Molecular Researches Of FLNB In Developmental Skeletal Malformations

Background:Non-randomly distributed missense mutations of Filamin B(FLNB)can lead to a spectrum of autosomal dominant-inherited skeletal malformations caused by bone hypoplasia,including Larsen syndrome(LS),atelosteogenesi-?(AO-?),atelosteogenesi-?(AO-?)and boomerang dysplasia(BD).Among this spectrum of diseases,LS causes a milder hypoplasia of the skeletal system,compared to BD's much more severe symptoms.Previous studies suggested us that FLNB mutations might cause cellular and molecular differences,which would be consistent with the differences in clinical phenotypes.There were little literature about the structural alterations of FLNB protein and differences in proteome,which were subsequent to the FLNB mutations.Apart from various reports of FLNB mutations associated with definitely diagnosed syndromes,it has not been reported whether FLNB mutations be associated with congenital scoliosis.Objective:From the cellular and molecular aspects,to verify whether FLNB mutations might cause functions,which would be consistent with the differences in clinical phenotypes,and to study the pathogenic mechanism of FLNB mutations.To verify association between FLNB mutations and congenital scoliosis and to determine new candidate gene for the research in etiology of congenital scoliosis.Methods:Using the ATDC5 cell model above,we compared cellular and molecular phenotypes of BD-associated FLBL171R and LS-associated FLBG1586R,including changes in distribution of mutated FLNB,aopotosis,migration and expression of Runx2.Computer molecular dynamic simulation analysis and SWATH were applied to study the structural changes in mutated FLNB and associated proteome.At last,gene burden analysis was used to study whole exome sequencing of 564 Chinese Han congenital scoliosis patients to verify the association between FLNB mutations and congenital scoliosis.Results:We found that while both phenotypes had an increased expression of Runx2,FLNBL171R-expressing ATDC5 cells presented globular aggregation of FLNB protein and increased cellular apoptosis rate while FLNBG1586R-expressing ATDC5 cells presented evenly distributed FLNB protein and decreased cellular migration.Molecular dynamic simulation revealed FLNBG1586R protein had decreased hydrogen bonds and stability and the secondary structure nearby the mutation site altered from bend to coil.Proteomic experiments indicated differences in osteogenesis associated proteins and signaling pathways between FLNBL171R-expressing ATDC5 cells and FLNBG1586R-expressing ATDC5 cells.Gene burden analysis verified that FLNB mutations were associated with congenital scoliosis.Conclusion:In this study,the main contradiction of significant clinical phenotypic differences between two extreme cases(LS and BD)in the disease spectrum caused by FLNB gene missense mutations was taken as a breakthrough point.With cellular experiments,molecular dynamic simulation and proteomic techonilogies,we provided a reasonable explanation for the phenotypic differences between LS and BD.Furterhmore,the correlation between the FLNB gene mutation and the congenital scoliosis in Chinese Han population was proved by whole exome sequencing and gene burden analysis.All results help to find out new directions to study the physiological role of FLNB gene in the process of endochondral osteogenesis and the pathological role of its variation in the occurrence of skeletal malformation,and to add new candidate gene for the etiology study of congenital scoliosis.