Tumor suppression in the absence of p53-mediated cell-cycle arrest, apoptosis, and senescence.

Cell-cycle arrest, apoptosis, and senescence are widely accepted as the major mechanisms by which p53 inhibits tumor formation. Nevertheless, it remains unclear whether they are the rate-limiting steps in tumor suppression. Here, we have generated mice bearing lysine to arginine mutations at one (p53(K117R)) or three (p53(3KR); K117R+K161R+K162R) of p53 acetylation sites. Although p53(K117R/K117R) cells are competent for p53-mediated cell-cycle arrest and senescence, but not apoptosis, all three of these processes are ablated in p53(3KR/3KR) cells. Surprisingly, unlike p53 null mice, which rapidly succumb to spontaneous thymic lymphomas, early-onset tumor formation does not occur in either p53(K117R/K117R) or p53(3KR/3KR) animals. Notably, p53(3KR) retains the ability to regulate energy metabolism and reactive oxygen species production. These findings underscore the crucial role of acetylation in differentially modulating p53 responses and suggest that unconventional activities of p53, such as metabolic regulation and antioxidant function, are critical for suppression of early-onset spontaneous tumorigenesis.

Testing for association with rare variants in the coding and non-coding genome: RAVA-FIRST, a new approach based on CADD deleteriousness score.

Rare variant association tests (RVAT) have been developed to study the contribution of rare variants widely accessible through high-throughput sequencing technologies. RVAT require to aggregate rare variants in testing units and to filter variants to retain only the most likely causal ones. In the exome, genes are natural testing units and variants are usually filtered based on their functional consequences. However, when dealing with whole-genome sequence (WGS) data, both steps are challenging. No natural biological unit is available for aggregating rare variants. Sliding windows procedures have been proposed to circumvent this difficulty, however they are blind to biological information and result in a large number of tests. We propose a new strategy to perform RVAT on WGS data: "RAVA-FIRST" (RAre Variant Association using Functionally-InfoRmed STeps) comprising three steps. (1) New testing units are defined genome-wide based on functionally-adjusted Combined Annotation Dependent Depletion (CADD) scores of variants observed in the gnomAD populations, which are referred to as "CADD regions". (2) A region-dependent filtering of rare variants is applied in each CADD region. (3) A functionally-informed burden test is performed with sub-scores computed for each genomic category within each CADD region. Both on simulations and real data, RAVA-FIRST was found to outperform other WGS-based RVAT. Applied to a WGS dataset of venous thromboembolism patients, we identified an intergenic region on chromosome 18 enriched for rare variants in early-onset patients. This region that was missed by standard sliding windows procedures is included in a TAD region that contains a strong candidate gene. RAVA-FIRST enables new investigations of rare non-coding variants in complex diseases, facilitated by its implementation in the R package Ravages.

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

RAVAQ: An integrative pipeline from quality control to region-based rare variant association analysis.

Next-generation sequencing technologies have opened up the possibility to sequence large samples of cases and controls to test for association with rare variants. To limit cost and increase sample sizes, data from controls could be used in multiple studies and might thus be generated on different sequencing platforms. This could pose some problems of comparability between cases and controls due to batch effects that could be confounding factors, leading to false-positive association signals. To limit batch effects and ensure comparability of datasets, stringent quality controls are required. We propose an integrative five-steps pipeline, RAVAQ, that (a) performs a specific three-step quality control taking into account the case-control status to ensure data comparability, (b) selects qualifying variants as defined by the user, and (c) performs rare variant association tests per genomic region. The RAVAQ pipeline is wrapped in an R package. It is user-friendly and flexible in its arguments to adapt to the specificity of each research project. We provide examples showing how RAVAQ improves rare variant association tests. The default RAVAQ quality control outperformed the widely used Variant Quality Score Recalibration method, removing inflation due to spurious signals. RAVAQ is open source and freely available at https://gitlab.com/gmarenne/ravaq.

Generation and application of a convolutional neural networks algorithm in evaluating stool consistency in diapers.

AIM: The aim of the study was to develop a deep convolutional neural networks (CNNs) algorithm for automated assessment of stool consistency from diaper photographs and test its performance under real-world conditions. METHODS: Diaper photographs were enrolled via a mobile phone application. The stool consistency was assessed independently according to the Brussels Infant and Toddler Stool Scale (BITSS) by paediatricians. These images were randomised into a training data set and a test data set. After training and testing, the new algorithm was used under real-world conditions by parents. RESULTS: There was an overall agreement of 92.9% between paediatricians and the CNN-generated algorithm. Post hoc classification into the validated 4 categories of the BITSS yielded an agreement of 95.4%. Spearman correlation analysis across the ranking of 7 BITSS photographs and validated 4 categories showed a significant correlation of rho = 0.93 (95% CI, 0.92, 0.94; p < 0.001) and rho = 0.92 (95% CI, 0.90, 0.93; p < 0.001), respectively. The real-world application yielded further insights into changes in stool consistency between age categories and mode of feeding. CONCLUSION: The new CNN-based algorithm is able to reliably identify stool consistency from diaper photographs and may support the communication between parents and paediatricians.

End-Truncated LAMB1 Causes a Hippocampal Memory Defect and a Leukoencephalopathy.

OBJECTIVE: The majority of patients with a familial cerebral small vessel disease (CSVD) referred for molecular screening do not show pathogenic variants in known genes. In this study, we aimed to identify novel CSVD causal genes. METHODS: We performed a gene-based collapsing test of rare protein-truncating variants identified in exome data of 258 unrelated CSVD patients of an ethnically matched control cohort and of 2 publicly available large-scale databases, gnomAD and TOPMed. Western blotting was used to investigate the functional consequences of variants. Clinical and magnetic resonance imaging features of mutated patients were characterized. RESULTS: We showed that LAMB1 truncating variants escaping nonsense-mediated messenger RNA decay are strongly overrepresented in CSVD patients, reaching genome-wide significance (p < 5 × 10-8 ). Using 2 antibodies recognizing the N- and C-terminal parts of LAMB1, we showed that truncated forms of LAMB1 are expressed in the endogenous fibroblasts of patients and trapped in the cytosol. These variants are associated with a novel phenotype characterized by the association of a hippocampal type episodic memory defect and a diffuse vascular leukoencephalopathy. INTERPRETATION: These findings are important for diagnosis and clinical care, to avoid unnecessary and sometimes invasive investigations, and also from a mechanistic point of view to understand the role of extracellular matrix proteins in neuronal homeostasis. ANN NEUROL 2021;90:962-975.

A homozygous splice variant in ATP5PO, disrupts mitochondrial complex V function and causes Leigh syndrome in two unrelated families.

Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar, severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with markedly reduced amounts of peripheral stalk proteins, and complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome.

Intra-rater Variability of the Brussels Infants and Toddlers Stool Scale (BITSS) Using Photographed Stools.

OBJECTIVES: The Brussels Infants and Toddlers Stool Scale (BITSS) was developed for the assessment of stool consistency in non-toilet-trained children. This study aimed to (1) investigate the intra-rater reliability of the BITSS among health care professionals (HCPs) and caregivers (CGs); (2) study a potential learning curve; (3) explore the impact of photo quality on intra-rater reliability. METHODS: Photos of diapers containing stool were assessed twice by 4 HCP (2432 photos) and 8 CGs (492 photos) using the BITTS. Intra-rater reliability was calculated by the percentage of exact agreement and a κ-value. A learning effect and the impact of photo quality was explored using mixed linear model and generalized estimating equations. RESULTS: HCPs generated 24,320 stool consistency ratings: 12.1% were scored as watery, 31.0% loose, 29.4% formed, and 27.6% hard. CGs performed 7872 ratings: 9.2% classified as watery, 34.6% loose, 28.9% formed, and 27.3% hard. Intra-rater reliability (κ) for HCPs ranged from 0.64 [95% confidence interval (CI) = 0.61-0.66] to 0.78 (95% CI = 0.76-0.80) and from 0.68 (95% CI = 0.63-0.73) to 0.94 (95% CI = 0.91-0.97) in the CG group. Both groups had <1% improvement in the odds of identical classification per 50 photos. The percentage of absolute agreement was higher in photos rated as good quality than those that were not (HCPs: 80.3% vs 69.5%, P < 0.001; CGs: 90.4% vs 86.3%, P < 0.001). CONCLUSIONS: The BITSS has an excellent intra-rater reliability for the stool consistency scoring of photographs of stools in diapers, but can be influenced by photo quality. A clinically meaningless learning effect was found.

Modeling Potential-Dependent Electrochemical Activation Barriers: Revisiting the Alkaline Hydrogen Evolution Reaction.

Accurate theoretical simulation of electrochemical activation barriers is key to understanding electrocatalysis and guides the design of more efficient catalysts. Providing a detailed picture of proton transfer processes encounters several challenges: the constant potential requirement during charge transfer, the different time scales involved in the processes, and the thermal fluctuation of the solvent. Hence, it is prohibitively expensive computationally to apply density functional theory (DFT) calculations in modeling the potential-dependent activation barrier at the electrode-solvent interface, and the results are dubious. To address these challenges, we have developed an analytical approach based on charge conservation and decoupled potential energy surfaces to compute charge transfer barriers. The method makes it possible to simulate an electrochemical process at different potentials and explicitly include thermal fluctuations of the solvent at the electrode-solvent interface. We use the Pt-catalyzed alkaline hydrogen evolution reaction (HER) as our benchmark reaction, and we model the microkinetics of HER with consideration of the spatial fluctuations between the metal surface and the first solvent layer at room temperature. The distribution of water-metal distances has a large effect on the barriers of the charge transfer processes, and an accurate account of the statistical fluctuation in the reaction network leads to a several orders of magnitude increase in HER current as compared to transfer from a static solvent. The trends of the different reaction mechanisms in HER were successfully simulated with our model, and the theoretical I-V curves obtained are in good qualitative agreement with experimental results.

Whole exome sequencing, a hypothesis-free approach to investigate recurrent early miscarriage.

RESEARCH QUESTION: Are there genetic determinants shared by unrelated women with unexplained recurrent early miscarriage (REM)? DESIGN: Thirty REM cases and 30 controls were selected with extreme phenotype among women from Eastern Brittany (France), previously enrolled in an incident case-control study on thrombophilic mutations. Cases and controls were selected based on the number of early miscarriages or live births, respectively. Peripheral blood was collected for DNA extraction at initial visit. The burden of low-frequency variants in the coding part of the genes was compared using whole exome sequencing (WES). RESULTS: Cases had 3 to 17 early miscarriages (20 cases: ≥5 previous losses). Controls had 1 to 4 live births (20 controls: ≥3 previous live births) and no miscarriages. WES data were available for 29 cases and 30 controls. A total of 209,387 variants were found (mean variant per patient: 59,073.05) with no difference between groups (P = 0.68). The top five most significantly associated genes were ABCA4, NFAM1, TCN2, AL078585.1 and EPS15. Previous studies suggest the involvement of vitamin B12 deficiency in REM. TCN2 encodes for vitamin B12 transporter into cells. Therefore, holotranscobalamin (active vitamin B12) was measured for both cases and controls (81.2 ± 32.1 versus 92.9 ± 34.3 pmol/l, respectively, P = 0.186). Five cases but no controls were below 50 pmol/l (P = 0.052). CONCLUSIONS: This study highlights four new genes of interest in REM, some of which belong to known networks of genes involved in embryonic development (clathrin-mediated endocytosis and ciliary pathway). The study also confirms the involvement of TCN2 (vitamin B12 pathway) in the early first trimester of pregnancy.

Safety and Tolerance of a Novel Anti-Regurgitation Formula: A Double-Blind, Randomized, Controlled Trial.

OBJECTIVES: A novel anti-regurgitation (AR) formula has been designed to support gut health and improve gastrointestinal (GI) symptoms beyond regurgitation. This study assessed the tolerance and safety of this new AR formula. METHODS: This was a 4-week double-blind, randomized, controlled trial with a 4-week extension in formula-fed infants with regurgitation. The new AR (Test) formula contained 0.4 g/100 mL locust bean gum (LBG) as thickener, partly fermented formula with postbiotics, and short-chain galacto-oligosaccharides (scGOS) and long-chain fructo-oligosaccharides (lcFOS) (0.4 g/100 mL, ratio 9:1). The Control AR formula contained LBG (0.4 g/100 mL) with postbiotics and has a history of safe use. The primary outcome was the Infant Gastrointestinal Symptom Questionnaire (IGSQ) sum score including stooling, spitting-up/vomiting, crying, fussiness and flatulence. RESULTS: All 182 infants screened were enrolled in the study. The primary analysis showed the equivalence of the IGSQ sum scores at Week 4 between groups. IGSQ sum scores improved significantly within 1 week (Mixed Model Repeated Measurement [MMRM], P < 0.001). Post-hoc analyses showed a bigger improvement of the IGSQ score in the Test (n = 38) versus Control (n = 44) group (MMRM, P = 0.008) in infants with more severe gastrointestinal (GI) symptoms (IGSQ score ≥35). Stool characteristics were comparable between groups. Growth related z scores were in line with the WHO child growth standards and both groups showed improvement of regurgitation. Adverse events did not show any safety concerns. CONCLUSIONS: The novel AR formula combining LBG, scGOS/lcFOS and postbiotics is well-tolerated, safe and supports adequate growth during the intervention. Post-hoc analyses suggest that the formula results in more improvement of GI symptom burden in infants with more severe symptoms.

Machine Learning Supports Automated Digital Image Scoring of Stool Consistency in Diapers.

BACKGROUND/AIMS: Accurate stool consistency classification of non-toilet-trained children remains challenging. This study evaluated the feasibility of automated classification of stool consistencies from diaper photos using machine learning (ML). METHODS: In total, 2687 usable smartphone photos of diapers with stool from 96 children younger than 24 months were obtained after independent ethical study approval. Stool consistency was assessed from each photo according to the original 7 types of the Brussels Infant and Toddler Stool Scale independently by study participants and 2 researchers. A health care professional assigned a final score in case of scoring disagreement between the researchers. A proof-of-concept ML model was built upon this collected photo database, using transfer learning to re-train the classification layer of a pretrained deep convolutional neural network model. The model was built on random training (n = 2478) and test (n = 209) subsets. RESULTS: Agreements between study participants and both researchers were 58.0% and 48.5%, respectively, and between researchers 77.5% (assessable n = 2366). The model classified 60.3% of the test photos in exact agreement with the final score. With respect to the 4-class grouping of the 7 Brussels Infant and Toddler Stool Scale types, the agreement between model-based and researcher classification was 77.0%. CONCLUSION: The automated and objective scoring of stool consistency from diaper photos by the ML model shows robust agreement with human raters and overcomes limitations of other methods relying on caregiver reporting. Integrated with a smartphone application, this new framework for photo database construction and ML classification has numerous potential applications in clinical studies and home assessment.

Rare variant association testing for multicategory phenotype.

Genetic association studies have provided new insights into the genetic variability of human complex traits with a focus mainly on continuous or binary traits. Methods have been proposed to take into account disease heterogeneity between subgroups of patients when studying common variants but none was specifically designed for rare variants. Because rare variants are expected to have stronger effects and to be more heterogeneously distributed among cases than common ones, subgroup analyses might be particularly attractive in this context. To address this issue, we propose an extension of burden tests by using a multinomial regression model, which enables association tests between rare variants and multicategory phenotypes. We evaluated the type I error and the power of two burden tests, CAST and WSS, by simulating data under different scenarios. In the case of genetic heterogeneity between case subgroups, we showed an advantage of multinomial regression over logistic regression, which considers all the cases against the controls. We replicated these results on real data from Moyamoya disease where the burden tests performed better when cases were stratified according to age-of-onset. We implemented the functions for association tests in the R package "Ravages" available on Github.

GEMPROT: visualization of the impact on the protein of the genetic variants found on each haplotype.

SUMMARY: When analyzing sequence data, genetic variants are considered one by one, taking no account of whether or not they are found in the same individual. However, variant combinations might be key players in some diseases as variants that are neutral on their own can become deleterious when associated together. GEMPROT is a new analysis tool that allows, from a phased vcf file, to visualize the consequences of the genetic variants on the protein. At the level of an individual, the program shows the variants on each of the two protein sequences and the Pfam functional protein domains. When data on several individuals are available, GEMPROT lists the haplotypes found in the sample and can compare the haplotype distributions between different sub-groups of individuals. By offering a global visualization of the gene with the genetic variants present, GEMPROT makes it possible to better understand the impact of combinations of genetic variants on the protein sequence. AVAILABILITY AND IMPLEMENTATION: GEMPROT is freely available at https://github.com/TaniaCuppens/GEMPROT. An on-line version is also available at http://med-laennec.univ-brest.fr/GEMPROT/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair.

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.

Solvation at metal/water interfaces: An ab initio molecular dynamics benchmark of common computational approaches.

Determining the influence of the solvent on electrochemical reaction energetics is a central challenge in our understanding of electrochemical interfaces. To date, it is unclear how well existing methods predict solvation energies at solid/liquid interfaces, since they cannot be assessed experimentally. Ab initio molecular dynamics (AIMD) simulations present a physically highly accurate, but also a very costly approach. In this work, we employ extensive AIMD simulations to benchmark solvation at charge-neutral metal/water interfaces against commonly applied continuum solvent models. We consider a variety of adsorbates including *CO, *CHO, *COH, *OCCHO, *OH, and *OOH on Cu, Au, and Pt facets solvated by water. The surfaces and adsorbates considered are relevant, among other reactions, to electrochemical CO2 reduction and the oxygen redox reactions. We determine directional hydrogen bonds and steric water competition to be critical for a correct description of solvation at the metal/water interfaces. As a consequence, we find that the most frequently applied continuum solvation methods, which do not yet capture these properties, do not presently provide more accurate energetics over simulations in vacuum. We find most of the computed benchmark solvation energies to linearly scale with hydrogen bonding or competitive water adsorption, which strongly differ across surfaces. Thus, we determine solvation energies of adsorbates to be non-transferable between metal surfaces, in contrast to standard practice.

A large-scale study to describe the prevalence, characteristics and management of functional gastrointestinal disorders in African infants.

AIM: To evaluate the pattern, impact on quality of life and management of common functional gastrointestinal disorders (FGIDs) and related signs and symptoms in infants below 1 year of age in Africa. METHODS: Multicentre, cross-sectional, observational study in 10 African countries. At the first paediatric consultation of children with gastrointestinal symptoms, the perception of paediatricians on FGIDs (infant colic, constipation and regurgitation) and gas/bloating, impact on infant quality of life and parental anxiety and patient management practices were evaluated by standardised questionnaires. RESULTS: Questionnaires were completed by 759 paediatricians for 10 812 infants. Overall, 49.9% of paediatricians reported ≥30% of first infant consultations each month for FGIDs or related symptoms. Infant colic was most commonly diagnosed (57.6% of infants), followed by gas/bloating (43.2%), regurgitation (39.7%) and constipation (31.4%). Overall, 53% presented >1 symptom. Mean scores for infant quality of life, sleep and parental anxiety were worse when children had multiple symptoms compared to children with a single symptom (P < .025). Prescription of medication was common (62.4%). There were no consistent differences between countries. CONCLUSION: Functional gastrointestinal disorder occurrence in Africa was high with a gap between expert recommendation that emphasises parental reassurance and nutritional advice and daily practice, particularly prescription of medication.

Functional gastrointestinal disorders in infants: Practice, knowledge and needs of Australian pharmacists.

AIM: To determine the nature and extent of interactions between retail pharmacists and families of infants concerned about functional gastrointestinal disorders. METHODS: A 15-question online survey was developed that could be completed by retail pharmacists in approximately 5 min. This survey aimed to obtain information relating to the frequency of interactions with parents of infants seeking advice and/or information about colic, gastro-oesophageal reflux (GOR) or constipation in pharmacies; what recommendations and/or advice was given by the pharmacists; from where the pharmacists obtained their information and what guidelines/recommendations they would value; and demographic information. RESULTS: A total of 362 pharmacists from every state and territory within Australia completed the survey. Conversations with parents/carers about constipation at least once a week were reported by 85% of pharmacists, with the equivalent percentages for GOR and colic both being 76%. In the case of constipation, medication was recommended in 70% of cases, and a nutritional approach was recommended in 67% of cases. Medication was recommended in 81% of cases of suspected colic, significantly greater than nutritional advice at 50%. For possible GOR, recommendations were similar, with medication being suggested in 66% and nutritional advice in 68%. GOR guidelines were the most sought after, with 42% of pharmacists placing such guidelines as their number one need. CONCLUSIONS: This survey indicates the need for greater emphasis to be given to reassurance by health-care professionals involved in the management of functional gastrointestinal disorders in infancy, as well as consideration of the construction of easily accessible, evidence-based national guidelines.

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair.

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.

Abrupt involution induces inflammation, estrogenic signaling, and hyperplasia linking lack of breastfeeding with increased risk of breast cancer.

BACKGROUND: A large collaborative analysis of data from 47 epidemiological studies concluded that longer duration of breastfeeding reduces the risk of developing breast cancer. Despite the strong epidemiological evidence, the molecular mechanisms linking prolonged breastfeeding to decreased risk of breast cancer remain poorly understood. METHODS: We modeled two types of breastfeeding behaviors in wild type FVB/N mice: (1) normal or gradual involution of breast tissue following prolonged breastfeeding and (2) forced or abrupt involution following short-term breastfeeding. To accomplish this, pups were gradually weaned between 28 and 31 days (gradual involution) or abruptly at 7 days postpartum (abrupt involution). Mammary glands were examined for histological changes, proliferation, and inflammatory markers by immunohistochemistry. Fluorescence-activated cell sorting was used to quantify mammary epithelial subpopulations. Gene set enrichment analysis was used to analyze gene expression data from mouse mammary luminal progenitor cells. Similar analysis was done using gene expression data generated from human breast samples obtained from parous women enrolled on a tissue collection study, OSU-2011C0094, and were undergoing reduction mammoplasty without history of breast cancer. RESULTS: Mammary glands from mice that underwent abrupt involution exhibited denser stroma, altered collagen composition, higher inflammation and proliferation, increased estrogen receptor α and progesterone receptor expression compared to those that underwent gradual involution. Importantly, when aged to 4 months postpartum, mice that were in the abrupt involution cohort developed ductal hyperplasia and squamous metaplasia. Abrupt involution also resulted in a significant expansion of the luminal progenitor cell compartment associated with enrichment of Notch and estrogen signaling pathway genes. Breast tissues obtained from healthy women who breastfed for < 6 months vs ≥ 6 months showed significant enrichment of Notch signaling pathway genes, along with a trend for enrichment for luminal progenitor gene signature similar to what is observed in BRCA1 mutation carriers and basal-like breast tumors. CONCLUSIONS: We report here for the first time that forced or abrupt involution of the mammary glands following pregnancy and lack of breastfeeding results in expansion of luminal progenitor cells, higher inflammation, proliferation, and ductal hyperplasia, a known risk factor for developing breast cancer.