Novel polymorphisms in the promoter region of the perforin gene among distinct Brazilian populations and their functional impact.

Cytotoxic T lymphocytes and natural killer cells play a crucial role in eliminating tumour and virus-infected cells. The perforin is a key part of the arsenal that these cells use to destroy their targets. In this study, we characterized single-nucleotide polymorphisms (SNPs) located in the promoter region of the perforin gene among distinct Brazilian ethnic groups. The study was carried out by sequencing this region in three groups: European, African and Asian descents. We demonstrated for the first time the occurrence of three new polymorphisms in the promoter region of gene PRF1: 494A/G (rs78058707), 720G/A (rs75925789) and 1176C/T (rs75183511). Three other SNPs already described in the literature 63A/G (rs35401316), 112A/G (rs10999428) and 1012C/T (rs35069510) were also detected. The SNPs are distributed differently in the ethnic groups studied. The 112G allele was observed at high frequency, especially among Asian descents (48.1%). The 1012T allele was detected only among European descents, the 494G allele only among Asian descents and 1176T allele only in African descents. Based on the association between the polymorphisms described, ten new haplotypes were originated. In functional analysis, we noticed that SNPs present in most common haplotypes cannot induce significant differences in expression levels of perforin alone. In conclusion, this study demonstrates for the first time the existence of three new polymorphisms in perforin promoter and, contrary to what was stated, the presence of these SNPs does not alter the levels of protein expression.

Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and ßCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.