Background:Congenital scoliosis(CS)is a birth defect with variable clinical and anatomical manifestations due to a spinal malformation.Congenital scoliosis may present as an isolated manifestation and/or as a part of clinical findings under syndromes occurring with other congenital malformations.It is one of the most reasons for disability and teratogenicity in patients and will also bring a high burden to both the family and society.Congenital scoliosis occurs as a result in altered development of the paraxial mesoderm,somite or axial skeleton.In human and other vertebrates,vertebrae derived from somite periodically is called somitogenesis.Somitogenesis is a process of complicated interactions of genes,signaling pathways as well as related effectors.Disruption of any factors in somitogenesis could lead to CS.Besides,environmental factors are also considered as an important cause of CS.Recently,more and more attention were paid to the genetic etiology of CS as a result of reports on the genetic mechanism of genes and changes of related factors.Whole Exome Sequencing(WES)is now widely applied to search for genetic etiology of congenital malformations clinically.Copy number variation(CNV),which may present as deletions or duplications within genome DNA5 could also contribute to congenital malformations.Thus,to systematically explore the genetic etiology of CS should focus on both single nucleotide variation(SNV)and CNV level.Previous studies revealed that the genetic etiology underlying about 10%of CS cases in the Chinese population is the compound inheritance of TBX6 variants.The compotxnd inheritance model of TBX6 refers to a eombination of TBX6 null variants(16p11.2 deletion/TBX6 nonsense or frameshift variants)with T-C-A hypomorphic haplotype,by which the gene dosage is reduced below that of haploinsufficiency.In this genetic model,the trait manifests as a result of the combined effect of a rare variant allele and a common pathogenic variant allele at a locus.CS patients in such compound inheritance model are defined as TBX6associated congenital scoliosis(TACS).However,variants of unknown significance(VUS)accounts for the majority of sequencing data of WES results,while no effective analysis strategy nor pipeline to evaluate the pathogenicity of VUS.In this study,we try to establish a systematic analysis and evaluation strategy to reveal the pathogenicity of TBX6 VUS,further to expand the mutational spectrum in congenital scoliosis.Objects:To systematically investigate the genetic etiology of TBX6 VUS,including the filter process of candidate variants,in vitro functional experiments,and phenotype-genotype correlation study.Methods:We performed whole exome sequencing data for 523 patients diagnosed clinically with scoliosis in Asia and analyzed data from exomes on mixed clinical cohorts in the USA.Sanger sequencing were used to confirm the variants.Haplotyping test was conducted to check the hypomorphic haplotype of candidate cases.CNV analysis was conducted by WES data-based software(XHMM and Conifer)to check the CNV in candidate cases.In vitro fimctional testing of dual-luciferase reporter assay,SDS-PAGE and immunoblotting,as well as immunofluorescence were performed to evaluate the pathogenicity of these variants.Phenotype-genotype correlation study was conducted to systematically evaluate the association of TBX6 VUS and CS.Results:We identified six TBX6 likely gene-disruptive variants from 11 unrelated CS patients,2 from Texas USA,via exome sequencing and in vitro functional testing.The in trans hypomorphic allele was identified in 10 out of the 11 subjects;as anticipated from our previous TACS studies these 10 shared a similar spinal deformity of hemivertebrae.The remaining case has a homozygous variant in TBX6(c.418C>T)and presents with a more severe spinal deformity phenotype.Conclusion:Evidence for decreased TBX6 transcriptional activity and abnormal cellular localization were found as molecular mechanisms for TBX6 missense and in-frame indel loss-of-function alleles.Expanding the mutational spectrum of TBX6 pathogenic alleles enabled an increased molecular diagnostic detection rate,provided further evidence for the gene dosage-dependent genetic model underlying CS?and refined clinical classification of scoliosis. |