| ObjectivesFacioscapulohumeral muscular dystrophy(FSHD)is one of the most common muscular genetic disorders,after duchenne muscular dystrophy and ankylosing muscular dystrophy.There are two clinical types of FSHD.FSHD type 1 and FSHD type 2.Of these,95%are FSHD type 1,which has a reduced number of D4Z4 repeats in the 4q35 region(less than 10)resulting in hypomethylation of the D4Z4 region and abnormal expression of the DUX4 gene.The disease has significant inter-and intra-familial heterogeneity and can manifest to varying degrees from asymptomatic carriers to wheelchair dependence.The first part of this study aims to clarify the copy number of the pathogenic D4Z4 repeat array in a family line of facioscapulohumeral muscular dystrophy type 1 by genetic testing and analyze it in terms of genetic characteristics,muscle function,severity of the disease and genotype-phenotype correlation,in order to provide an appropriate reference for the diagnosis of FSHD type 1 patients and prognosis of the disease.Aberrant expression of DUX4 in FSHD skeletal muscle causes transcriptional changes that ultimately lead to skeletal muscle cell death.DUX4 is a transcription factor whose misexpression is associated with the regulation of hundreds of genes.These DUX4 target genes are mainly associated with germline genes,reverse transcription factors,immune mediators and RNA metabolism,and trigger pathophysiological changes in FSHD by mediating a series of downstream pathways such as cytotoxicity,inflammatory response,and oxidative stress.Current research on therapeutic approaches for FSHD is mainly through the inhibition of DUX4 mRNA expression,but the downstream gene targets that lead to the toxic effects of DUX4 and the key pathways associated with FSHD pathogenesis are still not fully understood.Furthermore,the pathological skeletal muscle damage observed in FSHD may be driven not only by DUX4 expressed in myocytes,but also by an abnormal muscle microenvironment.such as persistent abnormal inflammation and pathological fibrotic states.The second part of this study aims to comprehensively characterize the cell lineage and transcriptional phenotype of the muscle microenvironment in FSHD and cell-cell interactions by single nuclei RNA sequencing(snRNA-seq),providing a more intuitive and novel perspective for understanding the pathogenesis of FSHD as well as drug-targeted therapy.Methods1 The study population was obtained from one patient with FSHD type 1 and his family members who came to the Cleft Lip and Palate Center of the Plastic Surgery Hospital of the Chinese Academy of Medical Sciences between August 2021 and April 2022,and the clinical data(name,sex,age,time of onset,clinical manifestations,and ancillary examinations)of each patient in the family were recorded in detail.2 The FSHD Comprehensive Clinical Evaluation Form(CCEF)was used to classify the different disease phenotypes of the family members.Muscle strength was assessed bilaterally by manual muscle testing(MMT).Disease severity was assessed using the FSHD clinical score(CSS).3 The Bionano single-molecule optical mapping technique was applied to genetic testing of the prior witnesses and their family members to determine the number of FSHD patients within the family and the genetic characteristics of the family,including the number of repeats of the D4Z4 sequence and the presence of the allele 4qA/4qB.4 Two patients with facioscapulohumeral muscular dystrophy and two healthy volunteers who visited our cleft lip and palate center from August 2021 to April 2022 were selected as study subjects,and the orbicularis oris muscle tissues of two FSHD patients with bilateral asymmetric lesions and two normal orbicularis oris muscle tissues were taken for single-cell nuclear transcriptome sequencing analysis.The clinical and pathological characteristics of each study subject were collected.5 Muscle tissue cell lineages of FSHD patients were constructed based on the cell types corresponding to the annotation of differentially expressed genes in different cell populations.Analysis was performed for myogenic cells,immune cells,fibrous-adipogenic progenitors(FAPs),and vascular-related cell populations to find differentially expressed genes and signaling pathways between disease and control groups in each cell type.The intercellular differentiation trajectory of FAPs was explored using pseudo-temporal trajectory analysis.6 Validation of key signaling pathways using previous Bulk RNA-seq datasets and validation of key differential expressed genes by quantitative real-time PCR(RT-qPCR).Results1 There were 11 patients with FSHD type Ⅰ in the family line,consistent with autosomal dominant inheritance.The presence of a gene mutation with a missing D4Z4 copy number on the 4qA allele and a D4Z4 repeat number of 4 in the family line was consistent with the diagnosis of FSHD type 1.Two patients,Ⅱ4 and Ⅱ5,carried both the two pathogenic genes with 4qA D4Z4 repeat number 4 and 4qA D4Z4 repeat number 7.The father of Ⅱ4 and Ⅱ5,Ⅰ 1,carried the pathogenic gene with 4qA D4Z4 repeat number 7,but did not show FSHD epiphenomenon during his lifetime,implying that Ⅰ 1 may be an asymptomatic carrier of FSHD.2 The clinical phenotypes of the members of this family line showed significant heterogeneity,with nine patients carrying the same pathogenic gene with a 4qA D4Z4 repeat number of 4 having different clinical phenotypic classifications and disease severity assessments.Ⅱ4 and Ⅱ5 also differed significantly in disease severity although both carried two pathogenic genes with D4Z4 repeat numbers of 4 and 7.We observed a positive correlation between disease severity and age in this family,with older patients tending to have more severe clinical manifestations.In addition,another characteristic of this family is that female patients generally have less severe disease than male patients.3 In this study,we successfully constructed a single-cell nuclear transcriptional profile of muscle tissue from FSHD patients.We identified a total of 10 cell types from six samples of muscle tissue:myoblasts,satellite cells,FAPs,macrophages,lymphoid T cells,mast cells,endothelial cells,vascular smooth muscle cells.adipocytes,and Schwann cells.Compared with the control group,the proportion of immune cells increased and the proportion of endothelial cells and vascular smooth muscle cells decreased in the disease group;the proportion of myocytes decreased significantly and the proportion of FAPs and adipocytes increased significantly in the severe disease group.4 We found that immune cells and FAPs play an important role in disease progression in FSHD.Immune cells in FSHD have upregulation of genes associated with immune inflammatory infiltration and downregulation of genes regulating immune function,resulting in a dysregulated immune microenvironment that is directly involved in the pathogenesis of FSHD.The increased proportion of Ml-type cells in macrophages in the severe group of FSHD promotes muscle inflammatory responses and lipid deposition,resulting in loss of muscle function and fat replacement.the expression profile of FAPs in FSHD is in an abnormal state of disturbed differentiation and development and activates a set of inflammatory and pro-fibrotic genes that target FAPs in a state that impairs regeneration and promotes fibrosis,fatty infiltration and atrophy.FAPs are likely are likely to be central players in the progression of FSHD and are associated with asymmetric bilateral muscle changes in FSHD.ConclusionThis study clarified the copy number of the pathogenic D4Z4 repeat array within a facioscapulohumeral muscular dystrophy type 1 family,and analyzed the genetic characteristics and genotype-phenotype correlation of patients within this family.SnRNA-seq analysis revealed the cell lineage composition and transcriptional heterogeneity of bilateral muscle samples in FSHD,explored the role of immune cells and FAPs in the muscle microenvironment of FSHD,and suggested potential therapeutic targets to provide appropriate references for diagnosis and disease prognosis in FSHD type 1 patients. |
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