DOCSLIB.ORG

Congenital Secretory Diarrhoea Caused by Activating Germline

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Congenital Secretory Diarrhoea Caused by Activating Germline Downloaded from http://gut.bmj.com/ on February 22, 2018 - Published by group.bmj.com Colon ORIGINAL ARTICLE Congenital secretory diarrhoea caused by activating germline mutations in GUCY2C Thomas Müller,1 Insha Rasool,2 Peter Heinz-Erian,1 Eva Mildenberger,3 Christian Hülstrunk,3 Andreas Müller,4 Laurent Michaud,5 Bart G P Koot,6 Antje Ballauff,7 Julia Vodopiutz,8 Stefan Rosipal,9 Britt-Sabina Petersen,10 Andre Franke,10 Irene Fuchs,1 Heiko Witt,11 Heinz Zoller,12 Andreas R Janecke,1 Sandhya S Visweswariah3 For numbered affiliations see ABSTRACT end of article. Objective Congenital sodium diarrhoea (CSD) refers to Significance of this study Correspondence to a form of secretory diarrhoea with intrauterine onset and Ass-Prof Dr Andreas Janecke, high faecal losses of sodium without congenital Department of Pedatrics I, malformations. The molecular basis for CSD remains What is already known on this subject? Medical University of unknown. We clinically characterised a cohort of infants ▸ Congenital diarrhoeal diseases represent a group Innsbruck, 6020 Innsbruck, with CSD and set out to identify disease-causing Austria; of rare inherited enteropathies with a [email protected] mutations by genome-wide genetic testing. disease-onset early in life. There is a broad range Design We performed whole-exome sequencing and of different monogenic conditions, and a number Prof Sandhya S Visweswariah, chromosomal microarray analyses in 4 unrelated patients, of patients have not been classified so far. Department of Molecular followed by confirmatory Sanger sequencing of the likely ▸ A GUCY2C mutation, p.S840I, has been Reproduction, Development and Genetics, Indian Institute disease-causing mutations in patients and in their family describedin32affectedmembersofalarge of Science, Biological Sciences members, followed by functional studies. Norwegian family with autosomal dominantly Building, GA09, Bangalore Results We identified novel de novo missense inherited diarrhoea of infantile onset, frequently 560012, Karnataka, India; mutations in GUCY2C, the gene encoding receptor associated with IBS, bowel obstruction and IBD. [email protected] guanylate cyclase C (GC-C) in 4 patients with CSD. One GUCY2C encodes guanylate cyclase C (GC-C), TM, IR and PH-E contributed patient developed severe, early-onset IBD and chronic and ligand binding to GC-C results in production equally. ARJ and SSV arthritis at 4 years of age. GC-C is an intestinal brush of cyclic guanosine monophosphate (cGMP). The contributed equally. border membrane-bound guanylate cyclase, which mutation conferred a higher cGMP production functions as receptor for guanylin, uroguanylin and Received 22 February 2015 upon ligand-binding to GC-C. Revised 9 April 2015 Escherichia coli heat-stable enterotoxin. Mutations in GUCY2C were present in different intracellular domains What are the new findings? Accepted 10 April 2015 ▸ Published Online First of GC-C, and were activating mutations that enhanced We identify the molecular basis of a clinical 20 May 2015 intracellular cyclic guanosine monophosphate entity, congenital sodium diarrhoea (CSD) as four accumulation in a ligand-independent and ligand- distinct de novo mutations of GUCY2C occurring stimulated manner, following heterologous expression in in four patients. GUCY2C mutations identified in HEK293T cells. patients with CSD caused increases in basal and Conclusions Dominant gain-of-function GUCY2C ligand-stimulated GC-C activities. mutations lead to elevated intracellular cyclic guanosine ▸ We report that patients with GUCY2C monophosphate levels and could explain the chronic mutations presented with intrauterine onset of diarrhoea as a result of decreased intestinal sodium and diarrhoea requiring ileostomata within the 1st water absorption and increased chloride secretion. Thus, month of age. One patient with a p.L775P mutations in GUCY2C indicate a role for this receptor in mutation in GC-C developed IBD and arthritis the pathogenesis of sporadic CSD. at 4 years of age thereby implicating GUCY2C as an IBD gene. How might it impact on clinical practice in the foreseeable future? INTRODUCTION ▸ This study provides a rationale for including Congenital diarrhoeal disorders are rare and het- GUCY2C into a fast, targeted or panel-based erogeneous enteropathies, generally with severe routine testing of patients suspected with CSD clinical manifestations. These disorders can be or patients with diarrhoeal disease. caused by defects in absorption and transport of ▸ Studying patient-derived tissues or organoids nutrients and electrolytes, in enterocyte differenti- with GUCY2C mutations might contribute to ation and polarisation, in enteroendocrine cell dif- the understanding of cGMP-mediated ferentiation, and by modulations of the intestinal stimulation of chloride secretion by cystic immune response. Clinical and histological criteria fibrosis transmembrane conductance regulator To cite: Müller T, Rasool I, and, increasingly, mutation detection in known (CFTR) and inhibition of sodium absorption by Heinz-Erian P, et al. Gut disease genes are used to classify these disorders.1 – the sodium-hydrogen exchanger 3 (NHE3). 2016;65:1306 1313. Some types of congenital diarrhoeal disorders are 1306 Müller T, et al. Gut 2016;65:1306–1313. doi:10.1136/gutjnl-2015-309441 Downloaded from http://gut.bmj.com/ on February 22, 2018 - Published by group.bmj.com Colon amenable to dietary treatment, while others require chronic human genome (hg19) with Burrows-Wheeler transformation,8 nutritional support or small bowel transplantation. sorted, converted to BAM format and indexed with SAMtools.9 Congenital sodium diarrhoea (CSD) refers to a secretory diar- PCR duplicates were removed with PICARD (http://picard. rhoea presenting with maternal polyhydramnios, prominent sourceforge.net) and SNPs and small indels were identified with abdominal distension due to dilated fluid-filled loops of the the SAMtools mpileup software. All variants were submitted to intestine, which may lead to intestinal obstruction at birth, and SeattleSeq (http://snp.gs.washington.edu/SeattleSeqAnnotation/) high faecal losses of sodium and chloride. CSD associated with for annotation, categorisation into synonymous and non- choanal atresia, corneal erosions and tufting enteropathy (syn- synonymous SNPs or indels, and for filtering using the data dromic CSD) can be distinguished from the originally described from dbSNP138, the 1000 Genomes Project, the National classic form of CSD.2 Syndromic CSD is caused by SPINT2 Heart, Lung, and Blood Institute Exome Sequencing Project. loss-of-function mutations,3 whereas the molecular basis for The exome aggregation consortium server (exac.broadinstitute. classic CSD remains unknown.34 org) was subsequently searched for known GUCY2C variants. Known genetic entities of CCDs are most often autosomal Single exons of GUCY2C were sequenced in the index cases and recessively inherited; however, a single heterozygous mutation in available family members to test for the presence of mutations in GUCY2C, encoding guanylate cyclase C (GC-C) has been detected in index patients by whole-exome sequencing. PCR and reported as the cause of a dominantly inherited form of secre- sequencing primer sequences were based on the ENSEMBL refer- tory diarrhoea (Familial Diarrhoea Syndrome) in 32 individuals ence entries for mRNA (NCBI reference sequence for mRNA of a Norwegian family.5 The mutation in the Norwegian study NM_004963.3), Ensembl reference entry (ENST00000261170) was a hyperactivating mutation present in the catalytic domain and for genomic DNA (ENSG00000070019); sequences are avail- of GC-C. This mutation resulted in increased sensitivity of able from the authors upon request. Codon numbering was based GC-C to its endogenous ligands guanylin and uroguanylin and on the published online amino acid and mRNA sequences of these to the heat-stable enterotoxin (ST) of Escherichia coli, giving genes (http://www.ncbi.nlm.nih.gov/) using the first methionine as rise to increased levels of cyclic guanosine monophosphate an initiation codon. GoTaq polymerase and buffer (Promega, (cGMP) in the epithelial cell, resulting in enhanced fluid and Mannheim, Germany) were used to PCR-amplify 20–50 ng of ion secretion, and diarrhoea. In contrast, inactivating mutations genomic DNA in 35 cycles of 20 s at 95°C, 20 s at 60°C and 30 s of GUCY2C in three Bedouin families resulted in meconium at 72°C in addition to a 7 min final extension, applied to all sets of ileus67as a consequence of diminished fluid and ion secretion primers (table 2). The amplicons were cleaned using ExoSap-IT that is normally maintained by the action of guanylin and uro- (USB, Vienna, Austria), and subsequently sequenced using an M13 guanylin on GC-C. universal primer on an ABI 3730s automated sequencer, with In the Norwegian kindred, the predominant phenotype of BigDye terminator mix (Applera, Vienna, Austria). Sequence chro- affected family members mimicked IBS, resulting in three to matographs were analysed using the Sequence Pilot computer four loose stools daily, while 8 out of the 32 affected family program ( JSI medical systems, Kippenheim, Germany). members already presented in infancy with severe dehydration. Small-bowel obstruction and/or IBD were/was diagnosed in Site-directed mutagenesis and characterisation of mutant 20–25% of these patients. As opposed to this relatively mild GC-C proteins type of diarrhoea, the four patients with CSD described here We generated mutant complementary DNA
Load more

Recommended publications
  • Meconium Ileus Caused by Mutations in GUCY2C, Encoding the CFTR-Activating Guanylate Cyclase 2C
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector REPORT Meconium Ileus Caused by Mutations in GUCY2C, Encoding the CFTR-Activating Guanylate Cyclase 2C Hila Romi,1,6 Idan Cohen,1,6 Daniella Landau,2 Suliman Alkrinawi,2 Baruch Yerushalmi,2 Reli Hershkovitz,3 Nitza Newman-Heiman,2 Garry R. Cutting,4 Rivka Ofir,1 Sara Sivan,1 and Ohad S. Birk1,5,* Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modi- fier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage. Meconium ileus (MI), intestinal obstruction by inspissated homozygosity on chromosome 12p13 (spanning 9.5 Mb meconium in the distal ileum and cecum, develops in between markers D12S366 and D12S310) that was utero and presents shortly after birth as failure to pass common to all affected individuals.
    [Show full text]
  • Supp Table 1.Pdf
    Upregulated genes in Hdac8 null cranial neural crest cells fold change Gene Symbol Gene Title 134.39 Stmn4 stathmin-like 4 46.05 Lhx1 LIM homeobox protein 1 31.45 Lect2 leukocyte cell-derived chemotaxin 2 31.09 Zfp108 zinc finger protein 108 27.74 0710007G10Rik RIKEN cDNA 0710007G10 gene 26.31 1700019O17Rik RIKEN cDNA 1700019O17 gene 25.72 Cyb561 Cytochrome b-561 25.35 Tsc22d1 TSC22 domain family, member 1 25.27 4921513I08Rik RIKEN cDNA 4921513I08 gene 24.58 Ofa oncofetal antigen 24.47 B230112I24Rik RIKEN cDNA B230112I24 gene 23.86 Uty ubiquitously transcribed tetratricopeptide repeat gene, Y chromosome 22.84 D8Ertd268e DNA segment, Chr 8, ERATO Doi 268, expressed 19.78 Dag1 Dystroglycan 1 19.74 Pkn1 protein kinase N1 18.64 Cts8 cathepsin 8 18.23 1500012D20Rik RIKEN cDNA 1500012D20 gene 18.09 Slc43a2 solute carrier family 43, member 2 17.17 Pcm1 Pericentriolar material 1 17.17 Prg2 proteoglycan 2, bone marrow 17.11 LOC671579 hypothetical protein LOC671579 17.11 Slco1a5 solute carrier organic anion transporter family, member 1a5 17.02 Fbxl7 F-box and leucine-rich repeat protein 7 17.02 Kcns2 K+ voltage-gated channel, subfamily S, 2 16.93 AW493845 Expressed sequence AW493845 16.12 1600014K23Rik RIKEN cDNA 1600014K23 gene 15.71 Cst8 cystatin 8 (cystatin-related epididymal spermatogenic) 15.68 4922502D21Rik RIKEN cDNA 4922502D21 gene 15.32 2810011L19Rik RIKEN cDNA 2810011L19 gene 15.08 Btbd9 BTB (POZ) domain containing 9 14.77 Hoxa11os homeo box A11, opposite strand transcript 14.74 Obp1a odorant binding protein Ia 14.72 ORF28 open reading
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown Et Al
    US 20030082511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0082511 A1 Brown et al. (43) Pub. Date: May 1, 2003 (54) IDENTIFICATION OF MODULATORY Publication Classification MOLECULES USING INDUCIBLE PROMOTERS (51) Int. Cl." ............................... C12O 1/00; C12O 1/68 (52) U.S. Cl. ..................................................... 435/4; 435/6 (76) Inventors: Steven J. Brown, San Diego, CA (US); Damien J. Dunnington, San Diego, CA (US); Imran Clark, San Diego, CA (57) ABSTRACT (US) Correspondence Address: Methods for identifying an ion channel modulator, a target David B. Waller & Associates membrane receptor modulator molecule, and other modula 5677 Oberlin Drive tory molecules are disclosed, as well as cells and vectors for Suit 214 use in those methods. A polynucleotide encoding target is San Diego, CA 92121 (US) provided in a cell under control of an inducible promoter, and candidate modulatory molecules are contacted with the (21) Appl. No.: 09/965,201 cell after induction of the promoter to ascertain whether a change in a measurable physiological parameter occurs as a (22) Filed: Sep. 25, 2001 result of the candidate modulatory molecule. Patent Application Publication May 1, 2003 Sheet 1 of 8 US 2003/0082511 A1 KCNC1 cDNA F.G. 1 Patent Application Publication May 1, 2003 Sheet 2 of 8 US 2003/0082511 A1 49 - -9 G C EH H EH N t R M h so as se W M M MP N FIG.2 Patent Application Publication May 1, 2003 Sheet 3 of 8 US 2003/0082511 A1 FG. 3 Patent Application Publication May 1, 2003 Sheet 4 of 8 US 2003/0082511 A1 KCNC1 ITREXCHO KC 150 mM KC 2000000 so 100 mM induced Uninduced Steady state O 100 200 300 400 500 600 700 Time (seconds) FIG.
    [Show full text]
  • Supplementary Table 2
    Supplementary Table 2. Differentially Expressed Genes following Sham treatment relative to Untreated Controls Fold Change Accession Name Symbol 3 h 12 h NM_013121 CD28 antigen Cd28 12.82 BG665360 FMS-like tyrosine kinase 1 Flt1 9.63 NM_012701 Adrenergic receptor, beta 1 Adrb1 8.24 0.46 U20796 Nuclear receptor subfamily 1, group D, member 2 Nr1d2 7.22 NM_017116 Calpain 2 Capn2 6.41 BE097282 Guanine nucleotide binding protein, alpha 12 Gna12 6.21 NM_053328 Basic helix-loop-helix domain containing, class B2 Bhlhb2 5.79 NM_053831 Guanylate cyclase 2f Gucy2f 5.71 AW251703 Tumor necrosis factor receptor superfamily, member 12a Tnfrsf12a 5.57 NM_021691 Twist homolog 2 (Drosophila) Twist2 5.42 NM_133550 Fc receptor, IgE, low affinity II, alpha polypeptide Fcer2a 4.93 NM_031120 Signal sequence receptor, gamma Ssr3 4.84 NM_053544 Secreted frizzled-related protein 4 Sfrp4 4.73 NM_053910 Pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 4.69 BE113233 Suppressor of cytokine signaling 2 Socs2 4.68 NM_053949 Potassium voltage-gated channel, subfamily H (eag- Kcnh2 4.60 related), member 2 NM_017305 Glutamate cysteine ligase, modifier subunit Gclm 4.59 NM_017309 Protein phospatase 3, regulatory subunit B, alpha Ppp3r1 4.54 isoform,type 1 NM_012765 5-hydroxytryptamine (serotonin) receptor 2C Htr2c 4.46 NM_017218 V-erb-b2 erythroblastic leukemia viral oncogene homolog Erbb3 4.42 3 (avian) AW918369 Zinc finger protein 191 Zfp191 4.38 NM_031034 Guanine nucleotide binding protein, alpha 12 Gna12 4.38 NM_017020 Interleukin 6 receptor Il6r 4.37 AJ002942
    [Show full text]
  • Strand Breaks for P53 Exon 6 and 8 Among Different Time Course of Folate Depletion Or Repletion in the Rectosigmoid Mucosa
    SUPPLEMENTAL FIGURE COLON p53 EXONIC STRAND BREAKS DURING FOLATE DEPLETION-REPLETION INTERVENTION Supplemental Figure Legend Strand breaks for p53 exon 6 and 8 among different time course of folate depletion or repletion in the rectosigmoid mucosa. The input of DNA was controlled by GAPDH. The data is shown as ΔCt after normalized to GAPDH. The higher ΔCt the more strand breaks. The P value is shown in the figure. SUPPLEMENT S1 Genes that were significantly UPREGULATED after folate intervention (by unadjusted paired t-test), list is sorted by P value Gene Symbol Nucleotide P VALUE Description OLFM4 NM_006418 0.0000 Homo sapiens differentially expressed in hematopoietic lineages (GW112) mRNA. FMR1NB NM_152578 0.0000 Homo sapiens hypothetical protein FLJ25736 (FLJ25736) mRNA. IFI6 NM_002038 0.0001 Homo sapiens interferon alpha-inducible protein (clone IFI-6-16) (G1P3) transcript variant 1 mRNA. Homo sapiens UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 15 GALNTL5 NM_145292 0.0001 (GALNT15) mRNA. STIM2 NM_020860 0.0001 Homo sapiens stromal interaction molecule 2 (STIM2) mRNA. ZNF645 NM_152577 0.0002 Homo sapiens hypothetical protein FLJ25735 (FLJ25735) mRNA. ATP12A NM_001676 0.0002 Homo sapiens ATPase H+/K+ transporting nongastric alpha polypeptide (ATP12A) mRNA. U1SNRNPBP NM_007020 0.0003 Homo sapiens U1-snRNP binding protein homolog (U1SNRNPBP) transcript variant 1 mRNA. RNF125 NM_017831 0.0004 Homo sapiens ring finger protein 125 (RNF125) mRNA. FMNL1 NM_005892 0.0004 Homo sapiens formin-like (FMNL) mRNA. ISG15 NM_005101 0.0005 Homo sapiens interferon alpha-inducible protein (clone IFI-15K) (G1P2) mRNA. SLC6A14 NM_007231 0.0005 Homo sapiens solute carrier family 6 (neurotransmitter transporter) member 14 (SLC6A14) mRNA.
    [Show full text]
  • Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer
    1521-0111/90/3/199–204$25.00 http://dx.doi.org/10.1124/mol.115.103192 MOLECULAR PHARMACOLOGY Mol Pharmacol 90:199–204, September 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics MINIREVIEW Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer Erik S. Blomain, Dante J. Merlino, Amanda M. Pattison, Adam E. Snook, and Scott A. Waldman Downloaded from Department of Pharmacology and Experimental Therapeutics, Thomas Jefferson University, Philadelphia, Pennsylvania Received December 29, 2015; accepted May 25, 2016 ABSTRACT Obesity has emerged as a principal cause of mortality worldwide, in mice and humans. Hormone loss reflects reversible calorie- molpharm.aspetjournals.org reflecting comorbidities including cancer risk, particularly in induced endoplasmic reticulum stress and the associated colorectum. Although this relationship is established epidemio- unfolded protein response, rather than the endocrine, adipo- logically, molecular mechanisms linking colorectal cancer and kine, or inflammatory milieu of obesity. Loss of intestinal obesity continue to be refined. Guanylyl cyclase C (GUCY2C), uroguanylin secretion silences the hypothalamic GUCY2C a membrane-bound guanylyl cyclase expressed in intestinal endocrine axis, creating a feed-forward loop contributing to epithelial cells, binds the paracrine hormones guanylin and hyperphagia in obesity. Importantly, calorie-induced guanylin uroguanylin, inducing cGMP signaling in colorectum and small loss silences the GUCY2C-cGMP paracrine axis underlying intestine, respectively. Guanylin is the most commonly lost obesity-induced epithelial dysfunction and colorectal tumor- gene product in sporadic colorectal cancer, and its universal igenesis. Indeed, genetically enforced guanylin replacement loss early in transformation silences GUCY2C, a tumor sup- eliminated diet-induced intestinal tumorigenesis in mice.
    [Show full text]
  • Dema and Faust Et Al., Suppl. Material 2020.02.03
    Supplementary Materials Cyclin-dependent kinase 18 controls trafficking of aquaporin-2 and its abundance through ubiquitin ligase STUB1, which functions as an AKAP Dema Alessandro1,2¶, Dörte Faust1¶, Katina Lazarow3, Marc Wippich3, Martin Neuenschwander3, Kerstin Zühlke1, Andrea Geelhaar1, Tamara Pallien1, Eileen Hallscheidt1, Jenny Eichhorst3, Burkhard Wiesner3, Hana Černecká1, Oliver Popp1, Philipp Mertins1, Gunnar Dittmar1, Jens Peter von Kries3, Enno Klussmann1,4* ¶These authors contributed equally to this work 1Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Robert- Rössle-Strasse 10, 13125 Berlin, Germany 2current address: University of California, San Francisco, 513 Parnassus Avenue, CA 94122 USA 3Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany 4DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Oudenarder Strasse 16, 13347 Berlin, Germany *Corresponding author Enno Klussmann Max Delbrück Center for Molecular Medicine Berlin in the Helmholtz Association (MDC) Robert-Rössle-Str. 10, 13125 Berlin Germany Tel. +49-30-9406 2596 FAX +49-30-9406 2593 E-mail: [email protected] 1 Content 1. CELL-BASED SCREENING BY AUTOMATED IMMUNOFLUORESCENCE MICROSCOPY 3 1.1 Screening plates 3 1.2 Image analysis using CellProfiler 17 1.4 Identification of siRNA affecting cell viability 18 1.7 Hits 18 2. SUPPLEMENTARY TABLE S4, FIGURES S2-S4 20 2 1. Cell-based screening by automated immunofluorescence microscopy 1.1 Screening plates Table S1. Genes targeted with the Mouse Protein Kinases siRNA sub-library. Genes are sorted by plate and well. Accessions refer to National Center for Biotechnology Information (NCBI, BLA) entries. The siRNAs were arranged on three 384-well microtitre platres.
    [Show full text]
  • Gene Symbol Accession Alias/Prev Symbol Official Full Name AAK1 NM 014911.2 KIAA1048, Dkfzp686k16132 AP2 Associated Kinase 1
    Gene Symbol Accession Alias/Prev Symbol Official Full Name AAK1 NM_014911.2 KIAA1048, DKFZp686K16132 AP2 associated kinase 1 (AAK1) AATK NM_001080395.2 AATYK, AATYK1, KIAA0641, LMR1, LMTK1, p35BP apoptosis-associated tyrosine kinase (AATK) ABL1 NM_007313.2 ABL, JTK7, c-ABL, p150 v-abl Abelson murine leukemia viral oncogene homolog 1 (ABL1) ABL2 NM_007314.3 ABLL, ARG v-abl Abelson murine leukemia viral oncogene homolog 2 (arg, Abelson-related gene) (ABL2) ACVR1 NM_001105.2 ACVRLK2, SKR1, ALK2, ACVR1A activin A receptor ACVR1B NM_004302.3 ACVRLK4, ALK4, SKR2, ActRIB activin A receptor, type IB (ACVR1B) ACVR1C NM_145259.2 ACVRLK7, ALK7 activin A receptor, type IC (ACVR1C) ACVR2A NM_001616.3 ACVR2, ACTRII activin A receptor ACVR2B NM_001106.2 ActR-IIB activin A receptor ACVRL1 NM_000020.1 ACVRLK1, ORW2, HHT2, ALK1, HHT activin A receptor type II-like 1 (ACVRL1) ADCK1 NM_020421.2 FLJ39600 aarF domain containing kinase 1 (ADCK1) ADCK2 NM_052853.3 MGC20727 aarF domain containing kinase 2 (ADCK2) ADCK3 NM_020247.3 CABC1, COQ8, SCAR9 chaperone, ABC1 activity of bc1 complex like (S. pombe) (CABC1) ADCK4 NM_024876.3 aarF domain containing kinase 4 (ADCK4) ADCK5 NM_174922.3 FLJ35454 aarF domain containing kinase 5 (ADCK5) ADRBK1 NM_001619.2 GRK2, BARK1 adrenergic, beta, receptor kinase 1 (ADRBK1) ADRBK2 NM_005160.2 GRK3, BARK2 adrenergic, beta, receptor kinase 2 (ADRBK2) AKT1 NM_001014431.1 RAC, PKB, PRKBA, AKT v-akt murine thymoma viral oncogene homolog 1 (AKT1) AKT2 NM_001626.2 v-akt murine thymoma viral oncogene homolog 2 (AKT2) AKT3 NM_181690.1
    [Show full text]
  • Effect of Guanylate Cyclase-C Activity on Energy and Glucose
    Page 1 of 19 Diabetes Effect of guanylate cyclase-C activity on energy and glucose homeostasis Denovan P. Begg1,2, Kris A. Steinbrecher3, Joram D. Mul1, Adam P. Chambers1, Rohit Kohli3, April Haller1, Mitchell B. Cohen3, Stephen C. Woods1 and Randy J. Seeley1 1 Metabolic Diseases Institute, University of Cincinnati, Cincinnati, OH, 45237 2 School of Psychology, University of New South Wales, UNSW Sydney, NSW, Australia, 2052 3 Division of Gastroenterology, Hepatology, and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45220 Running title: GC-C activation and energy balance Key words: obesity, glucose tolerance, uroguanylin, guanylate cyclase-C Word count: 2143 Number of figures: 3 Corresponding Author Denovan P. Begg School of Psychology, University of New South Wales, UNSW Sydney, NSW, Australia, 2052 Ph: +61-2-9385-2441 Fax: +61-2-9385-3641 Diabetes Publish Ahead of Print, published online June 4, 2014 Diabetes Page 2 of 19 Abstract Uroguanylin is a gastrointestinal hormone primarily involved in fluid and electrolyte handling. It has recently been reported that prouroguanylin, secreted postprandially, is converted to uroguanylin in the brain and activates the receptor guanylate cyclase-C (GC- C) to reduce food intake and prevent obesity. Here, we tested CNS administration of two GC-C agonists and found no significant reduction of food intake. We also carefully phenotyped mice lacking the GC-C receptor and found them to have normal body weight, adiposity and glucose tolerance. Interestingly, uroguanylin knockout mice had a small but significant increase in body weight and adiposity that was accompanied by glucose intolerance. Our data indicate that the modest effects of uroguanylin on energy and glucose homeostasis are not centrally mediated by central GC-C receptors.
    [Show full text]
  • Guanylate Cyclase C Reduces Invasion of Intestinal Epithelial Cells by Bacterial Pathogens Received: 11 October 2017 Surya Amarachintha1, Eleana Harmel-Laws1 & Kris A
    www.nature.com/scientificreports OPEN Guanylate cyclase C reduces invasion of intestinal epithelial cells by bacterial pathogens Received: 11 October 2017 Surya Amarachintha1, Eleana Harmel-Laws1 & Kris A. Steinbrecher1,2 Accepted: 20 December 2017 The guanylate cyclase C (GC-C) receptor regulates electrolyte and water secretion into the gut following Published: xx xx xxxx activation by the E. coli enterotoxin STa, or by weaker endogenous agonists guanylin and uroguanylin. Our previous work has demonstrated that GC-C plays an important role in controlling initial infection as well as carrying load of non-invasive bacterial pathogens in the gut. Here, we use Salmonella enterica serovar Typhimurium to determine whether GC-C signaling is important in host defense against pathogens that actively invade enterocytes. In vitro studies indicated that GC-C signaling signifcantly reduces Salmonella invasion into Caco2-BBE monolayers. Relative to controls, GC-C knockout mice develop severe systemic illness following oral Salmonella infection, characterized by disrupted intestinal mucus layer, elevated cytokines and organ CFUs, and reduced animal survival. In Salmonella- infected wildtype mice, oral gavage of GC-C agonist peptide reduced host/pathogen physical interaction and diminished bacterial translocation to mesenteric lymph nodes. These studies suggest that early life susceptibility to STa-secreting enterotoxigenic E. coli may be counter-balanced by a critical role of GC-C in protecting the mucosa from non-STa producing, invasive bacterial pathogens. Infectious diarrheal disease is a signifcant cause of morbidity and mortality in the developing world1. Diarrheal disease, such as that caused by enterotoxigenic Escherichia coli (ETEC), kills approximately 500,000 children each year2.
    [Show full text]
  • GDNF and GUCY2C Mutation Associated with Hirschsprung's Disease in a Trio Pedigree: a Case Report
    GDNF and GUCY2C mutation associated with Hirschsprung's disease in a trio pedigree: a case report Senmao Chai Xiangyang No.1 People's Hospital Kai Deng Hubei University of Medicine Xiaodong Sun Xiangyang No.1 People's Hostipal Qiang Zhao Xiangyang No.1 People's Hospital Pan Fu Xiangyang No.1 People's Hospital Qianqian Xu Xiangyang No.1 People's Hospital Ming Sang ( [email protected] ) Hubei University of Medicine https://orcid.org/0000-0001-7862-0603 Case report Keywords: Hirschsprung's disease, Exome sequencing, GDNF, GUCY2C, Ganglion cells Posted Date: April 6th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-21172/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/12 Abstract Background: Hirschsprung's disease (HSCR) caused by absence or dysfunction of ganglion cells in the submucosal plexus and intermuscular plexus of the intestinal wall. Studies have shown that homozygous mutations of glial cell-line derived neurotrophic factor ( GDNF ) can lead to the occurrence of HSCR. but heterozygous mutation must combine with other related abnormal genes could to cause the HSCR. The other Guanylyl cyclase C ( GUCY2C ) gene is rarely reported as a direct cause of HSCR. Our ndings Provides a new perspective for application of mutation screening in the genetic counseling. Case presentation: We report clinical and genetic ndings of a trio pedigree with Hirschsprung's disease. Whole exome sequencing and Sanger sequencing were performed for propositus and all members of trio pedigree, respectively. We also collected 74 healthy control samples and used Sanger sequencing to identify that found the mutation.
    [Show full text]