Molecular analysis of promoter region of the SMN2 gene in the patients of spinal musculatr atrophy.

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 SMN1 gene. The isoform to this gene, SMN2 gene, 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. We postulated that the difference in the promoter region of SMN2 gene produces the different level of FL-SMN protein. To verify this hypothesis, the DNA samples of 69 SMA patients who were referred to the Human Genome Center, USM were extracted from their blood. The SMN1 deletion analysis was performed, followed by the SMN2 copy no. analysis and NAIP deletion analysis to remove any clinical bias as NAIP gene deletion and SMN2 copy number have been reported to be associated with SMA disease severity. Only 10 SMA patients from different clinical types (type I=2, type II=3, type III=5) with homozygous deletion of the SMN1 and 2 copies of the SMN2 and deletion in NAIP were finally recruited. Primers were designed for the amplification of the SMN2 promoter region. Bioinformatics analysis was performed to identify the crucial transcription factor binding sites within the reported ~4.6 kb promoter region. As the core promoter region is still unknown (unreported), we analyzed 15 ORFs and 24 nested ORFs with 15 TATA boxes reflecting the diverse functional integrity of this region. The promoter prediction and core promoter prediction was also performed. Based on the bioinformatics analysis and the designed primers, PCR amplification was done for different regions in the promoter and the PCR products were subjected to direct DNA sequencing. The results were analyzed by Vector NTI suite 9, ClustalX and Gene Doc softwares. The molecular analysis confirmed the absence of any mutation in the promoter region of the SMN2 gene between normal healthy individuals (total 2) and SMA patients. In 4 patients and 1 normal healthy individual the CA repeats were found to be increased which we think cause no effect in disease progression and severity. In conclusion, there was no mutation found in the promoter region of the SMN2 gene among the SMA patients of different clinical types and normal controls. Further analysis involving the cloning of the promoter regions with highest probability of involvement in expression of the SMN2 gene using luciferase assay is ongoing. The results will be useful for the subsequent phase of the study involving the transcription initiation of the SMN2 gene