Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2cre/cre) develop precursor T-cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4-32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid, T and B lymphocyte. All Mcm2cre/creNHD13+ mice develop pre-T LBL, and 26% develop an unrelated, concurrent B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy Number Alteration (CNA) analysis demonstrated that pre-T LBL were characterized by homozygous deletions of Pten and Tcf3, and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALL were characterized by recurrent deletions involving Pax5 and Ptpn1, and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks, and resultant CNAs due to errors in DNA DSB repair. CNAs which involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts, due to a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.
Mianmian Yin, Timour Baslan, Robert L. Walker, Yuelin J. Zhu, Amy Freeland, Toshihiro Matsukawa, Sriram Sridharan, André Nussenzweig, Steven C. Pruitt, Scott W. Lowe, Paul S. Meltzer, Peter D. Aplan
Background. The presence of an early repolarization pattern (ERP) on the surface electrocardiogram (ECG) is associated with risk of ventricular fibrillation and sudden cardiac death. Family studies have shown that ERP is a highly heritable trait but molecular genetic determinants are unknown. Methods. To identify genetic susceptibility loci for ERP, we performed a GWAS and meta-analysis in 2,181 cases and 23,641 controls of European ancestry. Results. We identified a genome-wide significant (p<5E-8) locus in the KCND3 (potassium voltage gated channel subfamily D member 3) gene that was successfully replicated in additional 1,124 cases and 12,510 controls. A subsequent joint meta-analysis of the discovery and replication cohorts identified rs1545300 as the lead SNP at the KCND3 locus (OR 0.82 per minor T allele, p=7.7E-12), but did not reveal additional loci. Co-localization analyses indicate causal effects of KCND3 gene expression levels on ERP in both cardiac left ventricle and tibial artery. Conclusions. In this study we identified for the first time a genome-wide significant association of a genetic variant with ERP. Our findings of a locus in the KCND3 gene not only provide insights into the genetic determinants but also into the pathophysiological mechanism of ERP, discovering a promising candidate for functional studies. Funding. For detailed information per study, see Acknowledgments.
Alexander Teumer, Teresa Trenkwalder, Thorsten Kessler, Yalda Jamshidi, Marten E. van den Berg, Bernhard Kaess, Christopher P. Nelson, Rachel Bastiaenen, Marzia De Bortoli, Alessandra Rossini, Isabel Deisenhofer, Klaus Stark, Solmaz Assa, Peter S. Braund, Claudia Cabrera, Anna F. Dominiczak, Martin Gögele, Leanne M. Hall, M. Arfan Ikram, Maryam Kavousi, Karl J. Lackner, Christian Müller, Thomas Münzel, Matthias Nauck, Sandosh Padmanabhan, Norbert Pfeiffer, Tim D. Spector, Andre G. Uitterlinden, Niek Verweij, Uwe Völker, Helen R. Warren, Mobeen Zafar, Stephan B. Felix, Jan A. Kors, Harold Snieder, Patricia B. Munroe, Cristian Pattaro, Christian Fuchsberger, Georg Schmidt, Ilja M. Nolte, Heribert Schunkert, Peter Pramstaller, Philipp S. Wild, Pim van der Harst, Bruno H. Stricker, Renate B. Schnabel, Nilesh J. Samani, Christian Hengstenberg, Marcus Dörr, Elijah R. Behr, Wibke Reinhard
The acute respiratory distress syndrome (ARDS) is an inflammatory lung disorder that frequently complicates critical illness, and most commonly occurs in the setting of sepsis. Although a number of clinical and environmental risk factors for ARDS have been described, not all patients with risk factors develop the syndrome, raising the possibility of genetic underpinnings for ARDS susceptibility. We have previously reported that circulating cell-free hemoglobin (CFH) is elevated during sepsis, and higher levels are associated with worse outcomes. CFH is rapidly scavenged by the plasma protein haptoglobin (Hp). A common HP genetic variant HP2 is unique to humans and represents 60% of the HP allele frequency in populations of European ancestry. The HP2 gene product has reduced ability to inhibit CFH-mediated inflammation and oxidative stress compared to the alternative HP1. We hypothesized that the HP2 variant increases ARDS susceptibility during sepsis when plasma CFH levels are elevated. In a murine model of sepsis with elevated CFH levels, transgenic mice homozygous for Hp2 had increased lung inflammation, pulmonary vascular permeability, lung apoptosis, and mortality compared to mice homozygous for the alternative allele Hp1. We then tested the clinical relevance of our findings in a prospective observational cohort study of 496 septic critically ill adults, and found that the HP2 variant was significantly associated with increased ARDS susceptibility (odds ratio 1.41 per HP2 allele, 95% confidence interval 1.06 – 1.88, P = 0.018) after controlling for clinical risk factors and plasma CFH. This relationship between the HP2 genetic variant and ARDS risk was only seen in patients with elevated plasma CFH levels. These observations identify the HP2 variant as a novel genetic ARDS risk factor during sepsis, and may have important implications in the study and treatment of ARDS.
V. Eric Kerchberger, Julie A. Bastarache, Ciara M. Shaver, Hiromasa Nagata, J. Brennan McNeil, Stuart R. Landstreet, Nathan D. Putz, Wen-Kuang Yu, Jordan Jesse, Nancy E. Wickersham, Tatiana N. Sidorova, David R. Janz, Chirag R. Parikh, Edward D. Siew, Lorraine B. Ware
Kabuki syndrome 1 (KS1) is a Mendelian disorder of the epigenetic machinery caused by mutations in the gene encoding KMT2D, which methylates lysine 4 on histone H3 (H3K4). KS1 is characterized by intellectual disability, postnatal growth retardation, and distinct craniofacial dysmorphisms. A mouse model (Kmt2d+/bGeo) exhibits features of the human disorder and has provided insight into other phenotypes; however, the mechanistic basis of skeletal abnormalities and growth retardation remains elusive. Using high-resolution micro-computed tomography we show that Kmt2d+/bGeo mice have shortened long bones and ventral bowing of skulls. In vivo expansion of growth plates within skulls and long bones suggests disrupted endochondral ossification as a common disease mechanism. Stable chondrocyte cell lines harboring inactivating mutations in Kmt2d exhibit precocious differentiation, further supporting this mechanism. A known inducer of chondrogenesis, SOX9, and its targets show markedly increased expression in Kmt2d-/- chondrocytes. By transcriptome profiling, we identify Shox2 as a putative KMT2D target. We propose that decreased KMT2D-mediated H3K4me3 at Shox2 releases Sox9 inhibition and thereby leads to enhanced chondrogenesis, providing a novel and plausible explanation for precocious chondrocyte differentiation. Our findings provide insight into the pathogenesis of growth retardation in KS1 and suggest novel therapeutic approaches for this and related disorders.
Jill A. Fahrner, Wan-Ying Lin, Ryan C. Riddle, Leandros Boukas, Valerie B. DeLeon, Sheetal Chopra, Susan E. Lad, Teresa Romeo Luperchio, Kasper D. Hansen, Hans T. Bjornsson
Transcriptomic profiling classifies pancreatic ductal adenocarcinoma (PDAC) into several molecular subtypes with distinctive histological and clinical characteristics. However, little is known about the molecular mechanisms that define each subtype and their correlation with clinical outcome. Mutant KRAS is the most prominent driver in PDAC, present in over 90% of tumors, but the dependence of tumors on oncogenic KRAS signaling varies between subtypes. In particular, squamous subtype are relatively independent of oncogenic KRAS signaling and typically display much more aggressive clinical behavior versus progenitor subtype. Here, we identified that YAP1 activation is enriched in the squamous subtype and associated with poor prognosis. Activation of YAP1 in progenitor subtype cancer cells profoundly enhanced malignant phenotypes and transformed progenitor subtype cells into squamous subtype. Conversely, depletion of YAP1 specifically suppressed tumorigenicity of squamous subtype PDAC cells. Mechanistically, we uncovered a significant positive correlation between WNT5A expression and the YAP1 activity in human PDAC, and demonstrated that WNT5A overexpression led to YAP1 activation and recapitulated YAP1-dependent but Kras-independent phenotype of tumor progression and maintenance. Thus, our study identifies YAP1 oncogene as a major driver of squamous subtype PDAC and uncovers the role of WNT5A in driving PDAC malignancy through activation of the YAP pathway.
Bo Tu, Jun Yao, Sammy Ferri-Borgogno, Jun Zhao, Shujuan Chen, Qiuyun Wang, Liang Yan, Xin Zhou, Cihui Zhu, Seungmin Bang, Qing Chang, Christopher A. Bristow, Ya'an Kang, Hongwu Zheng, Huamin Wang, Jason B. Fleming, Michael Kim, Timothy P. Heffernan, Giulio F. Draetta, Duojia Pan, Anirban Maitra, Wantong Yao, Sonal Gupta, Haoqiang Ying
Lymphatic malformations (LMs) are congenital, non-neoplastic vascular malformations associated with post-zygotic activating PIK3CA mutations. The mutation spectrum within LMs is narrow, with the majority having one of three “hotspot” mutations. Despite this relative genetic homogeneity, clinical presentations differ dramatically. We used molecular inversion probes and droplet digital polymerase chain reaction to perform deep, targeted sequencing of PIK3CA in 271 affected and unaffected tissue samples from 81 individuals with isolated LMs and retrospectively collected clinical data. Pathogenic PIK3CA mutations were identified in affected LM tissue in 64 individuals (79%) with isolated LMs, with variant allele fractions (VAFs) ranging from 0.1 to 13%. Initial analyses revealed no correlation between VAF and phenotype variables. Recognizing that different mutations activate PI3K to varying degrees, we developed a metric, the genotype-adjusted VAF (GVAF), to account for differences in mutation strength, and found significantly higher GVAFs in LMs with more severe clinical characteristics including orofacial location or microcystic structure. In addition to providing insight into LM pathogenesis, we believe GVAF may have broad applicability for genotype-phenotype analyses in mosaic disorders.
Kaitlyn Zenner, Chi Vicky Cheng, Dana M. Jensen, Andrew E. Timms, Giridhar Shivaram, Randall Bly, Sheila Ganti, Kathryn B. Whitlock, William B. Dobyns, Jonathan Perkins, James T. Bennett
Clinical trials of high-dose androgen therapy for prostate cancer have shown promising efficacy but are limited by lack of criteria to identify likely responders. To elucidate factors that govern the growth-repressive effects of high-dose androgens we applied an unbiased integrative approach utilizing genetic screens and transcriptional profiling of prostate cancer cells with or without demonstrated phenotypic sensitivity to androgen-mediated growth repression. Through this comprehensive analysis, we identified genetic events and related signaling networks that determine the response to both high-dose androgen and androgen withdrawal. We applied these findings to develop a gene signature that may serve as an early indicator of treatment response and identify men with tumors amenable to high dose androgen therapy.
Michael D. Nyquist, Alexandra Corella, Osama Mohamad, Ilsa Coleman, Arja Kaipainen, Daniel A. Kuppers, Jared M. Lucas, Patrick J. Paddison, Stephen R. Plymate, Peter S. Nelson, Elahe A. Mostaghel
Chromatin modifiers act to coordinate gene expression changes critical to neuronal differentiation from neural stem/progenitor cells (NSPCs). Lysine-specific methyltransferase 2D (KMT2D) encodes a histone methyltransferase that promotes transcriptional activation, and is frequently mutated in cancers and in the majority (>70%) of patients diagnosed with the congenital, multisystem intellectual disability (ID) disorder Kabuki syndrome 1 (KS1). Critical roles for KMT2D are established in various non-neural tissues, but the effects of KMT2D loss in brain cell development have not been described. We conducted parallel studies of proliferation, differentiation, transcription, and chromatin profiling in KMT2D-deficient human and mouse models to define KMT2D-regulated functions in neurodevelopmental contexts, including adult-born hippocampal NSPCs in vivo and in vitro. We report cell-autonomous defects in proliferation, cell cycle, and survival, accompanied by early NSPC maturation in several KMT2D-deficient model systems. Transcriptional suppression in KMT2D-deficient cells indicated strong perturbation of hypoxia-responsive metabolism pathways. Functional experiments confirmed abnormalities of cellular hypoxia responses in KMT2D-deficient neural cells, and accelerated NSPC maturation in vivo. Together, our findings support a model in which loss of KMT2D function suppresses expression of oxygen-responsive gene programs important to neural progenitor maintenance, resulting in precocious neuronal differentiation in a mouse model of KS1.
Giovanni A. Carosso, Leandros Boukas, Jonathan J. Augustin, Ha Nam Nguyen, Briana L. Winer, Gabrielle H. Cannon, Johanna D. Robertson, Li Zhang, Kasper D. Hansen, Loyal A. Goff, Hans T. Bjornsson
BACKGROUND. Aberrant expression of RNA processing genes may drive the alterative RNA profile in lower-grade gliomas (LGGs). Thus, we aimed to further stratify LGGs based on the expression of RNA processing genes. METHODS. This study included 446 LGGs from The Cancer Genome Atlas (TCGA, training set) and 171 LGGs from the Chinese Glioma Genome Atlas (CGGA, validation set). The least absolute shrinkage and selection operator (LASSO) Cox regression algorithm was conducted to develop a risk-signature. The receiver operating characteristic (ROC) curves and Kaplan–Meier curves were used to study the prognosis value of the risk-signature. RESULTS. Among the tested 784 RNA processing genes, 276 were significantly correlated with the OS of LGGs. Further LASSO Cox regression identified a 19-gene risk-signature, whose risk score was also an independently prognosis factor (P<0.0001, multiplex Cox regression) in the validation dataset. The signature had better prognostic value than the traditional factors “age”, “grade” and “WHO 2016 classification” for 3‐ and 5‐year survival both two datasets (AUCs > 85%). Importantly, the risk-signature could further stratify the survival of LGGs in specific subgroups of WHO 2016 classification. Furthermore, alternative splicing events for genes such as EGFR and FGFR were found to be associated with the risk score. mRNA expression levels for genes, which participated in cell proliferation and other processes, were significantly correlated to the risk score. CONCLUSIONS. Our results highlight the role of RNA processing genes for further stratifying the survival of patients with LGGs and provide insight into the alternative splicing events underlying this role.
Rui-Chao Chai, Yi-Ming Li, Ke-Nan Zhang, Yu-Zhou Chang, Yu-Qing Liu, Zheng Zhao, Zhi-Liang Wang, Yuan-Hao Chang, Guan-Zhang Li, Kuan-Yu Wang, Fan Wu, Yong-Zhi Wang
Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.
Alessia De Caneva, Fabiola Porro, Giulia Bortolussi, Riccardo Sola, Michela Lisjak, Adi Barzel, Mauro Giacca, Mark A. Kay, Kristian Vlahoviček, Lorena Zentilin, Andrés F. Muro
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