DMD/BMD - Part 1

Question 1

What is one of the main clinical distinctions between BMD and DMD?

Answer 1

The age of wheelchair dependency. Usually less than 13 years for DMD and more than 16 years for BMD.

Question 2

What is the incidence of DMD?

Answer 2

1 in 3,500 males.

Question 3

What is the incidence of BMD?

Answer 3

1 in 18,000 males.

Question 4

What does the class of dystrophinopathies include?

Answer 4

DMD, BMD and X-linked cardiomyopathy.

Question 5

What is DMD caused by?

Answer 5

Mutations in the dystrophin gene at Xp21.2.

Question 6

What pattern of inheritance do the dystrophinopathies follow?

Answer 6

The dystrophinopathies follow an X-linked recessive pattern of inheritance - e.g. carrier women pass the disorder on to affected sons.

Question 7

What is the penetrance of dystrophinopathies in males?

Answer 7

Penetrance is 100% in males.

Question 8

Are females affected by dystrophinopathies?

Answer 8

• Women are not normally affected.
• 2.5% of female carriers have muscle symptoms according to a 1989 study. More recent studies estimate this figure to be between 5% and 10%.
• The incidence of cardiomyopathy my be higher than muscle symptoms in females.
• It is believed that non-random X-inactivation is responsible for clinically affected females.

Question 9

What is thought to be responsible for clinical symptoms of dystrophinoathies presenting in females?

Answer 9

It is believed that non-random X-inactivation is responsible for clinically affected females.

Question 10

What might a typical muscular dystrophy pedigree look like?

Answer 10

• Only males will usually be affected.
• In a typical DMD pedigree the mutation is carried through unaffected carrier females and maternal uncles or cousins may be affected.
• Each carrier female has a 50% chance of passing the mutation to her offspring.
• Males with DMD are unlikely to reproduce so they will not pass on their mutation.
• Due to the milder nature of BMD affected males may have children of their own. In BMD families where the father is affected and the mother is unaffected all female offspring will be carriers and all male offspring will be unaffected.

Question 11

What is the likelihood of a DMD carrier female passing on her mutation?

Answer 11

Each carrier female has a 50% chance of passing the mutation to her offspring.

Question 12

What is the mutation rate like in the dystrophin gene?

Answer 12

• The dystrophin gene has a high mutation rate.
• Approximately 1/3 of the mutations are de novo.
• This means 2/3 of mothers with a son with DMD and no other family history will be carriers.
• Germline mosaicism = 7-10%

Question 13

What is the rate of germline mosaicism for DMD in the mothers of affected children?

Answer 13

Germline mosaicism = 7-10%

Question 14

At what points might de novo mutations in the dystrophin gene occur?

Answer 14

1) . The mutation may occur in the egg at the time of the proband’s conception. This mutation would be present in every cell of the proband’s body. The mother does not carry the mutation so there is no recurrence risk.
2) . The mutation may occur after conception and not present in all cells of the proband. The proband is somatic mosaic. The mother does not carry the mutation and so there is no recurrence risk.
3) . The mutations is present in the mother of the proband’s egg cells. The mutation will not be detected in the DNA extracted from the blood. The mother has germline mosaicism and there is a risk to further children.

Question 15

In what circumstances may a female present with classical DMD?

Answer 15

1) . A translocation - the female may have an X-autosome translocation that could result in disruption of the gene. The normal X will be inactivated in all the cells otherwise there would be a lethal imbalance. There fore if the dystrophin gene is disrupted the female will have no normal dystrophin and no compensating mechanism.
2) . Turner Syndrome (X0) - if a female has Turner Syndrome she will have only 1 X. Therefore if this X has a mutation then they will be affected with DMD.
3) . Uniparental Isodisomy - maternal uniparental isodisomy is another method which could cause classical DMD. This is when a daughter inherits 2 copies of the same chromosome from her mother. If this chromosome has a DMD mutation then the individual will have no normal dystrophin and will be affected.
4) . Skewed X-inactivation - skewed x-inactivation resulting in an X-chromosome carrying a DMD mutation remaining active in a disproportionate number of cells can also cause classical DMD.
5) . Father affected with BMD and mother a carrier - father affected with BMD may have children with a carrier female and daughters would be at risk of inheriting a mutation on both X-chromosomes.

Question 16

Describe the dystrophin protein and the dystrophin-associated protein complex (DAPC).

Answer 16

• The dystrophin gene encodes the dystrophin protein. This protein is part of the dystrophin-associated protein complex (DAPC).
• The DAPC forms the link between the actin cytoskeleton and the ECM.
• This complex stabilises the sarcolemma during repeated rounds of contraction and relaxation. It is important in the maintenance of muscle integrity.
• There is also evidence that the DAPC is involved in cell signalling.

Question 17

What forms the link between the actin cytoskeleton and ECM?

Answer 17

The DAPC forms the link between the actin cytoskeleton and the ECM.

Question 18

What is the function of the DAPC?

Answer 18

• The dystrophin gene encodes the dystrophin protein. This protein is part of the dystrophin-associated protein complex (DAPC).
• The DAPC forms the link between the actin cytoskeleton and the ECM.
• This complex stabilises the sarcolemma during repeated rounds of contraction and relaxation. It is important in the maintenance of muscle integrity.
• There is also evidence that the DAPC is involved in cell signalling.

Question 19

Describe the structure of the dystrophin protein.

Answer 19

The dystrophin protein has 4 domains:

1) . Amino terminal - binds actin filaments
2) . Rod-like domain - 24 spectrin-like triple helical coiled coils (much of this is dispensable)
3) . Cysteine-rich domain
4) . Carboxy terminal - interacts with integrl membrane proteins such as sarcoglycan and dystroglycan

Question 20

Describe the pathogenesis of DMD and BMD. What effects does a lack of dystrophin have?

Answer 20

• In patients with DMD the dystrophin protein is virtually absent.
• In patients with BMD dystrophin levels of 10-40% of normal OR protein present but with reduced function.

Lack of dystrophin has a number of effects:

• A lack of dystrophin affects the formation of the DAPC and causes disruption of the link between cytoskeletal actin and ECM.
• The cell membrane is then more fragile and can be mechanically damaged during eccentric muscle contraction.
• It has also been suggested that looseness in the sarcolemma permits calcium channels to open - increase in calcium ions activates calpain proteases which digest contractile proteins = much weaker muscles.
• Studies indicate that dystrophin-deficiency disrupts subsarcolemmal mitochondria localisation, promotes mitochondrial inefficiency and restricts maximal mitochondrial ATP-generating capacity.

Other members of the DAPC have been implicated in other muscle wasting disorders.

Question 21

What other members of the DAPC have been implicated in muscle wasting disorders apart from dystrophin in DMD/BMD?

Answer 21

• Mutations in the genes encoding sarcoglycans can cause Limb Girdle Muscular Dystrophy (LGMD).
• Defects in laminin alpha 2 can cause merosin deficient congenital muscular dystrophy (MDC).

Question 22

Describe the dystrophin gene itself.

Answer 22

• The dystrophin gene is the largest known human gene at 2.4Mb.
• Only 0.3% of the genomic sequence is present in the mature transcript.
• The gene has 79 exons encoding 14kb of mRNA.
• There are at least 7 different promoters.
• There are 3 tissue-specific promoters that produce large dystrophin proteins of the same size but all have different exon 1s. These include a brain-specific promoter, a muscle-specific promoter and a Purkinje-specific promoter.
• At least 4 other promoters can be used which produce smaller proteins expressed in the retina, brain, schwann cells or ubiquitously.

Question 23

What size is the dystrophin gene?

Answer 23

The dystrophin gene is the largest known human gene at 2.4Mb.

Question 24

What type of mutation is the most frequent in DMD and BMD?

Answer 24

Deletions of 1 or more exons are the most frequent type of mutation accounting for about 65% of DMD mutations and 85% of BMD mutations.

Deletions can be spread throughout the gene but there are 2 hotspots. These include a proximal hotspot between exons 2 and 20 and a distal hotspot between exons 45 and 55.

Question 25

What percentage of mutations do deletions of 1 or more exons account for in DMD?

Answer 25

65%

Question 26

What percentage of mutations do deletions of 1 or more exons account for in BMD?

Answer 26

85%

Question 27

Where are deletions in the dystrophin gene most likely to occur?

Answer 27

Deletions can be spread throughout the gene but there are 2 hotspots. These include a proximal hotspot between exons 2 and 20 and a distal hotspot between exons 45 and 55.

Question 28

What is the frameshift hypothesis and what can it be used to predict? What cautions should one be aware of when using the frameshift hypothesis?

Answer 28

The Frameshift Hypothesis:

• The frameshift hypothesis can be used to predict whether a mutation is likely to cause a BMD or DMD phenotype.
• Deletions which disrupt the translational reading frame generally cause a severe DMD phenotype as they result in a premature termination codon leading to nonsense mediated decay of mRNA and no protein being produced.
• Deletions leaving the reading frame intact generally cause the milder BMD phenotype as although the protein is abnormal it retains some function.
• The frameshift hypothesis holds true in about 92% of cases.

Cautions:

• The prediction is based on assessment at the DNA level and it may be different at the RNA level. For example, a deletion may affect splicing and cause exon skipping of an exon that is not deleted. Therefore the prediction of whether the deletion is in frame may or may nor be true or there may be initiation of translation downstream of the the deletion.
• Deletions in protein-binding domains may severely affect dystrophin function even if they are in frame.
• Dystrophin can retain significant function even when missing large portions of its amino acid sequence. For example, deletions affecting the central rod domain may be associated with very mild or no manifestations.

Question 29

Deletions make up the majority of dystrophin mutations in DMD and BMD. What is the next most common type of mutation?

Answer 29

• Duplications of one or more exons occur in 5-10% of DMD/BMD cases.
• Non-contiguous duplications and complex rearrangements have been reported.
• Accurate assessment of the consequences of a duplication are important when applying the frameshift hypothesis to duplications because the orientation and position of the duplication is not usually known. It is normally applied assuming it is a tandem duplication.

Question 30

The majority of BMD and DMD mutations are deletions or duplications. What mutations make up the remainder of cases?

Answer 30

• The remaining types of mutations seen in the dystrophin gene are point mutations including Nonsense mutations, Splice site mutations, Small deletions/insertions, Missense mutations, and Intronic mutations.
• In DMD these account for about 25-35% of cases and they usually result in a premature termination codon.
• In BMD point mutations account for about 10-20% of cases. The point mutations in BMD cases may be missense or splice site mutations that don’t result in a change to the reading frame. However, you can also get nonsense mutations that cause exon skipping or are in the 3’ end of the gene and therefore avoid nonsense mediated decay.

Question 31

Summarise the types of mutation that are found in the dystrophin gene.

Answer 31

1). Deletions:

Deletions of 1 or more exons are the most frequent type of mutation accounting for about 65% of DMD mutations and 85% of BMD mutations.

Deletions can be spread throughout the gene but there are 2 hotspots. These include a proximal hotspot between exons 2 and 20 and a distal hotspot between exons 45 and 55.

2) . Duplications:
- Duplications of one or more exons occur in 5-10% of DMD/BMD cases.
- Non-contiguous duplications and complex rearrangements have been reported
3) . Point Mutations:
- The remaining types of mutations seen in the dystrophin gene are point mutations including Nonsense mutations, Splice site mutations, Small deletions/insertions, Missense mutations, and Intronic mutations.
- In DMD these account for about 25-35% of cases and they usually result in a premature termination codon.
- In BMD point mutations account for about 10-20% of cases. The point mutations in BMD cases may be missense or splice site mutations that don’t result in a change to the reading frame. However, you can also get nonsense mutations that cause exon skipping or are in the 3’ end of the gene and therefore avoid nonsense mediated decay.

Question 32

What mutations exclusively cause DMD-related cardiomyopathy?

Answer 32

• There are some mutations that exclusively cause DMD-related cardiomyopathy.
• This can happen if the mutations specifically disrupts the muscle-specific isoform of dystrophin. A deletion of the muscle-specific promoter has been reported in cases of cardiomyopathy. In other tissues compensation as a result of upregulation of other promoters occurs but this does not happen in the heart.