A novel mutation in CLCN1 associated with feline myotonia congenita

Abstract

Myotonia congenita (MC) is a skeletal muscle channelopathy characterized by inability of the muscle to relax following voluntary contraction. Worldwide population prevalence in humans is 1:100,000. Studies in mice, dogs, humans and goats confirmed myotonia associated with functional defects in chloride channels and mutations in a skeletal muscle chloride channel (CLCN1). CLCN1 encodes for the most abundant chloride channel in the skeletal muscle cell membrane. Five random bred cats from Winnipeg, Canada with MC were examined. All cats had a protruding tongue, limited range of jaw motion and drooling with prominent neck and proximal limb musculature. All cats had blepharospasm upon palpebral reflex testing and a short-strided gait. Electromyograms demonstrated myotonic discharges at a mean frequency of 300 Hz resembling the sound of a 'swarm of bees'. Muscle histopathology showed hypertrophy of all fiber types. Direct sequencing of CLCN1 revealed a mutation disrupting a donor splice site downstream of exon 16 in only the affected cats. In vitro translation of the mutated protein predicted a premature truncation and partial lack of the highly conserved CBS1 (cystathionine β-synthase) domain critical for ion transport activity and one dimerization domain pivotal in channel formation. Genetic screening of the Winnipeg random bred population of the cats' origin identified carriers of the mutation. A genetic test for population screening is now available and carrier cats from the feral population can be identified.

Figure 1. The hypertrophic tongue in a cat affected with myotonia congenita.

The tongue is very enlarged and constantly protrudes from the mouth.

Figure 2. Myotonic discharges in a cat with myotonia congenita.

EMG recorded from the biceps femoris muscle showed a sustained run of initially positive biphasic to triphasic spikes with a firing frequency of 240 Hz. Note also the waxing and waning in amplitude of the spike train.

Figure 3. Supramaximal repetitive nerve stimulation in a cat with myotonia congenita.

Stimulation of the right tibial nerve at 3 Hz shows a decrement of 48.3% between the 1st and 30th waveforms.

Figure 4. Pathological changes were limited to myofiber hypertrophy in cats with myotonia congenita.

a. H&E stained cryosections of the biceps femoris muscle. Hypertrophy involved both type 1 and type 2 fibers. b. H&E stain of biceps femoris muscle. c. Antibody staining for type 1 (brown color) and type 2 (red color) fibers. For comparison of fiber size, an unaffected approximately age matched control cat muscle is shown. b. H&E stain of biceps femoris muscle; d; antibody staining for type 1 and type 2 fibers. Magnification bar  = 100 µm for all images.

Figure 5. Electropherogram representing the genomic and RNA sequence of CLCN1 in wild-type and myotonia congenita cats.

a. Genomic sequence of three control cats and the five affected cats. The red rectangle indicates the c.1930+1G>T polymorphism in the first base of intron 16 associated with MC in the domestic cat. b. RNA sequence lacks exon 15 and 16, while no alterations are observed in the control cat at exon junctions 14–15 and 16–17.

Figure 6. Schematic representation of CLCN1 splicing in the wild-type cat and myotonia congenita cats.

In exon 15, the yellow line represents the third dimerization site (dim site), while the blue box represents the CBS1 domain, present in exon 16 and 17. The black triangle represents the identified mutation and the two red circles connected by a red dashed line represent the donor splice site and the acceptor splice site in the mutated protein. a. Normal splicing occurring in the wild-type subject, both dimerization sites and CBS1 are present. b. In the affected cat, the mutation in intron 16 is associated with the absence of exons 15 and 16, and, therefore the protein lack the dimerization domain.

Figure 7. Immunofluorescent staining for localization of antibodies against the N-terminus and C-terminus of CLCL1.

Cryosections of biceps femoris muscle from a representative myotonic cat and approximately age matched control. A similar pattern of punctate cytoplasmic staining was found with both antibodies in the myotonic and control cat (rhodamine filter, red color). An antibody against dystrophin was used to localize the muscle sarcolemma (FITC, green color). Bar  = 50 µm for all images.

Figure 8. Antibody markers against RYR, DHRPα1 and CLCN1.

To further investigate the subcellular localization of the chloride channels in myotonic cats, antibody markers for the sarcoplasmic reticulum (RYR; ryanodine receptor, green) and T-tubule system (DHRPα1, green) were co-stained with antibodies against CLCL1 (red) and compared to control muscle. The merge figures for both myotonic and control cats (yellow) show close proximity of CLC1 to the sarcoplasmic reticulum and T-tubule system. Bar  = 50 µm for all images.