| ObjectivesMicrotia is a common congenital craniofacial malformation,which has a great impact on the patients’ psychology and physical development because of the malformation is located in the face area.Because microtia is mainly characterized by hypoplasia of the auricle cartilage,this study selected patients with simple and non-syndromic microtia and used single-cell transcriptome sequencing technology to analyze the differentially developed ear cartilage,so as to explore the molecular mechanism of microtia.MethodsFrom December 2020 to June 2021,we selected two patients with non-syndromic microtia as the research objects.Their residual ear cartilage(case group)and normal ear cartilage tissue(control group)were sequenced by single-cell transcriptome sequencing technology,and then constructed the cell map of ear cartilage tissue.Cell types were identified based on differentially expressed genes from each cell cluster.For chondrocyte groups,we searched for disease-related differentially expressed genes in each group,and explored the trajectory of cells by pseudotime method.ResultIn this study,we successfully constructed the single-cell transcriptome map of human ear cartilage.In the study of tissue stem cells,we identified four types of stem cell,namely stromal progenitor cells,myofibroblasts,endothelial related stem cells and cartilage progenitor cell.The number of cartilage progenitor cells in the case group decreased significantly,and the genes related to oxidative phosphorylation were highly expressed,while the genes related to tissue development and extracellular matrix process were low expressed.And the transcriptional dysregulation of transcription factors PRRX2,PRRX1,MEOX2,AEBP1,and SOX10 in CPCs may lead to the hypoplasia of chondrocytes or extracellular matrix.Besides,in the study of chondrocytes,we identified nine chondrocyte types with different cell proportion and function,that is,five previously reported types(effector chondrocyte,prehypertrophic chondrocyte,homeostatic chondrocyte,fibrocartilage chondrocyte and mitochondrial chondrocyte)and four new groups(C1-C4).Pseudotime analysis found that two main trajectory branches,in which effector chondrocyte and C4 cluster were located at the root of the trajectory,C1,C2 and C3 were in the intermediate stage,and prehypertrophic chondrocyte,fibrocartilage chondrocyte and homeostatic chondrocyte were mainly distributed at the end of the trajectory 1.Besides,most chondrocytes in the case group were located at trajectory 1,while most cells in the control group were at trajectory 2.Compared to the control group,the differences of the case group are significant in groups of effector chondrocyte,prehypertrophic chondrocyte,homeostatic chondrocytes and fibrocartilage chondrocyte.And there were also significant differences in the expression of genes such as SPARC,ELN,COL2A1,IL6,COL1A1,COL1A2,CXCL2,COL3A1,LUM,CXCL12.The above results suggested that the changes in the number of such cell groups or the spatiotemporal transformation of gene expression may play important roles in the process of deformity.This study also found several signaling pathways related to microtia.ConclusionIn this study,the single-cell transcriptome map and the chondrocyte differentiation lineage of ear cartilage in microtia patients were successfully established.This study confirmed that the differential development of bilateral auricles was mainly related to chondrocyte progenitor cells,effector chondrocytes,prehypertrophic chondrocytes,homeostatic chondrocytes and fibrocartilage chondrocyte,especially in terms of changes of cell number and gene spatiotemporal expression.Signaling pathways such as cell metabolism and proliferation,inflammatory reaction and extracellular matrix organization were also related to cartilage dysplasia.Furthermore,it also proved that it was feasible to study the pathogenesis of microtia at the single cell level.ObjectivesMicrotia is a common craniofacial congenital disease,which occurrence is related to various genetic and environmental factors.In this study,patients with simple and nonsyndromic microtia were selected.By collecting their epidemiological data,the epidemiological characteristics and possible risk factors of microtia were discussed,and a nomogram model that can predict the risk of severe cases was established.MethodsThis retrospective study was conducted from July 2014 to July 2019 at Plastic Surgery Hospital in China.The detailed questionnaires concerning potential risk factors were finished and data were gathered.Using Chi-Square and Fisher’s tests to analyze the variables,and using the multivariate logistic regression model to select variables related to severe microtia,and then construct a nomogram.Then,evaluate the nomogram model by the concordance index(C-index),calibration plot,and receiver operating characteristics curve(ROCs).Bootstraps with 1000 resamples were applied to these analyses.ResultOf the 965 enrolled patients,629(65.2%)were male and 867(89.8%)were sporadic.it was observed more commonly in unilateral(83.1%)and right-sided(52.0%).And multiple malformations were observed in 392(40.6%)cases.Multivariate logistic regression analysis demonstrated that maternal age,miscarriage frequency,virus,infection,anemia,using progesterone,paternal alcohol intake,and topography of living areas were related to a higher risk of severe cases.All the significant variables were combined into a predictive nomogram(C-index=0.755,95%CI=0.703-0.807).Higher prediction accuracy(adjusted C-index=0.749)was further verified via bootstrap validation.The calibration plot showed good performance,and the ROCs curve analysis showed high specificity and sensitivity.ConclusionMost microtia patients are male,sporadic,and accompanied by other malformations,which are similar to the phenotypic analysis results of other studies.A nomogram predicting severe microtia was constructed to provide scientific guidance for individualized prevention in clinical practice. |
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