It was confirmed that results of Real-time PCR were consistent with the gene chips. Hsa-miR-126and hsa-miR-451were upregulated obviously in deformed external ear tissue and hsa-miR-203, hsa-miR-205and hsa-miR-200c were downregulated obviously.(3) The bioinformatics study:There were26target genes for hsa-miR-126and15target genes for hsa-miR-451. In some of the genes, the function were still unkown. Conclusions:(1) miRNA expression profile of deformed external ear tissue was established. There were7upregulated miRNAs and5downregulated miRNAs in deformed external ear tissue.(2) The12different expressed miRNAs were verified by Real-time PCR using specific primers.(3) The function of some target genes were still unkown, further functional research should be performed to clarified the relation to the microtia.