Abstract
Introduction
Intervertebral disc being an avascular tissue, the extracellular matrix protein play a major role in its structural stability. The pathophysiology underlying the process of Disc Herniation is still not clear but genetic factors play a vital role in the early occurrence of the disease. The study was initiated to determine the association of Collagen9A1, Collagen9A2, Collagen9A3 and TNF-α gene polymorphisms in Disc herniated patients in comparison to healthy individuals in Indian population.
Methods
Pre Operative blood samples were collected from 20 patients under going surgery for disc herniation between the age group of 20–60years and 10 samples from healthy individuals with no history of Intervertebral disc disease. DNA was isolated, primers designed for selected exonic regions in the triple helical binding sites for Collagen and for complete 2763 bp of TNF-α and VNTR in exon 12 for aggrecan, performed PCR and samples sequenced for analysis of polymorphic variants. Genotypic and phenotypic distributions were compared with Healthy controls. Hardy Weinberg calculations were done to correlate the allelic/genotypic frequencies in the both study groups.
Results
The PCR sample analysis resulted in identification of 11 variants in complete TNF-α gene, 8 variants in Col9A1, 6 variants in Col9A2 and 3 variants in Col9A3 gene.Polymorphic variants pro620thr, gln621arg and a splicesite variant at intron-exon29 A > C for Col9A1,gln326arg for Col9A2 and ser2 gly and ala16 to arg for TNF-α were statistically significant. These variants were predominant in lower age groups of 40 years. Aggrecan showed the allele 14 and 15 to be common among both the study groups. The healthy subjects for Collagen9 did not show any variant where as TNF-α showed 3 variants which were not statistically significant.
Conclusion
The SNP at amino acid proline 620, glutamine 621 in Col9A1, glutamine 326 in Col9A2 and arginine 103 for Col9A3 are important for the stability of the triple helical structure of collagen9 and can be directly corelated to the early occurrence of the disease in younger patients. There was no association between an aggrecan VNTR polymorphism and disc herniation. The TNF-α gene variants were not directly correlated to the onset or progression of the disease but could influence their expression levels which could trigger additional inflammatory response as in rheumatoid arthritis. PCR genetic markers can be used as a diagnostic tool to identify the progression of the disease as in Ankylosing spondilosis. This is a first report on Indian population in Disc Herniated patients.