Ng Dean Yew, Dennis. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 85-98). / Abstract --- p.i / Acknowledgments --- p.iii / Table of contents --- p.iv / List of figures --- p.vii / List of tables --- p.ix / List of abbreviation --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- History --- p.1 / Chapter 1.2. --- Classification and structure of Epstein-Barr Virus --- p.2 / Chapter 1.3. --- Genomic organization of EBV --- p.3 / Chapter 1.4. --- Replication cycle of EBV --- p.5 / Chapter 1.5. --- EBV latent and lytic cycle proteins --- p.6 / Chapter 1.6. --- Clinical diseases associated with EBV Infection --- p.11 / Chapter 1.7. --- Association of EBV and NPC --- p.13 / Chapter 1.8. --- EBV serological markers in the diagnosis of NPC --- p.13 / Chapter 1.9. --- Sources of EBV-specific DNase --- p.15 / Chapter 1.10. --- Characteristics of Epstein-Barr virus alkaline DNase --- p.15 / Chapter 1.11. --- Aim of the project --- p.18 / Chapter Chapter 2 --- Materials & Methods / Chapter 2.1. --- Molecular cloning --- p.19 / Chapter 2.1.1. --- Cell culture --- p.19 / Chapter 2.1.2. --- mRNA purification --- p.19 / Chapter 2.1.3. --- First strand cDNA synthesis --- p.21 / Chapter 2.1.4. --- Polymerase chain reaction (PCR) of cDNA --- p.21 / Chapter 2.1.5. --- Purification of PCR product after gel electrophoresis --- p.22 / Chapter 2.1.6. --- Ligation of PCR amplified DNase gene into pUC18 Sma/BAP vector --- p.23 / Chapter 2.1.7. --- Transformation by electroporation --- p.24 / Chapter 2.1.7.1. --- Cell preparation --- p.24 / Chapter 2.1.7.2. --- Electroporation procedure --- p.25 / Chapter 2.2. --- Extraction ofplasmid DNA --- p.28 / Chapter 2.2.1. --- Boiling preparation --- p.28 / Chapter 2.2.2. --- Plasmid digestion --- p.29 / Chapter 2.3. --- Large-scale purification ofplasmid --- p.29 / Chapter 2.4. --- Small-scale purification ofplasmid --- p.32 / Chapter 2.5. --- DNA sequencing --- p.33 / Chapter 2.5.1. --- Annealing of primer to template DNA --- p.33 / Chapter 2.5.2. --- Labelling reaction --- p.34 / Chapter 2.5.3. --- Sequencing termination reaction --- p.35 / Chapter 2.5.4. --- Prepartion of sequencing gel --- p.36 / Chapter 2.5.5. --- Autoradiography of sequencing gel --- p.38 / Chapter 2.6. --- Epitope mapping --- p.39 / Chapter 2.6.1. --- Processing of EBV- specific DNase peptides --- p.39 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Molecular cloning --- p.41 / Chapter 3.1.1. --- Cell culture --- p.41 / Chapter 3.1.2. --- mRNA purification --- p.42 / Chapter 3.1.3. --- PCR amplification --- p.42 / Chapter 3. 1.4 --- DNA purification of PCR product --- p.42 / Chapter 3.1.5. --- Molecular cloning of PCR amplified DNase gene into pUC18 SmaI/BAP vector --- p.44 / Chapter 3.1.6. --- Transformation by electroporation --- p.46 / Chapter 3.1.7. --- Extraction of plasmid DNA --- p.48 / Chapter 3.1.7.1. --- Boiling preparation --- p.48 / Chapter 3.1.8. --- Plasmid digestion --- p.51 / Chapter 3.2. --- DNA sequencing --- p.51 / Chapter 3.2.1. --- Comparison of B95-8 EBV-speicific DNase gene with gene sequence of EBV in GeneBank --- p.50 / Chapter 3.2.2. --- Comparison of 5' end of Raji & B95-8 EBV derived EBV-specific DNase gene --- p.57 / Chapter 3.2.3. --- Comparison of the 3'end of the Raji and B95-8 denved EBV-specific DNase gene --- p.63 / Chapter 3.2.4. --- Amino acid sequence homology between B95-8 & Raji EBV-specific DNase --- p.64 / Chapter 3.2.5. --- Amino acid sequence comparison between the 3' end of the B95-8 EBV DNase protein with that of the Raji EBV DNase protein --- p.62 / Chapter 3.3. --- Epitope mapping --- p.67 / Chapter 3.3.1. --- Amino acid key --- p.67 / Chapter 3.3.2. --- Amino acid sequence of peptides --- p.73 / Chapter 3.3.2. --- O.D. readings at 492nm of five histologically proven NPC sera --- p.74 / Chapter Chapter 4 --- Discussions / Chapter 4.1. --- Overall strategy --- p.75 / Chapter 4 2 --- Significance of EBV-specific DNase as marker for NPC --- p.76 / Chapter 4.3. --- Characterization of EBV-specific DNase --- p.76 / Chapter 4.4. --- Molecular cloning of PCR amplified gene into PUC18 SmaI/BAP vector --- p.77 / Chapter 4.4.1. --- Cell culture --- p.77 / Chapter 4.4.2. --- PCR amplification --- p.73 / Chapter 4.4.3. --- "Blunting,kinasing and ligation of EBV-specific DNase cDNA into pUC18 vector" --- p.78 / Chapter 4.4 .4 --- .Transformation by electroporation --- p.80 / Chapter 4.4.5. --- Restriction enzyme digestion of pUC18/EBV-DNase plasmid … --- p.81 / Chapter 4.5. --- DNA sequencing --- p.81 / Chapter 4.6. --- Epitope mapping --- p.83 / Reference --- p.85

Systemic lupus erythematosus is an autoinflammatory disorder driven by the development of autoantibodies to self-nucleic acids, in particular to DNA and to chromatin. Loss-of-function mutations of the secreted deoxyribonuclease DNASE1L3 have been implicated in the development of aggressive familial lupus. In addition, recent genome-wide association studies have linked a hypomorphic variant of DNASE1L3 to sporadic lupus. Studies in the lab determined that Dnase1l3-deficient mice develop rapid autoantibody responses against dsDNA and chromatin, and at older ages this leads to a lupus-like inflammatory disease. These disease manifestations were completely independent of the intracellular DNA sensor STING, which has been implicated in other examples of self-DNA driven autoinflammatory diseases. My project focused on developing assays to track the activity of DNASE1L3, as well as identifying the endogenous source of self-DNA normally processed by DNASE1L3. Using mouse models that allow the depletion of specific cell populations, we found that circulating DNASE1L3 is produced by hematopoietic cells, in particular by CD11c+ dendritic cells and by tissue macrophages. Taking into account the unique properties of DNASE1L3, we discovered that this enzyme is uniquely able to digest chromatin contained within and on the surface of apoptotic microparticles. Loss of DNASE1L3 activity in circulation results in elevated levels of DNA in plasma, in particular within microparticles. Microparticles are extensively bound by anti-chromatin autoantibodies isolated from both murine models of lupus as well as prototypical human clones. In addition, Dnase1l3-deficient mice have high levels of circulating IgG that bind to microparticles from young ages, and these titers increased as disease progressed in aged animals. Pretreatment of microparticles with DNASE1L3 largely abrogated this binding, demonstrating that DNASE1L3 directly reduces the immunogenicity of microparticles. We also studied two human patients with null mutations in DNASE1L3, and observed increased DNA circulating in plasma and, in particular, in their microparticles, demonstrating a conserved role for DNASE1L3 in mice and humans. Finally, we obtained plasma samples from a cohort of patients with sporadic SLE, and found that roughly 80% had circulating IgG that avidly bound microparticles. Roughly half of this group failed to bind to microparticles that had been pretreated with DNASE1L3, and this DNASE1L3-sensitive group also presented with lower levels of DNASE1L3 activity. We conclude that extracellular chromatin associated with microparticles acts as a potential self-antigen capable of causing loss of tolerance to self-DNA and inflammatory disease in both mice and humans. The secretion of a DNA-processing enzyme thus represents a novel, conserved tolerogenic mechanism by which dendritic cells restrict autoimmunity.

Bibliography: leaves 143-172 / vi, 172 leaves, [25] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1989

by Jose Victor Lopez. / Typescript. / Thesis (M.S.)--Florida State University, 1988. / Bibliography: leaves 98-109.

DNA double strand breaks (DSB) are the most toxic of all types of DNA lesions. In Saccharomyces cerevisiae, DNA DSBs are predominantly repaired by the homologous recombination repair (HRR) pathway. The initial step of HRR requires extensive processing of DNA ends from the 5' to 3' direction by specific endo-exonuclease(s) (EE) at the DSB sites, but no endo-exonuclease(s) has yet been conclusively determined for such processing of DSBs. S. cerevisiae TRM2/RNC1 is a candidate endo-exonuclease that was previously implicated for its role in the HRR pathway and was also shown to have methyl transferase activity primarily located at its c-terminus. / In this dissertation, we provided compelling biochemical and genetic evidence that linked TRM2/RNC1 to the DNA end processing role in HRR. Trm2/Rnc1p purified with a small calmodulin binding peptide (CBP) tag displayed single strand (ss) specific endonuclease and double strand (ds) specific 5' to 3' exonuclease activity characteristic of endo-exonucleases involved in HRR. Intriguingly, purified Trm2/Rnc1p deleted for its C-terminal methyl transferase domain retained its nuclease activity but not the methyl transferase activity indicating that the C-terminal part responsible for its methyl transferase function is not required for its nuclease activity. / Our genetic and functional studies with S. cerevisiae trm2/rnc1 single mutants alone or in combination with other DNA DSB repair mutants after treatment with the DNA damaging drug methyl methane sulfonate (MMS) or IR that is believed to produce DSBs, or with specific induction of DNA DSBs at the MAT locus by HO-endonuclease demonstrated an epistatic relationship of TRM2/RNC1 with the major recombination factor RAD52. These studies suggested that TRM2/RNC1 probably acts at an earlier step than RAD52 in the HRR pathway. The genetic evidence also indicated a possible functional redundancy with the bona fide endo-exonuclease EXO1 in the processing of DNA ends at the DSB sites. / In a recent report, the immuno-purified mouse homologue of TRM2/RNC1 exhibited similar enzymatic properties as the endo-exonucleases involved in HRR. A small molecular inhibitor pentamidine specifically inhibited the nuclease activity of the mouse EE and sensitized various cancer cells to DNA damaging agents commonly used in cancer chemotherapy. We specifically suppressed expression of the mouse EE using small interfering RNA (siRNA) that conferred sensitivity of B16F10 melanoma cells to a variety of DNA damaging drugs often used in cancer treatment. This further validated our earlier claim of the endo-exonuclease as a potential therapeutic target in treating cancer.

Orientadores: Stephen Hyslop, Carla Beatriz Collares-Buzato / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T23:21:13Z (GMT). No. of bitstreams: 1 Nascimento_JulianaMinardi_D.pdf: 8097756 bytes, checksum: 5350a8b542e9015bd5486f4418b36285 (MD5) Previous issue date: 2008 / Resumo: As peçonhas de serpentes Bothrops são responsáveis por diversos danos locais (na região da mordida) e sistêmicos durante o envenenamento. Dentre as manifestações sistêmicas, a insuficiência renal aguda e um dos mais importantes efeitos tóxicos causados por acidentes botropicos. Esta tese teve como objetivos: 1) investigar a ação da peçonha bruta de B. alternatus em células epiteliais renais MDCK; 2) proceder a purificação e caracterização de uma DNase acida presente nesta peçonha; e 3) determinar a possível ação apoptotica desta DNase sobre células MDCK em cultura. Com relação as ações in vitro do peçonha bruta de B. alternatus, observamos modificações no citoesqueleto, nas junções intercelulares e sobre a morte celular das células MDCK tratadas com 10 ou 100 µg/mL de peçonha bruta. Dentre as modificações observadas, houve uma diminuição da resistência transpitelial, uma redistribuição de algumas proteínas associadas as junções intercelulares acompanhada por um desarranjo do citoesqueleto envolvendo as fibras de estresse na superfície basal e adesão focal associada a F-actina na região de contato celula-matriz. A analise morfometrica mostrou uma diminuição do numero de células entrando em mitose e um aumento do numero de células com núcleos picnoticos ou morfologicamente alterados apos tratamento com o peçonha. Microscopia eletrônica de varredura revelou uma densidade de microvilosidades diminuída, assim como a alteração da morfologia celular normal, de poliédrico para um formato fusiforme, nas células tratadas. O tipo principal de morte celular induzido pelo tratamento com o peçonha bruta foi a necrose, com uma freqüência pequena de indução de apoptose. O pré-tratamento das células MDCK com catalase, superoxido dismutase e L-NAME inibiu os efeitos sobre morte celular causados pelo peçonha bruta, indicando o envolvimento de espécies reativas de oxigênio e nitrogênio nos danos causados pelo envenenamento pela peçonha de B. alternatus. As peçonhas ofídicas possuem uma grande variedade de enzimas que degradam ácidos nucléicos e seus constituintes. As desoxirribonucleases (DNases) são endonucleases presentes em peçonhas de serpentes que hidrolisam acido desoxirribonucleico (DNA). Sua ação no envenenamento ocorre em conjunto com outras enzimas que quebram ligações fosfato (ATPases, 5¿-nucleotidases, fosfodiesterases, ribonucleases). Vários estudos sobre endonucleases em mamiferos sugerem que a DNase II (DNase acida) seria responsável pela fragmentacao do DNA durante a apoptose. E também conhecido que peçonhas são capazes de induzir apoptose em células. Através de uma combinação de cromatografias de troca iônica, gel filtração, afinidade e HPLC, foi purificada uma DNase da peçonha de B. alternatus, com atividade especifica de 3.489 unidades/mg (atividade especifica da peçonha: 65 unidades/mg) e fator de purificação de ~54 vezes. As características bioquímicas desta enzima são: uma massa molecular de ~31 kDa, ausência de subunidades, pI de 4,4-5,2, pH otimo de atividade de 4,7, termo estabilidade ate 40oC. O Km e Vmax são 10.1 µg/mL e 352.5 U/mg, respectivamente. A enzima degrada preferencialmente DNA de dupla fita, com atividade menor sobre DNA desnaturado; também degrada DNA circular (dos plasmideos pGEM e pBR322) mas não degrada RNA ou poly A. E inibida por altas concentrações (100 mM) de cations (Ca2+, Mg2+ e Zn2+), e também por N-etilmaleimida, iodoacetamida, acido aurintricarboxilico e DTT, mas não por EDTA. A enzima e reconhecida por IgG antibotropica em Western blot e no ELISA e por anti-DNase II humana no Western blot. Em celulas MDCK, a DNase produz alterações morfologicas como vacuolacão do citoplasma, retração e destacamento das celulas em monocamada, presença de núcleos condensados e/ou fragmentados. Através de ensaios de viabilidade e citotoxicidade, verificamos que a DNase e citotoxica as células MDCK em doses a partir de 400 U/mL (~20 µg/mL). Alem disso, causa fragmentação de DNA e um aumento no numero de células apoptoticas, em menor proporção, de células em necrose. A via de apoptose estimulada pela enzima envolve a ativação das caspases 3, 8 e 9, e uma diminuição na expressão da proteína anti-apoptotica Bcl-2. Estes resultados mostram que a peçonha de B. alternatus e citotoxica as células MDCK, com parte desta toxicidade mediada pela DNase II. Esta enzima induz apoptose que poderia contribuir para a toxicidade geral da peçonha / Abstract: Bothrops snake venoms are cytotoxic to a variety of cells (endothelial, smooth muscle, renal and inflammatory cells), and may cause cell death by apoptosis. The venom components implicated in apoptosis include metalloproteinases and L-amino acid oxidase. In contrast, although acidic deoxyribonucleases (DNase II) have been implicated in DNA fragmentation during apoptosis in mammals, nothing is known of the involvement of venom deoxyribonucleases in this phenomenon. In this thesis, we 1) investigated the cytotoxicity of Bothrops alternatus venom in Madin-Darby canine kidney (MDCK) epithelial cells, 2) purified and characterized an acidic DNase from this venom, and 3) assessed the apoptotic activity of this DNase in MDCK cells. Treatment with B. alternatus venom (10 and 100 µg/mL) markedly decreased the transepithelial electrical resistance of cultured cells, and caused redistribution of some junctional proteins followed by cytoskeletal rearrangement involving stress fibers at the basal cell surface and focal adhesion-associated F-actin in the cell-matrix contact region. There was a decrease in the number of mitotic cells and an increase in the number of cells with pycnotic or morphologically altered nuclei. Scanning electron microscopy revealed a decrease in microvillar density and alteration in the normal cell morphology from polyhedric to fusiform. Staining with annexin V-FITC and electrophorese of cellular DNA suggested that cell death was predominantly by necrosis. Pretreating the cells with catalase, superoxide dismutase or L-NAME significantly attenuated the venom-induced cell death, indicating the possible involvement of reactive oxygen and nitrogen species in this phenomenon. DNase II was purified from B. alternatus venom by a combination of ion exchange and gel filtration chromatographies (specific activity = 1.9x103 units/mg vs. 36.1 units/mg for venom, purification factor = 51.2, with a protein yield of 1.75%). The molecular mass of 26.4 kDa (SDS-PAGE) was unaffected by dithiothreitol or i-mercaptoethanol, indicating a single-chain protein. Immunoblotting with affinity-purified IgG from commercial bothropic antivenom also yielded a single protein band with the same molecular mass. The enzyme was also recognized by antibothropic IgG in ELISA and crossreacted with anti-human DNase II in western blots. The isoelectric point determined by 2Dgel electrophoresis was ~5.0. DNase II cleaved double-stranded DNA, denatured DNA and circular DNA (from the plasmids pGEM and pBR 322), but there was no degradation of RNA. The enzyme was active in the pH range of 4.5-5.5, with an optimum at 4.7; activity was lost at >50oC. The Km and Vmax were 10.1 µg/mL and 352.5 U/mg, respectively. Enzymatic activity was inhibited by aurintricarboxylic acid (25 µM), iodoacetamide (1 mM), DTT (1 mM) and Ca2+, Mg2+ and Zn2+ (100 mM), but not by EDTA (5 mM). In MDCK cells, DNase II produced cytoplasmic vacuolization, cell shrinkage and cell detachment from the substrate, as well as condensed and/or fragmented nuclei. DNase was cytotoxic to MDCK cells at > 400 U/mL (~20 µg/mL), caused DNA fragmentation, and increased the proportion of apoptotic cells. The apoptotic pathway stimulated by this enzyme involved the activation of caspases 3, 8 and 9, and a decrease in the expression of the anti-apoptotic protein Bcl2. These results show that B. alternatus venom is cytotoxic to MDCK cells, with part of this toxicity probably being mediated by DNase II. This enzyme induces apoptosis that could contribute to the general cytotoxicity of the venom / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

No description available.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.

INTRODUÇÃO: A ventriculomegalia é a malformação congênita de sistema nervoso central (SNC) mais prevalente, e a malformação Dandy Walker (apesar de menos prevalente) é, assim como a ventriculomegalia, uma doença com um importante impacto pré e pós-natal na morbidade e mortalidade fetal. A etiologia dessas patologias é heterogênea e o fator genético está entre as principais causas. A técnica mais usada no período pré-natal para rastreamento genético é o cariótipo por banda G; contudo esta técnica não revela todas as anormalidades genéticas. Portanto, em fetos com alteração morfológica ultrassonográfica detectada e cariotipagem tradicional normal, o estudo molecular pode ser oferecido para uma investigação etiológica e aconselhamento genético. OBJETIVO: Este estudo visa investigar a presença de CNVs (do inglês, copy number variations) utilizando cariotipagem por array em amostras de DNA obtidas de fetos portadores de ventriculomegalia (VM) ou Malformação de Dandy-Walker (MDW), com resultado de cariótipo clássico normal. MÉTODO: Foram selecionadas 29 gestantes com fetos portadores de VM e MDW. Das 29 amostras colhidas, 2 foram excluídas devido PCR positivo para infecções congênitas e outras 3 excluídas devido cariótipo por banda G apresentando anomalias cromossômica. Um total de 24 fetos foram incluídos no estudo, sendo 19 portadores de ventriculomegalia e 5 casos de MDW diagnosticados na ultrassonografia pré-natal. Todos os casos apresentavam cariótipo por banda G normal e PCR negativo para infecções congênitas no líquido amniótico. O DNA fetal foi extraído do cordão umbilical através da cordocentese entre 20 e 34 semanas e foi analisado pelo SNP array. As CNVs encontradas foram comparadas com banco de dados e literatura e posteriormente classificadas patogênicas, significado clínico incerto (VUS; do inglês variation of uncertain clinical significance) ou benignas. RESULTADOS: Nos 5 fetos com MDW a média da idade gestacional do diagnóstico foi de 26,8 semanas (SD 1,78 semanas). Todos os fetos eram do sexo feminino. Encontradas 6 CNVs. Todas consideradas benignas. Nos 19 fetos portadores de VM, a média do diâmetro no nascimento foi de 29,9 mm (DP 15,21mm). A média da idade gestacional do diagnóstico foi de 27 semanas (DP: 3,41 semanas). O sexo masculino representou 57,8% dos casos e o feminino, 42,1%. Foram encontradas 41 CNVs (22 benignas e 16 VUS) além de 15 eventos de perda de heterozigosidade (LOH). Nenhuma CNV foi considerada patogênica. CONCLUSÃO: Foi possível detectar CNVs utilizando cariotipagem por array em 22, dos 24 casos selecionados de VM e MDW; determinar qual a região cromossômica alterada, além de associar essas regiões com genes envolvidos. Os genes envolvidos foram estudados e comparados com o banco de dados. Contudo para correlacionar os achados ultrassonográficos com os achados citogenômicos encontrados ainda necessitamos de mais estudos acumulativos para enriquecer os bancos de dados existentes e melhorar a assistência pré-natal, diagnóstico e prognóstico dos pacientes portadores de ventriculomegalia e malformação Dandy Walker / BACKGROUND: Ventriculomegaly is the most prevalent congenital central nervous system (CNS) anomalies. Dandy Walker malformation (DWM), although lower prevalent, is, like ventriculomegaly, a disease with a significant prenatal and postnatal impact on fetal morbidity and mortality. The etiology of these pathologies is heterogeneous and the genetic factor is among the main causes. The most used technique in the prenatal period for genetic tracing is the K-band karyotype. However, this technique does not reveal all genetic abnormalities. Therefore, in fetuses with detected ultrasound morphological alteration and normal traditional karyotyping, the molecular study may be offered for an etiological investigation and genetic counseling. OBJECTIVE: To investigate copy number variations (CNVs) using a single nucleotide polymorphism (SNP) array and identify changes in chromosomal regions in fetuses with ventriculomegaly (VM). METHOD: Twenty nine pregnant women with fetuses with ventriculomegaly and DWM were selected. Of the 29 samples collected, 2 were excluded due to positive PCR for congenital infections and another 3 excluded due to G-band karyotype presenting chromosomal abnormalities. A total of 24 fetuses were included in the study, 19 of whom had ventriculomegaly and 5 MDW cases diagnosed on prenatal ultrasonography. All cases presented normal G-band karyotype and negative PCR for congenital infections in the amniotic fluid. The fetal DNA was extracted from the umbilical cord by cordocentesis between 20 and 34 weeks and was analyzed by the SNP array. The CNV were compared with databases and literature and then classified into three groups: pathogenic CNVs, CNVs with uncertain clinical significance (VUS) and benign CNVs. RESULTS: In the 5 fetuses with MDW the mean gestational age of the diagnosis was 26.8 weeks (SD 1.78 weeks). All fetuses were female. Found 6 CNVs. All CNVs considered benign. In the 19 fetuses with MV, the mean of the cerebral ventricle diameter at birth was 29.9 mm (SD 15.21 mm). The mean gestational age of the diagnosis was 27 weeks (SD: 3.41 weeks). The male gender represented 57.8% of the cases and female, 42.1%. We found 41 CNVs (22 benign and 16 VUS) in addition to 15 heterozygosity loss (LOH). No CNV was considered pathogenic. CONCLUSION: It was possible to detect CNVs using array in 22 of the 24 selected cases of VM and DWM; determine the altered chromosomal region, and associate these regions with the genes involved. The genes involved were studied and compared to the database. However, to correlate ultrasonographic findings with the cytogenetic anomalies detected, we still need more cumulative studies to enrich existing databases and improve prenatal care, diagnosis and prognosis of patients with VM and DWM

INTRODUÇÃO: A ventriculomegalia é a malformação congênita de sistema nervoso central (SNC) mais prevalente, e a malformação Dandy Walker (apesar de menos prevalente) é, assim como a ventriculomegalia, uma doença com um importante impacto pré e pós-natal na morbidade e mortalidade fetal. A etiologia dessas patologias é heterogênea e o fator genético está entre as principais causas. A técnica mais usada no período pré-natal para rastreamento genético é o cariótipo por banda G; contudo esta técnica não revela todas as anormalidades genéticas. Portanto, em fetos com alteração morfológica ultrassonográfica detectada e cariotipagem tradicional normal, o estudo molecular pode ser oferecido para uma investigação etiológica e aconselhamento genético. OBJETIVO: Este estudo visa investigar a presença de CNVs (do inglês, copy number variations) utilizando cariotipagem por array em amostras de DNA obtidas de fetos portadores de ventriculomegalia (VM) ou Malformação de Dandy-Walker (MDW), com resultado de cariótipo clássico normal. MÉTODO: Foram selecionadas 29 gestantes com fetos portadores de VM e MDW. Das 29 amostras colhidas, 2 foram excluídas devido PCR positivo para infecções congênitas e outras 3 excluídas devido cariótipo por banda G apresentando anomalias cromossômica. Um total de 24 fetos foram incluídos no estudo, sendo 19 portadores de ventriculomegalia e 5 casos de MDW diagnosticados na ultrassonografia pré-natal. Todos os casos apresentavam cariótipo por banda G normal e PCR negativo para infecções congênitas no líquido amniótico. O DNA fetal foi extraído do cordão umbilical através da cordocentese entre 20 e 34 semanas e foi analisado pelo SNP array. As CNVs encontradas foram comparadas com banco de dados e literatura e posteriormente classificadas patogênicas, significado clínico incerto (VUS; do inglês variation of uncertain clinical significance) ou benignas. RESULTADOS: Nos 5 fetos com MDW a média da idade gestacional do diagnóstico foi de 26,8 semanas (SD 1,78 semanas). Todos os fetos eram do sexo feminino. Encontradas 6 CNVs. Todas consideradas benignas. Nos 19 fetos portadores de VM, a média do diâmetro no nascimento foi de 29,9 mm (DP 15,21mm). A média da idade gestacional do diagnóstico foi de 27 semanas (DP: 3,41 semanas). O sexo masculino representou 57,8% dos casos e o feminino, 42,1%. Foram encontradas 41 CNVs (22 benignas e 16 VUS) além de 15 eventos de perda de heterozigosidade (LOH). Nenhuma CNV foi considerada patogênica. CONCLUSÃO: Foi possível detectar CNVs utilizando cariotipagem por array em 22, dos 24 casos selecionados de VM e MDW; determinar qual a região cromossômica alterada, além de associar essas regiões com genes envolvidos. Os genes envolvidos foram estudados e comparados com o banco de dados. Contudo para correlacionar os achados ultrassonográficos com os achados citogenômicos encontrados ainda necessitamos de mais estudos acumulativos para enriquecer os bancos de dados existentes e melhorar a assistência pré-natal, diagnóstico e prognóstico dos pacientes portadores de ventriculomegalia e malformação Dandy Walker / BACKGROUND: Ventriculomegaly is the most prevalent congenital central nervous system (CNS) anomalies. Dandy Walker malformation (DWM), although lower prevalent, is, like ventriculomegaly, a disease with a significant prenatal and postnatal impact on fetal morbidity and mortality. The etiology of these pathologies is heterogeneous and the genetic factor is among the main causes. The most used technique in the prenatal period for genetic tracing is the K-band karyotype. However, this technique does not reveal all genetic abnormalities. Therefore, in fetuses with detected ultrasound morphological alteration and normal traditional karyotyping, the molecular study may be offered for an etiological investigation and genetic counseling. OBJECTIVE: To investigate copy number variations (CNVs) using a single nucleotide polymorphism (SNP) array and identify changes in chromosomal regions in fetuses with ventriculomegaly (VM). METHOD: Twenty nine pregnant women with fetuses with ventriculomegaly and DWM were selected. Of the 29 samples collected, 2 were excluded due to positive PCR for congenital infections and another 3 excluded due to G-band karyotype presenting chromosomal abnormalities. A total of 24 fetuses were included in the study, 19 of whom had ventriculomegaly and 5 MDW cases diagnosed on prenatal ultrasonography. All cases presented normal G-band karyotype and negative PCR for congenital infections in the amniotic fluid. The fetal DNA was extracted from the umbilical cord by cordocentesis between 20 and 34 weeks and was analyzed by the SNP array. The CNV were compared with databases and literature and then classified into three groups: pathogenic CNVs, CNVs with uncertain clinical significance (VUS) and benign CNVs. RESULTS: In the 5 fetuses with MDW the mean gestational age of the diagnosis was 26.8 weeks (SD 1.78 weeks). All fetuses were female. Found 6 CNVs. All CNVs considered benign. In the 19 fetuses with MV, the mean of the cerebral ventricle diameter at birth was 29.9 mm (SD 15.21 mm). The mean gestational age of the diagnosis was 27 weeks (SD: 3.41 weeks). The male gender represented 57.8% of the cases and female, 42.1%. We found 41 CNVs (22 benign and 16 VUS) in addition to 15 heterozygosity loss (LOH). No CNV was considered pathogenic. CONCLUSION: It was possible to detect CNVs using array in 22 of the 24 selected cases of VM and DWM; determine the altered chromosomal region, and associate these regions with the genes involved. The genes involved were studied and compared to the database. However, to correlate ultrasonographic findings with the cytogenetic anomalies detected, we still need more cumulative studies to enrich existing databases and improve prenatal care, diagnosis and prognosis of patients with VM and DWM