Extracting DNA from Embalmed Tissue to Study Genetic Predisposition to Degenerative Disc Disease

INTRODUCTION. According to a prominent study, 80-90% of people will show some evidence of degenerative disc disease (DDD) by age 50. Researchers have proposed that a Q326W substitution in the COL9A2 gene increases susceptibility to DDD. The role of collagen IX in intervertebral discs is not completely known, but due to its increased flexibility compared to other types of collagens, it is thought to connect collagen II with other components of the cartilage which are important to the integrity of the disc. Collagen II can be found within the nucleus pulposus and the inner portion of the annulus fibrosis. Researchers have also suggested that the heterozygous form of the TaqI polymorphism can increase susceptibility to DDD. Vitamin D plays a role in sulphate metabolism. Therefore, the extent to which the vitamin D receptor (VDR) is expressed can affect sulphation of glycosaminoglycans during proteoglycan synthesis which can impact the structural integrity of the extracellular matrix of the nucleus pulposus. Following previous research that demonstrated successful genomic DNA (gDNA) isolation from embalmed tissues, this project attempted to analyze the VDR and COL9A2 genes in samples collected from three embalmed female cadaver donors with obvious lumbar DDD. METHODS. Samples were isolated from four tissues for gDNA extraction: parotid gland, cerebellum, genioglossus muscle and occipital lobe following approval from the donor institution. DNA samples were subsequently analyzed for concentration and purity. COL9A2 and VDR specific primers were used for polymerase chain reaction (PCR). Following PCR amplification and DNA sequencing, samples were compared with published National Center for Biotechnology Information (NCBI) sequences using BLASTn analysis. SUMMARY. DNA was successfully extracted from each tissue, but cerebellar and parotid tissue yielded DNA of highest concentration. The mean purities of each tissue were not significantly different. Nucleotide BLAST alignment did not show evidence of the Q326W substitution; however, studies suggest that this substitution is present in only 4% of patients with a form of DDD. Evidence of the TaqI polymorphism was not observed either. CONCLUSION. While the Q326W substitution and TaqI polymorphism were not identified in these cadavers, the ability to successfully extract and amplify DNA from embalmed tissue was confirmed. This is a preliminary project and additional genes and donors will be studied in the future