MYOM2 Variations Are Associated With Congenital Scoliosis ——A Study Based On Whole-exome Sequencing And Zebrafish Model

Objective The prevalence of congenital scoliosis（CS）is about 1 in 1,000.CS is mainly manifested as three-dimensional deformity of the spine,and at the same time,it will cause an imbalance in the whole body during the growth of the spine,mainly due to congenital vertebral abnormalities.However,the exact pathogenic factors of CS are still unclear,and some previous studies have shown that both genetic and environmental factors are involved in the occurrence and development of CS.With the update of the technology,Whole Exome Sequencing（WES）provides a good means for us to study gene point mutation,insertion,deletion,and other mutations as well as single nucleotide variation.WES technology has fewer requirements on the number of samples and capital expenditure,and the data analysis technology has gradually improved,and now it is more and more widely used.Regarding skeletal development and related diseases,zebrafish have gradually been used in experiments in recent years.Because zebrafish move primarily in water,the spine is constantly under pressure.Compared with quadruped animals such as mice,the zebrafish spine bears more pressure similar to that of humans,so zebrafish is more suitable for studying spinal deformity-related diseases.Therefore,the purpose of this study was to explore the possible causative genes of CS through WES and zebrafish model animal studies.Methods For exome sequencing,we selected a discovery set consisting of 6 trios groups（consisting of a sporadic congenital scoliosis patient and two healthy parents）（Series 1）,and we selected an additional 2 trios group and 6 matched groups（including a person with sporadic congenital scoliosis and a healthy father or mother）（Series 2）.A total of8 families with congenital scoliosis and 6 sporadic cases from China were included in this study.Harmful annotations mainly include SIFT,Polyphen2＿HDIV,Polyphen2＿HVAR,LRT,Mutation Taster,Mutation Assessor,FATHMM,PROVEAN,VEST3,Meta SVM,Meta LR,M＿CAP,CADD,DANN,FATHMM＿MKL,Eigen,Geno Canyon,fit Cons,GERP,phylo P,phast Cons,Si Phy,REVEL,Re Ve.Gene function annotations are mainly performed through Genecard（https://www.genecards.org/）,Ontology（GO）,KEGG database,OMIM database,and Enrichr（https://maayanlab.cloud/Enrichr/）.The tissuespecific expression profiles of genes were mainly annotated through GTEx database（www.gtexportal.org）and Proteomics DB（www.proteomicsdb.org）.At the same time,the expression patterns of genes in developing spine bones were queried by examining Gene Paint（www.genepaint.org）and Allen Spine Column（mousespinal.brain-map.org）databases.These online servers provide photomicrographs of complete section series of 14.5-d-postcoitum（dpc）mouse embryos,P4 and P56 mouse spinal cord tissues processed for ISH with nonradioactive probes.Chimera 1.15 software was used to model the wild-type（WT）and mutant（Mut）domains of the gene,the WT template was downloaded from the SWISS-MODEL tool（https://swissmodel.expasy.org/）.Sanger sequencing was performed after screening candidate genes.The expression of related proteins in the plasma of mutant families was determined using ELISA.The mutant plasmids were constructed and introduced into HEK293 cells.Western blotting was performed and q RT-PCR was used to detect the effect of mutations on the m RNA expression of osteogenesis and chondrogenesis-related genes.For morpholine（MO,Gene Tools）gene knockdown,we synthesized antisense morpholine fragments.MO1 against the translation initiation sites of the zebrafish myom2 a and myom2 b genes,and the control 5MIS-MO against a 5-bp mismatch in the MO1 sequence.All morpholines need to be diluted in juvenile medium and microinjected when zygotes are fertilized at the onecell stage,and deformities are visualized by microscopy and skeletal staining.Results A total of six families were included,all with CS and no other syndromes,five with hemivertebrae and one with block vertebrae.The biological function annotation results of candidate genes（MYOM2,NOTCH2,CTBP2,LILRB3,LILRA6,MYH14）showed that these genes can regulate the development of osteoclasts,participate in the Notch pathway,participate in the Wnt pathway,and myosin structure organization.Harmful analysis showed that both LILRB3:c.1701T>A and LILRB3:c.428G>A were harmless,while the rest of the mutations were predicted to be harmful.In order to check whether the candidate gene is involved in the development of the spine,through the query,there is no data of Lilra6 in the database.In the sagittal section of the 14.5 dpc mouse embryo in the ISH image,it can be observed that Myom2 and Notch2 are expressed in the axial bone region of the spine,while no expression of Ctbp2,Lilrb3,and Myh14 was observed.In addition,ISH analysis of spinal tissue from 4-day-old pups in Allen Spinal Column cross-section showed that these five genes were expressed to a certain extent in spinal cartilage and soft tissue around the spinal cord.Among them,Myh14 was the most abundantly expressed,followed by Myom2.The Sanger sequencing verification results showed that there were no two mutations CTBP2: c.2929G>A and MYH14: c.6107A>C,and two mutations MYOM2: c.3872G>T and MYOM2: c.3997T>C were verified.After further bioinformatics analysis and validation by next-generation sequencing,we excluded five of them（CTBP2,MYH14,NOTCH2,LILRB3,and LILRA6）because they were either not expressed in the spine,were not functionally related,or were not mutated.Finally,we identified two mutations,MYOM2: c.3872G>T and MYOM2: c.3997T>C.Therefore,the conservation of its amino acids was further analyzed first,and the results showed that the 1291 position was relatively conservative,while the 1333 position was not conserved.It was F before mutation and L after mutation,which resulted in the same amino acid as the animal site.The ELISA results showed that MYOM2 in the plasma of the patients was decreased compared with that of the parents,and it was statistically significant,and there was no difference between the parents.The changes of MYOM2 protein expression were detected by Western blotting,and the results showed that the single mutation of MYOM2: c.3872G>T or MYOM2: c.3997T>C had less effect on the expression of MYOM2 protein,while MYOM2: c.3872G>T and MYOM2: c.3997T>C double mutation resulted in a significant decrease in MYOM2 protein expression.q RTPCR results showed that the double mutation of MYOM2: c.3872G>T and MYOM2:c.3997T>C reduced the m RNA expression of the markers（SP7 and OC）of late maturation of osteoblasts,but increased the m RNA expression of the markers（COL II and COL X）of chondrogenesis.In addition,double mutations also lead to slight increases in IHH,SOX9,COL I,and RUNX2.Embryonic morphological observations revealed that severe developmental defects in zebrafish,including deformed head,small eyes,scoliotic spine,and unconsumed yolk sac,were observed in the 2 ng myom2b-MO1-injected group.And the total number of abnormal and dead zebrafish was much higher compared to the WT group as well as the control 5MIS group with the same dose.However,the injection of myom2a-MO1 did not differ between the WT group and the control 5MIS group with the same dose.Rescue experiments using myom2 b m RNA showed that the phenotype of morpholine mutants was at least partially rescued after zebrafish myom2 b m RNA was coinjected with myom2b-MO1.Bone staining revealed that the mandible size was also significantly reduced in the myom2b-MO1 group,characterized by a reduced distance between the angular cartilage（ch）and the Meckel cartilage（Mk）,whereas the injection of myom2a-MO1 did not differ between the WT group and the control 5MIS group with the same dose.Conclusion Through WES,cell experiments,and the construction of a zebrafish model,we found that in this study,MYOM2 double missense mutations were associated with the occurrence of CS,which can be autosomal recessive.At the same time,we also found that MYOM2: c.3872G>T combined with MYOM2: c.3997T>C can lead to a decrease in the m RNA expression of some osteogenic genes（especially SP7 and OC）,but it can lead to an increase in the m RNA expression of some cartilage genes（especially COL II and COL X）,indicating that MYOM2 double missense mutations can affect the maturation and differentiation process of osteoblasts.In the study of zebrafish,it was found that the zebrafish myom2 a and myom2 b genes are highly conserved compared with their mammalian homologous genes.In early zebrafish development,the expression of myom2 b is mainly the main expression,and the knockdown of the zebrafish myom2 b gene will promote CS-like spinal deformity in zebrafish.Through the above experiments,we found that the occurrence of congenital scoliosis may be related to MYOM2 mutation,and the related mechanism is under further study.