PCR Real time Mismatch Amplification Mutation Assay (MAMA Real Time PCR) for evaluation of TNFα promoter gene polymorphism -308 G/A in patients with psoriasis.

BACKGROUND: Psoriasis is a common chronic inflammatory disease, the plaques are infiltrated by leukocytes producing high levels of proinflammatory cytokines and TNF-α. Single-nucleotide polymorphisms within the gene promoters have been shown to affect gene expression. The -308 G/A polymorphism could affect TNF synthesis at transcriptional level. The present study develops a MAMA Real Time PCR assay, in order to identify homozygosis or heterozygosis for TNF-α -308 G/A polymorphism. METHODS: Seventy patients with psoriasis and 235 controls were considered for the development of the real time PCR assay. Whole blood was processed for nucleic acid extraction. RESULTS: A percentage of 36.17% controls and 38.6% patients were heterozygosis, considering Amplification-refractory mutation system (ARMS)-PCR assay while 23% and 22.85% were heterozygosis using Mismatch Amplification Mutation Assay (MAMA)-PCR. On the contrary, 1.3% and 1.4% were homozygosis A, while 75.7% and 75.75% presented homozygosis G, taking into account the MAMA-PCR results. The two assays were significantly different (P=0.0004 at χ2 Test), but MAMA-PCR showed a better performance for TNF-α -308 G/A gene polymorphism investigation. CONCLUSIONS: Further studies are needed for a better comprehension of the role of this polymorphism, such as MAMA real time PCR assays development for other players in cellular immune response.