Genetics
Abstract
Single cancer cell sequencing studies currently use randomly-selected cells, limiting correlations between genomic aberrations, morphology and spatial localization. We laser-captured microdissected single cells from morphologically-distinct areas of primary breast cancer and corresponding lymph node metastasis and performed whole-exome or deep-target sequencing of greater than 100 such cells. Two major subclones co-existed in different areas of the primary tumor, and the lymph node metastasis originated from a minor subclone in the invasive front of the primary tumor with additional copy number changes including 8q gain, but no additional point mutations in driver genes. Lack of metastasis-specific driver events lead us to assess whether other clonal and subclonal genomic aberrations pre-existing in primary tumors contribute to lymph node metastasis. Gene mutations and copy number variations analyzed in five breast cancer tissue sample sets revealed that copy number variations in several genomic regions, including areas within chromosome 1p, 8q, 9p, 12q and 20q, harboring several metastasis-associated genes, were consistently associated with lymph node metastasis. Moreover, clonal expansion was observed in an area of morphologically-normal breast epithelia, likely driven by a driver mutation and a subsequent amplification in chromosome 1q. Our study illuminates the molecular evolution of breast cancer and genomic aberrations contributing to metastases.
Authors
Li Bao, Zhaoyang Qian, Maria B. Lyng, Ling Wang, Yuan Yu, Ting Wang, Xiuqing Zhang, Huanming Yang, Nils Brünner, Jun Wang, Henrik J. Ditzel
Abstract
Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of a particular aldolase, aldolase B, results in the accumulation of intracellular phosphorylated fructose thus leading to phosphate sequestration and depletion, increased ATP turnover and a plethora of conditions leading to clinical manifestations including fatty liver, hyperuricemia, Fanconi syndrome and severe hypoglycemia. Unfortunately, to date, there is no treatment for HFI and avoiding sugar and fructose in our society has become quite challenging. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI using aldolase B knockout mice. We thus provide evidence for the first time of a potential therapeutic approach for this condition. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.
Authors
Miguel A. Lanaspa, Ana Andres-Hernando, David J. Orlicky, Christina Cicerchi, Cholsoon Jang, Nanxing Li, Tamara Milagres, Masanari Kuwabara, Michael F. Wempe, Joshua D. Rabinowitz, Richard J. Johnson, Dean R. Tolan
Abstract
Metastatic breast cancers are still incurable. Characterizing the evolutionary landscape of these cancers, including the role of metastatic axillary lymph nodes (ALNs) in seeding distant organ metastasis, can provide a rational basis for effective treatments. Here, we have described the genomic analyses of the primary tumors and metastatic lesions from 99 samples obtained from 20 patients with breast cancer. Our evolutionary analyses revealed diverse spreading and seeding patterns that govern tumor progression. Although linear evolution to successive metastatic sites was common, parallel evolution from the primary tumor to multiple distant sites was also evident. Metastatic spreading was frequently coupled with polyclonal seeding, in which multiple metastatic subclones originated from the primary tumor and/or other distant metastases. Synchronous ALN metastasis, a well-established prognosticator of breast cancer, was not involved in seeding the distant metastasis, suggesting a hematogenous route for cancer dissemination. Clonal evolution coincided frequently with emerging driver alterations and evolving mutational processes, notably an increase in apolipoprotein B mRNA–editing enzyme, catalytic polypeptide-like–associated (APOBEC-associated) mutagenesis. Our data provide genomic evidence for a role of ALN metastasis in seeding distant organ metastasis and elucidate the evolving mutational landscape during cancer progression.
Authors
Ikram Ullah, Govindasamy-Muralidharan Karthik, Amjad Alkodsi, Una Kjällquist, Gustav Stålhammar, John Lövrot, Nelson-Fuentes Martinez, Jens Lagergren, Sampsa Hautaniemi, Johan Hartman, Jonas Bergh
Abstract
Genetic forms of vitamin D–dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR, however, using whole exome sequence analysis we identified a recurrent de novo missense mutation c.902T>C (p.I301T) in CYP3A4 in both subjects that alters the conformation of substrate-recognition-site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than wild-type CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide new insight into vitamin D metabolism, and demonstrate that accelerated inactivation of vitamin D metabolites represents a previously undescribed mechanism for vitamin D deficiency.
Authors
Jeffrey D. Roizen, Dong Li, Lauren O'Lear, Muhammad K. Javaid, Nicholas J. Shaw, Peter R. Ebeling, Hanh H. Nguyen, Christine P. Rodda, Kenneth E. Thummel, Tom D Thacher, Hakon Hakonarson, Michael A. Levine
Abstract
SCN5A encodes the voltage-gated Na+ channel NaV1.5 that is responsible for depolarization of the cardiac action potential and rapid intercellular conduction. Mutations disrupting the SCN5A coding sequence cause inherited arrhythmias and cardiomyopathy, and single-nucleotide polymorphisms (SNPs) linked to SCN5A splicing, localization, and function associate with heart failure–related sudden cardiac death. However, the clinical relevance of SNPs that modulate SCN5A expression levels remains understudied. We recently generated a transcriptome-wide map of microRNA (miR) binding sites in human heart, evaluated their overlap with common SNPs, and identified a synonymous SNP (rs1805126) adjacent to a miR-24 site within the SCN5A coding sequence. This SNP was previously shown to reproducibly associate with cardiac electrophysiological parameters, but was not considered to be causal. Here, we show that miR-24 potently suppresses SCN5A expression and that rs1805126 modulates this regulation. We found that the rs1805126 minor allele associates with decreased cardiac SCN5A expression and that heart failure subjects homozygous for the minor allele have decreased ejection fraction and increased mortality, but not increased ventricular tachyarrhythmias. In mice, we identified a potential basis for this in discovering that decreased Scn5a expression leads to accumulation of myocardial reactive oxygen species. Together, these data reiterate the importance of considering the mechanistic significance of synonymous SNPs as they relate to miRs and disease, and highlight a surprising link between SCN5A expression and nonarrhythmic death in heart failure.
Authors
Xiaoming Zhang, Jin-Young Yoon, Michael Morley, Jared M. McLendon, Kranti A. Mapuskar, Rebecca Gutmann, Haider Mehdi, Heather L. Bloom, Samuel C. Dudley, Patrick T. Ellinor, Alaa A. Shalaby, Raul Weiss, W.H. Wilson Tang, Christine S. Moravec, Madhurmeet Singh, Anne L. Taylor, Clyde W. Yancy, Arthur M. Feldman, Dennis M. McNamara, Kaikobad Irani, Douglas R. Spitz, Patrick Breheny, Kenneth B. Margulies, Barry London, Ryan L. Boudreau
Abstract
Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget’s disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid–phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S–persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.
Authors
YouJin Lee, Per Harald Jonson, Jaakko Sarparanta, Johanna Palmio, Mohona Sarkar, Anna Vihola, Anni Evilä, Tiina Suominen, Sini Penttilä, Marco Savarese, Mridul Johari, Marie-Christine Minot, David Hilton-Jones, Paul Maddison, Patrick Chinnery, Jens Reimann, Cornelia Kornblum, Torsten Kraya, Stephan Zierz, Carolyn Sue, Hans Goebel, Asim Azfer, Stuart H. Ralston, Peter Hackman, Robert C. Bucelli, J. Paul Taylor, Conrad C. Weihl, Bjarne Udd
Abstract
Immune evasion and the suppression of anti-tumor responses during cancer progression are considered hallmarks of cancer and are typically attributed to tumor-derived factors. Although the molecular basis for the crosstalk between tumor and immune cells is an area of active investigation, whether host-specific germline variants can dictate immunosuppressive mechanisms has remained a challenge to address. A commonly occurring germline mutation (c.1162G>A/rs351855 G/A) in the FGFR4 (CD334) gene enhances STAT3 signaling and is associated with poor prognosis and accelerated progression of multiple cancer types. Here, using rs351855 single nucleotide polymorphism (SNP) knock-in transgenic mice and Fgfr4 knockout mice, we reveal the genotype-specific gain of immunological function of suppressing the CD8/CD4+FOXP3+CD25+ve regulatory T cell ratio in vivo. Furthermore, using knock-in transgenic mouse models for lung and breast cancers, we establish the host-specific tumor-extrinsic functions of STAT3-enhancing germline variants in impeding the tumor infiltration of CD8 T cells. Thus, STAT3-enhancing germline receptor variants contribute to immune evasion through their pleiotropic functions in immune cells.
Authors
Daniel Kogan, Alexander Grabner, Christopher Yanucil, Christian Faul, Vijay Kumar Ulaganathan
Abstract
Congenital long QT syndrome (LQTS) is an inherited channelopathy associated with life-threatening arrhythmias. LQTS type 2 (LQT2) is caused by mutations in KCNH2, which encodes the potassium channel hERG. We hypothesized that modifier genes are partly responsible for the variable phenotype severity observed in some LQT2 families. Here, we identified contributors to variable expressivity in an LQT2 family by using induced pluripotent stem cell–derived cardiomyocytes (iPSC-CMs) and whole exome sequencing in a synergistic manner. We found that iPSC-CMs recapitulated the clinical genotype-phenotype discordance in vitro. Importantly, iPSC-CMs derived from the severely affected LQT2 patients displayed prolonged action potentials compared with cells from mildly affected first-degree relatives. The iPSC-CMs derived from all patients with hERG R752W mutation displayed lower IKr amplitude. Interestingly, iPSC-CMs from severely affected mutation-positive individuals exhibited greater L-type Ca2+ current. Whole exome sequencing identified variants of KCNK17 and the GTP-binding protein REM2, providing biologically plausible explanations for this variable expressivity. Genome editing to correct a REM2 variant reversed the enhanced L-type Ca2+ current and prolonged action potential observed in iPSC-CMs from severely affected individuals. Thus, our findings showcase the power of combining complementary physiological and genomic analyses to identify genetic modifiers and potential therapeutic targets of a monogenic disorder. Furthermore, we propose that this strategy can be deployed to unravel myriad confounding pathologies displaying variable expressivity.
Authors
Sam Chai, Xiaoping Wan, Angelina Ramirez-Navarro, Paul J. Tesar, Elizabeth S. Kaufman, Eckhard Ficker, Alfred L. George Jr., Isabelle Deschênes
Abstract
A modifier variant can abrogate risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin-homology-domain of GAB1, an essential scaffold in the MET/HGF pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescues the GAB1 associated morphant phenotype. In mouse, GAB1 and METTL13 co-localize in auditory sensory neurons, and METTL13 co-immunoprecipitates with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET-signaling provides potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET-signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET-signaling is likely to have broad clinical implications.
Authors
Rizwan Yousaf, Zubair M. Ahmed, Arnaud P.J. Giese, Robert J. Morell, Ayala Lagziel, Alain Dabdoub, Edward R. Wilcox, Sheikh Riazuddin, Thomas B. Friedman, Saima Riazuddin
Abstract
Profound hyperphagia is a major disabling feature of Prader-Willi syndrome (PWS). Characterization of the mechanisms that underlie PWS-associated hyperphagia has been slowed by the paucity of animal models with increased food intake or obesity. Mice with a microdeletion encompassing the Snord116 cluster of noncoding RNAs encoded within the Prader-Willi minimal deletion critical region have previously been reported to show growth retardation and hyperphagia. Here, consistent with previous reports, we observed growth retardation in Snord116+/–P mice with a congenital paternal Snord116 deletion. However, these mice neither displayed increased food intake nor had reduced hypothalamic expression of the proprotein convertase 1 gene PCSK1 or its upstream regulator NHLH2, which have recently been suggested to be key mediators of PWS pathogenesis. Specifically, we disrupted Snord116 expression in the mediobasal hypothalamus in Snord116fl mice via bilateral stereotaxic injections of a Cre-expressing adeno-associated virus (AAV). While the Cre-injected mice had no change in measured energy expenditure, they became hyperphagic between 9 and 10 weeks after injection, with a subset of animals developing marked obesity. In conclusion, we show that selective disruption of Snord116 expression in the mediobasal hypothalamus models the hyperphagia of PWS.
Authors
Joseph Polex-Wolf, Brian Y.H. Lam, Rachel Larder, John Tadross, Debra Rimmington, Fàtima Bosch, Verónica Jiménez Cenzano, Eduard Ayuso, Marcella K.L. Ma, Kara Rainbow, Anthony P. Coll, Stephen O’Rahilly, Giles S.H. Yeo
Copyright © 2018 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)