Molecular analysis of the promoter and the core promoter region Of the smn2 gene in circumscribing the clinical severity of sma

• Main Author: Baig, Atif Amin
• Format: Thesis
• Language: English
• Published: 2012
• Subjects: R Medicine (General); RA0421 Public health. Hygiene. Preventive Medicine; RA440-440.87 Study and teaching. Research
• Online Access: http://eprints.usm.my/60940/1/ATIF%20AMIN%20BAIG%20-%20e.pdf; http://eprints.usm.my/60940/
• Institution: Universiti Sains Malaysia

Spinal muscular atrophy (SMA) is a neurodegenerative disorder caused by the absence of the full length SMN protein (FL-SMN) as a result of mutation or deletion of SMN\ gene. The isoform to this gene, SMN2, with mutation in 1 base pair, encodes for 10% of FL-SMN protein and is reported to decrease the severity of the disease when there is an increase gene dosage. There are 3 clinical types of SMA; type I, type II and type III. Type I SMA is the most severe type and only a small amount of FL-SMN protein is present in these individuals. In this study it was hypothesized that the variation in the DNA sequence in the promoter region of the SMN2 gene could be the possible cause of difference in clinical severity of SMA. To verify this hypothesis, a total of ten (10) SMA patients from different clinical types were selected from the retrospective pool data of the SMA patients and their DNA was extracted. The promoter regions of the SACV2 gene in these patients were screened for the presence of any variation, firstly within the proximal promoter region (Pro) which also contains a reported CR.E-II element followed by the screening of the entire promoter region. The screening of the entire promoter was performed in two steps. Firstly, the regions within the promoter with computational cis acting elements were identified (using Cister analysis) and then the DNA sequencing was performed using specific designed primers. Secondly, the regions other than cis acting elements were screened for the presence of any variations. The bioinformatics analysis also revealed the probability for crucial transcriptional factor bindings sites, novel TSS at -557 bp and a TATA box at -411 bp within the 15 ORFs and 24 nested ORFs. The core promoter region of the SMN2 gene needs to be identified to study the diverse functional integrity of this region. For this purpose, the computational promoter prediction and core promoter prediction analysis was performed and the results of this analysis along with the previous reported literature were used to propose a model of the core promoter region of the SMN2 gene. To analyze this model biochemically, the entire Pro region of 720 bp was divided into five regions (Pro 720, Pro 500, Pro 400, Pro 200 and Pro 100). These five regions were PCR amplified and were cloned into pTOPO-2.1 e cloning vector. After confirming the insert and the vector backbone by PCR, restriction analysis and DNA sequencing, the Pro regions were subcloned into pGL4.74 reporter vector by high throughput directional cloning using EcoRI and HindWl restriction sites to be transfected directly into HeLa cell lines. Before the transfection the optimization of the concentration of the test vector pGL4.74 and the control vector pGL4.10 was performed beside the optimum concentration of test to control vectors for the co-transfection. The transfected HeLa cell lines were subjected to lysis followed by the dual luciferase assay. After the normalization of data of the luciferase assay, it was found that the fold change in the mean percent activity of luciferase (from 1.03 to 2.71) between Pro constructs highly suggested the role of newly identified computational TSS (-557 bp) in TATA (-411 bp) specific CREB induced expression of downstream reporter gene through structural and functional role of CRE-II element. Since, there was no variation identified in the promoter region of the SMN2 gene in the SMA patients from different clinical severities, it showed that the promoter region of the SMN2 gene has no role in circumscribing the clinical severity of SMA.