Duncan - osteogenesis, DMD and MPLA Flashcards

Question

what are muscular dystrinopathies? genetics progression size of their impact

Answer 1

A group of hereditary, germline (effect every cell) disorders, there are over 40 types All with a genetic basis, but the fault can lie in different genes They typically are progressive - get worse with age, and involve the degeneration/death of muscle fibres Muscle is everywhere (most abundant body tissue, ~23% females and ~40% males weight) so impact is large and multisystem, including the heart (cardiomyopathy) and intellectual disability (lungs - muscles - lack of oxygen to brain)

Answer 2

Patients with muscular dystrophy had balanced translocations between an autosome and X chromosome. Breakpoints on the X chromosome are always at Xp21. Hypothesis: A disrupted gene at Xp21 causes muscular dystrophy. Cloning and Identification: The DMD gene was cloned, and dystrophin protein was identified

Answer 3

DMD gene encodes dystrophin, at position Xp21.2 (on the X chromosome) Largest gene in the human genome, 2.3 megabases (0.08% of the HG). takes 16 hrs to transcribe. Mature mRNA = 14 Kb Made up of 79 exons - only 0.6% of the gene; so >99% = introns Translocations easy to spot due to how large the gene is Its X-linked recessive, so almost never seen in females - males only have one X chromosome so are hemizygous. it is found in 1 in 3,500 male infants.

Answer 4

Located in skeletal and cardiac muscle fibres, the protein is a middle man, connecting F-actin (major components of cell cytoskeleton) to ECM proteins like laminin, via the dystroglycan complex (so you've got F-actin → dystrophin → dystroglycan complex spanning cell membrane → ECM/laminins) Strengthen muscle fibres and protects them from injury, holding cells in place Prevents membrane damage during muscle contractions - transmits force generated during muscle contraction to the ECM

Answer 5

Skeletal & cardiac muscle cells with absence of or reduced expression of functional dystrophin The dystroglycan complex is disassembled The interaction between F-actin (cytoskeleton) and laminin (extracellular matrix is lost so no protection against force of muscle contraction Cells become damaged as the muscles repeatedly contract & relax with use The damaged cells weaken & die over time. So muscle becomes increasingly depleted, causing characteristic muscle weakness & heart problems seen in DMD and BMD

Answer 6

Both BMD and DMD - mostly caused by deletions, typically large scale/more than one exon (70% of the 66%) Hot spot for deletions from exons 3-8 and between 44-50 Point mutations and duplications are in the minority of causes

Answer 7

Deletion = loss of genetic information (especially in X chromosome in males) causes severe phenotype. Duplication = extra copy of information often tolerated, e.g. down syndrome is an extra entire chromosome and is arguably milder than many single gene deletions (or even a few exon deletions). As long as reading frame is preserved duplications - still likely to be (but not definitely) pathogenic. the ones we do see in DMD and BMD must interrupt exons, with the breakpoints of the duplication probably occurring within an exon note - duplications are also hard to identify, as genetic testing doesn't tell you where your duplication is

Answer 8

2/3 pathogenic variants are de novo (not seen in either parent, makes sense as severe phenotype often prevents reproduction so rarely see inherited cases) ~70% have deletion of >1 exon

Answer 9

Frameshift mutations - As we know, they cause truncation (early stop codon bound to happen within 100 codons) so the mRNA is destroyed by NMD, and dystrophin isn't produced. This is DMD - when you get NO dystrophin produced BMD - occurs when the variant does not alter the reading frame. I..e the deletion/duplication is in frame, possibly including a number of exons. Not destroyed by NMD as the sequence that is there is correct So dystrophin is produced, it's just typically a shorter protein so is only partially functional (hence why BMD is less severe than DMD) NOTE - using this idea to distinguish BMD and DMD mutations is only 92% accurate, so not necessarily reported straight to patients

Answer 10

Age of onset 3 to 5 years old Delayed walking Muscle weakness, lower limbs Pseudohypertrophy- enlarged calves Cardiomyopathy – heart disease (by 14 years) Breathing problems - caused by deformed bones and muscle weakness Wheelchair bound by 12 Reduced life span, <30 years

Answer 11

Milder (but still severe) Later-onset skeletal muscle weakness Wheelchair dependency (after age 16 years); although some remain ambulatory (capable of walking) into their 30s – key difference from DMD Cardiomyopathy (diagnosed at ~14 years) Life span to mid 40s

Answer 12

one mutated allele, one wild type allele... 76% of DMD and 81% of BMD have NO symptoms others can have: Mild to moderate muscle weakness (20% of DMD, 15% of BMD) Cardiomyopathy (8% in DMD, none in BMD) range in effect due to: Features vary dependent on X-inactivation X-inactivation may be skewed favouring either wild type or variant allele XX - one of the X’s in every cell gets inactivated. The proportion throughout the body of which X is inactivated differs. Women with some symptoms are likely to have a greater proportion of their normal/WT X gene being inactivated

Answer 13

the quickest strategy to identify pathogenic variant in the DMD is to first use MLPA to exclude the exonic deletions and duplications, which will detect over 70% of positive cases. This takes 3 days vs 2 weeks for NGS if the test was negative, you'd do NGS analysis to identify any point mutations/sequence variants (if they cause a frameshift, they are likely to be pathogenic)

Answer 14

ratio would be zero if there had been a deletion, not 0.5, because they only have one X as it is Deletions Exon to whole gene deletions generally considered pathogenic Where reading frame is disrupted, DMD is likely diagnosis Where reading frame is maintained, BMD is likely diagnosis Literature/databases for similar deletion variants is helpful

Answer 15

No cure Steroids (Corticosteroids) to maintain muscle function & slow muscle weakening But has side effects: weight gain, decreased bone mineralization (weakened bones) & behavioural disturbances Physical therapy - maintain muscle strength Assisted ventilation Surgeries, Cardiac transplantation

Answer 16

Antisense oligonucleotides (AONs) are small modified pieces of DNA or RNA that specifically hybridize to a target exon during the pre-mRNA splicing process This hides the target exon from the splicing machinery, So the exon will be spliced out with its flanking introns This may make deletion larger, but restores reading frame Allows production of a partially functional dystrophin like those found in BMD

Answer 17

it's a drug containing AON (antisense oligonucleotides) that causes exon 51 to be skipped, which can only treat the 14% of DMD patients with exon 51 truncating variants

Answer 18

Induced pluripotent stem cells - Induced pluripotent stem cells (iPSC) are derived from the patient’s mature cells that have been reprogrammed to embryonic? stem cell like state Can differentiate to any human cell type Therapy – use patients own cells, gene edit the pathogenic variant, transplant the cells back Huge potential not just in DMD but other conditions e.g. Parkinson's In DMD - Generate iPSC from e.g. fibroblast Gene edit DMD pathogenic variant - CRISPR/Cas9 or zinc finger nucleases Differentiate iPSCs to myogenic progenitors Transplant back into patient Now has self derived cells producing normal dystrophin

Duncan - osteogenesis, DMD and MPLA Flashcards

(42 cards)