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A novel mutation in CLCN1 associated with feline myotonia congenita.

Gandolfi B, Daniel RJ, O'Brien DP, Guo LT, Youngs MD, Leach SB, Jones BR, Shelton GD, Lyons LA - PLoS ONE (2014)

Bottom Line: 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).Muscle histopathology showed hypertrophy of all fiber types.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Medicine and Surgery, School of Veterinary Medicine, University of Missouri - Columbia, Columbia, Missouri, United States of America.

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.

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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.
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pone-0109926-g006: 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.

Mentions: The entire CLCN1 coding sequence (GeneBank accession no. KJ561451) and partial 5′ and 3′ UTRs were analyzed for six cats (Table 1) representing the five affected cats and one random bred control. In humans, CLCN1 has one isoform and the length of the coding region within the transcript is 2967 bp. In the cat, CLCN1 has 23 exons (Table S1), the boundaries were confirmed by genomic sequencing of the five cats used for the analyses of CLCN1, producing a 2,970 bp coding sequence (CDS) that translates into 989 amino acids (Figure S1). The average CDS identity between humans and cats is 87.5% and at the protein level, CLCN1 in felines is 89.5% identical to humans. Seven of the seventeen identified polymorphisms are exonic mutations (Table 1). Two mutations in exon 18 result in an amino acid substitutions, however, they were not concordant with the disease phenotype. One of the ten intronic mutations segregated concordantly with the phenotype and affects an exon/intron splice site in affected cats. The c.1930+1G>T transversion altered the 5′ splice site at the junction of exon 16 and intron 16 (Figure 5a). The effect of this polymorphism was investigated at the RNA level (Figure 5b). All the affected cats showed the same haplotype across all the identified polymorphisms (Table 1). All the mutations were confirmed by the RNA transcript sequence obtained from muscle tissues of affected and wildtype cats. Interestingly, the identified mutation is associated with the upstream absence of exons 15 and 16 (Figure 6). Intron 14 sequence showed polymorphisms in the control cat only, in a heterozygous state, while the affected cat sequence was identical to the wildtype sequence. To exclude retrotranscription artifacts, the RNA experiment was conducted twice, beginning at the RNA isolation step. In silico translation of the altered transcript predicts the lack of 116 amino acids, from residues 557 to residue 643 of the protein (Figure S1). The predicted premature CLCN1 protein truncation would cause the partial absence of the first highly conserved CBS1 (cystathionine β-synthase) domain and the third dimerization domain p.578Y. The strength of the 5′ splicing sites (5′ss) was calculated for exon-intron 14, 15 and 17 boundaries using the Maximum Entropy Model score, the Maximum Dependence Decomposition Model score, the first-order Markov Model and the Weight Matrix Model score. The strength of the 5′ss in all the tested boundaries was similar in each method when compared, suggesting exon-intron 15 5′ss as the strongest. Complete donor splice site strength is confirmed in the mutated sequence (Table S2).


A novel mutation in CLCN1 associated with feline myotonia congenita.

Gandolfi B, Daniel RJ, O'Brien DP, Guo LT, Youngs MD, Leach SB, Jones BR, Shelton GD, Lyons LA - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214686&req=5

pone-0109926-g006: 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.
Mentions: The entire CLCN1 coding sequence (GeneBank accession no. KJ561451) and partial 5′ and 3′ UTRs were analyzed for six cats (Table 1) representing the five affected cats and one random bred control. In humans, CLCN1 has one isoform and the length of the coding region within the transcript is 2967 bp. In the cat, CLCN1 has 23 exons (Table S1), the boundaries were confirmed by genomic sequencing of the five cats used for the analyses of CLCN1, producing a 2,970 bp coding sequence (CDS) that translates into 989 amino acids (Figure S1). The average CDS identity between humans and cats is 87.5% and at the protein level, CLCN1 in felines is 89.5% identical to humans. Seven of the seventeen identified polymorphisms are exonic mutations (Table 1). Two mutations in exon 18 result in an amino acid substitutions, however, they were not concordant with the disease phenotype. One of the ten intronic mutations segregated concordantly with the phenotype and affects an exon/intron splice site in affected cats. The c.1930+1G>T transversion altered the 5′ splice site at the junction of exon 16 and intron 16 (Figure 5a). The effect of this polymorphism was investigated at the RNA level (Figure 5b). All the affected cats showed the same haplotype across all the identified polymorphisms (Table 1). All the mutations were confirmed by the RNA transcript sequence obtained from muscle tissues of affected and wildtype cats. Interestingly, the identified mutation is associated with the upstream absence of exons 15 and 16 (Figure 6). Intron 14 sequence showed polymorphisms in the control cat only, in a heterozygous state, while the affected cat sequence was identical to the wildtype sequence. To exclude retrotranscription artifacts, the RNA experiment was conducted twice, beginning at the RNA isolation step. In silico translation of the altered transcript predicts the lack of 116 amino acids, from residues 557 to residue 643 of the protein (Figure S1). The predicted premature CLCN1 protein truncation would cause the partial absence of the first highly conserved CBS1 (cystathionine β-synthase) domain and the third dimerization domain p.578Y. The strength of the 5′ splicing sites (5′ss) was calculated for exon-intron 14, 15 and 17 boundaries using the Maximum Entropy Model score, the Maximum Dependence Decomposition Model score, the first-order Markov Model and the Weight Matrix Model score. The strength of the 5′ss in all the tested boundaries was similar in each method when compared, suggesting exon-intron 15 5′ss as the strongest. Complete donor splice site strength is confirmed in the mutated sequence (Table S2).

Bottom Line: 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).Muscle histopathology showed hypertrophy of all fiber types.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Veterinary Medicine and Surgery, School of Veterinary Medicine, University of Missouri - Columbia, Columbia, Missouri, United States of America.

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.

Show MeSH
Related in: MedlinePlus