Duchenne Muscular Dystrophy

Mutations causing DMD

60-65% patients have deletions 5-15% duplications 20-35% small mutations, intron deletions, exon insertions of repetitive sequences Generally... Frameshift deletions in dystrophin cause DMD - alters the reading frame »site and size of the deletion is very heterogeneous »often a number of exons are deleted » always causes no detectable dystrophin expression - severe clinical symptoms Frame neutral deletions in dystrophin cause BMD - deletion - may be larger than those causing DMD - may be in same region as those causing DMD - but it doesn't disrupt the reading frame -Always results in expression of a lower molecular weight form of dystrophin - less severe symptoms

The DMD gene

Characteristics: largest human gene (2.5 million bp) - so hard to clone located on Xp21 contains multiple introns (99%) and 86 exons many isoforms are formed from these gene so they all have: - different promoters - splice variants takes 14 hrs for it to be transcripted gene product = DYSTROPHIN Mutations cause: Duchenne (severe) or Beckers (mild)

Control of dystrophin splicing

DMD mutations are mostly frameshift antisense mediated exon skipping uses antisense oligonucleotides - synthetic RNA or DNA - targeted to splice site of mutation-containing exon - can cause the exon to be skipped to restore the reading frame - To generate BMD like truncated but partially functional dystrophin Initially tried in - Mouse model of DMD Lu et al. 2003 Nature medicine 9(8):1009 - In vitro in patients cells van Deutekom et al. Hum Mol Genet. 2001 Jul 15;10(15):1547-54.

Genetic testing for DMD

Difficult Many different mutations cause DMD site and type of mutation vary very large gene Mutations 'hotspots' in exons 2-19 & 45-55 - 2 MULTIPLEX PCR's will reveal 98% of all deletions Cloning of the dystrophin gene: 1) characterise mutations associate with DMD 2) predict function of protein encoded by gene 3) reveal molecular pathology underlying DMD 4) generate diagnostic tests 5) design new treatments to correct effect of mutation

Exon skipping with AONs

Experimentally successful in cells from 1) the mdx mouse - model mouse for DMD 2) DMD patients muscle cells Two proof‐of‐concept studies in DMD patients Phase 2 trials - variable increase of sarcolemmal dystrophin (<20%) - slower disease progression based on 6 min walk distance test (6MWD) exon‐skipping strategies limited by low efficacy in cardiac muscle, poor cellular uptake of AON, and rapid clearance of AON from the circulation. repeated administrations required new chemistries and alternative delivery methods essential intrinsically exon specific and therefore beneficial for only a subset of patients. targeting exon 51 or exon 45 could treat 13% or 8% of patients - multi‐exon‐skipping strategies is theoretically possible. - for exon 45-55 skipping theoretically applicable to 63% of DMD patients

Molecular pathology of DMD

Part of DMD gene in mutation 'hot-spot' - regions of the gene which are more susceptible in BMB the number of exons deleted doesn't matter as long as the reading frame isn't deleted in DMD similarly to BMB the number of exons deleted doesn't matter as the reading frame has been disrupted

Gene Therapy to combat DMD

Problems -Dystrophin mRNA is very large -muscle cells post mitotic Potential solutions include -Use minigenes containing dystrophin crucial domains, would give a BMB phenotype -Substitute dystrophin with utrophin - this is expressed prenatal but is switched off - Correct reading frame using exon skipping, would give a BMB phenotype

advances in genetic therapeutic strategies

Problems with DMD Muscle fibres (myofibres) are long, post mitotic multinucleate cells cannot use retroviruses can use adeno associated viruses (AAVs), but they have: - Limited capacity (4.6Kb DNA/dystrophin cDNA is 11-14Kb) - are non-integrating, means repeated treatments are needed Gene-replacement approaches can treat all DMD patients regardless of the mutation. Only 20% of the wild-type level of dystrophin is needed to correct muscle pathology Requires delivery to all muscles. micro- and mini-dystrophin have been delivered using AAVvectors 2006 - the first gene therapy trial for 6 DMD boys using mini-dystrophin - transgene expression was undetectable, - immune response to mini-dystrophin and AAV - adverse effects could be minimized by transient immunosuppression

Identifying the DMD gene

Several groups attempted to isolate DNA encoding gene responsible for DMD Cytogenetics - involves the examination of chromosomes to identify structural abnormalities. revealed deletions on X chromosome in some patients two X chromosomal abnormalities - used to clone DMD gene: 1) DMD boy with visible deletion at Xp21 in chromosomal banding pattern 2) DMD female with X:21 translocation - swap between chromosome 21 and the X chromosome which disrupts the DMD gene

using patient with Xp21 deletion to clone the DMD gene

boy with cytogenetically visible deletion at Xp21 with DMD plus retinitis pigmentosa, chronic granulomatous disease as he had lost a lot of genetic material at this site Used a method called subtraction cloning - Selectively clone DNA from a normal individual that is absent in the patient Generate a series of clones representing deleted parts of patients X chromosome Within this DNA, the gene responsible for DMD was located

Exon-skipping and DMD

exon 45 is the most frequently deleted exon in DMD exon (45+46) deletions cause only a mild form of BMD induced exon 46 skipping in cultured myotubes from 2 DMD patients with exon 45 deletion AON induced skipping of exon 46 in 15% of the mRNA - properly localized dystrophin in at least 75% of myotubes. - could be applicable to >65% of DMD mutations a similar thing occurs on exon 51 - exon 50 deletion causes frameshift, early stop codon, non-sense mediated mRNA decay and DMD - Antisense Oligonucleotides (AON) have been designed to cause skipping of exon 51 to restore the reading frame

Why do females with X:autosome translocation have DMD?

in only one X chromosome so would expect females to be carriers BUT 'X inactivation' favours DMD

what is dystrophin?

is a 427 kDa protein expressed at the sarcolemma in skeletal muscle maintains the strength, flexibility and stability of muscle fibres link between the dystrophin‐associated protein complex at the sarcolemma and the cytoskeleton the crucial domains (red) for dystrophin to have it function include: the domains which interact with the cytoskeleton, those that interact with the B-dystroglycan and the syntrophins

what is DMD?

is a genetic disorder characterized by progressive muscle degeneration and weakness due to the alterations of a protein called dystrophin that helps keep muscle cells intact Clinical phenotype »progressive muscular weakness »death in 3rd decade from cardiac issues »mostly males affected but occasionally females »occurs in families (1 in 5000 male births) identified via Gower's sign -patient that has to use their hands and arms to "walk" up body from a squatting position due to lack of hip and thigh muscle strength. - Presents at around 3-5 yrs of age. Wheelchair by 12 yrs, death by 30yrs (cardiac and respiratory failure). Treatment »None »Manage cardiac and respiratory symptoms and corticosteroids Genetics »X-linked disorder »mendelian inheritance (single gene disorder) - recessive Beckers Muscular Dystrophy - involves the same gene »Much milder disease »Patients still walking at 60yrs

what is a loss of function mutation?

loss of function gene product has reduced or none of normal function any mutation that inactivates gene product will result in the same clinical symptoms eg: Duchenne Muscular Dystrophy (DMD)

dystrophin in DMD

loss of the dystrophin‐associated protein complex, this causes: - Continual inflammation - cycles of necrosis and regeneration - satellite cell depletion - necrosis and fibrosis - contraction‐induced injury in myofibres - muscle wasting the muscles are enable to function normally

what is X- inactivation?

process that occurs in all female mammals - dosage compensation mechanism to ensure only one X chromosome is active in each cell - in the early zygote either the paternal or maternal X is randomly inactivated - so some cells have active paternal X, others active maternal X Occurs in human females with X-autosomal translocation causing DMD so they have a: - normal X chromosome - translocated X chromosome but they aren't equally expressed

Dystrophin domain structure

red = crucial domains these include the actin binding, dystroglycan binding and the dystrobrevin & syntrophin binding domains

how was X:autosome Translocation used to find the DMD gene?

the tips of the X chromosome and the normal 21 chromosome swap forming an X chromosome with the top of chromosome 21 - this is a translocation from this they construct gDNA library from patient screened for clones containing both chromosome 21 and X chromosome sequences X-junction clones - marked position and sequence of DMD gene

X inactivation in DMD

there is sporadic X:21 translocation which creates the an X, X:21, 21:X and 21 chromosome chromosome 21 is very important as the tip encodes rDNA so in the blastocyst there is random inactivation of X and X:21 inactivation of X:21 leaves X, 21:X and 21 expressed - this means these cells are non-viable as they only have one copy of the DNA which encodes rDNA inactivation of X leaves X:21, 21:X and 21 expressed - these cells are still viable as both copies of rDNA are present all cells have this genotype meaning that X chromosome is disrupted and DMD occurs

using a patient with an X-autosome translocation to clone the DMD gene

used the patient which has undergone X-autosome translocation DMD is an X-linked recessive disorder - females with DMD are very rare (~20 worldwide) occur sporadically in families with no DMD history - new spontadius mutation all carry X-autosomal translocations in each case the X breakpoint has disrupted the DMD gene - therefore marks the position of the DMD gene