Optimizing a Method for Screening Interleukin-1β (+3954) Gene Polymorphisms

Objectives: IL-1β is a proinflammatory cytokine involved in initiating and propagating immune and inflammatory reactions. IL-1β polymorphisms have been implicated in chronic periodontitis, root resorption, oral aphthous stomatitis, burning mouth syndrome, and osteosarcoma. Specifically, the single-nucleotide polymorphisms (SNPs) of the IL-1β gene at +3954 have been reported to alter the amount of IL-1β production. Models have been proposed where alterations in the level of IL-1β expression may result in an individual’s predisposition to a particular disease. Objectives: To optimize a DNA isolation and analysis protocol to identify IL-1β +3954 allelic variations on a large scale. A standardized and efficient technique will allow for large scale adoption in both large medical and dental care facilities and small practice settings.
Methods: Samples for DNA analysis were collected by scraping the inside of the cheek with a sterile nylon bristle brush. Genomic DNA was obtained from these samples with the Puregene method (Gentra Systems). PCR amplification of a region encompassing the +3954 site was performed, followed by TaqI digest. PCR products and digested fragments were separated by agarose gel electrophoresis, stained with ethidium bromide and visualized under ultraviolet light.
Results: Isolation of genomic DNA from buccal cheek cells resulted in a higher yield than isolation from saliva. The digested PCR products were detectable and specific to each allele at +3954 C/T. The fragments of 85 bp and 97 bp corresponded to +3954 C and a single 182 bp fragment corresponded to +3954 T.
Conclusions: This optimized protocol is suitable for identification of homozygous and heterozygous polymorphisms at IL-1β +3954 C/T. This method is suitable for use on a large scale and will serve as the basis to develop protocols to detect other inflammatory cytokine polymorphisms that have been implicated in other diseases.