Haploinsufficiency of factors governing genome stability underlies hereditary breast and ovarian cancer. Homologous recombination (HR) repair is a major pathway disabled in these cancers. With the aim of identifying new candidate genes, we examined early onset breast cancer patients negative for BRCA1 and BRCA2 pathogenic variants. Here, we focused on CtIP (RBBP8 gene) that mediates HR repair through the end-resection of DNA double-strand breaks (DSB). Notably, the patients exhibited a number of rare germline RBBP8 variants, and functional analysis revealed that these variants did not affect DNA DSB end-resection efficiency. However, expression of a subset of variants led to deleterious nucleolytic degradation of stalled DNA replication forks in a manner similar to cells lacking BRCA1 or BRCA2. In contrast to BRCA1 and BRCA2, CtIP deficiency promoted the helicase-driven destabilization of RAD51 nucleofilaments at damaged DNA replication forks. Taken together, our work identifies CtIP as a critical regulator of DNA replication fork integrity, which when compromised, may predispose to the development of early onset breast cancer.
Reihaneh Zarrizi, Martin R. Higgs, Karolin Voßgröne, Maria Rossing, Birgitte Bertelsen, Muthiah Bose, Arne N. Kousholt, Heike I. Rösner, Bent Ejlertsen, Grant S. Stewart, Finn Cilius Nielsen, Claus Sørensen
Several missense mutations in the orphan transporter FLVCR2 have been reported in Fowler syndrome. Affected subjects exhibit signs of severe neurological defects. We identified the mouse ortholog Mfsd7c as a gene, which is expressed in the blood brain barrier. Here, we report the characterizations of Mfsd7c knockout (KO) mice and compare it to phenotypic findings in humans with bi-allelic FLVCR2 mutations. Global KO of Mfsd7c in mice resulted in late gestation lethality, likely due to central nervous system (CNS) phenotypes. We found that the angiogenic growth of CNS blood vessels in the brain of Mfsd7c KO embryos was inhibited in cortical ventricular zones and ganglionic eminences. Vascular tips were dilated and fused resulting in glomeruloid vessels. Nonetheless, CNS blood vessels were intact without haemorrhage. Both embryos and humans with bi-allelic FLVCR2 mutations exhibited reduced cerebral cortical layers, enlargement of the cerebral ventricles, and microcephaly. Transcriptomic analysis of Mfsd7c knockout (KO) embryonic brains revealed upregulation of genes involved in glycolysis and angiogenesis. The Mfsd7c KO brain exhibited hypoxia and neuronal cell death. Our results indicate MFSD7c is required for the normal growth of CNS blood vessels and ablation of this gene results in microcephaly-associated vasculopathy in mice and humans.
Pazhanichamy Kalailingam, Kai Qi Wang, Xiu Ru Toh, Toan Q. Nguyen, Madhuvanthi Chandrakanthan, Zafrul Hasan, Clair Habib, Aharon Schif, Francesca Clementina Radio, Bruno Dallapiccola, Karin Weiss, Long N. Nguyen
Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler Syndrome by inactivating Flvcr2 in mice. We show that Flvcr2 is necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood brain barrier. Endothelial cells lacking Flvcr2 have altered expression of angiogenic factors, fail to adopt tip-cell properties and display reduced sprouting leading to vascular malformations similar to those seen in humans with Fowler Syndrome. Brain hypo-vascularization is associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier is maintained in Flvcr2 mutant mice. Our new Fowler syndrome models therefore define the pathobiology of this disease, and provide new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
Nicolas Santander, Carlos Omar Lizama, Eman Meky, Gabriel L. McKinsey, Bongnam Jung, Dean Sheppard, Christer Betsholtz, Thomas D. Arnold
The microbiome provides resistance to infection. However, mechanisms for this are poorly understood. Here we demonstrate in a murine model that colonization with the intestinal bacterium Clostridium scindens provided protection from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte-progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone-marrow from C. scindens colonized-mice into naïve-mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Because of the known ability of the Lachnoclostridia C. scindens to metabolize the bile salt cholate, we measured deoxycholate and discovered that it was increased in the sera of C. scindens colonized specific pathogen free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone (in the absence of C. scindens) increased GMPs and provided protection from amebiasis. We have discovered a mechanism by which C. scindens and the microbially-metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with Entamoeba histolytica.
Stacey L. Burgess, Jhansi L. Leslie, Md. Jashim Uddin, David Noah Oakland, Carol A. Gilchrist, G. Brett Moreau, Koji Watanabe, Mahmoud M. Saleh, Morgan Simpson, Brandon A. Thompson, David T. Auble, Stephen D. Turner, Natasa Giallourou, Jonathan Swann, Zhen Pu, Jennie Z. Ma, Rashidul Haque, William A. Petri, Jr.
Hepatocellular carcinoma (HCC) is difficult to detect, carries a poor prognosis, and is one of few cancers with an increasing yearly incidence. Molecular defects in complement factor H (CFH), a critical regulatory protein of the complement alternative pathway (AP), are typically associated with inflammatory diseases of the eye and kidney. Little is known regarding the role of CFH in controlling complement activation with the liver. While studying aging CFH-deficient (fH–/–) mice, we observed spontaneous hepatic tumor formation in more than 50% of aged fH–/– males. Examination of fH–/– livers (3–24 months) for evidence of complement-mediated inflammation revealed widespread deposition of complement activation fragments throughout the sinusoids, elevated transminase levels, increased hepatic CD8+ and F4/80+ cells, overexpress of hepatic mRNA associated with inflammatory signaling pathways, steatosis and increased collagen deposition. Immunostaining of human HCC biopsies revealed extensive deposition of complement fragments within the tumors. Interrogation of the Cancer Genome Atlas also revealed that increased CFH mRNA expression is associated with improved survival in HCC patients, whereas mutations are associated with worse survival. These results indicate that CFH is critical for controlling complement activation in the liver, and in its absence, AP activation leads to chronic inflammation and promotes hepatic carcinogenesis.
Jennifer Laskowski, Brandon Renner, Matthew C. Pickering, Natalie J. Serkova, Peter M. Smith-Jones, Eric T. Clambey, Raphael A. Nemenoff, Joshua M. Thurman
There are more than 7000 described rare diseases, most lacking specific treatment. Autosomal-dominant hyper-IgE syndrome (AD-HIES, Job’s syndrome) is caused by mutations in signal transducer and activator of transcription 3 (STAT3). These patients present with immunodeficiency accompanied by severe non-immunological features including skeletal, connective tissue and vascular abnormalities, poor post-infection lung healing, and subsequent pulmonary failure. No specific therapies are available for these abnormalities. Here we investigated underlying mechanisms in order to identify therapeutic targets. Histological analysis of skin wounds demonstrated delayed granulation tissue formation and vascularization during skin wound healing in AD-HIES patients. Global gene expression analysis in AD-HIES patient skin fibroblasts identified deficiencies in a STAT3 controlled transcriptional network regulating extracellular matrix (ECM) remodeling and angiogenesis, with hypoxia inducible factor 1α (HIF1α) being a major contributor. Consistent with this, histological analysis of skin wounds and coronary arteries from AD-HIES patients showed decreased HIF1α expression, and revealed abnormal organization of the ECM and altered formation of the coronary vasa vasorum. Disease modeling utilizing cell culture and mouse models of angiogenesis and wound healing confirmed these predicted deficiencies and demonstrated therapeutic benefit of HIF1α stabilizing drugs. The study provides mechanistic insights into AD-HIES pathophysiology and finds new treatment option for this rare disease.
Natalia I. Dmitrieva, Avram D. Walts, Dai P. Nguyen, Alex Grubb, Xue Zhang, Xujing Wang, Xianfeng Ping, Hui Jin, Zhen Yu, Zu-Xi Yu, Dan Yang, Robin Schwartzbeck, Clifton L. Dalgard, Beth A. Kozel, Mark D. Levin, Russell H. Knutsen, Delong Liu, Joshua D. Milner, Diego B. López, Michael P. O'Connell, Chyi-Chia R. Lee, Ian A. Myles, Amy P. Hsu, Alexandra F. Freeman, Steven M. Holland, Guibin Chen, Manfred Boehm
Molecular mechanisms governing the development of mammalian cochlea, the hearing organ, remain largely unknown. Through genome sequencing in three subjects from two families with non-syndromic cochlear aplasia, we identified homozygous 221 KB and 338 KB deletions in a non-coding region on chromosome 8 with an ~200 KB overlapping section. Genomic location of the overlapping deleted region was starting from ~350 KB downstream of GDF6. Otic lineage cells differentiated from induced pluripotent stem cells derived from an affected individual show reduced expression of GDF6 compared to control cells. A mouse knock-out of Gdf6 reveals cochlear aplasia closely resembling the human phenotype. We conclude that GDF6 plays a necessary role in early cochlear development controlled by cis-regulatory elements located within ~500 KB region of the genome in humans and that its disruption leads to deafness due to cochlear aplasia.
Guney Bademci, Clemer Abad, Filiz Basak Cengiz, Serhat Seyhan, Armagan Incesulu, Shengru Guo, Suat Fitoz, Emine Ikbal Atli, Nicholas C. Gosstola, Selma Demir, Brett M. Colbert, Gozde Cosar Seyhan, Claire J. Sineni, Duygu Duman, Hakan Gurkan, Cynthia Casson Morton, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
Retinitis pigmentosa (RP) is a genetically heterogenous group of eye diseases in which initial degeneration of rods triggers secondary degeneration of cones, leading to significant loss of daylight, color, and high-acuity vision. Gene complementation with adeno-associated viral (AAV) vectors is one strategy to treat RP. Its implementation faces substantial challenges, however — e.g., the tremendous number of loci with causal mutations. Gene therapy targeting secondary cone degeneration is an alternative approach that could provide a much-needed generic treatment for many RP patients. Here, we show that microglia are required for the upregulation of potentially neurotoxic inflammatory factors during cone degeneration in RP, creating conditions that might contribute to cone dysfunction and death. To ameliorate the effects of such factors, we used AAV vectors to express isoforms of the anti-inflammatory cytokine transforming growth factor-beta (TGF-β). AAV-mediated delivery of TGF-β1 rescued degenerating cones in three mouse models of RP carrying different pathogenic mutations. Treatment with TGF-β1 protected vision, as measured by two behavioral assays, and could be pharmacologically disrupted by either depleting microglia or blocking the TGF-β receptors. Our results suggest that TGF-β1 may be broadly beneficial for patients with cone degeneration, and potentially other forms of neurodegeneration, through a pathway dependent upon microglia.
Sean K. Wang, Yunlu Xue, Constance L. Cepko
Medications that target catecholamine-associated inflammation may prevent cytokine storm syndrome associated with COVID-19 and other diseases.
Maximilian F. Konig, Michael A. Powell, Verena Staedtke, Ren-Yuan Bai, David L. Thomas, Nicole M. Fischer, Sakibul Huq, Adham M. Khalafallah, Allison Koenecke, Ruoxuan Xiong, Brett Mensh, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Joshua T. Vogelstein, Susan Athey, Shibin Zhou, Chetan Bettegowda
Improved pandemic preparedness could be achieved by proactively managing emerging virus threats using available technologies.
Barney S. Graham, Kizzmekia S. Corbett
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