A study of adrenocortical tumors in children and adolescents by a comparative genomic hybridization technique

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1-p15.3 and 20p11.2-p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1-q24.3 and the more frequent losses being 3q21-q26.2, 20q12-qter, and 22q11.2-q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21-p31.2, 2p12-p21 and loss of 20p11.2-p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

In situ hybridization to mRNA: from black art to guiding light

In situ hybridization to mRNA in embryo sections or wholemount embryos is one of the most powerful analytical tools available to the molecular developmental biologist. For many workers, this procedure provides the first insights into the function of newly isolated genes, allowing the formulation of hypotheses and setting the course for further research. This paper presents a personal historical perspective of the development of in situ hybridization, looks at the theory and practice of the technique, summarizes the current state of the art, and speculates on possible directions for the future as a tool in functional genomics.

Specificity of PCR and in situ hybridization assays designed for detection of Marteilia sydneyi and M-refringens

Primers and DNA probes designed for use in the specific detection of the paramyxean parasites Marteilia sydneyi and Marteilia refringens were tested for their potential to cross-react with closely related species in Polymerase Chain Reaction (PCR) and in situ hybridization. PCR primers and a DNA probe designed within the ITS1 rRNA of M. sydneyi were specific for M. sydneyi when compared with related species of Marteilia and Marteilioides. PCR primers designed within the 18S rRNA of M. refringens were specific in the detection of this species in PCR while a DNA probe (named Smart 2) designed on the same gene cross-reacted with M. sydneyi in tissue sections of Saccostrea glomerata as well as Marteilioides sp. infecting Striostrea mytiloides. Though not species specific, the Smart 2 probe provided a stronger signal in detection of all stages of M. sydneyi than the ITS1 probe. The ITS probe is proposed for use as a confirmatory diagnostic too] for M. sydneyi.

Combining the analyses of introgressive hybridisation and linkage mapping to investigate the genetic architecture of population divergence in the lake whitefish (Coregonus clupeaformis, Mitchill)

Adaptation and reproductive isolation, the engines of biological diversity, are still elusive when discussing the genetic bases of speciation. Namely, the number of genes and magnitude of selection acting positively or negatively on genomic traits implicated in speciation is contentious. Here, we describe the first steps of an ongoing research program aimed at understanding the genetic bases of population divergence and reproductive isolation in the lake whitefish (Coregonus clupeaformis). A preliminary linkage map originating from a hybrid cross between dwarf and normal ecotypes is presented, whereby some of the segregating AFLP markers were found to be conserved among natural populations. Maximum-likelihood was used to estimate hybrid indices from non-diagnostic markers at 998 AFLP loci. This allowed identification of the most likely candidate loci that have been under the influence of selection during the natural hybridisation of whitefish originating from different glacial races. As some of these loci could be identified on the linkage map, the possibility that selection of traits in natural populations may eventually be correlated to specific chromosomal regions was demonstrated. The future prospects and potential of these approaches to elucidate the genetic bases of adaptation and reproductive isolation among sympatric ecotypes of lake whitefish is discussed.

Chromosomal gains and losses in ocular melanoma detected by comparative genomic hybridization in an Australian population-based study

To define the location of potential oncogenes and tumor suppressor genes in ocular melanoma we carried out comparative genomic hybridization (CGH) analysis on a population-based series of 25 formalin-fixed, paraffin-embedded primary tumors comprising 17 choroidal, 2 ciliary body, 4 iris, and 2 conjunctival melanomas. Twelve (48%) of the 25 melanomas showed no chromosomal changes and 13 (52%) had at least one chromosomal gain or loss. The mean number of CGH changes in all tumors was 3.3, with similar mean numbers of chromosomal gains (1.5) and losses (1.8). The highest number of chromosomal changes (i.e., nine) occurred in a conjunctival melanoma and included four changes not observed in tumors at any other ocular site (gains in 22q and 11p and losses in 6p and 17p). The most frequent gains in all primary ocular melanomas were on chromosome arm 8q (69%), 6p (31%) and 8p (23%) and the most frequent losses were on 6q (38%), 10q (23%), and 16q (23%). The most common pairing was gain in 8p and gain in 8q, implying a whole chromosome copy number increase; gains in 8p occurred only in conjunction with gains in 8q. The smallest regions of copy number alteration were mapped to gain of 8q21 and loss of 6q21, 10q21, and 16q22. Sublocalization of these chromosomal changes to single-band resolution should accelerate the identification of genes involved in the genesis of ocular melanoma.

Nitrifying microbial community analysis of nitrite accumulating biofilm reactor by fluorescence in situ hybridization

Biological nitrogen removal via nitrite pathway in wastewater treatment is very important especially in the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulations were carried out in a biofilm reactor. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.2 kg N/m3/d. Nitrite accumulation was achieved by the selective inhibition of nitrite oxidizers by free ammonia and oxygen limitation. Nitrite oxidation activity was recovered as soon as the inhibition factor was removed. Fluorescence in situ hybridization studies of the nitrite accumulating biofilm system have shown that genus Nitrosomonas which is specifically hybridized with probe NSM 156 was the dominant nitrifying bacteria while Nitrospira was less abundant than those of normal nitrification systems. Further FISH analysis showed that the combinations of Nitrosomonas and Nitrospira cells were identified as important populations of nitrifying bacteria in an autotrophic nitrifying biofilm system.

Caracterização molecular de três espécies de Trachycephalus (Anura: Hylidae) : investigando potenciais híbridos interespecíficos

Animais híbridos representam um desafio à taxonomia e sistemática, pois correspondem a unidades evolutivas geralmente sem clara delimitação morfológica, comportamental e genética. Híbridos podem ser morfologicamente intermediários aos parentais ou, devido à introgressão e retrocruzamentos, suas características podem se misturar tornando difícil sua identificação. Uma das formas de identificação de híbridos é por meio de ferramentas de biologia molecular, que ao utilizarem marcadores de DNA mitocondrial (herança exclusiva materna) e DNA nuclear (herança materna e paterna), permitem a comparação entre informações genéticas. Além da hibridização existem outras fontes de conflito entre dados moleculares provenientes do DNA mitocondrial e DNA nuclear, como por exemplo a retenção de polimorfismos ancentrais. Em localidades do Espírito Santo, Brasil, foram coletados indivíduos de morfologia distinta de Trachycephalus mesophaeus e T. nigromaculatus, que são as únicas espécies do gênero conhecidas nesse estado. Porém, estudos piloto usando o gene mitocondrial Citocromo Oxidase subunidade I (COI) agruparam esses espécimes com amostras de T. typhonius. Devido a estas incongruências, foram sequenciados fragmentos de dois genes mitocondriais - COI e Nicotinamida Desidrogenase subunidade 2 (ND2) e um exon nuclear (tirosinase) de 173 indivíduos de Trachycephalus, de forma a esclarecer as identificações taxonômicas e investigar a correspondência entre caracteres morfológicos e genéticos nesta linhagem, na sua área de ocorrência As filogenias moleculares, divergências genéticas, redes de haplótipos e polimorfismos de nucleotídeos únicos (SNPs) confirmaram as três espécies acima mencionadas como linhagens evolutivas distintas e revelaram mais sete indivíduos potencialmente híbridos, mas morfologicamente assinalados a T. mesophaeus, T. nigromaculatus ou T. typhonius.. Devido à taxa de evolução lenta da tirosinase, as espécies mais recentes T. typhonius e T. nigromaculatus parecem não terem sido sorteadas completamente nesse gene. Já T. mesophaeus, que é a espécie mais antiga das três, foi recuperada inequivocamente em todas as análises. De forma inédita, as análises moleculares evidenciaram a ocorrência de introgressão bidirecional entre T. nigromaculatus e T. typhonius e entre T. nigromaculatus e T. mesophaeus, sendo que há indícios de indivíduos F1 (cruzamentos entre espécies parentais puras gerando híbridos). A utilização do gene ND2 mostrou-se mais eficiente do que o gene COI nas filogenias e, apesar da tirosinase ser um gene nuclear de evolução lenta, contribuiu para a identificação de incongruências citonucleares. Nossos resultados mostram que a história filogenética de Trachycephalus é complexa e que o uso de marcadores nucleares de evolução mais rápida e ampliação dessas análises para outras espécies do gênero podem revelar mais eventos de hibridização.

Gold electrode modified by self-assembled monolayers of thiols to determine DNA sequences hybridization

The process of immobilization of biological molecules is one of the most important steps in the construction of a biosensor. In the case of DNA, the way it exposes its bases can result in electrochemical signals to acceptable levels. The use of self-assembled monolayer that allows a connection to the gold thiol group and DNA binding to an aldehydic ligand resulted in the possibility of determining DNA hybridization. Immobilized single strand of DNA (ssDNA) from calf thymus pre-formed from alkanethiol film was formed by incubating a solution of 2-aminoethanothiol (Cys) followed by glutaraldehyde (Glu). Cyclic voltammetry (CV) was used to characterize the self-assembled monolayer on the gold electrode and, also, to study the immobilization of ssDNA probe and hybridization with the complementary sequence (target ssDNA). The ssDNA probe presents a well-defined oxidation peak at +0.158 V. When the hybridization occurs, this peak disappears which confirms the efficacy of the annealing and the DNA double helix performing without the presence of electroactive indicators. The use of SAM resulted in a stable immobilization of the ssDNA probe, enabling the hybridization detection without labels. This study represents a promising approach for molecular biosensor with sensible and reproducible results.

Human papillomavirus detection in cervical dysplasias or neoplasias and in condylomata acuminaata by in situ hybridization with biotinylated DNA probes

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with bioti-nylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.

Cytogenetics of Triatominae: III - A study on male sterility induced through hybridization of Triatoma sórdida and Triatoma pseudomaculata

Males from bilateral crosses between Triatoma sórdida and Triatoma pseudomaculata were unable to give offspring, as shown by subsequent backcrosses (BC) between hybrid males and parental females. This kind of sterility indueed through interspecific hybridization seems to be due to lack of sperm migration from the bursa copulatrix to the spermateca, thus suggesting primarily failure on the part of hybrid males to produce and/or to incorporate male accessory secretions into the spermatophore bulb. Addicional proof that sterility induced in hybrid males is at the sperm level has been afforded by the spermatogenesis herein studied. The anomalous processes like; 1) prophases of spermatogonia with the chromosomes scattered in the cytoplasm, 2) first metaphases with unpaired tetrades, 3) spermatids differing in size and 4) spermatozoa of abnormal shape and generdlly of giant size, can be taken as an indicator of the degree of departure from the normal course of spermatogenesis.

Desenvolvimento de um ensaio de hibridação múltipla (MHA-MULTIPLE REGION HYBRIDIZATION ASSAY) para identificação presumível de vírus da imunodeficiência humana do tipo 1 (HIV-1) dos subtipos B, G e de formas recombinantes CRF14_BG e CRF02_AG circulantes em Portugal

A maioria dos métodos utilizados na caracterização genética do HIV-1 baseia-se na análise de regiões específicas do genoma viral, fornecendo informação parcial sobre o mesmo e, por consequência, revelando-se inadequados para a identificação de vírus recombinantes. O único método que permite uma caracterização integral do genoma viral passa pela sua sequenciação completa. No entanto, este é um método dispendioso, laborioso e de difícil implementação quando se pretende a análise de elevados números de amostras. Como alternativa a este último, o conjunto de métodos genericamente designados de MHA (Multiple Region Hybridization Assay) baseiam-se na amplificação, por PCR em tempo-real, de várias regiões ao longo do genoma viral e na sua caracterização com sondas específicas (TaqMan). Tendo este modelo por base, o objectivo deste estudo foi o desenvolvimento de um ensaio de hibridação múltipla (MHABG0214) passível de ser aplicado ao estudo de um elevado número de amostras. Este método foi desenvolvido tendo como objectivo a genotipagem as estirpes circulantes dominantes na epidemia Portuguesa, nomeadamente os subtipos B, G e formas genéticas recombinantes CRF02_AG e CRF14_BG. Com base em alinhamentos de sequências de referência de genoma completo, delinearam-se primers universais e subtipo-específicos para a amplificação de diversas regiões codificantes distribuídas ao longo do genoma do HIV-1 (Gag, Protease, Transcriptase Reversa, Integrase, Rev, Gp120 e Gp41). A optimização foi efectuada, inicialmente, para um conjunto de amostras de referência e seguidamente avaliada num conjunto de 50 amostras clínicas. O MHABG0214 foi implementado numa estratégia de PCR em tempo-real, numa detecção dependente de SYBR® Green I para todas as regiões ou, como alternativa, usando sondas TaqMan (Gp41). Apresentamos ainda uma estratégia em que a análise de resultados se baseia, simplesmente, numa abordagem usando PCR/gel de agarose convencional. Estas abordagens constituem ferramentas úteis na identificação das estirpes de HIV-1 em Portugal.

Renal and urinary findings in 20 patients with Williams-Beuren syndrome diagnosed by fluorescence in situ hybridization (FISH)

PURPOSE: Williams-Beuren syndrome is a rare multiple anomalies/mental retardation syndrome caused by deletion of contiguous genes at chromosome region 7q11.23. The aim of this work was to determine the frequency and the types of renal and urinary tract anomalies in 20 patients with Williams-Beuren syndrome. METHODS: The fluorescence in situ hybridization test using a LSI Williams syndrome region DNA probe was performed for all 20 patients to confirm the diagnosis of Williams-Beuren syndrome. A prospective study was performed in order to investigate renal and urinary aspects using laboratory assays to check renal function, ultrasonography of the kidneys and urinary tract, voiding cystourethrogram and urodynamics. RESULTS: Deletion of the elastin gene (positive fluorescence in situ hybridization test) was found in 17 out of 20 patients. Renal alterations were diagnosed in 5 of 17 (29%) the patients with the deletion and in 1 of 3 patients without the deletion. Fourteen patients with the deletion presented dysfunctional voiding. Arterial hypertension was diagnosed in 3 patients with deletions and 1 of these presented bilateral stenosis of the renal arteries. CONCLUSIONS: Due to the high incidence of renal and urinary abnormalities in Williams-Beuren syndrome, performing a systematic laboratory and sonographic evaluation of the patients is recommended.