| Myofibril myopathy is a set of clinical phenotype and complex,with rich clinical heterogeneity and genetic heterogeneity of the disease.This group of myopathy has similar pathological manifestations,namely,abnormal deposition of proteins associated with myofibril,especially the Z disc.The pathogenic genes found at present are DES,CRYAB,MYOT,LDB3,FLNC,BAG3,FHL1,TTN,DNAJB6,PLEC,HSPB8 A,ACTA1,etc.The clinical manifestations of this group were from distal myopathy,limb muscle weakness syndrome,congenital myopathy and other characteristic muscle groups,to shoulder fibular syndrome,foot drop,axial muscle fatigue,asymmetrical limb weakness and other asymmetrical myopathy.The pathogenic mechanism is mainly related to the structural or functional abnormality related to myofibrillar fibrinolytic protein and z-line related protein,resulting in abnormal myofibrosis.Therefore,the muscle fiber is less damaged,and the blood creatine kinase is slightly elevated or normal.Electromyography can manifest various abnormal manifestations such as myogenic,neurogenic,myogenic,neurogenic,etc.Myopathologic diagnosis of MFMs relies primarily on optical microscopy.The typical pathological manifestations of light microscopy include: HE staining,eosinophilic negri or eosinophilic substance;MGT staining with amorphous or granular heterochromate,rimmed vacuoles or vacuoles;In the abnormal myofibrillar region(especially in the eosinophilic region),the oxidase activity is partially weakened or absent: At the same time,the neurogenic changes such as angle muscle fiber and muscle fiber grouping were present.Similar pathologic features of the pathogeny and the complex clinical phenotypes have presented a challenge for the diagnosis of MFMs,and the diagnosis must be based on the pathogenic gene analysis.Using traditionalSanger forensic analysis,there are different limitations in the diagnosis of positive rate,differential diagnosis,timeliness,etc.Next generation sequencing technology(NGS)targeted gene capture technology analysis of multiple genes at the same time,to identify the cause of disease mutations.Directed sequencing provides not only high coverage(99%)and cover(read depth > 100)the accuracy of the DNA sequencing,but also reduce the cost of each nucleotide.In addition,around the genome in clinical and all exons is unrealistic,due to the time,cost and complexity of analysis and generation and interpretation of sequence information,obtained from sequencing the entire genome.This study screened the skeletal muscle genetic resources of the third hospital of hebei medical university(2005.3-2016.12,1598 cases): 1)myofibrosis(9 cases);2)other types of congenital myopathy without special pathological changes(11 cases);3)immunohistochemical staining of negative limb girdle muscular dystrophy,12 patients with clinical and pathological analysis,capture the second generation gene sequencing to pathogenic gene screening,bioinformatics analysis it is concluded that the locus of the patients and relatives Sanger method validation can be obtained.Part one Clinical,pathological and molecular biological study of myofibrillar myopathyObjective: By skeletal muscle pathological biopsy and history of myofibril myopathy in 9 patients through the purpose gene to capture the second generation sequencing of molecular biology research,analysis of the possible pathogenic genes,to summarize the clinical and pathologic phenotypic expression and pathogenic genes have relevance,to investigate the diagnostic value of NGS in myofibrillar myopathy.Methods:1.Filter into the group of patients with clinical and pathological data: 1)clinical manifestations: conforms to the clinical characteristics of myofibrillar myopathy,slow increase of symmetric/asymmetric limbs distal proximal muscle weakness,muscle atrophy;2)the creatine kinase is normal or slightlyelevated;Electromyography suggests muscle origin and/or neurogenic damage;3)skeletal muscle histochemistry staining: visible muscle cell membrane in the cytoplasm under abnormal sediments,the abnormal muscle fibril area(especially with eosinophilic corpuscle area),visible oxidase activity of local diminished or absent;2.The clinical data of patients were collected,including gender,age of onset,age of treatment,evaluation of clinical muscle strength,results of serum creatine kinase,electromyography and neurogram results,nuclear magnetic results of skeletal muscles of the lower limbs,and the results of myocardial nuclide imaging.3.The pathological analysis of the chemical staining of skeletal muscle tissue,including light and electron microscopy.4.Target gene capture,second-generation sequencing,possible pathogenesis mutation by molecular biology analysis,Sanger sequencing verification.Results:1.Clinical manefestation :There were 6 males and 3 females in 9 cases,and 4 patients with distal muscle weakness and 5 cases of myasthenia of limb,Serum creatine kinase was elevated by 6 cases and normal in 3 cases.Electromyography examination showed 5 cases of pure myogenetic abnormality,and 3 cases of neurogenic abnormality combined with myogenicity,1case of pure neurogenic abnormality.In 5 patients,the myocardial nuclide imaging was examined,3 of which were normal and 2 were abnormal.7routine bilateral skeletal muscle MRI examination patients had different muscle group fat deposition,which were consistent with the imaging manifestations of myopathy.2.Pathological analysis of skeletal muscle: 9 cases showed pathological manifestations of myopathy,and the diameter of muscle fibers was different with some degeneration fibers.Necrosis and regenerated muscle fibers are rare;The connective tissue was mildly or moderately hyperplasia,and granular,filamentous,bead-like,and other abnormal material deposits were observed in the cytoplasmic intramuscular membrane,and the myofibrillar network wasdisordered by NADH,SDH and COX enzymes.3.Targeted enrichmengt and NGS,Sanger verified:Four of the nine patients did not find myofibrosis related gene mutation;1 case of TTN missense mutation(c.90211 t > c,Cys30071Arg),1 case of DES missense mutation(c.1255C>T,Pro419Ser),1 case of MYH7 new shearing mutation(c.3337-2-> c,splicing);New composite heterozygous mutation of PLEC(c.13001G>A,p.R4334H;C.10879G>A,p.D3627N)1 case,MYH7 reported pathogenic mutation(c.5533C>T,p.R1845W)1 case.Conclusion:1.The clinical phenotype of myofibril myopathy including distal myopathy,limb syndrome,asymmetric muscle involvement,it has no fixed form of onset and muscle involvement model,clinical manifestation is not exact directivity for disease diagnosis.But asymmetrical muscle weakness is relatively unique in clinical manifestations.2.Pathology of the skeletal muscle biopsy with similar performance,namely intracytoplasmic abnormal material deposition,monoclonal protein involved in immunohistochemical staining hyperchromatic has directed to the disease diagnosis.3.A variety of pathogenic mutations lead to interact with protein Z line or play an important role in maintaining integrity myofibril protein structural or functional change can cause the disease.4.The pathogenic gene was complex,and some patients with clinical and muscle pathology were not found to have the pathogenic gene in the patients with MFMs performance,so the patients with negative genetic test could not rule out the MFMs diagnosis.5.It is helpful for that molecular biology diagnosis of the group to improve the recognition of abnormal sedimentary disease in the cytoplasm,enlarge the spectrum of disease,and enrich the clinical phenotype and clinical external phenotype of pathogenic gene.Part two Clinical,pathological and molecular biological study of atypical congenital myopathyObjective: Congenital myopathy(Congenital myopathies,CMs)is a group of single-gene genetic disease of the skeletal muscle,with clinical and genetic heterogeneity,biopsy specific structure found in skeletal muscle pathological changes.However,the diagnosis and differential diagnosis of congenital myopathy with no special pathological changes in skeletal muscle biopsy are relatively complicated.In this study,11 patients with atypical congenital myopathy were selected for clinical and skeletal muscle pathology,and molecular biological studies were carried out through the analysis of the pathogenic genes in the second generation sequencing,with a view to strengthening the understanding of the patients in the group with no specific pathological changes in skeletal muscle biopsy,and identifying the pathogenic pedigree,summarizing the clinical characteristics and identifying the pathogenic genes.Methods:1.Filter into the group of patients with clinical and pathological data: 1)clinical manifestations: conforms to the clinical characteristics of congenital myopathy,childhood onset,raw athletic ability after developmental delays,with/without intelligence dysplasia,electromyography prompt muscle damage source sex or neurogenic damages or muscle source sex combined neurogenic damages,moderately elevated serum creatine kinase or normal,skeletal muscle biopsy pathology without typical congenital myopathy pathology,except the rod body myopathy,central nucleus myopathy,empty disease characteristic such as the central axis of congenital myopathy.2.The clinical data of patients were collected,including gender,age of onset,age of treatment,evaluation of clinical muscle strength,results of serum creatine kinase,electromyography and neurogram results,nuclear magnetic results of skeletal muscles of the lower limbs,and the results of myocardial nuclide imaging.3.The pathological analysis of the chemical staining of skeletal muscle tissue,including light and electron microscopy.4.Target gene capture,second-generation sequencing,possible pathoge-nesis mutation by molecular biology analysis,Sanger sequencing verification.5.Gene detection indicated COLA6 gene abnormal patients,monoclonal antibody against COLA6 immunofluorescence staining.Result:1.Clinical data: among the 9 patients,1 female and 8 male patients,all of them were children with retarded motor ability,and the severe ones were not able to walk,and the light could run and jump.Five patients had joint contracture,and 8 patients had distal joint extension.2.Laboratory and auxiliary examination: that serum creatine kinase fluctuate in 86-1335u/L,6 patients show muscular source-induced fatigue,1case is myogenic combined neurogenic change,the musculogenic change is dominant,2 cases are mainly neurogenic change,and 5 routine bilateral skeletal muscle MRI examination patients have different muscle group fat deposition,and no obvious specific muscle group is affected.3.Skeletal muscle biopsy pathology analysis: Both patients showing myogenic changes,5 patients with obvious nucleus,6 cases with muscle fiber connective tissue increased moderately or seriously.2 cases with cytochrome C oxidase obviously missing,6 patients Ⅵ collagen and collagen Ⅳfluorescent monoclonal antibody immunohistochemical staining,prompt Ⅵcollagen proteins in the membrane and connective tissue of muscle fiber is absent or reduced.4.Targeted enrichment and NGS,Sanger verified: Among the patients in this group,6 genes were found to be abnormal,including RYR1,LMNA,COL6A1,COL6A2,,MYH7,etc.Among them,2 were missing mutations,1was splicing,and 13 were missense mutations,among which 12 were new gene mutations.As has been reported to the pathogenic mutation in 4patients.Conclusion:1.The hereditary skeletal muscle diseases of children are similar to the clinical phenotype: muscle weakness,muscle atrophy,soft child syndrome,bone joint contracture,deformity,etc.,the same clinical manifestations are thevarious pathogenic genes,and skeletal muscle biopsy has a certain role in the diagnosis of diseases.However,when skeletal muscle pathology has no specific changes,it is necessary to rely on molecular biological technology for further diagnosis.2.The clinical phenotypes of collagen related myopathy include Ullrich congenital muscular dystrophy,intermediate type VI collagen disease and Bethlem myopathy,among which the intermediate type can be characterized by congenital myopathy,which is difficult to differentiate clinically.3.Center nuclear phenomena appeared in the congenital myopathy caused by multiple genes,in addition to the characteristic center nuclear myopathy,collagen protein Ⅵrelated myopathy,MYH7 related myopathy may appear.4.Objective gene capture NGS technology has the value of diagnosis and differential diagnosis of congenital myopathy and congenital muscular dystrophy with no specific pathologic manifestations,and the clinical phenotype of the pathogenic gene is enriched,and the recognition of the disease is enhanced,and the pedigree of differential diagnosis is expanded.Part three The diagnostic value of second-generation sequencing technology in limb girdle syndrome.Objective: Many different kinds of myopathy can lead to limb syndrome-the expression of muscle weakness in the proximal segment of the limb and the muscle weakness of the belt,and the common types of muscular dystrophy,congenital myopathy,myofibromatosis,etc.,among which the dystrophy of the limb is rich in genetic heterogeneity,and the simple pathological changes cannot provide the direction for the diagnosis of molecular biology.In this study,12 patients with dystrophy were selected from clinical and skeletal muscle pathology,and molecular biological studies were carried out through the analysis of the pathogenic genes in the second generation sequencing,and the clinical/pathological characteristics were summarized.Methods:1.A total of 12 cases with LGMD were screened by clinical andbiopsies.Criteria: 1)clinical manifestations: the clinical features of the muscular dystrophy of the extremities,the muscle weakness and atrophy of the extremities of the extremities,and the progressive aggravation of the muscles,the myogram of the muscle,and the serum creatine kinase,which can be significantly elevated,the serum creatine kinase value is significantly increased,and the pathological changes of the muscular dystrophy are indicated by the pathological changes of skeletal muscle biopsy,while the immunohistochemical staining of monoclonal antibodies against Dydtrophin,anti-dysferlin,and anti-sacoglycan are all normal.2.The clinical data of patients were collected,including gender,age of onset,age of treatment,evaluation of clinical muscle strength,results of serum creatine kinase,electromyography and neurogram results,nuclear magnetic results of skeletal muscles of the lower limbs,and the results of myocardial nuclide imaging.3.The pathological analysis of the chemical staining of skeletal muscle tissue,including light and electron microscopy.4.Target gene capture,second-generation sequencing,possible pathogenesis mutation by molecular biology analysis,Sanger sequencing verification.Result:1.Clinical data: among the 12 patients,6 were male,6 were female,and all of them had muscle weakness in the proximal limb of the limb and the muscle of the belt,and 4 patients with cervical muscle weakness.Two cases were significantly increased after pregnancy.The serum creatine kinase was significantly increased,and the myogram examination indicated myogenetic changes.5 routine myocardial radionuclide imaging showed abnormal expression of myocardial perfusion.MRI of skeletal muscle in 8 patients with bilateral lower limbs revealed different degrees of fat infiltration.2.Biopsy skeletal muscle histochemistry staining: all the patients were pathological changes in different degree of muscular dystrophy,2 cases of a large number of typical leaf fibers.3.Capture molecular biology gene analysis: 12 patients found in seven different genetic mutations: carry CAPN3 gene mutations found in 4 patients,1 patient TCAP homozygous mutations,1 case of POMT2 compound heterozygous mutations;1 case of FKRP complex heterozygous mutation;1case of PLEC complex heterozygous mutation,1 case of GMPPB complex heterozygous mutation,1 case LAMA2 complex heterozygous mutation.Conclusion:1.There is a rich genetic heterogeneity in the limb girdle muscular dystrophy,and there is little difference in clinical manifestations.2.The limb girdle muscular dystrophy and congenital muscular dystrophy virulence genes are overlapping.3.The second generation sequencing technology can help to expand the clinical phenotype of known genes and enrich the differential diagnosis pedigree of similar clinical symptoms. |
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