LABORATORY TEST for MUSCULAR DYSTROPHY
1. Blood test
When blood tests are performed for muscular dystrophy, the levels of enzyme, creatine phosphokinase (CPK) is estimated. It is found mainly in the brain, heart, skeletal muscles, and other tissues which help cells produce a biochemical reaction that results in high-energy molecules that cells use to perform normal functions. When creatine kinase combines with adenosine triphosphate (ATP) it produces phosphocreatine and adenosine triphosphate (ATP). The muscles use these energy molecules to contract muscle fibers. This enzyme rises in the blood due to muscle damage and may reveal some forms of muscular dystrophy before any physical symptoms appear.
Electromyography (EMG) measures muscle response or electrical activity in response to a nerve’s stimulation of the muscle. The test is used to help detect neuromuscular abnormalities. A needle is inserted into the muscle at rest and during contraction. By measuring the muscle’s response, the muscle damage which has occurred can be measured.
Increased insertional activity or abnormal spontaneous activity may be present if there has been a substantial amount of muscle fiber necrosis.
When the muscle fiber membrane excitability is increased further, these electrical activities may become spontaneous. Although, abnormally increased insertional activity is a well-known abnormality in a denervation process or with muscle fiber necrosis, reduced insertional activity is also abnormal. This may occur in a number of settings, including muscle fibrosis and fatty infiltrates. An important clue to muscle fiber replacement by fibrosis or fatty tissue is that the consistency of the muscle and resistance to the advancing needle are changed. In the case of fatty infiltrate, resistance to the needle is reduced.
3. Muscle biopsy
A muscle biopsy is a procedure used to diagnose, diseases involving muscle tissue. It is one of the most reliable tests to confirm the diagnosis of muscular dystrophy. The tissue sample is obtained by inserting a biopsy needle into the muscle and a small piece of muscle tissue is removed and then examined microscopically. If muscular dystrophy is present, changes in the structure of muscle cells and other characteristics of the different forms of muscular dystrophy can be detected. The sample can also be stained using immunohistochemistry, to detect the presence or absence of particular proteins. The muscle selected for the biopsy depends on the location of symptoms which may include pain or weakness. The muscles often selected for sampling are the biceps (upper arm muscle), deltoid (shoulder muscle), or quadriceps (thigh muscle).
4. DNA test
Genetic testing is often the best way to confirm a diagnosis in a patient with signs or symptoms suggestive of a muscular disease. Availability of genetic tests make it possible to diagnose these disorders early and also avoid invasive procedures like muscle biopsy. A muscle biopsy is needed only if the DNA-based test is negative. Genetic tests are available for DMD/ BMD, Myotonic dystrophy, FSHD and few forms of LGMD.
Testing for dystrophin gene deletions or structural inversions in coding regions with the use of DNA-based technology is now the preferred diagnostic test for DMD when clinical signs and symptoms suggest the diagnosis. Deletions account for 60-65% cases in DMD; duplications for 5-6% and point mutations for the remaining cases. Using primers targeting 18 hotspot exons in the dystrophin gene, 98% of deletions can be detected. The proximal hotspot encompasses exons 3-7 and the distal hotspot exons 45-51. A negative test occurs in about 30 percent of patients with DMD because some mutations in the dystrophin gene are not detected by current DNA tests available like the multiplex PCR and will require whole gene sequencing. (7-11). In our statistical analysis of 51 DMD children, maximum deletions were observed in the distal 43-53 exons i.e. 82% (42 out of 51) , 6% (3 out of 51 ) showed proximal deletions in 3-7 exons ,6% deletions were seen in 8-10 exons where as remaining 3 children showed deletions in 12-23 exons.
The genetic test for FSHD locates and measures the size of DNA deletion on chromosome 4. The size of the DNA in the normal individual is greater than 51 kilo base pairs while that of the affected one is less than 35 kilo base pairs. Approximately 5% of individuals that have the clinical symptoms of FSHD do not have the DNA deletion on chromosome 4.
In Myotonic dystrophy type 1, the mutation is a DNA expansion or an increase in the amount of DNA that is normally located on a chromosome. A section of DNA on the dystrophia myotonica protein kinase (DMPK) gene contains a repeated sequence of three DNA nucleotide bases, CTG. The additional DNA is located on chromosome 19. This test has nearly a 100% rate of accuracy in detecting the genetic mutation. An affected individual has approximately 50 to 2000 CTG repeats depending on the severity of the condition wherein a normal individual has 38 repeats. In Myotonic Dystrophy type 2, the additional DNA is located on chromosome 3. The Zinc Finger Protein 9 (ZnF9) gene contains a section of DNA that contains a repeated sequence of four DNA nucleotide bases, CCTG. The number of CCTG base pairs in affected individuals averages approximately 20,000.
Genetic tests are also available for all the subtypes of LGMD except type 2G, 2H, 2J, 1D, 1E and 1F.
5. Magnetic Resonance Imaging (MRI)
MRI is a readily available, noninvasive method of monitoring tissue structure in muscular dystrophy patients. Several investigators have used MRI as an additional option to physical examination and to investigate the increase in fat tissue in dystrophic muscles. MRI does not use ionizing radiation, produces high-resolution images and can be used for quantitative tissue characterization by measuring the T2 relaxation time of the muscles. In muscular dystrophy patients, the T2 relaxation time in peripheral muscles is significantly different from that measured in healthy control subjects, essentially reflecting differences in fat and water composition between diseased and healthy muscles. Because the T2 relaxation time changes as the disease progresses, it could be used to monitor disease progression and possibly response to therapy in these patients. Developments in MRI techniques have prompted investigators to explore the advantages of better spatial and contrast resolution. MRI has been used to show the pattern of age-related changes in muscle bulk and fatty infiltration in the lower extremities of untreated patients. Other small studies in which MRI was used showed abnormalities in muscle size and structure in patients with DMD.
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