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Nemaline myopathy is a disease that primarily affects skeletal muscles due to mutations in the Actin Protein family. The purpose of analysis was to determine the potential regions on the ACTA1 protein that have maximum probability of mutations. The identification of the disordered regions was performed using various tools like DISEMBL, GLOB PLOT and RONN. From the results of the disorder prediction the regions are identified which are further processed for the identification of sites within the regions that have maximum chances of mutability and thus termed as mutational hotspots. To identify the mutational hotspots RONN results were considered followed by the use of the tools like Polyphen and I Mutant. The effect of point substitution mutations on the stability of the protein was calculated using Imutant. Polyphen would detect the effect of the mutations on the function of the proteins. The complete mutational site annotation and structure prediction was carried out. The work was extended to identify the restriction sites present in the gene sequence of the study protein. Once the restriction sites were identified using Nebcutter the results were employed for the construction of Plasmid vector that can carry the mutant or wild peptide so as to express the activity of the peptide. This data can be of immense use I the application r DNA technology in the analysis of this protein.
Nemaline myopathy, Mutation Analysis, ACT1 Protein, ACTA1 protein, r DNA Technology.
Myopathy is the neuromuscular disorders in which the primary symptom is the muscle weakness  caused due to dysfunction of muscle fiber. Myopathy has been classified in to many disorders; they are Inflammatory Myopathies, Muscular dystrophies, Myotonic Syndromes, Congenital myopathies, metabolic myopathies, Endocrine myopathies, Endocrine myopathies etc. Mypathic disorders generally produce proximal arm and leg weakness a so called limb girdle pattern . Congenital myopathies are muscle diseases present at birth (congenital) that result from genetic defects in our muscle development. Congenital myopathies may result in muscle weakness, breathing and eating problems. Congenital Myopathies include Central core disease, Centronuclear myopathies, congenital fiber type disproportion myopathy, Nemaline myopathy, Multiminicore disease, Myotubular myopathy. Congenital myopathies has led to the recognition that diverse disorders are often associated with the same causative gene, and is challenging traditional classifications . Missense mutations of the skeletal muscle alpha-actin gene (ACTA1) often results in the onset of Ccngenital myopathies . In view of the importance of this protein in the onset of the disease the current work aims to study the mutational hot spot regions within the ACTA1 protein followed by the gene analysis and construction of suitable vector to carry the gene using Insilco methodology.
The current study aimed to analyze the complete protein of ACTA1 mutations in which results in the nemaline myopathy. The work involves the analysis of the normal sequence structure and functional properties of the protein from Homo sapiens. Once the properties of the native proteins were studied the work focused on the identification of the probable mutational regions present in the sequence based on the tools like GLOB PLOT, DISEMBL. Once the region was identified the study was extended to select the specific sites base on the tools like RONN and parameters like hydropathicity and mutation probability. The site 54 has been identified as the mutational hot spot for the ACTA1 protein from Homo sapiens. The effect of substitution of wild amino acid by any of the other 19 possible amino acids at the potion 54 was analyzed using various tools. Io mutant was used to analyze the effect on the stability of the protein where as Polyphen was used to analyze the effect on the functionality o the protein. Finally the protein was reverse translated to obtain the corresponding gene sequence which was used to assess the restriction sites in the gene. The results of neb cutter for restriction site identification were used to design the vector for carrying the ACTA1 gene. This was done using the tool PLASMAPPER. The final out come of the work is the identification of the mutational hot spot and study the effect of mutation in this site. This was followed by designing a suitable vector that can act as a carrier for the query gene sequence.
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