Genetic And Molecular Investigation For Distinct Scoliotic Phenotypes Conveyed By Allelic Mutations Implicated In FBN1

Background:FBN1 encodes fibrillin-1,located on chromosome 15q21 and associated with many developmental defects or abnormalities in the musculoskeletal system.The most common is FBN1-caused Marfan syndrome(MFS),which refers to a heritable autosomal dominant disease trait of fibrous connective tissue.The cardinal phenotypic features primarily occur in the skeletal,ocular,and cardiovascular systems.Marfanoid-progeroid-lipodystrophy syndrome(MPLS)is a recently defined fibrillinopathy,and also a complex disease characterized by accelerated aging and postnatal lipodystrophy,poor postnatal weight gain,and characteristic dysmorphic facial features that have very rarely been clinically recognized and reported.Recent studies have implicated a potential hormone,named asprosin,encoded by the FBN1 locus as a mediator of the lipodystrophy phenotype.All previously reported MPLS individuals consistently harbor heterozygous truncating mutations in the penultimate exon,which leads to the formation of premature stop codons in the C-terminal domain of FBN1.In addition,we also focus on the non-syndromic scoliosis,especially congenital scoliosis(CS),which is a form of scoliosis caused by congenital vertebral malformations potentially resulting from formation failure,segmentation defects,or a combination of both.Genetic predisposition has been demonstrated in CS.Objectives:To systematically investigate the genetic association between FBN1 and scoliotic diseases,encompassing the filtering process of candidate variants,in vitro functional assays,and genotype-phenotype association study.Methods:615 Chinese CS and 3 MD/MFS included scoliosis subjects were recruited.Whole exome sequencing(WES)was performed on all the participants.Orthogonal Sanger sequencing were used to confirm the variants.Mutational burden analyses of FBN1 were implemented.In vitro functional assay,including construction of expression plasmids,cell culture and transfection,real-time quantitative polymerase chain reaction,western blot analyses were performed to evaluate the pathogenicity and molecular mechanism of candidate variants.Results:We conducted trio-based WES analysis and allele-based mutational burden analysis,and provided evidence for the genetic association between FBN1 and CS.In vitro functional assays for FBN1 variant and revealed the upregulation of TGF-β signaling caused by FBN1 variant.We also reported a novel de novo FBN1 mutation for the first Chinese MPLS subject.We further predict that the MPLS truncating mutation is prone to escape the nonsense-mediated decay(NMD),while the MFS mutation is predicted to trigger NMD.Also,the MPLS mutation occurs within the glucogenic hormone asprosin domain of FBN1.In vitro experiments showed that the single MPLS mutation p.Glu2759Cysfs*9 appears to perturb proper FBN1 protein aggregation as compared with the classical MFS mutation p.Tyr2596Thrfs*86.Both mutations appear to upregulate SMAD2 phosphorylation in vitro.Conclusion:Association between FBN1 and CS phenotype was investigated.Evidence for upregulation of TGF-β signaling was found as a potential molecular mechanism for FBN1 missense loss-of-function alleles in CS.We provide direct evidence of dominant-negative effects of truncating FBN1 variants predicted to escape NMD in MPLS subjects.Taken together,our study expands the mutational spectrum of FBN1 and highlights the potential molecular mechanism for MPLS subjects,which facilitates our understanding of genotype-phenotype correlations in FBN1 to provide effective genetic counseling,implementation and timing of therapy(e.g.mitigation of TGF-β hypersignaling,surgical intervention for cardiovascular complications or for scoliosis),or early intervention.