Characterization of an individual's sample with idiopathic intellectual disability : clinical, cytogenetic and molecular aspects

Orientadores: Antonia Paula Marques de Faria, Maricilda Palandi de MeloTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A deficiência intelectual (DI) ou transtorno do desenvolvimento intelectual, isolada ou associada a anomalias congênitas, corresponde a uma das categorias mais amplas de distúrbios, acometendo de 1% a 3% da população nos países industrializados, enquanto nos países em desenvolvimento estima-se prevalência cerca de três vezes maior. Devido à heterogeneidade de fatores causais associados a essa condição, sua investigação diagnóstica pode ser complexa e 40% dos casos não têm sua origem determinada. Recentemente, com a aplicação da tecnologia genômica, alterações cromossômicas crípticas, mutações gênicas e rearranjos genômicos diversos vêm sendo relacionados à DI, modificando as estratégias de pesquisa nessa área. Entre as mesmas, destacam-se as de Microarray-based Copy Number Variation (CNV), Multiplex Ligation Probe-dependent Amplification (MLPA), a Hibridização In Situ por Fluorescência (FISH), além de Next-Generation Sequencing (NGS). Em projeto anterior, a técnica de MLPA foi aplicada na investigação de rearranjos subteloméricos em 62 pacientes, com alteração em 3,2% deles. Diante da perspectiva de prosseguir na triagem por MLPA e, quando negativa, indicar a análise por microarray cromossômico ou o sequenciamento do exoma, foi proposta a continuidade do projeto, visando caracterizar uma amostra de indivíduos com DI de origem indeterminada quanto a aspectos clínicos e fatores causais. Sendo assim, 300 indivíduos foram avaliados clinicamente, submetidos a triagem por MLPA subtelomérico, com identificação de alterações patogênicas em 22 (7,3%) [del1p (2 casos), del1q, del4p, del6p, del18q, delXYp, dup7p, dup7q, dup11p, dup15p, dup17p, dupXYp (2 casos), del1p/dup4p, del4p/dup8p, del4p/dup12p, dup4p/del8p, dup4p/del13q, del5p/dup9p, del9p/dup19q e dup9p/del18p]. Entre os 278 restantes, 21 pacientes foram estudados pela a técnica de microarray, com detecção de variantes patogênicas em 11 [del1p36.33-p36.32/dup4p16.3-p15.33; del7p15.3-p15.2; del7q11.23; del15q21.2-q22.2; del22q11.21; delXq21.1-q21.33; dup6p11.2/dup10q26.3; dup12p13.2-p13.1; dup15q11.2-q13.1; dupXp22.33 e dupXq26.2). O sequenciamento do exoma foi realizado para outros 13 indivíduos, com alterações observadas em sete casos concluídos, envolvendo os genes KIRREL3/SHANK2, CTTNBP2, TCF4, PRKCG, RAI1, HTT e KMT2D. Considerando as 40 alterações identificadas pelas três técnicas utilizadas, em 36 casos foi estabelecida correlação genótipo-fenótipo, corroborando o diagnóstico laboratorial. Conforme verificado na literatura, a aplicação desses métodos, incluídos nos algoritmos atuais de investigação diagnóstica da DI, se mostram efetivos e complementares, ampliando consideravelmente as perspectivas de determinação do diagnóstico etiológico dessa condiçãoAbstract: The intellectual disability (ID) or intellectual development disorder isolated or associated with congenital anomalies, is the most frequent disability and occurs 1% to 3% of general population in industrialized world, while in developing countries it is considered to be three times higher. The etiology is heterogeneous and includes multiple genetic and environmental factors, although its cause remains undetermined in about 40% of the cases. Recently, with the application of genomic technology, cryptic chromosomal alterations, gene mutations and several genomic rearrangements have been related to ID, modifying research strategies in this area. Among them, the most important are microarray, Multiplex Ligation Probe-dependent Amplification (MLPA), Fluorescent In Situ Hybridization (FISH), and Next Generation Sequencing (NGS). Lincoln-de-Carvalho (2009) applied the MLPA technique to investigate 62 patients for subtelomeric rearrangements and found copy number changes in 3.2% of cases. Considering the perspective of continuing the search for CNVs by MLPA and indicate the analysis by microarray or exome sequencing for negative cases, the present project followed with the propose of characterizing a sample with idiopathic ID regarding clinical aspects and genetic etiology. Therefore, the clinical characterization of 300 patients was performed based on their medical records. After screening using subtelomeric MLPA, pathogenic alterations were identified in 22 (7.3%) cases [del1p (2 cases), del1q, del4p, del6p, del18q, delXYp, dup7p, dup7q, dup11p, dup15p, dup17p, dupXYp (2 cases), del1p/dup4p, del4p/dup8p, del4p/dup12p, dup4p/del8p, dup4p/del13q, del5p/dup9p, del9p/dup19q e dup9p/del18p]. The microarray technique was performed for 20 patients, and pathogenic variants were detected in 11 of them [del1p36.33-p36.32/dup4p16.3-p15.33; del7p15.3-p15.2; del7q11.23; del15q21.2-q22.2; del22q11.21; delXq21.1-q21.33; dup6p11.2/dup10q26.3; dup12p13.2-p13.1; dup15q11.2-q13.1; dupXp22.33 and dupXq26.2). Whole exome sequencing was performed for 13 individuals with alterations observed in seven concluded cases involving the KIRREL3/SHANK2, CTTNBP2, TCF4, PRKCG, RAI1, HTT and KMT2D genes. Considering the total of 40 alterations detected by combining the three tecniques, the genotype-fenotype correlations were concluded in 36 cases, corroborating the laboratory diagnosis. As verified in the literature, the application of these methods, included in the current diagnostic algorithms of DI, are effective and complementary, considerably broadening the prospects for determining the etiological diagnosis of this conditionDoutoradoGenetica MedicaDoutora em Ciências Médica

The MLPA technique : an alternative for subtelometric rearrangenents investigation in developmental delay or idiopathic mental retardation

Orientadores: Antonia Paula Marques de Faria, Maricilda Palandi de MelloDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: A deficiência mental (DM) corresponde a uma das categorias mais amplas de distúrbios, acometendo de 1% a 3% da população nos países industrializados, enquanto nos países em desenvolvimento estima-se prevalência cerca de três vezes maior. Devido à heterogeneidade de fatores causais associados a essa condição, sua investigação diagnóstica pode ser complexa e 40% dos casos não têm sua origem determinada. Recentemente, foi demonstrado que rearranjos subteloméricos são responsáveis por 5% a 7% dos casos de DM idiopática. Entre as técnicas mais promissoras para a identificação dessas alterações, está a Multiplex Ligationdependent Probe Amplification (MLPA), que possibilita a quantificação relativa, quanto ao número de cópias, de mais de 50 sequências de ácidos nucléicos em um único experimento, sendo capaz de detectar deleções e duplicações de diversos genes, além de mutações de ponto conhecidas. É baseada na hibridização do DNA genômico a uma mistura de sondas específicas para cada região estudada, com amplificação dos produtos de ligação pela técnica de PCR, utilizando um par de primers universal. A visualização dessas amplificações pode ser feita por eletroforese capilar e a análise por programas específicos de genotipagem. Entre as vantagens do método estão o custo relativamente baixo, simplicidade, rapidez e sensibilidade, justificando a implantação em serviços direcionados para o estudo da etiologia da DM. Assim, o propósito deste trabalho foi implantar o método em nosso laboratório, utilizando o kit SALSA MLPA P036 HUMAN TELOMERE para pesquisa de rearranjos subteloméricos em indivíduos com DM idiopática. Na padronização, foram empregados controles com e sem alteração subtelomérica, sendo avaliados os parâmetros de tempo de estocagem das reações em refrigerador; quantidade de DNA utilizado em cada reação; forma de normalização dos dados, e possibilidade de redução do volume das soluções empregadas para metade ou dois terços do preconizado no protocolo original, cujo volume final de reação é de 50 µL, dos quais somente 0,75 µL são utilizados na eletroforese capilar, com descarte do restante. A análise dos resultados, confirmados pela utilização de controles com e alterações subteloméricas, mostrou que a implantação do método foi bem-sucedida. Além disso, foi verificado que o tempo máximo de estocagem das reações em refrigerador sem prejuízo dos resultados é de aproximadamente três meses; que a quantidade de DNA satisfatória para o procedimento está entre 150-250 ng por reação; que não é aconselhável a utilização de protocolos com volumes reduzidos, pois a análise de reações realizadas dessa forma mostrou resultados falso-positivos quando comparados às reações feitas segundo o protocolo original; que a forma de normalização dos dados mais adequada para a análise dos resultados é a combinação dos programas GeneScan®, Genotyper® e planilha específica para cada kit ao invés do programa Coffalyser®, indicado pelo fabricante do produto. Após essa padronização, o método foi aplicado no estudo de 62 indivíduos com atraso do desenvolvimento ou DM de causa indeterminada, sendo identificadas alterações em seis deles. Duas foram constatadas também pelo kit de confirmação SALSA MLPA P070, sendo uma del(1p36) e outra um rearranjo del(5p)/dup(9p), ambas validadas pela análise por FISH. As quatro restantes [del(12p)] não foram observadas pelo kit de confirmação, sendo realizado sequenciamento automático que identificou o SNP (rs60220187). Concluindo, a técnica de MLPA foi implantada com sucesso, mostrando-se adequada e eficiente para a investigação de rearranjos subteloméricos.Abstract: Mental retardation (MR) is the most frequent disability and affects 1% to 3% of general population in industrialized world, while in developing countries it is considered to be three times higher. The etiology is heterogeneous and includes multiple genetic and environmental factors, although in about 40% of the cases its cause remains undetermined. Recently, several studies suggest that chromosomal rearrangements involving subtelomeric regions are common causes of idiopathic MR and occur in approximately 5% to 7% of patients. The Multiplex Ligationdependent Probe Amplification (MLPA) technique has been proposed as one of the alternatives to identify these abnormalities. It is based on hybridization of genomic DNA to a mixture of standardized probes for each region studied. All of the probe ligation products are amplified by PCR using only one universal primer pair. Separation and visualization of the fragments can be developed and the analysis by means of specific softwares for genotyping. Relatively low cost, simplicity and sensitivity are among the advantages of this method, which has been considered as an alternative for diagnostic screening of individuals with undetermined MR or developmental delay. The present study aimed to implement this technique in our laboratory, using the SALSA MLPA P036 human telomere test kit for detecting subtelomeric rearrangements in idiopathic MR patients evaluated in our service. For validation study, eleven normal controls and two positive controls were utilized, as well as the following parameters were evaluated: period of storage of reaction, amount of DNA used for each reaction, procedures to data normalization and reduction of reaction volume. The validation was successful; in addition, it was verified that maximum period for storage of reaction was about three months; adequate amount of DNA for each reaction was approximately 150ng-250 ng; Reduction in volume reaction produced false-positive results; the more suitable method for data normalization was a combination of GeneScan®, Genotyper® softwares and a the use of a specific Microsoft Excel® spreadsheet for each kit, instead of the Coffalyser® program, recommended by the manufacturer. After validation, a pilot study was carried out with 62 patients considered as idiopathic MR using initially the SALSA MLPA P036 kit. MLPA analyses have indicated abnormal copy number in six patients. SALSA MLPA P070 kit confirmed 2 out of the 6 alterations: a 1p36 deletion and a 5p deletion / 9p duplication rearrangement, which were validated by FISH analysis. Other four alterations corresponded to a same 12p deletion, which was not further confirmed. Sequencing the correspondent fragment a known as SNP rs60220187 was identified. In conclusion, we demonstrated that MLPA is an appropriate and efficient technique and should be considered as a routine screening in the evaluation of individuals with unexplained mental retardation or developmental delay.MestradoCiencias BiomedicasMestre em Ciências Médica

A de novo mutation in CYP21A2 gene in a case of in vitro fertilization

Congenital adrenal hyperplasia, one of the most frequent autosome recessive disorders, is caused by defects in steroidogenic enzymes involved in the cortisol biosynthesis. Approximately 95% of the cases are caused by abnormal function of the 21-hydroxylase enzyme. This deficiency leads to androgen excess, consequently, to virilization and rapid somatic growth with accelerated skeletal maturation. Mutations in CYP21A2 are responsible for different forms of 21-hydroxylase deficiency. Mild impairment in the enzymatic activity causes the non-classic or late-onset congenital adrenal hyperplasia that is observed with a prevalence of 1 in 1000 subjects in different populations. The present paper describes a de novo mutation that occurred in the paternal meiosis. The child, who was conceived by in vitro fertilization, presented with precocious puberty and diagnosed with non-classical 21-hydroxylase deficiency. DNA sequencing showed the compound heterozygosis for a de novo CYP21A1P/A2 chimeric gene and the p.Val281Leu mutation inherited from her mother, who was heterozygous for the mutation. The chimeric gene showed pseudogene-derived sequence from 5′-end to intron 3 and CYP21A2 sequences from intron 3 to 3′-end of the gene. Sequencing analysis of the father did not show any mutation. The multiplex ligation-dependent probe amplification (MLPA) assay did not indicate loss of DNA discarding gene deletion but confirmed the chimeric gene. In addition, supernumerary copies of CYP21A1P were observed for both parents and for the affect child. Since paternity has been confirmed, those results suggest that a de novo large gene conversion in the paternal meiosis could have occurred by misalignment of alleles bearing different copy numbers of genes in CYP21 locus

Neotropical freshwater fisheries : A dataset of occurrence and abundance of freshwater fishes in the Neotropics

The Neotropical region hosts 4225 freshwater fish species, ranking first among the world's most diverse regions for freshwater fishes. Our NEOTROPICAL FRESHWATER FISHES data set is the first to produce a large-scale Neotropical freshwater fish inventory, covering the entire Neotropical region from Mexico and the Caribbean in the north to the southern limits in Argentina, Paraguay, Chile, and Uruguay. We compiled 185,787 distribution records, with unique georeferenced coordinates, for the 4225 species, represented by occurrence and abundance data. The number of species for the most numerous orders are as follows: Characiformes (1289), Siluriformes (1384), Cichliformes (354), Cyprinodontiformes (245), and Gymnotiformes (135). The most recorded species was the characid Astyanax fasciatus (4696 records). We registered 116,802 distribution records for native species, compared to 1802 distribution records for nonnative species. The main aim of the NEOTROPICAL FRESHWATER FISHES data set was to make these occurrence and abundance data accessible for international researchers to develop ecological and macroecological studies, from local to regional scales, with focal fish species, families, or orders. We anticipate that the NEOTROPICAL FRESHWATER FISHES data set will be valuable for studies on a wide range of ecological processes, such as trophic cascades, fishery pressure, the effects of habitat loss and fragmentation, and the impacts of species invasion and climate change. There are no copyright restrictions on the data, and please cite this data paper when using the data in publications

Search for Higgs and ZZ Boson Decays to J/ψγJ/\psi\gamma and Υ(nS)γ\Upsilon(nS)\gamma with the ATLAS Detector

A search for the decays of the Higgs and $Z$ bosons to $J/\psi\gamma$ and $\Upsilon(nS)\gamma$ ($n=1,2,3$) is performed with $pp$ collision data samples corresponding to integrated luminosities of up to $20.3\mathrm{fb}^{-1}$ collected at $\sqrt{s}=8\mathrm{TeV}$ with the ATLAS detector at the CERN Large Hadron Collider. No significant excess of events is observed above expected backgrounds and 95% CL upper limits are placed on the branching fractions. In the $J/\psi\gamma$ final state the limits are $1.5\times10^{-3}$ and $2.6\times10^{-6}$ for the Higgs and $Z$ bosons, respectively, while in the $\Upsilon(1S,2S,3S)\,\gamma$ final states the limits are $(1.3,1.9,1.3)\times10^{-3}$ and $(3.4,6.5,5.4)\times10^{-6}$, respectively

Search for Scalar-Charm pair production in pp collisions at s=8\sqrt{s}=8 TeV with the ATLAS detector

The results of a dedicated search for pair production of scalar partners of charm quarks are reported. The search is based on an integrated luminosity of 20.3 fb$^{-1}$ of pp collisions at $\sqrt{s}=8$ TeV recorded with the ATLAS detector at the LHC. The search is performed using events with large missing transverse momentum and at least two jets, where the two leading jets are each tagged as originating from c-quarks. Events containing isolated electrons or muons are vetoed. In an R-parity-conserving minimal supersymmetric scenario in which a single scalar-charm state is kinematically accessible, and where it decays exclusively into a charm quark and a neutralino, 95% confidence-level upper limits are obtained in the scalar-charm-neutralino mass plane such that, for neutralino masses below 200 GeV, scalar-charm masses up to 490 GeV are excluded

Finska tingsdomares bedömningar av partsutlåtanden givna på plats i rätten eller via videokonferens

Professionals within the judicial system sometimes believe they can assess whether someone is lying or not based on cues such as body language and emotional expression. Research has, however, shown that this is impossible. The Finnish Supreme Court has also given rulings in accordance with this demonstrated fact. There has also been previous research on whether party or witness statements are assessed differently in court depending on whether they are given live, via videoconference, or via prerecorded video. In the present study, we investigated how a Finnish sample of district judges (N=47) assigned probative value to different variables concerning the statement or the statement giver, such as body language and emotional expression. We also investigated the connection between the judges’ beliefs about the relevance of body language and emotional expression and their preference for live statements or statements via videoconference. The judges reported assigning equal amounts of probative value to statements given live and statements given via videoconference. However, judges found it easier to detect deception live, and this preference correlated with how relevant they thought body language is when assessing the probative value of the statement. In other words, a slight bias to assess live statements more favorably than statements given via videoconference might still exist. More effort needs to be put into making judges and Supreme Courts aware of robust scientific results that have been the subject of decades of research, such as the fact that one cannot assess whether someone is lying or not based on cues such as body language

Search for Higgs and Z Boson Decays to J/ψγJ/\psi\gamma and Υ(nS)γ\Upsilon(nS)\gamma with the ATLAS Detector

A search for the decays of the Higgs and Z bosons to J/ψγ and ϒ(nS)γ (n=1,2,3) is performed with pp collision data samples corresponding to integrated luminosities of up to 20.3 fb-1 collected at s=8 TeV with the ATLAS detector at the CERN Large Hadron Collider. No significant excess of events is observed above expected backgrounds and 95% C.L. upper limits are placed on the branching fractions. In the J/ψγ final state the limits are 1.5×10-3 and 2.6×10-6 for the Higgs and Z boson decays, respectively, while in the ϒ(1S,2S,3S)γ final states the limits are (1.3,1.9,1.3)×10-3 and (3.4,6.5,5.4)×10-6, respectively