NDMs can be easily misdiagnosed due to the fact that it clinical features are common to a host of other myotonia disease which associated with a number of skeletal muscle channelopathies, especially the DMs. As a group of autosomal dominant/recessive disorders caused by mutations in the CLCN-1 or SCN4A gene, NDMs are caused that delay muscle relaxation after a voluntary or evoked muscle contraction. Clinically, myotonia is the main symptom which affected the muscle system; however, NDMs show marked clinical variability. This suggests that the elucidation of the genetic etiology of these disorders in addition to clinical and electrophysiological features may help to distinguish NDMs from DMs or skeletal muscle diseases with myotonia [3-4].
The average age of disease onset for the 17 patients in our study was 8 years. According to the neurological examinations and EMG evaluations, all of these patients received a clinical diagnosis of NDMs. Muscle biopsies stained with H&E showed typical myogenic changes with only minor alterations in all samples. The results showing few atrophic fibers, internal nuclei and small angular fibers are consistent with previous studies . 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. A specific pathological change was found in myosin ATPase. Myosin ATPase characterized by grouping and the predominance of type 2 fibers in the patients. And all patients with CLCN-1 gene mutations, type 2B fibers were absent and type 2A fibers were predominant. There are two hypotheses that could explain these phenomena. First, a biochemical abnormality in the nerves of patients with CLCN-1 mutations may preclude the development of type 2B motor units. The second hypothesis was that due to the repetitive electrical activity associated with myotonia – analogous to that occurring in the conversion of fast muscle fibers to slow fibers by repetitive stimulation of nerves – type 2B fibers become type 2A fibers. This characteristic can be used to distinguishing PMC from MC .
Due to “clinical phenotype overlap”, the diagnosis of NDMs is dependent on gene analyses. Moreover, numerous studies pointed out that found CLCN-1 mutations in 75% of NDMs suggested that this was a large percentage. By simultaneously sequencing genes about, we detected mutations in most of our patients; CLCN-1 gene mutations were found in 8 patients, and SCN4A gene mutations in 4 patients. However, we failed to detect any mutations in 5 patients even though they fulfilled the diagnostic criteria for NDMs. It is plausible that deletions or other types of mutations deep within the intron or the promoter region of a gene may underlie the disease in these cases [7.8]. Nevertheless, our study showed that the analysis of the genes for CLCN-1 together with SCN4A resulted in the detection of high levels of mutations in Chinese individuals with NDMs and that it is helpful to also identify the mutations for KCNE3 and CACNA1S in these people with mutations in CLCN-1 and SCN4A. We conclude that the application of second-generation sequencing technology is important in diagnosing NDMs .
Eight patients with CLCN-1 mutations were clinically diagnosed with MC. Normally, the CLCN-1 gene, having a highly conserved domain, encodes the voltage-dependent chloride channel CLC1, which is responsible for the large chloride resting potential of skeletal muscle . Molecular and genetic researchs on MC showed that skeletal muscle chloride gene mutations (CLCN-1) mapped to chromosome 7q35. These were “loss of function mutations” that inhibited the depolarization of chloride currents into activated chloride currents to prolong the muscle relaxation process . A large number of studies on the mutation expression of CLCN-1 in DMC have shown that they could produce an effect on the common gate with dominant negative effect on the wild-type subunit through voltage dependent changes, and the RMC mutation involves a fast gate showing complete loss of two monomer funtions . An explanation of these changes in mutations, which was helpful to understand mutations in the same mutation could lead to both dominant and recessive diseases and why RMC is more seroous . In our study, the 2 patients who presented with myotonia showed mild improvement with repetitive activity, and their muscle strength changed slightly without the hypertrophy and tendon reflexes that are characteristic of the DMC gene mutation. Six patients presented with typical myotonia and muscle hypertrophy. Secondary dystonia caused mechanical straining in these patients, and with development of the disease, this could lead to joint contracture and scoliosis, clinical features consistent with RMC. Consequently, the incidence rate in RMC was higher than DMC in our study, and certain clinical manifestations were more common in RMC than in DMC. Some of our patients also presented with accompanying arrhythmia, pre-excitation syndrome or left ventricular enlargement. We postulate that ion channels in cardiomyocytes may contribute to this variable myocardial involvement. To date, more than 100 missense, non-sense, insertions, deletions and splice site mutations have been reported in the CLCN-1 gene. The mutations are spread throughout the entire gene and show no location specificity for mutations leading to DMC or RMC . Interestingly, the mutations that we detected in CLCN-1 were novel, except for the c.1262 on exon 12, which was reported by Koch . This finding suggested that CLCN-1 variations exist for race, region or height. The mutations detected in our study [c.1012C>T(p.R338X), c.1872G>C(p.E6 24X), c.2330delG and c.1389insT] were nonsense, deletion or insertion mutations, which are predicted to cause premature translation termination codons and lead to frame shift or splice site-interrupting mutations. Both of the latter mutations are typically associated with RMC . Missense mutations can also lead to either RMC or DMC depending on their location and the effect of the amino acid substitution on channel gating. In our study, five missense mutations were found, with two of these (p.V286E and p.A298T) in CLCN-1 occurring in exon 8. Duffield et al.  reported that exon 8 encodes the H and I helix, the H-I interlink and part of the I-J interlink that form the channel dimer. Thus, mutations occurring in exon 8 affect the formation of the channel dimer, which affects the conductivity of the chloride channel. Both of the missense mutations that we detected in exon 8 have been reported to be compounded with other mutations (nonsense/deletion/insertion/missense). We speculate that these mutations are pathogenic variations because they are truncating mutations. The screening outcome for CLCN-1 showed that the form and location of the mutations contribute to the genotype-phenotype relationship at the clinical level.
Four patients who were characterized by marked, typical myotonia exacerbated by cold temperatures, the presence of clear episodes of weakness and with SCN4A mutations were clinically diagnosed with PMC. PMC is an autosomal-dominant disorder caused by a mutation in the SCN4A gene, which encodes the α-subunit of the skeletal muscle sodium channel. Normally, this channel is responsible for forming and conducting the action potential. Thus, mutations in SCN4A lead to “gain of channel function” defects, impairing channel inactivation or enhancing channel activation. To date, more than 50 different SCN4A gene mutations have been reported from several populations, and many of these mutations are distributed in exons 13, 19, 22, 23 and 24. Exons 22 and 24 have been recognized as mutation hot-spot regions in PMC . Three novel missense mutations occurred in exon 24, and 1 missense mutation has been reported in exon 22, both of which are located in hot-spot regions. Studies using different gene mutations in the functional domains of the sodium channel complex have confirmed that they form the structural basis for the deactivation mechanism. For example, the mutation c.3877G>A(p.V1293I) located in the cytoplasmic region of membrane domains Ⅲ/Ⅳ lead to the inactivation of the sodium channel and result in a unique temperature-sensitive phenotype. In our study, a two-year-old girl who carried this mutation showed myotonia that was exacerbated by cold and accompanied by muscle hypertrophy and joint contracture, consistent with a previous report . The 3 novel mutations in exon 24 detected in our study were located in the voltage-sensing transmembrane S4 segment in domain Ⅳ of the sodium channel and affected the rapid depolarization process. Interestingly, analysis of the family members determined that the myotonia syndrome was relieved with age. This finding suggested that PMC has a mild impact on the quality of life. These findings indicated that single mutations in the SCN4A gene often affect the processes of slowing fast inactivation, impairing slow inactivation, hastening recovery from inactivation and slowing deactivation, eventually leading to different clinical phenotypes. Our findings also suggest that SCN4A mutations may have height, race and region specificity [21-22].
We were unable to detect any mutations in 5 patients using simultaneous sequencing of CLCN-1, SCN4A, KCNE3 and CACNA1S. We postulate that mutations or deletions in introns or novel genes may correlate with the disease. Therefore, genetic testing may become the gold standard for the definitive diagnosis of patients with NDM, and in the future, DNA chip technology may replace the time-consuming electrodiagnostic studies currently required in the initial evaluation.
After clinical, electrophysiological, skeletal muscle pathology and genetic analyses, all patients were administered carbamazepine (100 mg, 2–3 times/day) and mexiletine (50 mg, 3 times/day). Clinical symptoms improved significantly, consistent with previously published reports [21, 22]. In vitro studies have identified that these pharmacological agents preferentially block sodium channels in the open state, thereby targeting persistent sodium currents [23, 24].