Genetics
Abstract
Hereditary cardiomyopathies are the prototypic forms of heart failure and major causes of sudden cardiac death. The genome in cardiomyopathies is exposed to internal stressors, which damage the DNA and activate the DNA damage response (DDR) pathways. We set to determine whether the DDR pathways were activated and pathogenic in an established mouse model of desmoplakin (DSP)-cardiomyopathy generated upon deletion of the Dsp gene in cardiac myocytes (Myh6-McmTam:DspF/F). The mice exhibited premature death, cardiac dysfunction, myocardial cell death, fibrosis, and increased expression levels of the pro-inflammatory cytokines, consistent with the phenotype of human DSP-cardiomyopathy. Cytosolic nuclear self-DNA (nDNA) and mitochondrial DNA (mtDNA) were increased in cardiac myocyte cytosol in the Myh6-McmTam:DspF/F mice. Likewise, the DDR pathway proteins, including the cyclic GMP-AMP synthase (CGAS), stimulator of interferon response 1 (STING1) were upregulated as were the transcript levels of interferon response factor 3 (IRF3) and the nuclear factor κB (NFκB) target genes. Deletion of the Mb21d1 gene encoding CGAS in the Myh6-McmTam:DspF/F mice prolonged survival, improved cardiac function, attenuated fibrosis, and reduced cell death. Thus, cytosolic nDNA and mtDNA are increased and the DDR pathways are activated and pathogenic in a mouse model of DSP-cardiomyopathy, whereas genetic blockade of CGAS is salubrious.
Authors
Weiyue Wang, Benjamin Cathcart, Quoc D. Nguyen, Loi Q. Lao, Amelia Bryans, Sara E. Coleman, Leila Rouhi, Priyatansh Gurha, Ali J. Marian
Abstract
β-Thalassemia is a genetic disorder arising from mutations in the β-globin gene, leading to ineffective erythropoiesis and iron overload. Ineffective erythropoiesis, a hallmark of β-thalassemia, is an important driver of iron overload, which contributes to liver fibrosis, diabetes, and cardiac disease. Iron homeostasis is regulated by the hormone hepcidin; BMP6/hemojuvelin–mediated (BMP6/HJV-mediated) signaling induces hepatic hepcidin expression via SMAD1/5, with transmembrane serine protease 6 (TMPRSS6) being a negative regulator of HJV. Individuals with loss-of-function mutations in the TMPRSS6 gene show increased circulating hepcidin and iron-refractory iron-deficiency anemia, suggesting that blocking TMPRSS6 may be a viable strategy to elevate hepcidin levels in β-thalassemia. We generated a human mAb (REGN7999) that inhibits TMPRSS6. In an Hbbth3/+ mouse model of β-thalassemia, REGN7999 treatment led to significant reductions in liver iron, reduced ineffective erythropoiesis, and showed improvements in RBC health, running distance during forced exercise, and bone density. In a phase I, doubleblind, randomized, placebo-controlled study in healthy human volunteers (NCT05481333), REGN7999 increased serum hepcidin and reduced serum iron with an acceptable tolerability profile. Our results suggest that, by both reducing iron and improving RBC function, inhibition of TMPRSS6 by REGN7999 may offer a therapy for iron overload and impaired erythropoiesis in β-thalassemia.
Authors
Heinrich E. Lob, Nikhil Singh, Kusha Mohammadi, Larisa Ivanova, Beth Crowell, Hyon J. Kim, Leah Kravets, Nanditha M. Das, Yonaton Ray, Jee Hae Kim, Sylvie Rottey, Emily Labriola-Tompkins, Hazem E. Hassan, Lorna Farrelly, Harvey F. Chin, Marilena Preda, Leigh Spencer Noakes, Kei Saotome, Matthew Franklin, Marc W. Retter, Elif Karayusuf, John J. Flanagan, William Olson, Kalyan C. Nannuru, Vincent Idone, Michael E. Burczynski, Olivier A. Harari, Lorah Perlee, Griet Van Lancker, Andrew J. Murphy, Aris N. Economides, Sarah J. Hatsell
Abstract
The gain-of-function MUC5B promoter variant is the dominant risk factor for the development of idiopathic pulmonary fibrosis (IPF). However, its impact on protein expression in both non-fibrotic control and IPF lung specimens have not been well characterized. Utilizing laser capture microdissection coupled to mass spectrometry (LCM-MS), we investigated the proteomic profiles of airway and alveolar epithelium in non-fibrotic controls (n = 12) and IPF specimens (n = 12), stratified by the MUC5B promoter variant. Through qualitative and quantitative analyses, as well as pathway analysis and immunohistological validation, we have identified a distinct MUC5B-associated protein profile. Notably, the non-fibrotic control alveoli exhibited substantial MUC5B-associated protein changes, with an increase of IL-3 signaling. Additionally, we found that epithelial cells overlying IPF fibroblastic foci cluster closely to alveolar epithelia and express proteins associated with cellular stress pathways. In conclusion, our findings suggest that the MUC5B promoter variant leads to protein changes in alveolar and airway epithelium that appears to be associated with initiation and progression of lung fibrosis.
Authors
Jeremy A. Herrera, Mark Maslanka, Rachel Z. Blumhagen, Rachel Blomberg, Nyan Ye Lwin, Janna Brancato, Carlyne D. Cool, Jonathan P. Huber, Jonathan S. Kurche, Chelsea M. Magin, Kirk C. Hansen, Ivana V. Yang, David A. Schwartz
Abstract
Loss-of-function (LOF) variants in IL6ST, encoding GP130, can cause hyper-IgE syndrome (HIES). Monoallelic LOF variants in IL6ST lead to HIES when located in the intracellular domain downstream of box 1/2 and upstream of the STAT3 phosphorylation sites and the recycling motif, due to their dominant negative (DN) activity. In this region, two previously unreported IL6ST variants, p.K702Sfs7* and p.Y759Wfs26*, were identified in two families with autosomal dominant (AD) HIES. Both variants were LOF, exhibited DN effects, leading to the accumulation of mutant GP130 on the cell surface. The p.K702Sfs7* mutation was the most upstream N-terminal mutation linked to HIES caused by heterozygous IL6ST variants. Comprehensive screening of IL6ST mutants revealed that most premature terminations downstream of amino acid F641, at the end of the transmembrane domain, resulted in LOF and DN effects via GP130 accumulation on the cell surface. The absence of the recycling motif (positions 782-787) in surface-expressed LOF GP130 led to its accumulation, contributing to the DN effect. The importance of intracellular truncating IL6ST variants can possibly be predicted based on the location of the premature stop codon. GP130 accumulation on the cell surface is a characteristic and potentially diagnostic finding in HIES patients with heterozygous IL6ST variants.
Authors
Kosuke Ashihara, Takaki Asano, Kanako Takeuchi, Kosuke Noma, Miyuki Tsumura, Wenjie Wang, Wei-Te Lei, Hisao Higo, Toshio Kubo, Yoko Mizoguchi, Shuhei Karakawa, Aurélie Cobat, Clément Conil, Etsushi Toyofuku, Akimasa Sekine, Kohsuke Imai, Dusan Bogunovic, Jean-Laurent Casanova, Cheng-Lung Ku, Vivien Béziat, Satoshi Okada
Abstract
Human Caspase Recruitment Domain Containing Protein 9 (CARD9) deficiency predisposes to invasive fungal disease, particularly by Candida spp. Distinctly, CARD9-deficiency causes chronic central nervous system (CNS) candidiasis. Currently, no animal model recapitulates the chronicity of disease, precluding a better understanding of immunopathogenesis. We established a knock-in mouse homozygous for the recurring p.Y91H mutation (Y91HKI) and, in parallel to Card9-/- mice, titrated the intravenous fungal inoculum to the CARD9-genotype to develop a model of chronic invasive candidiasis. Strikingly, CARD9-deficient mice had predominantly CNS involvement, with neurological symptoms appearing late during infection and progressive brain fungal burden in the absence of fulminant sepsis, reflecting the human syndrome. Mononuclear cell aggregation at fungal lesions in the brain correlated with increased MHCII+Ly6C+ monocyte numbers at day 1 post-infection in WT and Y91HKI mice, but not in Card9-/- mice. At day 4 post-infection, neutrophils and additional Ly6C+ monocytes were recruited to the CARD9-deficient brain. As in humans, Y91HKI mutant mice demonstrated cerebral multinucleated giant cells and granulomata. Subtle immunologic differences between the hypomorphic (p.Y91H) and null mice were noted, perhaps explaining some of the variability seen in humans. Our work established a disease-recapitulating animal model to specifically decipher chronic CNS candidiasis due to CARD9 deficiency.
Authors
Marija Landekic, Isabelle Angers, Yongbiao Li, Marie-Christine Guiot, Marc-André Déry, Annie Beauchamp, Lucie Roussel, Annie Boisvert, Wen Bo Zhou, Christina Gavino, Julia Luo, Stéphane Bernier, Makayla Kazimerczak-Brunet, Yichun Sun, Brendan Snarr, Michail S. Lionakis, Robert T. Wheeler, Irah L. King, Salman Qureshi, Maziar Divangahi, Donald C. Vinh
Abstract
Many chemotherapeutic agents impair cancer growth by inducing DNA damage. The impact of these agents on mutagenesis in normal cells, including sperm, is largely unknown. Here, we applied high-fidelity duplex sequencing to 94 samples from 36 individuals exposed to diverse chemotherapies and 32 controls. We found that many of the sperm samples from men exposed to chemotherapy, the mutation burden was elevated as compared to controls and the expected burden based on trio studies, with one subject having >10-fold increase over expected for age. Saliva from this same individual also had a markedly higher mutation burden. We then validated this finding using other tissues, also finding an increased mutation burden in the blood and liver of many subjects exposed to chemotherapy as compared to unexposed controls. Similarly, mice treated with three cycles of cisplatin had an increased mutation burden in sperm but also in the liver, and hematopoietic progenitor cells. These results suggest an association between cancer therapies and mutation burden, with implications for counseling cancer patients considering banking sperm prior to therapy and for cancer survivors considering the tradeoffs of using banked sperm as compared to conceiving naturally.
Authors
Shany Picciotto, Camilo Arenas-Gallo, Amos Toren, Ruty Mehrian-Shai, Bryan Daly, Stephen Rhodes, Megan Prunty, Ruolin Liu, Anyull Bohorquez, Marta Grońska-Pęski, Shana Melanaphy, Pamela Callum, Emilie Lassen, Anne-Bine Skytte, Rebecca C. Obeng, Christopher Barbieri, Molly Gallogly, Brenda Cooper, Katherine Daunov, Lydia Beard, Koen Van-Besien, Joshua Halpern, Quintin Pan, Gilad D. Evrony, Viktor A. Adalsteinsson, Jonathan E. Shoag
Abstract
Inherited retinal degenerations (IRDs) are important causes of progressive, irreversible blindness. Hereditary macular diseases in particular are significant in their effect on the specialized, central cone photoreceptor-rich macula responsible for high resolution vision. Autosomal dominant Best vitelliform macular dystrophy (BVMD), caused by variants in the BEST1 gene, is one of the most common inherited macular dystrophies. Gene therapies have emerged as promising treatments for IRDs, but a lack of suitable animal models has hindered progress both in treatments and in understanding the mechanisms underlying macular diseases. Here, we report a Macaca fascicularis carrying a heterozygous potential pathogenic BEST1p.Q327E variant that disrupts the BEST1 ion channel by destabilizing the A195 helix, mirroring the structural perturbations seen in certain human pathological mutants. Longitudinal imaging over two years revealed progressive macular changes, including subfoveal cleft enlargement, lipid-rich deposit accumulation, retinal pigment epithelium (RPE) disruption, and central-to-peripheral photoreceptor degeneration, recapitulating early human BVMD pathology. Histopathology demonstrated diminished BEST1 expression, attenuation of the RPE-photoreceptor interface, and two distinct types of lipid deposits, including heretofore unappreciated cone mitochondrial-enriched lesions, highlighting selective cone mitochondria vulnerability. This first non-human primate model of inherited macular dystrophy links BEST1 mutations, mitochondrial dysfunction, and progressive macular degeneration, offering new insights into BVMD pathophysiology and highlighting its utility for studying disease progression and potential therapeutic interventions.
Authors
Wei Yi, Mingming Xu, Ying Xue, Yingxue Cao, Ziqi Yang, Lingli Zhou, Yang Zhou, Le Shi, Xiaomei Mai, Zehui Sun, Wenjie Qing, Yuying Li, Aolun Qing, Kaiwen Zhang, Lechun Ou, Shoudeng Chen, Elia J. Duh, Xialin Liu
Abstract
Non-small cell lung cancer (NSCLC) largely consists of lung squamous (LUSC) and lung adenocarcinoma (LUAD). Alterations in the TRP53 and PTEN tumor suppressors are common in both subtypes, but their relationship with SOX2 is poorly understood. We deleted Trp53 or Pten in a C57BL/6J-Sox2hi;Nkx2-1-/-;Lkb1-/- (SNL) genetic background and generated a highly metastatic LUSC cell line (LN2A; derived from a Sox2hi mouse model, followed by Trp53, Pten, and Cdkn2a deletion). Histologic and single-cell RNAseq analyses corroborated that SNL mice developed mixed tumors with both LUAD and LUSC histopathology while SNL-Trp53 and SNL-Pten mice developed LUAD and LN2A tumors retained LUSC morphology. Compared with SNL mice, additional loss of Trp53 or Pten resulted in significantly reduced survival, increased tumor burden and altered tumor mucin composition. We identified a sub-cluster of CD38+ tumor-associated inflammatory monocytes in the LN2A model that significantly enriched for activation of the classical and alternative complement pathways. Complement Factor B (CFB) is associated with poor survival in LUSC patients, and we observed the LN2A model had significantly improved survival on a Cfb-/- background. Our findings demonstrate a cooperative role of Trp53 and Pten tumor suppressors in Sox2-mediated NSCLC tumor progression, mucin production, and remodeling of the immune tumor microenvironment.
Authors
Nisitha Sengottuvel, Kristina M. Whately, Jennifer L Modliszewski, Rani S. Sellers, William D. Green, Weida Gong, Allison T. Woods, Eric W. Livingston, Katerina D. Fagan-Solis, Gabrielle Cannon, Lincy Edatt, Hong Yuan, Aaron C. Chack, Yazmin Sanchez, Katherine Zhou, Alyaa Dawoud, Jarred M. Green, Virginia Godfrey, J Justin Milner, Gaorav P. Gupta, Chad V. Pecot
Abstract
It is well documented that impaired DNA damage repair (DDR) induces genomic instability that can efficiently increase the sensitivity of prostate cancer (PCa) cells to PARP inhibitors; however, the underlying mechanism remains elusive. Here, we found profound genomic instability in PCa cells with SPOP gene mutations and confirmed the sensitivity of SPOP-mutated PCa cells to olaparib-induced apoptosis. Mechanistically, we identified olaparib-induced CK2-mediated phosphorylation of PIAS1-S468, which in turn mediated SUMOylation of SPOP, thus promoting its E3 ligase activity in the DDR. Moreover, an abnormal CK2/PIAS1/SPOP axis due to SPOP mutations or defects in CK2-mediated phosphorylation of PIAS1, as well as SPOP inhibitor treatment, led to impaired DDR, thus increasing olaparib-induced apoptosis of PCa cells and enhancing olaparib sensitivity in animal models and patient-derived organoids. This suggested that disruption of the CK2/PIAS1/SPOP signaling axis could serve as an indicator for targeted therapy of PCa using a PARP inhibitor.
Authors
Hui Zhang, Lili Kong, Jinhui Li, Zhihan Liu, Yiting Zhao, Xiuyi Lv, Liangpei Wu, Lin Chai, Hongjie You, Jiabei Jin, Xinyi Cao, Zhong Zheng, Yadong Liu, Zejun Yan, Xiaofeng Jin
Abstract
Mutations in genome maintenance factors drive sporadic and hereditary breast cancers. Here, we searched for potential drivers based on germline DNA analysis from a cohort consisting of early-onset breast cancer patients negative for BRCA1/BRCA2 mutations. This revealed candidate genes that subsequently were subjected to RNAi-based phenotype screens to reveal genome integrity impacts. We identified several genes with functional roles in genome maintenance, including Glucose-6-Phosphatase Catalytic Subunit 3 (G6PC3), SMC4, and CCDC108. Notably, G6PC3-deficient cells exhibited increased levels of γH2AX and micronuclei formation, along with defects in homologous recombination (HR) repair. Consistent with these observations, G6PC3 was required for the efficient recruitment of BRCA1 to sites of DNA double-strand breaks (DSBs). RNA sequencing analysis revealed that G6PC3 promotes the expression of multiple homologous recombination repair genes, including BRCA1. Through CRISPR-Select functional-genetic phenotype analysis of G6PC3 germline mutations, we identified two germline G6PC3 variants displaying partial loss-of-function. Furthermore, our study demonstrated that G6pc3 deficiency accelerates mammary tumor formation induced by Trp53 loss in mice. In conclusion, our cohort-based functional analysis has unveiled genome maintenance factors and identified G6PC3 as a potential candidate tumor suppressor in breast cancer.
Authors
Xin Li, Maria Rossing, Ana Moisés da Silva, Muthiah Bose, Thorkell Gudjónsson, Jan Benada, Jayashree Thatte, Jens Vilstrup Johansen, Judit Börcsök, Hanneke van der Gulden, Ji-Ying Song, Renée Menezes, Asma Tajik, Lucía Sena, Zoltan Szallasi, Morten Frödin, Jos Jonkers, Finn Cilius Nielsen, Claus Storgaard Sørensen
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