18.03.15 RNA mediated GoF in DM1 and 2

Question 1

What is the locus and mutation associated with DM1?

Answer 1

CTG expansion in the 3’UTR of DMPK (19q13.3)

Question 2

Give some of the clinical features of myotonic dystrophy.

Answer 2

Distal muscle weakness and wasting
Dysmotility of GI muscles and biliary tree
Dementia
Swan-like neck
Testicular atrophy
Relflexes depressed or absent
Ocular cataract and ptosis
Pilomatrixomas (skin,) pulmonary infections
Haggard or Hatchet facies, Hair loss (frontal balding)
Intrinsic muscles of hand wasting. IQ low, IBS
Cardiomyopathy and arrthymia.

Question 3

What is the function of DMPK?

Answer 3

serine-threonine kinase, mainly expressed in heart and skeletal muscle

Question 4

What are the repeat classes for DM1?

Answer 4

Normal alleles 5-36 repeats
Premutation 37-50
Full penetrance alleles 51-150 (minimal or classical DM1) >150 classical, juvenile or congenital DM1

Question 5

What is the locus and mutation associated with DM2?

Answer 5

Expansion of complex repeat motif (TG)n(TCTG)n(CCTG)n in intron 1 of the CNBP (formally known as ZNF9) gene (3q21) encoding CNBP protein – cellular nucleic acid binding protein (zinc finger protein 9)

The largest number of uninterrupted CCTG repeats is 9

Question 6

Why do molecular based tests (other than sequencing) give sizes based on bp and now number of repeats?

Answer 6

All three repeat tracts are present as a complex motif on all normal and pathogenic alleles, additionally, the CCTG repeat tract in normal alleles typically contains one or more tetranucleotide interruptions (TCTG or GCTG).

The overall length of the (TG)n(TCTG)n(CCTG)n complex repeat in normal alleles ranges from 104 to 176 base pairs.

Because TG and TCTG repeat tracts are highly polymorphic, allele sizes determined by methods other than sequencing are reported in overall base-pair length rather as a definable number of CCTG repeats

Question 7

Give a difference between DM1 and DM2?

Answer 7

1. Less severe phenotype in DM2

2. DM2 does not show anticipation: Length of repeat correlated with age at onset but not severity

Question 8

What are the repeat classes for DM2?

Answer 8

Normal CCTG repeat tract, including any GCTC or TCTG interruptions ranges from 11 to 26 tetranucleotide repeats

In 85% of unaffected individuals, the overall lengths of the complex repeat tract clearly differ on the two alleles and are distinct on analysis of PCR amplicons

Full penetrance, mutation alleles are greater than 75 repeats, up to 11,000 repeats.

Question 9

What are the three proposed models for the pathogenicity of DM alleles?

Answer 9

The mechanism by which the expanded CTG repeat leads to the multisystemic clinical phenotype of DM1 is not clear. Because of the location of the (CTG)n in the 3’ UTR, the gene’s coding region remains intact in the mutant DMPK gene, however the (CTG)n repeat is transcribed into the mRNA as a (CUG)n repeat.

1. Haploinsufficiency
2. Chromatin structure
3. RNA gain of function

Question 10

Describe the proposed haploinsufficiency model for DM pathogenesis. What is the evidence for/against?

Answer 10

Due to decreased levels of DMPK mRNA and protein in the adult DM1 tissue, DMPK deficiency was proposed originally.

Knockout -/- DMPK mice develop mild, age-onset, progressive skeletal myopathy, as well as cardiac conduction abnormalities and metabolic impairment. However symptoms were mild suggesting DM1 phenotype is not caused simply by DMPK haploinsufficiency.

Also no DMPK point mutations identified in any DM1 cases

Question 11

Describe the proposed chromatin structure model for DM pathogenesis. What is the evidence for/against?

Answer 11

This model is based on the effect exerted by CTG expanded repeats on chromatin structure which may lead to partial silencing of neighbouring DMWD and SIX5 genes (3’ end of DMPK contains regulatory region of SIX5).

DMWD levels have been reported as decreased in DM1 patients and decreased SIX5 may participate in muscle-wasting and mesodermal components of DM1.

Doesn’t explain entire phenotype or similarity between DM1 and DM2

Question 12

Describe the proposed RNA GoF model for DM pathogenesis. What is the evidence for/against?

Answer 12

1. Proposes that CUG repeats in the pathogenic range fold into RNA hairpins that are not exported from the nucleus but accumulate within the ribonuclear foci, acquiring a new toxic function by trapping essential cellular RNA-binding proteins including alternatively-spliced modulators and transcription factors (TF’s).
2. The expansions cause DM pathologies through accumulation of mutant RNAs that alter RNA metabolism in patients’ tissues by targeting RNA-binding proteins such as CUG-binding protein 1 (CUGBP1) and Muscle blind-like protein 1 (MBNL1).
3. MBNL1 and CUGBP1 have antagonistic splicing activities, and function to regulate spliced isoforms during normal skeletal muscle and heart development (Ranum and Cooper, 2006).
4. In DM1, a reduction in MBNL1 splicing activity and increase in CUGBP1 splicing activity results in accumulation of embryonic-specific transcripts and their protein products in adult muscle, affecting muscle function and leading to muscle atrophy
5. MBNL1 knockout mice show whole range of DM phenotypes

Question 13

What are the therapeutic approaches for DM1 and DM2?

Answer 13

Two major approaches have been used to reduce the toxicity of the mutant CUG repeats in pre-clinical studies of DM1.

1. The first approach is based on reduction of the levels of mutant CUG RNA by antisense oligonucleotides (ASOs)
2. The second involves techniques that “normalize” the activities of the RNA-binding proteins affected by the mutant CUG repeats, such as MBNL1 and CUGBP1. The main challenge lies in efficient delivery to the multiple target tissues.