DM1 1 and 2

Question 1

What is DM1 stand for?

Answer 1

Myotonic dystrophy

Question 2

What is myotonic dystrophy?

Answer 2

Multisystem NMD, characterized by muscle weakness, wasting, & myotonia, Microsatellite repeat expansion in DMPK 3’ UTR (DM1) or CNBP 1st intron (DM2)

Question 3

What does the toxic gain of function cause?

Answer 3

Toxic gain-of-function causes widespread dysregulation of pre-mRNA splicing (spliceopathy)

Question 4

What type of genetic mutation of DM?

Answer 4

autosomal dominant

Question 5

If one parent has one affected allele what is the percentage kids will be affected?

Answer 5

50%

Question 6

Prevalence of DM?

Answer 6

DM is second most common MD after DMD, most common adult MD, prevalence = ~1/8,000

Question 7

What % DM1 and its future?

Answer 7

98% of DM is DM1
• ~15% of DM1 is fetal-onset congenital DM1
• ~10% of DM1 is childhood-onset, beginning 1-10 yrs old
• ~75% of DM1 is adult-onset, with patients developing symptoms in 2nd, 3rd, 4th decade

Question 8

where is DM highly prevalent?

Answer 8

High prevalence in PQ, particularly in Charlevoix and Saguenay–Lac St-Jean regions, ~1/600

Question 9

Diagnosis of DM1?

Answer 9

• DM1 patients usually have characteristic thin, long face, hollow temples and, in men, early frontal balding
• DM causes muscle weakness and wasting, usually beginning in face, neck, and distal limbs
• Myotonia = delayed relaxation (prolonged contraction)
• Myotonic myopathy includes action (grip & release) and percussion myotonia
• When clinical signs point to DM1, genetic testing definitive
• Histological evidence of central nuclei, fiber size variability

Question 10

What causes DM1?

Answer 10

• DM1 caused by CTG repeat expansion in the 3’ UTR of the DMPK gene
• Mutation causes formation of CUG)n in DMPK transcripts, nuclear aggregation of DMPK mRNA (foci), sequestration of RNA binding proteins = DMPK mRNA toxic gain-of-function
• DMPK protein reduced, however this loss-of-function mechanism does not cause DM1

Question 11

at DNA level what is expanded?

Answer 11

CTG expansion in 3 pime uTR area of DMPK gene

Question 12

at mRNA level?

Answer 12

CUG is added

Question 13

In healthy individuals how many CTG repeats do we have? And what is the threshold

Answer 13

5 is most common, ranges from 5-28, largest number of repeats in healthy individual is 37

Question 14

Do carries of pre-mutation present symptoms?

Answer 14

No or at least very mild sympotoms such as cateracts

Question 15

Explain correlation b/w repeats, severity and age of onset for DM1

Answer 15

INCREASED repeats = increase servierty and early onset

Question 16

Important aspects of DM1 pathobiology:

Answer 16

anticipation and instability

Question 17

Repeat instability

Answer 17

• Variations in CTG)n lengths between tissues can occur during multiple stages of development
• Against expectations, CTG)n more unstable in nondividing cells of muscle, heart, brain, than in proliferating blood cells
• In skeletal muscle, DM1 expansion typically grows to >2,000 repeats by 20 yrs old, and in patients over 40, average repeat length in muscle >4,000, 3- to 25-fold larger than in blood
• Thus, CTG)n expansion size depends on tissue sampled
& age

Question 18

Genetic anticipation

Answer 18

Genetic anticipation a phenomenon in which disease severity increases and/or age of disease onset decreases from one generation to the next
• In DM1, anticipation often occurs from generation to next, with larger CTG expansions, younger onset, & more severe symptoms in each subsequent
generation
• CTG)n increases >200 repeats per generation
• Anticipation not inevitable, occasionally CTG)n undergoes intergenerational contraction

Question 19

What do the CUG expansions on mRNA DMPK do?

Answer 19

CUG expansions form nuclear foci caused by folding of CUG)n into double-stranded hairpin structures that sequester muscleblind (MBNL1) & upregulate CELF1 (aka CUGBP1) RNABPs

Question 20

Microsatellite expansion within DMPK 3’ UTR sequesters MBNL1, what does MBNL1 do?

Answer 20

mitigates MBNL1- dependent splicing, as well as promotes nuclear retention of DMPK mRNA (nuclear foci)

Question 21

what does PKC activation by expanded CUG do?

Answer 21

induces CUGBP1 (CELF1) hyperphosphorylation, which stabilizes protein & enhances alternative splicing, stability, translation of CUGBP1 targets

Question 22

In spinal cord sections of DM1 patients (A and B), FISH and IF analyses demonstrates:

Answer 22

nuclear (blue) foci of CUG)n mRNA (red) in ChAT-positive (green) α-motoneurons of anterior horn, in DM1 patients, muscle blind and cug foci are centrally located (seeing how it traps MBNL-1

Question 23

Fluorescence in situ hybridization (FISH) and immunofluorescence of tibialis anterior muscle section shows:

Answer 23

myonuclear (blue) colocalization of CUG)n mRNA foci (red) with MBNL1 protein (green) in DM1 patients

Question 24

Pathogenic model of DM1

Answer 24

• Decreased MBNL1 and increased CUGBP1 activity alter splicing of transcripts involved in DM1 pathology, e.g., RNAs encoding chloride channel (CLCN1) and insulin receptor α subunit (IRα)
• Genes responsible for some clinical features, such as cataracts, not yet been identified

Question 25

What does MBNL1 do?

Answer 25

Exon 7a skipping on CLCN-1 gene in the premrna for adult splicing patterns

Question 26

what does the reduced CLCN protein look like?

Answer 26

• DM1 patients exhibit reduced CLCN1 protein to < 10% of levels in healthy individuals
• Loss of CLCN1 protein in skeletal muscle results in augmented excitability & myotonia

Question 27

What leads to the insulin resistance in DM1 patients?

Answer 27

Missplicing of IRa subunit (contains exon 11 and is predominantly expressed in healthy skeletal muscle)

Question 28

What does missplicing of IRa do?

Answer 28

• Missplicing of IRα results in fetal IRα-A isoform with lower signaling capability, causing
impaired insulin-stimulated glucose uptake and insulin resistance (IR) in DM1 skeletal muscle
• IR in DM1 correlates with failure to express mature, functional IRα-B splice variant

Question 29

Is splicing in DM1 patients similar or different

Answer 29

Dysregulated alternative splicing in DM1 skeletal muscle (different for everyone)