R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

Transcription poses a threat to genomic stability through the formation of R-loops that can obstruct progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA–DNA hybrid with a displaced non-template DNA strand. We developed RNA–DNA Proximity Proteomics to map the R-loop proximal proteome of human cells using quantitative mass spectrometry. We implicate different cellular proteins in R-loop regulation and identify a role of the tumor suppressor DDX41 in opposing R-loop and double strand DNA break accumulation in promoters. DDX41 is enriched in promoter regions in vivo, and can unwind RNA–DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied with replication stress, an increase in the formation of double strand DNA breaks and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia in adulthood. We propose that R-loop accumulation and genomic instability-associated inflammatory response may contribute to the development of familial AML with mutated DDX41.

Iodine containing porous organosilica nanoparticles trigger tumor spheroids destruction upon monochromatic X-ray irradiation: DNA breaks and K-edge energy X-ray

X-ray irradiation of high Z elements causes photoelectric effects that include the release of Auger electrons that can induce localized DNA breaks. We have previously established a tumor spheroid-based assay that used gadolinium containing mesoporous silica nanoparticles and synchrotron-generated monochromatic X-rays. In this work, we focused on iodine and synthesized iodine-containing porous organosilica (IPO) nanoparticles. IPO were loaded onto tumor spheroids and the spheroids were irradiated with 33.2 keV monochromatic X-ray. After incubation in CO2 incubator, destruction of tumor spheroids was observed which was accompanied by apoptosis induction, as determined by the TUNEL assay. By employing the γH2AX assay, we detected double strand DNA cleavages immediately after the irradiation. These results suggest that IPO first generate double strand DNA breaks upon X-ray irradiation followed by apoptosis induction of cancer cells. Use of three different monochromatic X-rays having energy levels of 33.0, 33.2 and 33.4 keV as well as X-rays with 0.1 keV energy intervals showed that the optimum effect of all three events (spheroid destruction, apoptosis induction and generation of double strand DNA breaks) occurred with a 33.2 keV monochromatic X-ray. These results uncover the preferential effect of K-edge energy X-ray for tumor spheroid destruction mediated by iodine containing nanoparticles.

P–577 Impaired double strand DNA repair in isolated Primary Ovarian Insufficiency with homozygous nonsense mutation of SPIDR

Study question To identify the etiology of isolated Primary Ovarian Insufficiency (POI) in a patient from an Indian consanguineous family. Summary answer A homozygous nonsense mutation of SPIDR in the patient yielded chromosomal instability: first evidence of a role of this gene in DNA repair. What is known already POI, affecting 1% of women under 40, is a public health problem. To date ∼ 70% of cases remain idiopathic. The leap due to exome sequencing, led to the identification of ∼ 80 genes, often in single or few cases. SPIDR was recently identified as a scaffolding protein connecting RAD51, a central player in homologous recombination, to BLM, a helicase implicated in the integrity of the genome. But its precise role is still unknown. A SPIDR mutation was previously associated with POI. However, contradictory conclusions were reported on the mechanism of SPIDR action and on its pathogenic role in POI. Study design, size, duration Prospective genetic study of a cohort of 150 pateints with POI worldwide using a custom-made targeted next generation sequencing (NGS) panel comprising 60 known POI-causing genes. A single patient was found mutated in SPIDR. Cytogenetic studies were performed to analyse the consequences of the mutation on DNA repair and sister chromatide exchanges (SCE). Participants/materials, setting, methods The patient with SPIDR mutation had POI with primary amenorrhea, delayed puberty and streaks ovaries. She was born to consanguineous Indian parents. No other mutation was detected in our cohort of 150 patients with POI. Targeted NGS was performed in the proposita. Familial segregation was performed by Sanger sequencing. Mitomycin C (MMC)-induced chromosomal breakages were studied and a sister chromatid exchange (SCE) assay was performed in patient’s peripheral lymphocytes. Main results and the role of chance We identified a novel homozygous nonsense mutation in the exon 7 of SPIDR (KIAA0146) c.814C>T, R272*, predicted to yield either a truncated protein, or a non-sense-mediated mRNA decay. The patient’s cells display increased chromosomal fragility with high MMC-induced chromosomal breaks when compared to a control. Remarkably, there was no increased SCE. In the previous report of a SPIDR mutation in POI, no cytogenetic studies were performed, and contradictory results were obtained on a homologous recombination test between the two sisters, either enhanced or reduced. In conclusion, we show here that inactivation of SPIDR results in a defect of double strand DNA damage repair, similar to alteration of the RAD51 pathway. There was no increased SCE, the hallmark of the BLM pathway. This observation has major consequences for this patient’s care : indeed mutations of DNA-repair genes may also yield to tumors/cancers. A long follow-up of the patient is needed in a multidisciplinary team to detect possible comorbidities. Indeed, even in the absence of somatic symptomatology, the patient has enhanced chromosomal instability highlighted by cytogenetic studies, that may yield tumor-predisposition. Limitations, reasons for caution No other mutation of SPIDR in the replication cohort of 150 POI patients. SPIDR mutation are thus very rare world-wide. Wider implications of the findings: This is the first evidence of chromosomal instability associated with SPIDR defect, providing strong evidence for a role of SPIDR in double strand DNA damage repair in humans and for its causal role in POI. Our study improves the knowledge on SPIDR function and confirms its involvement in POI worldwide. Trial registration number Not applicable

Meiotic Cas9 expression mediates genotype conversion in the male and female mouse germline

Highly efficient genotype conversion systems have potential to facilitate the study of complex genetic traits using laboratory mice and to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of genotype conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double strand DNA break is feasible in the female mouse germline. In the male germline, however, all double strand breaks were instead repaired by end joining mechanisms to form an 'insertion/deletion' (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate genotype conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient double strand DNA break formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of genotype conversion and further increase the rate of 'super-Mendelian' inheritance from both male and female mice.