DMD/BMD - Part 2 Flashcards
What is usually the first non-genetic tests performed for muscular dystrophinopathies?
- Testing for serum creatine kinase (CK) levels is usually one of the first tests performed.
- CK is an enzyme found in muscle and brain and there is normally very little in blood.
- CK levels are elevated when there is muscle damage. Therefore CK testing is a general test for muscle disease.
- CK testing is not specific - will also get elevated CK levels where there has been injury/trauma to muscles or after excessive exercise etc.
- CK levels decrease with advancing age due to progressive elimination of dystrophic muscle fibres.
- Levels can be taken into account in Baysian calculations of carrier risk.
- In DMD patients CK levels are usually >10x the normal level.
- In BMD patients CK levels are usually >5x the normal levels.
- CK levels will also be increased in patients with DMD-related cardiomyopathy.
- In approximately 50% of cases female carriers of DMD will have CK levels of 2-10x the normal levels.
- In approximately 30% of cases female carriers of BMD will have CK levels of 2-10x the normal levels.
What do elevated creatine kinase (CK) levels indicate?
- CK levels are elevated when there is muscle damage. Therefore CK testing is a general test for muscle disease.
- CK testing is not specific - will also get elevated CK levels where there has been injury/trauma to muscles or after excessive exercise etc.
What are CK levels likely to be in DMD patients in comparison to normal levels?
- In DMD patients CK levels are usually >10x the normal level.
What are CK levels likely to be in BMD patients in comparison to normal levels?
- In BMD patients CK levels are usually >5x the normal levels.
What are CK levels likely to be in patients with DMD-related cardiomyopathy in comparison to normal levels?
- CK levels will also be increased in patients with DMD-related cardiomyopathy.
What are the CK levels likely to be in female carriers if DMD in comparison to normal levels?
- In approximately 50% of cases female carriers of DMD will have CK levels of 2-10x the normal levels.
What are the CK levels likely to be in female carriers if BMD in comparison to normal levels?
- In approximately 30% of cases female carriers of BMD will have CK levels of 2-10x the normal levels.
What non-genetic methods can be used to test for dystrophinopathies?
1) . Creatine Kinase (CK) testing
2) . Muscle biopsy followed by chemical staining
3) . Muscle biopsy followed by IHC with anti-dystrophin antibody
How can muscle biopsies be used to test for muscular dystrophies?
- Muscle cell histology differs in patients with a dystrophinopathy and muscle biopsy can therefor be a very useful tool.
- Characterisation of muscle fibres can be carried out using a number of chemical stains. In DMD patients you may see an increase in the variation in fibre size diameter, an increase in fibrous connective tissue and fat, the presence of larger rounded hyaline fibres, and foci of degeneration and regeneration.
- IHC using anti-dystrophin antibody should show and absence of staining in DMD patients and reduced staining in BMD patients.
- As it is not possible to refute a diagnosis of a dystrophinopathy based on the results of genetic testing as no testing is 100% sensitive a muscle biopsy and dystrophin analysis by IHC
On muscle biopsy followed by chemical staining what features might you see in DMD patients?
In DMD patients you may see an increase in the variation in fibre size diameter, an increase in fibrous connective tissue and fat, the presence of larger rounded hyaline fibres, and foci of degeneration and regeneration.
On muscle biopsy followed by IHC with anti-dystrophin antibody what might you expect to see in DMD and BMD patients?
IHC using anti-dystrophin antibody should show and absence of staining in DMD patients and reduced staining in BMD patients.
What genetic techniques are usually used to test for deletions and duplications in cases of dystrophinopathies?
Due to high proportions of deletions found in dystrophinopathy patients, methods used for diagnosis are focussed on techniques that can detect deletions.
1) . Southern Blotting
2) . Multiplex PCR
3) . Fluorescent Quantitative Dosage PCR
4) . MLPA
5) . Array CGH
6) . NGS (Future)
1) . Southern Blotting - One of the first methods utilised for DMD testing was Southern blotting. However, this technique is laborious and time consuming and uses radioactivity. Also, depending on the probes used it may not be possible o detect small changes and sensitivity for carrier testing is low.
2) . Multiplex PCR - Was developed and used for a number of years in diagnostic laboratories and can still sometimes be of use today. This technique amplifies the exons most commonly deleted in DMD patients using PCR. Primers are multiplexed to reduce the number of PCRs required. The PCR products are then separated by agarose gel electrophoresis. The absence of a band suggests that the exon is deleted. 2 common multiplex PCRs were developed that enabled the the detection of 98% of all DMD deletions. There are however several limitations to this technique. Firstly, it cannot characterise the end points of all deletions as it does not contain all exons. Furthermore, it does not detect duplications. It also cannot be used for carrier testing as it is not quantitative. Multiplex PCR led onto the development of fluorescent quantitative dosage PCR.
3) . Fluorescent Quantitative Dosage PCR - Based on the same principle as dosage PCR, however, the primers are tagged with fluorescent dye so that the products can be separated by a capillary analyser. Absence of a peak on the electropherogram indicates a deletion of that exon. The advantage of this technique is that the heights of the PCR products can be measured and compared because the number of cycles of PCR is kept low. A reduction in peak height is evidence that the patient is heterozygous for a deletion and an increase in peak height suggests a duplication of that exon.
4) . Multiplex Ligation-dependant Probe Amplification (MLPA) - This is the most widely used technique for testing for BMD and DMD. This technique can test for deletions and duplications and can be used for diagnostic and carrier testing. MLPA uses probes consisting of 2 parts that hybridise to adjacent sequences of target DNA. When the 2 parts of the probe bind to the DNA next to each other they are ligated and form a probe ligation product that can then be amplified by PCR. However, if the target sequence is not present there will be no ligation and no PCR product. MLPA uses control probes specific to other chromosomal locations as internal controls. Normal control samples are also included to allow comparison to expected ratios, thereby allowing the identification of deletions and duplications in male and female samples. MRC Holland makes all MLPA kits. As the dystrophin gene is so large, 2 MLPA kits are required to cover all the exons for the gene. P034 includes probes for exons 1-10, 21-30, 41-50 and 61-70. P035 includes probes for exons 11-20 + promoter, 31-40, 51-60 and 71-79. The sensitivity of MLPA is approximately 72% as it does not detect point mutations. Results showing single exon deletions should be interpreted with caution as they could be false positives caused by a variant under the probe hybridisation site. Therefore, all single exon deletions should be confirmed by another technique such as multiplex PCR. Alternatively the probe hybridisation region should be sequenced to ensure there is not a variant in the sequence that could stop the probe binding and cause a reduction in peak height. It should be noted that these variants can be neutral polymorphisms, variants of unknown clinical significance, or pathogenic mutations. This means that in some cases MLPA can indirectly detect pathogenic point mutations.
5) . Array CGH - High resolution array CGH of the dystrophin gene has been developed but is not in general use. Can detect loss or gin of sequences at intronic break points associated with some inversions and complex rearrangements It has a slightly higher pickup rate than MLPA and can give the approximate intronic boundaries of mutation events. However, MLPA is quicker, easier and cheaper to perform.
6) . NGS - In the future NGS may be available to assess copy number and could therefore be used to simultaneously detect deletions and duplications and point mutations.
Describe multiplex PCR for dystrophinopathy testing. What are the limitations of this technique?
- Multiplex PCR was developed and used for a number of years in diagnostic laboratories and can still sometimes be of use today.
- This technique amplifies the exons most commonly deleted in DMD patients using PCR.
- Primers are multiplexed to reduce the number of PCRs required.
- The PCR products are then separated by agarose gel electrophoresis.
- The absence of a band suggests that the exon is deleted.
- 2 common multiplex PCRs were developed that enabled the the detection of 98% of all DMD deletions.
- There are however several limitations to this technique. Firstly, it cannot characterise the end points of all deletions as it does not contain all exons. Furthermore, it does not detect duplications. It also cannot be used for carrier testing as it is not quantitative.
Describe fluorescent quantitative dosage PCR for dystrophinopathy testing. What are some advantages of this method over normal multiplex PCR?
Fluorescent Quantitative Dosage PCR - Based on the same principle as dosage PCR, however, the primers are tagged with fluorescent dye so that the products can be separated by a capillary analyser. Absence of a peak on the electropherogram indicates a deletion of that exon. The advantage of this technique is that the heights of the PCR products can be measured and compared because the number of cycles of PCR is kept low. A reduction in peak height is evidence that the patient is heterozygous for a deletion and an increase in peak height suggests a duplication of that exon.
What is currently the main genetic testing method used to identify dystrophinopathies? Describe this technique.
- Multiplex Ligation-dependant Probe Amplification (MLPA) is the most widely used technique for testing for BMD and DMD.
- This technique can test for deletions and duplications and can be used for diagnostic and carrier testing.
- MLPA uses probes consisting of 2 parts that hybridise to adjacent sequences of target DNA.
- When the 2 parts of the probe bind to the DNA next to each other they are ligated and form a probe ligation product that can then be amplified by PCR. However, if the target sequence is not present there will be no ligation and no PCR product.
- MLPA uses control probes specific to other chromosomal locations as internal controls.
- Normal control samples are also included to allow comparison to expected ratios, thereby allowing the identification of deletions and duplications in male and female samples.
- MRC Holland makes all MLPA kits.
- As the dystrophin gene is so large, 2 MLPA kits are required to cover all the exons for the gene. P034 includes probes for exons 1-10, 21-30, 41-50 and 61-70. P035 includes probes for exons 11-20 + promoter, 31-40, 51-60 and 71-79.
- The sensitivity of MLPA is approximately 72% as it does not detect point mutations.
- Results showing single exon deletions should be interpreted with caution as they could be false positives caused by a variant under the probe hybridisation site. Therefore, all single exon deletions should be confirmed by another technique such as multiplex PCR. Alternatively the probe hybridisation region should be sequenced to ensure there is not a variant in the sequence that could stop the probe binding and cause a reduction in peak height.
- It should be noted that these variants can be neutral polymorphisms, variants of unknown clinical significance, or pathogenic mutations. This means that in some cases MLPA can indirectly detect pathogenic point mutations.
