Targeted Next Generation Sequencing in patients with Myotonia Congenita
Introduction
Myotonia Congenita (MC; MIM #118425) is a nondystrophic skeletal muscle disease characterized by muscle stiffness: all striated muscle groups including the extrinsic eye muscles, facial muscles, and tongue may be involved. Stiffness is usually relieved by repeated contractions of the muscle (the “warm-up” phenomenon). MC is characterized by an exaggerated response to stimulation of certain muscle fibers (hyperexcitability) and as a result, there is painless spasm (tonic spasm) of affected muscle after forceful voluntary contraction and difficulty in relaxing muscle (myotonia). Characteristic symptoms may include difficulties in relaxing hand grip, in opening completely the eyes after forcibly closing them, in arising from a chair, or starting to walk or run, climbing stairs and/or arising from bed at night. Most patients also develop hypertrophy, resulting in an athletic appearance [1].
The disease is distinguished into Thomsen disease (THD; MIM #160800), inherited as an autosomal dominant and Becker disease (GM; MIM #255700), inherited as autosomal recessive. Both diseases are caused by mutation within CLCN1 gene (MIM #118425), spanning 36 kb and containing 23 exons with a transcript length of 3093 nucleotides. The gene encodes for the voltage-gated chloride channel ClC-1 located within the sarcolemma [2], [3], present in virtually all tissues and organisms, and widely expressed in most mammalian cells [4]. It is now recognized that chloride channels regulate a variety of important physiological and cellular functions [5], such as pH, cell volume, electrical impulses, transport of salts across cells, and voltage stabilization of excitable muscle cells [6].
The muscular ClC-1 channel contributes to approximately 80% of the total resting conductance and determines membrane excitability.
MC patients may have progressive, minor distal weakness and attacks of transient weakness brought on by movement after rest. Occasionally, proximal weakness or distal myopathy is reported [4]. Extra muscular manifestations such as early cataracts, abnormal cardiac conduction, or endocrine dysfunction are absent. The age of onset is variable: in THD patients the onset of symptoms is usually in infancy or early childhood; in GM the average age of onset is slightly older. Electromyography (EMG) usually reveals frequent myotonic discharges, which are helpful in confirming the myotonic disorder, but do not distinguish between the recessive and dominant variants.
Originally, the prevalence of the disease has been estimated with a frequency of 1:23,000 for the dominant form and 1:50,000 for the recessive one; later studies suggested that the recessive form is more common than the dominant one.
More than 130 CLCN1 pathogenic variants identified to date are associated with Becker disease [7], while only 20 with THD phenotype. Surprisingly approximately 12 pathogenic variants associate with both autosomal and autosomal segregation, making it difficult to predict the mode of inheritance, without knowing both the clinic and the family pedigree [2].
The CLCN1 mutations include small deletions, insertions, frame-shifts, stop codons, missense, deletions or duplications and splice-site mutations [2], [8], [9], [10], distributed widespread along the coding sequence. Usually “private” mutations, segregating only in one family, have been described [2], [11], [12] making molecular analysis long and difficult, as well as the genotype-phenotype correlation. On the other hand, for some mutations (for example F484L within exon 13) a founder effect has been reported [13]. Moreover, complex alleles made of more than one variant with or without clinical significance and located in cis have been described [14].
Due to all these aspects, molecular analysis of CLCN1 are to be performed by sequencing the entire coding region (23 exons), including intron/exon boundaries, an expensive and time consuming approach.
Thus we developed a Next Generation Sequencing (NGS) approach allowing us to process a high number of patient samples simultaneously in much less time [15], [16]. To do this a specific panel for CLCN1 gene has been designed for sequencing the entire gene coding region, including intron/exon junctions, in patients with clinical diagnosis of congenital myotonia. Specifically, the protocol has been firstly validated on 40 samples already characterized by Sanger method, in order to compare both sensitivity and specificity of the two approaches. Successively additional 15 patients have been analyzed in a first time by NGS and successively the variants identified have been confirmed by Sanger method.
This work has allowed us to characterize novel variants responsible for Myotonia Congenita, broadening the spectrum of CLCN1 pathological alleles in Italy.
Section snippets
Patients
We analyzed 55 MC patients, selected both by Medical Genetics Unit of Tor Vergata Hospital and by other Italian clinical centers over the last 15 years. All patients reported clinical signs of MC. The informed consent was obtained from each patient.
DNA extraction
The DNA was extracted from peripheral blood with the kit of Qiagen FlexiGene following manufacture's protocol. DNA was analyzed by Nanodrop to evaluate the quality by measurement of the levels of absorbance at different wavelengths. At the same time,
Results
Upon Sanger sequencing the CLCN1 gene in a group of 40 unrelated and selected MC patients, using NGS we characterized 31 pathogenic variants confirming the clinical diagnosis in all patients (Table 1). As reported, the allele frequency of each mutation described confirms the allelic heterogeneity of CLCN1 gene. After PGM sequencing we obtain, on average, 500,000 sequences for 314v2 chip loaded with up to 16 MC samples. In Fig. 1 an example of final report is shown, evidencing the percentage of
Discussion
NGS technology is widely used in many areas of clinical genetic research, enough to take a role as a diagnostic tool, thereby replacing most other molecular diagnostic technologies [28], [29], [30], [31].
The decrease in run times and costs have turned reading an individual's DNA from a multi-annual, multi-laboratory undertaking into a feasible effort for individual laboratories within a reasonable time frame. Thus many molecular diagnostic laboratories have adopted NGS as the preferred
Acknowledgments
We thank all neurologists for clinical classification and sample collection.
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Exome sequencing identifies a novel pathogenic CLCN1 mutation in an Iranian family with Myotonia Congenita: A case report
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Next-generation sequencing application to investigate skeletal muscle channelopathies in a large cohort of Italian patients
2021, Neuromuscular DisordersCitation Excerpt :The NGS analysis of CLCN1 gene revealed 14 different missense mutations, 4 splice site mutations, 3 nonsense mutations, 1 frameshift and 1 synonymous variant (Fig. 1, Table 3). The most frequent mutations in the CLCN1 gene were c.180+3A>T, p.F167L, p.G190S and p.I290M, in intron 1 and exon 4, 5 and 8, respectively, confirming the high-frequency mutation rate of these regions [16,18–21]. The p.G395Cfs*32, p.L843P, p.V829M, p.E258E and c.1471+4delTCAAGAC variants were classified by ACMG guidelines (Table 6).
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