The Pathogenic Mechanism Studies And The Genotype-Phenotype Correlation Of MYH3 Associated Congenital Scoliosis

Background:Spinal deformity is a kind of orthopedic disease caused by various causes that seriously affects the daily life and appearance of patients.Among them,congenital scoliosis(CS)is a complex three-dimensional deformity of the spine which is caused by abnormal somitogenesis during the embryonic period.The characteristic of CS is that coronal Cobb angle is more than 10°.Severe spinal deformity affects patients' daily activities and even leads to paralysis.At the same time,abnormal appearance of the patients affects the physical and mental health of children,causing a great burden on families.Clinically,patients with CS can only present as congenital vertebral malformation(CVM);CS can also be associated with other organ malformations,such as intraspinal,cardiac,urogenital,musculoskeletal and gastrointestinal malformations;sometimes CS can be a part of clinical manifestations of syndromic disease.Because the etiology of CS remains unknown,the progress of CS cannot be effectively predicted clinically.The treatment is still based on conservative treatment or passive and traumatic surgery.Therefore,exploring the causes of CS is of great significance for controlling the morbidity from the etiology,early diagnosis and early intervention for severe spinal deformity,and alleviating the burden on the family and society.Exploring early diagnosis methods and effective etiological intervention targets of CS is the current international research hotspot.With the advance of genetic testing technology,more and more candidate genes have been identified as the causative genes of CVM,and genetic factors considered to be important causes of CS.The MYH3 encodes myosin heavy chain 3,also known as myosin heavy chain-embryonic(MyHC-emb),which is a skeletal muscle-specific contraction protein expressed during the embryonic development of bones and muscles.The MYH3 is not expressed in adult muscle cells and osteoblasts,which is expressed when skeletal muscle is regenerated after injury.Recent studies have confirmed that the pathogenic MYH3 variants can cause a variety of congenital skeletal deformities,including Arthrogryposis,distal,type 1(DA1),Arthrogryposis,distal,type 2A(DA2A),Arthrogryposis,distal,type 2B3(DA2B3),Contractures,Pterygia,and Spondylocarpotarsal Fusion Syndrome 1A(CPSFS1A),Contractures,Pterygia,and Spondylocarpotarsal Fusion Syndrome 1B(CPSFS1B).The MYH3 is specifically transcribed and expressed in the spinal vertebrae and paravertebral muscle during embryonic period.MYH3 pathogenic variants were screened and identified in the distal arthrogryposis(DA)cohort and SCTs cohort.There are no studies on the pathogenicity assessment of the MYH3 variants in the CS cohort.Objects:To systematically investigate the genetic etiology of MYH3 variants based on DISCO cohort(http://www.discostudy.org/)based on PUMCH,including the filter process of candidate variants,in vitro functional experiments,and phenotype-genotype correlation study.Methods:According to strict inclusion and exclusion criteria,based on 447 patients with CS in the DISCO http://www.discostudy.org/)cohort of PUMCH,we performed whole exome sequencing and data analysis to evaluate and screen for pathogenic variants of MYH3 and performed Sanger sequencing fo verification.In vitro experiments were used to evaluate the expression of MYH3 protein and its effect on phosphorylation of key proteins in TGF-? signaling pathway by Western blot and qPCR.Minigene experiments were used to examine the pathogenicity of MYH3 splicing variants.The association between MYH3 mutation and the clinical phenotype of CS were studied by genotype-phenotype correlation analysis.Results:Among the 447 patients with CS enrolled in this study,through whole exon sequencing(WES)and preliminary bioinformatics analysis,we screened 2 cases of dominant de novo missense mutations of MYH3 and 2 cases of recessive compound heterozygous mutations of MYH3 based on CS trio cases;Among the sporadic subjects of CS,14 cases with potential pathogenic MYH3 variants were identified,including 1 frameshift mutation,1 splicing site mutation,and 12 missense mutations.According to the ACMG variants interpretation standards and guidelines,4 MYH3 variants based on trio,including c.841G>A(p.Glu281Lys),c.1400A>C(p.Glu467Ala),c.3249-9₃249-5del/c.1582-6A>G and c.3062del/c.4244T>G,are judged as possible pathogenic(LP).The 14 sporadic MYH3 variants was determined as variant of unknown significance(VUS).MYH3 missensevaraints,including c.841G>A,c.1400A>C and c.4244T>G were verified by in vitro that did not affect the MYH-emb expression,Instead,MYH3 mutant protein down-regulates the phosphorylation of key proteins in the TGF-? signaling pathway;c.841G>A and c.1400A>C both reduces the phosphorylation of Smad3(p-Smad3)in the canonical TGF-? signaling pathway,while c.4244T>G down-regulated the phosphorylation of p38(p-p38)in the non-canonical TGF-? signaling pathway.The splicing-site variants,c.3249-9₃249-5del and c.1582-6A>G completely inactivate the original splicing sites examined by minigene assay,causing a frameshift variant and resulting in a range of abnormal transcripts.A genotype-phenotype correlation analysis was conducted in 19 CS patients carried MYH3 variants.CS patients carried MYH3 variants exhibited vertebral malformations involving the middle and lower part of thoracic spine(especially the T6-T10 region)with higher incidence of segmentation defect.Patients affected MYH3 mutation are more likely to have maxillofacial deformities and additional musculoskeletal deformities.Conclusion:MYH3 may be an important candidate pathogenic gene for congenital scoliosis.The mutation spectrum of CS has been expanded by the dominant de novo missense mutation of MYH3 and recessive compound heterozygous mutation of MYH3.Perturbation of vertebral development may be caused by MYH3 mutant protein affecting the phosphorylation in the classical/non-classical TGF-? signaling pathway.CS patients carried MYH3 variants exhibited vertebral malformations involving the middle and lower part of thoracic spine(especially T6-T10 region)with higher incidence of segmentation defect identified by the genotype-phenotype correlation analysis.We expanded the phenotype spectrum of MYH3,and initially explored the pathogenic mechanism of MYH3 varaints in CS.