DMD Genetics

What is the mainstay of the current treatment for DMD?

*Steroids is the main stay of treatment:* - Slow progression of muscle weakness and prolong ambulation - Thought to possible up regulate genes in muscle fibres - May also up regulate utrophin Surgery - scoliosis PT, OT Promoting bone health - vitamin D and calcium

What are cell based therapies? What are the pro's and con's?

Cell-based strategies involve transplantation of healthy myoblasts into patients, and as such are handicapped by issues of immune rejection and poor systemic delivery and viability of transplanted cells. Different cell types have been candidates.

How is a diagnosis of DMD made?

Clinical Genetic testing (MLPA and full dystrophin gene sequencing) Muscle biopsy - High CK Genetic counselling DMD is usually clinically defined as producing no detectable dystophin and loss of ambulation before the age of 12. However, due to genetic heterogeneity - this phenotype is wide.

Why did they consider viral mediated gene therapy as an option?

Conventional gene-based strategies aiming to deliver functional copies of DMD in patients have turned out problematic, mostly due to poor delivery (owing to the large, complex structure of the gene) and the activation of an immune response in cases when a viral vector is used. However, the identification of patients with a mild phenotype who carry large deletions led to a suggestion that delivery of specific domains of dystrophin could be functional. Vector-mediated gene therapy for DMD consists of delivering functional domains of dystrophin via viral or non-viral vectors to restore dystrophin.

What role does dystrophin play in other tissues such as the brain or eye?

Dystrophin is required for the normal functioning of the retina. Often electro retinopathy is abnormal in DMD and BMD patients Dystophin also plays a key role in the brain. Those with DMD have lower than normal IQ's and have associated disorders such as ADHD, anxiety and cognitive difficulties. Dystrophin normally modulates synaptic terminal integrity, distinct forms of synaptic plasticity and regional cellular signal integration. At a systems level, dystrophin may regulate essential components of an integrated sensorimotor attentional network.

How does Eteplirsen work?

Exon skipping is a therapeutic approach that can correct the disrupted reading frame in patients with DMD. The reading frame is corrected by skipping a targeted exon with pre-designed antisense oligonucleotides. This restores the reading frame of DMD by modulating the dystrophin pre-mRNA splicing process. Eteplirsen is a drug that acts to promote dystrophin production by restoring the translational reading frame of DMD through specific skipping of exon 51 in defective gene variants. Eteplirsen is applicable for approximately 14% of patients with DMD mutations. In this approach, the translational reading frame of a gene is restored using synthetic nucleic acid analogs called antisense oligonucleotides (AOs) to interfere with pre-messenger RNA (mRNA) splicing. AOs are employed to bind target complementary sequences in the pre-mRNA, which influence the splicing machinery to exclude an exon (or exons) from the final transcript. Eteplirsen (green bar) specifically recognizes exon 51 of the DMD gene. Upon binding, it influences the splicing machinery to skip exon 51 from the mature mRNA transcript. This restores the reading frame of DMD, allowing for successful translation of a shortened but functional dystrophin protein.

Give a therapy that is used to cause exon skipping

Exon skipping: Eteplirsen:

Can females have clinical features of DMD? What are the most common causes of death for DMD? What is the general 'natural history' of the disease

Females are often asymptomatic, but can have mild symptoms such as calf hypertrophy. Moreover, cardiac screening in >45 females is recommended. Causes of death - Respiratory complications - Cardiac related conditions - Dilated cardiomyopathy

How many mutations are known to occur in the DMD gene? What sort of mutations are they? Where in the DMD gene are the mutational hotspots?

More than 5000 mutations in the DMD gene have been identified in people with DMD and BMD. 2/3rds of DMD mutations are deletions that span one or multiple exons. These tend to cluster around two mutational hotspots (exon 45 to exon 55 and exon 3 to 19). If the reading frame is affected, the phenotype is severe. They are loss of reading frame mutations due to; -Out-of-frame intragenic deletions (~65%) -Out of frame intragenic duplications (~10%) -Nonsense mutations (~10%) -Splice site mutations and other small mutations (~15%)

List some classical clinical features of DMD

Muscular dystrophy is a collection of inherited diseases, characterised by skeletal muscle weakness and degeneration. DMD is one of the most common forms of this DMD presents in childhood with delayed motor milestones. BMD is characterised with later onset skeletal muscle weakness, with a larger phenotypic spectrum. Most patients die of heart complications.

What is BMD? Why is the phenotype less severe?

Note that Becker Muscular dystrophy is the result of an in-frame deletion within the dystrophin gene. Because it is in frame more of the gene can be read, and therefore, a more functional protein can be produced. The two exons can be read next to each other. It does produce a truncated and less stable protein, but the protein works. The reading frame rule is not always true - and may suggest that particular regions of the gene are more important. This is why the phenotype of Becker's is much less severe. The BMD phenotype occurs when some dystrophin is produced, usually resulting from deletions or duplications that juxtapose in-frame exons, some splicing variants, and most non-truncating single-base changes that result in translation of a protein product with intact N and C termini. The shorter-than-normal dystrophin protein molecule, which retains partial function, produces the milder BMD phenotype

Does the penetrance vary in DMD?

Penetrance in males is complete. Penetrance in females varies, often depending on the patterns of x-chromosome inactivation (skewed inactivation or other chromosomal conditions).

What are read through therapies? Give an example of read through therapies for DMD

Read through therapies is an approach available to patients with nonsense mutations. This results in a premature stop codon leading to a truncated protein which is susceptable to NMD. Ataluren is thought to make ribosomes less sensitive to premature stop codons (referred to as "read-through") by promoting insertion of certain near-cognate tRNA at the site of nonsense codons with no apparent effects on downstream transcription, mRNA processing, stability of the mRNA or the resultant protein, thereby making a functional protein similar to the non-mutated endogenous product. It seems to work particularly well for the stop codon 'UGA'. Therefore it causes the ribosome to bypass a premature stop codon and allows formation of functional protein For DMD/BMD, nonsense mutations are causative in ~15% of patients Seems to be beneficial in stopping the progression of losing ambulation

What do you see in the histology of DMD?

See the muscle fibres are separated, with fat and connective tissue The nuclei become centralised On the lower right - you can see big necrotic regions, lots of fat and lots of fibrotic tissue

Where is the DMD gene? Is it special?

The DMD gene, encoding the dystrophin protein, is one of the longest human genes known, covering 2.3 megabases (0.08% of the human genome) at locusXp21 and has 79 exons. Due to this HUGE gene - it is susceptible to mutations Depending on where the mutation is, the phenotype may be different

What determines the genotype phenotype correlation?

The distinction between BMD and DMD can sometimes be difficult. Reading frame rule - This states that pathogenic variants that do not alter the reading frame generally correlate with a milder phenotype. The degree of expression of the dystrophin gene is correlated with phenotype

How can viral based gene therapy work in DMD?

The dystrophin gene is too big for viral vectors - it's impossible to put the entire gene inside. Instead, certain more important bits of the gene are being inserted - to create a micro-dystrophin (which is smaller and less stable). This may modify the phenotype but not cure the disease. Below, you can see that there is an improvement in phenotype in dog studies The issue is that antibodies develop against the vector (~4/5 years). You may have to give immunosuppression to counteract this. However, in 2006 the human trials were initiated - with unfavourable results (particularly around lack of transgene expression).

List some different possible therapeutic approaches to DMD Restore vs compensate

Therapeutic approaches can be split into those that aim to restore dystrophin expression and those that aim to compensate for the lack of dystrophin. *Restoring dystrophin expression:* ○ Read through therapy ○ Exon skipping therapy ○ Vector mediated gene therapy ○ Cell therapy *Compensate for lack of dystrophin/limit progression:* ○ Anti-inflammatory ○ Anti-fibrotic ○ Anti-oxidants ○ Utrophin upregulation therapy ○ More.

What is dystrophin? What is its function in the muscle fibre?

This is a protein with a major structural role in muscle Connects the cytoskeleton of the muscle fibre to the surrounding ECM through the cell matrix Links actin filaments to other support proteins and protects the sacrolemma from contraction-induced injury. If it isn't working properly (as in DMD), the more you use the muscle, the more it gets damaged. This produces a scar in the muscle with fat replacement. It also supports concentric exercise (Bicep curl) Dystophin may also play a role in delivering oxygen to the muscle in microcirculation.

What is the inheritance pattern of DMD?

X-linked recessive disorder

How may CRISPR be used in gene therapy for DMD?

Young et al., 2016 Cell stem cell.