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Block 6- SHANE > Duchenne Muscular Dystrophy > Flashcards

Flashcards in Duchenne Muscular Dystrophy Deck (24)

What is the mode of inheritance and prevalence of DMD?

X linked; 1:3500 males


What are some of the symptoms of DMD?

delayed motor milestones, progressive weakness and muscle loss, hypertrophic calf muscles, respiratory complications; Gower maneuver to stand; cardiac muscle may be involved


When is the typical onset of DMD and outlook?

The disease presents in early childhood and follows a progressive course. Males with DMD rarely survive past their 20s


Is Becker muscular dystrophy milder or more severe than DMD?

BMD is an allelic variant characterized by a milder course

By allelic variant, I mean that both disorders are caused by mutations in the same gene (DMD gene).


T or F. There is a correlation between severity of mutations and disease severity, where complete loss of function causes the more severe disease course seen in DMD



What is the product of normal DMD gene?

dystrophin, which functions in cytoskeletal function of skeletal and cardiac muscle


What is the primary distinction between DMD and BMD?

the level of residual dystrophin. DMD patients have a (near) complete absence of protein (0-5%), while BMD have somewhere between 20-100%

Note that there is an intermediate category. I include this to make the point that, at a molecular level, phenotype is more of a spectrum than it is two completely separate categories. (However, patients receive a diagnosis of Duchenne or Becker.)


How is the dystrophin of BMD patients affected?

Normal dystrophin protein has an actin-binding domain at one end and a domain at the other end that binds other membrane proteins. In between those two functional domains is a large rod domain. Typical BMD patients have dystrophin with a shortened rod domain but with the two terminal domains intact; this shortened protein retains some functional capacity.


T or F. DMD is the largest known gene, which makes it a prominent target for mutations

T. It is so large that it spans multiple recombination hotspots and includes a multitude of repetitive sequence elements within its long introns

79 exons spanning 2.2 MB (small exons surrounded by large introns)


Does DMD have a high de novo mutation rate?

Yes, 1/3 of simplex males are new mutations; more than 5000 mutations identified


What are the major molecular causes of DMD or BMD?

65% large deletions spanning one or more exon

10% large duplications

25% point mutations or small insertions/deletions


T or F. Deletion breakpoints tend to occur within introns, eliminating at least one exon, and, in many cases, eliminating a large portion of the gene



What is a frame-neutral shift?

eliminates multiple of 3 bp and maintains the reading frame


Patients with a frame-neutral shift mutation have which type of muscular dystrophy?

BMD. Typically a large deletion occurs but the frame is maintained and the sequence is not truncated


Patients with a frameshift mutation have which type of muscular dystrophy?

DMD. The sequence tends to be truncated here leading to loss of the protein

The overall size of the deletion is much less critical than the reading frame


Are nonsense mutations more common in DMD or BMD?

DMD. nonsense mutations do not preserve the C-terminal functional domain


T or F. Splice site mutations and small insertions and deletions are observed in both DMD and BMD

T. Mutations that disrupt normal splicing may have diverse effects on the transcript. Just to consider one scenario, a splice site mutation that causes exon skipping may be frame-neutral or may introduce a frameshift, depending on the number of bases omitted (multiple of 3?). Similarly, deletion (or insertion) of one to a few bases may or may not maintain the reading frame. In considering in/del mutations, think of them as variants that are contained entirely within one exon. In other words, they are closer in size to a point mutation than to exon-spanning deletions. Importantly, in/del mutations are too small to be detected by a deletion array


T or F. Missense mutations are not a common cause of DMD/BMD.

T. Certainly, they occur, but in most instances, replacement of a single amino acid doesn’t cause DMD/BMD.


Is scanning or targeting more effective for testing for DMD/BMD? Why?

scanning because in DMD, we have extensive allelic heterogeneity and high mutation rate. Thus, it is necessary to look through the entire gene to identify a patient’s disease-causing mutation (this is the definition of a scanning approach to genetic testing)


What is a case where a targeted approach may be better for DMD?

If a DMD mutation already has been identified in a family member, then a targeted approach can be used to test for the presence or absence of that specific mutation

For example, if a specific deletion has been identified in an affected male, then his mother can have a targeted test – Is that specific deletion present or absent in her? (Is she a carrier? Or does her son have a new mutation?)


How can CMA be used to scan for DMD?

CMA is a testing platform that uses oligonucleotides (“features”) fixed to the array to scan the entire genome (excepting repetitive sequences, down to a resolution of several hundred thousand basepairs). While that’s what CMA is designed to do, the fact is that a microarray can be designed for just about any genomic query. The array I’ll discuss next is a targeted*, high-density array that is designed to find large deletions or duplications (affecting one or more entire exons) anywhere in the DMD gene. The test will identify almost all of the 75% of DMD mutations caused by large deletion/duplication. It will NOT identify mutations in any other gene. It will NOT identify small in/dels (~~100 bp) or point mutations. (*Don’t be confused by use of the word “targeted” as in “targeted array”. In this context, the target is one very large gene, which will be SCANNED for deletions anywhere within it.)


How does the DMD array test (just described) work?

The DMD array test is very good at what it is designed to do. Using the patient’s genomic DNA, it will detect almost all deletions or duplications spanning one or more exons anywhere within the DMD gene. Again, it does not query any other genes, and it is not designed to detect small point mutations in DMD. No test is perfect, and there is always the possibility to miss a deletion, but the test has impressive accuracy in testing both males and females.


What would be the first test performed on a suspected DMD?

Since the majority of DMD mutations are large deletions and duplications, the deletion array test is done first


What happens to patients who are negative on a DMD array test but still suspected of DMD?

In the next tier of testing, a patient’s DMD gene is sequenced, including the entire coding sequence, splice junctions, the large promoter region, and a few specific intron segments where mutations are known to occur. Just as the array test scanned the entire gene for large deletions, sequencing now scans the majority of the gene for nucleotide-level variants. Deviations from the normal consensus sequence are interpreted for their pathogenic potential. As with any test, there are limitations to gene sequencing. It’s not a good way to find large deletions and other large-scale genomic rearrangements. The level of resolution isn’t right (by analogy to microscopy – the deletion array is like a low-resolution dissecting scope, whereas sequencing is more like a compound microscope with a 100x objective.). The vast majority of intronic sequences are NOT sequenced, so rare pathogenic variants within introns will not be detected.

Another limitation is possible challenges in interpretation. For example, if sequencing reveals a previously undescribed missense mutation, it may be very difficult to assess the pathogenic potential of the variant. I will say more about clinical sequencing and “variants of uncertain significance” (VUS) in a future recording. The approach to analysis I will describe then also applies to DMD sequencing.
In spite of limitations, use of both approaches for DMD testing is very good. In combination with the deletion array test, combined analysis detects 98-99% of DMD mutations.