Identication of Novel Mutations of the CLCN-1 and SCN4A Genes in Non-dystrophic Myotonia in China

Background: The aim of our study was to characterize the genetic, pathological and clinical alterations of 17 patients in China presenting with non-dystrophic myotonia (NDM). Methods: We rst sequenced the CLCN-1 gene in patients having clinical features and muscle pathology indicative of NDM. If no mutations were detected, we subsequently analyzed the SCN4A, KCNE3 and CACNA1S genes. Results: As determined by needle electromyography, patients may have accompanying atypical myopathy as well as muscle hypertrophy, secondary dystonia and joint contracture All participants in this study were administered mexiletine in combination with carbamazepine and showed signicant improvements in their myotonia symptoms. Routine pathological examinations showed mild abnormalities in muscle pathology. Oxidative enzyme activity was decreased in many bers. ATPase studies of ber subtypes demonstrated a predominance of type 2A bers and a complete absence of type 2B muscle bers in patients with CLCN-1 mutations. CLCN-1 gene mutations were found in 8 cases diagnosed with myotonia congenital by gene screening. The detected mutations included 5 missense, 2 nonsense, 1 deletion and 2 insertions, and these CLCN-1 mutations were concentrated in exons 8 and 12. Further gene analysis showed 4 mutations in the SCN4A gene in patients diagnosed with paramyotonia congenita. One of these mutations was consistent with a previously reported mutation, whereas 3 mutations were novel. All of these novel mutations occurred within “hot spots” of exons 22 and 24. Five patients with NDM lacked any identiable mutations in CLCN-1, SCN4A, CACNA1S or KCNE3. Conclusions: Myotonia congenita and paramyotonia congenita are the predominant forms of NDM in China. NDM may be best diagnosed using genetic analysis in combination with clinical features. New mutations of the CLCN-1 and SCN4A genes in patients with NDM were detected, we postulate that novel pathogenic genes for NDM occur in China.

The genetic and phenotypic heterogeneities in NDM create complications in distinguishing patients with sodium channel myotonia from those with myotonia congenita (MC) and underscore the need for these patients to undergo genetic screening. Due to the incomplete dominance of certain mutations with variable penetrance and expression as additional compounding factors, 25% of the patients with NDM examined in one study lacked an identi able gene mutation [2] .
Therefore, we conducted this study to characterize the genetic, skeletal muscle pathology, clinical manifestations of 17 patients with NDM in China.

Clinical data
We analyzed 38 people, including 17 patients who had been admitted to the Department of prenatal disgnosis at our hospital from 2015 to 2019 and 5 of their family members. Characteristics of included studies: patients who diagnosed with typical myotonia accompanied by grip or percussion myotonia, an absence of muscular atrophy and an EMG showing typical myotonic discharges; and an absence of myotonic dystrophy.

Skeletal muscle histopathology
After obtaining informed consent from the patients and their family members, muscle biopsy specimens were collected from the biceps brachii. For histopathological analysis, serial frozen sections (7 μm) were stained with hematoxylin and eosin (H&E), modi ed Gomori trichrome, oil red O (ORO) and periodic acid-Schiff (PAS) or were treated using histochemical reactions with NADH-TR, succinate dehydrogenase (SDH), adenosine monophosphate (AMP), deaminase, cytochrome C oxidase (COX), acid phosphatase (Acid) and myosin ATPase (pH 4.2, 9.98).

Mutation screening
The genomic DNA was extracted from peripheral blood of 17 patients and their family members. Meanwhile, 100 healthy and unrelated Chinese subjects were selected as the control group. The research of applying Primer Primer 5.0 to design PCR primers which capture the entire coding regions and anking sequence, included 4 genes: CLCN-1, SCN4A, KCNE3 and CACNA1S. 50 ng of genomic DNA with these primers was used a hot-start PCR for ampli cation of polymorphic markers. PCR products were puri ed and sequenced using dye terminator chemistry with an ABI Prism 377 DNA Sequencer (Applied Biosystems). The Sequencher 4.90 software was used to analyze the sequences. The relationship between the gene and disease phenotype was analyzed, and a new mutation detected in the 100 control subjects.

Clinical features analysis
A total of 17 patients were recruited, including 14 boys and 3 girls. Case 1 and case 2 from the same family, and case 3 and 4 from the same family. The age of disease onset was 1-17 years with an average onset of 8 years. Serum CK and K + levels were normal in all 17 patients. All patients suffered from muscular stiffness. Muscle strength and re exes were assessed as follows. Weakness in the limbs was observed in 9 patients with muscle strength scores from + to -.
Weakness in the trunk was observed in 9 patients with a muscle strength score of -. Ten patients showed secondary muscle guarding, 12 patients showed muscular hypertrophy, 3 patients showed scoliosis accompanied by contracture of the Achilles tendon, 1 patient showed contracture of the Achilles tendon and elbow joint and 5 patients showed contracture of only the Achilles tendon. Five patients presented with myotonic discharges accompanied by atypical myopathic discharges upon EMG examination. Three patients had sinus arrhythmia, 1 patient had pre-excitation syndrome, 1 patient had sinus bradycardia and 5 patients had left ventricular enlargement and ventricular myocardial perfusion (Fig. 1, Table 1). There was no patient performance muscle deterioration, testicular atrophy or cataracts. Myotonia was improved by the administration of mexiletine (50 mg, 3 times/day) in combination with cabamazepine (100 mg, 2-3 times/day).

Muscle pathology
In the patients with NDMs, the skeletal muscle biopsies showed minor variations in stained with H&E. The muscle histopathology was characterized by variations in ber size with the presence of atrophic bers and by increases in connective tissue elements. Six patients showed occasional degeneration or necrosis, 4 patients exhibited small angular bers and 2 patients displayed an increase in the number of internal nuclei. Myosin ATPase was characterized by ber grouping and a predominance of type 2 bers, which showed that type 2B bers demonstrated an absence and a predominance of type 2A bers in all patients who with CLCN-1 mutations; the type 1 bers in these patients that either normal or showed mild atrophy. The results from the oxidative enzyme reactions indicated a focal decrease in some bers with changes predominantly occurring in type 1 bers (Fig. 2).

Molecular genetic analyses
To obtain an overview of the clinical features, CLCN-1 was rst sequenced in all patients. Mutations in CLCN-1 genes were identi ed in 8 patients. SCN4A was subsequently sequenced in the remaining 9 patients, and mutations in this gene were identi ed in 4 patients. The KCNE3 and CACNA1S genes were sequenced in the remaining 5 patients. We were unable to nd any mutations in the KCNE3 and CACNA1S genes, although these 5 patients ful lled the diagnostic criteria for NDMs.

CLCN-1 gene mutations in 8 patients
Ten different CLCN-1 mutations were identi ed. A mutation (c.1262_1263insC) detected in one family was consistent with a previously reported mutation. We  Table 2).

Discussion
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 bers, internal nuclei and small angular bers are consistent with previous studies [5] . The differences between NDMs and DMs are that NDMs lack the typical nuclear pyknotic clumps, sarcoplasmic masses, muscle ber 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 speci c pathological change was found in myosin ATPase. Myosin ATPase characterized by grouping and the predominance of type 2 bers in the patients. And all patients with CLCN-1 gene mutations, type 2B bers were absent and type 2A bers 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 bers to slow bers by repetitive stimulation of nerves -type 2B bers become type 2A bers. This characteristic can be used to distinguishing PMC from MC [6] .
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 ful lled 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 voltagedependent chloride channel CLC1, which is responsible for the large chloride resting potential of skeletal muscle [9] . 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 [10] . 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 [11] . 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 [12] . 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 re exes 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 speci city for mutations leading to DMC or RMC [13] . Interestingly, the mutations that we detected in CLCN-1 were novel, except for the c.1262 on exon 12, which was reported by Koch [14] . This nding 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 [15] . 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, ve missense mutations were found, with two of these (p.V286E and p.A298T) in CLCN-1 occurring in exon 8. Du eld et al. [16] 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 [17] . 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 con rmed that they form the structural basis for the deactivation mechanism [18] . For example, the mutation c.3877G>A(p.V1293I) located in the cytoplasmic region of membrane domains / lead to the inactivation of the sodium channel and result in a unique temperature-sensitive phenotype [19] . In our study, a twoyear-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 [20] . The 3 novel mutations in exon 24 detected in our study were located in the voltage-sensing transmembrane S4 segment in domain 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 nding suggested that PMC has a mild impact on the quality of life. These ndings 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 ndings also suggest that SCN4A mutations may have height, race and region speci city [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 de nitive 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 signi cantly, consistent with previously published reports [21,22] . In vitro studies have identi ed that these pharmacological agents preferentially block sodium channels in the open state, thereby targeting persistent sodium currents [23,24] .

Conclusion
Page 5/10 MC and PMC have similar but heterogeneous clinical phenotypes. Analysis of the CLCN-1 and SCN4A genes can identify a large number of mutations in patients with NDMs. Most of the abundant gene mutations detected in CLCN-1 and SCN4A in our study were novel. In addition, we found a signi cant racial difference for the mutations in CLCN-1 and SCN4A genes. Because new gene mutations are likely to be discovered in patients with NDMs, DNA chip technology will become invaluable for diagnosing NDMs. The administration of carbamazepine combined with mexiletine offered signi cant bene ts to patients, indicating the clinical merit of these drugs. All experimental protocols were approved by a named institutional and/or licensing committee.

Abbreviations
All methods were carried out in accordance with relevant guidelines and regulations.      Pedigree 5 from 3 generations of one family with CLCN-1 gene mutations. Right: Con rmation of novel heterozygous mutations in CLCN-1; A>G mutation at nucleotide 795 in the mother, and G>C mutation at nucleotide 1872 in the father. Sporadic case: Con rmation of heterozygous mutations in the CLCN-1gene: C>T mutation at nucleotide 138 in one patient, and G>A mutation at nucleotide 891 in one patient. Pedigrees and sequence analyses from families with PMC: Left: Con rmation of a novel heterozygous mutation in the SCN4A gene; G>A mutation at nucleotide 4916 in one patient. Right: Con rmation of a novel heterozygous mutation in the SCN4A gene; C>T mutation at nucleotide 5283 in one patient. Left: Con rmation of a novel heterozygous mutation in the SCN4A gene; C>G mutation at nucleotide 5468 in one patient. Right: Con rmation of a novel heterozygous mutation in SCN4A; G>A mutation at nucleotide 3877 in one patient.