Cytogenomic assessment of the diagnosis of 93 patients with developmental delay and multiple congenital abnormalities: The Brazilian experience

OBJECTIVE: The human genome contains several types of variations, such as copy number variations, that can generate specific clinical abnormalities. Different techniques are used to detect these changes, and obtaining an unequivocal diagnosis is important to understand the physiopathology of the diseases. The objective of this study was to assess the diagnostic capacity of multiplex ligation-dependent probe amplification and array techniques for etiologic diagnosis of syndromic patients. METHODS: We analyzed 93 patients with developmental delay and multiple congenital abnormalities using multiplex ligation-dependent probe amplifications and arrays. RESULTS: Multiplex ligation-dependent probe amplification using different kits revealed several changes in approximately 33.3% of patients. The use of arrays with different platforms showed an approximately 53.75% detection rate for at least one pathogenic change and a 46.25% detection rate for patients with benign changes. A concomitant assessment of the two techniques showed an approximately 97.8% rate of concordance, although the results were not the same in all cases. In contrast with the array results, the MLPA technique detected ∼70.6% of pathogenic changes. CONCLUSION: The obtained results corroborated data reported in the literature, but the overall detection rate was higher than the rates previously reported, due in part to the criteria used to select patients. Although arrays are the most efficient tool for diagnosis, they are not always suitable as a first-line diagnostic approach because of their high cost for large-scale use in developing countries. Thus, clinical and laboratory interactions with skilled technicians are required to target patients for the most effective and beneficial molecular diagnosis.

Efeitos do pareamento no perfil de expressão gênica do parasita Schistosoma mansoni / Effects of pairing on the gene expression profiles of the parasite Schistosoma mansoni

Esquistossomose é uma doença crônica e debilitante. Schistosoma representa a única classe de trematódeos com vida dióica. Um contínuo pareamento com o macho é essencial para a maturação sexual do sexo feminino. Fêmeas adultas provenientes de infecções uni-sexuadas são subdesenvolvidas, apresentam atrofia do tamanho e um sistema reprodutivo imaturo. Para estudar os mecanismos envolvidos no pareamento de vermes adultos foram utilizadas duas plataformas de microarranjos distintas: uma composta por 4 mil sondas de cDNA dupla fita produzida pelo nosso grupo de pesquisas e outra composta por 44 mil sondas de oligonucleotideos desenhadas pelo nosso grupo e produzida pela empresa Agilent Technologies. Com a plataforma de 4 mil sondas detectamos 113 transcritos diferencialmente expressos em fêmeas adultas mantidas separadas de seus respectivos pares durante 24 horas de cultivo in vitro quando comparadas com fêmeas adultas pareadas; para 10 destes genes obtivemos uma confirmação adicional da expressão diferencial por transcrição reversa fita específica seguida de PCR em Tempo Real. Observamos também os efeitos do pareamento no perfil de expressão gênica de machos adultos mantidos separados de seus respectivos pares durante 24 horas de cultivo in vitro; foram encontrados 152 transcritos diferencialmente expressos. Com a plataforma de 44 mil sondas foi detectada a expressão de 5.798 genes transcricionalmente ativos em verme adulto, em um conjunto de 19.907 genes únicos representados nesta plataforma. A análise do conjunto de genes "no match" mostrou que em 156 genes ocorria expressão senso e anti-senso; para 6 destes transcritos obtivemos uma confirmação adicional da expressão nas duas fitas por transcrição reversa fita específica seguida de PCR em Tempo Real. Adicionalmente foram identificados 2717 transcritos diferencialmente expressos em fêmeas separadas de seus respectivos pares durante 13 dias de cultivo in vitro, quando comparadas com fêmeas mantidas pareadas. Para as análises com machos separados durante 13 dias foram encontrados 243 transcritos diferencialmente expressos. Por fim, realizamos estudos com o objetivo de observar os genes que podem estar correlacionados com o contato físico do pareamento (macho e fêmea) e genes que podem ser regulados pela possível difusão de proteínas e hormônios secretados no meio, para os quais a mudança do nível de expressão não dependa da necessidade de contato entre o macho e a fêmea. Sabe-se que o contato direto da fêmea com o macho é necessário para manter a atividade reprodutiva feminina e observamos que o re-pareamento pode restabelecer o perfil de expressão gênica de fêmeas ou machos separados. Além disso, observamos que fêmeas separadas e depois mantidas na presença do macho, porém sem re-pareamento, apresentam uma expressão gênica diferente das fêmeas separadas e depois mantidas na ausência de machos, sugerindo que algum fator secretado pelo macho no meio regula a expressão. Este trabalho representa uma importante contribuição no entendimento da relação macho-fêmea em nível molecular

Schistosomiasis is a chronic and debilitating disease. Schistosoma represents the only class of trematodes with a dioecious life. A continuous pairing with the male is essential for female sexual maturation. Adult females from uni-sexual infections are underdeveloped, have body atrophy and an immature reproductive system. To study the mechanisms involved in pairing of adult worms two microarray platforms were used: one comprised by 4000 cDNA probes and printed by our research group and another comprised by 44 000 oligonucleotide probes designed by our group and printed by Agilent Technologies Company. With the 4000-probes platform we detected 113 transcripts differentially expressed in adult females kept separated from their mates during 24 hours in vitro when compared with paired adult females; for 10 of these genes we obtained additional confirmation of differential expression by Real Time RT-PCR. We also observed the effects of pairing on the gene expression profile of adult males kept separate from their mates during 24 hours in vitro, where we found 152 differentially expressed transcripts. With the 44 000-probes platform we detected the expression of 5798 genes in adult worms, out of a set of 19 907 unique genes represented on this platform. Analysis of the "no match" genes showed that 156 have transcription from the sense and anti-sense strands; for 6 of them we obtained additional confirmation of expression by strand specific Real Time RT-PCR. Additionally, we identified 2717 differentially expressed transcripts in females separated from their mates during 13 days in vitro when compared to females that remained paired. In the analysis of males separated for 13 days we found 243 differentially expressed transcripts. Finally, we performed a study aimed at observing genes which might be correlated to physical contact pairing (male and female) and compared to genes that might be regulated by the possible diffusion of secreted proteins and hormones in the medium, for which the change of expression level does not depend on physical contact between male and female. It is known that direct female-male contact is needed to keep the female reproductively activity and we observed that repairing can restore the gene expression profile of females or males that were kept separated. Furthermore, we observed that females separated and then maintained in the presence of male, but without re-pairing, have a different gene expression from the separated females kept without males, suggesting that some male secreted factors might be involved in gene regulation. This work represents an important contribution to the understanding of male-female relation at the molecular level

SpotWhatR: a user-friendly microarray data analysis system

SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella fastidiosa Microarray Projects. SpotWhatR is freely available at http://blasto.iq.usp.br/~tkoide/SpotWhatR, in English and Portuguese versions. In addition, the user can choose between [quot ]single experiment[quot ] and [quot ]batch processing[quot ] versions.

Development of an angiogenesis-focused cDNA chip and validation of its functionality

DNA chip has been used as a powerful tool to study the genetic reprogramming of cells and its link to cellular phenotype such as angiogenesis. To evaluate the angiogenesis related genetic reprogramming more efficiently, we here developed an angiogenesis- focused cDNA chip containing 153 angiogenesis related genes arrayed in duplicate on a slide glass. In order to validate the functionality of the angiogenesis-focused cDNA chip, we examined gene expression profiles in HT1080 cells treated with either fetal bovine serum, a well known pro-angiogenic factor, or trichostatin A, a known angiogenesis inhibitor, using the cDNA chip. All duplicate data from the analysis are well matched with each other and gene expression profiles are well consistent with previously reported data. These results demonstrate that the angiogenesis-focused cDNA chip developed here can be a useful tool towards angiogenesisrelated researches.