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Loss of BRCC3 Deubiquitinating Enzyme Leads to Abnormal Angiogenesis

ARTICLE

Loss of BRCC3Deubiquitinating Enzyme

Leads to Abnormal Angiogenesis

and Is Associated with Syndromic Moyamoya

Snaigune Miskinyte,1,10Matthew G.Butler,2,10Dominique Herve′,3,10Catherine Sarret,4Marc Nicolino,5 Jacob D.Petralia,6Francoise Bergametti,1Minh Arnould,1Van N.Pham,2Aniket V.Gore,2 Konstantinos Spengos,7Steven Gazal,8France Woimant,3Gary K.Steinberg,6Brant M.Weinstein,2 and Elisabeth Tournier-Lasserve1,9,*

Moyamoya is a cerebrovascular angiopathy characterized by a progressive stenosis of the terminal part of the intracranial carotid arteries and the compensatory development of abnormal and fragile collateral vessels,also called moyamoya vessels,leading to ischemic and hemorrhagic stroke.Moyamoya angiopathy can either be the sole manifestation of the disease(moyamoya disease)or be associated with various conditions,including neuro?bromatosis,Down syndrome,TAAD(autosomal-dominant thoracic aortic aneurysm),and radiotherapy of head tumors(moyamoya syndromes).Its prevalence is ten times higher in Japan than in Europe,and an estimated 6%–12%of moyamoya disease is familial in Japan.The pathophysiological mechanisms of this condition remain obscure.Here,we report on three unrelated families affected with an X-linked moyamoya syndrome characterized by the association of a moyamoya angiopathy,short stature,and a stereotyped facial dysmorphism.Other symptoms include an hypergonadotropic hypogonadism, hypertension,dilated cardiomyopathy,premature coronary heart disease,premature hair graying,and early bilateral acquired cataract. We show that this syndromic moyamoya is caused by Xq28deletions removing MTCP1/MTCP1NB and BRCC3.We also show that brcc3 morphant zebra?sh display angiogenesis defects that are rescued by endothelium-speci?c expression of brcc3.Altogether,these data strongly suggest that BRCC3,a deubiquitinating enzyme that is part of the cellular BRCA1and BRISC complexes,is an important player in angiogenesis and that BRCC3loss-of-function mutations are associated with moyamoya angiopathy.

Introduction

Moyamoya(MIM252350,MIM607151,MIM608796)is a cerebrovascular angiopathy characterized by a progressive stenosis of the terminal part of the intracranial internal carotid arteries(ICA)and their proximal branches within the circle of Willis.1,2This disease is associated with the compensatory development of abnormal,thin,and fragile collateral vessels(‘‘moyamoya’’vessels)at the base of the brain.Reduced cerebral blood?ow and rupture of the fragile collateral vessels cause ischemic and hemorrhagic stroke in children and adults with moyamoya.The preva-lence of this disorder has been estimated to be close to 3/100000in Japan and is ten times less prevalent in Europe.1,2

Histopathological analysis of affected carotids shows a marked decrease in the outer diameters of the carotid terminations,a?brocellular thickening of the intima con-taining proliferating smooth-muscle actin-positive cells, luminal thrombosis,and thinning of the media vascular layer.Collateral moyamoya vessels display fragmented elastic lamina,thinned media in the vessel wall,and mi-croaneurysms.3Despite a great deal of investigation,the molecular etiology and pathogenesis of moyamoya angi-opathy remains unclear.4

Moyamoya angiopathy can be associated with different conditions,including tuberculous meningitis,atheroscle-rosis,radiotherapy of head tumors,and various diseases of known genetic etiology,such as Down syndrome (MIM190685),neuro?bromatosis type1(MIM162200), sickle cell disease(MIM603903),and autosomal-dominant thoracic aortic aneurysm disease,also called TAAD(MIM 102620).1,5–7These conditions are known as moyamoya syndromes.They are distinct from moyamoya disease (MMD),which occurs as an idiopathic disorder in which the sole manifestation of the disease is the moyamoya angiopathy.

MMD occurs predominantly in patients of Asian origin, and the highest prevalence of the disease is found in Japan, especially among pediatric patients.2However,it can occur

1INSERM UMR-S-740;Universite′Paris,7Denis Diderot,10Avenue de Verdun,75010Paris,France;2Program in Genomics of Differentiation,National Insti-tute of Child Health and Human Development,National Institutes of Health,Bethesda,MD20892;3Assistance Publique des Ho?pitaux de Paris,Groupe Hospitalier Lariboisie`re-Saint-Louis,Service de Neurologie,Centre de Re′fe′rence des Maladies Vasculaires Rares du Cerveau et de l’Oeil,F-75010Paris,France; 4Hospices Civils de Lyon,Groupe Hospitalier Est,Ho?pital Femme-Me`re-Enfant,Service de Neurologie Pe′diatrique,69677Bron,France;5Division of Pedi-atric Endocrinology,Lyon University Pediatric Hospital,INSERM U.870,Centre d’Investigation Clinique201,Universite′Claude Bernard Lyon1,Hospices Civils de Lyon,Lyon,France;6Department of Neurosurgery,Stanford Stroke Center and Stanford Institute for Neuro-Innovation and Translational Neuro-sciences,Stanford University School of Medicine,Stanford,California,USA;7First Department of Neurology,Eginition Hospital,National and Kapodestrian University of Athens,School of Medicine,11528Athens,Greece;8Assistance Publique des Ho?pitaux de Paris,Plateforme de Ge′nomique Constitutionnelle du Groupe Hospitalo Universitaire Nord,Ho?pital Bichat,F-75010Paris,France;9Assistance Publique des Ho?pitaux de Paris,Groupe Hospitalier Lariboisie`re-Saint-Louis,Laboratoire de Ge′ne′tique,Centre de Re′fe′rence des Maladies Vasculaires Rares du Cerveau et de l’Oeil,F-75010Paris,France

10These authors contributed equally to this work

*Correspondence:tournier-lasserve@univ-paris-diderot.fr

in people of all ethnicities and in all age groups.Although most cases of MMD appear to be sporadic,an estimated 6%à12%of all reported cases in Japan are familial.The inheritance has been suggested to follow an autosomal-dominant,autosomal-recessive,X-linked-recessive,or multifactorial inheritance pattern.8Several whole-genome linkage studies have been aimed at identi?cation of MMD genetic loci.Linkage analyses conducted in Japanese fami-lies suggested that MMD candidate loci might be located at 3p24.2-p26,6q25,8q23,12p12,and 17q25.9–12No mutated gene has yet been identi?ed within those linked regions.Interestingly,a recent genome-wide association study suggested that RNF213(MIM 613768),a gene that is located at 17q25and encodes a ring ?nger protein,is an MMD susceptibility gene.13Additionally,mutations in ACTA2(MIM 102620),which encodes the vascular smooth-muscle-cell-speci?c isoform of a -actin,have been reported in rare MMD patients.14

Herein,we report on three unrelated families affected by an X-linked moyamoya syndrome in which an overlap-ping deletion at Xq28removes MTCP1/MTCP1NB (MIM 300116)and BRCC3(MIM 300617)and cosegregates with the affected phenotype.Morpholino-mediated knockdown of the BRCC3ortholog in zebra?sh led to

defective angiogenesis that was rescued by endothelial-speci?c expression of brcc3,strongly suggesting that this deubiquitinating enzyme is a novel important player in angiogenesis.

Material and Methods

Patients and Families

Three families (F1–F3)including a total of ten males affected by a syndromic moyamoya condition were included in this study.All patients and family members provided written informed consent.This study was conducted in accordance with ethical recommen-dations in France for genetic disorders.The geneological trees are shown in Figure 1.Some of the clinical and magnetic reso-nance imaging (MRI)features of family F1have previously been reported.6Additional information on family F1as well as clinical,biological,and MRI features of F2family members and of two of the affected F3family members is provided in the results section.Three additional unrelated families including two offspring showing pure,nonsyndromic MMD were also included in this study (F4:two brothers.F5:brother and sister.F6:two sisters).

DNA and RNA Extraction

Genomic DNA from probands and consenting relatives was extracted from peripheral blood leukocytes by standard proce-dures.F1–F3family members whose blood was sampled are indi-cated in Figure 1.Total RNA was isolated from lymphoblastoid Epstein-Barr virus (EBV)cell lines via the Trizol method and tran-scribed into cDNA via dT primers.RNA was available for probands of families F1,F2,F4,and F5.

Microsatellite Markers and Chromosome X Haplotyping

Family F1was genotyped with a panel of 19microsatellite markers spanning the X chromosome (average genetic distance ?20cM).Additional microsatellite markers,DXS984,DXS8106,DXS8045,DXS1193,and DXS8061,were used for Xq27-q28haplotyping in family F1.Parametric multipoint and two-point linkage anal-yses were carried out with the MINX program of MERLIN v1.1.2package;a recessive X-linked model with complete penetrance,disease allele frequency of 0.0001,and a phenocopy frequency of 0.0001were assumed.

Copy-Number Analysis with Affymetrix SNP 6.0Arrays

On the basis of the hypothesis that a deletion might be involved in the disease mechanisms,we used high-density Affymetrix SNP 6.0arrays (Affymetrix Santa Clara,CA)to search for putative deletions located within the linked region on the X chromosome in family F1.Genomic DNA from the mother (F1I-2)and two of her affected sons (F1II-3and F1II-4)was used for genotyping and copy-number analysis.Sample processing and labeling were performed according to the manufacturer’s instructions (Affymetrix Cytoge-netics Copy Number Assay User Guide,P/N 702607Rev.2).Hybridization was performed with a GeneChip Hybridization oven 640at 50 C and 60rpm for 16hr,washed with a GeneChip Fluidics Station 450,and scanned with a GeneChip Scanner 3000-7G.The image data were processed with the Affymetrix Genotyping Console (GTC v4.0)for determination of SNP call and copy-number

variation.

Figure 1.Genealogical Trees of Families F1,F2,and F3

Circles:females.Squares:males.Black-?lled symbols:affected individuals.Empty symbols:healthy individuals.Members whose blood has been sampled are indicated by an asterisk.

UCSC databases,the HapMap project,and the Database of Genomic Variants were used for?ltering copy-number polymor-phisms within the region of interest.

STS Markers and Oligonucleotides Used for Walking PCR

To con?rm the putative deletion in family F1and re?ne the critical interval,we used a panel of12STS markers selected from NCBI build36.3or designed with Primer3software(Table S1,available online).Two of these STS markers,ECD13623and ECD22603, closely?anked the putative deletion.Three of these STS markers (ECD07678,ECD07752,and ECD15347)map within FUNDC2, MTCP1/MTCP1NB,and BRCC3.Standard PCR was carried out with DNA from all affected males,two obligatory female carriers (F1I-2and F1II-5),and healthy males(F1III-2and F1III-3).Addi-tional oligonucleotides were used for walking PCR in family F2 (Table S1).

Long-Range PCR and Sequencing

In order to identify the deletion breakpoints,we performed long-range PCR with primers ECD21451(forward)and ECD02005R (reverse)in family F1and STSR7(forward)and BRCC3R4(reverse) in families F2and F3(Table S1and S2).Long-range PCR was carried out on DNA from all affected individuals and each obliga-tory carrier female.Unaffected spouses were included as negative controls.The reactions were carried out with the Expand 20kb PLUS PCR system(Roche)according to the manufacturer’s instructions.Puri?ed PCR products were sequenced with BigDye Terminator3.1(Applied Biosystems,Foster City,CA)chemistry on an ABI3130DNA sequencing apparatus according to the manufacturer’s instructions.

Screening for BRCC3and MTCP1/MTCP1NB

in Familial MMD Cases

All coding exons and?anking splice sites of BRCC3,MTCP1,and MTCP1NB were ampli?ed and sequenced with the genomic DNA of probands from families F4–F6(RefSeq accession numbers: BRCC3,NM_001018055.1;MTCP1,NM_001018025.3;and MTCP1NB,NM_001018024.2).Sequencing primers are shown in Table S2.

Immunoblot Analyses

A polyclonal antibody speci?c for human BRCC3was purchased from ProSci(reference number4331F).A polyclonal antibody speci?c for human p8MTCP1N

B was kindly provided by M.H.Stern (Institut Curie,France).

Immunoblot analysis of p8MTCP1NB and BRCC3expression in EBV-transformed lymphoblastoid cell lines from patients F1II-2 and F2II-2and two healthy controls was conducted with10m g of total protein lysates.

In addition,10m g of protein lysates from HUVEC endothelial cells,40m g of total protein from human adult arterial tissue(Gen-taur Molecular Products P1234013),and total protein from human adult vein tissue(Gentaur Molecular Products P1234020)were used in screens for p8MTCP1NB and BRCC3protein levels.

Zebra?sh Lines and Husbandry

EK strain wild-type?sh were used for in situ hybridization,and both Tg(?i1-eGFP)y1and Tg(?k1a-eGFP)were used in morpholino knockdown experiments.15All lines were maintained according to established convention.16brcc3and mtcp1nb cDNA Cloning and In Situ Hybridization

Total RNA was isolated from24hpf wild-type zebra?sh embryos with TRIzol(Invitrogen,Carlsbad,CA)and reversed transcribed with the SuperScript III First Strand Synthesis System(Invitrogen). brcc3and mtcp1nb cDNAs were ampli?ed from cDNA by PCR with the following primers:

brcc3-F:50-TTCGCGATGGCTGTCAACGCGGTGCATTT-30

brcc3-R:50-TTACAATGCCGCCAGCTCTTGCGTCAGC-30

mtcp1nb-F:50-AGCTCGAAGTCGGAGTTCATTTC-30

mtcp1nb-R:50-GACAGTGTTTAATGATGTGTAGCAGCA-30

The resulting PCR products were cloned into pCRaII-TOPOa(In-vitrogen)according to manufacturer’s protocol.Riboprobes were generated using the mMESSAGE mMACHINEaT7Kit(Life Tech-nologies Corp.,Carlsbad,CA).Riboprobes were generated with the Roche DIG RNA Labeling Kit(Indianapolis,IN).Whole-mount in situ hybridization was performed as previously described.17 Morpholino,mRNA,and DNA Injections

Morpholinos were designed to disrupt splicing of brcc3or mtcp1nb (Gene Tools,LLC.Philomath,OR).The brcc3morpholino (50-GTGATGCAGGAATAAAGCACATTCA-30)or mtcp1nb morpho-lino(50-AGCTGATAAACACAAAAGTCACACA-30)was injected into one-cell-stage Tg(?i1-eGFP)y1or Tg(?k1a-eGFP)embryos.The effectiveness of the brcc3morpholino was veri?ed by reverse tran-scriptase PCR(RT-PCR)aimed at detecting loss of the appropriately spliced gene product(data not shown).The brcc3and mtcp1nb morpholinos were injected at doses of9.5pg and4.0pg,respec-tively.For mRNA rescue experiments,wild-type brcc3was cloned into pCS-DEST and transcribed with the Ambion mMESSAGE mMACHINE SP6Kit.brcc3was injected(375pg)into control or brcc3morpholino-injected embryos.18Injection of brcc3mRNA into control animals had no effect on vessel growth(data not shown).Endothelium-speci?c rescue of brcc3morphants was per-formed by mosaic expression of brcc3via a6.4kb?k1promoter.19 Detailed methods for construction of?k1-mCherry-2A-brcc3and ?k1-mCherry transgenes are provided in Figure S5.SceI homing endonuclease was coinjected with the mcherry-2a-brcc3transgene (30pg)so that mosaicism would be increased.20

Microscopy and Imaging

The vascular endothelium of injected Tg(?i1-eGFP)y1and Tg(?k-eGFP)was illuminated with a488nm argon laser and visu-alized with an Olympus BX61WI confocal microscope.Embryos were mounted laterally in Nusieve GTG agarose(0.5%in Embryo water),and optical sections were collected through the trunk at 1.5m m intervals.Confocal Z series were rendered into three-dimensional representations with Olympus Fluoview software. For quantitation of phenotypes,ten ISVs from each of12different injected embryos were scored for each control and experimental condition.Statistical signi?cance was determined by a two-sided student’s t test calculation at a99.9%con?dence interval. Results

Clinical Features of Families F1–F3

Within families F1and F2,a total of seven males showed a strikingly similar phenotype characterized by the

association of a moyamoya angiopathy,a hypergonado-tropic hypogonadism,and several multisystemic manifes-tations (Figure 1and Figure 2).The main clinical,biolog-ical,and morphological features of this condition are summarized in Table 1.

In family 1,?ve male members related through a maternal lineage over two generations were clinically symptomatic,strongly suggesting an X-linked pattern of inheritance.Moyamoya angiopathy was responsible for cerebral infarcts or hemorrhages in four of these affected individuals,and the age of individuals at onset of acute neurological symptoms ranged from 4to 32years.A progressive worsening of arterial narrowing associated with additional strokes was observed in two affected members,F1II-1and F1II-2,(Figure 3).One member (F1III-1)had no intracranial artery abnormality,but he was

only 27years at the time of the last examination.Short stature of postnatal onset was constant and associated with azoospermia at adulthood.All patients had increased levels of gonadotropic hormones as a result of a primary gonadal failure.GH de?ciency was partial when observed and was never associated with any pituitary gland abnor-mality detectable by MRI.Another hallmark observed in all affected members was a stereotyped facial dysmorphism with hypertelorism,long philtrum,mild ptosis,and premature white hair locks.The hands were small and had short and broad ?ngers.Involvement of the heart varied from isolated and asymptomatic left ventricular enlargement to severe nonspeci?c dilated cardiomyopathy responsible for heart failure and recurrent neurologic de?-cits due to low cardiac output.Other manifestations of the disease in this family included a right aortic arch in one affected individual and a nonspeci?c bilateral early-onset cataract in four affected individuals.High-resolution kar-yotyping performed for four of the patients was normal.In family 2,two male siblings born from nonconsangui-neous healthy parents with unremarkable family,prenatal,and perinatal histories were affected (Figure 1,Table 1).At the age of 12years,they were both diagnosed with short stature and delayed puberty.Biologically,the results suggested a partial de?ciency of growth hormone and a hypergonadotropic hypogonadism with preferential dysfunction of Sertoli cells (Table 1).Neither pituitary gland nor brain abnormalities were observed on a cerebral MRI scan.Case F2II-1received a substitutive therapy by rhGH from age 13.The growth rate remained low despite a high dose of rhGH (55m g/kg/day).At age 17,the delayed puberty resolved progressively during treatment.He never had neurological symptoms,but cerebral MRI detected a moyamoya angiopathy with severe stenosis of the terminal portion of ICAs.Case F2II-2suffered from dys-praxia during childhood.At age 14,he had a diagnostic work-up prior to rhGH treatment.Imaging of the brain revealed a moyamoya angiopathy with occlusion of both distal ICA and an extensive neovascularization in the terri-tory of lenticulostriate arteries.Arterial hypertension was suspected and con?rmed by blood-pressure monitoring.Ultrasound examination excluded stenosis of the renal arteries.In both siblings,hypertelorism,mild bilateral ptosis,long philtrum,and ?ared nares were observed.Neither cataract nor dilated cardiomyopathy were present at the last examination (when the siblings were 17and 14years old).

In family 3,there were three affected males,including two maternal cousins for whom clinical information was available for the study (Figure 1).At the ages of 18and 21,both cousins were diagnosed with short stature (160.0cm).Neither received hormone therapy.A maternal family history of dwar?sm was present.Both mothers measured 147.3cm.Symptoms of delayed puberty or hy-pergonadotropic hypoganadism were not reported.Case F3III-7reported speech and behavioral developmental delays of 15–16months.He was diagnosed with

infantile

Figure 2.Facial Dysmorphy and Moyamoya Angiopathy of Cases F1II-2,F1III-1,and F2II-2

(A)facial dysmorphy observed in three affected members from families F1and F2.Hypertelorism is obvious in F1III-1and F2II-2,and a long philtrum is observed in all three patients.Prema-ture white hair locks are present in F1II-2and F1III-1(note that F1III-1dyed his hair).

(B and C)Moyamoya angiopathy.In case F2II-2,conventional angiography showed an occlusion of a distal internal carotid artery (purple arrow),and MRI revealed neovascularization (yellow arrows)associated with bilateral small deep infarcts (red arrows).Magnetic resonance angiography (MRA)of case F1II-2showed a stenosis of the terminal portion of the internal carotid arteries and of the origin of anterior and middle cerebral arteries associated with a left cortical infarct on FLAIR images (red arrow).Cerebral MRA and MRI did not detect any moyamoya angiopathy nor parenchymal lesion in F1III-1,the youngest patient of family F1.

febrile seizures at the age of1and epilepsy at the age of10. He is hypertensive and has hypercholesterolemia,which is present among his immediate family members.He pre-sented with right-hemisphere transient ischaemic attacks (TIAs),and an MR head scan demonstrated bilateral frontal watershed infarcts.Digital subtraction angiography showed bilateral supraclinoid internal carotid artery (ICA)occlusion and extensive moyamoya collateral vessels bilaterally(Figure S1).Case F3III-2had developmental delays in walking,speaking,and social behavior at an early age,was diagnosed with an enlarged heart at the age of8, and suffered a myocardial infarction at the age of21as measured by elevated troponin levels.Subsequent cardiac catheterization showed signi?cant coronary artery disease. He was also diagnosed with hypertension.He suffered left-hemisphere TIAs,and an MR head scan showed bilateral watershed infarcts.Digital subtraction angiography showed a right cervical ICA occlusion;reconstitution of the cavernous ICA from multiple collateral vessels, including moyamoya vessels;and in the left cervical ICA, multiple areas of irregularity and narrowing.These?ndings suggested prior occlusion with partial recanalization and occlusion of both vertebral artery origins with reconstitu-tion from collateral vessels(Figure S2).He also had dysmor-phic facial features,including a narrow cranial vault,thick eyebrows,wide nose,deeply set eyes,and low-set ears,as

Table1.Main Characteristics of Symptomatic Members of Families F1and F2

Family1Family2

III-1II-1II-2II-3II-4II-2II-1 Gender M M M M M M M

Current age(age at death),yr28(34)4145481417

Short stature of postnatal onset yes yes yes yes yes yes yes

Facial dysmorphism yes yes yes yes yes yes yes Premature canities yes yes yes yes yes no no Azoospermia yes yes yes n.a.n.a.n.a.n.a. Hormonal testing

Age at testing,yr17293044461412.5 Testis volume—Tanner Stage n.a.T1T2n.a.n.a.T1T1 Testosterone,nmol/liter a 3.47(Y)23.619.8 5.7 6.60.290.11(Y)

LH plasma levels,IU/liter b9([)14([)10([)11([)20.3([)0.3([)0.25([) FSH plasma levels,IU/liter c71([)37([)36([)38([)48.6([) 2.4 4.1([) AMH,pmol/liter d n.a.n.a.n.a.<0.35<0.35n.a.358 Inhibin B,pg/ml e n.a.<15(Y)<15(Y)<15(Y)<15(Y)n.a.n.a.

IGF-1,mcg/liter f n.a.n.a.n.a.n.a.n.a.116(Y)n.a.

Partial GH insuf?ciency g yes yes no n.a.n.a.yes yes

Stroke no yes yes yes yes yes no

Age at neurological onset,yr-223534410-

Moya-Moya angiopathy h no yes yes yes yes yes yes

History of hypertension no no no no no yes i no

Dilated Cardiomyopathy j yes yes no yes no no no

Right aortic arch n.a.no n.a.yes n.a.n.a.no

Early onset cataract yes no yes yes yes no no

a Normal values:5.4–30.4for tests performed in family1,0.2850.01for tests performed in family2.

b Normal values:1–7for family1,0.17–0.19for family2.

c Normal values:1.5–14for family1,0.44–3.1for family2.

d Normal values:15–90for family1,250–1500for family2.

e Normal values:80–270.

f Normal values:230–876.

g The de?nition of complete and partial GH de?ciency was a GH peak response of less than3and10ng/ml,respectively,after a stimulation test.

h Moyamoya angiopathy on MR Angiography or Conventional Angiography.

i No renal artery stenosis was detected on ultrasound examination.

j The de?nition of dilated cardiomyopathy was an association of left ventricular enlargement with reduction of left ventricular fraction shortening.A down arrow indicates a decrease,and an up arrow indicates an increase.

well as signi?cant graying (over 75%)of the hair at the age of 26.

Cosegregation of an Xq28Deletion in Affected Members of Family F1

The segregation of the disease in family F1strongly sug-gested an X-linked recessive pattern of inheritance.To con?rm this hypothesis,we conducted a genetic-linkage analysis in this family by using a panel of microsatellite markers spanning the X chromosome.A maximum multi-point point LOD score of 2.1(maximum theoretical LOD score in family F1)was obtained at four closely linked markers,DXS8106,DXS8045,DXS1193,and DXS8061,mapping on Xq27–q28.Haplotype analysis mapped the gene as being distal to DXS984(centromeric boundary)as the result of a recombination event in the affected indi-vidual III-1(Figure S3).None of the telomeric markers,down to the telomere,detected any recombinant event.The physical size of the linked region has been estimated to be close to 15.5Mb.

Because the pleiotropism of the clinical manifestations observed in family F1was possibly consistent with a contig-uous gene-deletion syndrome,we used an Affymetrix SNP 6.0array to screen for putative loss of genetic material within the linked region on chromosome X.We analyzed three members,including the mother (F1I-2)and two of her affected sons (F1II-3and F1II-4).

Ten consecutive copy-number probes detected putative null alleles in the two affected sons,and hemizygosity for these markers was observed in the mother (Table S3).These consecutive probes spanned a 46.3Mb interval bracketed by CN_923665and CN_923676copy-number probes (Table S3and Figure 4C).Ampli?cation of four STS markers,including ECD13623,ECD07678,ECD07752,and ECD15347,within this interval failed in

all affected males of family F1,con?rming the existence of a deletion within the linked interval.Flanking STS markers ECD21451(cen)and ECD20005(tel)were success-fully ampli?ed.We then used walking PCR with additional STS markers in combination with long-range PCR and sequencing to identify the centromeric and telomeric breakpoints.In brief,for all affected individuals we ampli-?ed a 3416bp fragment that was absent in control individ-uals and present in the heterozygous state in the obligate female carriers.The sequencing of this PCR product re-vealed that the deletion size was 41007bp long.Interest-ingly,a 32bp insertion was detected at the breakpoint junction.This insert corresponded to a small part of the ?rst intron of MTCP1/MTCP1NB .

This deletion interrupted two genes,FUNDC2and BRCC3,and deleted entirely MTCP1/MTCP1NB .The break-point within FUNDC2occurred within exon 2,and the breakpoint in BRCC3occurred within intron 3.

Altogether,these data established the existence of a40kb Xq28deletion that cosegegated with the affected pheno-type in family F1.

Detection of Additional Xq28Deletions in Families F2and F3

Family F2includes two affected brothers (F2II-1and F2II-2)whose phenotypes were strikingly similar to the one observed in family F1.The blood of both brothers and their two parents was sampled.We failed to amplify the ?rst exon of MTCP1/MTCP1NB or exons 1–3of BRCC3in proband F2II-2and his brother F2II-1,strongly suggesting the existence of a genomic deletion.Walking PCR,long-range PCR,and sequencing identi?ed a 5295bp deletion in these two affected brothers.Their mother was heterozy-gous for this deletion.The deleted fragment removed 691bp of intron 1and exon 1of MTCP1/MTCP1NB ,exons 1–3of BRCC3,and 1866bp of intron 3of BRCC3.

Family F3includes three affected males.The blood of two of them (F3III-2,F3III-7)as well as that of the mother of F3III-7(F3II-4)has been sampled.A 4218bp deletion removing 197bp of exon 1of MTCP1/MTCP1NB ,exons 1–3of BRCC3,and 1860bp of intron 3of BRCC3has been identi?ed in both cousins and the carrier mother F3II-4.

The Critical Region of Overlap between F1,F2,

and F3Deletions Includes MTCP1/MTCP1NB Exon 1and the First Three Exons of BRCC3

The three deletions observed in families F1,F2,and F3overlapped over an interval of 3362bp,which is thus de?ned as a new critical interval that includes MTCP1/MTCP1NB exon 1and the ?rst three exons of BRCC3.FUNDC2is located outside of this region,strongly suggest-ing that it is not involved in the disease.

MTCP1was until recently annotated as a single locus expressing alternatively spliced transcripts;it is now annotated as two genes,MTCP1and MTCP1NB ,sharing exon 1and encoding two distinct proteins,p13

MTCP1

Figure 3.Progression of the Moyamoya Angiopathy in Case F1II-2over 8Years

At baseline,MRA shows a bilateral stenosis of the terminal portion of the ICAs and the origin of ACAs and MCAs (yellow arrows).Three years later,a progression of the arterial stenoses is visible on MRA and punctuate ischemic lesions are detected on MRI (red arrows).A 8years follow-up examination reveals a progression of the angiopathy with left ICA occlusion (green arrow)and larger ischemic lesions.

and p8MTCP1NB .The protein p13MTCP1is not detected in normal tissues but is restricted to a rare form of mature-T-cell-proliferation leukemia.P8MTCP1NB encodes a mito-chondrial 8kDa protein of unknown function.BRCC3encodes a 36kDa ubiquitous deubiquitinating enzyme.MTCP1and BRCC3Screening in Patients with Nonsyndromic Familial Moyamoya Disease

The screening of the coding regions and intron-exon boundaries of BRCC3,MTCP1,and MTCP1NB in the probands of the three pure MMD-unrelated F4–F6families did not detect any deleterious

mutation.

Figure 4.Xq28Deletions in Families F1–F3

(F1)The deletion in family F1begins after the 153,914,898th nucleotide of chromo-some X (build 36.3)within the second exon of FUNDC2.It removes the remain-ing part of FUNDC2,MTCP1/MTCP1NB ,and three exons of BRCC3.A fragment of 32bp is inserted at the breakpoint junc-tion.

(F2)The deletion identi?ed in family F2begins after the 153,951,471th nucleotide within intron 1of MTCP1/MTCP1NB .It removes exon 1of MTCP1and three exons and part of intron 3of BRCC3.

(F2)The deletion identi?ed in family F3begins after the 153,952,544th nucleotide,within exon 1of MTCP1/MTCP1NB .It re-moves part of exon 1of MTCP1and three exons and part of intron 3of BRCC3.

(Lower panel)Schematic view of the region involved in the deletion.Approxi-mate positions of Affymetrix SNP 6.0array probes and STS markers used for deletion mapping are shown.The copy-number probes ?anking the deletion in family F1are boxed.The STS markers showing the null alleles are represented as empty bars;the ?lled bars indicate that the sequence is not deleted.The dashed black lines show the deleted region in families F1–F3.Black arrows indicate the breakpoints.The genes interrupted by the deletion are represented as vertical,differently colored bars,and arrows indicate their transcrip-tional direction.The 3362bp critical interval is represended by a dashed line,and the exons lying within this region are denoted by empty bars.

p8MTCP1NB and BRCC3Analysis in Patient Lymphoblastoid Cell Lines and in Human Vascular Tissues

Immunoblot analysis of EBV lym-phoblastoid cell lines from affected individuals F1II-2and F2II-2did not detect BRCC3or p8MTCP1NB ,whereas these proteins were readily detected in healthy control EBV cell lysates (Figure S5).

p8MTCP1NB and BRCC3were also readily detectable in HUVEC endothelial cells as well as in arterial and venous lysates from normal human individuals (Figure S4).Morpholino-Mediated Knockdown of brcc3Leads to Defective Angiogenesis in Zebra?sh

We decided to further examine the function of the genes inactivated by the deletions in families F1–F3by using the zebra?sh.The region syntenic to the portion of the genome removed by the overlapping deletion of these families is found on zebra?sh chromosome 21.The human and zebra?sh syntenic regions share similar organization;

there is a small intergenic GC island located between the mtcp1nb and brcc3loci,which are transcribed in opposite directions (Figure 5A).A mtcp1homolog is not found in ze-bra?sh.Mtcp1homologs are present in both mammals and sauropsids but are not detected in amphibians,suggesting that Mtcp1arose in the last common tetrapod ancestor shared by mammals and sauropsids (Figure S6).In situ hybridization of 24hr postfertilization (hpf)zebra?sh embryos reveals low-level ubiquitous expression of brcc3ortholog with somewhat higher levels of expression in the head and in the ventral trunk adjacent to developing vessels (Figure 5B).Expression of mtcp1nb is not detectable by in situ hybridization in 24hpf embryos (data not shown).

The role of brcc3and mtcp1nb in vascular development was analyzed by morpholino-mediated knockdown in Tg (?i1-eGFP)y1transgenic zebra?sh embryos,which permit easy visualization of blood vessels by ?uorescence micros-copy because of eGFP expression within endothelial cells of the transparent animal 15(Figure 6).Injection of mor-pholinos targeting brcc3inhibits angiogenesis of trunk intersegmental vessels (Figure 6C).This defect is amelio-rated by coinjection of wild-type brcc3mRNA (Figures 6D and 6E),suggesting that defective angiogenesis in brcc3morpholino-injected animals is due to a speci?c functional requirement for this gene.As noted above,brcc3shows a low level of ubiquitous expression,but increased expres-sion is noted in the vicinity of the developing axial blood vessels from which the intersegmental vessels sprout and

grow.To determine whether the defects in brcc3morpho-lino-injected animals re?ect a cell-autonomous require-ment for brcc3function within endothelial cells,we gener-ated ?k1-mCherry-2A-brcc3and ?k1-mCherry constructs for endothelium-speci?c expression,driven by the ?k1promoter,of either brcc3plus mCherry or mCherry alone.Injection of these constructs into zebra?sh embryos results in mosaic endothelial expression of their respective transgenes (Figures 6F–6I).Expression of brcc3in ISVs of injected Tg (?i1-eGFP)y1animals is suf?cient to rescue angiogenic sprouting and growth (arrows);in comparison,in transgenic ?k1-mCherry -expressing endothelial cells (arrowheads),no rescue was observed (Figures 6F–6J).Unlike brcc3morpholino-injected animals,mtcp1nb mor-pholino-injected animals showed no vascular phenotype at either 24hpf or 48hpf (data not shown).These results indicate that brcc3ortholog function is required cell-autonomously within the endothelium for angiogenesis.

Discussion

We report herein on three unrelated families affected by a disorder characterized by the association of a moyamoya angiopathy,short stature,and a stereotyped facial dysmor-phism.In addition,other symptoms included a hypergona-dotropic hypogonadism (7/9patients),hypertension (3/9patients),a dilated cardiomyopathy (3/9patients),prema-ture coronary heart disease (1/9patients),premature hair graying (6/9patients),and early bilateral acquired cataract (4/9patients).In these three families,we identi?ed three overlapping Xq28deletions cosegregating with the affected phenotype.The critical interval includes exon 1of MTCP1/MTCP1NB and exons 1–3of BRCC3.This dele-tion leads to a complete loss of BRCC3and MTCP1NB (the sole MTCP protein expressed in normal tissues)in affected patients.In order to investigate the in vivo conse-quences of the loss of each of these two proteins,we con-ducted morpholino knockdown experiments for both genes in zebra?sh.Knockdown of mtcp1nb did not lead to any vascular phenotype at either 24hpf or 48hpf,whereas injection of morpholinos targeting brcc3lead to defective angiogenesis,which was rescued by endothelial expression of brcc3.

The defective angiogenesis observed in brcc3morphant zebra?sh and the endothelial-speci?c brcc3rescue of these defects strongly suggest that this protein plays an impor-tant role in vascular physiology and may therefore be involved in the moyamoya angiopathy of these patients.BRCC3is a ubiquitously expressed K63-speci?c deubiqui-nating (DUB)enzyme containing a JAMM (JAB1/MPN/Mov34metalloenzyme)domain.21–24It is a member of two protein complexes,the nuclear BRCA1/Rap80/Abraxas/Merit40/BRCC45DNA repair complex and the cytoplasmic BRISC complex.25After cell irradiation,BRCC3is readily detected at DNA double-strand breaks,where it interacts with abraxas and is thought to

Figure 5.Zebra?sh Orthologs of Moyamoya Candidate Genes (A)Schematic drawing depicting the moyamoya deletion critical region in the human genome and comparing it to the syntenic region of the zebra?sh genome.An untranslated exon (cyan)is alternatively spliced (dashed lines)into two transcripts encoding the unrelated MTCP1(green)and MTCP1NB (blue)proteins.The mtcp1nb (blue)and brcc3(black)loci are conserved in zebra?sh,but mtcp1is absent.

(B)The whole-mount in situ hybridization of a 24hpf zebra?sh embryo probed for brcc3,showing lower level ubiquitous expres-sion with somewhat higher expression in the head and around the trunk axial vessels (arrows).The scale bar represents 200m m.

a positive regulator of BRCA1E3ligase activity.It is impli-cated in G2checkpoint and viability response through its DUB activity.How an alteration of DNA repair could possibly lead to moyamoya angiopathy would be entirely speculative at this point.However,several lines of evidence suggest a possible link between genomic maintenance and moyamoya.Irradiation of the neck is one of the most frequent environmental causes of moyamoya,and the clinical,radiological,and histological features of irradia-tion-induced cerebral vasculopathy and those of MMD are strikingly similar.5In addition,moyamoya angiopathy is a frequent complication of MOPDII (MIM 210720,Ma-jewski Osteodysplastic Primordial dwar?sm)and Seckel syndrome (MIM 210600,606744,613676,608664)which are recessive disorders caused by mutations of pericentrin,a protein involved in DNA-damage signaling.26–28Other clinical features,such as early acquired cataract and prema-ture hair graying,encountered in these patients are also present in several disorders caused by mutations in DNA repair genes.BRCC3is also a member of the BRISC complex (BRCC36Isopeptidase Complex),a cytoplasmic complex containing BRCC3,BRCC45,MERIT40,and ABRO1,a complex whose function was until recently totally unknown.25,29,30Recent data suggest that ABRO1and the BRISC complex might have a cardioprotective function.31Indeed,ABRO1is strongly expressed in cardiomyocytes and is upregulated in myocardial infarction.Its induction is associated with a K63-linked deubiquitination of cardiomyocyte proteins associated with a cardio-protective effect.An exciting hypothesis is that loss of the integrity of the BRISC complex could lead to the cardiomyopathy and premature coronary heart disease detected in several of our patients.In addition to moyamoya angiopathy,all patients have a short stature (and most have partial GH de?ciency).Seven out of nine patients showed a hypergonadotropic hypogonadism,suggesting a preferential dysfunction of Sertoli cells.BRCC45(also called BRE)is mostly expressed in the testis;there is a strong mRNA signal in germ cells and Sertoli cells.32Because BRCC45is a member of

both

Figure 6.Loss of brcc3Function in the Endothelium Results in Defective Angiogenesis

(A)Drawing of 24hpf zebra?sh embryo (adapted from Kimmel et al.37).The region shown in subsequent panels is highlighted in blue.(B–D)Confocal imaging of intersegmental vessels (ISVs)in 24hpf Tg (?k1a-eGFP)transgenic zebra?sh embryos injected with either control morpholino (B),brcc3morpholino (C),or brcc3morpholino plus brcc3mRNA (D).

(E)Quantitation of the percentage of ISVs that sprout and grow at least half-way up the trunk (to the horizontal myoseptum)in morpholino-and mRNA-injected animals.

(F–I)Confocal imaging of ISVs in 24hpf Tg (?k1a-eGFP)transgenic zebra?sh embryos injected with either ?k1-mCherry-2A-brcc3(F and G)or ?k1a-mCherry (H and I)transgene DNA,showing EGFP-positive vessels (F and H)and mCherry-positive transgene-expressing cells (G and I).

(J)Quantitation of the percentage of ISVs that sprout and grow at least half-way up the trunk (to the horizontal myoseptum)in ?k1a-mCherry-2A-brcc3or ?k1a-mCherry transgene-injected animals.For the quantitative data in panels (E)and (J),ten ISVs from each of 12different injected embryos were scored for each control and experimental condition.Error bars show the standard error of the mean for these data.Scale bars represent 25m m (B–D)and 25m m (F–I).

BRCC3complexes,either of these two complexes could also be involved in this endocrine phenotype.Interest-ingly,hypergonadotropic hypogonadism and infertility are important clinical features encountered in several chro-mosome-breakage and-instability syndromes,including Fanconi anemia.33

Because both MTCP1/MTCP1NB and BRCC3are inter-rupted in all three families,a role for MTCP1/MTCP1NB loss in the disease phenotype cannot be ruled out at this point.MTCP1,also called c6.1B,was initially identi?ed because of its involvement in the t(X;14)(q28;q11)translo-cation,which is associated with a rare subset of T cell pro-lymphocytic leukemia,involving proliferation of mature T cells.34–36It leads to two types of transcripts,MTCP1 and MTCP1NB,with two distinct open reading frames. The longest transcript encodes p13MTCP1,a protein that is not found in any normal tissue but only in T cell prolym-phocytic leukemic cells,in which the t(X;14)translocation event activates a cryptic promoter sequence within MTCP1 intron 1.p8MTCP1NB encodes a mitochondrial8kDa protein whose function is unknown and which is detected in various normal tissues,including arteries and veins,and is overexpressed in mature T cell leukemia cells.This unusually small68amino acid protein is expressed in the mitochondria.However,at present we have no idea as to the putative pathophysiological links between its loss and the phenotype,or part of the phenotype,reported herein.

In summary,we report herein on a novel X-linked syndromic moyamoya disorder whose investigation leads to the identi?cation of a deletion removing two genes with a previously unsuspected role in vessel physiology. Several lines of evidence strongly suggest that one of these two genes,BRCC3,might play an important role in angio-genesis and vessel maintenance.

Supplemental Data

Supplemental Data include six?gures and three tables and can be found with this article online at https://www.wendangku.net/doc/d68418148.html,/AJHG/. Acknowledgments

We thank affected individuals and their families for their participa-tion in this reasearch program.We also thank the French Tanguy moyamoya association of patients.We are also indebted to N.Al-lili,M.G.Bousser,S.Chabrier,A.Gaudric,E.Houdart,V.Krivosic, D.Logeart,J.P.Metzger,P.Touraine,and A.Verloes for excellent clinical evaluation of some of the patients,to M.H.Stern for help-ful discussions and the MTCP1NB polyclonal antibody,to Marc Nomaksteinsky for microsatellite analysis,and to Audrey Dela-forge for excellent technical help in preparing the samples. Research was supported by the INSERM and the Leducq Founda-tion(grant to E.T.L.and B.M.W.),Foundation Leducq Transat-lantic Network of Excellence(07CVD02Hemorrhagic Stroke), the intramural program of the Eunice Kenndy Shriver National Institute of Child Health and Human Development(NICHD;to B.M.W.),and the Huber Family Moyamoya Fund(G.K.S.).Received:March17,2011

Revised:April18,2011

Accepted:April26,2011

Published online:May19,2011

Web Resources

The URLs for data presented herein are as follows:

Database of Genomic Variants,http://project.tcag.ca/variation/ International HapMap Project,https://www.wendangku.net/doc/d68418148.html,/ https://www.wendangku.net/doc/d68418148.html,/

NCBI BLAST,https://www.wendangku.net/doc/d68418148.html,/Blast.cgi/

NCBI Entrez Gene,https://www.wendangku.net/doc/d68418148.html,/gene/http:// https://www.wendangku.net/doc/d68418148.html,/gene

NCBI Single Nucleotide Polymorphism Database,http://www. https://www.wendangku.net/doc/d68418148.html,/snp/

NCBI UniSTS,https://www.wendangku.net/doc/d68418148.html,/unists/

Online Mendelian Inheritance in Man,https://www.wendangku.net/doc/d68418148.html,/ UCSC Genome Browser,https://www.wendangku.net/doc/d68418148.html,/

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Schilling,T.F.(1995).Stages of embryonic development of the zebra?sh.Dev.Dyn.203,253–310.

网络文明演讲稿3篇

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INTERNET演讲稿

so there are more and more people who have became internet users. do you know how many internet users in the world? let’s see the chart! 3 this is a questionnaire [?kw?st??n??r, ?kwest???ne?] (调查卷) about world internet users and population stats in 2009. the users has grown [gr?un] at a remarkable [ri?mɑ:k?bl] rate 。the rising line(上升的曲线） is not this ,not this ,is this. let us see the next pictures. 4 who is he？yes .our respectable premier [?premi?] wen .how old is he? a ha .this i dont know ,but i know he is very old , he is also surfing the internet and caring about current events and politics .so how we do ? 5 yes .the man may be watch a funny movie. the mother is telling her children learning some knowledge through the internet. in this picture .the people from different nations also learn the same thing-------how to use the internet. even the lovely dog is also want to use the internet. why are many people attracted by internet? now im not surprised to see the results, for i can find the reason just from my own life. 6 the internet changed my life; there is no doubt about that. to spend a part of our day on the internet is quite normal for many people. 7 second. the internet is a database ?deit?beis] full of latest information and offers me a lot of services. i can read the daily newspapers, movies and music from all over the world. 8 when i saw this news, my mood just like this picture—angry birds .how cruel the passed people are .ok, let me back to the topic .this is an online game--- angry birds. that’s another important part for internet .the online games are always attractive and challenging. in my opinion its more exciting to play with friends than playing alone. 9 third. to improve our english, through internet i often find professional english learning methods. is internet good or bad we should admit that there are so many advantages brought by the internet. firstly, the internet affords us lots of convenience. for example, we can shop, have meetings and even study on-line. furthermore, the internet has improved our working efficiency. we can contact colleagues on the other side of the world to talk about the working project via the internet easily. piles of files can be sent by e-mails with the help of the internet. in addition, the internet makes information conveyance much easier. just clipping “google”, related information will boom out explosively within several seconds. while applauding for those benefits the internet brings to us, we need to worry about disadvantages of the internet as well. to begin with, it is easy for the young to indulge themselves in the so-called “cyber romance”, which is full of dangers, cheatings

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文明上网演讲稿【五篇】【篇一】文明上网演讲稿各位领导，各位老师，同学们：大家早上好！我今天演讲的题目是《告别痴迷，文明上网》。现在随着中国社会的不断发展，人们的生活也逐渐富裕起来，网络也跟着在社会中流行，融入到了我们生活中去，而且扩张的范围很大，尤其是对于中学生来说更是对“网络”爱不释手。为此，上个星期，老师叫我写有关网络文明的演讲稿，我想就写“告别网吧，做文明健康中学生”为题的演讲稿吧，于是我就中学生告别网吧一事专门调查了几位同学，他们中的一些坚定的摇了头，并说出了一大堆上网的好处，而且反问了我：“你也不是经常上网吗？”，于是，产生了我的几个思考：告别网吧，是不是就等于告别了网络？告别网吧现实吗？我们的中学生在接触网络时，该如何把持自己呢？老师说过，21世纪是知识经济的时代，是网络时代，是人类数字化生存的时代，电脑和网络，是每一个学生都必须掌握的一门课程。在美国等西方国家，四五年级的学生都能够熟练地使用电脑、网络、查阅资料，学习知识。相比之下，让我们中学生告别网络的做法，我想在座的同学没有一个会同意的。但我们法律为什么又规定“禁止未成年人进入网吧”？首先，我们不能把“网络”等同于“网吧”。不接触网吧，我们同样可以接触网络。比如通过：家庭个人电脑、学校电脑室等，当然，这方面的开发和利用，有待学校、老师、家长和我们同学的共同认识基础上逐步实现。第二，表面禁止的同时，深含对我们未成年人的身心保护。我们中学生迷恋网吧，不能自拔，导致学业无成，甚至是猝死网吧的现象时有耳闻，一旦事件发生，我们总认为这是因无知而犯下的错，但也为时已晚。“禁止未成年人进入网吧”是为了让我们的学生少犯或不犯同样的错误，让更多的人来关爱我们这些未成人。但是，目前那些黑网吧，像一个个美丽的陷阱，使许多同学丢魂失魄、丧失意志、无心学习、前途废弃。据调查，学生上网80%以上是打游戏，15%左右是交友聊天，真正查询资料用于学习的为数极少。有13%以上的男生很喜欢上网或迷恋上网，达到了严重影响学习的地步。一到寒暑假，学生们更是肆无忌惮，不

文明上网演讲稿

理性面对网络诱惑尊敬的领导、老师，亲爱的同学们：大家早上好！我是来自八（6）班的贺靖，今天，演讲的题目是：理性面对网络诱惑。非洲原野上有一种花，它色彩斑斓，芳香扑鼻，路过的飞虫往往经不起这种诱惑，扑上去贪婪地吮吸，并不知道这它分沁的是一种毒液。很快，花的黏液将贪吃的飞虫牢牢地粘住，花瓣悄悄的合拢，把这些小虫都关闭起来，变成了腹中之物。学习、生活中，我们每个人都经历着大大小小的诱惑。迷茫时，网络中虚拟世界的五光十色是一种诱惑；疲惫时，网络游戏的惊险刺激也是一种诱惑。互联网拓宽了我们的求知途径，为我们打开了认识世界的一扇窗，更为我们创造了一个展现自我个性的空间，当然也存在着传播不健康信息、提供不文明服务等严重危害社会的问题，对学生们身心健康的危害尤其严重。有调查显示，11.4%的学生有长时间玩电子游戏的习惯。随着学习阶段的上升，长时间玩电子游戏的比例呈上升趋势。网络既能造就人才，也能毒害人类，有的同学因沉迷网络，导致学习成绩一落千丈；更有同学因长时间玩游戏，视力严重下降；还有同学受不良信息的影响，走上犯罪的道路。每次放假，父母在家里备好了饭菜，对我们的归来翘首以盼。有些同学却三五成群地在网吧污浊的空气里挥汗如雨，忘乎所以。虚拟世界在一步步隔断我们的亲情，减退我们的交流能力。更严重的是，许多同学沉迷于网络游戏，放假的时间全泡在网络游戏里，荒废了学业，浪费了青春。一旦上瘾，甚至会不顾一切翻墙出校，最后被学校开除，留下终身的遗憾与忏悔。所有这些情景，令人心痛令人警醒。我们中小学生是祖国的未来，家庭的希望，是最具科技意识和创新能力的一代。如果在上网时，我们不抵制不良信息、不理性面对网络游戏、不能够做到文明上网和健康上网，那网络不仅网住了我们的心，更会毁掉我们的一生！为此，为践行“文明上网，理性上网，拒绝网络诱惑”的行动，我提倡： 1、利用自动关机软件或者闹钟来提示和控制自己的上网时间。让外力成为一副让人清醒的良药。 2、详细记录下自己每次上网所做的事，看看哪些是有益的，哪些是在浪费时间，从中改变自己的不良习惯，形成不看无用信息，抵制网络游戏的习惯。 3、在学校或在家里上网时，自觉遵守网络文明，上网时不使用侮辱、谩骂性的语言聊天。不轻易和网友约会，自觉遵守互联网规范，自觉抵制不良网络信息的侵害。 4、我们上网要做到“三上”和“三不上”。“三上”即不进营业性网吧，不进色情、垃圾网站，不沉迷网络游戏。“三上”是健康上网，把网络作为获取知识的园地；文明上网，听听网络专家的视频课堂，拓宽我们的求知途径，正确利用这个展现自我个性的空间；绿色上网，熟悉上网的安全通道。同学们，如果你能正确使用网络，可以学到比课本更多的知识；如果正确使用网络，你会获得一个更广阔的发展空间。同学们，请让我们告别网络不良行为，学会辨明是非，提高自身控制力，理性面对网络诱惑，让人类的科技发明助力我们成长，让我们的学习更上一层楼！我的发言完毕，谢谢大家！

网络文明演讲稿范文

网络文明演讲稿范文尊敬的老师，同学们：大家好! 我将要给大家演讲《网络——把锋利的双刃剑》。众所周知，随着时代的时步，科技的更新，更多的新事物涌入我们的世界，其中也包括了互联网。自90年代后期互联网进入我国以来，网络就如同雨后春笋般，发展迅速，如今已遍布城乡。作为一种新的传媒手段，互联网在经济建设和人们的各项活动中起着不可忽视的作用，同时也为青春少年的学习.交流以及娱乐提供了更为广阔的天地。但是对于为成年人来说，网络则是一把锋利的“双刃剑”，一朵带刺的玫瑰，很多青少年正是因为过度沉迷于网络，沉迷于网吧，缺乏自我保护意识和对危险的预防能力，对其学业.健康和思想都造成了巨大的危害。由此可见，网络可以造就人才，也可以把人才引向无底深渊，以下是我总结的三点上网的危害：一.网络对青少年的人生观.世界观和价值观形成的构成潜在威胁。

二.网络使许多青少年沉溺于虚拟的世界脱离现实，也使一些青少年荒废学业。很多人把游戏称作“电子海络因”是不无道理的。三.互联网中的不良信息和网络犯罪对青少年的身心健康和安全构成危害和威胁。许多学生因此耽误了自己的学习.废弃了美好的前程。据一项调查显示，上网学生超过80%是在打游戏， 15%左右是在交友聊天，真正查询资料用于学习的人数少之又少。沉迷于网络的学生，别说是上课学习，就连吃饭睡觉也能省就省，一天到晚迷迷糊糊，欲罢不能，可换来的是什么呢?在此，我呼吁那些还于网络游戏，沉侵在虚幻甜蜜中的同学，当你替身意意正酣的时候，你可曾想到焦急的父母在正在等待你回家的消息?当你陶醉于刀光剑影的时候，你可曾想辛苦的老师为你耕耘的身影?当你于新识的网友高谈阔论的'时候，你可曾想到同学间真挚的话语才是真正的友情?当你沉溺在那些略带灰色的网络文学中的时候，你可曾想到现实世界正在渐渐离你远去?同学们，快从虚幻的世界解脱出来吧，一样最宝贵的东西——时间，正从你点击鼠标，轻巧键盘的手指尖悄悄溜走，无法挽回。同学们，一个人的青春能有几年?莫待白了少年

“健康上网文明上网”演讲稿

“健康上网文明上网”演讲稿 “健康上网文明上网”演讲稿亲爱的老师、同学们，大家好！这里是九中青春校园广播站，我是高一6班的董晓骐。今天我演讲的题目是《远离网吧，健康上网、文明上网》现在互联网越来越普及当今社会已进入以互联网为标志的信息时代。网络给我们带来许多方便，我们可以在网上查阅资料；看最新的消息还可以开阔我们的视野促进我们的学业。我们的老师也充分地利用网上资源努力提高我们的学习成绩。但网络也是一把双刃剑，有的同学过度依赖电脑整天沉迷于网络游戏之中，课也不上学习成绩直线下降有的同学做了网络的俘虏成了“小网虫”还

有的同学因整天看着电脑眼睛受到严重损坏成了高度近视。甚至有的同学上网的目的只是打游戏或者聊天，一刻也不停歇地上网、上瘾甚至酿成了惨剧。 17岁的少年吴x治2001年第一次在网吧接触了电脑游戏后就不可收拾从自己省钱发展到主动要钱最后就是偷钱，2004年6月15日吴x治想打游戏向奶奶要钱，奶奶没有答应他就残忍地将自己奶奶杀害。最近某县城也发生了一件类似的事件，一位女孩因父母不在家连续上网四天四夜，最后昏倒在电脑旁幸好抢救及时避免了悲剧的发生。当前青少年上网人数较多，在我国2650万上网者中25岁以下的青少年占了百分之八十五以上，并且正在以每年翻一番的惊人速度增长。这当中约2/3的人在网吧上网，在网吧上网的人中99%以上的都是沉迷于网络游戏，绝大多数学生因此学业荒废，毁掉了自己美好的前程。在我看来我们学生应当上好现代信息技术课，掌握基本的电脑技能，

条件允许的话在家长或老师的监督和指导下可适度上网，并做到“五要五不要”即：要善于网上学习，不浏览不良信息；要诚实友好交流，不侮辱欺诈他人；要增强自我保护意识，不随意约会网友；要维护网络安全，不破坏网络秩序；要有益身心健康，不沉溺虚拟时空。但决不能到网吧上网要相信只要你学业有成将来是一定能在网上冲浪和遨游的。所以希望同学们远离网吧在家长和老师的指导下健康上网、文明上网，使网络成为我们成长的好帮手而不是扼杀我们生命的毒瘤。今天九中青春广播到此结束，谢谢大家收听。

“健康上网文明上网”演讲稿

“健康上网文明上网”演讲稿尊敬的读友您好：本文由网络收集而来，分享到本网站是为了能够帮助到大彖大家如果雕之后是自己需要的文档可以点击下载本文档，下我文档是收费的（所以请先阅读再下载■谢谢各位读友’本人在此祝各位读友工作顺利’事事如意。健康上网文明上网”演讲稿亲爱的老师、同学们，大家好！这里是九中青春校园广播站，我是咼一一6 班的董晓骐。今天我演讲的题目是《远离网吧，健康上网、文明上网》现在互联网越来越普及当今社会已进入以互联网为标志的信息时代。网络给我们带来许多方便，我们可以在网上查阅资料；看最新的消息还可以开阔我们的视野促进我们的学业。我们的老师也充分地利用网上资源努力提高我们的学习成绩。但网络也是一把双刃剑，有的同学过度依赖电脑整天沉迷于网络游戏之中，课也不上学习成绩直线下降有的同学做了网络的俘虏成了小网虫”还有的同学因整天看着电脑眼睛受到严重

损坏成了高度近视。甚至有的同学上网的目的只是打游戏或者聊天，一刻也不停歇地上网、上瘾甚至酿成了惨剧。仃岁的少年吴x治2001年第一次在网吧接触了电脑游戏后就不可收拾从自己省钱发展到主动要钱最后就是偷钱，2004年6月15日吴x治想打游戏向奶奶要钱，奶奶没有答应他就残忍地将自己奶奶杀害。最近某县城也发生了一件类似的事件，一位女孩因父母不在家连续上网四天四夜，最后昏倒在电脑旁幸好抢救及时避免了悲剧的发生。当前青少年上网人数较多，在我国2650万上网者中25岁以下的青少年占了百分之八十五以上，并且正在以每年翻一番的惊人速度增长。这当中约2/3的人在网吧上网，在网吧上网的人中99%以上的都是沉迷于网络游戏，绝大多数学生因此学业荒废，毁掉了自己美好的前程。在我看来我们学生应当上好现代信息技术课，掌握基本的电脑技能，条件允许的话在家长或老师的监督和指导下可适度上

文明上网演讲稿

( 演讲稿 ) 单位：_________________________姓名：_________________________日期：_________________________ 精品文档 / Word文档 / 文字可改文明上网演讲稿 Civilized Internet Speech

文明上网演讲稿【篇一】文明上网演讲稿各位领导，各位老师，同学们：大家早上好！我今天演讲的题目是《告别痴迷，文明上网》。现在随着中国社会的不断发展，人们的生活也逐渐富裕起来，网络也跟着在社会中流行，融入到了我们生活中去，而且扩张的范围很大，尤其是对于中学生来说更是对“网络”爱不释手。为此，上个星期，老师叫我写有关网络文明的演讲稿，我想就写“告别网吧，做文明健康中学生”为题的演讲稿吧，于是我就中学生告别网吧一事专门调查了几位同学，他们中的一些坚定的摇了头，并说出了一大堆上网的好处，而且反问了我：“你也不是经常上网吗？”，

于是，产生了我的几个思考：告别网吧，是不是就等于告别了网络？告别网吧现实吗？我们的中学生在接触网络时，该如何把持自己呢？老师说过，21世纪是知识经济的时代，是网络时代，是人类数字化生存的时代，电脑和网络，是每一个学生都必须掌握的一门课程。在美国等西方国家，四五年级的学生都能够熟练地使用电脑、网络、查阅资料，学习知识。相比之下，让我们中学生告别网络的做法，我想在座的同学没有一个会同意的。但我们法律为什么又规定“禁止未成年人进入网吧”？首先，我们不能把“网络”等同于“网吧”。不接触网吧，我们同样可以接触网络。比如通过：家庭个人电脑、学校电脑室等，当然，这方面的开发和利用，有待学校、老师、家长和我们同学的共同认识基础上逐步实现。第二，表面禁止的同时，深含对我们未成年人的身心保护。我们中学生迷恋网吧，不能自拔，导致学业无成，甚至是猝死网吧的现象时有耳闻，一旦事件发生，我们总认为这是因无知而犯下的错，但也为时已晚。“禁止未成年人进入网吧”是为了让我们的学生少犯或不犯同样的错误，

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