Preliminary Study On The Pathogenesis Of Congenital Muscular Torticollis By RNA Sequencing And Whole Genome Sequencing

Objective: To study the genes and molecular mechanisms involved in the pathogenesis of congenital muscular torticollis(CMT),the transcriptomic differences between the muscle tissues of sternocleidomastoid muscle(SCM)and platysma from operated patients with CMT were studied by RNA Sequencing.The Whole Genome Sequencing was used to screen the pathogenic gene mutations of CMT families and to explore the possible genetic pathogenesis of CMT.Methods: Before the RNA sequencing was performed,the muscle blocks were collected from the SCM myotomy of CMT and divided into four groups by visual assessment of their appearance.The red platysma muscle obtained from the operation area was named "Platysma group",the fibrotic white SCM tissue was named "Whitish SCM group",the adjacent nonfibrotic red SCM tissue was named "Reddish SCM group",and mixed red and white SCM tissue adjacent to the fibrosis is named "Whitish and Reddish SCM group".The RNaseH method was used to construct the library after the total RNA extraction.The differentially expressed genes(p<0.05,|log2FoldChange|>1)were analyzed by functional annotation,GO enrichment analysis and KEGG pathway enrichment analysis.In the genomic study,peripheral blood DNA was collected from CMT patients and their families to complete the whole genome sequencing.As the genetic model of CMT has not been completely determined in literatures,a candidate gene list was constructed based on related genes reported in other muscle diseases,and the genetic variation of all samples from normal and diseased CMT families was compared with the candidate gene list.According to the sequence variation interpretation guidelines developed by the American Medical Genetics and Genomics Society,the degree of gene variations was evaluated.Results: At transcriptome level,35 samples of muscle tissue were obtained from 16 patients in this study.Among them,3 were “Platysma group”,5 were “Reddish SCM group”,9 were “Whitish SCM group”,and 18 were “Whitish and Reddish SCM group”,respectively.By obtaining differentially expressed genes that are compared in each pairs between the four groups,it was found that the extracellular matrix(ECM)may play an important role in the pathogenesis :(1)The up-regulation of SFRP1 gene expression may inhibit SCM fibrosis by inhibiting Wnt/Catenin signaling pathway.(2)The up-regulation of Wnt7 b gene expression may promote SCM regeneration through Wnt/PI3 K signaling pathway.(3)The activation of PPAR signaling pathway may inhibit intramuscular fat infiltration by promoting SCM lipid metabolism and inhibiting adipose differentiation.(4)The pathogenesis of CMT may be related to autophagy signal pathway.At the genomic level,13 members of three families which are the CMT2101 family,the CMT2106 family,and the CMT2114 family were go through Whole Genome Sequencing.By Interpretation of mutations in related genes,four pathogenic or Likely Pathogenic gene variants were obtained,including the variant of MTHFR gene in CMT2101 family,the variant of TRNT1 gene in CMT2106 family,the variant of LIPE gene in CMT2106 family and the variant of PLN gene in CMT2114 family.However,these gene mutations are all belong to the recessive genetic diseases.And these relevant samples were all the carriers of the corresponding gene mutations which are not directly related to the pathogenesis of CMT.Conclusion: At the transcriptome level,it was found that the ECM might play an important role in the pathogenesis of CMT,The evidence of the results were:(1)The Wnt/?-catenin pathway might be associated with fibrosis.(2)The Wnt/PI3 K pathway might be associated with SCM skeletal muscle regeneration.(3)The PPAR signaling pathway may be related to intramuscular fat infiltration of SCM.Besides of that,the pathogenesis of CMT may be related to autophagy signaling pathway.The Results need to be further verified by functional experiments.At the genomic level,epidemiologic studies found the CMT had a familial aggregation,referring that CMT may be related to genetic factors.In this study,we did not obtain obvious evidence of CMT-associated mutations in whole genome sequencing of these families.It required to expand the samples and choose different sequencing technologies and analysis in the future.