To Explore The Molecular Mechanism Of Pathogenic Copy Number Variable And Auricular Reconstruction For Low Hairline In Congenital Microtia

Background and ObjectiveCongenital microtia is defined as morphological abnormity or defect of the external ear in different degrees,partly accompanied by developmental deficits of the middle ear and hearing loss,which seriously affects the physical and mental health of patients China is a region with high incidence about 3.06 per 10,000 births of microtia and its rate is still rising recently.The exploration of the pathogenesis and the improvement of the treatment methods for microtia are always the focus of the research on congenital craniofacial malformation.Genetic factors are considered to be the most important pathogenic factor of microtia,but the research on the pathogenic genes and mechanisms related to isolated microtia is still lacking.In our previous work,a shared pathogenic copy number variation(CNV)of three microtia pedigrees which is located in non-coding region(chr4:8701900-8702500(hg19))was oriented through Genome-wide linkage analysis,target capture and second-generation sequencing technology.And two kinds of different genotypes gene-editing mice models,the pathogenic fragment inserted(Duplication,DUP)type and knocked out(KO)type,were constructed using CRISPR/Cas9 technology.Among them,homozygous DUP type mices successfully replicated the phenotype of the bilateral microtia in the pedigree patients which manifested bilateral microtia,and the expression level of Hmx1 in the external ear was measured to be high,while homozygous KO type mice showed a phenotype of bilateral big ears.Base on this evidence,we carried this study in order to further understand the biological characteristics of this animal model and the pathogenic mechanism of congenital auricular deformity due to this CNV.In addition,aiming at microtia patients acompanied with low hairline,which are clinically difficult to treat,we introduced and reviewed a modified ear reconstruction technique with a single expanded scalp flap in the third part.Method1.Back mating,test mating and other hybridization methods were applied to establish mice pedigrees with the same genetic background of DUP,KO and WT(wild type).The phenotypic characteristics and genetic rules of each genotype were compared through standard photos,Magnetic Resonance Imaging and genetic balance analysis.Combined with the genomic information analysis of the loci segment,the possible pathogenesis of this segment was identified.2.Transcriptome sequencing was performed on the external ear tissues from mices of each genotype at different age.Differential expressed genes(DEGs)analysis,DEGs enrichment analysis and Protain Protain Interaction(PPI)analysis was used to screen candidate genes and relevant pathogenic molecular signal pathways.4.The medical records of microtia patients with extremely low hairline who were treated with total auricular reconstruction using a singer expanded scalp flap and intense pulsed light treatments(IPLT)by our team from January 2015 to April 2019 was reviewed.The operation steps were detailed,the curative effect and complication management were concluded,and the experience was summarized.Results1.The first pathogenic CNV gene-editing mice pedigree based on the study of isolated congenital microtia in human was constructed.And the incomplete dominantly inherited triangular ear malformation in DUP gene type mice and the recessively inherited"disciform" big ear malformation in KO mice were identified.2.The pathogenic fragment is an evolutionarily conserved region(ECR)cis-acting enhancer downstream of Hmx1.This ECR,which is necessary for the normal expression of Hmx1 in multiple species,can specifically regulate the expression level of Hmx1 in the neural-crest-derived craniofacial mesenchyme(CM)with gene dose effect.3.In transcriptome sequencing,we screened out 8 candidate pathogenic genes,including Hmx1,whose expression level was related to the number of CNV,as well as related signaling pathways,such as ECM receptor interaction pathway,Wnt signaling pathway,MAPK signaling pathway,Notch signal pathway and Cardiac muscle contraction pathway.4.A total of 41 microtia patients with very low hairline admitted to our team were included in this study.The follow-up time was 10 months to 4 years.The satisfaction rate for the treatment was 90.2%and 9.8%were partially satisfied.No unsatisfactory cases.5.Compared with patients who started IPLT treatment before surgery,patients who started IPLT during expansion period had fewer times of depilations and achieved more lasting and satisfactory depilation effects,P<0.001.ConclusionIn this study,the target segment is a tissue specific ECR enhancer of Hmx1,which might specifically regulate the expression of Hmx1 in the neural-crest-derived CM,and its CNV might cause isolated congenital ear malformation in multiple species.Hmx1,Tcap,Sln,Mb,Gapdh,Nell2,rp121-ps10,and Gm42047 are pathogenic candidate genes,and MAPK signaling pathway,Wnt signaling pathway,ECM receptor interaction pathway,and myocardial contraction pathway are related signaling pathways.Nsun7 and Nr5a2 might be cofactors of Hmx1 at different expression levels.The candidate genes and related signaling pathways screened out in this study have a broad spectrum and complex interactions.Further identification and verification of their role in the development of the external ear would be very significant for the understanding of pathogenesis of microtia.In addition,total ear reconstruction using an expanded scalp flap combined with IPLT is a safe and effective treatment for microtia patients with very low hairline.Compared with IPLT performed before surgery,IPLT performed during expansion has a more efficient and lasting depilation effect.