Five patients with myotonia congenita due to CLCN1 gene mutations were enrolled from the Department of Neurology, Children's Hospital of Fudan University in September, January 2011, and March 2021. The clinical presentation, laboratory data, electrophysiological tests, pathology feature, and genetic results were collected and reviewed.

Ethical approval for the study was obtained from the health authority ethical committee of Children's Hospital of Fudan University. All the blood samples were collected after obtaining verbal consent from the parents of each patient in compliance with the Declaration of Helsinki.

Genomic DNA of the children and their parents was extracted from whole blood using a QIAamp DNA Blood Mini Kit (Catalog no. 51106). Nucleic acid preparation and high-throughput sequencing (whole-exome sequencing or next-generation sequencing based on a neuromuscular disorder panel) were performed according to standard protocols in the Clinical Laboratory Improvement Amendments (CLIA) compliant sequencing laboratory in Wuxi NEXTCODE (288 Fute Zhong Road, Waigaoqiao Free Trade Zone Shanghai 200,131, China CLIA ID 99D2064856). Exome capture was performed using an Agilent Sure Select Human All Exon 50 Mb Kit (Agilent Technologies, Santa Clara, CA, USA) followed by sequencing as 150-bp paired-end runs on an Illumina XTen (Illumina, San Diego, CA, USA) platform. Segregation of the CLCN1 variant within the family was confirmed by Sanger sequencing on the ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).

Sequence data were mapped to the human reference genome (GRCh37/hg19). Variant calling used the Genome Analysis Toolkit Best Practices Pipeline (Version 3.2.2). Data filtering, variant prediction, and interpretation followed the ACMG guidelines (5) and those from our previous work (6). Variant analysis was based on the 1,000 Genomes database (http://www.internationalgenome.org/), the gnomAD (http://gnomad-sg.org/), and an internal database (more than 30 k samples). The variants were predicted by the online software platforms PolyPhen2.2 (http://genetics.bwh.harvard.edu/pph2/), SIFT (http://sift.jcvi.org/), and Mutation Taster (http://www.mutationtaster.org/).

A muscle biopsy was conducted in case 2. Muscle samples were appropriately oriented and frozen as we previously reported (7). Cryostat sections (8–10 μm) were cut from transversely oriented muscle blocks. Staining was performed with hematoxylin and eosin, modified Gomori trichrome (MGT), succinate dehydrogenase (SDH), cytochrome c oxidase (COX), and oil red O.

Previously reported cases of Chinese population with CLCN1-related myotonia congenita were identified through search in available national database and international public data such as PubMed. The clinical, biochemical, electrophysiological, and molecular data were obtained from the respective references, reviewed, and compared with those of our present cases.

The GraphPad Prism software (version 6.01) was used for statistical analysis. For variables distributed in a normal fashion, mean ± standard deviation was calculated. For non-normally distributed variables, medians were calculated. Mann–Whitney U-test was used to compare quantitative variables and Chi-square test to compare qualitative variables (Fisher's exact test was used to analyze for component proportion in groups). A value of p < 0.05 was considered statistically significant.

Five unrelated patients were enrolled including four boys and one girl. The clinical presentation and electromyography (EMG) results are shown in Table 1 (case 1–5). All patients developed normally before onset, except case 5, who had a slightly delayed motor milestone. There was no family history. The median onset age of five patients was 3.0 years old, ranging from 1.0 to 5.0 years old, while the median age of admission was 5.0 years old, ranging from 3.5 to 8.8 years old. Five patients complained of muscle stiffness when rising from chairs or starting to climb stairs (5/5, 100.0%), and four patients presented with delayed relaxation of their hands after a forceful grip (4/5, 80%), all of which improved with exercise (warm-up phenomenon) (5/5, 100%). The muscle stiffness aggravated in the morning (1/6, 16.7%), coldness (1/6, 16.7%). Laboratory tests showed slightly elevated creatine kinase (CK) level (278 IU/L, normal range: 210 IU/L) in one patient (1/5, 20%). Electrocardiogram (ECG) was conducted in four patients and echocardiography (ECHO) in three patients, which all showed normal (100%). Nerve conduction study was conducted in five patients which showed normal, while needle EMG test showed remarkable myotonic burst in five patients (5/5, 100%) and myopathic change in one patient (1/5, 20%) (Table 3).

Muscle biopsy was conducted in the biceps brachii muscle of patient 2, which showed only few angular fibers, without variation in muscle fibers, degeneration, regeneration, internal nuclear, or enzymatic deficiency (Figure 1).

Trio-whole exome sequencing was conducted in case 1 and 3, while NGS-based genetic test of neuromuscular panel was performed in the rest of the patients. The mean depth of the trio-whole-exome sequencing data was ~120× . Molecular tests revealed nine potential disease-causing mutations in CLCN1 gene (Figure 2), including seven reported variants: c.139C>T(p.R47W), c.892G>A (p.A298T), c.1657A>T (p.I553F), c.1649C>T (p.T550M), c.350A>G (p.D117G), c.762C>G (p.C254W), c.1277C>A (p.T426N), and two novel variants: c.962T>A (p.V321E) and c.1250A>T(p.E417V)(NM 000083), which have been submitted to the public database ClinVar (http://www.ncbi.nlm.nih.gov/clinvar, submission number: SUB 10199359).

In family 1, three reported pathogenic variants in the CLCN1 gene were revealed in the proband, which were inherited from his parents, respectively.

In family 2, the proband was clinically diagnosed as myotonia congenita based on typical myotonia and tonic burst in EMG test (Table 3). Muscle biopsy excluded common metabolic myopathy and most congenital myopathy (Figure 1). Panel-based next-generation sequencing test revealed one reported pathogenic c.1649C>T (p.T550M) variant, which was inherited from his asymptomatic father who refused to receive physical examination and EMG test. Threonine residue is highly conserved, and c.1649C>T (p.T550M) is predicted to be probably damaging by software such as SIFT and Polyphen. Additionally, a previous experimental study has shown that it affects the gating of CLC-1 channels (24). Incomplete penetrance of this variant has been reported in some patients with a heterozygous state. Thus, we consider it to be pathogenic.

In family 3, the c.962T>A (p.V321E) variant has never been reported in the 1000 Genomes Project, once reported in the GnomAD database as heterozygous state, and is predicted to be probably damaging by software such as SIFT and Polyphen. According to the ACMG 2015 criteria (5), it is likely pathogenic (PM2 + PM3 + PP3 + PP4).

In family 4, the proband harbored compound heterozygous variants in CLCN1 gene: a reported c.1277G>A (p.T426N) variant and a novel c.1250A>T (P.E417V) variant, which was inherited from his father and mother, respectively. The c.1250A>T (p.E417V) variant has never been reported in the 1000 Genomes Project, once reported in the GnomAD database as a heterozygous state, and is predicted to be probably damaging by software such as SIFT and Polyphen. According to the ACMG 2015 criteria (5), it is likely pathogenic (PM2 + PM3 + PP3 + PP4).

In family 5, molecular test in the proband revealed compound heterozygous variants in CLCN1 gene, including a reported c.762C>G (p.C254W) variant and a reported c.892G>A (p.A298T) variant, which was inherited from his mother and father, respectively.

Overall, 43 probands from unrelated families have been reported including our five patients (Table 1). The inheritance pattern of 14 patients was not clear because of invalid data of genetic results from the parents of the probands. Fourteen patients were classified as automatic dominant Thomsen's disease (14/29, 48.3%), while 15 patients were classified as automatic recessive Beck's disease (15/29, 51.7%).

The median onset age was 8 years old (ranging from 0.5 to 24 years old). The most common involved muscles with myotonia were lower limb muscle, followed by upper limb muscle and facial muscle. Warm-up phenomenon was observed in 93.1% patients and myotonic change in 100% patients. Creatine kinase was elevated in three patients (3/21, 9.7%), while cardiac problem was revealed in three patients (3/38, 7.9%) (Table 2).

Overall, 47 variants of CLCN1 gene in Chinese population were reported, which consisted of 33 missense mutations, 6 nonsense mutations, 5 frame-shift mutations, and 3 splicing mutations. Mutations in exons 8, 15, 12, and 16 were most prevalent, while the most common variants were c.892G>A(p.A298T) (n = 9), c.139C>T(p.R47W) (n = 3), c.1205C>T(p.A402V) (n = 3), c.1657A>T(p.I553F) (n = 3), c.1679T>C(p.M560T) (n = 3), c.350A>G(p.D117G) (n = 2), c.762C>G(p.C254W) (n = 2), c.782A>G(P.Y261C) (n = 2), and c.1277C>A(p.T426N) (n = 2).