Myotonia and chloride channel Chen Sun Haukeland Universitetssykehus

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Myotonia and chloride channel
Chen Sun Haukeland Universitetssykehus
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Myotonia and chloride channel

• Myotonia
• Chloride channel
• Myotonia congenita
• Dystrophic myotonia

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Myotonia

• Muscle stiffness
• Percusion and action myotonia

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Action myotonia
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Myotonia

• Muscle stiffness
• Percusion and action myotonia
• EMG myotonic runs
• (Latent myotonia)

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EMG
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Myotonia

• A characteristic clinical phenomenon,
  not a disease in itself.
• Shared by a group of neuromuscular diseases.
• Not a single genetic defect, rather a common
  final pathway.

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Myotonic disorders

• Two major categories
• Non-dystrophic myotonias
• Dystrophic myotonias

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Myotonic disorders

• Two major categories
• Non-dystrophic myotonias
• Involve solely muscle system
• channelopathies
• Dystrophic myotonias

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Myotonic disorders

• Two major categories
• Non-dystrophic myotonias
• Involve solely muscle system
• channelopathies
• Dystrophic myotonias
• Multisystem involvement
• Nucleotide repeat expansion

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Myotonic disorders
AD autosomal dominant inheritance AR autosomal
recessive inheritance Promm proximal myotonic
myopathy PDM proximal dystrophic myotonia.
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Myotonia

• Hyperexcitability of muscle membrane
• Pathomechanisms
• a decrease in muscle chloride conductance
• alterations of sodium channel activity

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Myotonia

• Hyperexcitability of muscle membrane
• Pathomechanisms
• a decrease in muscle chloride conductance
• myotonia congenita
• dystrophic myotonia
• alterations of sodium channel activity

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Myotonia

• Hyperexcitability of muscle membrane
• Pathomechanisms
• a decrease in muscle chloride conductance
• myotonia congenita
• dystrophic myotonia
• alterations of sodium channel activity
• myotonia congenita
• sodium-channel myotonias
• dystrophic myotonia

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Neuromuscular junction
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Action potential
Depolarization - Na
Repolarization Hyperpolarization - K
Resting potential of muscle cell membrane
- Cl-
closed
open
inactivated
closed
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Chloride channels ClCs

• A large conservative family
• A wide variety of physiological functions
• cell volume regulation and ionic homeostasis
• transepithelial transport
• regulation of electrical excitability

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Chloride channels ClCs

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Chloride channels ClCs

• A large conservative family
• A wide variety of physiological functions
• cell volume regulation and ionic homeostasis
• transepithelial transport
• regulation of electrical excitability
• Still a relatively young research field
• torpedo ray electric organ
• 1980 recordings
• 1991 identification on a molecular level
• 2004 crystal structure

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Chloride channels ClCs

• Unique structure

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Chloride channels ClCs

• Unique structure
• Homodimer - two identical subunits

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Chloride channel subunit
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Chloride channels ClCs

• Unique structure
• Homodimer - two identical subunits
• Double-barreled architecture
• Fast gating and slow gating

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Chloride channels ClCs

• Unique structure
• Homodimer - two identical subunits
• Double-barreled architecture
• Fast gating and slow gating
• Fast gating
• Independent
• Strongly dependent on extracellular chloride
• Slow gating
• Common
• less understood, inactive
• exceedingly slow and very sensitive to
  temperature

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Myotonia and chloride channel

• Myotonia
• Chloride channel
• Myotonia congenita
• Dystrophic myotonia

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Myotonia congenita (MC)

• Thomsen autosomal dominant
• Becker autosomal recessive
• Prevalence
• Worldwide 0.2-0.9100,000
• Northern Scandinavia
• Northern Finland 7.2100,000
• Northern Norway 9.4100,000
• Northern Sweden ?

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Clinical features

• Myotonia
• Hypertrophy

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Clinical features

• Myotonia
• More pronounced after rest
• Underextremities
• Warm-up
• Hypertrophy
• Calve muscle

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Clinical features

• Myotonia
• More pronounced after rest
• Underextremities
• Warm-up
• Hypertrophy
• Calve muscle
• Transient muscle weakness
• Becker type

Variability
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Molecular genetics

• CLCN1
• encodes the major muscle chloride channel protein
  ClC-1 (988 aa)
• 7q35
• 23 exons, coding sequence 2964 bp

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Molecular genetics

• CLCN1
• CLCN1 mutations gt 60
• All types of mutations
• Majority recessively inherited
• Dominantly inherited 7
• Both 9
• Spread over the whole CLCN1 gene
• no cluster for dominantly or recessively
  inherited mutations

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CLCN1 mutations
missense
deletion
nonsense
insertion
splicing
blue- - dom/rec
red - dominant
white - recessive
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Molecular genetics

• CLCN1
• CLCN1 mutations
• CLCN1 mutations in Northern Scandinavia
• High heterogeneity
• Novel mutation for the population
• Founder effect

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Spectrum of CLCN1 mutations and polymorphisms
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Molecular genetics

• CLCN1
• CLCN1 mutations
• CLCN1 mutations in Northern Scandinavia
• High heterogeneity
• Novel mutation for the population
• Founder effect
• Recessive mutations in dominant pedigree
• Reclassification of MC?

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Molecular genetics

• CLCN1
• CLCN1 mutations
• CLCN1 mutations in Northern Scandinavia
• Some issues
• Reclassification
• Other genes or modified genetic factors
• Location of ClC-1
• Clinical heterogeneity, esp. intergenerational
• Not 100 detection-rate

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Myotonia and chloride channel

• Myotonia
• Chloride channel
• Myotonia congenita
• Dystrophic myotonia

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Dystrophic myotonias (DM)

• The most common form of adult muscular dystrophy
• 2 types DM1 / DM2
• Autosomal dominant inheritance
• Nucleotide repeat expansion in non-coding region
• Multisystemic
• Progressive muscle degeneration

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Dystrophic myotonias (DM)

• Prevalence
• DM1
• Steinerts disease, 1909
• Worldwide 18,000
• Europe 125,000
• DM2
• PROMM, PMD
• 1994
• Same prevalence ?

99 of all DM
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Common clinical features DM1 and DM2

• Highly heterogenous, multisystemic symptoms
• Muscle
• myotonia, progressive muscle weakness and wasting
• Eye
• cataract
• Genital
• testicular atrophy
• Endocrine
• increased g-GT/CK, insulin resistance
• Heart
• cardiac conduction defect
• Others
• Disease severity correlates with repeats length

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Common clinical features DM1 and DM2

• Highly heterogenous, multisystemic symptoms
• Muscle
• (myotonia, progressive muscle weakness and
  wasting)
• Eye (cataract)
• Genital (testicular atrophy)
• Endocrine
• increased g-GT/CK, insulin resistance
• Heart
• cardiac conduction defect
• Others
• Disease severity correlates with repeats length

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Molecular genetics

• DM1
• DMPK, encodes for myotonin protein kinase
• Signal pathway
• triplet nucleotide repeat expansion disease
• (CTG)n, 3 untranslated region

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Molecular genetics

• DM1
• DMPK, encodes for myotonin protein kinase
• Signal pathway
• triplet nucleotide repeat expansion disease
• (CTG)n, 3 untranslated region
• Normal 5-37 repeats
• Premutation 38-50
• Affected gt50
• Congenital gt1000

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Molecular genetics

• DM2
• ZNF9, encodes the zinc finger protein 9
• Function unknown, DNA/RNA binding ?
• first tetra-nucleotide repeat expansion disease
• (CCTG)n, intron 1

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Molecular genetics

• DM2
• ZNF9, encodes the zinc finger protein 9
• 3q13
• 500 aa
• Function unknown, DNA/RNA binding ?
• first tetra-nucleotide repeat expansion disease
• (CCTG)n, intron 1
• Normal unclear
• Affected 75-11,000 repeats
• 5,000 in average?
• - without interrupted non-repeated sequences

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Dystrophic myotonias (DM)
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Molecular pathophysiology

• How could nucleotide expansions in non-coding
  region of a gene give a clinically heterogenous,
  multisystemic disorder ?

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The Central Dogma of Molecular Biology
NUCLEUS
CYTOPLASM
RNA
DNA
Protein
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The Central Dogma of Molecular Biology
NUCLEUS
CYTOPLASM
2. Transcription (RNA synthesis)
RNA
DNA
4. Translation (protein synthesis)
1. DNA replication (DNA duplicates)
3. RNA editing
Protein

• Intron
• PolyA tail
• Alternative splicing

Structural Enzymatic Immunological
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Molecular pathophysiology

• How could nucleotide expansions in non-coding
  region of a gene give a clinically heterogenous,
  multisystemic disorder ?
• Model mechanisms
• Haploinsufficiency
• Chromatin structure change
• Interfere with RNA processing

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Haploinsufficiency

• Cytoplasmic DMPK was reduced in DM1 patients
• Knockout mice
• DMPK-/DMPK- mice
• Late onset mild myopathy
• DMPK-/DMPK- and DMPK/DMPK- mice
• Display cardiac conduction defects
• Isolated skeletal mescle cells and cardiac
  myocytes
• Abnormalities in Na and Ca cycling akin
• Other models are necessary for full explaination

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Chromatin structure change

• Chromatin - gene density - activity
• Nucleotide repeat motifs - hair-pin structure
• DMPK is located in a gene-rich region
• DMWD, SIX5(DMAHP), RSHL1...
• Flanked by nuclear matrix attachment regions
  (MARs)
• Knock out mice
• SIX5- premature cataract
• cardiac condection defects anf testicular atrophy
  

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Interfere with RNA processing

• DM1 cells were associated with multiple nuclear
  foci of mutant DMPK RNA
• Toxic gain-of-function at the RNA level
• Discovery of DM2
• No similarity between DMPK and ZNF9
• No similarity between genes flanking DMPK and
  ZNF9
• Common
• Clinical
• Nucleotide expansion disorders

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Interfere with RNA processing

• CUG-BP1
• Muscleblind-like proteins (MBNL)

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Interfere with RNA processing

• CUG-BP1
• Regulates alternative splicing of cardiac
  troponin(TNNT2), CLCN1 and INSR
• (CTG)n , DMPK or HSA
• Ribonuclear inclusion, myotonia, myopathy
• Muscleblind-like proteins (MBNL)

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Interfere with RNA processing

• CUG-BP1
• Regulates alternative splicing of cardiac
  troponin(TNNT2), CLCN1 and INSR
• (CTG)n , DMPK or HSA
• Ribonuclear inclusion, myotonia, myopathy
• Muscleblind-like proteins (MBNL)
• Splicing irregularities in CLCN1, TNNT2, and
  skeletal muscle troponin T(TNNT3)

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Interfere with RNA processing

• Splicing antagonist
• CUG-BP1/embryonic isoforms,
• MBNL/adult forms
• Do not directly compete with each other
• Require different RNA binding site
• Their expression levels are independently
  regulated

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Cardiac arrythmia
Tau protein
CNS affect
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Takk for oppmerksomheten
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