R-loops that accumulate at transcription sites pose a persistent threat to genome integrity. PSIP1 is a chromatin protein associated with transcriptional elongation complex, possesses histone ...chaperone activity, and is implicated in recruiting RNA processing and DNA repair factors to transcription sites. Here, we show that PSIP1 interacts with R-loops and other proteins involved in R-loop homeostasis, including PARP1. Genome-wide mapping of PSIP1, R-loops and γ-H2AX in PSIP1-depleted human and mouse cell lines revealed an accumulation of R-loops and DNA damage at gene promoters in the absence of PSIP1. R-loop accumulation causes local transcriptional arrest and transcription-replication conflict, leading to DNA damage. PSIP1 depletion increases 53BP1 foci and reduces RAD51 foci, suggesting altered DNA repair choice. Furthermore, PSIP1 depletion increases the sensitivity of cancer cells to PARP1 inhibitors and DNA-damaging agents that induce R-loop-induced DNA damage. These findings provide insights into the mechanism through which PSIP1 maintains genome integrity at the site of transcription.
The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk ...individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.
Abstract Body mass results from a complex interplay between genetics and environment. Previous studies of the genetic contribution to body mass have excluded repetitive regions due to the technical ...limitations of platforms used for population scale studies. Here we apply genome-wide approaches, identifying an association between adult body mass and the copy number (CN) of 47S-ribosomal DNA (rDNA). rDNA codes for the 18 S, 5.8 S and 28 S ribosomal RNA (rRNA) components of the ribosome. In mammals, there are hundreds of copies of these genes. Inter-individual variation in the rDNA CN has not previously been associated with a mammalian phenotype. Here, we show that rDNA CN variation associates with post-pubertal growth rate in rats and body mass index in adult humans. rDNA CN is not associated with rRNA transcription rates in adult tissues, suggesting the mechanistic link occurs earlier in development. This aligns with the observation that the association emerges by early adulthood.
Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified ...in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knockdown cells and abolished ERCCGL2 traffic to the mitochondria and nucleus in response to this DNA-damaging agent. Collectively, these observations identify a distinct bone-marrow-failure syndrome due to mutations in ERCC6L2, a gene implicated in DNA repair and mitochondrial function.
Dyskeratosis congenita is a highly pleotropic genetic disorder. This heterogeneity can lead to difficulties in making an accurate diagnosis and delays in appropriate management. The aim of this study ...was to determine the underlying genetic basis in patients presenting with features of dyskeratosis congenita and who were negative for mutations in the classical dyskeratosis congenita genes. By whole exome and targeted sequencing, we identified biallelic variants in genes that are not associated with dyskeratosis congenita in 17 individuals from 12 families. Specifically, these were homozygous variants in USB1 (8 families), homozygous missense variants in GRHL2 (2 families) and identical compound heterozygous variants in LIG4 (2 families). All patients had multiple somatic features of dyskeratosis congenita but not the characteristic short telomeres. Our case series shows that biallelic variants in USB1, LIG4 and GRHL2, the genes mutated in poikiloderma with neutropenia, LIG4/Dubowitz syndrome and the recently recognized ectodermal dysplasia/short stature syndrome, respectively, cause features that overlap with dyskeratosis congenita. Strikingly, these genes also overlap in their biological function with the known dyskeratosis congenita genes that are implicated in telomere maintenance and DNA repair pathways. Collectively, these observations demonstrate the marked overlap of dyskeratosis congenita with four other genetic syndromes, confounding accurate diagnosis and subsequent management. This has important implications for establishing a genetic diagnosis when a new patient presents in the clinic. Patients with clinical features of dyskeratosis congenita need to have genetic analysis of USB1, LIG4 and GRHL2 in addition to the classical dyskeratosis congenita genes and telomere length measurements.
Abstract Background The bone marrow failure syndromes are a diverse group of rare genetic conditions. Mutations in telomere maintenance genes cause a large proportion of cases of dyskeratosis ...congenita. Our aim was to determine whether TA65, marketed as a telomerase activator, can correct this defect in vitro. We also investigated danazol, which improves peripheral blood counts in dyskeratosis congenita and Fanconi's anaemia by an unknown mechanism. Methods We isolated T lymphocytes from the peripheral blood of 13 patients with bone marrow failure (seven dyskeratosis congenita, two Fanconi's anaemia, four uncharacterised) and ten age-matched controls. All 23 T lymphocyte cultures were incubated with TA65 (0·5 μg/mL and 0·05 μg/mL). Lymphocytes from nine patients and five controls were also incubated with danazol (dose range 0·048 ng/mL to 3 μg/mL). Cell proliferation was measured by neutral red assay (days 6 and 8) and nucleocounter (day 8). Telomerase activity of cells from six patients with dyskeratosis congenita was measured by TRAP (telomeric repeat amplification protocol) assay on day 8. Findings We observed increased proliferation of T lymphocytes after treatment with TA65 0·5μg/mL and danazol 6 ng/mL (starting cell count 1 × 106 , mean final count 7·64 × 106 with TA65 and 7·66 × 106 with danazol vs 7·31 × 106 with dimethyl sulfoxide DMSO only, as the control condition). Dyskeratosis congenita cells showed the most consistent results with TA65 treatment, with increased growth of five out of six cultures and overall relative proliferation of 1·033 (95% CI 1·001–1·064). Danazol improved cell growth most markedly in patients with Fanconi's anaemia (1·4 times increased proliferation over DMSO control). There was 1·23 times increased telomerase activity of T lymphocytes from patients with dyskeratosis congenita treated with TA65 0·5 μg/mL compared with DMSO controls. Treatment with danazol 1·2 ng/mL resulted in 1·45 times increased telomerase activity. Interpretation This is the first time, to our knowledge, that these drugs have been studied in vitro in patients with bone marrow failure. Our results show a trend to increased proliferation of T lymphocytes from patients with different bone marrow failure syndromes treated with TA65 and danazol. In addition, our data show increased telomerase activity in dyskeratosis congenita cells treated with these drugs. Our study is limited by small patient numbers from this rare heterogeneous bone marrow failure group. However, these preliminary results provide an exciting basis for further studies to help establish the clinical utility of these drugs. Funding Barts Charity (Academic Clinical Fellowship Support Grant).
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