Clinical And Molecular Biology Of Patients With Myotonic Myopathies

Myotonic myopathies are an important group of neuromuscular disorders characterized by dysfunction or structure of ion channels(CL-/Na+/ Ca2+/ K+) that primary/secondary regulate membrane excitability. Clinical manifestations vary and include myotonia, dystrophic muscle degenerate, muscle weakness and the EMG shows typical myotonic discharges, partly patients accompanied by myopathic discharges. The syndrome can be categorized into myotonic dystrophies(DMs) and non-dystrophic myotonias(NDMs).Myotonic dystrophies are autosomal dominant disorders characterized by myotonia, the combined prevalence is estimated at 2.45~5.5/100,000. Based on clinical findings, two genetically distinct entities have been identified: myotonic dystrophy type1(DM1; OMIM#160900) and myotonic dystrophy type 2(DM2; OMIM# 602668). DM1 is caused by expansion of a CTG-repeat within the 3’ untranslated region of DMPK in chromosome 19q13.3, the pathogenic mechanism of DM1 is mediated through abnormal nucleotide repeat amplification inducing “trans-dominant toxic gain of function” to reduce the conductance of the chloride channels. Meanwhile, the disease is characterized by missplicing of several downstream effector genes, which are thought to account, at least in part, for multiorgan involvement. DM2 is caused by expansion of a CCTG-repeat within the first intron of ZNF9 in chromosome 3q21.3. It should be noted that DM1 and DM2 share the same pathogenic mechanism.NDMs are a group of autosomal dominant/recessive disorders caused by mutations in the ion channels(CLCN-1/SCN4 A gene) that delay muscle relaxation after a voluntary or evoked muscle contraction. The prevalence is estimated at 1 in 100,000. The most characteristic symptoms are muscle stiffness generated by voluntary movement, muscle hypertrophy, muscle weakness and fatigue, heart and respiratory function also can be involvement. Clinically, NDMs are classified as dominant and recessive myotonia congenital(MC), paramyotonia congenita(PC(OMIM 168300)), potassium-aggravated myotonias(PAMs(OMIM608390)), Hyperkalemic periodic paralysis(Hyper PP) and Hypro kalemic periodic paralysis with myotonia(Hypro PP).According to the various modes of inheritance, MC can be divided into autosomal-dominant Thomsen’s myotonia(DMC) and Becker’s recessive generalized myotonia(RMC). The prevalence is estimated at 0.3~0.6 in 100,000. Genetic studies have demonstrated that skeletal muscle chloride gene mutations(CLCN-1) mapped to chromosome 7q35 is associated with MC. These were “loss of function mutations” that inhibited the depolarization of chloride currents into activated chloride currents to prolong the muscle relaxation process. Patients with MC exhibit classic myotonia, a “warm-up” phenomenon and muscular hypertrophy on examination.PC is an autosomal-dominant disorder caused by a mutation in the SCN4 A gene, which encodes the α-subunit of the skeletal muscle sodium channel. The prevalence is estimated at 1 in 350, 000-380,000 and rare caused in Aisa. Mutations in SCN4 A lead to “gain of channel function” defects, impairing channel inactivation or enhancing channel activation. Furthermore, patients with PC demonstrate cold sensitivity and muscle function worsens with continuing exercise.Hyper PP, PAM, and PC are allelic variants of sodium channelopathy. They are inherited as autosomal dominant traits, and are associated with mutations in the SCN4 A gene located on chromosome 17q23. Mutations in SCN4 A cause gain of channel function(increased sodium current) in a variety of ways that include slowing fast inactivation, impairing slow inactivation, hastening recovery from inactivation, slowing deactivation(closure of the channel upon re‐polarization) and shifting the voltage dependence of activation to more negative potentials. However, as in Cl- channel myotonia, phenotypic variation has been observed in association with the same SCN4 A genotype, for myotonia, cold-sensitivity and no episodic weakness. Both categories of NDMs are clinically highly heterogeneous with a wide variety of phenotypes, and it is difficult to differentiate CLCN-1 from SCN4 A mutations solely on a clinical basis.Familial Hyper PP, Anderson-Hyper PP, Hypro PP, Malignant Hyper thermia and central core disease are allelic variants of calcium channelopathy. They are inherited as autosomal dominant traits, and are associated with mutations in the CACNA1 S gene located on chromosome 1q31-32. CACNA1 S gene encodes the subunit of CaV1.1 of the skeletal muscle calcium channel, which distribute in cardiac, skeletal muscle, peripheral nervous system and central nervous system. Muscle fibers tend to be depolarized relative to the normal range, but the mechanisms underlying this process are poorly understood.EMGs were used to establish the diagnosis of myotonic disorders. Although EMGs show typical myotonic discharges, different compound muscle action potential amplitude after exercise changes thus different gene mutations. For example, Patients with MC can show an immediate CMAP decrement after exercise which recovers quickly and diminishes with repetition; Patients with PC typically have a gradual and prolonged decrement in CMAP after exercise; patients with DMs show small-amplitude, short-duration motor units in addition to the electrical myotonia.Skeletal muscle biopsy confirms: patients with DMs show myogenic changes and revealed typical alterations, such as central nuclei, pyknotic clumps, core-like, sarcoplasmic masses, and small angular fibers that appeared inconsistently in both subtypes. Different molecular genetics affects muscle fibers that preferential type 1 fiber atrophy and a higher prevalence of central nucleation among type 1 fibers were observed in all DM1, and high rates of atrophic and hypertrophic type 2 fibers were observed in the DM2 cases. Patients with NDMs show typical myogenic changes with only minor. But the skeletal muscle pathological changes are not a specific group of pathological changes of the disease. So it is difficult to make the diagnosis and differential diagnosis of the diseases of skeletal muscle biopsy pathology.The second generation sequence technology to bring effective, accurate technology for diagnosis. The genetic and phenotypic heterogeneities in NDMs create complications in distinguishing patients with sodium channel myotonia from those with MC and underscore the need for these patients to undergo genetic screening. DMs are repeat nucleotide diseases, PCR as a simple, specific and sensitive method has been suggested as an ideal method to identify the number of CTG/CCTG-repeat for DMs diagnosis, but fails to detect larger expansions(>100) due to the limitation that great length and secondary structures which formed by GC-rich domains. Above all, the concise, efficient DMs diagnosis is still difficult.In order to characterize the clinical, skeletal muscle, pathology and genetic features of Chinese patients with myotonic myopathies, we analyzed patients with DMs(25 cases, included 6 pedigrees) and patients with NDMs(17 cases, included 5 pedigrees) who had been admitted to the Third Hospital of the Hebei Medical University from 2004 to 2014. Meanwhile, carbama zepine and mexiletine are administered for myotonia myopathies to relieve the clinical symptoms, provide molecular basis for prenatal diagnosis. Part 1 Optimization PCR for diagnosising of Myotonic dystrophiesObjective: Myotonic dystrophies(DMs) are a group of autosomal dominant neuromuscular disorders which caused by large CTG/CCTG-repeat expansions in untranslated regions of DMPK/ZNF9 gene. The mutation of DM1 is an expansion of the unstable CTG trinucleotide repeat in the 3’untranslated region(UTR) of the DMPK gene,(CTG)n expansions range from 51 repeats to several thousands, whereas healthy individuals carry 5-37 repeats. Repeat lengths of 38-50 are considered premutation alleles, whereas 51-100 repeats are protomutations, 100-40000 repeats are mutation alleles, both of which shows increased instability towards expansion. Carriers of premutations or protomutations(n<100) present no or few mild symptoms; Patients with adult-onset myotonic dystrophy type 1 carry more than 100 repeats. This disor der shows a striking anticipation and parental-gender affection upon trans- mission in DM1 pedigrees, which can be used for genetic counseling and estimated patient conditions in time. To distinguish from DM1, DM2 is rare caused in Asia but in European, the CCTG-repeat tract is part of(TG)n(TCTG)n(CCTG) n, normal individuals show 10-27 repeats and patients with larger CCTG-repeat range from 75 to more than 11 000 repeats, the average of(CCTG)n-repeat is 5000. But the genotype-phenotype correlation shows low quotient in DM2.The “clinical phenotype overlap” and a large number of individuals with milder symptoms remain been misdiagnosed in clinical, which made genetic test is necessary. It is well known that GC-rich regions within a template generate a complex secondary structure thereby prohibiting the denaturation, annealing and extension processes during PCR amplification. In conclusion, optimization PCR is a suitable alternative technique for DMs diagnostic. Thus, specific amplification of large numbers CTG/CCTG-repeat can be completed successfully by following our strategy, which included primer modifications, application of shorter the annealing time, enhancer and longer denaturation with extensive conditions in PCR cycles.Methods:1 The whole study has been approval by the Institution Review Board. We analyzed 42 individuals from the northern of China; included 11 pedigrees who had been consecutive attended to department of neuromuscular disorders of our hospital from 2004 to 2014. Clinical data and a detailed family history were collected. A clinical diagnosis of Myotonic myopathies based on the guideline of the International Myotonic Dystrophy Consotium.2 Molecular analyze①After obtaining informed consent from the patients and their family members, genomic DNA from 42 patients and their family members was extracted from the peripheral blood, which was obtained using standard methods. Control genomic DNA was obtained from unrelated healthy Chinese subjects. We designed primer sets to capture the whole sequences of DMPK. The primer of ZNF9 was described by John Day from Espacenet.② To optimize the denaturation, annealing and extension processes during PCR amplification.③The Optimization PCR reaction were performed, All PCR products were separated on 2.5% Ready Agarose gel. Detect homozygous/ heterozygous samples by DHPLC then use optimization PCR to amplify the objective fragment.④The PCR products were purified and sequenced using dye terminator chemistry with an ABI Prism 377 DNA Sequencer(Applied Biosystems, Foster City, CA). The Sequencher 4.90 software(Gene Codes, Ann Arbor, MI) was used to analyze the sequences.Results:1 Primers①DMPK(450bp):Forward primer: 5’-CGA CTC CGG GGC CCC GTT GGA AGA CT-3’;Reverse primer: 5’-CTC CCC AGA GCA GGG CGT CAT GCA CAA G-3’;②ZNF9(295bp):Forward primer: 5’-GGC CTT ATA ACC ATG CAA AT-3’;Reverse primer: 5’-GCC TAG GGG ACA AA GTGAGA-3’;2 The optimization method for the PCR amplification① Optimization method for DM1The following conditions were used for amplification: 5 minutes at 95°C; 30 cycles of amplification(95°C for 45 s, 66°C for 8 s, and 78°C for 3min) and 10 minutes at 72°C for the last extension step.② Optimization method for DM2The following conditions were used for amplification: 5 minutes at 95°C; 30 cycles of amplification(95°C for 45 s, 65°C for 8 s, and 75°C for 3min) and 10 minutes at 72°C for the last extension step.③The Optimization PCR reaction was performed in 25 ul volume, reaction mixture included: 1x PCR buffer, 200mmol/L d NTP, 1.6 units of Dy NAzyme EXT DNA polymerase(Finnzymes, Espoo, Finland), 10 pmol of the DMKF and DMKR primers, 50 ng of template DNA.④We identified homozygous/ heterozygous alleles in all patients by DHPLC for DMPK locus. The results showed 22 homozygous patients and 20 heterozygous patients for the DMPK locus.3 GenotypingTo use the optimization method for the PCR amplification. All expanded alleles were separated by 2.5% agarose gel electrophoresis, normal individuals were detected as bands of 450 bp, and the average of CTG-repeat was about 20. 22 homozygous patients for CTG-repeat number was detected about from 53-683 and the average number was 535. Meanwhile, 17 family members of them were confirmed the CTG-repeat expansions by the optimization PCR amplification, 11 of the 17 individuals had permutated or pathogenic CTG-repeat allele. The 11 individuals from 5 families, both son and their mother were clinically suspected for DM1 and confirmed by optimization PCR. Subsequently, 3 heterozygous patients and 4 normal Chinese control subjects were confirmed CCTG-repeat by the optimization PCR amplification in ZNF9 gene. Products were separated by 2.5% agarose gel electrophoresis showed the number was detected about from 400-450 and the average number was 416. 17 patients the expanded alleles were not visible by optimum PCR analysis.Conclusion:1 Designed primer with high Tm to adjust △G free energy levels, thereby reducing primer self-dimer and hairpin formation.2 Optimization PCR under the optimal conditions of primer design, modified annealing and extension conditions can be used for efficient PCR in DMs diagnosis.3 Optimization PCR can greatly improve the positive detection of DMs, the method not only used for DMs but also for other disorders caused by nucleotide repeat expansions.Part 2 Climical and skeletal muscle biopsy characteristics of Myotonic dystrophiesObjective: Myotonic dystrophies(DMs) are a group of autosomaldominant disorders that are characterized by myotonia, dystrophic muscledegeneration, and other variably associated multisystemic manifestations, such as lens opacities, cardiac, gastrointestinal, endocrine and central nervous system abnormalities. These disorders most frequently manifest as adolescence. The global prevalence is estimated at 2.45-5.5/100,000. The relative frequency of affected DMs families from different racial backgrounds(white, black, Asian) was in accordance with the frequency of each of these racial groups in our population. EMGs show small-amplitude, short-duration motor units in addition to the electrical myotonia. Skeletal muscle biopsy shows myogenic changes and revealed typical alterations, such as central nuclei, pyknotic clumps, core-like, sarcoplasmic masses, and small angular fibers that appeared inconsistently in both subtypes. There are two genetically distinct types of myotonic dystrophy: DM1 and DM2. DM1 causes distal muscle weakness and atrophy, as opposed to prominent proximal muscleweak ness, often as a presenting symptom in DM2.Thus, the purpose of our study was to characterize the clinical, skeletal muscle pathology and genetic features of 25 patients with DMs in China.Methods:1 Patient selectionTo obtain an overview of the clinical, molecular and muscle biopsyinformation from 25 patients who were examined from part 1.2 Skeletal muscle histopathologyAfter obtaining informed consent from the patients and their familymembers, muscle biopsy specimens were collected from the biceps brachii.For histopathological analysis, serial frozen sections(7μm) were stained withhematoxylin and eosin(H&E), modified Gomori trichrome(MGT), oil red O(ORO) and periodic acid-Schiff(PAS) and were treated using histochemicalreactions with NADH-TR, succinate dehydrogenase(SDH), adenosine monophosphate(AMP), deaminase cytochrome C oxidase(COX), acid phosphatase(Acid) and myosin ATPase(p H4.2, 4.6, 9.98). Part 2 Clinical and skeletal muscle biopsy characteristics of MyotonicdystrophiesResults:1 Of the 25 DMs patients, 22 patients exhibited DM1, including 9(41%) males and 13(59%) females; the age of disease onset ranged from 9-43 years, and the average age of onset was 30 years. Eighty percent of affected male children had affected female parents. Muscle strength and reflexes were assessed as follows. Weakness in the distal dominant limb was observed, and the muscle strength was rated from 1 to 4. Variable degrees of atrophy were observed. A total of 60% of patients showed sternocleidomastoid involvements, “hatchet face” and baldness were observed in 27% of patients. Cardiac disease remains a common complication of DMs related to this issue, 4 patients showed atrioventricular blockages, 4 patients exhibited dilated patients showed arrhythmias. In addition, several patients presented with dysarthria, cataracts(7), cognitive decline(6) and endocrine abnormalities(1). Three patients presented DM2, including 1male and 2famles; the age of disease onset ranged from 22–39 years, and the average age of was 33 years. Muscle strength and reflexes were assessed as follows. Weakness in the proximal dominant limb was observed, and muscle strength was rated from 3+ to 5; untypical atrophy was also observed in some patients. One patient experienced muscle pain, and 2 patients showed sternocleidomastoid involvement. Three patients exhibited cardiac involvement.2 The affected muscles showed typical central nuclei and pyknotic clumps in all DM patients, particularly those with more severe cases of DM1. Structural changes in our study mainly consisted of sarcoplasmic masses, small angular fibers and “core-like” fibers. Changes in our study mainly consisted of sarcoplasmic masses, small angular fibers and “core-like” fibers frequent in DM1 patients with early onset disease. Ring fibers and rimmed vacuolar fibers were not encountered in our study. Increase connective tissue and fibers with inflammatory responses were most common in patients with mild to moderate disease severity, but two severely also presented these characteristics. An examination of myosin ATPase, characterized by fiber grouping and a predominance of type 2 fibers, demonstrated the atrophy of type 1 fibers in patients with DM1.It was interesting that type 2 fiber atrophy was found in DM2 patients, but type 1fiber atrophy was absent in DM1 patients. The results from the oxidative enzyme reactions indicated a focal decrease in certain fibers, with changes predominantly occurring in type 1 fiber.Conclusion:1 Our study showed the high levels of DM1 in Chinese individuals with DMs, and patients with DMs show typical myotonia, muscle weakness and multisystemic involvement. There is no significant difference between previous reports.2 Skeletal muscle biopsy is an essential tool for making a definite and differential diagnosis of DMs, for preferential type 1 fiber atrophy and a higher prevalence of central nucleation among type 1 fibers were observed in all DM1, and high rates of atrophic and hypertrophic type 2 fibers were observed in the DM2 cases.3 There is straightly relationship between(CTG)n repeats with clinical phenotype and skeletal muscle biopsy; but onset age and disease duration to DM2. Part 3 Molecular, clinical and skeletal muscle biopsy characteristics of patients with non-dystrophic myotoniasObjective:Myotonia congenital(MC) and paramyotonia congenita(PC) are important subtypes with non-dystrophic myotonias(NDMs). Oppose to DMs, NDMs are a group of neuromuscular disorders caused by mutations in the genes encoding the skeletal muscle chloride(CLCN-1) and sodium(SCN4A) channels. Skeletal muscle chloride gene mutations(CLCN-1) mapped to chromosome 7q35 are associated with MC. Patients with MC have a muscular appearance, action myotonia, percussion myotonia and “warm up” phenomenon. According to the various modes of inheritance, MCs can be divided into autosomal-dominant Thomsen’s myotonia(DMC) and Becker’s recessive generalized myotonia(RMC). Skeletal muscle sodium gene mutations(SCN4A) mapped to chromosome 17q23-25 are associated with PC. Cold-induced, prolonged, painful myotonia, and episodic weakness are the hallmarks of PC. In contrast to myotonia congenita, facial stiffness and eye closure myotonia are more common in PC; paradoxical eye closure myotonia is exclusively seen in PC. Muscle weakness in PC can last from several hours to 2 days whereas it may last only seconds to minutes in MC.Due to the incomplete dominance of certain mutations with variable penetrance and expression as additional compounding factors, patients with NDMs examined in one study lacked an identifiable gene mutation. The identification of disease-specific genetic and histopathological patterns for NDMs could help clinicians successfully incorporate the findings of genetic studies.Thus, the purpose of our study was to characterize the clinical, skeletal muscle pathology and genetic features of 17 patients with NDMs in China.Methods:1 Patient selectionWe analyzed 17 Chinese patients who had been consecutive attended to department of neuromuscular disorders in our Hospital from 2004 to 2014 and 21 of their family members(from 5 pedigrees). All patients were gave their written informed consent prior to their participation. Clinical data and a detailed family history were collected. Included: Gender, onset age, family history, percussion myotonia(±), grip myotonia(±), “warm up”(±), muscle strength, muscular, contracture of tendon(±), other system involvement, creatine kinase(CK), EMG and treatment.2 Skeletal muscle histopathologySame to the first part.3 Mutation screeningWe designed primers set to capture the entire coding regions and flanking sequences of the following 4 genes: CLCN-1, SCN4 A, KCNE3 and CACNA1 S. We amplified the coding exons from 50 ng of genomic DNA with these primers using a hot-start PCR method for the amplification of polymorphic markers.Results:1 Nine different mutations in the CLCN-1 gene were identified in 8 patients, including 4 missense, 3 nonsense, 2 insertion and 1 deletion mutations. Six patients were heterozygous and two patients were homozygous. SCN4 A was subsequently sequenced in the remaining 4 patients, and 4 independent mutations were identified in the SCN4 A gene; 3 were novel missense mutations and 1 was a previously reported mutation.2 The age of disease onset was 5-10 years with an average onset of 8years. All patients suffered from typical myotonia and typical warm-up phenomenon, patients presented with myotonic discharges upon EMG examination. Secondary scoliosis accompanied and contracture of the Achilles tendon can be observed in partly patients. All patients were administered carbamazepine and mexiletine, clinical symptoms improved significantly3 The muscle biopsies stained with hematoxylin and eosin(H&E) appeared normal or showed only minor variations in the patients with NDMs. The patients presented occasional degeneration, necrosis and occasional small angular fibers. Examination of the myosin ATPase showed fiber grouping and a predominance of type 2 fibers. Moreover, an absence of type 2B fibers and a predominance of type 2A fibers observed in patients with CLCN-1 mutations; the type 1 fibers in these patients were either normal or showed mild atrophy. The differences between NDMs and DMs are that NDMs lack the typical nuclear pyknotic clumps, sarcoplasmic masses, muscle fiber degeneration and severe necrosis of connective tissue. These differences are useful in diagnosing mild cases of myotonic dystrophy, when weakness has not fully developed into pathology.Conclusion:1 Analysis of the CLCN-1 and SCN4 A genes can identify a large number of mutations in patients with NDMs. Most of the abundant gene mutations detected in CLCN-1 and SCN4 A in our study were novel. In addition, we found a significant racial difference for the mutations in CLCN-1 and SCN4 A genes.2 New gene mutations are likely to be discovered in patients with NDMs, DNA chip technology will become invaluable for diagnosing NDMs.