DMD/BMD Flashcards
What is the incidence for DMD and BMD?
- DMD: 1/3500 males
- BMD: 1/18000 males
DMD and BMD are caused by mutations in what gene?
Dystrophin gene (Xp21.2)
What is the mode of inheritance for DMD/BMD?
X-linked recessive (100% penetrance in males)
Is there a high mutation rate in the dystrophin gene?
- Yes: approx 1/3 of mutations are de novo
What are the three ways de novo mutations can arise in the context of DMD/BMD?
- Mut may occur in egg at time of proband’s conception (Mut in every cell of proband’s body, mother does not carry Mut so no recurrence risk)
- Mut may occur after conception and not present in all cells of proband (proband = somatic mosaic, mother doesn’t carry Mut so no recurrence risk)
- Mut present in mother of the proband’s egg cells (Mut not detected in DNA extracted from blood, mother = germline mosaic - risk to further children)
What are the five ways in which females can manifest with classical DMD?
- X:autosome translocation affecting inactivation resulting in no active dystrophin gene
- Turner syndrome - only one X, if it has mutation they have DMD
- UPD - daughter inherits two copies of the X with the mut and therefore has no normal dystrophin
- Skewed X-inactivation - X containing the Mut remains active in disproportionate number of cells
- Father affected with BMD and mother a carrier - daughter inherits Mut on both X chromosomes
What is the difference between levels of dystrophin protein in DMD vs BMD individuals?
- DMD: dystrophin virtually absent
- BMD: dystrophin levels 10-40% of normal OR protein present but with reduced function
What is the effect of dystrophin deficiency in DMD/BMD?
- Affects formation of dystrophin associated protein complex (DAPC)
- disruption of link between cytoskeletal actin and extracellular matrix
- Cell membrane more fragile and can be mechanically damaged during eccentric muscle contraction
- Looseness of sarcolema permits calcium channels to open
- increase in calcium ions activates proteases that digest contractile proteins resulting in much weaker muscle
Provide some details on the dystrophin gene
- Largest known human gene
- 2.4Mb but only 0.3% of genomic sequence is present in mature transcript
- 79 exons
- at least 7 different promoters : produce tissue specific transcripts of differing sizes
What percentage of DMD/BMD cases are caused by deletions/dups of one or more exons?
Dels - DMD = 65% - BMD = 85% Dups - 5-10% for both
What are the deletion hotspots in the dystrophin gene?
- Proximal = exons 2-20
- Distal = exons 45-55
What is the Frameshift hypothesis for DMD/BMD?
- Deletions which disrupt translational reading frame generally cause severe DMD phenotype: premature termination codon leads to nonsense mediated decay = no protein
- Deletions leaving reading frame intact cause milder BMD: abnormal version of dystrophin but retains some function
- Consistent in approx 90% of cases
What are some things to be cautious about with regards to the Frameshift hypothesis in DMD/BMD?
- Prediction based on assessment at DNA level: may be different at RNA level (e.g. may affect splicing)
- Deletions in protein binding domains may severely affect dystrophin function even if in frame
- Dystrophin can retain significant function even when missing large portions of amino acid sequence: e.g. Dels affecting central rod domain may be associated with mild/no manifestations
What proportion of DMD/BMD cases are caused by dystrophin point mutations?
- DMD: 25-35% (usually result in premature termination codon)
- BMD: 10-20%
What is the major non-genetic testing method for DMD/BMD?
- Measuring serum creatine kinase (CK) levels (non specific screening test)
- Enzyme mainly found in muscle and brain
- Elevated levels indicate muscle damage
What are the expected levels of serum CK in DMD/BMD?
- DMD males: more than 10x normal level
- BMD males: more than 5x normal level
- Males with DMD-related cardiomyopathy tend to have increased levels
- DMD/BMD carrier females: ~50%/30% have more than 2-10 times normal level
What are two important points to remember surrounding serum CK levels in DMD/BMD?
- Levels can decrease with advancing age due to progressive elimination of dystrophic muscle fibres
- Levels can be taken into account for Bayes
Aside from serum CK levels, what is another non-genetic test employed for DMD/BMD?
- Muscle biopsy
- characterisation of muscle fibres using number of chemical stains or IHC with anti-dystrophin antibody
- IHC = absence (DMD)/reduction (BMD) of stain
What is the main genetic testing method for DMD/BMD?
- MLPA: detects dels and dups
- Two probe sets needed to cover gene (P034 and P035)
- Sensitivity approx. 72% as can’t detect point mutations
What aspect of MLPA should be approached with caution in the context of DMD/BMD?
- Variants under probe hybridisation site can cause reduction in peak height = false positives
- All single exon deletions should be confirmed by another technique (e.g. Multiplex PCR or sequencing)
What were three previous genetic testing methods employed for DMD/BMD?
- Southern blotting (time consuming, laborious, can’t detect small changes)
- Multiplex PCR: amplify exons known to be most commonly deleted
- Fluorescent quantitative dosage PCR: limited number of exons included
What is the future method for genetic testing of DMD/BMD?
NGS - when copy number analysis routinely available using NGS then will be able to detect dels/dups/point mutations
Provide details on linkage analysis in the context of DMD/BMD testing
- Required when mutation in family not known
- microsatellite Markers near and within dystrophin gene are genotyped in number of family members to determine haplotype associated with DMD/BMD
- Can enable prenatal diagnosis to be performed
What are the limitations of linkage analysis for DMD/BMD?
- Risk of recombination between markers: 10% for dystrophin gene
- Markers may be uninformative
- Assumes affected family member has DMD/BMD
- Need appropriate family members to identify high risk haplotype: ideally affected males are required but may be possible to perform exclusion testing based on presence of unaffected males in pedigree
When reporting diagnostic male DMD/BMD results, what does it mean if you have a normal MLPA result?
- Does not exclude diagnosis as dels/dups only account for approx 72% of dystrophin mutations
- If clinical diagnosis confirmed (CK levels or muscle biopsy) then can offer point mutation analysis/linkage studies
When reporting diagnostic male DMD/BMD results, what does it mean if you have a positive deletion or duplication identified by MLPA?
- Single exon deletion must be confirmed by another method
- Need to check if predicted to be in frame or out of frame using Leiden Muscular Dystrophy site
- Result confirms diagnosis of DMD/BMD
- If first/last exon deleted then could extend into neighbouring genes and alter phenotype
- If individual has children all daughters are obligate carriers
When reporting diagnostic female DMD/BMD results, what does it mean if you find a duplication or deletion in your MLPA result?
- If the female has symptoms of DMD/BMD then this result is consistent with clinical phenotype of a manifesting DMD/BMD carrier
- Further testing should be offered such as cytogenetic analysis to detect if translocation/Turner syndrome
- X inactivation studies may be appropriate
What does it mean if you do not identify a mutation in DMD/BMD carrier testing?
- If familial mutation is known then (1) if individual is not mother of affected child they are unlikely to be a carrier but (2) if they are mother then still risk of germline mosaicism
- If familial mutation is not known then carrier risk is reduced depending on detection rate of the test performed (can perform a Bayes)
What are some of the prior risks involved in Bayes calculation for DMD/BMD?
- 4u (mutation rate) is risk that a female is carrier of DMD when no other info taken into account (prior population risk)
- this is 18u for BMD (due to biological fitness)
- risk for mother of sporadic case = 2/3
- risk for grandmother of sporadic case =1/3
Provide details on prenatal diagnosis for DMD/BMD
- Usually only test male foetus as females not usually affected and ethical issues surround identification of carrier female prior to age of onset
- Can sex free fetal DNA prior to prenatal diagnosis
- MCC will need to be ruled out
- If mutation is unknown then PND may be performed by linkage analysis
- PGD now available for DMD
Can prenatal diagnosis be performed for DMD/BMD when mother is not known to be carrier?
Yes - due to the risk of germline mosaicism
What are the common clinical features of DMD?
- Onset before 5yrs
- Dev delay and LD (30-50%)
- Progressive muscular weakness
- Dilated cardiomyopathy
- Loss of ambulation before 12 years
- Mean age of death 25 years due to cardiomyopathy and respiratory insufficiency
What are the common clinical features of BMD?
- May be late learning to walk
- Muscle weakness around 11 years
- Muscle cramps
- Lose ability to walk around 40-50 years
- No LD
- Survive into middle age and beyond
What are the common clinical symptoms for female carrier of DMD/BMD?
- Approx 5-10% have cramps or mild muscle weakness (proximal and asymmetric)
- Usually noticed in 30s
- Carriers of BMD are less affected than DMD
- 20% have evidence of dilated cardiomyopathy
What are the reproductive risks for a mother not found to be carrying the mutation of her affected son?
- 10% risk of having affected future son due to gonadal mosaicism
- 10% carrier risk for future daughter for same reason
What is germline mosaicism and what is the prevalence of this in DMD/BMD?
- More than one set of genetic information is found specifically within the gamete cells
- In DMD/BMD = approx 7-10%
