Susceptibility Genes For Nonsyndromic Cleft Lip And Palate In China Detected By Whole Exome Sequencing And Treatment Analysis Of Secondary Deformity Of Nostril Sill

Purpose1.By sequencing the whole exome of nonsyndromic cleft lip and palate(NSCLP)multiplex families to characterize variants of coding region and regulatory region,and to identify the susceptibility gene of NSCLP in China.2.By validating the variants in other NSCLP families to determine the reliability of susceptibility genes.3.Using three-dimensional finite element analysis(FEA)to analyze the relationship between the first auxiliary tension line group and nasolabial morphology,and to explore the feasibility of the first auxiliary tension line group reconstruction in secondary nostril sill deformity repair.4.By retrospectively analyzing the effects of secondary nostril sill deformity repair to affirm clinical value of the first auxiliary tension line group reconstruction in cleft lip repair.Methods1.In this study,we analyzed the NSCLP multiplex families,collected the epidemiological information,and mapped the pedigree.The genomic DNA were obtained from whole blood sample.Exome sequencing and genotyping were performed to achieve variants.Based on hg19,software of Cutadapt,FastQC,BWA,SAMtools,PICARD,and GATK were used to map the variants,SnpEff,SeattleSeq,SIFT,DAVID,cl invar,and cadd were used to annotate the variants,and databases of NCBI dbSNP,HapMap,ESP,and ExAC were used to screen the variants.The candidate genes were selected according to the location of the variants,allele frequencies in ESP database,function,conservation,disease risk,as well as their association with craniofacial development and cleft lip and palate.2.The candidate genes were verified by the exome sequencing data of other families to find out whether the related genes had variants that were consistent with the inherited model in other families.3.A patient with typical nostril sill deformity after cleft lip surgery was selected to carry out multiple angle photographs and 3D laser scanning images for reconstructing the nasolabial model.The FEA model was established by selecting appropriate material properties and element types.According to the three dimensional configuration model of nasolabial muscle fibers,the lines were described in the model along the approximate direction of the muscle fibers.The force was loaded according to the direction of the first auxiliary tension line group,and the result of displacement was calculated.4.The effect of the first auxiliary tension line group reconstruction in secondary nostril sill deformity repair was analyzed retrospectively.The satisfaction of the patients was investigated by subjective scale,as well as the facial images were obtained by 3D laser scanning and the symmetry was analyzed by Mimics software.Results1.From July 2015 to July 2017,we accomplished exome sequencing of 3 multiplex families(M1,M2,M3),and identified 3 variants as candidate mutations for NSCLP including a variant in the TFBS region of TFAP2A(6:10406995,C>T)from M1,a missense of AXIN2(c.2018G>A,p.S673F)from M2,and a missense of C5orf42(c.3548G>A,p.T1183M)from M3.2.We found variants of the candidate genes in 6 families from 15 NSCLP families,and these variants were in consistent with the autosomal dominant inherited model.A variant of TFAP2A and AXIN2 were found in 1 family.Variants of C5orf42 gene including variant(c.3548G>A,p.T1183M)were found in 5 family.Finally,TFAP2,AXIN2,and C5orf42 were identified as susceptibility genes of NSCLP.3.After loading the FEA model in the direction of the first auxiliary tension line group,the maximum displacement of the model occurred in the lateral foot of the nasal alar,and the displacements of the nasal floor and the nasal alar were found in all directions.In macroscopic view,the floor was raised and the alar rolled inward.4.From January 2012 to December 2016,a total of 437 patients with secondary nostril sill deformities completed repair.From 6 months to 3 years after operation,378 patients completed the questionnaire,and 84%of the patients considered that the shape of the nostril sill had been significantly or moderately improved.24 randomly selected patients were analyzed by 3D measurement.There was no significant difference in the nasal alar and nasal floor between the affected side and the normal side(P>0.05),which indicated that the post-operative nasal symmetry was good.Conclusion1.We identified TFAP2A,AXIN2,and C5orf42 as the susceptibility genes of NSCLP,which provided new genetic evidence for elucidating the pathogenesis of orafacial cleft in China.2.It was proved that reconstruction of the first auxiliary tension line group was an effective method to repair the secondary nasal deformity,which could greatly restore the nasal symmetry.The study provided evidence for clinical application and popularization of this technique.