The Role And Mechanism Of MYH3 Gene Mutation In The Pathogenesis Of Congenital Vertebral Malformation

BackgroundCongenital vertebral malformations（CVM）are a group of diseases with spinal deformity caused by abnormal development of the spine during embryonic period.Epidemiological investigations have found that the incidence rate in newborns is around 0.5-1‰.Because teratogenic factors exist before the peak period of growth,the abnormality usually developes rapidly.When the patients enters the peak stage of spinal development,physical deformity and pain are easy to cause serious psychological problems and high operation cost,therefore,they impose a huge burden on families and society.Until now,the etiology of CVM is not well known,and genetic factors are generally considered to play important roles in the pathogenesis of CVM.Through genetic analysis,such as linkage analysis of CVM families,it was found that the copy number variations of specific regions in the chromosome,such as 16p11.2,10q24.31,17p11.2,21p11,22q11.2 areas,was closely related to CVM.According to the candidate gene strategy,it was found that rare variations of PAX1,WNT3A,DLL3,SCL35A3,T（Brachyury）and other genes might be related to the occurrence of CVM.The association analysis based on genome-wide association analysis（GWAS）and whole exon sequencing（WES）also suggested that the polymorphism and rare mutations of TBX6,LMX1A,HES7 were closely related to CVM.But the coverage ratio of these chromosomal and genetic abnormalities in CVM patients is still low,and the number of cases is also limited,the interral relation between patients’characteristic imaging findings,malformation progression,other clinical features,genotypes is also not clear till now,there are no specific molecular markers for early screening and diagnosis.Therefore,deep research CVM pathogenesis is expected to provide new ideas for diagnosis,treatment and even early intervention.Screening of known genetic variants and new mutations which are based on genetic heterogeneity still remains one of the current focuses of CVM.The pathological mechanism of CVM mainly includes bone formation,development and abnormality of bone structure maintenance process.Screening for the genes on such genetic diseases can help us to improve our understanding of the relevant gene functions and related regulatory mechanisms during bone development.The exon region,which occupies only 1%of the entire human genome,contains most of the phenotypic-related variants of hereditary diseases,and is a key area in next-generation sequencing（NGS）.With the continuous advancement of whole-exome sequencing（WES）technology in recent years,mutation prediction software based on large sample database and experimental-based structure and function of mutant proteins have been spawned.The mature method of conducting research,so in the screening of pathogenic genes in the family,WES shows great advantages in both economic and efficiency aspects.ObjectiveTo explore the disease-causing gene from a Chinese Han-race pedigree who presented with autosomal dominant inherited CVM,to verify the function of this gene and to study the possible pathogenic mechanisms.From our work,we hope to expand the spectrum of gene-related diseases and provide a basis for early diagnosis and early intervention of CVM in the field of etiology.Materials and methods1.Blood samples of members from a Chinese Han-race CVM family were collected and subjected to WES and data analysis.The target genes were further identified in the sporadic cases and normal controls after targeting the disease-causing gene MYH3.The clinical phenotypes of the family members were summarized and compared with the clinical phenotype of patients who also carried the gene mutation.2.The expression of MYH3 in spine tissues at different stages of development was observed by immunohistochemical staining of wild-type C57B/L mice at different time during embryonic and postnatal period.3.The expression of MYH3 was observed in clinical diagnosed bone tumor specimens of different cell origins.4.Human umbilical cord-derived mesenchymal stem cells were cultured in vitro and induced into osteogenic/chondrogenic differentiation.Total RNA and cellular proteins were extracted at different time to observe the temporal and spatial patterns of MYH3 expression during stem cell osteogenic/chondrogenic differentiation.5.The three-dimensional structure simulation of protein was used to predict the effect of MYH3 gene mutation（c.695G>A:p.G232E）on the structure and function of the encoded protein.MYH3 plasmids carrying wild type sequence and sequence containing the above mutation were constructed and transfected into cells.After the cytoskeleton staining with the phalloidin peptide,the effect of the mutation on cytoskeleton was observed.The ATP kit was used to detect the change of ATP concentration in each group to evaluate the binding and hydrolysis ability of the protein to ATP.Western Blotting was applied in evaluting proteins and key proteins in the TGF-β/BMP signaling pathway.6.Myh3^G232E/+mutation transgenic C57BL/6J mouse model was constructed by ES targeting technique.The bone and cartilage formation process in the mice spinal region were observed.Results1.The missense mutation（c.695G>A:p.G232E）on exon 8 of myosin heavy chain 3（MYH3）gene may be the cause of the pathogenesis of this Han-race CVM family.Simultaneously,via 15 sporadic CVM patients’WES,we found 2 respectively carry rare mutations（5375A>G,4324G>A）located at exon 31 and exon 37.No mutations were observed in 120 controls,implying that this is a very rare mutation.Prediction of the effects of the three loci mutations all showed"Damaging",suggesting that mutations at these sites may have a great impact on the structure and function of the encoded protein.2.Although the mutation sites were different,the phenotypes of cervical and thoracic fusion and scoliosis without intramedullary dysplasia could also be observed in two sporadic CVM patients with MYH3 gene mutation,which were similar to those in pedigree patients.Suggesting that patients with MYH3 mutations may have genotype-phenotype-related characteristics.3.Younger patients in the family can only be observed on the reduction of intervertebral space height,partial vertebral body fusion with mild scoliosis,but extensive fusion and scoliosis are more common in adult patients.These results suggested that as the age increases,changes in spinal deformity and fusion in the family may continually develop.4.It is confirmed that during embryonic and early postnatal period the Myh3 in the spinal region of the mouse,especially in such areas as the small muscles joining the neural arches of the spine,and in the chondrocyte hypertrophic zone of the growth plate and the neurocentral synchondrosis（NCS）of the vertebral body.And the spatial colocalization of MYH3 and ALP protein can be observed in this spinal region.5.Immunohistochemical staining of spinal tumor tissue sections revealed that the protein was mainly expressed in osteoblasts.With the increase of differentiation and maturation,ALP activity increased,while MYH3 expression showed a downward trend.It implied suggesting that MYH3 may also participate in osteogenic differentiation and bone maturation procedure.6.In the middle stage of osteogenic differentiation（2-3w）induced by hUCMSCs,the MYH3 mRNA increased dramatically,and was consistent with the expression trend of MYH3 detected by Western Blot.At the same time,up-regulation of MYH3 mRNA was earlier than that of the OCN mRNA,which suggests that this gene may be involved in osteogenesis especially in cartilage osteogenesis.It may act in the early stages of the osteogenic differentiation,thus may play a key regulative role in the upstream of osteogenesis-related genes.7.The 232th amino acid residues of MYH3 is highly conserved among different species.The change from non-polar amino acid glycine to acidic amino acid glutamate will result in the change of amino acid residue-residue interaction near this site.Furthermore,the secondary structure of the protein changes in a spatial position,which ultimately changes the overall structure of the MyHC.This may have an effect on the function of the protein.8.At the protein level,mutation of MYH3 may affect the ability of MyHC binding and hydrolysis ATP.Then exert force on the actin of cytoskeleton to maintain cytoskeleton morphology;At the cellular level,it will cause intracellular distribution pattern changes of MyHC and then interactions with other intracellular protein.Utimately it may have effects on the polarity of the cells.9.MYH3 can activate canonical and noncanonical TGFβsignaling pathway,and this ability will be significantly reduced after mutation at the c.695G>A locus.It suggests that MyHC protein may be one of the regulatory proteins in the TGF-β/BMP signaling pathway.10.Compared with wild-type mice,Myh3 mutation transgenic mice were smaller in body weight and body length at 5 to 8w after birth;tail bending was observed from gross and X-ray films early in life,the thoracic deformity of spine was also observed;In addition,compared with wild-type mice,the body length,rib,and costal cartilage of the mutant mice was abnomal.Mild scoliosis can also be observed in the thoracic rigion at the first day after birth of the mutant mice.Bone fusion can be aslo observed in the cervical and thoracic rigion of the mutant mice.11.Spinal tissue section staining showed that in the early postnatal period of the mutant mice（P1）,abnormal appearance occurred,for example,（1）the upper cervical spine deformity,（2）multiple vertebral body wedge deformation in the thoracic rigion,and（3）the asymmetrical atrophy in the chondrocyte hypertrophic zone of the growth plate located at the vertebral ends.The intrinsic mechanism may due to the inhibition of osteogenic differentiation of chondrocytes after Myh3 gene mutation.The process of cartilage osteogenesis was affected.The proliferation kinetics in the growth plate of the vertebral ends are different,ultimately lead to spinal deformity.ConclusionIn conclusion,we confirmed that the rare mutation of MYH3（c.695G>A;p.G232E）is the cause of disease in this CVM family.The mutation of this gene affected the contractile ability of the paravertebral muscles,leading to asymmetric contraction stress of bilateral paravertebral muscles.In addition,it also affected the cell energy metabolism,the maintenance of the cytoskeleton,the cell polarity and the non-classical TGF-βsignaling pathway,inhibiting the osteogenic differentiation of chondrocytes;affecting the proliferation of the growth plate of the secondary ossification center and leading to longitudinal growth asymmetry and wedge deformation on both sides of the vertebral body.Ultimately it resulted observed phenotype of spinal deformity in transgenic mice.In this study we conducted a study based on a Chinese Han-race CVM pedigree,whose disease-causing gene was the rare variant of the MYH3 gene.Our finding suggest that,on one hand,the mechanism of MYH3 mutation that leads CVM may reduce the contractile function of the the small muscles joining the neural arches of the spine,on the other hand it affect the osteogenesis of the vertebral body.Screening for pathogenic genes in bone dysplastic genetic diseases has increased our understanding of related gene functions and their related regulatory mechanisms during bone development.However,we should also recognize that bone formation,bone development,and bone structure maintenance are extremely complex processes.At present,we have very limited understanding of it.Further systematic and further research have a long way to go.