Allelic Dropout During Polymerase Chain Reaction Due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

Total Page:16

File Type:pdf, Size:1020Kb

Allelic Dropout During Polymerase Chain Reaction Due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci INVESTIGATION Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci Aaron J. Stevens,* Millie G. Taylor,* Frederick Grant Pearce,† and Martin A. Kennedy*,1 *Department of Pathology, University of Otago, Christchurch 8140, New Zealand and †Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand ORCID IDs: 0000-0001-9148-8045 (A.J.S.); 0000-0002-6445-8526 (M.A.K.) ABSTRACT Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as KEYWORDS allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound G-quadruplex implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic PCR amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation allelic dropout and G-quadruplex formation was previously described for a differentially methylated region of the human methylation imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can poten- polymerase chain tially occur at several other genomic regions that display genomic imprinting and have propensity for reaction G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the ge- nome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations. DNA amplification by polymerase chain reaction (PCR) is an enzymatic Saunders et al. 2010; Wenzel et al. 2009). ADO is an insidious problem technique for the in vitro synthesis of targeted DNA regions, mediated that is difficult to recognize because the PCR appears successful, but by thermally stable DNA polymerases. PCR is a prerequisite for most half of the genetic information is missing. ADO can have significant experimental procedures that involve DNA detection, sequencing, implications in both research and clinical applications, where there is a cloning, and genotyping (Mullis and Faloona 1987; Saiki et al. 1988; requirement for high sensitivity and accurate PCR genotyping. Incor- Bevan et al. 1992), and is of widespread application in genetic research rect genotyping can have substantial negative consequences and may and molecular diagnostic applications (Desforges and Eisenstein 1990). result in the misdiagnosis of genetic diseases, loss of the ability to Despite extensive optimization and near ubiquitous usage, PCR is still differentiate between individuals, and false assumptions about parent- prone to failure under certain circumstances. For diploid organisms, age or genetic diversity (Boán et al. 2004; Landsverk et al. 2012; the failure of one allele to amplify can result in allelic dropout (ADO), Saunders et al. 2010; Tomaz et al. 2010; Wenzel et al. 2009). causing apparent homozygosity (Askree et al. 2011; Boán et al. 2004; We previously characterized a novel mechanism of ADO that Lam and Mak 2013; Landsverk et al. 2012; Piyamongkol et al. 2003; occurred during genotyping of the imprinted human gene, MEST (Stevens et al. 2014). Extensive PCR analysis of a short region in the Copyright © 2017 Stevens et al. MEST promoter invariably led to non-Mendelian genotype patterns for doi: 10.1534/g3.116.038687 three single nucleotide polymorphisms (SNPs), which could not be Manuscript received December 22, 2016; accepted for publication January 23, resolved by primer redesign or standard PCR optimization strategies. 2017; published Early Online January 30, 2017. We established that both cytosine methylation and DNA structures This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ known as G-quadruplexes (G4s) in this region contributed to licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction ADO, leading to incorrect genotyping (Stevens et al. 2014). G4 in any medium, provided the original work is properly cited. are secondary DNA structures that can form in G-rich regions Supplemental material is available online at www.g3journal.org/lookup/suppl/ due to the self-association of guanine through Hoogsteen bonds. Four doi:10.1534/g3.116.038687/-/DC1. 1Corresponding author: Department of Pathology, University of Otago, guanine nucleotides can adopt a square planar arrangement, referred Christchurch, P.O. Box 4345, Christchurch, 8140 New Zealand. E-mail martin. to as a G-quartet, and multiple G-quartets can then stack upon one [email protected] another to form a G4 (Sen and Gilbert 1988; Sundquist and Klug Volume 7 | March 2017 | 1019 1989). G4 formation is stabilized by the integration of a cation, like potassium (Ambrus et al. 2005; Biffi et al. 2012; Maizels 2015; Rhodes and Lipps 2015; Simonsson 2001), making PCR buffer an optimal environment for G4 formation. G4 structures may then act as a steric block to Taq polymerase (Boán et al. 2004; Chambers et al. 2015; Saunders et al. 2010; Weitzmann et al. 1996), an effect that is exac- erbated when the G4 region is methylated (Stevens et al. 2014). During amplification of the imprinted MEST promoter region, we observed consistent ADO of the maternally inherited, methylated allele. Correct genotypes from this locus were only obtained using extraordi- nary modifications of PCR, including methylation-specific PCR, allele- specific enzymatic digestion of genomic DNA, and PCR buffers lacking potassium. We demonstrated that ADO resulted from the combination of both cytosine methylation and guanine Hoogsteen bonds in the template DNA, which can form G4 structures, and that neither factor in isolation was sufficient to cause complete ADO (Stevens et al. 2014). The novel form of ADO observed at the MEST promoter region was Figure 1 Synthetic template ADO assay (A) Two templates (black bars) intriguing and problematic, but it was unclear if it was a phenomenon were generated by targeted PCR amplification of genomic DNA (gray). restricted solely to this genomic region or a more general occurrence These assay templates differed by a single base pair, which was introduced throughout the genome. In this report, we describe the design of an into one template (right) through primer-directed mutagenesis (small assay and its application to test for the potential occurrence of ADO green arrows and enlarged view). (B) An aliquot of each template was during amplification of differentially methylated DNA. The templates then methylated (blue circles) with M.SssI, and reciprocal mixing exper- used in this assay were generated by PCR from a range of imprinted iments were performed on each template combination as shown. In total, genes, and in vitro methylation with the enzyme M.SssI was used to three template mixing experiments were performed per amplicon, for vs. vs. mimic differentially methylated alleles. After demonstrating potential example: (1) methylated G nonmethylated A; (2) methylated A vs. ADO using the assay on multiple synthetic model templates, we then nonmethylated G; and (3) nonmethylated A nonmethylated G. demonstrated that this type of ADO could be observed in genomic DNA analysis of an imprinted human locus other than MEST. Sequencing reaction products were run on an AB3130xl fragment anal- ysis system equipped with a 50-cm capillary, using POP7 polymer. MATERIALS AND METHODS Synthetic DNA templates Selection of G4s for analysis Selected G4-containing amplicons (Figure S1 in File S1)wereamplified Imprinted genes with a confirmed parent-of-origin methylation status by PCR, and as described below (ADO assay on synthetic templates), a (Jirtle 2012; Morison et al. 2001) were analyzed for G4-forming motifs single nucleotide difference was introduced to allow alleles to be dis- using the bioinformatic software, QGRS Mapper (Kikin 2006). G4 tinguished. In vitro methylation and digestion experiments were per- motifs that contained runs of three or more guanines and a loop length formed on PCR products generated from genomic DNA, using between 0 and 7 nt were considered for analysis, as these were most enzymes purchased from New England Biolabs Inc., (Ipswich, MA). likely to adopt G4 structure. This corresponded with a QGRS mapper In vitro methylation was carried out by incubation with M.SssI for score of at least 37, which was therefore selected as an arbitrary thresh- 120 min at 37°, followed by heat inactivation at 65° for 20 min, as old for G4 cutoff. PCR amplicons of 300 bp were designed from these recommended by the manufacturer’s protocol. To assess the extent of regions, to contain a single G4 motif and a CCGG endonuclease rec- in vitro methylation achieved, each methylated DNA template was ognition sequence for HpaII and MspI endonucleases (Supplemental then incubated with the restriction enzyme MspIanditsmethylation- Material, Figure S1 and Table S1 in File S1). sensitive isoschizomer HpaII. The resulting digestion products were analyzed by gel electrophoresis and compared, to ensure successful PCR methylation (data not shown). PCR was carried out in a Mastercycler pro thermal cycler (Eppendorf, Stevenage, UK) with Fisher Taq-ti polymerase (Fisher Biotec, WA, ADO assay on synthetic templates Australia). The initial denaturation step consisted of 95° for 2 min, The assay for modeling methylation specific ADO
Recommended publications
  • Genomic Imprinting at the Porcine PLAGL1 Locus and the Orthologous Locus in the Human
    G C A T T A C G G C A T genes Article Genomic Imprinting at the Porcine PLAGL1 Locus and the Orthologous Locus in the Human Jinsoo Ahn 1 , In-Sul Hwang 2 , Mi-Ryung Park 2, Seongsoo Hwang 2 and Kichoon Lee 1,* 1 Functional Genomics Laboratory, Department of Animal Sciences, The Ohio State University, Columbus, OH 43210, USA; [email protected] 2 Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju, Jeonbuk 55365, Korea; [email protected] (I.-S.H.); [email protected] (M.-R.P.); [email protected] (S.H.) * Correspondence: [email protected]; Tel.: +1-614-688-7963 Abstract: Implementation of genomic imprinting in mammals often results in cis-acting silencing of a gene cluster and monoallelic expression, which are important for mammalian growth and function. Compared with widely documented imprinting status in humans and mice, current understanding of genomic imprinting in pigs is relatively limited. The objectives of this study were to identify DNA methylation status and allelic expression of alternative spliced isoforms at the porcine PLAGL1 locus and assess the conservation of the locus compared to the orthologous human locus. DNA methylome and transcriptome were constructed using porcine parthenogenetic or biparental control embryos. Using methylome, differentially methylated regions between those embryos were identified. Alternative splicing was identified by differential splicing analysis, and monoallelic expression was examined using single nucleotide polymorphism sites. Moreover, topological boundary regions were identified by analyzing CTCF binding sites and compared with the boundary of human orthologous locus. As a result, it was revealed that the monoallelic expression of the PLAGL1 Citation: Ahn, J.; Hwang, I.-S.; Park, M.-R.; Hwang, S.; Lee, K.
    [Show full text]
  • Zinc-Finger Protein 471 Suppresses Gastric Cancer Through
    Oncogene (2018) 37:3601–3616 https://doi.org/10.1038/s41388-018-0220-5 ARTICLE Zinc-finger protein 471 suppresses gastric cancer through transcriptionally repressing downstream oncogenic PLS3 and TFAP2A 1 1 1 2 1 3 Lei Cao ● Shiyan Wang ● Yanquan Zhang ● Ka-Chun Wong ● Geicho Nakatsu ● Xiaohong Wang ● 1 3 1 Sunny Wong ● Jiafu Ji ● Jun Yu Received: 28 June 2017 / Revised: 23 December 2017 / Accepted: 23 February 2018 / Published online: 3 April 2018 © The Author(s) 2018. This article is published with open access Abstract Zinc-finger protein 471 (ZNF471) was preferentially methylated in gastric cancer using promoter methylation array. The role of ZNF471 in human cancer is unclear. Here we elucidated the functional significance, molecular mechanisms and clinical impact of ZNF471 in gastric cancer. ZNF471 mRNA was silenced in 15 out of 16 gastric cancer cell lines due to promoter hypermethylation. Significantly higher ZNF471 promoter methylation was also observed in primary gastric cancers compared to their adjacent normal tissues (P<0.001). ZNF471 promoter CpG-site hypermethylation correlated with poor 1234567890();,: survival of gastric cancer patients (n = 120, P = 0.001). Ectopic expression of ZNF471 in gastric cancer cell lines (AGS, BGC823, and MKN74) significantly suppressed cell proliferation, migration, and invasion, while it induced apoptosis in vitro and inhibited xenograft tumorigenesis in nude mice. Transcription factor AP-2 Alpha (TFAP2A) and plastin3 (PLS3) were two crucial downstream targets of ZNF471 demonstrated by bioinformatics modeling and ChIP-PCR assays. ZNF471 directly bound to the promoter of TFAP2A and PLS3 and transcriptionally inhibited their expression. TFAP2A and PLS3 showed oncogenic functions in gastric cancer cell lines.
    [Show full text]
  • PLAGL1 (ZAC1/LOT1) Expression in Clear Cell Renal Cell Carcinoma: Correlations with Disease Progression and Unfavorable Prognosis
    ANTICANCER RESEARCH 36: 617-624 (2016) PLAGL1 (ZAC1/LOT1) Expression in Clear Cell Renal Cell Carcinoma: Correlations with Disease Progression and Unfavorable Prognosis JANUSZ GODLEWSKI1, BARTLOMIEJ E. KRAZINSKI1, ANNA E. KOWALCZYK1, JOLANTA KIEWISZ1, JACEK KIEZUN1, PRZEMYSLAW KWIATKOWSKI1, AGNIESZKA SLIWINSKA-JEWSIEWICKA1, ZBIGNIEW MASLOWSKI2 and ZBIGNIEW KMIEC1,3 1Department of Human Histology and Embryology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland; 2Department of Oncological Surgery, Warmia and Mazury Oncological Center, Olsztyn, Poland; 3Department of Histology, Medical University of Gdansk, Gdansk, Poland Abstract. Background: Pleiomorphic adenoma gene-like 1 Clear cell renal cell carcinoma (ccRCC), the prevailing form (PLAGL1) protein was originally shown to induce cell-cycle of RCC, is characterized by the most aggressive behavior and arrest and promote apoptosis in several types of human poor prognosis among all types of kidney cancer (1-3). tumors and therefore it was considered a candidate tumor Although ccRCC tumors can be removed surgically, suppressor. The involvement of PLAGL1 gene in the etiology haematogeneous metastases frequently occur in the early and pathogenesis of clear cell renal cell carcinoma (ccRCC) stage of the disease and 35-65% of patients develop has not been evaluated. The purpose of the present study metastatic disease after nephrectomy (3). Over the past was to determine the expression level of PLAGL1 in ccRCC decade, the background of ccRCC pathogenesis has been and to investigate its potential utility as a prognostic factor. extensively screened for molecular biomarkers and relevant Materials and Methods: We applied quantitative real-time gene signatures, however, only few have significant polymerase chain reaction (QPCR), western blotting and prognostic value which can be used in clinical practice (3-5).
    [Show full text]
  • Altered Expression of the PLAGL1 (ZAC1/LOT1) Gene in Colorectal Cancer: Correlations to the Clinicopathological Parameters
    INTERNATIONAL JOURNAL OF ONCOLOGY 47: 951-962, 2015 Altered expression of the PLAGL1 (ZAC1/LOT1) gene in colorectal cancer: Correlations to the clinicopathological parameters ANNA E. KowalcZYK1, Bartlomiej E. KRAZINSKI1, JANUSZ GODLEWSKI1, Jolanta KIEWISZ1, PRZEMYSlaw KwiatkowSKI1, AGNIESZKA SLIWINSKA-JEWSIEWICKA1, JACEK KIEZUN1, PIOTR M. WIERZBICKI2, GABRIEL BODEK3, MARIAN SULIK4 and ZBIGNIEW KMIEC1,2 1Department of Human Histology and Embryology, Faculty of Medical Sciences, University of Warmia and Mazury, 10-082 Olsztyn; 2Department of Histology, Medical University of Gdansk, 80-210 Gdansk; 3In Vitro and Cell Biotechnology Laboratory, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-243 Olsztyn; 4Department of Pathomorphology, Faculty of Medical Sciences, University of Warmia and Mazury, 10-561 Olsztyn, Poland Received April 23, 2015; Accepted June 2, 2015 DOI: 10.3892/ijo.2015.3067 Abstract. Pleomorphic adenoma gene-like 1 gene (PLAGL1) late significantly with patient overall survival; however, the encodes a zinc-finger nuclear transcription factor which hazard ratio for patients whose tumor tissues showed reduced promotes apoptosis and cell cycle arrest. Loss or downregu- PLAGL1 immunohistochemical staining was twice higher lation of its expression has been observed in various human than in patients with increased PLAGL1 immunoreactivity. In neoplasms. This study compared PLAGL1 expression in conclusion, these results suggest that dysregulation of PLAGL1 colorectal cancer (CRC) tissue and colon mucosa of healthy expression may be involved to some extent in the progression subjects at the mRNA and protein levels, and estimated its of CRC, but the so far collected patient survival data do not prognostic value. The PLAGL1 mRNA levels were also deter- confirm applicability of the PLAGL1 expression level as a mined in CRC cell lines.
    [Show full text]
  • Genome-Wide DNA Methylation Dynamics During Epigenetic
    Gómez‑Redondo et al. Clin Epigenet (2021) 13:27 https://doi.org/10.1186/s13148‑021‑01003‑x RESEARCH Open Access Genome‑wide DNA methylation dynamics during epigenetic reprogramming in the porcine germline Isabel Gómez‑Redondo1*† , Benjamín Planells1†, Sebastián Cánovas2,3, Elena Ivanova4, Gavin Kelsey4,5 and Alfonso Gutiérrez‑Adán1 Abstract Background: Prior work in mice has shown that some retrotransposed elements remain substantially methylated during DNA methylation reprogramming of germ cells. In the pig, however, information about this process is scarce. The present study was designed to examine the methylation profles of porcine germ cells during the time course of epigenetic reprogramming. Results: Sows were artifcially inseminated, and their fetuses were collected 28, 32, 36, 39, and 42 days later. At each time point, genital ridges were dissected from the mesonephros and germ cells were isolated through magnetic‑ activated cell sorting using an anti‑SSEA‑1 antibody, and recovered germ cells were subjected to whole‑genome bisulphite sequencing. Methylation levels were quantifed using SeqMonk software by performing an unbiased analysis, and persistently methylated regions (PMRs) in each sex were determined to extract those regions showing 50% or more methylation. Most genomic elements underwent a dramatic loss of methylation from day 28 to day 36, when the lowest levels were shown. By day 42, there was evidence for the initiation of genomic re‑methylation. We identifed a total of 1456 and 1122 PMRs in male and female germ cells, respectively, and large numbers of transpos‑ able elements (SINEs, LINEs, and LTRs) were found to be located within these PMRs. Twenty‑one percent of the introns located in these PMRs were found to be the frst introns of a gene, suggesting their regulatory role in the expression of these genes.
    [Show full text]
  • Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and The
    Robbins et al. Journal of Biomedical Science 2012, 19:95 http://www.jbiomedsci.com/content/19/1/95 RESEARCH Open Access Expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine Katherine Marie Robbins, Zhiyuan Chen, Kevin Dale Wells and Rocío Melissa Rivera* Abstract Background: Beckwith-Wiedemann syndrome (BWS) is a loss-of-imprinting pediatric overgrowth syndrome. The primary features of BWS include macrosomia, macroglossia, and abdominal wall defects. Secondary features that are frequently observed in BWS patients are hypoglycemia, nevus flammeus, polyhydramnios, visceromegaly, hemihyperplasia, cardiac malformations, and difficulty breathing. BWS is speculated to occur primarily as the result of the misregulation of imprinted genes associated with two clusters on chromosome 11p15.5, namely the KvDMR1 and H19/IGF2. A similar overgrowth phenotype is observed in bovine and ovine as a result of embryo culture. In ruminants this syndrome is known as large offspring syndrome (LOS). The phenotypes associated with LOS are increased birth weight, visceromegaly, skeletal defects, hypoglycemia, polyhydramnios, and breathing difficulties. Even though phenotypic similarities exist between the two syndromes, whether the two syndromes are epigenetically similar is unknown. In this study we use control Bos taurus indicus X Bos taurus taurus F1 hybrid bovine concepti to characterize baseline imprinted gene expression and DNA methylation status of imprinted domains known to be misregulated in BWS. This work is intended to be the first step in a series of experiments aimed at determining if LOS will serve as an appropriate animal model to study BWS.
    [Show full text]
  • Neonatal Diabetes Mellitus and Congenital
    Topiol et al. International Journal of Pediatric Endocrinology 2012, 2012:21 http://www.ijpeonline.com/content/2012/1/21 CASE REPORT Open Access Neonatal diabetes mellitus and congenital diaphragmatic hernia: coincidence or concurrent etiology? Emmanuelle S Topiol1, Laurie A Minarich1, Charles A Williams2, Roberto T Zori2, David W Kays3 and Michael J Haller1* Abstract Neonatal diabetes mellitus (NDM) is a rare metabolic disorder, affecting approximately 1 in 500,000 live births. The management of NDM is challenging, as the benefits of controlling hyperglycemia must be balanced with the risks of iatrogenic hypoglycemia. NDM occurs in both permanent and transient forms, which have been genetically and phenotypically well characterized. Herein, we present the previously unreported combination of transient NDM (TNDM) and congenital diaphragmatic hernia (CDH). In addition to reviewing the management and genetics of NDM we discuss the potential for overlapping genetic or embryologic abnormalities to explain the concurrence of CDH and NDM. Keywords: Neonatal diabetes mellitus, Congenital diaphragmatic hernia, PLAGL1, Imprinting, Duplication Background Congenital diaphragmatic hernia (CDH) occurs in ap- Neonatal diabetes mellitus (NDM) is a rare disorder of proximately 1 in 2,500 children and results from a devel- glucose metabolism (affecting 1 in 500,000 live births) opmental defect in the diaphragm that allows abdominal and may be either transient or permanent [1]. Affected viscera to penetrate the chest [4]. Because the herniating neonates frequently present with hyperglycemia, intra- spleen, liver, and intestines may compress lung tissue uterine growth retardation, and variable degrees of dehy- during critical periods of lung organogenesis, CHD dration. Despite the relative severity of insulin results in varying degrees of irreversible pulmonary deficiency, ketoacidosis is uncommon.
    [Show full text]
  • Gene Expression Profiling Identifies the Role of Zac1 in Cervical Cancer
    www.nature.com/scientificreports OPEN Gene expression profling identifes the role of Zac1 in cervical cancer metastasis Hui‑Chen Su1,6, Sheng‑Cheng Wu2,6, Li‑Chen Yen3, Li‑Kang Chiao4, Jehng‑Kang Wang4, Yi‑Lin Chiu4,6*, Ching‑Liang Ho5 & Shih‑Ming Huang4,6* The zinc‑fnger protein which regulates apoptosis and cell cycle arrest 1 (Zac1), encoded by Plagl1 gene, is a seven‑zinc‑fnger containing transcription factor belonging to the imprinted genome and is expressed in diverse types of embryonic and adult human tissues. Zac1 is postulated to be a tumor suppressor by inducing cell cycle arrest and apoptosis through interacting and modulating transcriptional activity of p53 as it was named. Correspondingly, the reduction or loss of Zac1 expression is associated with the incidence and progression of several human tumors, including cervical cancer, breast cancer, ovarian cancer, pituitary tumors, and basal cell carcinoma, implying the rationality of utilizing Zac1 expression as novel a biomarker for the evaluation of cervical cancer prognosis. However, to date, it has not been elucidated whether Zac1 expression is related to the prognosis of patients in clinical cervical cancer tumor samples. To address the questions outlined above, we report here a comprehensive investigation of Zac1 expression in biopsies of clinical cervical carcinoma. By analyzing Zac1 expression in various gene expression profling of cervical cancer databases, we show the association between high Zac1 expression and poor prognosis of cervical cancer. Functional enrichment analysis showed that high Zac1 expression was associated with epithelial‑mesenchymal transition (EMT), which was further observed in clinical characteristics and metastatic carcinoma samples using immunohistochemical staining.
    [Show full text]
  • PLAGL1 Gene Function During Hepatoma Cells Proliferation
    www.oncotarget.com Oncotarget, 2018, Vol. 9, (No. 67), pp: 32775-32794 Research Paper PLAGL1 gene function during hepatoma cells proliferation Ana F. Vega-Benedetti1, Cinthia N. Saucedo1, Patrizia Zavattari2, Roberta Vanni2, Felix Royo3, Francisco Llavero4,5,6, José L. Zugaza4,5,6 and Luis A. Parada1 1Institute of Experimental Pathology, CONICET-UNSa, Salta, Argentina 2Biochemistry, Biology and Genetics Unit, Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato SP 8, Monserrato, Cagliari, Italy 3CIC BioGUNE-CIBERehd, Bizkaia Technology Park, Derio, Spain 4Achucarro Basque Center for Neuroscience, UPV/EHU Technology Park, Leioa, Spain 5Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Medicine and Dentistry, University of the Basque Country, Leioa, Spain 6IKERBASQUE, Basque Foundation for Science, Bilbao, Spain Correspondence to: Luis A. Parada, email: [email protected] Keywords: PLAGL1; hepatocellular carcinoma; cell proliferation; chromosome; methylation Received: March 26, 2018 Accepted: July 31, 2018 Published: August 28, 2018 Copyright: Vega-Benedetti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Hepatocellular carcinoma develops as a multistep process, in which cell cycle deregulation is a central feature, resulting in unscheduled proliferation. The PLAGL1 gene encodes a homonym zinc finger protein that is involved in cell-proliferation control. We determined the genomic profile and the transcription and expression level of PLAGL1, simultaneously with that of its molecular partners p53, PPARγ and p21, in cell-lines derived from patients with liver cancer, during in vitro cell growth.
    [Show full text]
  • Transient Neonatal Diabetes, a Disorder of Imprinting I K Temple,Jphshield
    872 REVIEW ARTICLE J Med Genet: first published as 10.1136/jmg.39.12.872 on 1 December 2002. Downloaded from Transient neonatal diabetes, a disorder of imprinting I K Temple,JPHShield ............................................................................................................................. J Med Genet 2002;39:872–875 Transient neonatal diabetes (TND) is a rare but distinct risk factor for diabetes in later life. In 1995, Muh- type of diabetes. Classically, neonates present with lendahl and Herkenhoff8 conducted a long term follow up of published cases and identified 13 growth retardation and diabetes in the first week of life. infants with TND with later recurrence. Apparent remission occurs by 3 months but there is a tendency for children to develop diabetes in later life. THE SEARCH FOR THE TND GENE Evidence suggests it is the result of overexpression of an Although there were occasional reports in sibs (Coffey et al9 reported TND in paternal half sibs, imprinted and paternally expressed gene/s within the and Fergusson et al10 reported diabetes in sibs), as TND critical region at 6q24. Two imprinted genes, ZAC the majority were sporadic a genetic cause was (zinc finger protein associated with apoptosis and cell not suspected. The first lead came in 1995 when two children with TND were discovered to have cycle arrest) and HYMAI (imprinted in hydatidiform paternal uniparental isodisomy of chromosome 6 mole) have been identified as potential candidates. (pat UPD(6)).11 The first case was part of a Three genetic mechanisms have been shown to result in systematic study to identify the incidence of UPD in carriers of supernumerary marker chromo- TND, paternal uniparental isodisomy of chromosome 6, somes (SMCs).12 The patient had few develop- paternally inherited duplication of 6q24, and a mental or dysmorphic features and was karyo- methylation defect at a CpG island overlapping exon 1 typed because of intrauterine retardation and a large tongue.
    [Show full text]
  • 393LN V 393P 344SQ V 393P Probe Set Entrez Gene
    393LN v 393P 344SQ v 393P Entrez fold fold probe set Gene Gene Symbol Gene cluster Gene Title p-value change p-value change chemokine (C-C motif) ligand 21b /// chemokine (C-C motif) ligand 21a /// chemokine (C-C motif) ligand 21c 1419426_s_at 18829 /// Ccl21b /// Ccl2 1 - up 393 LN only (leucine) 0.0047 9.199837 0.45212 6.847887 nuclear factor of activated T-cells, cytoplasmic, calcineurin- 1447085_s_at 18018 Nfatc1 1 - up 393 LN only dependent 1 0.009048 12.065 0.13718 4.81 RIKEN cDNA 1453647_at 78668 9530059J11Rik1 - up 393 LN only 9530059J11 gene 0.002208 5.482897 0.27642 3.45171 transient receptor potential cation channel, subfamily 1457164_at 277328 Trpa1 1 - up 393 LN only A, member 1 0.000111 9.180344 0.01771 3.048114 regulating synaptic membrane 1422809_at 116838 Rims2 1 - up 393 LN only exocytosis 2 0.001891 8.560424 0.13159 2.980501 glial cell line derived neurotrophic factor family receptor alpha 1433716_x_at 14586 Gfra2 1 - up 393 LN only 2 0.006868 30.88736 0.01066 2.811211 1446936_at --- --- 1 - up 393 LN only --- 0.007695 6.373955 0.11733 2.480287 zinc finger protein 1438742_at 320683 Zfp629 1 - up 393 LN only 629 0.002644 5.231855 0.38124 2.377016 phospholipase A2, 1426019_at 18786 Plaa 1 - up 393 LN only activating protein 0.008657 6.2364 0.12336 2.262117 1445314_at 14009 Etv1 1 - up 393 LN only ets variant gene 1 0.007224 3.643646 0.36434 2.01989 ciliary rootlet coiled- 1427338_at 230872 Crocc 1 - up 393 LN only coil, rootletin 0.002482 7.783242 0.49977 1.794171 expressed sequence 1436585_at 99463 BB182297 1 - up 393
    [Show full text]
  • Gli3 Utilizes Hand2 to Synergistically Regulate Tissue-Specific
    RESEARCH ARTICLE Gli3 utilizes Hand2 to synergistically regulate tissue-specific transcriptional networks Kelsey H Elliott1,2,3, Xiaoting Chen4, Joseph Salomone1,3,5, Praneet Chaturvedi1, Preston A Schultz1,2, Sai K Balchand1,2, Jeffrey D Servetas6, Aime´ e Zuniga7, Rolf Zeller7, Brian Gebelein1, Matthew T Weirauch1,4, Kevin A Peterson6*, Samantha A Brugmann1,2,8* 1Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States; 2Division of Plastic Surgery, Department of Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States; 3Graduate Program in Molecular and Developmental Biology, Cincinnati Children’s Hospital Research Foundation, Cincinnati, United States; 4Center for Autoimmune Genomics and Etiology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States; 5Medical-Scientist Training Program, University of Cincinnati College of Medicine, Cincinnati, United States; 6Jackson Laboratory, Bar Harbor, United States; 7Developmental Genetics, Department of Biomedicine, University of Basel, Basel, Switzerland; 8Shriners Children’s Hospital, Cincinnati, United States Abstract Despite a common understanding that Gli TFs are utilized to convey a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. Using the mouse model (Mus musculus), we demonstrated that rather than being driven by a Hh *For correspondence: threshold, robust Gli3 transcriptional activity during skeletal and glossal development required [email protected] (KAP); interaction with the basic helix-loop-helix TF Hand2. Not only did genetic and expression data [email protected] support a co-factorial relationship, but genomic analysis revealed that Gli3 and Hand2 were (SAB) enriched at regulatory elements for genes essential for mandibular patterning and development.
    [Show full text]