ATM allelic variants associated to hereditary breast cancer in 94 Chilean women: susceptibility or ethnic influences?

Artículo de publicación ISIBesides BRCA1 and BRCA2, two genes accounting for a small proportion of breast cancer cases, ATM has been widely proposed as a low-penetrance susceptibility gene. Several nucleotide changes have been proposed to be associated with breast cancer, still remaining a high controversy in this sense. We screened the ATM gene in 94 breast cancer patients selected from 78 high-risk families, not presenting a mutation in BRCA1 or BRCA2. We found three novel allelic variants: IVS64 + 51delT and p.L752L, not showing association with hereditary breast cancer, and p.L694L found in one family in two breast cancer patients. Two amino acid substitutions p.S707P and p.F858L, previously reported to be associated with breast cancer, were present in our study in cases and controls, lacking of association with breast cancer. A positive association of c.5557G>A (p.D1853N) was found (OR 2.52, P = 0.008), when analyzed alone and in combination with an intronic variant IVS24-9delT (OR 3.97; P = 0.0003). We postulate that our discrepancies with other reports related to the associated ATM alleles to hereditary breast cancer, as well as discrepancies in the literature between other groups, could be explained by the diversity in the ethnic origins of families gathered in a sole study, and the selection of the control group. In relation to this issue, and based on genetic markers, we found that the Chilean group of breast cancer families in this study has a stronger European genetic component than our control sample selected randomly from the Chilean population

A cis-acting element present within the Gag open reading frame negatively impacts on the activity of the HIV-1 IRES.

Translation initiation from the human immunodeficiency virus type-1 (HIV-1) mRNA can occur through a cap or an IRES dependent mechanism. Cap-dependent translation initiation of the HIV-1 mRNA can be inhibited by the instability element (INS)-1, a cis-acting regulatory element present within the gag open reading frame (ORF). In this study we evaluated the impact of the INS-1 on HIV-1 IRES-mediated translation initiation. Using heterologous bicistronic mRNAs, we show that the INS-1 negatively impact on HIV-1 IRES-driven translation in in vitro and in cell-based experiments. Additionally, our results show that the inhibitory effect of the INS-1 is not general to all IRESes since it does not hinder translation driven by the HCV IRES. The inhibition by the INS-1 was partially rescued in cells by the overexpression of the viral Rev protein or hnRNPA1

Secondary structure model of the 5′UTR of selected HIV-1 VAR sequences.

<p>The HIV-1 5′leader recovered from the dl VAR constructs (followed by 58 nucleotides of fLuc gene) was probed using Dimethyl Sulfate (DMS), N-Cyclohexyl-N0- [N-Methylmorpholino)-ethyl]-Carbodiimide-4-Toluolsulfonate (CMCT) and RNAse V1 as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos1" target="_blank">[20]</a>. (A) Secondary structure model of the control (pNL4.3) HIV-1 5′UTR according to DMS, CMCT and RNAse V1 structure probing <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Berkhout1" target="_blank">[6]</a>, a key indicating the respective reactivity to the different probes is provided (box). Data included in the figure have been extracted from reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos1" target="_blank">[20]</a>. (B) Typical examples of DMS probing. The HIV-1 5′leader was probed using (+) DMS. Reverse transcription (RT) products were separated on a 8% gel as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Weill1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-James1" target="_blank">[48]</a>. Sequencing lanes were also included. Note that DMS induces a premature RT stop one nucleotide before the hit. Therefore the DMS induced stops migrate faster than the corresponding sequence product <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Weill1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-James1" target="_blank">[48]</a>. The RT pattern of the modified RNA was compared to the profile obtained with an unmodified RNA. Some hits are indicated in the figure. The asterisks on the gel denote the FLuc initiation codon (AUG). Results for VAR 1 (C), VAR 2 (D), VAR 3 (E), and VAR 4 (F) were fitted in a model of the HIV-1 5′ leader <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Berkhout1" target="_blank">[6]</a> as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos1" target="_blank">[20]</a>. Numbering in A-F is with respect to clone pNL4.3, here considered the wt sequence. Insertions are indicated in red as independent numbers (iN, were N is the number). Nucleotide changes with respect to clone pNL4.3 are indicated in green. The nucleotide located before a deletion is highlighted in yellow, in this case numbering with respect to pNL4.3 is not altered.</p

Analysis of a promoterless bicistronic construct containing the HIV-1 5′UTR sequences recovered from clinical samples.

<p>(<b>A</b>) Schematic representation of the bicistronic constructs. The SV40 promoter from dl ΔEMCV (lane 4), dl HIV-1 IRES, or dl VAR was removed to generate the equivalent promoterless (ΔSV40) vectors. (<b>B</b>) HeLa cells were transfected with DNA (200 ng) corresponding to the vectors depicted in (A) as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos2" target="_blank">[23]</a>. Total DNA was extracted from transfected cells and the presence of the transfected plasmids was confirmed by PCR (upper panel). The dl HIV-1 IRES plasmid (100 ng) was used as an amplification control. Total RNA was extracted from transfected cells and the presence of transcripts for the ΔSV40-dl ΔEMCV, ΔSV40-dl HIV-1 IRES, or the ΔSV40-dl VAR plasmids was evaluated by a one step RT-PCR designed to detect the bicistronic RNA (depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone-0035031-g002" target="_blank">Fig. 2B</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos2" target="_blank">[23]</a>. <i>In vitro</i> transcribed RNA (100 ng) generated from plasmids dl HIV-1 IRES (lane 2) were used as amplification controls. (<b>C</b>) HeLa cells were transfected with either the 200 ng of SV40 or ΔSV40 version of dl ΔEMCV, dl HIV-1 IRES, or the different dl VAR plasmids as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035031#pone.0035031-Vallejos2" target="_blank">[23]</a>. Cells were processed and RLuc and FLuc activities were measured. For each data point the [RLuc/(total protein)] (left panel) and the [FLuc/(total protein)] (right panel) for the SV40 positive plasmids was arbitrary set to 100%. Values are the means +/- SD from three independent experiments.</p