Bioethical issues of preventing hereditary diseases with late onset in the Sakha Republic (Yakutia)

Background: Prenatal diagnosis of congenital and hereditary diseases is a priority for the development of medical technologies in Russia. However, there are not many published research results on bioethical issues of prenatal DNA testing. Objective: The main goal of the article is to describe some of the bioethical aspects of prenatal DNA diagnosis of hereditary diseases with late onset in genetic counselling practice in the Sakha Republic (Yakutia) – a far north-eastern region of Russia. Methods: The methods used in the research are genetic counselling, invasive chorionic villus biopsy procedures, molecular diagnosis, social and demographic characteristics of patients. Results: In 10 years, 48 (76%) pregnant women from families tainted with hereditary spinocerebellar ataxia type 1 and 15 pregnant women from families with myotonic dystrophy have applied for medical and genetic counselling in order to undergo prenatal DNA testing. The average number of applications is 7–8 per year. There are differences in prenatal genetic counselling approaches. Conclusion: It is necessary to develop differentiated ethical approaches depending on the mode of inheritance, age of manifestation, and clinical polymorphism of hereditary disease

Effect of Low-Dose Ionizing Radiation on the Expression of Mitochondria-Related Genes in Human Mesenchymal Stem Cells

The concept of hormesis describes a phenomenon of adaptive response to low-dose ionizing radiation (LDIR). Similarly, the concept of mitohormesis states that the adaptive program in mitochondria is activated in response to minor stress effects. The mechanisms of hormesis effects are not clear, but it is assumed that they can be mediated by reactive oxygen species. Here, we studied effects of LDIR on mitochondria in mesenchymal stem cells. We have found that X-ray radiation at a dose of 10 cGy as well as oxidized fragments of cell-free DNA (cfDNA) at a concentration of 50 ng/mL resulted in an increased expression of a large number of genes regulating the function of the mitochondrial respiratory chain complexes in human mesenchymal stem cells (MSC). Several genes remained upregulated within hours after the exposure. Both X-ray radiation and oxidized cfDNA resulted in upregulation of FIS1 and MFN1 genes, which regulated fusion and fission of mitochondria, within 3–24 h after the exposure. Three hours after the exposure, the number of copies of mitochondrial DNA in cells had increased. These findings support the hypothesis that assumes oxidized cell-free DNA as a mediator of MSC response to low doses of radiation

Satellite III (1q12) Copy Number Variation in Cultured Human Skin Fibroblasts from Schizophrenic Patients and Healthy Controls

Background: The chromosome 1q12 region harbors the genome’s largest pericentromeric heterochromatin domain that includes tandemly repeated satellite III DNA [SatIII (1)]. Increased SatIII (1) copy numbers have been found in cultured human skin fibroblasts (HSFs) during replicative senescence. The aim of this study was to analyze the variation in SatIII (1) abundance in cultured HSFs at early passages depending on the levels of endogenous and exogenous stress. Methods: We studied 10 HSF cell lines with either high (HSFs from schizophrenic cases, n = 5) or low (HSFs from healthy controls, n = 5) levels of oxidative stress. The levels of endogenous stress were estimated by the amounts of reactive oxygen species, DNA damage markers (8-hydroxy-2′-deoxyguanosine, gamma-H2A histone family member X), pro- and antioxidant proteins (NADPH oxidase 4, superoxide dismutase 1, nuclear factor erythroid 2-related factor 2), and proteins that regulate apoptosis and autophagy (B-cell lymphoma 2 [Bcl-2], Bcl-2-associated X protein, light chain 3). SatIII (1) copy numbers were measured using the nonradioactive quantitative hybridization technique. For comparison, the contents of telomeric and ribosomal RNA gene repeats were determined. RNASATIII (1 and 9) were quantified using quantitative Polymerase Chain Reaction (PCR). Results: Increased SatIII (1) contents in DNA from confluent HSFs were positively correlated with increased oxidative stress. Confluent cell cultivation without medium replacement and heat shock induced a decrease of SatIII (1) in DNA in parallel with a decrease in RNASATIII (1) and an increase in RNASATIII (9). Conclusions: During HSF cultivation, cells with increased SatIII (1) content accumulated in the cell pool under conditions of exaggerated oxidative stress. This fraction of cells decreased after the additional impact of exogenous stress. The process seems to be oscillatory

Genome instability in MCF-7 cells exposed to gDNA^OX at final concentration 50 ng/mL for 24 hours.

<div><p>A – multiple micronuclei [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>], chromatin bridges [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>], M-phase chromatin decondensation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>], non-treated control cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B4" target="_blank">4</a>] (x100). </p> <p>B – proportions of cells with micronuclei in non-treated control cells, cells exposed to gDNA, cells exposed to gDNA<b>^OX</b>. Grey columns: non-confluent, actively proliferating MCF-7 culture. Black columns: MCF-7 cells at high confluency. *p < 0.05 against control group of cells, non-parametric U-test.</p> <p>С - Exposure to gDNA<b>^OX</b> (50 ng/mL, 2 hours) induces formation of 8-oxodG-containing micronuclei (x100). </p></div

Changes of KEAP1/NRF2 and IKB/NF-κB Expression Levels Induced by Cell-Free DNA in Different Cell Types

Cell-free DNA (cfDNA) is a circulating DNA of nuclear and mitochondrial origin mainly derived from dying cells. Recent studies have shown that cfDNA is a stress signaling DAMP (damage-associated molecular pattern) molecule. We report here that the expression profiles of cfDNA-induced factors NRF2 and NF-κB are distinct depending on the target cell’s type and the GC-content and oxidation rate of the cfDNA. Stem cells (MSC) have shown higher expression of NRF2 without inflammation in response to cfDNA. In contrast, inflammatory response launched by NF-κB was dominant in differentiated cells HUVEC, MCF7, and fibroblasts, with a possibility of transition to massive apoptosis. In each cell type examined, the response for oxidized cfDNA was more acute with higher peak intensity and faster resolution than that for nonoxidized cfDNA. GC-rich nonoxidized cfDNA evoked a weaker and prolonged response with proinflammatory component (NF-κB) as predominant. The exploration of apoptosis rates after adding cfDNA showed that cfDNA with moderately increased GC-content and lightly oxidized DNA promoted cell survival in a hormetic manner. Novel potential therapeutic approaches are proposed, which depend on the current cfDNA content: either preconditioning with low doses of cfDNA before a planned adverse impact or eliminating (binding, etc.) cfDNA when its content has already become high

The exposure to gDNA^OX leads to an increase in the production of ROS.

<p>А – Microscopy-based evaluation of MCF-7 cells sequentially treated with DNA (50 ng/mL) and H2DCFH-DA (control, gDNA, gDNA^ox [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]) and incubated for 30 minutes (x100). Alternatively, MCF-7 cells were incubated with DNA (50 ng/mL) for 1 hour followed by addition of H2DCFH-DA and photography 30 minutes later (gDNA^ox [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]). B - MCF-7 cells exposed to gDNA^ox (0.5h; 50ng/mL), were sequentially treated with Mito-tracker TMRM (15 min) and H2DCFH-DA (15 min) (x200). C - Co-detection of labeled probe gDNA^red (50 ng/mL) and DCF after 30 minutes of incubation. D - The results of the quantification of fluorescence using plate reader [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. The time kinetics of fluorescence outputs in cells sequentially treated with H2DCFH-DA and, three minutes later, a DNA sample at final concentration of 5 or 50 ng/mL [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. The same for cells pretreated with DNA (final concentration 5 ng/mL) for one hour, with subsequent addition of H2DCFH-DA. *) p < 0.05 against control group of cells, non-parametric U-test.</p

The exposure to gDNA^OX (50 ng/mL) leads to a transient increase in expression cytoplasmic DNA sensor AIM2, while not changing expression levels of TLR9.

<div><p>A - intracellular localization of AIM2 (FITC-conjugated antibodies) and labeled probe gDNA<b>^red-ox</b> (x40). B – the ratio of the levels of AIM1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] and TLR9 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>] – encoding RNAs to the levels TBP-encoding reference mRNA in cells exposed to gDNA or gDNA^OX for 2 hrs (grey columns) and 48 hrs (black columns).</p> <p>C and D – Flow cytometry detection of AIM2 (C) and TLR9 (D) expression in MCF-7. Cells were stained with AIM2 (C) or TLR9 (D) antibody (secondary PE-conjugated antibodies). Panels D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] and E [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] – control cells plots: FL2 versus SSC. R: gated area. Panels C [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>] and D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]: median signal intensity of FL2 (R) in MCF-7 cells (mean value for three independent experiments). Panels C [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>] and D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>]: relative proportions of AIM2- or TLR9-positive cells in R gates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. Background fluorescence was quantified using PE-conjugated secondary antibodies. </p> <p>*p < 0.05 against control group of cells, non-parametric U-test.</p></div

The analysis of 8-oxodG content in cells exposed to either gDNA or gDNA^OX (50 ng/mL).

<p>A - Cells stained with PE-labeled anti-8-oxodG antibodies and DAPI (x20). B - Three types of anti-8-oxodG stain distribution observed in cells treated with gDNA<b>^OX</b> (x100). Cell were incubated with DNA samples for 1 hour, fixed with 3% formaldehyde, permeated with 0,1 % triton X100 and stained with anti-8-oxodG (PE-conjugated secondary antibodies). C – colocalization of 8-oxodG with mitochondria. Cells were incubated with <b>gDNA^OX</b> for 0.5 hour, обработаны Mito-tracker (30 nM, 15 min), photographed, then fixed with 3% formaldehyde, permeated with 0,1 % triton X100, stained with anti-8-oxodG antibodies (FITC-conjugated secondary antibodies) and photographed again. D - 8-oxodG content in DNA exposed cells pre-treated with NAC (FACS analysis). Cells were incubated with NAC (0.15 mM) for 30 min, then exposed to gDNA^OX for 1 hour and analyzed using anti-8-oxodG antibodies (PE-conjugated secondary antibodies). Background fluorescence was quantified using PE-conjugated secondary antibodies. E - Relative proportions of nuclei stained for 8-oxodG in non-treated control cells, cells exposed to gDNA, cells exposed to gDNA^OX (grey columns). Light grey column reflects cells pre-treated with NAC and exposed to gDNA^OX. *p < 0.05 against control group of cells, non-parametric U-test.</p

Cell death in MCF-7 cultures exposed to either gDNA or gDNA^OX at final concentration 50 ng/mL for 48 hours.

<div><p>A. Total number of cells in studied cell population.</p> <p>B. (FACS) – enumeration of cells with sings of early apoptosis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>]. - the distribution of fluorescence intensities of the cells stained with Annexin V-FITC (green color) или FITC-conjugated secondary antibodies (grey color) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>]. - control cells plots: FL1 versus SSC. R: gated area [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B3" target="_blank">3</a>]. - the proportion of Annexin V -positive cells in total cell population. </p> <p>C. Evaluation of modified nuclei in three studies typed of MCF-7 cultures. (1) -Example of Hoechst33342 staining; (2) - Graph of the proportion of cells with modified nuclei in three studied types of MCF-7 cultures. </p> <p>D. Electrophoresis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B1" target="_blank">1</a>] and evaluation of ecDNA concentrations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077469#B2" target="_blank">2</a>] in the media of non-treated control cells and cells exposed to either gDNA or gDNA^OX. Dashed line indicates amounts of ecDNA that should be present in the media when exogenous DNA is taken into account. *p < 0.05 against control group of cells, non-parametric U-test.</p></div