Evaluation of IFN-gamma polymorphism+874 T/A in patients with recurrent tonsillitis by PCR Real Time Mismatch Amplification Mutation Assay (MAMA Real Time PCR)

摘要

Interferon gamma (IFN-gamma) is an important cytokine that plays a crucial role in the balance between normal and pathological immune response. Defect of IFN-gamma can give a predisposition to infectious disease, autoimmune pathologies and turnouts. Different polymorphisms in this gene have been described, in particular the single nucleotide polymorphism (SNP) + 874 (*) T/A that may affect IFN-gamma gene expression. Several techniques can be used for the detection of SNPs. In this work two PCR Real Time assays were developed, an Amplification Refractory Mutation System (ARMS) and a Mismatch Amplification Mutation Assay (MAMA). Twenty-seven samples from patients (tonsillectomy) and 85 from donor's blood bank were considered. As a result, 78/85 controls (91.7%) and 25/27 patients (92.6%) were heterozygosis, considering the ARMS-PCR; 55/85 (64.7%) and 14/27 (51.9%) were heterozygosis using MAMA-PCR assay. Fourteen of 85 (16.5%) and 8/27 (29.6%) were homozygosis A, 16/85 (18.8%) and 5/27 (18.5%) presented homozygosis T, taking into account the MAMA-PCR. There are statistically difference between the two assay with p < 0.0001 at Chi-square test. Our preliminary data suggest that tonsillectomy patients had a statistical trend to possess the low IFN-gamma polymorphism when compared with control subject (p = 0.3) but is not statistically significant. In conclusion the Real time MAMA-PCR assay has several advantages over other SNP identification techniques such as rapidity, reliability, easily to perform in one working day and applicable in clinical molecular diagnostic laboratories, although sequencing remains the gold standard. (C) 2014 Elsevier Ltd. All rights reserved.