The Accuracy of Immunohistochemistry in Duchenne Muscular Dystrophy
Duchene Muscular Dystrophy or DMD is the most severe of the dystrophies, and begins in early childhood, usually before the age of 4, but with a range of 1 to 10 years. Walking is delayed in about half of patients, and when they start walking, they do so clumsily, with frequent falls. Early weakness of the gluteal and hip extensor muscles causes the characteristic waddling gait and compensatory excessive lumbar lordosis. In addition, enlargement of muscles is often present from birth and is more evident in the calves. Proximal weakness of the arms becomes apparent later, but cranial nerves and sphincter functions are consistently spared (2003). DMD results from failure of synthesis of the muscle cytoskeletal protein, dystrophin, whose gene is located on the X chromosome, Xp12. Approximately, 1 in 4,000 male children get affected, being passed on from the mother (2002). It has been reported that women who carry the defective gene passes an abnormal X on to their sons, and since males have an X from their mother and a Y from their father, there is no second X to make up for the defective gene from the carrier mother. In this regard, the sons of the carrier mother each have a 50% chance of having the disease, and the daughters each have a 50% chance of being carriers. Thus, daughters of men with DMD will always be carriers, since they inherit an affected X chromosome from their father (2006). In relation to this disorder is the mutation of the gene for dystrophin. The gene for dystrophin is very large and a common target for mutation. Large deletions or duplications can be detected in the gene for dystrophin in 65% of cases. Demonstration of such mutation provides a useful genetic marker for the disorder, but diagnosis and prognosis require muscle biopsy and immunohistochemistry for the dystrophin protein (2002). In this sense, it can be perceived that in order to effectively detect Duchenne Muscular Dystrophy or DMD, immunohistochemistry must be used. As such, this paper would be discussing the accuracy of immunohistochemistry in DMD through a specific study or experiment.
To be able to attest to the accuracy of immunohistochemistry in DMD, a specific study must be discussed and examined. The particular experiment referred to was written by (2007), and was entitled, “Duchenne and Becker Muscular Dystrophy: A Molecular and Immunohistochemical Approach”. The aim of this experiment is to report the frequency of dystrophin gene deletions in a group of patients with clinical evidences of DMD/BMD. To be able to determine the accuracy of immunohistochemistry, the experiment studied 106 unrelated patients referred for DNA analysis of the dystrophin gene between 1999 and 2005. Such patients had a diagnosis of DMD or BMD based on findings through electromyography and elevated serum creatinine kinase activity. All patients underwent DNA analysis and muscle biopsy. The DNA of the patients was isolated from peripheral blood leukocytes using the standard PCR analysis or phenol/chloroform method. The exons studied include having the numbers 3, 4, 6, 8, 12, 13, 17, 19, 42, 43, 44, 45, 47, 78, 50, 51, 52, 53, 60, and Pm. On the other hand, the muscle biopsy was done in 51 patients, which was freshly frozen and cut on cryostat into 8 micron sections and stained with hematoxylin-eosin, modified Gomori trichrome, oil-red O, PAS, cresyl violet, and Sirius red.
Results of the experiment indicate that at least one deletion in the 20 dystrophin-gene exons were detected in 76 cases. A total of 121 deletions were found in such cases. 82 deletions, corresponding to 68% were detected in exons 42 to 63; 39 deletions, corresponding to 32% were detected in exons 3 to 19, and the most frequent deletions found in the patients were in exons 45, corresponding to 19.05%, exons 19, corresponding to 13.10%, and exons 43, having 11.9%. In addition, the diagnosis of DMD or BMD was confirmed by dystrophin analysis in 39/51 muscular biopsies, 21/39 of which had deletions in the dystrophin gene. One symptomatic carrier showed an absence of the carboxyl region of dystrophin and 35 DMD patients, including one female, showed no reaction for the carboxyl- and amino-terminal and rod domains. Only one BMD patient and two symptomatic female carriers had large gaps or punctuated discontinuous immunostains for dystrophin in the sarcolemma of muscle fibers. 12 of 51 muscle biopsies analyzed showed the presence of other muscle diseases. 2 had severe denervation in the muscle biopsy compatible with spinal muscular atrophy; one had alterations in muscular fibers compatible with congenital myopathy; 2 had sarcoglycan deficiency; and 7 had alterations compatible with muscular dystrophy but had normal immunohistochemistry.
The results of the experiments were said to be compatible with other studies, as the 20 studied exons of the dystrophin gene and detected deletions in 71.7% of the patients were detected in exons 45 and 43. Because the samples of the DNA in this experiment were obtained from the leukocytes of the patients, its results were different to another study, with which samples were obtained from impaired muscle tissues of patients. In this regard, the study may have found out that deletions caused by somatic mosaicism or germ-line cells were not obtained using leukocytes. In relation to immunohistochemistry, it has been emphasized that 30 samples for which DNA deletions were not found were submitted to muscle biopsy and immunohistochemistry for dystrophin, sarcoglycans and dysferlin, thus, showing alterations in muscle fibers. In this regard, immunohistochemistry of muscle biopsy serves to be the gold standard for the effective and efficient diagnosis of DMD/BMD, but is a more invasive procedure and should only be performed in those cases where PCR blood screening does not detect deletions. To spare more patients from muscle biopsy, other techniques to investigate mutations and other methodologies, such as quantitative real-time PCR, should be used, particularly when the patient symptoms, family history, and clinical findings suggest DMD or BMD.
From the results and findings of the experiment, it can be perceived that immunohistochemistry in relation to the detection of BMD or DMD is the most accurate test, thus, allowing precise, effective, and efficient analysis of samples. In this regard, if immunohistochemistry would be used in the detection of the disease, then practical solutions and assistance can be provided to the patient suffering from the disease. Although cure for the disease cannot be provided, temporary assistance for the patient can be given, such as steroids, with the help and support of the family and peers of the patient.
Credit:ivythesis.typepad.com
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