Pathological Gene Screening And The Applicational Study Of Chinese Patients With Neuromuscular Diseases

Hereditary neuromuscular diseases includ muscular, neuromuscularjunction, peripheral nerve and cornu anterius medullae spinalis diseasescaused by abnormality in genetic material. Hereditary muscular diseases andhereditary peripheral nerve diseases are the most popular hereditaryneuromuscular diseases.Hereditary muscular diseases have multiple inheritance modes includingautosomal dominant inheritance, autosomal recessive inheritance, X-linkageinheritance and sporadic cases. Mutations in gene cause coding proteins(skeleton protein, membrane protein, basilar membrane protein and ionicchannel protein) absence or dysfunction, which induce membrane broke,plasmosin abnormal, metabolic disorder and membrane channels dysfunction.The main clinical features of hereditary muscular diseases are weakness,muscle atrophy and muscle myotonic accompanied with less involvement ofbrain, heart, liver, peripheral nerve, bone and joint systems. Hereditarymuscular diseases mainly consist of muscular dystrophy, myotonic syndrome,distal myopathy, congenital myopathy, mitochondrial diseases, glycogenstorage disease and deposition of lipid disorder. Clinical manifestationsanalysis and pathological analysis of biopsies of skeletal muscle are the basicof diagnosis of these diseases; application of genetics, molecular biologytechniques make it reality to molecular diagnosis hereditary muscular disease.Most of hereditary muscular diseases have muscle weakness and atrophyat childhood or young adults' period, then the symptoms progress aggravated,result in trapped in the wheelchair. Because of lacking effective treatment, thediseases bring heavy burden to patients, their family and society. It is essentialto molecular diagnosis, research the pathogenesis and explore the effectivetherapy methods. More and more new diseases were identified accompanied with new disease-causing gene and protein being found. Currently, basicmethods for diagnosing hereditary diseases include: involved musclesjudgement, electrophysiological study, pathological analysis of biopsiedmuscle, biological and chemical detection; molecular pathological,ultrastructural pathological and molecular study are now used in clinicaldiagnosis. Muscle MRI is now widely used in clinic; it is a reliable method tojudge involved muscles and the severity. Involved muscles type is animportant way to decide phenotype, which can provide evidence in objectivegene. Hereditary motor and sensory neuropathy (HMSN) is the most popularhereditary perpheral nerve disease. It was first reported in1886by Charcot,Marie and Tooth respectively; after that the disease was named asCharcot-Marie-Tooth (CMT). Until now more than20genes related to CMTwere found, most of them inherited with autosomal dominant mode.Mutations in disease-causing genes lead to synthesis decrease or dysfunctionof proteins in myelin, axon and Schwann cells, which disturb integrity ofmyelin and cytoskeleton, induce disorders in axoplasmic transport and energymetabolism. CMT is charactered by distal weakness, atrophy with/withoutsensory abnormality. Currently the diagnosis of CMT was based on clinicalfeature, electrophysiological finding, pathological analysis of peripheral nerve;genetic analysis is the reliable typing diagnosis method.CMT is divided into three subtypes: CMT1, CMT2and intermediateCMT according to nerve conduction study. Electrophysiology study is animportant method for CMT diagnosis. Because the high degree of clinical andgenetic heterogeneity and there are more than20CMT-causing genes, it isdifficult to do gene sequence directly. The application of gene chip technologymakes it easier to screen the pathogenic gene and direct objective genesequence; makes more and more CMT patients receive molecular diagnosis.So far there are more than4000hereditary diseases, more than60percentof which are hereditary neurological diseases. The basic study of hereditaryneurological disease is relatively slow in our country. In China there areplenty of patients with hereditary neurological disease, but systematic research on hereditary neuromuscular diseases is relativly insufficient, so it isessentially important to perform research on neuromuscular diseases, todiscover Chinese genetic variant informations as soon as possible.Muscle biopsy and peripheral nerve biopsy have fewer side effects, it isclinical easily obtained, which makes pathological analysis of biopsiedspecimen possible, the pathological study promote the research process forhereditary neuromuscular diseases in the past decades. Hereditaryneuromuscular diseases are caused by gene mutations, so genetic diagnosis inthe most accurate diagnosis, genetic diagnosis platform is important forhereditary neuromuscular diseases.In this study, we selected37dysferlinopathy patients,19distalmyopathy/muscular dystrophy patients and one distal hereditary motorneuropathy family from the specimen bank of muscle, nerve and DNAestablished from2004to2011. We did clinical, pathological and molecularanalysis among these patients in order to obtain the clinical, pathological andmolecular character of Chinese patients.Part1Clinical, pathological diagnosis and pathological gene screening ofdysferlinopathy patientsObjective: Progressive muscular dystrophy (PMD) is a group ofhereditary muscular diseases charactered by progressively aggravatedmuscular weakness and atrophy. Dysferlinopathy inherited with autosomalrecessive mode, mutations in DYSF gene cause dysferlin protein absent, whichnormally expressed in muscular and myocardial cell membrane, lead tomembrane stability broken. Clinical character of dysferlinopathy isprogressive muscle weakness and atrophy, but initial involved muscles weredifferent among patients, according to initial involved muscles,dysferlinopathy is subtyped into three subtypes: Limb-girdle musculardystrophy2B (LGMD2B) mainly influences proximal muscles, Miyoshi distalmuscular dystrophy (MM) affects gastrocnemius muscle at the beginning anddistal myopathy with tibialis anterior onset (DMAT) involves tibialis anteriormuscle initially. We have about100PMD patients from2004to2011, among these patients about40percent were LGMD2B conformed by anti-dysferlinmonoclon immunohistochemical stain. In Europe and Japan the percentagewere about20percent lower than LGMD2A. This difference initiates us to dosystematic study of the chatacter of Chinese dysferlinnopathy clinically,pathologically and molecularly, in order to find the special feature of Chinesedysferlinopathy.Methods:1. Inclusion criteria of selected patients:1) Disease onset at juvenile;weakness and atrophy at proximal muscles and pelvic girdle muscles or distalmuscles; serum creatine kinase increased; electrophysiology study showedmyogenic changes;2) Histochemical stains of biopsied muscle specimenshowed muscle fibers degenerating, necrosis and regenerating, connectivetissue element increased at different degrees, which suggested muscularatrophy;3) Anti-dysferlin monoclon antibody immunohistochemical stainsshowed dysferlin protein absolutely absent or decreased severely.2. Collect and analyze the clinical data of selected patients, includinggender, onset age, involved muscles, CK level, electrophysiological studyresults and some muscle MRI in lower limbs.3. Reperform histochemical stains and immunohistochemical stains onbiopsied muscle specimen and pathological analysis.4. Perform DYSF gene sequence within selected patients with dysferlinprotein absolutely absent or decreased severely.5. Summarizing the clinical, pathological and molecular features withinChinese dysferlinopathy patients.Results:1. Among37dysferlinopathy patients, there were20male,17femalepatients, no significant gender difference existed; two patients had positivefamily history; onset age ranged from12to51years old, mean at14.76years,which consistented with previous reported. Clinical phenotypes accordinginvolved muscles:26LGMD2Bs,10MMs,1DMAT. Serum CK severelyincreased from997to25770U/L. 2. Electrophysiological study was done within all selected37patients,35patients showed typical myogenic changes, one showed peripheral nervechanges accompanied with myogenic change, one definitely showedperipheral nerve changes.3. Two cases accompanied with chronic and extensive peripheral nerveinvolvement indentifined by anti-dysferlin monoclonal antibodyimmunohistochemical stains and DYSF gene, suggested a new phenotype.And there are no similar reports.4. Skeletal muscle MRI of11patients:4cases showed proximal muscleinvolved;6cases involved gastrocnemius muscle mainly;1case involvedanterior tibial muscle initialy.5. Biopsied muscle pathology: all37cases showed typical musculardystrophy change, anti-dysferlin monoclonal antibody immunohistochemicalstain showed dysferlin completely absent.6. We did DYSF gene sequence in14patients,4patients found nopathogenic variations, seven different mutations were identified, including onenonsense mutation, two insertion mutations, two deletion mutations, and onesplice site mutation, with six of them were novel mutations.Conclusions:1. Clinical character of dysferlinopathy was progressive muscleweakness and atrophy; pathological features of dysferlinopathy weredegeneration, necrosis, regeneration and connective tissue element increased;immunohistochemical stain showed dysferlin protein completely absent.2. Muscle MRI of lower limbs makes it clear to judge the involvedmuscles, which is essential for clinical phenotype and selecting muscle biopsy.3. Immunohistochemical stain with anti-dysferlin monoclonal antibodyshowed dysferlin protein completely absent is an important method to clinicalscreens for dysferlinopathy and differ it from other PMD.4. DYSF gene sequencing and exploring pathogenic mutations are thebest for molecular diagnosis.5. We first reported dysferlinopathy accompanied with peripheral nerve involved confirmed with molecular diagnosis. More research needed toexplore the mechanism.Part2The study of clinical diagnosis system for19distalmyopathy/muscular dystrophyObjective: Distal myopathy/muscular dystrophy consist of a group ofhereditary muscular diseases and inherit with different modes. Distalmyopathy/muscular dystrophy have disease onset at the second decade oradult, charactered by progressive distal muscle weakness and atrophy,accompanied with heart muscle, facial muscles and throat muscle involvement.Electrophysiological study show myogenic, neurogenic or mixed change,pathological analysis of biopsied muscle show myogenic or musculardystrophy change. So far more than20genes were found in distalmyopathy/muscular dystrophy, and this group of diseases was suggested toclassify by location and function of coding proteins of pathogenic genes, themain classifications including structural components of the sarcomere, Z-diskproteins, membrane signal transduction and repair proteins, sarcoplasmicenzyme proteins nucleoprotein defects diseases.We selected19distal myopathy/muscular dystrophy patients and didclinical, pathological and molecular analysis among these patients in order toexplore the diagnosis protocol and obtain the direction of further objectivegene analysis.Methods:1. Inclusion criteria of selected patients:1) main clinical character wasmuscle weakness and atrophy in distal of the limbs;2) electrophysiologicalstudy showed normal, myogenic or mixed changes, and exclude other diseasesfor example: anterior horn of the spinal cord diseases, peripheral nervediseases and other definitely defined myopathies (facioscapulohumeralmuscular dystrophy, myotonic dystrophy and oculopharyngeal musculardystrophy);3) pathological analysis of biopsied muscle showed myogenicchange.2. Collect and analyze the clinical data of selected patients, including gender, onset age, involved muscles, CK level, electrophysiological studyresults and some muscle MRI in lower limbs.3. Perform histochemical stains and immunohistochemical stains onbiopsied muscle specimen and pathological analysis.4. We summarize and explore the diagnosis and differential diagnosisprotocol.Results:1. Clinical data: there were7male,12female patients within these19distal myopathy/muscular dystrophy. Two patients were from one family(mother and son), one patient had a symptom similar brother, other16patientshad no positive family history; onset age ranged from14to38years old.Serum CK severely increased in11patients and normal or slightly increasedin8cases.2. Electrophysiological study was done,13patients showed typicalmyogenic change,3cases showed mixed change, one normal. Skeletal muscleMRI of19patients:11cases showed gastrocnemius muscle involved;2casesinvolved anterior tibialis muscle;1case involved anterior tibialis muscle andsoleus muscle;5cases involved anterior and posterior muscle.3. Biopsied muscle pathology:11cases showed typical musculardystrophy change, anti-dysferlin monoclonal antibody immunohistochemicalstain showed dysferlin completely absent;4cases had many typical rimmedvacuoles; other3cases showed myopathy without characteristic pathologicalchange.Conclusions:1. Electrophsyological study is a reliable method to diagnose anddifferential diagnose distal myopathy/muscular dystrophy from motor neurondisease and peripheral nerve disease.2. Initial involved muscles and serum CK level offer clues to diagnosis;muscle MRI shows clearly involved muscles and severity, which do help inselecting biopsied muscle.3. Pathological features of distal myopthy/muscular dystrophy were different among various types, mainly including muscular dystrophy change,typical rimmed vacuoles and myopathy with abnormal material deposited inthe cytoplasm, and myogenic fiber network disorder. Bulk of typical rimmedvacuoles suggested DMRV. Immunohistochemical stain showed dysferlinprotein completely absent suggested MM or DMAT.4. Other distal myopathy/muscular dystrophy were difficult tomolecularly diagnose.5. Therefore it is very needed to do large chart of patients' researches, inorder to deepen the recognitions of distal myopathy/muscular dystrophy andestablish the diagnosis protocol.Part3Alanyl-tRNA synthetase mutation in a family with dominant distalhereditary motor neuropathyObjective: CMT is a group of hereditary peripheral nerve diseases,dHMN is known as a pure motor variant of CMT. We selected one AD3generations' dHMN family from28patients clinical diagnosed as CMT. Toidentify the genetic cause of this dHMN family.Methods:1. Clinical data collection of this dHMN family.2. Detailed neurological examination and electrophysiological study weredone; skeletal muscle MRI of the lower limbs was performed in proband.3. After singing the consents genomic DNA of eight members wereextracted; we screened for the mutations of28CMT or related pathogenicgenes using an originally designed microarray resequencing DNA chip.4. To confirm the mutation revealed by our DNA chip, the proband andseven members of the family underwent genetic analysis by direct sequencing.Results:1. The clinical features of the family included mild weakness and wastingof the distal muscles of the lower limb and foot deformity, without clinicalsensory involvement.2. Investigation of the family history revealed an autosomal dominanttransmission pattern. 3. Electrophysiological studies revealed motor neuropathy.4. Magnetic resonance imaging of the lower limbs showed accentuatedfatty infiltration of the gastrocnemius and vastus lateralis muscles.5. All4affected family members had a heterozygous missense mutationc.2677G>A (p.D893N) of alanyl-tRNA synthetase (AARS), which was notfound in other4unaffected members and controls.Conclusion:1. dHMN is known as subtype of CMT2; these two diseases are highlygenetically and clinically heterogeneous and have many causative genes.dHMN is charactered by distal weakness and atrophy without sensoryinvolvement. In some cases, dHMN and CMT phenotypes have been found tohave the same gentic genes. Electrophysiological study is the reliable methodto distinguish dHMN from CMT.2. CMT chip is a quick and convenient method to screen the pathogenicgene, and to confirm the molecular diagnosis, direct sequencing was needed.3. An AARS heterozygous mutation (c.2677G>A D893N) was found in aChinese dHMN family. AARS mutations result in not only a CMT phenotypebut also a dHMN phenotype.