A complex CLCN1 variant associated with hereditary myotonia in a mixed-breed dog

Abstract

Hereditary myotonia (HM) is characterized by delayed muscle relaxation after contraction as a result of a mutation in the CLCN1 gene. We describe here a complex CLCN1 variant in a mixed-breed dog with clinical and electromyographic signs of HM. Blood samples from the myotonic dog, as well as from his male littermate and parents, were analyzed via amplification of the 23 exons encoding CLCN1. After sequencing the CLCN1 gene, a complex variant was found in exon 6 c.[705T>G; 708del; 712_732del], resulting in a premature stop codon in exon 7 and a protein that was 717 amino acids shorter than the normal CLC protein. The myotonic dog was identified as homozygous recessive for the complex CLCN1 variant; its parents were heterozygous, and its male littermate was homozygous wild-type. Knowledge of the CLCN1 mutations responsible for the development of hereditary myotonia allows greater clarification of this condition.

Hereditary, or congenital, myotonia (HM) is marked by the impairment of skeletal muscle relaxation after voluntary contraction and electrical or mechanical stimulation. HM is caused by a decrease in the membrane permeability of chloride channels as a result of mutations in the CLCN1 gene; both recessive and dominant forms of HM have been described in humans. ⁶

Myotonia was first described in dogs in 1970, at which time it was suspected that genetic alterations were associated with the development of this clinical condition. ¹¹ Mutations in the CLCN1 gene associated with myotonia in dogs were first reported in 1999. ¹⁶

Clinical signs of HM have been documented in goats,^4,5 horses, ¹⁴ cats,^9,22 dogs,^7,8,12,20 buffaloes, ³ and pigs. ¹ Clinical signs are characterized by muscle stiffness after initiation of movement (or startle) and a bunny-hop type movement when running, with gait improvement after warm-up.^20,22 Nonpainful muscle spasms, generalized muscle hypertrophy, and episodes of recumbency after stimulation can also be observed. ¹⁶

The diagnosis of myotonia is confirmed by electromyography based on the observation of repetitive high-frequency myotonic discharges that initially increase in amplitude and frequency and then decrease. A distinct sound that waxes and wanes, described as the “dive bomber sound,” is produced. ⁷ Myotonic discharges may be evoked by voluntary movement, muscle percussion, or insertion of a needle electrode. DNA tests based on the PCR technique, used for screening in breeds known to have this disease, can be adopted as a detection tool. The CLCN1 mutations responsible for congenital myotonia in animals have been described in rats, ¹⁹ buffaloes, ³ pigs, ¹ horse, ²¹ sheep, ¹³ and cats.^9,22 Six studies have described CLCN1 mutations in dogs, namely in the Miniature Schnauzer^2,16 and Jack Russell Terrier ¹² (C to T transition in the ClC-1 allele), Australian Cattle dog (insertion of a single [deoxy] adenylate at position 2665 in CLCN1), ⁸ Labrador Retriever (variant c.2275A>T in CLCN1), ¹⁵ and the American Bulldog (frameshift mutation c.436_437insCTCT in CLCN1). ¹⁷ In addition to these breeds, HM has been described clinically in Cocker Spaniels ¹⁰ and Chow Chows,^7,11,18but no other breeds to date.

We describe here the clinical signs, electromyographic findings, and genetic characteristics of a mixed-breed dog with HM. The samples obtained and images used in our study were part of a clinical and genetic evaluation authorized by the owner of the affected dog.

A 6-mo-old male mixed-breed dog was referred to our veterinary hospital with generalized muscle stiffness when initiating movement or during excitement (with improvement after a few seconds) since 2 mo of age. The owner also described dysphagia when the dog was fed large pieces of food. The animal was the only one in the litter of 5 dogs with this clinical presentation; the parents of the litter were consanguineous (siblings) and lacked a history of similar signs.

On physical examination, vital signs were normal, and generalized muscle hypertrophy was observed during palpation. There was also transient stiffness of the 4 limbs and a fall to lateral recumbency immediately after stimulation or startle, with improvement after a few seconds (Suppl. Video 1). Hypertrophy and exaggerated contractile movements of the tongue after stimulation were also observed (Suppl. Video 2).

CBC, albumin, creatinine, alanine aminotransferase, aspartate aminotransferase, creatine kinase, and urinalysis were all within RIs. Measurements of ionic calcium, potassium, sodium, and chloride were also all within RIs. ELISA and indirect immunofluorescence tests for serologic detection of canine leishmaniasis were negative (the disease is endemic in this region of Brazil). Heart rhythm and electrocardiographic waves were within normal limits.

Given the suspicion of skeletal muscle dysfunction, electromyographic evaluation was performed (Neuro-MEP-Micro; Neurosoft) with a bandpass from 20 to 10,000 Hz and a concentric-type needle. Myotonic discharges were seen in the appendicular muscles and notably in the axial muscles, confirming the diagnosis of myotonia (Suppl. Video 3, Fig. 1A).

Figure 1.

Hereditary myotonia (HM) in a 6-mo-old male mixed-breed dog. A. Electromyography tracing obtained from the semitendinosus muscle. Note the high-frequency discharges characteristic of HM. Sustained runs of positive waves with initial sharp positivity followed by a slow negative component are also present. Vertical divisions represent 150 μV; horizontal divisions represent 40 ms. B. Partial capillary sequencing chromatogram for CLCN1 exon 6 in mutated homozygous myotonic dog 1, wild-type homozygous dog 5, and heterozygous dam (dog 3). The complex CLCN1 variant is a nonsynonymous single-nucleotide variation (SNV) at nucleotide 7 of exon 6 (c.705T>G; arrow) that resulted in the change of a phenylalanine to a valine (p.Phe234Val); the deletion of a guanine (c.708del; arrowhead); and the deletion of 21 bases (c.712_732del; Geneious 10.0 software, Biomatters). C. Schematic of the coding gene and amino acid sequences in the myotonic dog (above; sequence NC_0518520.1) and a normal dog (below). The c.705T>G SNV is represented in green, the c.708del and c.712_732del variants in red, and the new stop codon (TGA) in blue.

Given that the dog was presented with myotonia and a warm-up phenomenon (gait and muscle stiffness improvement after exercise), ⁷ we suspected a hereditary form of myotonia associated with mutations in the CLCN1 gene. To search for a CLCN1 genetic mutation, blood samples were obtained from the myotonic dog (dog 1), his parents (dogs 2 and 3), one of his male littermates (dog 4), and an unaffected, unrelated dog (dog 5, animal from the DNA sample bank of the Molecular Biology Laboratory of Veterinary Medicine). Genomic DNA was purified from blood samples (ReliaPrep blood gDNA miniprep system; Promega) according to the manufacturer’s instructions.

Specific primers were designed to amplify the 23 exons of CLCN1 and the 5′ and 3′ intron–exon junctions (Suppl. Table 1; PrimerQuest; IDT) The PCR was standardized to a total volume of 25 μL containing 5 μL of template DNA, 0.75 μL of each primer (10 pmol/μL), 12.5 μL of PCR master mix (Promega), and 6.0 μL of nuclease-free water. Amplicons were analyzed by 1.5% agarose gel electrophoresis, purified, and subjected to Sanger sequencing. The obtained sequences and electropherograms were examined (Geneious software) and compared with the reference sequence CLCN1 Canis lupus familiaris (GenBank NM_001003124.2).

Alignments of the sequences obtained for the affected dog, dog 3 (dam), the unaffected nonrelated dog (Fig. 1B), and the reference GenBank sequence revealed a complex variant c.[705T>G; 708del; 712_732del] in exon 6 (i.e., a nonsynonymous variant at the seventh nucleotide of exon 6 [c.705T>G] that resulted in the change of a phenylalanine to a valine [p. Phe234Val], Fig. 1C); 3 bases downstream, there was a deletion of a guanine (c.708del); and further downstream, 21 bases were deleted from positions 712–732 (c.712_732del). The first deletion caused a premature stop codon in exon 7 (c.800–802 positions), which led to the production of a truncated protein product of 259 amino acids, which was 717 amino acids shorter than the normal CLC protein (NP_001003124.1).

The myotonic dog (dog 1) was identified as homozygous for the complex CLCN1 variant; his parents (dogs 2 and 3) were heterozygous for this mutation. However, his littermate was wild-type homozygous for the complex variant. In the affected dog, we did not observe any of the mutations that have been associated with myotonia in dogs.^2,8,12,15,17

Our patient had clinical signs of myotonia, including stiff gait observed after starting movement, or in situations of fear and excitement; generalized muscle hypertrophy; bunny-hop movements; rigidity; difficulty swallowing; and hypertrophy of the tongue. These signs were first noticed when the dog was 2-mo-old, and are similar to those reported in dogs with hereditary myotonia.^12,15,17 The diagnosis was confirmed through electromyography, which showed myotonic discharges in the thoracic limbs, pelvic limbs, and tongue muscles, especially in the pelvic limbs.^1,12,17,20 The patient did not exhibit upper airway stridor or continuous wheezing sounds, which are also reported in some dogs with myotonia.

All genomic variants in our patient occurred in exon 6, namely, a nonsynonymous variant (c.705T>G), causing a substitution from a phenylalanine to a valine (p. Phe234Val); deletion of a guanine (c.708del); and 21-bp deletion (c.712_732del). The final result was a deletion of 717 amino acids from the normal CLC protein. The nonsynonymous variant (c.705T>G), with a change from a phenylalanine to a valine (p. Phe234Val) sequence, was compared to that of variants in a cat (NC_058369.1), dog (NC_051820.1), pig (NC_010460.4), horse (NC_009147.3), sheep (NC_056057.1), water buffalo (NC_059164. 1), goat (NC_030811.1), mouse (NC_000072.7), mouse (NM_013147.1), and human (NG_009815.2). The modification was not observed in these species.

In humans, several pathogenic mutations have been described in the CLCN1 gene, ¹⁷ with both dominant and recessive inheritance. In animals, no mutation in exon 6 has previously been reported as associated with myotonia. Exon 6 is translated into the F membrane loop, which contributes to the transport of ions to the intracellular medium. Although HM is extremely rare in mixed-breed dogs, it can arise occasionally. ²³ In our case, the parents of the dog were consanguineous, and the complex CLCN1 variant described was homozygous in the affected animal. Molecular analysis showed that his parents were heterozygous, and his male littermate was homozygous wild-type. These 3 dogs did not have any other abnormal clinical signs; however, electromyographic studies were not performed in these animals.

Mexiletine hydrochloride has shown the most promising results for the treatment of dogs with congenital myotonia, leading to a decrease in dysphagia and inspiratory sounds, although episodes of muscle collapse and stiffness can still occur. ¹⁵ Nevertheless, because this disease tends to stabilize at 12 mo of age, no treatment was given in our case. The dog was in stable condition in follow-up assessments, with no progression of muscle hypertrophy and myotonic crisis at the last evaluation when 2-y-old; no limitation of its quality of life was reported. The identification of a complex variant in a gene known to be associated with clinical signs is sufficient to predict that the identified variant is likely pathogenic.