Introduction: Duchenne Muscular Dystrophy (DMD) is one of the most prevalent neuro-genetic disorders in Iran. DMD is an X-linked recessive disorder characterized by severe, progressive, and muscle-wasting symptoms. The onset of DMD symptoms is between 2-5 years and consists of hypertrophy in the calf muscles, waddling walk, trouble climbing stairs, walking on toes, repeated falls, Gower’s sign, and progression of muscular degeneration. The Dystrophin gene is the largest known gene in human genomes and consists of 79 exons. The main cause of this disorder is deletion (66%), then duplication (7.5%). Deletions are more common in the two hotspot regions of the DMD gene which include exons 1–22 and exons 43–55. Based on vast and recent research in Iran, the most frequent deletions occur in exons 50 (6.8%), 48 (6.4%), 46 (6.2%), 49 (6.2%), 47 (6.0%), 51 (6.0%) and 45 (6.0%) respectively. Multiplex-PCR and MLPA are the most common tests in order to identify the deletions. Using isothermal techniques for the detection of the mutations could be a point of care and alternative approaches for the diagnosis of the disease. The aim of the present study was to identify the exon 47 deletion of the DMD gene using Gap-LAMP (Loop-mediated isothermal amplification) method.
Methods: The DNA sample of one patient that has exon 47 deletion in the DMD gene was collected from Kariminejad-Najmabadi Pathology & Genetics Center. The genomic DNA of the healthy individuals was extracted using the phenol-chloroform DNA extraction method.
We designed 6 primers for detecting exon 47 of the DMD gene and 6 primers for the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Gene as internal control by LAMP primer designing software PrimerExplorer-5 (https://primerexplorer.jp/e/). The LAMP reaction was performed on a total of 20µl containing Bst-2 DNA Polymerase manufactured by New England Biolabs. The reaction mix was incubated for 30 minutes at 65° centigrade. The agarose gel electrophoresis was applied for the Visualization of the LAMP products.
Results: The results of the study showed that the designed method could amplify the target region of the control group while this region was not detected in the patients due to the deletions. The GAPDH region was successfully detected in all Samples.
Conclusion: LAMP is an isothermal nucleic acid amplification technique in which the reaction is carried out at a constant temperature, and does not require a thermal cycler. This method has usually been used for the identification of infectious agents. The present study showed that this method can be used for detection of deletions in human DMD genes. Since LAMP is performed in an isothermal condition that does not require a thermal cycler device it can be used as a screening method for the detection of the deletions in DMD patients. Further studies are recommended to design Gap-LAMP approaches for detection of deletions in all exons of DMD gene.