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Types of diagnosis for Muscular Dystrophy

Muscle Biopsy:
Through outpatient surgery a small piece of muscle is removed (usually from the arm or leg) and evaluated with a variety of biochemical tests. Researchers are attempting to match results of muscle biopsies with DNA tests to better understand how variations in the genome present themselves in tissue anomalies.
Studying a small piece of muscle tissue taken from an individual during a muscle biopsy can sometimes tell a physician whether a disorder is muscular dystrophy and which form of the disease it is.  In Duchenne and Becker muscular dystrophy, a muscle protein called dystrophin is either missing, deficient or abnormally formed. This protein can be examined in the muscle sample.

DNA Test :

The reason for the flawed or deficient muscle protein is a flawed gene for dystrophin. A test that involves looking at this gene -- DNA testing -- can be done to diagnose or rule out Duchenne or Becker muscular dystrophies.

EMG (Electromyogram):

Another diagnostic test is the electromyogram (EMG). This test measures the electrical activity in the muscle. To do this test, small electrodes are put into the muscle, which allows the doctor to measure the electrical impulses coming from the muscle. The test is uncomfortable.

NCV (Nerve Conduction Velocity) Test:

Another test often performed measures nerve conduction velocity (NCV).  This test measures how fast signals travel from one part of a nerve to another. The nerve signals are measured with surface electrodes (similar to those used for an electrocardiogram).  During this test, electrical impulses are sent down the nerves of the arms and legs. By measuring the speed of these impulses with electrodes placed on the skin, the doctor can determine whether the nerves are functioning normally. This test is also uncomfortable.

Blood Enzyme - CPK Test:

Blood enzyme tests are helpful because degenerating muscles become "leaky." They leak enzymes (proteins that speed chemical reactions), which can then be detected in the blood. The presence of these enzymes in the blood at higher than normal levels may be a sign of muscular dystrophy. One such enzyme is creatine kinase, or CK. The CK level is elevated in many forms of muscular dystrophy, some forms resulting in a higher level than others.

Molecular genetic testing

Molecular genetic testing is now the mainstay of diagnosis in most centers. A multiplex polymerase chain reaction (PCR), covering 18 exons at the deletion hotspots developed by Chamberlain and Beggs detected 90-98% of all deletions, although duplications were not identified by this method.  More recently, the development of multiplex ligation-dependent probe amplification (MLPA) has provided a more sensitive technique for detecting deletions. All 79 exons are covered by two sets of probes, with individual exons depicted as a single peak. This allows gene dosage abnormalities to be detected, allowing detection of duplications and testing of carrier individuals as well as for deletions.Occasionally, point mutations will also be detected as single exon deletions, with further analysis allowing more specific delineation of the point mutation.

If MLPA testing is negative, the DUCHENNE MUSCULAR DYSTROPHY (DMD) gene can be tested for point mutations. Direct sequence analysis of the DUCHENNE MUSCULAR DYSTROPHY (DMD) gene is generally available on a research basis only, due to its labor-intensive and costly nature. Several groups have developed strategies to target exonic regions for direct sequencing after the use of initial screening methods.

A targeted high-density oligonucleotide comparative genomic hybridization microarray that allows high-resolution analysis of the DUCHENNE MUSCULAR DYSTROPHY (DMD) gene has also been developed recently, allowing identification of deletions and duplications but also previously unidentified deep intronic mutations.  

DUCHENNE MUSCULAR DYSTROPHY (DMD) is caused by mutations in the DUCHENNE MUSCULAR DYSTROPHY (DMD) gene, one of the largest known genes in humans, spanning 2.3 mega bases and accounting for 0.1% of the total human genome.  This gene encodes a protein called dystrophin, which localizes to the cytoplasmic face of the sarcolemma (plasma membrane) of the skeletal muscle,  forming one component of a large glycoprotein complex (dystrophin-associated glycoprotein complex).Dystrophin consists of an N-terminal actin-binding domain, 24 spectrin-like repeat units interspersed by four hinge regions, followed by a cysteine-rich domain and a C-terminal domain.  The cysteine-rich domain binds to laminin-2 via alpha and beta dystroglycan, and therefore acts as mechanical link between actin in the cytoskeleton, and the extra cellular matrix.

The DUCHENNE MUSCULAR DYSTROPHY (DMD) gene contains 79 exons, but accounts for only 0.6% of the gene; the rest made of large introns. The large size of the DUCHENNE MUSCULAR DYSTROPHY (DMD) gene makes it susceptible to mutations, with one-third of all mutations arising de novo.

Mutations in the DUCHENNE MUSCULAR DYSTROPHY (DMD) gene result in loss of function of dystrophin, resulting in a prematurely truncated, unstable dystrophin protein. The reading-frame rule explains the phenotypic differences between DUCHENNE MUSCULAR DYSTROPHY (DMD) and Becker muscular dystrophy (BMD): mutations that disrupt the open reading frame, resulting in an abnormal and truncated dystrophin cause DUCHENNE MUSCULAR DYSTROPHY (DMD), whilst mutations which maintain the open reading frame, resulting in a shorter lower molecular weight, but partly functional dystrophin, cause Becker Muscular Dystrophy. Ninety per cent of cases of DUCHENNE MUSCULAR DYSTROPHY (DMD) and Becker Muscular dystrophy conform to this reading-frame rule.

The majority of mutations are intragenic deletions, which account for 65-72% of all DUCHENNE MUSCULAR DYSTROPHY (DMD) patients.  Most deletions occur in the 'hotspot' region, spanning Exons 45-53, with the most common deletions being of Exon 45 and Exons 45-47.  Single or multiexon duplications are found in 7% of patients, most located in a minor hotspot spanning Exons 2-20. Point mutations, small deletions or insertions account for 20% of patients without deletions or duplications. Most are nonsense, frame shift or splice site mutations; missense mutations are extremely rare.

The precise mechanism of how dystrophin deficiency leads to degeneration of muscle fibers remains unclear. Absence of dystrophin at the plasma membrane leads to delocalization of dystrophin-associated proteins from the membrane, disruption of the cytoskeleton with resultant membrane instability and increased susceptibility to mechanical stress. In addition, altered membrane permeability and abnormal calcium homeostasis are thought to play a role, with increased cytosolic calcium concentration leading to activation of proteases such as calpains.  The absence of nitric oxide synthase, delocalized from the subsarcolemmal membrane, may contribute to damage, but is not thought to directly cause dystrophic features.

Various animal models of DUCHENNE MUSCULAR DYSTROPHY (DMD) exist; the most commonly studied and used is the mdx mouse. Several strains of mdx mouse have been characterized; the most commonly used strain has a nonsense mutation in Exon 23 of the DUCHENNE MUSCULAR DYSTROPHY (DMD) gene.

Serum muscle enzymes

The characteristic finding in DUCHENNE MUSCULAR DYSTROPHY (DMD) is a markedly raised serum CK level, at least 10 to 20 times (and often 50 to 200 times) the upper limit of normal before the age of five years. Serum CK concentrations are high even in newborns and prior to any symptoms. The high CK levels at birth can form the basis of neonatal screening for DUCHENNE MUSCULAR DYSTROPHY (DMD). Levels peak at two to three years of age and then decline with increasing age, due to progressive loss of dystrophic muscle fibers.  A serum CK less than 10 times normal in a child with suspected DUCHENNE MUSCULAR DYSTROPHY (DMD) in the first three years of life should raise the question of an alternate diagnosis.

Serum alanine transaminase and aspartate transaminase levels are raised in DUCHENNE MUSCULAR DYSTROPHY (DMD) and tend to correlate with CK levels.  Other enzymes raised in DUCHENNE MUSCULAR DYSTROPHY (DMD) include aldolase and lactate dehydrogenase.  Most of these are not specific for muscle and are generally not useful in the diagnosis of DUCHENNE MUSCULAR DYSTROPHY (DMD).

Courtesy: Muscular Dystrophy Campaign

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