DOCSLIB.ORG

Deciphering the Genetics of Hereditary Non-Syndromic Colorectal Cancer

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Deciphering the Genetics of Hereditary Non-Syndromic Colorectal Cancer European Journal of Human Genetics (2008) 16, 1477–1486 & 2008 Macmillan Publishers Limited All rights reserved 1018-4813/08 $32.00 www.nature.com/ejhg ARTICLE Deciphering the genetics of hereditary non-syndromic colorectal cancer Eli Papaemmanuil1,13, Luis Carvajal-Carmona2,13, Gabrielle S Sellick1,13, Zoe Kemp2,13, Emily Webb1, Sarah Spain2, Kate Sullivan1, Ella Barclay2, Steven Lubbe1, Emma Jaeger2, Jayaram Vijayakrishnan1, Peter Broderick1, Maggie Gorman2,3, Lynn Martin4,5, Anneke Lucassen6, D Timothy Bishop7, D Gareth Evans4, Eamonn R Maher5, Verena Steinke8, Nils Rahner8, Hans K Schackert9, Timm O Goecke10, Elke Holinski-Feder10, Peter Propping8, Tom Van Wezel11, Juul Wijnen11, Jean-Baptiste Cazier12, Huw Thomas3, Richard S Houlston*,1,14 and Ian Tomlinson*,2,14, The CORGI Consortium 1Section of Cancer Genetics, Institute of Cancer Research, Sutton, Surrey, UK; 2Molecular and Population Genetics Laboratory, Cancer Research UK, London, UK; 3Colorectal Cancer Unit, Cancer Research UK, St Mark’s Hospital, Harrow, UK; 4Medical Genetics, St Mary’s Hospital, Manchester, UK; 5Department of Medical and Molecular Genetics, University of Birmingham School of Medicine and West Midlands Genetics Service, Birmingham Women’s Hospital, Edgbaston, Birmingham, UK; 6Wessex Clinical Genetics Service, Princess Anne Hospital, Southampton, UK; 7Genetic Epidemiology Laboratory, Cancer Research UK, St James’s University Hospital, Leeds, UK; 8Institute of Human Genetics, University of Bonn, Bonn, Germany; 9Department of Surgical Research, Technische Universita¨t Dresden, Dresden, Germany; 10Institute of Human Genetics, University of Du¨sseldorf, Germany; 11Department of Pathology, Leiden Medical Centre, Leiden, The Netherlands; 12Bioinformatics and Biostatistics, London Research Institute, Cancer Research UK, London, UK Previously we have localized to chromosome 3q21–q24, a predisposition locus for colorectal cancer (CRC), through a genome-wide linkage screen (GWLS) of 69 families without familial adenomatous polyposis or hereditary non-polyposis CRC. To further investigate Mendelian susceptibility to CRC, we extended our screen to include a further GWLS of an additional 34 CRC families. We also searched for a disease gene at 3q21–q24 by linkage disequilibrium mapping in 620 familial CRC cases and 960 controls by genotyping 1676 tagging SNPs and sequencing 30 candidate genes from the region. Linkage analysis was conducted using the Affymetrix 10K SNP array. Data from both GWLSs were pooled and multipoint linkage statistics computed. The maximum NPL score (3.01; P ¼ 0.0013) across all families was at 3q22, maximal evidence for linkage coming from families segregating rectal CRC. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under a dominant model (HLOD ¼ 2.79; P ¼ 0.00034), with an estimated 43% of families linked. In the case–control analysis, the strongest association was obtained at rs698675 (P ¼ 0.0029), but this was not significant after adjusting for multiple testing. Analysis of candidate gene mapping to the region of maximal linkage on 3q22 failed to identify a causal mutation. There was no evidence for linkage to the previously reported 9q CRC locus (NPL ¼ 0.95, P ¼ 0.23; HLODdominant ¼ 0.40, HLODrecessive ¼ 0.20). Our findings are consistent with the hypothesis that variation *Correspondence: Dr Professor R Houlston, Section of Cancer Genetics, Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5 NG, UK. Tel: þ 44 208 722 4175; Fax: þ 44 208 722 4359; E-mail: [email protected] or Professor I Tomlinson Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK. Tel: þ 44 207 269 2884; Fax: þ 44 207 269 3093; E-mail: [email protected] 13These authors contributed equally to this work 14These authors have joint PI status Received 26 March 2008; revised 29 May 2008; accepted 29 May 2008; published online 16 July 2008 Deciphering the genetics of HNSCRC E Papaemmanuil et al 1478 at 3q22 contributes to the risk of CRC, but this is unlikely to be mediated through a restricted set of alleles. European Journal of Human Genetics (2008) 16, 1477–1486; doi:10.1038/ejhg.2008.129; published online 16 July 2008 Keywords: colorectal cancer; linkage; chromosome 3q22 Introduction pedigrees as phase 2. As before, familial CRC cases were Family history is well established to be one of the strongest ascertained through the COloRectal tumour Gene Identi- risk factors for the development of colorectal cancer (CRC).1 fication (CORGI) Study Consortium.8 Briefly, pedigrees Although germline mutations in APC,themismatchrepair informative for linkage had at least three affected indivi- (MMR) genes, MUTYH/MYH, SMAD4, ALK3 and STK11/LKB1 duals (confirmed by pathology reports). Individuals were are associated with Mendelian susceptibility to CRC, classed as affected if they had CRC at age r75 years or collectively, these do not account for the observed familial ‘significant’ adenomas (three or more synchronous or aggregation of the disease.2,3 Direct evidence for unchar- metachronous, and/or villous morphology, and/or severe acterized high/moderate-penetrance CRC genes is provided dysplasia, and/or diameter 41 cm and/or presentation at by CRC families showing evidence against linkage to known r45 years of age). No patient had clinical features of loci and by kindreds that fulfil the clinical (Amsterdam) Peutz-Jeghers syndrome (PJS), juvenile polyposis (JPS), criteria for a diagnosis of hereditary non-polyposis colo- hereditary mixed polyposis (HMPS) or inflammatory bowel rectal cancer (HNPCC) but whose CRCs do not display MMR disease. Germline MMR gene mutations were excluded by deficiency.4 Familial CRC risks in relatives of colorectal microsatellite instability (MSI) testing of BAT25 and BAT26 adenoma (CRA) cases parallel those seen in relatives of CRC in two CRCs from each family; kindreds in which both cases.1 It is therefore likely that a significant proportion of cancers were unstable were excluded. Where one MSI þ inherited predisposition to CRC is mediated through tumour was found or if the only available CRC was susceptibility to CRAs. These observations have provided a unstable, direct mutation screening of all coding regions strong rationale for searching of novel predisposition genes of MSH2 and MLH1 was undertaken. In addition, all through genome-wide linkage screens (GWLSs) of heredi- families with an attenuated familial adenomatous poly- tary non-FAP/HNPCC CRC families (hereditary non- posis (FAP)-like phenotype (45 adenomas) were tested for syndromic colorectal cancer – HNSCRC). mutations in APC, and the entire data set was tested for the Using a high-density SNP array, we have previously two common MYH variants (Y165C and G382D) using performed a GWLS of 69 HNSCRC families in which fluorescence-SSCP analysis. involvement of known predisposition genes had been The case–control study was based on 620 CRC cases (280 excluded and a novel CRC susceptibility locus at 3q21– male, 340 female) with at least one affected first-degree q24 was identified.5 Other workers have proposed relative ascertained through CORGI. As with the linkage additional loci for CRC susceptibility genes on the basis families, none had clinical features of FAP, PJS, JPS, HMPS of linkage, most notably on 9q22.2–31.2.6,7 or inflammatory bowel disease. In addition, none had a To further examine the impact of unknown moderate/ family history compatible with a diagnosis of HNPCC. high-penetrance genes on CRC risk, we conducted a Samples collected from unaffected spouses/partners of further GWLS on an additional 34 HNSCRC families using cases served as a source of 965 controls (439 male, 526 the same analytical platform employed in our first analysis. female). We then pooled data from scans. In an effort to predict the Genomic DNA was extracted from whole blood using the most likely location of the putative CRC gene on 3q21– Chemagic Magnetic Separation Module 1t. All samples q24, we undertook linkage disequilibrium (LD) mapping at were obtained with informed consent and local ethical 1676 tagging SNPs in 620 HNSCRC cases and 961 controls. review board approval in accordance with the tenets of the In addition, we have sought to identify disease-causing Declaration of Helsinki. variants by direct mutational analysis of candidate genes localizing to the region of maximal linkage on 3q22. Genome-wide linkage screen The current analysis was based on 34 families segregating CRC and/or CRA in which involvement of known loci had Materials and methods been excluded. A GWLS of families was undertaken using Ascertainment and collection of families and cases the GeneChips Mapping 10K Xba 142 Array (Affymetrix For clarity, we refer to our previously reported GWLS of 69 Inc., Santa Clara, CA). SNP genotypes were obtained by pedigrees as phase 18 and the current analysis of 34 following the Affymetrix protocol. Briefly, for each sample, European Journal of Human Genetics Deciphering the genetics of HNSCRC E Papaemmanuil et al 1479 250 ng of genomic DNA was digested with the restriction and 0.638 with corresponding phenocopy rates of 0.00004, endonuclease XbaI for 2.5 h, mixed with Xba adapters and 0.0003, 0.0026 and 0.023. ligated using T4 DNA ligase for 2.5 h. Ligated DNA was A second analysis was undertaken in which individuals added to four separate PCRs, cycled, pooled and purified were classed as affected if they had either CRC or to
Load more

Recommended publications
  • Genome-Wide Analysis of 5-Hmc in the Peripheral Blood of Systemic Lupus Erythematosus Patients Using an Hmedip-Chip
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 35: 1467-1479, 2015 Genome-wide analysis of 5-hmC in the peripheral blood of systemic lupus erythematosus patients using an hMeDIP-chip WEIGUO SUI1*, QIUPEI TAN1*, MING YANG1, QIANG YAN1, HUA LIN1, MINGLIN OU1, WEN XUE1, JIEJING CHEN1, TONGXIANG ZOU1, HUANYUN JING1, LI GUO1, CUIHUI CAO1, YUFENG SUN1, ZHENZHEN CUI1 and YONG DAI2 1Guangxi Key Laboratory of Metabolic Diseases Research, Central Laboratory of Guilin 181st Hospital, Guilin, Guangxi 541002; 2Clinical Medical Research Center, the Second Clinical Medical College of Jinan University (Shenzhen People's Hospital), Shenzhen, Guangdong 518020, P.R. China Received July 9, 2014; Accepted February 27, 2015 DOI: 10.3892/ijmm.2015.2149 Abstract. Systemic lupus erythematosus (SLE) is a chronic, Introduction potentially fatal systemic autoimmune disease characterized by the production of autoantibodies against a wide range Systemic lupus erythematosus (SLE) is a typical systemic auto- of self-antigens. To investigate the role of the 5-hmC DNA immune disease, involving diffuse connective tissues (1) and modification with regard to the onset of SLE, we compared is characterized by immune inflammation. SLE has a complex the levels 5-hmC between SLE patients and normal controls. pathogenesis (2), involving genetic, immunologic and envi- Whole blood was obtained from patients, and genomic DNA ronmental factors. Thus, it may result in damage to multiple was extracted. Using the hMeDIP-chip analysis and valida- tissues and organs, especially the kidneys (3). SLE arises from tion by quantitative RT-PCR (RT-qPCR), we identified the a combination of heritable and environmental influences. differentially hydroxymethylated regions that are associated Epigenetics, the study of changes in gene expression with SLE.
    [Show full text]
  • Title a New Centrosomal Protein Regulates Neurogenesis By
    Title A new centrosomal protein regulates neurogenesis by microtubule organization Authors: Germán Camargo Ortega1-3†, Sven Falk1,2†, Pia A. Johansson1,2†, Elise Peyre4, Sanjeeb Kumar Sahu5, Loïc Broic4, Camino De Juan Romero6, Kalina Draganova1,2, Stanislav Vinopal7, Kaviya Chinnappa1‡, Anna Gavranovic1, Tugay Karakaya1, Juliane Merl-Pham8, Arie Geerlof9, Regina Feederle10,11, Wei Shao12,13, Song-Hai Shi12,13, Stefanie M. Hauck8, Frank Bradke7, Victor Borrell6, Vijay K. Tiwari§, Wieland B. Huttner14, Michaela Wilsch- Bräuninger14, Laurent Nguyen4 and Magdalena Götz1,2,11* Affiliations: 1. Institute of Stem Cell Research, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 2. Physiological Genomics, Biomedical Center, Ludwig-Maximilian University Munich, Germany. 3. Graduate School of Systemic Neurosciences, Biocenter, Ludwig-Maximilian University Munich, Germany. 4. GIGA-Neurosciences, Molecular regulation of neurogenesis, University of Liège, Belgium 5. Institute of Molecular Biology (IMB), Mainz, Germany. 6. Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas and Universidad Miguel Hernández, Sant Joan d’Alacant, Spain. 7. Laboratory for Axon Growth and Regeneration, German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. 8. Research Unit Protein Science, Helmholtz Centre Munich, German Research Center for Environmental Health, Munich, Germany. 9. Protein Expression and Purification Facility, Institute of Structural Biology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 10. Institute for Diabetes and Obesity, Monoclonal Antibody Core Facility, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany. 11. SYNERGY, Excellence Cluster of Systems Neurology, Biomedical Center, Ludwig- Maximilian University Munich, Germany. 12. Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, USA 13.
    [Show full text]
  • High-Throughput, Pooled Sequencing Identifies Mutations in NUBPL And
    ARTICLES High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency Sarah E Calvo1–3,10, Elena J Tucker4,5,10, Alison G Compton4,10, Denise M Kirby4, Gabriel Crawford3, Noel P Burtt3, Manuel Rivas1,3, Candace Guiducci3, Damien L Bruno4, Olga A Goldberger1,2, Michelle C Redman3, Esko Wiltshire6,7, Callum J Wilson8, David Altshuler1,3,9, Stacey B Gabriel3, Mark J Daly1,3, David R Thorburn4,5 & Vamsi K Mootha1–3 Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases. Complex I of the mitochondrial respiratory chain is a large ~1-MDa ­assembly factors are probably required, as suggested by the 20 factors macromolecular machine composed of 45 protein subunits encoded necessary for assembly of the smaller complex IV9 and by cohort by both the nuclear and mitochondrial (mtDNA) genomes.
    [Show full text]
  • Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins
    International Journal of Molecular Sciences Article Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins M. Quadir Siddiqui 1,† , Maulik D. Badmalia 1,† and Trushar R. Patel 1,2,3,* 1 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; [email protected] (M.Q.S.); [email protected] (M.D.B.) 2 Department of Microbiology, Immunology and Infectious Disease, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive, Calgary, AB T2N 4N1, Canada 3 Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB T6G 2E1, Canada * Correspondence: [email protected] † These authors contributed equally to the work. Abstract: Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and func- tions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share Citation: Siddiqui, M.Q.; Badmalia, similarities with transcriptional regulators and have positively charged electrostatic patches, which M.D.; Patel, T.R.
    [Show full text]
  • The Basal Bodies of Chlamydomonas Reinhardtii Susan K
    Dutcher and O’Toole Cilia (2016) 5:18 DOI 10.1186/s13630-016-0039-z Cilia REVIEW Open Access The basal bodies of Chlamydomonas reinhardtii Susan K. Dutcher1* and Eileen T. O’Toole2 Abstract The unicellular green alga, Chlamydomonas reinhardtii, is a biflagellated cell that can swim or glide. C. reinhardtii cells are amenable to genetic, biochemical, proteomic, and microscopic analysis of its basal bodies. The basal bodies contain triplet microtubules and a well-ordered transition zone. Both the mother and daughter basal bodies assemble flagella. Many of the proteins found in other basal body-containing organisms are present in the Chlamydomonas genome, and mutants in these genes affect the assembly of basal bodies. Electron microscopic analysis shows that basal body duplication is site-specific and this may be important for the proper duplication and spatial organization of these organelles. Chlamydomonas is an excellent model for the study of basal bodies as well as the transition zone. Keywords: Site-specific basal body duplication, Cartwheel, Transition zone, Centrin fibers Phylogeny and conservation of proteins Centrin, SPD2/CEP192, Asterless/CEP152; CEP70, The green lineage or Viridiplantae consists of the green delta-tubulin, and epsilon-tubulin. Chlamydomonas has algae, which include Chlamydomonas, the angiosperms homologs of all of these based on sequence conservation (the land plants), and the gymnosperms (conifers, cycads, except PLK4, CEP152, and CEP192. Several lines of evi- ginkgos). They are grouped together because they have dence suggests that CEP152, CEP192, and PLK4 interact chlorophyll a and b and lack phycobiliproteins. The green [20, 52] and their concomitant absence in several organ- algae together with the cycads and ginkgos have basal isms suggests that other mechanisms exist that allow for bodies and cilia, while the angiosperms and conifers have control of duplication [4].
    [Show full text]
  • Differential Expression Profile Prioritization of Positional Candidate Glaucoma Genes the GLC1C Locus
    LABORATORY SCIENCES Differential Expression Profile Prioritization of Positional Candidate Glaucoma Genes The GLC1C Locus Frank W. Rozsa, PhD; Kathleen M. Scott, BS; Hemant Pawar, PhD; John R. Samples, MD; Mary K. Wirtz, PhD; Julia E. Richards, PhD Objectives: To develop and apply a model for priori- est because of moderate expression and changes in tization of candidate glaucoma genes. expression. Transcription factor ZBTB38 emerges as an interesting candidate gene because of the overall expres- Methods: This Affymetrix GeneChip (Affymetrix, Santa sion level, differential expression, and function. Clara, Calif) study of gene expression in primary cul- ture human trabecular meshwork cells uses a positional Conclusions: Only1geneintheGLC1C interval fits our differential expression profile model for prioritization of model for differential expression under multiple glau- candidate genes within the GLC1C genetic inclusion in- coma risk conditions. The use of multiple prioritization terval. models resulted in filtering 7 candidate genes of higher interest out of the 41 known genes in the region. Results: Sixteen genes were expressed under all condi- tions within the GLC1C interval. TMEM22 was the only Clinical Relevance: This study identified a small sub- gene within the interval with differential expression in set of genes that are most likely to harbor mutations that the same direction under both conditions tested. Two cause glaucoma linked to GLC1C. genes, ATP1B3 and COPB2, are of interest in the con- text of a protein-misfolding model for candidate selec- tion. SLC25A36, PCCB, and FNDC6 are of lesser inter- Arch Ophthalmol. 2007;125:117-127 IGH PREVALENCE AND PO- identification of additional GLC1C fami- tential for severe out- lies7,18-20 who provide optimal samples for come combine to make screening candidate genes for muta- adult-onset primary tions.7,18,20 The existence of 2 distinct open-angle glaucoma GLC1C haplotypes suggests that muta- (POAG) a significant public health prob- tions will not be limited to rare descen- H1 lem.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Supplemental Information Proximity Interactions Among Centrosome
    Current Biology, Volume 24 Supplemental Information Proximity Interactions among Centrosome Components Identify Regulators of Centriole Duplication Elif Nur Firat-Karalar, Navin Rauniyar, John R. Yates III, and Tim Stearns Figure S1 A Myc Streptavidin -tubulin Merge Myc Streptavidin -tubulin Merge BirA*-PLK4 BirA*-CEP63 BirA*- CEP192 BirA*- CEP152 - BirA*-CCDC67 BirA* CEP152 CPAP BirA*- B C Streptavidin PCM1 Merge Myc-BirA* -CEP63 PCM1 -tubulin Merge BirA*- CEP63 DMSO - BirA* CEP63 nocodazole BirA*- CCDC67 Figure S2 A GFP – + – + GFP-CEP152 + – + – Myc-CDK5RAP2 + + + + (225 kDa) Myc-CDK5RAP2 (216 kDa) GFP-CEP152 (27 kDa) GFP Input (5%) IP: GFP B GFP-CEP152 truncation proteins Inputs (5%) IP: GFP kDa 1-7481-10441-1290218-1654749-16541045-16541-7481-10441-1290218-1654749-16541045-1654 250- Myc-CDK5RAP2 150- 150- 100- 75- GFP-CEP152 Figure S3 A B CEP63 – – + – – + GFP CCDC14 KIAA0753 Centrosome + – – + – – GFP-CCDC14 CEP152 binding binding binding targeting – + – – + – GFP-KIAA0753 GFP-KIAA0753 (140 kDa) 1-496 N M C 150- 100- GFP-CCDC14 (115 kDa) 1-424 N M – 136-496 M C – 50- CEP63 (63 kDa) 1-135 N – 37- GFP (27 kDa) 136-424 M – kDa 425-496 C – – Inputs (2%) IP: GFP C GFP-CEP63 truncation proteins D GFP-CEP63 truncation proteins Inputs (5%) IP: GFP Inputs (5%) IP: GFP kDa kDa 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl 1-135136-424425-4961-424136-496FL Ctl Myc- 150- Myc- 100- CCDC14 KIAA0753 100- 100- 75- 75- GFP- GFP- 50- CEP63 50- CEP63 37- 37- Figure S4 A siCtl
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • Noelia Díaz Blanco
    Effects of environmental factors on the gonadal transcriptome of European sea bass (Dicentrarchus labrax), juvenile growth and sex ratios Noelia Díaz Blanco Ph.D. thesis 2014 Submitted in partial fulfillment of the requirements for the Ph.D. degree from the Universitat Pompeu Fabra (UPF). This work has been carried out at the Group of Biology of Reproduction (GBR), at the Department of Renewable Marine Resources of the Institute of Marine Sciences (ICM-CSIC). Thesis supervisor: Dr. Francesc Piferrer Professor d’Investigació Institut de Ciències del Mar (ICM-CSIC) i ii A mis padres A Xavi iii iv Acknowledgements This thesis has been made possible by the support of many people who in one way or another, many times unknowingly, gave me the strength to overcome this "long and winding road". First of all, I would like to thank my supervisor, Dr. Francesc Piferrer, for his patience, guidance and wise advice throughout all this Ph.D. experience. But above all, for the trust he placed on me almost seven years ago when he offered me the opportunity to be part of his team. Thanks also for teaching me how to question always everything, for sharing with me your enthusiasm for science and for giving me the opportunity of learning from you by participating in many projects, collaborations and scientific meetings. I am also thankful to my colleagues (former and present Group of Biology of Reproduction members) for your support and encouragement throughout this journey. To the “exGBRs”, thanks for helping me with my first steps into this world. Working as an undergrad with you Dr.
    [Show full text]
  • Feeling the Force: Role of Amotl2 in Normal Development and Cancer
    Department of Oncology and Pathology Karolinska Institute, Stockholm, Sweden FEELING THE FORCE: ROLE OF AMOTL2 IN NORMAL DEVELOPMENT AND CANCER Aravindh Subramani Stockholm 2019 All previously published papers were reproduced with permission from the publisher. Front cover was modified from ©Renaud Chabrier and illustrated by Yu-Hsuan Hsu Published by Karolinska Institute. Printed by: US-AB Stockholm © Aravindh Subramani, 2019 ISBN 978-91-7831-426-3 Feeling the force: Role of AmotL2 in normal development and cancer. THESIS FOR DOCTORAL DEGREE (Ph.D.) J3:04, Torsten N Wiesel, U410033310, Bioclinicum, Karolinska University Hospital, Solna, Stockholm Friday, April 12th, 2019 at 13:00 By Aravindh Subramani Principal Supervisor: Opponent: Professor. Lars Holmgren Professor. Marius Sudol Karolinska Institute National University of Singapore Department of Oncology-Pathology Mechanobiology Institute (MBI) Co-supervisor(s): Examination Board: Docent. Jonas Fuxe Docent. Kaisa Lehti Karolinska Institute Karolinska institute Department of Microbiology, Tumor and Cell Department of Microbiology and Tumor Biology Biology (MTC) center (MTC) Associate Professor. Johan Hartman Docent. Ingvar Ferby Karolinska Institute Uppsala University Department of Oncology-Pathology Department of Medical Biochemistry and Microbiology Docent. Mimmi Shoshan Karolinska Institute Department of Oncology-Pathology To my mom and dad “Real knowledge is to know the extent of one's ignorance.” (Confucius) ABSTRACT Cells that fabricate the body, dwell in a very heterogeneous environment. Self-organization of individual cells into complex tissues and organs at the time of growth and revival is brought about by the combinatory action of biomechanical and biochemical signaling processes. Tissue generation and functional organogenesis, requires distinct cell types to unite together and associate with their corresponding microenvironment in a spatio-temporal manner.
    [Show full text]
  • Is Cep70, a Centrosomal Protein with New Roles in Breast Cancer Dissemination and Metastasis, a Facilitator of Epithelial-Mesenchymal Transition?
    Is Cep70, a centrosomal protein with new roles in breast cancer dissemination and metastasis, a facilitator of epithelial-mesenchymal transition? Pedro A. Lazo 1,2 1 Experimental Therapeutics and Translational Oncology Program, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Salamanca, Spain 2 Instituto de Investigación Biomédica de Salamanca (IBSAL), Hospital Universitario de Salamanca, Salamanca, Spain Running title: Cep70 and EMT Disclosures: None declared. Contact address: [email protected] 1 Introduction Microtubules are driving mechanisms of chromosomes, intracellular organelle movement, and cell shape and motility. Microtubules are organized on centrosomes, which are assembled in microtubule-organizing centers (MTOC). In mitosis there is no nuclear envelope and centromeres associated to microtubules are mainly involved in chromosome redistribution into daughter cells. In differentiated cells the microtubule- organizing centers are dispersed in the cytoplasm (non centrosomal (ncMTOC)) and interact with the minus end of microtubules through γ-tubulin. 1 However, it is not known if the microtubule contribution to tumor biology is only by facilitating tumor aneuploidy. During tumor dissemination, a process not linked to cell division, important changes take place in cell shape and motility. Microtubules are very dynamic because of their inherent structural instability, and this plasticity facilitates their reorganization during the epithelial-mesenchymal
    [Show full text]