Clinical,pathological And Molecular Biological Study Of Congenital Myopathies

Congenial myopathies（CMs） are a clinically and genetically heterogeneous group of monogenic hereditary skeletal muscle disorders characterized by early onset weakness, hypotonia, with often static or minimally progressive weakness and characteristic, divided into subtypes based on the predominant histopathologic findings. Historically the congenital myopathies have been classified on the basis of the major morphological features seen on muscle biopsy-e.g., 1) core myopathies（central core disease and multiminicore disease, cores）; 2) centronuclear myopathies（centronuclear/myotubular myopathy, central nuclei）; 3) nemaline myopathy（rods）; 4) myosin storage myopathy（hyaline body） and 5) congenital fibre type disproportion（selective hypotrophy of type I fibres）. The common pathology features of CMs are: structural abnormalities in muscle fibrosis include variation in fibre size and alterations in the proportion of fibre types;necrosis and fibre regeneration are not typical features, glycogen and lipids are nomal, dislike muscular dystrophy myopathies and metabolic myopathies.CMs have multiple inheritance modes including autosomal dominant inheritance, autosomal recessive inheritance, X-linkage inheritance and sporadic cases. To date, more than thirty genes are associated with CMs. NEB（nebulin）, TPM2（β-tropomyosin）, TPM3（low a-tropomyosin）, ACTA1（a-skeletal actin）, TNNT1（slow troponin T）, CFL2（muscle-specific cofilin）,KBTBD13（muscle-specific ubiquitin ligase）, KLHL40（kelch-like family member 40） and KLHL41 have been identified for nemaline myopathy; RYR1（ryanodine receptor）, MYH7（slow skeletal β-cardiac myosin）, SEPN1（selenoprotein 1） are associated with core myopathies; MTM1（myotubularin）,BIN1（amphiphysin）, DNM2（dynamin-2）, TTN（titin） et al. are associated with centronuclear myopathies; TPM2, TPM3, RYR1, ACTA1, MYH7 are associatedwith congenital fibre type disproportion. Moreover, there is significant genetic heterogeneity within the main groups of congenital myopathies. Furthermore,mutations in a single gene may cause diverse clinical phenotypes and several forms of CMs. For example, mutations in the RYR1 may cause central core disease, multiminicore disease, centronuclear myopathy, congenital fiber type disproportion; mutations in the ACTA1 may cause nemaline myopathy（including cap myopathy）, actin filament aggregate myopathy, and congenital fiber type disproportion.The genetic diagnosis of congenital myopathies is challenging as following: 1) Clinical and pathological overlaps in this group of conditions highlight the complexities of classification and diagnosis; 2) Different genetic entities can share similar pathological findings; 3) The causative genes overlaps with myofibfillar myopathy; 4) Some gene candidates belong to the largest genes of the human genome. It is now recognised that the same pathological feature can be associated with a defect in more than one gene;while mutations in the same gene can give rise to varying pathological features and clinical phenotypes. Understanding these complexities is paramount to accurate diagnosis by gene analysis.Targeted gene capture with next generation sequencing（NGS）technology analyzes multiple genes simultaneously to identify mutations causing disease. Targeted sequencing not only provides high coverage（up to99%） and coverage（read depth>100） accuracy of DNA sequencing but also lowers the cost per nucleotide. Furthermore, unlike whole-genome and whole-exome sequencing in the clinical setting is not currently practical,owing to the time, cost, and complexity of analysis associated with generating and interpreting the large amount of sequence information obtained from sequencing the entire genome. Targeted sequcencing has improved diagnosis of CMs comparing to Sanger sequencing.We retrospectively reviewed clinical and pathological features of 1,471 muscle biopsies obtained at the Third Hospital of Hebei Medical University in China between March 2005 and December 2015 from patients with suspectedmyopathy.1) patients with central core disease（9 cases）; 2) patients with centronuclear myopathies（7 cases）. We performed targeted NGS in patients with CMs. All variants were verified by Sanger sequencing in patients and unaffected family members（22 cases）. We did clinical, pathological and molecular analysis among these patients in order to obtain the clinical,pathological and molecular character of Chinese patients with CMs.Part 1 Clinical, pathological and gene analysis of central core diseaseObjective: We screened 9 Chinese patients with clinicopathological diagnosis of central core disease（CCD）. Genetic analysis was carried out by targeted NGS to identify causative genes. We aimed to characterize the clinical, skeletal muscle pathology and genetic features of Chinese patients with CCD and discuss the value of targeted NGS in CCD diagnosis.Methods:1 Inclusion criteria of selected patients: 1) clinical phenotype consistent with congenital myopathy, including hypotonia during infancy, delayed motor development, axial/limb muscle weakness, and a static or slowly progressive clinical course; 2) serum creatine kinase normal or mild increased;electrophysiology study showed myogenic changes; and 3) biopsies demonstrating absent oxidative enzymatic activity in central core regions, and without dystrophic pattern. Muscle fiber glycogen and lipid contents appear normal.2 Collect and analyze the clinical data of selected patients, including gender, onset age, involved muscles, CK level, electrophysiological study results and Computerized Tomography scan of the hip joint（cases 1 and 7）.3 Histochemical stains biopsied muscle specimen and pathological analysis. Ultrastructural examination was performed in 4 patients（cases 2-4and 8） by standard techniques.4 Genetic analysis was carried out by targeted NGS to identify causative genes. Variants were assessed for pathogenicity using bioinformatic approaches, and NGS results were confirmed by Sanger sequencing（9 patients and 9 asymptomatic family members）.Results:1 Among 9 CCD patients, there were 3 males and 6 females; 3 patients had positive family history; age ranged from 2 to 32 years old. All patients were characterized by mild hypotonia during early childhood, and moderate non-progressive or slowly progressive proximal muscle weakness. Five patients presented with mild facial weakness（long face, high arched, a tented upper lip）. Six patients had skeletal complications（distal joint hypermobility,Achilles tendon contractures, scoliosis, bilateral congenital hip dislocation）.CK activity levels were mildly elevated in 1 patient while normal in 8 patients.Myopathic changes were found on electromyography（EMG） in 6 patients,whereas EMG was normal in 3.Computed tomography scan of the hip joint in 2 patients showed atrophy of the gluteal and muscles.2 Biopsied muscle pathology: All patients showed fiber size variability,and without dystrophy change. Areas devoid of oxidative activity（peripheral,central, or eccentric cores） were observed in staining for oxidative enzymes in all patients. All patients showed type I fiber atrophy, and 5 patients had exclusively type I muscle fibers, and 4 showed predominantly type I fibers.Electron microscopy demonstrated a variable degree of disintegration of the contractile apparatus within core regions, with variable degrees of sarcomere disorganization and absence of mitochondria.3 Targeted enrichmengt and NGS, Sanger verified: Sequencing showed that 8 patients had RYR1 heterozygous mutations. Of these, 2 was novel, and 3had been described previously. Three patients had novel mutations（c.13732T>G and c.13915₁3923del）, 3 had c.14582G>A, 1 had c.14581C>T,1 had c.13919T>G. Of those, 2 were de novo（c.13919T>G and c.13915₁3923del）.In patient 6, no pathogenic mutations were found in RYR1, MYH7, or SEPN1.Conclusion:1 These results support that CCD is consistent with clinicalmanifestations of congenital myopathy.2 Histopathological findings the presence of central cores could make differentiation from other CMs and inherited muscular diseases.3 RYR1 is considered the most common cause of Chinese CCD patients.In addition, mutations in RYR1 were generally enriched in exons 94-101 hotspot region.4 Our findings also indicate that NGS is an efficient and cost-effective strategy to improve CCD diagnosis.Part 2 Clinical, pathological and molecular biological study of centronuclear myopathiesObjective: Three main forms of centronuclear myopathys（CNMs） were identified according to its mode of inheritance and severity of clinical phenotypes: the X-linked recessive inheritance（MTM1）, the autosomal dominant form（e.g., DNM2, CCDC78） and the autosomal recessive form（e.g.,BIN1, RYR1）. Female patients carrying heterozygous MTM1 mutations could also develop XLMTM, which might be due to skewed X-chromosome inactivation（SXCI）.In this section, we used targeted NGS technology to identify the causative mutations in seven patients and their families with clinicopathological diagnoses of CNMs. We also investigated the X-chromosome inactivation（XCI） patterns in two female patients carrying MTM1 mutations. Together with data from physical examination, histology, and MRI, we characterized clinical features of all patients, trying to provide a more comprehensive view of the characterizations of different forms of CNMs, and discuss the value of targeted next NGS in diagnosis of CNMs.Methods:1 Inclusion criteria of selected patients: 1) clinical phenotype consistent with congenital myopathies, including hypotonia during infancy, delayed motor development, axial/limb muscle weakness, orthopaedic complications（contractures, scoliosis） and facial weakness（long face, ophthalmoplegia）, and a static or slowly progressive clinical course; 2) serum creatine kinase normalor mild increased; electrophysiology study showed myogenic changes; and 3)histological confirmation of increased number of fibers with nuclei internalization and centralization, and without dystrophic pattern. Muscle fiber glycogen and lipid contents appear normal.2 Collect and analyze the clinical data of selected patients, including gender, onset age, involved muscles, CK level, and electromyography study results. Five patients（2-4, 6 and 7） had magnetic resonance imaging（MRI）scans of their lower limbs.3 Histochemical stains biopsied muscle specimen and pathological analysis. Ultrastructural examination was performed in 2 patients（cases 1 and7） by standard techniques.4 Genetic analysis was carried out by targeted NGS to identify causative genes. Variants were assessed for pathogenicity using bioinformatic approaches, and NGS results were confirmed by Sanger sequencing（7 patients and 12 asymptomatic family members）.5 In two female patients carrying MTM1 mutations, the X chromosome inactivation（XCI） pattern was determined by analyzing DNA methylation at androgen receptor（AR） gene. The site is methylated on the inactive X-chromosome and therefore resists cleavage by methylation-sensitive enzyme Hpa II, and a PCR product is obtained from the inactive X-chromosome only.Results:1 Among 7 CNMs patients, there were 3 males and 4 females; Patient 1had a positive family history, whose maternal uncle had generalized severe weakness and died in childhood; the remaining six cases didn’t have a family history of neuromuscular disease. All patients developed their first symptoms during early childhood, characterized by hypotonia of variable severities,motor developmental delay, difficulties in performing anti-gravity movements such as climbing stairs, and moderate, non-progressive or slowly progressive axial and limb muscle weakness. Patients 4, 5 and 6 presented weakness initially more pronounced in distal muscle groups, while the remainingpatients exhibited proximal dominant limb weakness. Five patients presented with mild facial weakness（long face, high arched）; three patients had ptosis;four patients had skeletal complications（scoliosis, Achilles tendon contractures, long finger flexor contracture and articular distal joint hypermobility）. CK activity levels were mildly elevated in 1 patient while normal in 6 patients. Myopathic changes were found on EMG in 7 patients.Muscle MRI of lower limbs: proximal pattern of involvement presented in both MTM1-CNM and RYR1-CNM patients and a distal pattern of involvement presented in DNM2-CNM patients. In RYR1-CNM, the posterior group was more severe than anterior group in thigh. Muscles of the lower legs that were predominantly affected included the medial gastrocnemius and soleus muscles. In DNM2-CNM, the posterior was more severe than the anterior of the lower legs. Of the thigh muscles, the vastus intermedius was the most involved.2 Muscle pathology: Muscle fibers with centrally located nuclei were markedly increased to various degrees in all seven patients. Variation in fiber size could be seen in all patients. Connective tissue, together with excess fat,was abundant in patient 2. Two patients had uniform type I fibers, and two showed predominant type I fibers. Type I fiber hypotrophy was presented in all patients. In addition, the presence of necklace fibers was observed in MTM1-CNM, We observed radial sarcoplasmic strands（RSS） on NADH-TR staining in DNM2-CNM（patients 4, 5 and 6） and RYR1-CNM（patient 7）.Electron microscopy revealed the occurrence of nuclear bags and nuclei arranged in rows, in addition, the central internuclear spaces are occupied by mitochondria, rough endoplasmic reticulum, Golgi complex, and glycogen particles.3 Targeted enrichmengt and NGS, Sanger verified: Three patients had MTM1 mutations（patient 1 was c.1054-1G>A, patient 2 was c.286 dup A and patient 3 was c.1405C>G）, therefore belonged to XLMTM. Three patients（patient 4 was c.1106G>A, patient 5 was c.1102G>A, and patient 6 was c.1105C>T） carried mutations in DNM2 and therefore belonged to ADCNM.Patient 7 carried two heterozygous mutations in RYR1（c.12237 del C,c.12536G>A）, belonged to ARCNM. Of those, the mutations in MTM1（c.1405C>G） and RYR1（c.12237 del C） were novel.The mutations in patients 4and 5 were de novo.4 XCI analysis: Patients 1 & 2 had a mean ratio of 84:16 and 83:17respectively. The results showed that the two patients had skewed pattern in muscle DNA.Conclusion:1 These results support that CNMs is consistent with clinical manifestations of CMs. DNM2-CNM present weakness initially more pronounced in distal muscle groups.2 MRI provided great value in interpreting the genetic results. Meanwhile,it could also guide the precise locations for muscle biopsy to improve the diagnosis, especially for milder CNM patients.3 Histopathological findings the markedly increased muscle fibers with centrally located nuclei could orient genetic analysis. Further more, the presence of necklace fibers present XLMTM, and RSS present DNM2-CNM.4 Chinese CNMs patients inherit with different modes. Our findings also indicate that NGS is an efficient and cost-effective strategy to improve CNMs diagnosis.5 The heterogeneous MTM1 mutations in female patients who were symptomatic highly suggested the containing of a skewed XCI pattern. And our findings still highlighted that females showing features of XLMTM should be analyzed for their X-inactivation patterns.6 The genetic diagnosis of different forms CNMs could decrease or prevent birth defects and provide genetic counseling and eugenic advice.