Clinical,Pathological And Genetic Analysis Of Centronuclear Myopathy

Background:Centronuclear myopathy（centronuclear myopathy,CNM,MIM#310400）was first reported by Spiro in 1966[1]Patients with central nuclear myopathy mainly manifest as craniofacial muscle weakness,weakened limb muscle strength,and mild muscle atrophy[2].Genetic testing and muscle biopsy pathological analysis are the most important means of diagnosing CNM.Up to now,the mutation genes reported in domestic and foreign literatures that can cause central nuclear myopathy include MTM1（myotubularin1）,DNM2（dynamin2）,BIN1（amphiphysin2）,RRY1（skeletal muscle ryanodine receptor）,TTN（titin）,MTMR14（myotubularin related proteinl4）and CCDC78（coiled-coil domain containing protein78）[3].Common pathogenic genes of CNM include BIN1,DNM2,RYR1,and MTM1,which respectively cause autosomal recessive genetic CNM（autosomal recessive centronuclear myopathy,ARCNM）,autosomal dominant genetic CNM（autosomal dominant centronuclear myopathy,ADCNM）,autosomal dominant genetic CNM,and X linkage Recessive genetic CNM（X-linked myotubular myopathy,XLMTM）,these four gene mutations account for 70%of all CNM[4]Among them,the clinical manifestations of myotube myopathy with X chromosome-linked recessive inheritance caused by MTM1 gene mutation are the most serious,often occurring in the neonatal period or infancy.A soft child with hypotonia at birth.Can’t maintain spontaneous breathing;the clinical manifestations of autosomal dominant inherited CNM caused by mutations in the DNM2 gene are relatively mild,mostly in adulthood;the clinical manifestations of autosomal recessive inherited CNM caused by BIN1 mutations are in between,often Onset in infancy or childhood[5].The pathological feature of muscle biopsy in CNM patients is that the proportion of central nucleus muscle fibers is significantly increased.Type I fiber predominance is accompanied by type I fiber atrophy.NADH staining shows a grid structure between spoke-like myofibrils around the central nucleus of more central nucleus muscle fibers.Cytochrome oxidase（COX）staining and succinate dehydrogenase staining（SDH）showed abnormal aggregation of mitochondria around the central nucleus[6].XLMTM is an X-linked congenital myopathy caused by mutations in the MTM1 gene.Compared with other congenital myopathy,XLMTM patients have severe clinical symptoms and less heterogeneity.Herman GE et al[7]studied 35 male patients and found that 24%of patients died before the age of 1 and 80%of survivors needed respiratory support.In 1990,Thomas NS et al[8]conducted a study on XLMTM patients,indicating that the disease-causing gene located in the Xq28 region.In 1996,Laporte J et al.[9]discovered that the pathogenic gene of XLMTM is the myotubularin gene.The MTM1 gene contains 15 exons.The MTM1 gene encodes myotubulin,which belongs to the family of specific phosphatases.The MTM1 protein is composed of 4 domains:protein tyrosine phosphatase（PTP）,glucosyltransferases（GRAM）,Rac-induced recruitment domain（RID）,SET The interaction domain（SET-interaction domain,SID）,whose main role is to participate in vesicle transport and maturation[10].DNM2-related ADCNM is a rare congenital myopathy,characterized by progressive distal muscle weakness and wasting,usually beginning in childhood or adolescence.Patients with DNM2-related ADCNM often present with ptosis,facial abnormalities,and cognitive impairment.Bitoun et al[11]found in 2005 that the disease-causing gene of ADCNM is dynamin（dynamin2,DNM2）.The DNM2 gene at 19p13 contains 22 exons,and the encoded DNM2 protein is a large guanosine triphosphate protein（GTPase）.The domain（pleckstrin homology domain,PH domain）,GTPase effector domain（GTPase effector domain,GED）and the C-terminal proline-rich domain（Proline rich domain,PRD）are composed of 5 domains,the main function is to form on the biofilm And the function of releasing new vesicles is involved in membrane transport[12].Up to now,a total of 19 cases of ADCNM caused by DNM2 mutations have been reported in China,of which 2 cases were reported by the Wang Guoxiang team of the Department of Neurology,China-Japan Friendship Hospital[13];1 case reported by the Department of Anesthesiology,Xiangya Hospital Central South University[14].4 cases were reported by the team of Pu Chuanqiang of the Department of Neurology,Chinese PLA General Hospital[15];3 cases were reported by the team of Hu Jing,The Third Hospital of Hebei Medical University[16];the team of Yan Chuanzhu,Department of Neurology,Qilu Hospital of Shandong University reported a group of 7 patients DNM2-related CNM families[17]and 2 sporadic patients with DNM2-related CNM[18].5 cases of XLMTM caused by MTM1 mutations,3 cases were reported by the team of Hu Jing,The Third Hospital of Hebei Medica1 University[16],and 2 cases were reported by Taiwan,China[19].Here,we conducted the following two parts of research on the clinical phenotype,muscle pathology and genetic analysis of centronuclear myopathy in our research center:Part I:Clinical,Muscle Pathology and Gene Mutation Study of X-linked Centronuclear Myopathy Caused by MTM1 Gene MutationObjective:The Neuromuscular Pathology Laboratory of Qilu Hospital of Shandong University diagnosed a pedigree with centronuclear myopathy caused by MTM1 gene mutation through clinical manifestations and muscle pathological biopsy in February 2019.Then we studied the clinical manifestations,muscle strength,creatine kinase（CK）and gene mutation of all patients and their family members,and combined with previous literature reports to provide references for the diagnosis of XLMTM.Materials and methods:In February 2019,the Neuromuscular Pathology Laboratory of Qilu Hospital of Shandong University confirmed a family with X-linked recessive inheritance through clinical manifestations and muscle pathology biopsy.We performed detailed neurological examinations on three male patients and other family members,performed muscle biopsy on probands,and performed general chemical,enzymatic,and immunohistochemical staining.The peripheral blood of the probands was drawn and sent to Beijing Jinzhun Medical Laboratory performed second-generation sequencing,and extracted the peripheral blood of another 2 patients and other family members,extracted DNA,designed primers for PCR amplification and first-generation sequencing for mutation detection of MTM1 gene,and used software to carry out mutation pathogenicity analysis.Results:1.This pedigree can be traced back to 3 generations,3 male patients,3 patients with weakness of both lower limbs,gradually appeared craniofacial muscle weakness,ptosis,weak eye closure,weak muscle strength of extremities,progressive aggravation and systemic muscle atrophy.The serum CK level of the patients was normal.EMG showed myogenic changes.2.The muscle examined of the proband（Ⅲ 1）was replaced by connective tissue.The sizes of muscle fibers are obviously different,and most of the small fibers are round;the central nuclear muscle fibers are obviously increased accounts for 60%of all muscle fibers;type I fibers are dominant and atrophy;reduced coenzyme I tetrazolium reductase（NADH）staining shows the activity of wheel spoke enzyme centered on the central nucleus of atrophic muscle fibers is enhanced.Cytochrome oxidase（COX）staining and succinate dehydrogenase staining（SDH）showed abnormal aggregation of muscle fiber mitochondria,especially around the atrophic central nucleus fibers.3.Gene mutation detection showed that there was a missense mutation c.251A>T（transcribed version NM₀00252）in exon 5 of MTM1 gene inⅢ 1,Ⅲ 2 and Ⅲ 3,which caused the 84 position aspartic acid encoded of myotubulin replaced by valine,and the patient’s mother Ⅱ 4 and Ⅱ7 were carriers of the mutation.4.It was confirmed by searching HGMD database（http://www.hgmd.cf.ac.uk/ac/validate.php）that the mutation had not been reported so far,and the software of PolyPhen-2（http://gene tics.bwh.harvard.edu/pph2/）,SIFT（http://provean.jcvi.org/index.php）、and Mutation Taster（http://www.mutationtaster.org/）predicted that the mutation was a possible pathogenic mutation.Based on the comprehensive analysis of the phenotype of 3 patients in this family,the results of muscle pathological biopsy,the prediction of the pathogenicity of the mutation by software and the co-segregation between the mutation and the patient’s phenotype in the family,we determined that the c.251A>T missense mutation in exon 5 of MTM1 gene was the pathogenic mutation in this family.Conclusion:1.All three male patients in this family showed symmetrical muscle weakness in the limbs and craniofacial muscle weakness that progressed slowly since childhood.2.The results of muscle biopsy of the proband showed typical pathological manifestations of centronuclear myopathy.3.The results of gene detection showed that there was a missense mutation of MTM1 gene c.251 A>T in Ⅲ1,Ⅲ2 and Ⅲ3,and the motherⅡ4 and Ⅱ7 were carriers of the mutation.4.The point mutation of MTM1 gene c.251A>T is reported for the first time at home and abroad which expands the mutation spectrum of MTM1 gene.Part Ⅱ:Study on Clinical,Muscle Pathology and Gene Mutation in Patients with Autosomal Dominant Centronuclear Myopathy Caused by DNM2 Gene Mutation.Objective:In this study,the clinical phenotype,muscle pathology and gene mutation of 3 patients with DNM2-related ADCNM diagnosed in Qilu Hospital of Shandong University from 2011 to 2019 were analyzed,and compared with the clinical features,muscle pathology and gene mutation of such patients reported abroad,combined with the existing research in treatment,to provide some reference for the diagnosis of such patients.Materials and methods:The subjects were 3 ADCNM patients who were treated in Qilu Hospital of Shandong University from 2011 to 2019.Detailed medical history collection,medical and neurological examination,serological examination,electrophysiology,muscle pathological biopsy and gene detection were performed in 3 patients.Results:1.All the 3 patients were male,and all the patients began to develop the disease at an early age.All the 3 patients were able to walk from 1.5 to 2 years old,and they were easy to fall when they walked unsteadily.The age of patients was 22 years old,6 years old and 28 years old.The first symptoms of 3 patients were weakness of lower limbs,progressive aggravation of symptoms,gradual involvement of bilateral upper limbs,and craniofacial muscle weakness in 2 patients.One patient had high arch of foot,one patient had high palatal arch,one patient had joint contracture and one patient had muscle atrophy.The proximal muscle strength of upper limb was 4-5 in 3 patients,the muscle strength of distal upper limb was 4,the muscle strength of proximal lower limb was 4,and the muscle strength of distal lower limb was 2-4.The distal muscle of lower limb was seriously involved in 2 patients,and the muscle strength of extremities in 1 patient was 4.The reflexes of biceps brachii triceps and knee all disappeared in 3 patients.All the 3 patients had no respiratory and cardiovascular symptoms such as chest tightness,breath holding,chest pain,dyspnea,arrhythmia and so on.The serum creatine kinase（Creatine kinase,CK）levels of 3 patients were 53-149 U/L at the time of treatment,all of which were in the normal range.The EMG examination of 3 patients showed myogenic damage.2.Biopsies were performed in all 3 patients,including left biceps muscle in 2 cases and left tibialis anterior muscle in 1 case.The muscle fibers of 3 patients were obviously different in size,and the small fibers were round;in 2 patients,the central nuclear muscle fibers accounted for more than 90%of the total muscle fibers,and in 1 patient,the central nuclear muscle fibers accounted for 20%of the total muscle fibers（it was considered normal that the central nuclear muscle fibers were less than 5%）;2 patients had selective dominance and atrophy of type Ⅰfibers,and 1 patient could not distinguish between type Ⅰ and type Ⅱfibers by ATP enzyme staining.The myometrium of 3 patients had mild to severe hyperplasia,1 patient showed more phagocytosis,regeneration,hypertrophy and cleavage muscle fibers,NADH staining showed more target-like fibers in addition to radial enzyme activity enhanced muscle fibers centered on the central nucleus,NADH staining showed more muscle fibers in 2 patients,mainly in atrophic muscle fibers,occasionally in large fibers.The activity of radial enzyme centered on the central nucleus was increased in all the large and small fibers of 1 patient.3.All 3 patients underwent high-throughput sequencing,focusing on genes related to congenital myopathy and performed the verification on the discovered mutations.The results of gene sequencing showed that all the 3 patients carried DNM2 gene（transcribed version NM₀01005360.2）mutations,which were c.1393C>T（R465W）,c.1565G>A（R522H）and c.1106G>A（R369Q）heterozygous mutations,respectively.Conclusion:1.All the 3 patients suffered from weakness of both lower limbs from childhood.The main clinical manifestations were that both upper and lower limbs were involved and the distal lower limbs were more heavily involved.2.Skeletal muscle is mainly involved.at present,there are no clinical manifestations of respiratory muscle and myocardial involvement.3.The common feature of muscle biopsies in 3 patients was the increase of central nucleus muscle fibers and the enhancement of perinuclear enzyme activity of central nucleus fibers by NADH staining.4.The results of high throughput sequencing showed that 3 patients carried heterozygous mutations of DNM2 gene c.1393C>T,c.1565G>An and c.1106G>A.