Duchenne Muscular Dystrophy

Question 1

What is the mode of inheritance and prevalence of DMD?

Answer 1

X linked; 1:3500 males

Question 2

What are some of the symptoms of DMD?

Answer 2

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

Question 3

When is the typical onset of DMD and outlook?

Answer 3

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

Question 4

Is Becker muscular dystrophy milder or more severe than DMD?

Answer 4

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).

Question 5

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

Answer 5

T

Question 6

What is the product of normal DMD gene?

Answer 6

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

Question 7

What is the primary distinction between DMD and BMD?

Answer 7

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.)

Question 8

How is the dystrophin of BMD patients affected?

Answer 8

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.

Question 9

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

Answer 9

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)

Question 10

Does DMD have a high de novo mutation rate?

Answer 10

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

Question 11

What are the major molecular causes of DMD or BMD?

Answer 11

65% large deletions spanning one or more exon

10% large duplications

25% point mutations or small insertions/deletions

Question 12

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

Answer 12

T

Question 13

What is a frame-neutral shift?

Answer 13

eliminates multiple of 3 bp and maintains the reading frame

Question 14

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

Answer 14

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

Question 15

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

Answer 15

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

Question 16

Are nonsense mutations more common in DMD or BMD?

Answer 16

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

Question 17

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

Answer 17

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

Question 18

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

Answer 18

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

Question 19

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

Answer 19

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)

Question 20

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

Answer 20

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?)


Question 21

How can CMA be used to scan for DMD?

Answer 21

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.)


Question 22

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

Answer 22

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.


Question 23

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

Answer 23

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

Question 24

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

Answer 24

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.