Duchenne muscular dystrophy (DMD)
Duchenne muscular dystrophy (DMD) is a severe and progressive muscle degenerative disease. DMD is one of the most common and well-known genetic disorders. It was first described in 1860 by Duchenne de Boulogne. This disease, like other muscular dystrophies, is caused by degeneration of muscle cells. The first symptoms of DMD, include difficulties with walking and climbing stairs and frequent falls. These symptoms start at the age of 2-3. Most of these patients need a wheelchair between the ages of 10-12. In the best care conditions, DMD Patients die between the ages of 30 and 40 due to cardiac and respiratory problems.
DMD is caused by mutations in DMD gene. these mutations can also lead to a milder form of the disease, Becker muscular dystrophy (BMD). BMD symptoms occurs later than DMD and its progresses is slowly. DMD is the largest human gene with 2.5 M base pairs and 79 exons. Mutations in DMD lead to deficiency of dystrophin protein in the muscle cells. Dystrophin is a large protein that is expressed in skeletal and cardiac muscle cells and contributes to proper muscle function. This protein is located at the intracellular surface of muscle cell membrane and binds the F-actin cytoskeleton to the extracellular matrix. Lack of dystrophin in DMD and its abnormal function in BMD leads to abnormality in muscle cell membranes, destruction and eventually cell death. In DMD, Frameshift mutations (deletion and duplication) and nonsense mutations lead to abnormal, unstable, and inactive dystrophin; In BMD, on the other hand, deletion and duplication mutations maintaining the reading frame and produce proteins with partial activity.
About 65% of DMD cases and 85% of BMD cases cause by deletion of one or more exons in the DMD gene. duplication mutations in one or more exons, accounting for approximately 6-10% of DMD and BMD cases. The rest of cases include point mutations, small deletion and insertion. Complex rearrangements and large intronic changes also account for 2% of DMD cases.
DMD is a recessive, X-linked disorder; So it mostly affects boys. The prevalence of this disease in men is 1 in 3500-6300 live born. per 100,000 boys, 15.9 cases in the USA and 19.5 cases in the UK are reported. The prevalence of DMD in women is very rare (˃ 1 per million) and only has been reported in cases with Turner syndrome. Carriers who have a mutation in DMD on one X chromosome and no mutation on another X chromosome usually do not show symptoms, but in some cases have symptoms similar to BMD.
The DMD Diagnosis Guideline provides an algorithm for diagnosing the disease. The algorithm includes biochemical tests, genetic tests and morphological tests. According to this algorithm, plasma biochemical markers, including creatine kinase (CK), should be tested first for suspect boys aged 2-4 years who have symptoms such as muscle weakness, difficulties with walking and speaking. CK is elevated in the blood plasma of patient with DMD. However, CK may decrease due to progression of the disease and decrease of muscle cells. also this marker may increase for various reasons other than DMD. Thus, the gold standard for DMD diagnosis is genetic testing to detect DMD mutations. In people with physical symptoms and high plasma CK levels, genetic tests should be performed to detect mutations. The lowest level of genetic testing for DMD diagnosing is the use of methods that can detect exon deletions.
The most basic molecular method is to use the Multiplex PCR technique to simultaneously detect common deletion mutations. This method is relatively easy, but it is not able to detect duplication mutations and some deletions, and also it is not effective for carrier screening. For these reasons, newer methods are now being used to diagnose DMD. Newer methods include quantitative analysis of all gene exons that are capable of detecting all deletions and duplications. These methods also have the ability to detect mutations in female carriers.
One of the quantitative methods that is widely used in DMD diagnosis is multiplex ligation-dependent probe amplification (MLPA). The MLPA technique was first used in 2004 to DMD diagnosis. In that study, the DMD-MLPA kit was used, which uses two sets of primers (P034 & P035) to screen mutations in the DMD gene. MLPA is fast, easy, reliable, with high sensitivity and specificity and can amplify all 79 exons of the DMD gene. This technique allows the amplification and quantification of up to 40 nucleotide sequences simultaneously in one tube. MLPA can detect copy numbers of alleles, deletions and duplication mutations in all gene exons. However, in both above mentioned methods, due to the possibility of SNP state, if a mutation is detected by one method, it is necessary to confirm the existence of mutation with an alternative method.
A resent development in quantitative analysis include the use of oligonucleotide-based arrays (CGH arrays); Using this method, it is possible to analyze the variability of copy number along the entire gene, which also makes it possible to detect complex rearrangements and large intronic changes. In this method, most mutations are detected with multiple probes, which allow to control the false positive results due to SNPs.
If the deletion or duplication mutation is not detected in the suspected cases using MLPA and CGH array methods, a complete sequencing analysis should be performed. sequencing can be done for genomic DNA or muscle cDNA. However, using this method, it is not possible to detect 2% of DMD cases, which involve complex rearrangements and large interonic changes. If a mutation is not detected by MLPA, CGH array, and sequencing, a muscle biopsy should be performed. Using this morphological test, it is possible to determine dystrophin localization, size or quantity.
If DMD is diagnosed in a patient, women in the patient’s family should be screened for carriers. If a woman is diagnosed as a carrier, genetic mapping before pregnancy or prenatal DMD screening should be taken. Newborn screening is also done by measuring the level of CK using the newborn blood spots and subsequently genetic testing.
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