The Phenotype-genotype Analysis Of Wheelchair-dependent Patients With FSHD As Well As Preliminary Study In A FSHD Mouse Model Of DUX4-cre

Background and purposesFacioscapulohumeral muscular dystrophy（FSHD）is one of the most common types of muscular dystrophy.It is inherited in autosomal dominance,and about30%of the patients are De novo.Clinically,the disease presents significant inter-family and intra-family heterogeneity,ranging from asymptomatic carriers to disability（wheelchair dependency）.According to recent statistics,the incidence of the disease in the Netherlands is about 1:8,333.The clinical manifestations are progressive,asymmetric（or symmetrical）muscle weakness and muscular atrophy,usually involving the face and scapulae first,and gradually involving the upper arm,abdomen,pelvic band,and lower limb muscles.Although the disease progresses relatively slowly,it has a high disability rate,with about 20%of the patients in wheelchairs,which seriously affects the quality of life.At present,there is no effective treatment drug.The etiology of FSHD1 is mainly associated with the loss of multiple copies of the 3.3 KB D4Z4 large satellite tandem repeats（DRs）in an Eco R1 fragment in the sub-telomere region of the long arm of chromosome 4（4q35）.Patients are reduced to 1-10（<38 KB）and carry the downstream 4QA/4QA-L allele,compared to 11-100（>38 KB）in normal humans.The 4QA allele that leads to DUX4 expression works together to reduce the level of methylation,which in turn acts through epigenetic mechanisms,and ultimately leads to the disinhibition of DUX4 re-expression in skeletal muscle,leading to disease.Because FSHD is a unique and complex genetic disease,there is no effective targeted treatment at present.Animal model can be used as a carrier for the study of pathogenesis and treatment.Based on this understanding of epigenetic pathogenicity,researchers are able to study the design of preclinical models to further understand the mechanisms of FSHD and explore the pathways associated with FSHD and its treatment.Because DUX4 protein has high embryonic lethality,general AAV-DUX4,D4Z42.5,i DUX 2.7 mouse model because of its short life span or the lack of appreciable muscle phenotype is not suitable for malnutrition as muscle disease research for a long time,and FLEx DUX4 mice model,by using the FLEx orientation of switch system design,to by-pass the embryo DUX4 embryonic lethality of genetically modified expression.Cre was activated to express low dose DUX4-FL by mating with tool mice with the muscle-specific promoter ACTA1-Cre.Different DUX4expression levels were induced by low,medium and high doses of tamoxifen（TMX）to simulate different stages of the disease to deepen the understanding of the disease and identify potential therapeutic targets.Although the progression of FSHD is relatively slow and generally does not affect the life span,the disability rate is high.20%of the patients need to rely on wheelchairs in the later stage,which brings heavy burden to the society and family and seriously affects the quality of life of individuals.Therefore,the purpose of the first part of this study was to investigate the genotypic and phenotypic relationship between wheelchair-dependent FSHD patients and the severity of the disease,and to provide counseling for more severe FSHD patients and predict the future progression of the disease.However,the DUX4 protein expression is difficult to explain the pathogenesis of FSHD,and DUX4 gene may be involved in the occurrence and development of the disease by influencing the potential pathways.In recent years,due to the application of flow cytometry,Fibro/Adipogenic Progenitors（FAPs）have been selected from skeletal muscle cells to mediate muscle homeostasis and regeneration of myoplasmic progenitor cells mediated by promyogenic signaling.The expression of its most representative marker,PDGFRα,was positively correlated with the fibrosis of muscle tissue.In muscle diseases,the autocrine/paracrine control of FAP fat production and release leads to fat infiltration,highlighting the importance of Wnt/GSK/β-catenin signaling as an important pathway to regulate FAP fat production by stimulating islet signaling.In addition,inhibition of GSK3effectively stimulates the promyogenic effects of FAP by stimulating follicle secretion and differentiation of muscle satellite cells（MUSC）into mature muscle tubes.At present,the related pathways targeting FAPs and MUSC cells in FSHD have not been reported.The second part of this study aims to observe the phenotypic and pathological changes related to the muscle tissue of mice through the preliminary study of the mouse model with FSHD and the use of the molecular GSK3βinhibitor LY2090314 in the Wnt signaling pathway in the primary cells of patient-derived FSHD.Inhibiting GSK3 in the Wnt/GSK/β-catenin signaling pathway may help us develop new ideas for the treatment of myopathy.Methods1.Eighty-four patients with wheelchair-dependent FSHD were enrolled in a prospective,single-center,case-control observational study.Mann-Whitney U test（non-parametric test）was used to compare the measurement data,Chi-square test or Fisher’s exact test was used to compare the classification data,and Spearman’s rank correlation test was used to evaluate the correlation between phenotypic and genotype.P<0.05 was statistically significant.The statistical software was SPSS26.0.2.A mouse model of Flex DUX4 was introduced from The Jackson Laboratory（Stock No.028710）,USA,and DUX4 gene expression was induced by intraperitoneal injection（IP）of tamoxifen once daily for 5 consecutive days at an injection dose of approximately 100ul/20g.Doubly-transgenic mice induced by TMX at the age of 12 weeks were used as experimental subjects（n=3）.The experimental group was injected with LY2090314（GSK3βinhibitor）（25mg/kg）for 3 days,while the control group was injected with DMSO at the same dose for 3days.The experimental group was continuously injected with LY2090314（GSK3βinhibitor）for 3 days（the injection dose was 25mg/kg）,while the control group was continuously injected with DMSO for 3 days.The muscle strength of mice was measured by limb climbing test,and the muscle content was evaluated by weight measurement.The pathological changes of muscle tissue were observed by HE staining and SIRIUS staining,and the expression of DUX4 gene was detected by q PCR,DUX4 protein expression was detected by Western blot.3.This study was in accordance with the regulations of the ethics committee of the hospital,and informed consent was obtained.Results1.Among the 84 patients with wheelchair-dependent FSHD,34 cases were male（40%）and 50 cases were female（59%）,among which 2 cases（2%）began to wheelchair-dependent after their symptoms worsened during pregnancy and delivery.The median age at diagnosis was 40 years（16-81 years）;The median age at onset was 13 years（5-81 years）;The median age at Age at Independent ambulation loss was 33 years（13-82 years）;The median age at lower limb involvement was 20 years（5-55 years）,and the median age at wheelchair-dependent was 34 years（16-82 years）.Forty-one patients（39%）had upper extremities.In 12 patients（11%）,the onset was in the lower extremities;34cases（40%）showed scapular winging.26 patients（31%）with early onset FSHD;44 patients（52%）with typical FSHD;12 patients（14%）with later-onset FSHD;The median clinical score was 10（6-15）;The median clinical severity score（CSS）was 4（1-5）;The median age-adjusted clinical severity score（ACSS）was 183（50-370）;The median MRC score was 93（58.5-133）;The median DRS was 3（2-8）;Median D4Z4 methylation levels were 39%（16%-63%）;14 patients（16.6%）had De novo;There were 11 patients with abnormal respiratory function（13participants）,including 5 patients（38.4%）with severe restrictive ventilation dysfunction and 3 patients（23.1%）with moderate restrictive ventilation dysfunction.3 cases（23.1%）had mild restrictive ventilation dysfunction.33patients participated in the spinal examination,19 of them（57.5%）had spinal deformity;The myocardial enzyme creatine kinase（CK）was slightly higher than the normal.The EMG of the extremities of the 20 patients participating in the detection showed basically myogenic damage,and the muscle pathology of the 4patients participating in the muscle biopsy showed characteristic muscle pathology.The most common ECG abnormalities were incomplete right bundle branch block and sinus insufficiency.Hearing and visual acuity tests were performed in 35patients,including 7 patients（20%）with abnormal vision and 5 patients（14%）with abnormal hearing.Correlation analysis showed that the number of D4Z4repeat units was positively correlated with the age at wheelchair-dependent and the age at lower limb involvement（P1=0.0006;P2=0.0399）;The age at onset was positively correlated with the age at wheelchair-dependent and the age at lower limb involvement（P3<0.0001;P4<0.0001）.Single factor analysis found that 47cases of age at wheelchair independency FSHD patients（year≤35）compared with34 cases of age at wheelchair independency FSHD patients（year>35）,the age at onset,lower limb involvement of earlier onset age,D4Z4 more repeat unit quantity is short,MRC score is low,FSHD clinical severity score is higher,and ACSS also is higher,the contemporary higher mutation rate.2.There was a statistically significant difference in the limb climbing experiment between the two groups（P=0.0078）.Pathologically,after the continuous injection of TMX 75mg/kg/d for 7 days,HE staining showed muscle fibers of different sizes,muscle nucleus migration,cell membrane integrity destruction,and inflammatory cell infiltration around the cells.In the control group,the space between muscle fibers was normal,the nucleus was located in the surrounding muscle fibers,and no inflammatory cell infiltration was observed.DUX4-fl m RNA expression was detected 16 days after intraperitoneal injection（IP）of TMX,and the relative expression level of DUX4-fl m RNA in the control group was statistically significant compared with that in the experimental group（P=0.0078）.Compared with the control group injected with DMSO for 3 days,both muscle fiber atrophy and hypertrophy were observed by HE staining of pathological sections of the muscle.Basophilic muscle fibers:that is,muscle fibers are regenerated by satellite cells;Regenerated muscle fibers are smaller than the surrounding normal muscle fibers,and the muscle is halophilic.Microscopy of DUX4-cre mice in the control group and 3-month-old DUX4-Cre+LY2090314 mice in the experimental group showed significantly higher content of red fibers in the gastrocnemius muscle with Sirius staining than in the control group（n=5,P=0.0079）;q PCR showed that the expression level of TRIM36-m RNA,the downstream target gene of DUX4,was 1in the control group and 0.847 in the experimental group;The expression level of fibroblast gene COL1A1-m RNA in the control group was 1,the experimental group was 0.395（n=3）.Western blot was used to detect the total protein of GSK3-β,phosphorylated p-GSK-3β（Ser9 site）,and the expression level of DUX4protein,and the results showed（Fig.12）that there was no significant difference in GSK-3β/GAPDH（P=0.95）.The differences of p-GSK-3/GAPDH and DUX4/GAPDH were statistically significant（P₁=0.0008,P₂=0.0002）,（n=3）.Muscle satellite cells（MUSC）and Fibro/Adipogenic Progenitors（FAPs）were separated by flow cytometry between normal control and patients,respectively.MUSC of normal control（CD56）:FAPs（CD140A）83.1%:2.18%,and MUSC of FSHD patients（CD56）:FAPs（CD140A）0.37%:94.9%.Conclusion1.In 84 patients with wheelchair-dependent FSHD,the age at onset,early onset age of lower limb involvement,and large range of 4Q35-D4Z4 repeats were the possible factors for early development of wheelchair dependency;47 cases of age at wheelchair independency FSHD patients（year≤35）or less than 34 cases of age at wheelchair independency FSHD patients（year>35）,the onset age,lower limb involvement of the earlier onset age,D4Z4 number of repeat units smaller,MRC score is low,FSHD clinical severity score is higher,and ACSS also higher,contemporary mutations in patients was higher.The age of onset and the age of lower limb involvement can be used as potential prognostic indicators of disease severity.2.Flex Dux4 mice induced by TMX can develop parts of clinical and pathological phenotypes of FSHD,which can be used to study the pathophysiological mechanism of DUX4 pathogenicity and as a reasonable model for targeted intervention of DUX4 in vivo;The Wnt/GSK/β-catenin signaling pathway GSK3βplays a role in regulating the formation of FAPS fibers,and can promote the regeneration of muscle satellite cells（MUSC）to some extent.The depletion of the muscle satellite cell pool in the muscle cells of patients with FSHD and the imbalance of the ratio of FAPs may be one of the reasons for the decompensation of the muscle cells in patients with FSHD because the regeneration rate of the muscle cells is not as fast as the degeneration and necrosis rate of the muscle cells.