CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

[Confined placental mosaicisms a priori from meiotic origin: analysis of 10 cases]. / Anomalies chromosomiques limitées au placenta a priori d'origine méiotique : à propos de dix cas.

This retrospective monocenter study focused on confined placental mosaicisms a priori from meiotic origin (i.e. non-mosaic type 3 confined placental mosaicisms). From a series of 14,967 chorionic villus samplings performed in our Fetal Medicine Center, 10 non-mosaic type 3 confined placental mosaicisms were identified. These abnormalities only involved chromosomes 15, 16 or 22. Pregnancies complicated by these confined placental mosaicisms were associated with prematurity and neonatal hypotrophy. Thus, when a confined placental mosaicism is suspected, this retrospective study highlighted the need to characterize the type of confined placental mosaicism to prevent the probable intra-uterine growth retardation and to adapt the obstetrical monitoring if necessary.

First-trimester prenatal diagnosis performed on pregnant women with fetal ultrasound abnormalities: the reliability of interphase fluorescence in situ hybridization (FISH) on mesenchymal core for the main aneuploidies.

OBJECTIVES: To examine the reliability of interphase FISH analysis of the main aneuploidies performed on mesenchymal core when prenatal diagnosis was performed on pregnant women with first-trimester fetal abnormalities on ultrasound. STUDY DESIGN: 386 first-trimester prenatal examinations were investigated from chorionic villus samplings for increased nuchal translucencies or other fetal ultrasound abnormalities. Interphase fluorescence in situ hybridization (FISH) for the main aneuploidies (trisomies 13, 18, 21 and gonosomal aneuploidies) was performed on the mesenchymal core of villi. Molecular cytogenetic results were always complemented by conventional cytogenetic results on long-term cultured villi (LTC-villi). Short-term cultured villi (STC-villi) preparations were retrospectively performed only when a chromosomal abnormality was observed with interphase FISH and/or LTC-villi. RESULTS: 88 chromosomal abnormalities (88/386=22.8% of first-trimester diagnoses) which could discuss subsequent abortions were observed after LTC-villi preparations. All cases possibly detectable by interphase FISH were detected. Thus, 85 aneuploidies (85/386=22.0% of first-trimester diagnoses; 85/88=96.6% of chromosomal abnormalities) were detected by interphase FISH, allowing early abortion by curettage before week 14 amenorrhea. No discrepancy occurred between interphase FISH and LTC-villi results for the aneuploidies studied. Three false-negative results (3/386=0.77% of first-trimester diagnoses; 3/88=3.41% of chromosomal abnormalities) were observed with STC-villi. CONCLUSION: We observed a high rate of false-negative results on cytotrophoblast cells. Conversely, interphase FISH of the main aneuploidies on the mesenchymal core provided rapid and reliable results, and therefore should be preferred to STC-villi in first-trimester prenatal diagnosis performed on pregnant women with fetal abnormalities on ultrasound.

The association between the T309G polymorphism of the MDM2 gene and sensitivity to anticancer drug is dependent on the p53 mutational status in cellular models.

BACKGROUND: We investigated, in the panel of 60 human tumour cell lines of the National Cancer Institute (NCI-60), whether the R72P polymorphism of TP53 and the T309G polymorphism of MDM2 were associated to the in vitro cytotoxicity of anticancer agents, extracted from the NCI database. For validation, the same study was performed independently on a second panel of tumour cell lines, JFCR-45. METHODS: Both SNPs were identified in cell DNA using PCR-RFLP techniques confirmed by direct sequencing and by pyrosequencing. For the analysis of the results, the mutational status of p53 was taken into account. RESULTS: In the NCI-60 panel, the TP53 rare-allele frequency was 32% and the MDM2 rare-allele frequency 39%. The MDM2 alleles were distributed according to Hardy-Weinberg equilibrium whereas this was only found, for the TP53 alleles, in p53 non-mutated cell lines. Comparable results were obtained in the JFCR-45 validation set. The TP53 SNP had low impact on anticancer drug cytotoxicity in either panel. In contrast, the MDM2 gene polymorphism had a major impact on anticancer drug cytotoxicity, essentially in p53 non-mutated cell lines. Presence of the rare allele was associated to significantly higher MDM2 protein expression and to increased sensitivity to DNA-interfering drugs. In the JFCR-45 panel, a similar effect of the MDM2 gene polymorphism was observed, but was less dependent on the p53 mutational status. CONCLUSIONS: We hypothesised that cell lines harbouring the MDM2 G allele presented a lower availability of p53 for DNA repair, translating into higher sensitivity to DNA-damaging agents.

Expression of the gene encoding the pro-apoptotic BNIP3 protein and stimulation of hypoxia-inducible factor-1alpha (HIF-1alpha) protein following focal cerebral ischemia in rats.

Hypoxia is a common cause of cell death and is implicated in many disease processes including stroke and chronic degenerative disorders. In response to hypoxia, cells express a variety of genes which allow adaptation to altered metabolic demands, decreased oxygen demands, and the removal of irreversibly damaged cells. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that regulates the adaptive response to hypoxia in cells. In this study, we reported an early, time-related, gradual up-regulation of HIF-1alpha, and a moderate increase in vascular endothelial growth factor (VEGF)- and erythropoietin (Epo)-levels following transient focal ischemia. Moreover, we demonstrated, for the first time a specific localization of the pro-apoptotic regulator BNIP3 in striatal and cortical neurons after transient focal ischemia in rats. Prolonged intranuclear BNIP3 immunoreactivity was associated with delayed neuronal death. Experiments showed protein increases on Western blots of brain tissue with peaks at 48h after ischemia. Epo responds to ischemia in an early stage, whereas VEGF and BNIP3 accumulate in cells at later times after ischemia. This suggests the possibility that BH3-only proteins might be one of the major downstream effectors of HIF-1alpha in hypoxic cell death. These findings open the possibility that the hypoxia-regulated pro-apoptotic protein BNIP3 enters the nucleus and could interact with other proteins involved in DNA structure, transcription or mRNA splicing after focal brain ischemia.

Direct quantification of protein partitioning in oil-in-water emulsion by front-face fluorescence: avoiding the need for centrifugation.

The quantification of proteins adsorbed at the oil-in-water interface is often difficult since it requires separation of fat globules from the aqueous phase that may damage the fat globule size and/or modify the interfacial composition. Front-face fluorescence spectroscopy was used to characterize the protein partitioning between the aqueous and oil phases of emulsions without separating these two phases. Different emulsions based on skim milk powder (SMP), two mono- and di-glyceride (MDG) mixtures (saturated and partially unsaturated), and three fats (hydrogenated and refined coconut oils and refined palm oil) were studied. The impact of an ageing period (24 h at 4 degrees C) was also investigated to typify the first step of ice cream processing. The emulsions were characterized for protein partitioning, immediately following emulsification and after ageing, using the Bradford spectrophotometric method, applied to the aqueous phase recovered after emulsion centrifugation. In parallel, the emulsions were characterized by their tryptophan emission fluorescence spectra. The area of the peaks at 333 nm, of the fourth-derivative fluorescence spectra corresponding to the amount of proteins present in the aqueous phase of emulsions, was well correlated with the Bradford measurements (r2=0.91). This amount was also calculated from the fluorescence calibration curve obtained with SMP in solution. In conclusion, front-face fluorescence spectroscopy appeared to be a powerful and simple technique allowing the quantification of different populations of protein in an emulsified system, i.e., in the aqueous phase and loaded at the fat globule interface.