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

Phase III Trial Evaluating Letrozole As First-Line Endocrine Therapy With or Without Bevacizumab for the Treatment of Postmenopausal Women With Hormone Receptor-Positive Advanced-Stage Breast Cancer: CALGB 40503 (Alliance)

PURPOSE: To investigate whether anti-vascular endothelial growth factor therapy with bevacizumab prolongs progression-free survival (PFS) when added to first-line letrozole as treatment of hormone receptor-positive metastatic breast cancer (MBC). PATIENTS AND METHODS: Women with hormone receptor-positive MBC were randomly assigned 1:1 in a multicenter, open-label, phase III trial of letrozole (2.5 mg orally per day) with or without bevacizumab (15 mg/kg intravenously once every 3 weeks) within strata defined by measurable disease and disease-free interval. This trial had 90% power to detect a 50% improvement in median PFS from 6 to 9 months. Using a one-sided α = .025, a target sample size of 352 patients was planned. RESULTS: From May 2008 to November 2011, 350 women were recruited; 343 received treatment and were observed for efficacy and safety. Median age was 58 years (range, 25 to 87 years). Sixty-two percent had measurable disease, and 45% had de novo MBC. At a median follow-up of 39 months, the addition of bevacizumab resulted in a significant reduction in the hazard of progression (hazard ratio, 0.75; 95% CI, 0.59 to 0.96; P = .016) and a prolongation in median PFS from 15.6 months with letrozole to 20.2 months with letrozole plus bevacizumab. There was no significant difference in overall survival (hazard ratio, 0.87; 95% CI, 0.65 to 1.18; P = .188), with median overall survival of 43.9 months with letrozole versus 47.2 months with letrozole plus bevacizumab. The largest increases in incidence of grade 3 to 4 treatment-related toxicities with the addition of bevacizumab were hypertension (24% v 2%) and proteinuria (11% v 0%). CONCLUSION: The addition of bevacizumab to letrozole improved PFS in hormone receptor-positive MBC, but this benefit was associated with a markedly increased risk of grade 3 to 4 toxicities. Research on predictive markers will be required to clarify the role of bevacizumab in this setting

Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations

A Sensitive and Quantitative Polymerase Chain Reaction-Based Cell Free In Vitro Non-Homologous End Joining Assay for Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are responsible for sustaining hematopoietic homeostasis and regeneration after injury for the entire lifespan of an organism. Maintenance of genomic stability is crucial for the preservation of HSCs, which depends on their efficient repair of DNA damage, particularly DNA double strand breaks (DSBs). Because of the paucity of HSCs and lack of sensitive assays, directly measuring the ability of HSCs to repair DSBs has been difficult. Therefore, we developed a sensitive and quantitative cell free in vitro non-homologous end joining (NHEJ) assay using linearized plasmids as the substrates and quantitative polymerase chain reaction (qPCR) technique. This assay can sensitively detect DSB repair via NHEJ in less than 1 µg 293T cell nuclear proteins or nuclear extracts from about 5,000 to 10,000 human BM CD34+ hematopoietic cells. Using this assay, we confirmed that human bone marrow HSCs (CD34+CD38− cells) are less proficient in the repair of DSBs by NHEJ than HPCs (CD34+CD38+ cells). In contrast, mouse quiescent HSCs (Pyronin-Ylow LKS+ cells) and cycling HSCs (Pyronin-Yhi LKS+ cells) repaired the damage more efficiently than HPCs (LKS− cells). The difference in the abilities of human and mouse HSCs and HPCs to repair DSBs through NHEJ is likely attributed to their differential expression of key NHEJ DNA damage repair genes such as LIG4. These findings suggest that the qPCR-based cell free in vitro NHEJ assay can be used to sensitively measure the ability of human and mouse HSCs to repair DSBs

Evaluation of unclassified variants in the breast cancer susceptibility genes BRCA1 and BRCA2 using five methods: results from a population-based study of young breast cancer patients

Introduction Efforts are ongoing to determine the significance of unclassified variants (UVs) in the breast cancer susceptibility genes BRCA1/BRCA2, but no study has systematically assessed whether women carrying the suspected deleterious UVs have characteristics commonly seen among women carrying known deleterious or disease-causing mutations in BRCA1/BRCA2. Methods We sequenced BRCA1/BRCA2 in 1,469 population-based female breast cancer patients diagnosed between the ages of 20 and 49 years. We used existing literature to classify variants into known deleterious mutations, polymorphic variants, and UVs. The UVs were further classified as high risk or low risk based on five methods: allele frequency, Polyphen algorithm, sequence conservation, Grantham matrix scores, and a combination of the Grantham matrix score and sequence conservation. Furthermore, we examined whether patients who carry the variants classified as high risk using these methods have risk characteristics similar to patients with known deleterious BRCA1/BRCA2 mutations (early age at diagnosis, family history of breast cancer or ovarian cancer, and negative estrogen receptor/progesterone receptor). Results We identified 262 distinct BRCA1/BRCA2 variants, including 147 UVs, in our study population. The BRCA1 UV carriers, but not the BRCA2 UV carriers, who were classified as high risk using each classification method were more similar to the deleterious mutation carriers with respect to family history than those carriers classified as low risk. For example, the odds ratio of having a first-degree family history for the high-risk women classified using Polyphen was 3.39 (95% confidence interval = 1.16 to 9.94) compared with normal/polymorphic BRCA1 carriers. The corresponding odds ratio of low-risk women was 1.53 (95% confidence interval = 1.07 to 2.18). The odds ratio for high-risk women defined by allele frequency was 2.00 (95% confidence interval = 1.14 to 3.51), and that of low-risk women was 1.30 (95% confidence interval = 0.87 to 1.93). Conclusion The results suggest that the five classification methods yielded similar results. Polyphen was particularly better at isolating BRCA1 UV carriers likely to have a family history of breast cancer or ovarian cancer, and may therefore help to classify BRCA1 UVs. Our study suggests that these methods may not be as successful in classifying BRCA2 UVs

MKLN1 splicing defect in dogs with lethal acrodermatitis

Lethal acrodermatitis (LAD) is a genodermatosis with monogenic autosomal recessive inheritance in Bull Terriers and Miniature Bull Terriers. The LAD phenotype is characterized by poor growth, immune deficiency, and skin lesions, especially at the paws. Utilizing a combination of genome wide association study and haplotype analysis, we mapped the LAD locus to a critical interval of similar to 1.11 Mb on chromosome 14. Whole genome sequencing of an LAD affected dog revealed a splice region variant in the MKLN1 gene that was not present in 191 control genomes (chr14:5,731,405T>G or MKLN/:c.400+3A>C). This variant showed perfect association in a larger combined Bull Terrier/Miniature Bull Terrier cohort of 46 cases and 294 controls. The variant was absent from 462 genetically diverse control dogs of 62 other dog breeds. RT-PCR analysis of skin RNA from an affected and a control dog demonstrated skipping of exon 4 in the MKLN1 transcripts of the LAD affected dog, which leads to a shift in the MKLN1 reading frame. MKLN1 encodes the widely expressed intracellular protein muskelin 1, for which diverse functions in cell adhesion, morphology, spreading, and intracellular transport processes are discussed. While the pathogenesis of LAD remains unclear, our data facilitate genetic testing of Bull Terriers and Miniature Bull Terriers to prevent the unintentional production of LAD affected dogs. This study may provide a starting point to further clarify the elusive physiological role of muskelin 1 in vivo.Peer reviewe

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

Functional Connection between Rad51 and PML in Homology-Directed Repair

The promyelocytic leukemia protein (PML) is a tumor suppressor critical for formation of nuclear bodies (NBs) performing important functions in transcription, apoptosis, DNA repair and antiviral responses. Earlier studies demonstrated that simian virus 40 (SV40) initiates replication near PML NBs. Here we show that PML knockdown inhibits viral replication in vivo, thus indicating a positive role of PML early in infection. SV40 large T antigen (LT) induces DNA damage and, consequently, nuclear foci of the key homologous recombination repair protein Rad51 that colocalize with PML. PML depletion abrogates LT-induced Rad51 foci. LT may target PML NBs to gain access to DNA repair factors like Rad51 that are required for viral replication. We have used the SV40 model to gain insight to DNA repair events involving PML. Strikingly, even in normal cells devoid of viral oncoproteins, PML is found to be instrumental for foci of Rad51, Mre11 and BRCA1, as well as homology-directed repair after double-strand break (DSB) induction. Following LT expression or external DNA damage, PML associates with Rad51. PML depletion also causes a loss of RPA foci following γ-irradiation, suggesting that PML is required for processing of DSBs. Immunofluorescent detection of incorporated BrdU without prior denaturation indicates a failure to generate ssDNA foci in PML knockdown cells upon γ-irradiation. Consistent with the lack of RPA and BrdU foci, γ-irradiation fails to induce Chk1 activation, when PML is depleted. Taken together, we have discovered a novel functional connection between PML and the homologous recombination-mediated repair machinery, which might contribute to PML tumor suppressor activity

Effect of the BRCA2 CTRD domain on RAD51 filaments analyzed by an ensemble of single molecule techniques

Homologous recombination is essential for the preservation of genome stability, thereby preventing cancer. The recombination protein RAD51 drives DNA strand exchange, which requires the assembly, rearrangement and disassembly of a RAD51 filament on DNA, coupled to ATP binding and hydrolysis. This process is facilitated and controlled by recombination mediators and accessory factors. Here, we have employed a range of single molecule techniques to determine the influence of the C-terminal RAD51 interaction domain (CTRD) of the breast cancer tumor suppressor BRCA2 on intrinsic aspects of RAD51-DNA interactions. We show that at high concentration the CTRD entangles RAD51 filaments and reduces RAD51 filament formation in a concentration dependent manner. It does not affect the rate of filament disassembly measured as the loss of fluorescent signal due to intrinsic RAD51 protein dissociation from double-stranded DNA (dsDNA). We conclude that, outside the context of the full-length protein, the CTRD does not reduce RAD51 dissociation kinetics, but instead hinders filament formation on dsDNA. The CTRDs mode of action is most likely sequestration of multiple RAD51 molecules thereby rendering them inactive for filament formation on dsDNA

Role for the Mammalian Swi5-Sfr1 Complex in DNA Strand Break Repair through Homologous Recombination

In fission yeast, the Swi5-Sfr1 complex plays an important role in homologous recombination (HR), a pathway crucial for the maintenance of genomic integrity. Here we identify and characterize mammalian Swi5 and Sfr1 homologues. Mouse Swi5 and Sfr1 are nuclear proteins that form a complex in vivo and in vitro. Swi5 interacts in vitro with Rad51, the DNA strand-exchange protein which functions during HR. By generating Swi5−/− and Sfr1−/− embryonic stem cell lines, we found that both proteins are mutually interdependent for their stability. Importantly, the Swi5-Sfr1 complex plays a role in HR when Rad51 function is perturbed in vivo by expression of a BRC peptide from BRCA2. Swi5−/− and Sfr1−/− cells are selectively sensitive to agents that cause DNA strand breaks, in particular ionizing radiation, camptothecin, and the Parp inhibitor olaparib. Consistent with a role in HR, sister chromatid exchange induced by Parp inhibition is attenuated in Swi5−/− and Sfr1−/− cells, and chromosome aberrations are increased. Thus, Swi5-Sfr1 is a newly identified complex required for genomic integrity in mammalian cells with a specific role in the repair of DNA strand breaks