| Myopathy with rimmed vacuoles is a group of skeletal muscle disease characterized by rimmed vacuoles（RVs）in muscle biopsy.RVs are many granular substances adhered on the wall of vacuoles that is dyed by hematoxylin-eosin（HE）or modified gomori trichrome（MGT）in the frozen section of fresh muscle tissue.Most of RVs are located in atrophy and denatured muscle fibers and distributed in the subsarcolemmal or in the myofibroplasm.There are various forms such as fissures,irregular shapes and circles.The particles of RVs are basophilic stained blue in HE and purple red in MGT.The activity of acid phosphatase is increased in vacuolar site.RVs are not a real vacuole in muscle fibers,but an artificial product in dyeing process.Myopathy with RVs is a group of skeletal muscle disease caused by abnormal primary lysosome,the muscle fibers appearing RVs highly expressed cathepsin B,D,H,L and gridin that were related to lysosomes.Abnormal autophagy/autophagosome in RVs disease is related to the follow-ing mechanisms:（1）normal lysosomes could not degrade excessive substrates;（2）oxidative stress reaction;（3）endoplasmic reticulum stress activation;（4）deficiency of lysosomal enzyme function;（5）substrate conformation abnor-mal degradation;（6）degradation of autophagosome degradation ability;（7）the process of autophagy no longer progresses.RVs is not a specific pathological change of skeletal muscle biopsy.Its appearance is often suggestive of diagnostic/differential diagnosis.Myopathy with RVs is divided into hereditary/non hereditary according to the cause of disease,which almost involves all types of skeletal muscle disease.The patients whose biopsy of the skeletal muscle appearing RVs,detailed family history,personal history inquiry,combined with the patient’s clinical examination and skeletal muscle biopsy are necessary to exclude drug induced,infection and sporadic inclusion body myopathy（sIBM）.Hereditary myopathy with RVs,involved a large number of genes,including a variety of genetic patterns.Genes reported in the literature can cause RVs in skeletal muscle biopsy including:TIA1,TTN,LDB3,myotilin,MATR3,VCP,DES,FLNC,MYH7,GNE,PAPBN1,DNAJB6,TNPO3,HNRNPDL,TCAP,FHL1,MYH2.In addition,in the biopsy of light Becker muscular dystrophy（Becker muscular dystrophy,BMD）,facial scapulohumeral muscular dystrophy（facioscapulohumeral muscular dystrophy,FSHD）and multiple systemic protein related inclusion body myopathy,RVs can be found.Myopathy with RVs is faced with the following problems:（1）myopathy with RVs are inherited or not;（2）genetic myopathy with RVs in biopsy involves many genes,how to carry out gene detection and increase the positive rate;（3）how much is the value of RVs in the diagnosis/differential diagnosis of skeletal myopathy;（4）why RVs occurs in many kinds of hereditary skeletal muscle diseases,and whether the same mechanism leads to the atrophy and apoptosis of skeletal muscle cells,and the final manifestation of weakness.With the development of sequencing technology,the next generation sequencing（NGS）can sequence multiple genes at the same time,which is of great value for the diagnosis and differential diagnosis of hereditary skeletal muscle disease.Compared with all exon sequencing,target sequencing has little time,low cost and simple result analysis,and is widely used in clinic,which greatly promotes the diagnosis of hereditary myopathy with RVs.In this study,35 cases of hereditary myopathy with RVs was selected from the specimen bank of Department of Neuromuscular of the Third Hospital of Hebei Medical University from 2005 to 2018,including:（1）1families of VCP myopathy,3 patients;（2）16 cases of GNE myopathy;（3）16patients with other types of myopathy with RVs.We performed clinical and pathological analysis to identify the characteristics of different types of patients,so as to identify the value of RVs in diagnosis and the spectrum of inherited RVs disease.Part 1 A study of ophthalmoplegia,ptosis,dysphagia and distal weakn-ess of VCP myopathy in a familyObjective:The common types of myopathy with RVs involving the extraocular muscles and pharynx and larynx are common types of blepharop-tosis,extraocular muscle paralysis and dysphagia:vocal cord and pharyngeal distal myopathy,oculopharyngeal muscular dystrophy（OPMD）,oculophary-ngodistal myopathy（OPDM）and MYH2 myopathy.The inclusion body myopathy caused by the mutation of valosin-containing protein（VCP）,is mainly manifested limb-girdle weakness or distal weakness.VCP rarely involve facial muscle,and without relationship with eyelid ptosis,extraocular muscle paralysis and dysphagia.Method:1.Informed consent was obtained from all patients and their legal representatives.Afert that,all family members’data were collected through outpatient medical records,medical racords and medical history enquiries.Then the blood was collected of family members.2.We obtained clinical data from 6 family members including clinical signs and symptoms,and performed supplementary examinations including serum creatine kinases（CK）and alkaline phosphatase（ALP）levels,plain X-ray of bone,MRI of the brain and lower limbs,and electromyogram（EMG）.3.Pathological analysis and ultrastructural analysis of skeletal muscle biopsy.4.The proband performed Sanger sequencing of PABPN1 to exclud OPMD.5.NGS of the proband identified a reported heterozygous VCP c.463C>A（p.R155S）.Sanger sequencing confirmed the segregation of this variant in an autosomal-dominant pattern.WES did not show other pathogenic genes.Result:1.The proband:A 27-year-old woman,presented water reflux from nasal cavity when she was 8-year-old,presented ptosis and walked slowly when she was 14-year-old.When she was 17years old,she presented dysphagia,squat down and stand up difficultly and easily fell down whether she was walking on a level road,walking upstairs and downstairs.When she was 25 years old,her ability of audition and cognitive declined seriously but she still had the ability to communicate with her family members.Her wheelchair-bond age wad 26.When she was 21 years old,she was admitted to our department and went through related examination.The neurological examination of eldest sister revealed mild weakness（Medical Research Council grade 4）upper limb muscles;moderate weakness（Medical Research Council grade 3+）of proximal lower limbs muscles and ankle evertors;and severe weakness（Medical Research Council grade 1-2）of ankle dorsiflexors.The serum CK level was 460U/L.Electrophysiological studied of the left limbs,sternocleid-omastoid muscle,and orbicularis oculi muscle revealed myopathic change.The sister of the proband died when she was 21 years old.One year before she died,she lost the ability to walk around.When she was in school,she has got 0 marks on the exam.When she was 7 years old,she presented water reflux from nasal cavity.When she was 11 years old,she presented ptosis and foot drop.When she was 17 years old,she presented squat down and stand up difficultly,easily fell down,declined audition and cognitive and whole body peaky.Electrophysiological studied of the left limbs in her 17years old,sternocleidomastoid muscle,and orbicularis oculi muscle revealed myopathic change.The brother of the proband:A 18-year-old Chinese adolescent male presented with a 10-year history of dysphagia which initially presented water reflux from nasal cavity and 6-year history of ptosis,ophthalmoplegia and distal myopathy.The proband has no fluctuating weakness.The neurological examination of proband revealed mild weakness（Medical Research Council grade 4）of the neck and proximal upper limb muscles;moderate weakness（Medical Research Council grade 3）of proximal lower limbs muscles,wrist flexors,wrist abductors and hands’muscles;and severe weakness（Medical Research Council grade 1-2）of ankle evertors and dorsiflexors.The weakness was slightly asymmetric and more pronounced of the right side.The muscles in the distal lower limbs were atrophic.There was on winging of the scapulae bilaterally.There was on percussion-and no action-induced myotonia.There was severe binocular ptosis.The width of the palpebral fissure was 4mm of right and 2mm of left respectively.The movements of the eyeball were limited in all directions.Tendon reflexes were diminished.He did not have Babinski and Hoffmann signs on either side.Sensory and coordination exams were normal.The Portuguese version of MoCA testing was 24 out of 30.The deduct items:three-item confrontation naming task with low-familiarity-1,repetition of two syntactically complex sentences-2,a two-item verbal abstraction task-1,the short-term memory recall task-1,orientation to time and place-1.The serum CK level was ranged from 958-1340U/L（normal<300U/L）.Serum ALP was normal.Plain X-ray of the hips,head the spine showed normal.Brain MRI showed sulcus increased in the right cerebellum,but no atrophy in the frontal lobes.Electrophysiological studied of the right limbs at the age of 15 revealed normal nerve conduction studies（NCS）,except for no waves of superficial peroneal nerve.Needle EMG revealed fibrillation potentials and myopathic motor units in both proximal and distal muscles of right lower and upper limbs.The proband’s mother got generalized bone pain when she was 40 years old.At her 43 year old,she got dementia.When she was 46 years,she lost the ability to walk around.One year later,she died.Before she died,no ptosis and dysphagia.The proband’s uncle presented squat down and stand up difficultly when he was 45 years old while no ptosis,dysphagia,dementia and bone pain.The proband’s grandparents presented dementia after 45 years old and died before 50 years old.His grandmother presented limbs weakness.The proband’s niece and little uncle were no clinical manifestation.2.Skeletal muscle MRI of lower limb the proband and her brotherAnalysis of lower limb muscle MRI of 2 patients showed patchy regions of fat tissue replacing muscle tissue and varying degrees of involvement of different muscle groups.MRI of the lower limbs showed serious fat replacement in the posterior group of the thigh,anterior group and posterior group of the legs.The fat replacement of the leg was more marked compared with the thigh.The anterior muscle group of the thigh showed some involve-ment.3.Pathological analysis of skeletal muscleLight microscopy:H&E staining revealed variability of muscle fiber size.Some degenerated fibers and fat in the fibers were observed.Typical RVs were present in the cytoplasm of muscle fibers.Connective tissue was moderately increased.Focal devoid of oxidative activity was observed in oxidative enzymes and absent in RVs.Acid staining revealed high activity of rimmed vacuoles.Glycogen and fat components were normal in the muscle fibers.Pathological changes in the proband’s skeletal muscle biopsy were more serious than her brother’s,who presented with more obvious connective tissue hyperplasia and more RVs.Electron microscopy:The endoplasmic reticulum is dilated and the attached ribosome falls off and blurs;the mitochondrial ridge is blurred and dilated;the abnormal secondary lysosomes are aggregated;RVs is multi-layer membrane structure;and the contraction of the myocytes is visible.4.Sanger sequencing showed the proband was homozygous for the normal（GCG）6 allele in PABPN1.5.NGS identified a heterozygous missense variant NM-007126c.463C>A（p.R155S）in exon 5 of VCP.Sanger sequencing was performed in nine related family members,which confirmed the variant in all six patients in an autosomal dominant segregation pattern.WES did not indicate other pathogenic genes related to clinical manifestations.Conclusion:1.VCP-myopathy is the most common clinical manifestation of VCP associated diseases,with mainly limb/distal-type muscle weakness,rarely facial muscle involvement,no extraocular and throat muscle involvement.This study expands the clinical performance spectrum of VCP-myopathy.2.The mutation of VCP is characterized by incomplete penetrance and high clinical heterogeneity,involving the central nervous system,skeletal muscles,and bones.Members of the same family may have completely different clinical phenotypes.Severe cases may die in middle or middle age.3.The skeletal muscle group is highly selective involved in muscle MRI,which has a clue role to the diagnosis.4.RVs in skeletal muscle biopsy lack specific pathological diagnosis value for this disease.The pathogenic gene get from next generation sequencing is an important basis for diagnosis.Part 2 Clinical,pathological and molecular biological study of GNE myopathyObjective:GNE myopathy is the most common hereditary myopathy with RVs of the distal weakness.The typical clinical manifestation is foot drooping as the first symptom,the quadriceps is relatively spared.The age of the onset is mainly in early adulthoods.Some patients presented a rapid deterioration during pregnancy.Some patients had atypical clinical manifest-ations,with onset of proximal muscle weakness or weakness of the hand.GNE myopathy associated with congenital thrombocytopenia has been reported.16 GNE patients were diagnosed by Sanger sequencing,and their clinical features,skeletal muscle MRI manifestations and pathological features were summarized.The characteristics of genetic mutations of GNE myopathy in Chinese population were identified combining with the previous reports of Chinese population.Method:1.The criteria for admission:1)onset from early adulthood;2)initial symptom:weakness of distal limbs,combined with proximal limbs’weakness after long course;3)sporadic or autosomal recessive;4)normal to mild elevation of the serum CK;5)EMG mainly showed myopathic findings,and some patients combined neurogenic findings;6)muscle biopsy showing RVs.2.We collect the patient’s clinical data,including family history,clinical manifestations,complete blood count,CK,EMG,muscle biopsy,and skeletal muscle MRI.3.Pathological analysis of skeletal muscle biopsy.4.Sanger sequencing was performed to all patients.The pathogenic mutations were summarized.Result:1.There were 16 patients in the study,13 were female and 3 male.Patient2 and patient 3 are siblings.Patient 6 is the only sick offspring of consanguineous parents.Patient 8 had an autosomal recessive family history of distal myopathy.The other 12 cases were all sporadic cases.Onset age of the patients ranged from 16 to 39 years,average age of onset was 31 years.The interval from disease onset to diagnosis was 1 to 20 years.Among the 16patients,12 patients showed classic phenotype.Their initial symptom was the weakness of anterior compartment as they often complained of ankle weakness.With progression of the disease,both proximal and distal muscles of the upper limbs,neck flexors,and pelvic muscles were involved.Physical examination showed weakness of the tibialis anterior muscles in call cases（MRC grade 0-3）,the gastrocnemius muscles in call cases（MRC grade 1-5）,while the strength of their quadriceps femoris was MRC grade 4-5.4 patients showed weakness in the cervical muscles,and two of them are siblings.P5initially showed weakness in distal upper limbs.She could not hold things tightly at first,the weakness of the tibialis anterior developed five years later.And at the same time she had decreased sensation in the left lower extremities.P6 patients suffered from left hand weakness and showed muscle and pelvic muscles after 2 years.P13 is accompanied by thrombocytopenia.The first symptom of P16 was weakness of the proximal extremities of the lower extremities showing squatting hard.Physical examination showed weakness:The strength of proximal lower limbs muscle was MRC grade 3-4,the strength of ankle dorsiflexion and extensor muscle was MRC grade 5,the strength of hallux dorsiflexionws MRC grade 0,but dorsiflexion was MRC grade.4female patients presented rapid deterioration during pregnancy.Before pregnancy all 4 patients did not show prominently weakness,just a little weakness after along distance walk.But after delivery they all had difficulty to walk around.Serum creatine kinase levels were normal to slightly elevated,ranging from 62 to 1060 U/L（normal value 25-130U/L）,less than 10 folds of upper normal limites.The number of platelets in P13 patients was 4×109/L-26×109/L（normal value 100-300×109/L）.The number of megakaryocytes in bone marrow was 48/（1.5 x 3cm）,with normal morphology and PAIgG（+）.EMC showed myogenic findings in 16 patients,and 4 cases also showed neurogenic findings.Using MRI,we analyzed the fatty involvement of the skeletal muscle in lower extremities.Adductor longus,semitendinosus and soleus were the most frequently affected,followed by semimembranosus,biceps femoris,tibialis anterior,adductor magnus,and gastrocnemius.Gastrocnemius medialis was affected more seriously than the lateral gastrocnemius muscle.In the advanced stage of the disease,all the muscle in the lower legs were fatty infiltration completely.The vastus lateralis was the only spared muscle,while the rectus femoris,vastus intermedius and medialis of the quadriceps showed variable sings of fatty replacement.2.Muscle pathologyAll patients underwent muscle biopsy.RVs were the main pathological manifestations in all cases generally without signs of inflammation.Marked variation in fiber size with scattered fiber necrosis and degeneration was observed in some patients.Electron microscopy revealed myelin figures of RVs.3.Results of genetic analysisMutation analysis identified that all patients carried GNE mutations.Two patients were homozygous,with c.830G>A（p.R277Q）and c.649T>C（p.Y217H）.14 patients were compound heterozygous.In total,there were 14different mutations in this study.Of these,7 were novel mutations:c.541A>T（p.I181L）,c.125G>A（p.P42Q）,c.226G>A（p.V76I）,c.155A>G（p.D52G）,c.970C>G（p.H324D）,c.1055G>A（p.R352H）,c.859G>A（p.A287T）.All of the novel mutations were absent in 200 control chromosomes from healthy Chinese individuals.Functional prediction using SIFT and Polyphen2software also indicated that novel mutations were damaging or probably damaging.Among these mutations,p.D207V was detected in 9 patients,giving an allele frequency of 28.1%（9/32）.The second most common mutation was p.R277Q.Except patient 10 and patient 13,mutations of the other patients were located only in epimerase domain.In patient 10 and patient 13,comp-ound heterozygous mutations located in the epimerase and kinase domains.Conclusion:1.The clinical manifestations of 16 cases of GNE myopathy in the present study are consistent with the current research reports.Cervical muscle involvement,thrombocytopenia,and aggravation of pregnancy are clinical manifestations that are worthy of attention.The involvement of respiratory muscles and myocardium in some reports were not found in this study.2.Selective involved of muscle groups in MRI is an important feature in the differential diagnosis of other types of hereditary skeletal muscle diseases.3.When there are a large number of RVs in skeletal muscle biopsy,combined with clinical distal muscle weakness as the first symptom,GNE myopathy should be highly suspicious.RVs formation is associated with abnormal protein deposition in the cells.4.In the general case,patients with typical clinical symptoms can perform GNE sanger sequencing,which can reduce the cost of sequencing and shorten the time of sequencing.Next generation sequencing is needed when distinguishing from other types of skeletal muscle diseases associated with RVs.5.In this study,there were 7 new GNE mutations in 16 patients,which combined with more than 160 mutations in the GNE,indicating that there is no hotspot mutation in GNE and GNE is highly conserved among species.Part 3 Study of other types of hereditary myopathy With RVsObjective:It is generally believed that the formation of RVs is related to the abnormal function of lysosome function or abnormal autophagy process caused by protein deposition.The immunohistochemical staining of muscle fiber containing RVs show a variety of abnormal protein deposits such as APP,A beta-42,polyubiquitin,p62,TDP-43,tau protein and so on.Factors that can cause the dysfunction of the lysosome function can cause RVs formation,including chloroquine/hydroxychloroquine drug poisoning,HIV/HCV virus infection,the gene mutation affect cell sialic acid metabolism and of myosin structure.RVs does not have the specificity of pathological diagnosis.In the hereditary skeletal muscle disease,the significance of the appearance of RVs is still not clear.Therefore,we carry out clinical data and pathological collection of patients with hereditary myopathy with RVs.Firstly,we excluded GNE myopathy was by Sanger sequencing,NGS was carried out to identify the gene of the other hereditary myopathy with RVs to make clear the value of RVs in diagnostic/differential diagnostic of skeletal muscle disease.Method:1.The criteria for admission1)clinical manifestations:progressive weakness and atrophy of the extremities,proximal/distal limbs weakness;2)elevated CK,normal erythrocyte sedimentation,normal C reactive protein;3)EMG mainly showed myopathic findings,and some patients combined neurogenic findings;4)skeletal muscles MRI of the lower limbs showed fatty infiltration,but no inflammatory changes;5)skeletal muscle biopsy:RVs,no inflammatory cell infiltration.2.We collect the patient’s clinical data,including family history,clinical manifestations,CK,EMG and skeletal muscle MRI.3.Pathological analysis of skeletal muscle biopsy.4.NGS for all patients.The suspicious DMD exon deletion mutation was detected by MLPA and the X chromosome inactivation（XCI）analysis was performed in female patients.The pathogenic mutations were summarized and verified by Sanger sequecing.Result:1.There were 16 patients in the study,5 were female and 11 male.Patient14 and patient 15 are mother and son.The other patients were all sporadic cases.Onset age of the patients ranged from 14 to 54 years,average age of onset was 34.43 years.The interval from disease onset to diagnosis was 1 to20 years.Among the 16 patients,9 patients showed proximal extremities weakness as the first manifestation,the rest of the 7 cases showed distal extremities weakness as the first manifestation.The weakness of 2 patients were not asymmetrical.P2 showed weakness of the right upper and lower extremities,and P4 only had left upper limb weakness.P3 patients comp-licated with dysphagia.Apart from P4 and P5,the Serum creatine kinase levels were normal to slightly elevated,ranging from 149 to 947 U/L（normal value 25-130U/L）,less than 10 folds of upper normal limites.EMG showed myogenic findings in 16patients,and P3,P10 also showed neurogenic findings.We performed a skeletal muscle MRI examination in 4 patients.MRI of P6 showed severe infiltration of fat in quadriceps femoris,tibialis anterior,adductor magnus,musculus membranae sartorius and sartorius muscle.Both P14 and P15 were severe fatty infiltration in the anterior calf group.P14combined fatty infiltration in the lateral head of the gastrocnemius,while the lateral head of the gastrocnemius in P15 was better.The MRI of the P14 thigh was fatty infiltration,and the posterior group was relatively heavy.P16skeletal muscle MRI showed severe fat in the thigh group.Only the gracilis muscle was relatively preserved,while anterior muscles of the thigh and muscles of lower leg were slightly involved.2.Pathological analysis of skeletal muscle biopsyAll patients underwent skeletal muscle biopsy.There are varying amounts of RVs are associated with degeneration,necrosis and regeneration muscle fibers.6 cases had varying degrees of muscle adipose.Many cases’biopsy accompanied by central nucleus,nuclear aggregation and muscle fiber division.Except for P12,there were connective tissue hyperplasia.Activities of NADH,SDH,COX enzyme are decreased in degrees,and showed myofibrillar disorder.2 cases presented rubbing change and the other 2 cases showed lobular fibers.The activity of acid phosphatase increased in the denatured,necrotic muscle fibers,and around RVs.The composition of glycolipid was normal or secondary increased.4 patients with type I muscle fiber superiority,1 patients with type II muscle fiber superiority,the rest were the normal distribution of type two muscle fiber type.3.Results of genetic analysisBy NGS,a total of 5 patients had pathogenic mutations.The mutation of P4 was DMD exon 3-12 deletion,the ratio of inactivation of XCI between80:20 and 90:10,with moderate tilt mode.The mutation of P12 was DNAJB6c.287C>G（p.P96R）heterozygous mutation;The mutation of P13 was DYSF c.31143115insCGGC（p.R1038fs）,c.5231G>A（p.G1744D）compound heterozygosity;The mutation of P14 and P15 was TTN c.95134T>C（p.Cys31712Arg）.The pathogenicity of 2 cases’gene mutation was inaccurate.The mutation of P3 was FLNC c.4342T>G（p.C1448G）heterozygous mutation;the mutation of P10 was TTN c.82815T>A（p.Y27605X）heterozygous mutation.No pathogenic mutation was found in the other 9 patients.Conclusion:1.Hereditary myopathy with RVs is not only manifested as distal myopathy,but also limb-type muscle weakness syndrome.With the exception of GNE myopathy,there is no significant difference in proximal and distal muscle involvement in patients with hereditary myopathy of RVs.2.RVs are scattered in muscle biopsy of various hereditary skeletal muscle diseases.Pathological diagnosis needs to be combined with other pathological phenomena such as muscular dystrophy.To differentiate diagnosis,monoclonal antibody immunohistochemical staining at the molecular pathological level can be used to detect relevant proteins if necessary.3.MRI of skeletal muscle is an important clinical phenotype of inherited RVs myopathy,which is of great value for diagnosis and differential diagnosis.4.The targeted gene panels of inherited RVs myopathy should be further expanded to simplify clinical diagnosis and differential diagnosis. |
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