Increased RPA1 gene dosage affects genomic stability potentially contributing to 17p13.3 duplication syndrome

A novel microduplication syndrome involving various-sized contiguous duplications in 17p13.3 has recently been described, suggesting that increased copy number of genes in 17p13.3, particularly PAFAH1B1, is associated with clinical features including facial dysmorphism, developmental delay, and autism spectrum disorder. We have previously shown that patient-derived cell lines from individuals with haploinsufficiency of RPA1, a gene within 17p13.3, exhibit an impaired ATR-dependent DNA damage response (DDR). Here, we show that cell lines from patients with duplications specifically incorporating RPA1 exhibit a different although characteristic spectrum of DDR defects including abnormal S phase distribution, attenuated DNA double strand break (DSB)-induced RAD51 chromatin retention, elevated genomic instability, and increased sensitivity to DNA damaging agents. Using controlled conditional over-expression of RPA1 in a human model cell system, we also see attenuated DSB-induced RAD51 chromatin retention. Furthermore, we find that transient over-expression of RPA1 can impact on homologous recombination (HR) pathways following DSB formation, favouring engagement in aberrant forms of recombination and repair. Our data identifies unanticipated defects in the DDR associated with duplications in 17p13.3 in humans involving modest RPA1 over-expression

Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be define

Eye movements and the perceptual span in disordered reading: A comparison of schizophrenia and dyslexia

Increasing evidence of a common neurodevelopmental etiology between schizophrenia and developmental dyslexia suggests that neurocognitive functions, such as reading, may be similarly disrupted. However, direct comparisons of reading performance in these disorders have yet to be conducted. To address this gap in the literature, we employed a gaze-contingent moving window paradigm to examine sentence-level reading fluency and perceptual span (breadth of parafoveal processing) in adults with schizophrenia (dataset from Whitford et al., 2013) and psychiatrically healthy adults with dyslexia (newly collected dataset). We found that the schizophrenia and dyslexia groups exhibited similar reductions in sentence-level reading fluency (e.g., slower reading rates, more regressions) compared to matched controls. Similar reductions were also found for standardized language/reading and executive functioning measures. However, despite these reductions, the dyslexia group exhibited a larger perceptual span (greater parafoveal processing) than the schizophrenia group, potentially reflecting a disruption in normal foveal-parafoveal processing dynamics. Taken together, our findings suggest that reading and reading-related functions are largely similarly disrupted in schizophrenia and dyslexia, providing additional support for a common neurodevelopmental etiology

Germline Mutation in ATR in Autosomal- Dominant Oropharyngeal Cancer Syndrome

ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a ∼16.8 cM interval in chromosomal region 3q22–24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain—which can activate p53—of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans

Human bone marrow stem/stromal cell osteogenesis is regulated via mechanically activated osteocyte-derived extracellular vesicles

Bone formation or regeneration requires the recruitment, proliferation, and osteogenic differentiation of stem/stromal progenitor cells. A potent stimulus driving this process is mechanical loading. Osteocytes are mechanosensitive cells which play fundamental roles in coordinating loading-induced bone formation via the secretion of paracrine factors. However, the exact mechanisms by which osteocytes relay mechanical signals to these progenitor cells are poorly understood. Therefore, this study aimed to demonstrate the potency of the mechanically stimulated osteocyte secretome in driving human bone marrow stem/stromal cell (hMSC) recruitment and differentiation, and characterize the secretome to identify potential factors regulating stem cell behavior and bone mechanobiology. We demonstrate that osteocytes subjected to fluid shear secrete a distinct collection of factors that significantly enhance hMSC recruitment and osteogenesis and demonstrate the key role of extracellular vesicles (EVs) in driving these effects. This demonstrates the pro-osteogenic potential of osteocyte-derived mechanically activated extracellular vesicles, which have great potential as a cell-free therapy to enhance bone regeneration and repair in diseases such as osteoporosis

WT ATRIP cDNA but not cDNA encoding p.Arg760* ATRIP complements the G2/M checkpoint defect in CV1720 cells, and p.Arg760*ATRIP impairs ATR–ATRIP protein interaction.

<p>A) Analysis of the G2/M checkpoint defect in CV1720 cells following expression of ATRIP cDNA. G2/M checkpoint arrest was examined 2 h post exposure to 5 Jm⁻² UV. As shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen-1002945-g001" target="_blank">Figure 1A</a>, WT cells showed proficient checkpoint arrest whilst DK0064 (ATR–SS) and CV1720 (patient) cells are unable to undergo arrest. Expression of WT ATRIP cDNA restored the ability of CV1720 (patient) and DK0064 (ATR–SS) to undergo checkpoint arrest but this was not observed following transfection of cDNA encoding R760* ATRIP. Significantly, expression of ATRIP R760* did not impair checkpoint arrest in WT cells verifying that it does not exert a dominant negative impact. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#s2" target="_blank">Results</a> represent the mean and SD of three experiments. WT cells were GM2188. ATR–SS represents DK0064 and patient, CV1720. B) R760* ATRIP impairs ATR–ATRIP interaction. Crude lysates were prepared from HEK293T cells and either mock transfected (lane1), transfected with HA-tagged WT <i>ATRIP</i> cDNA (lane2), or R760* <i>ATRIP</i> cDNA (lane3) (generating p.Arg760* ATRIP protein) together with <i>ATR</i> cDNA. The extracts were immunoprecipitated with agarose-conjugated rabbit anti-HA-tag antibody (MBL). Interaction with ATR was examined by immunoblotting with ATR antibodies (left panel). Immunoblotting using the HA-tag (ATRIP; right panel) verified expression of the appropriately sized ATRIP in the samples. 33% of the crude lysate was loaded; IP, immunoprecipitate.</p

LBLs from patient 27-4BI and 19-8BI showed impaired ATR–dependent damage responses.

<p>A) 27-4BI cells were examined for their ability to activate G2/M checkpoint arrest at 4 h following exposure to 7 Jm⁻² UV. In contrast to WT cells (GM2188), no significant arrest was observed in 27-4BI cells. The checkpoint response to ionizing radiation, which is ATM rather than ATR dependent, was normal. B) LBLs derived from patients 27-4BI and 19-8BI were examined for their ability to phosphorylate the indicated ATR substrates at 1 h following exposure to 0.5 mM HU. WT represents IM257. 27-4BI and control LBLs have a similar cell cycle profile demonstrating that the lack of ATR substrate phosphorylation cannot be attributed to the lack of S phase cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945.s002" target="_blank">Figure S2</a>).</p

CV1720 cells show impaired ATR–dependent DNA damage responses.

<p>A) WT, DK0064 (ATR–SS), CV1720 (patient), CV1780 (patient's mother) and CV1783 (patient's father) cells were exposed to 5 Jm⁻² UV and the mitotic index (MI) assessed 2 h post exposure. A greater than two fold decrease in mitotic index is observed in WT and both paternal cell lines but not in DK0064 (ATR–SS) or CV1720 (patient) cells. B) Cells were exposed to 5 mM HU for 2 h and the percentage of p-H2AX (γ-H2AX) positive cells assessed by immunofluorescence. Note that HU causes pan nuclear p-H2AX formation rather than defined foci as observed after exposure to ionising radiation. Thus, the percentage of γ-H2AX positive cells was scored. C) Cells were exposed to UV (5 Jm⁻²) and subjected to Western Blotting (WB) using p-Chk1 (p-Ser317) antibodies at 2 h. Chk1 expression was shown to be similar in WT and patient cells (lower panel). D) Cells were exposed to 3 mM HU for 2 h and whole cell extracts analysed by WB using FANCD2 antibodies. The ubiquitylation of FANCD2, detectable by a product with reduced mobility, is diminished in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. E) Cells were exposed to 5 mM HU and examined for the percentage of cells showing >5 53BP1 foci at 2 h post exposure. 53BP1 foci formation is reduced in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. F–I) The indicated cells were processed by WB using ATRIP or ATR antibodies. MCM2 was used as a loading control. F shows the analysis of a range of protein levels for accurate comparison. CV1720 (patient) cells show markedly reduced ATR and ATRIP protein levels. G shows that both parental lines have approximately half the level of ATR and ATRIP compared to two WT cell lines. DK0064 (ATR–SS) and CV1720 cells, in contrast, have more dramatically reduced ATR and ATRIP protein levels. 50 ug protein was loaded. WT in all panels was GM2188. Patient, mother and father were as shown in panel A. H and I show the quantification of ATRIP and ATR protein levels from at least three independent WB experiments.</p

Patients 27-4BI and 19-8BI have reduced ATR and ATRIP expression and mutations in ATR.

<p>A) Photographs of patient included with informed consent of parent. B) Cell extracts (50 µg) from LBLs derived from WT (IM257), patient 27-4BI or patient 19-8BI were immunoblotted using the indicated antibodies. Reduced expression of ATR was observed in both patients. 27-4BI also had reduced ATRIP expression. C) Structure of ATR showing the site of the mutations identified and the UME domain. D) The UME domain is conserved between species and the methionine residue within this domain is conserved in yeast.</p