Myotonic Dystrophy

Question 1

What is the most common adult form of muscular dystrophy?

Answer 1

Myotonic dystrophy

Question 2

There are two types of myotonic dystrophy - what are they and which is most common?

Answer 2

• DM1 and DM2

- approx 98% is DM1

Question 3

What are some common symptoms seen in DM?

Answer 3

• Myotonia
• Cardiac conduction defects
• Cataracts
• Muscle weakness
• Insulin insensitivity
• insomnia
• Lethargy

Question 4

What is the prevalence of myotonic dystrophy type 1?

Answer 4

1 in 8,000

Question 5

What are additional clinical symptoms observed only in male DM patients?

Answer 5

Testicular atrophy and male pattern baldness

Question 6

What is the inheritance pattern for myotonic dystrophy?

Answer 6

• Autosomal dominant

- Anticipation may also occur: increasing disease severity and decreasing age of onset in successive generations

Question 7

What is the cause of myotonic dystrophy type 1?

Answer 7

• Expansion of CTG rpt in DMPK gene (chr19)
• DMPK protein mainly expressed in heart and skeletal muscle and codes for a kinase involved in development of muscle fibres

Question 8

Does the number of CTG repeats correlate with DM phenotype?

Answer 8

• Number of CTG rpts positively correlates to severity of phenotype and negatively correlates with age of onset

Question 9

If genetic testing for DM1 is normal, what else could you consider?

Answer 9

• DM2
• Weakness mostly proximal, mild myotonia, muscle pain
• Caused by CCTG expansion in first intron of the CNBP gene (previously ZNF9)

Question 10

What investigations tend to be carried out once a genetic diagnosis of DM1 is confirmed?

Answer 10

• ECG for heart block
• Fasting blood glucose for diabetes
• Opthamology if cataracts suspected

Question 11

What are the rpt allele size categories for DM1?

Answer 11

• Normal: 5-35 rpts (no phenotype and low prob of expansion on transmission)
• Intermediate: 36-50 rpts (no phenotype but may be unstable on transmission and potentially expand into affected range)
• Affected: greater than 51 rpts (classic DM usually between 100-1000 rpts and over 1000 likely to cause congenital DM)

Question 12

What are the allele size categories for DM type 2?

Answer 12

• Normal alleles have up to 26 repeats

- Affected individuals have between 75 and 11,000 repeats

Question 13

Provide some details on repeat expansion in DM1

Answer 13

• Alleles between 50-70 rpts will be transmitted stably in approx 25% of cases (majority expand to less than 200 rpts)
• Alleles between 70-90 rpts are very unlikely to be transmitted stably (approx 60% will expand above 200 rpts)
• Mutations with 100 or more rpts have high risk of expanding into congenitally affected range

Question 14

Provide details on parent of origin for repeat expansion in DM1

Answer 14

• If parental allele is relatively small the sex of the parent does not affect the risk of expansion
• Larger expansions are predominantly of maternal origin
• Males with adult onset DM1 will rarely have congenitally affected children

Question 15

What characterises congenital DM at birth?

Answer 15

• Hypotonia
• Severe generalised weakness at birth
• Often respiratory compromise and early death
• survivors often have sig. dev delay and develop progressive myopathy

Question 16

How may congenital DM present antenatally?

Answer 16

• Polyhydramnios
• Reduced foetal movements
• Talipes

Question 17

How are the problems observed with amplification if DM1 alleles overcome?

Answer 17

A combination of techniques are used: repeat sizing PCR and TP-PCR. Often run in parallel to save time

Question 18

What are the advantages and limitations of repeat sizing PCR?

Answer 18

Advantages: cheap and easy to set up, quick method to rule out DM in patients with two normal alleles of different sizes, accurate sizing of these normal alleles

Limitations: SNP under primer site could give false negative, can’t detect expansions over ~80rpts, can’t differentiate between homozygous alleles and one normal allele with a large undetectable expansion

Question 19

Describe the three primer system involved with TP-PCR

Answer 19

1. Forward primer - binds to sequence upstream of the rpt. Fluorescently labelled for capillary electrophoresis
2. Reverse primer - 3’ end is complementary to the rpt so primes at multiple locations. 5’ end is non-specific. Present at a limited conc.
3. Tail primer - specific to the 5’ end of the reverse primer. Allows further amplification of products from FW and RV primers.

Question 20

Why is the TP-PCR reverse primer present at a limited conc?

Answer 20

So that it is exhausted after first few PCR cycles to prevent priming from the PCR products that would lead to a decreasing length of products in final pool

Question 21

What are the advantages and limitations of TP-PCR?

Answer 21

• Advantages: cheap and simple, quicker than southern blotting, can be run in parallel with sizing PCR
• Limitations: Interruptions in rpt or SNP under primer site can give false negative, does not size the expansion, sensitive to MCC if used for PND

Question 22

What are the three main theories for the pathogenesis of DM?

Answer 22

1. Haploinsufficiency
2. Change to chromatin structure
3. RNA toxicity/gain of function (thought to account for majority of phenotype)

Question 23

Provide details on the RNA gain of function theory of DM pathogenesis

Answer 23

• mRNA containing the expanded rpt is not exported to the cytoplasm and forms aggregates/foci within the nucleus
• Abnormal interaction with RNA foci and RNA binding proteins leads to DM phenotype
• Muscleblind-like (MNBL) family of RNA binding proteins have been shown to be sequestered by the CUG containing RNA foci resulting in a functional depletion of MNBL proteins in cell (involved in regulation of alternative splicing)

Question 24

What is the evidence supporting the RNA toxic gain of function theory of DM pathogenesis?

Answer 24

• Similarity between DM1 and DM2 phenotypes due to common mutation type rather than the function of the mutated gene
• Links in to mice models knocked out for the MNBL proteins that show a wide range of DM-associated phenotypic features

Question 25

Provide some details on prenatal diagnosis for DM

Answer 25

• One parent must be known to have an expansion
• Requires both sizing PCR and TP-PCR
• Important to exclude MCC
• due to sensitivity of TP-PCR this MCC result must be backed up with linked microsatellite markers/southern blot if mother carries the expansion