Técnicas de citogenética y biología molecular para el diagnóstico y seguimiento de la leucemia promielocítica / Cytogenetic and molecular biology techniques for the diagnosis and monitoring of promyelocytic leukemia

Introducción: La leucemia promielocítica es un subtipo de leucemia mieloide aguda que se presenta frecuentemente con una coagulopatía potencialmente mortal, por lo que representa una emergencia médica. En la gran mayoría de los pacientes ocurre la t(15;17)(q24;q21) que genera el gen aberrante PML-RARA. Mediante diferentes técnicas de citogenética y de la biología molecular que detectan dichas aberraciones es posible diagnosticar la entidad de manera inequívoca y estudiar la enfermedad mínima residual. Objetivo: Describir, comparar y analizar las técnicas de citogenética y de la biología molecular que son útiles para el diagnóstico y el seguimiento del paciente con leucemia promielocítica. Así como señalar sus ventajas y limitaciones. Métodos: Se realizó revisión de la bibliografía científica de los últimos cinco años relacionada con el tema a través de PUBMED. Se realizó análisis y resumen de la información. Análisis y síntesis de la información: Se describen dos técnicas de citogenética y tres moleculares basadas en la aplicación de la reacción en cadena de la polimerasa. Se comparan y analizan sus ventajas y limitaciones. Conclusiones: Algunas de estas técnicas son útiles únicamente para el diagnóstico, mientras que otras, por su alta sensibilidad, se recomiendan para el seguimiento del paciente con leucemia promielocítica(AU)

Introduction: Promyelocytic leukemia (PML) is a subtype of acute myeloid leukemia that frequently presents with a potentially fatal coagulopathy, therefore it represents a medical emergency. In the vast majority of patients, the t (15; 17) (q24; q21) occurs, which generates the aberrant gene PML-RARA. Using different cytogenetic and molecular biology techniques that detect these aberrations, it is possible to unequivocally diagnose the entity and study minimal residual disease. Objective: To describe, compare and analyze cytogenetics and molecular biology techniques that are useful for diagnosis and follow-up of the patient with Promyelocytic leukemia. As well as pointing out its advantages and limitations. Methods: A review of the scientific bibliography of the last five years related to the subject was carried out through PUBMED. An analysis and summary of the information was made. Analysis and synthesis of the information: Two cytogenetic and three molecular techniques are described based on the application of the polymerase chain reaction. Its advantages and limitations are compared and analyzed. Conclusions: Some of these techniques are only useful for diagnosis, while others, due to their high sensitivity, are recommended for monitoring the patient with Promyelocytic leukemia(AU)

Síndrome de Down y su pesquisa durante el primer trimestre de la gestación / Down Syndrome and Screening During Pregnancy First Trimester

RESUMEN Introducción: La pesquisa prenatal de anomalías cromosómicas, mediante el uso de marcadores epidemiológicos y ecográficos del primer trimestre permite identificar gestantes con riesgo incrementado de síndrome de Down. Objetivos: Analizar la edad materna, la translucencia nucal, el ductus venoso y el hueso nasal, durante el cribaje del primer trimestre, en las gestantes que se realizaron diagnóstico prenatal citogenético, con el fin de evaluar la efectividad del mismo en la detección temprana del síndrome Down y su utilidad para la reducción del número de pruebas invasivas. Métodos: Se realizó un estudio descriptivo retrospectivo de corte transversal y se analiza una muestra de 3439 gestantes a las que se realizó el estudio citogenético indicado en el Centro Provincial de Genética Médica de La Habana, en el período comprendido entre el 3 de enero de 2006 y el 30 de diciembre de 2008. Resultados: La edad materna avanzada mostró una sensibilidad de un 87 por ciento del test y una tasa de falsos positivos de 99 por ciento. La translucencia nucal se comportó con una sensibilidad de 10 por ciento. El hueso nasal no mostró asociación con los cariotipos positivos para síndrome de Down. Al no realizarse sistemáticamente la presencia del ductus venoso, no se pudo establecer una asociación estadística. La estimación de riesgo de síndrome de Down basada únicamente en la edad materna avanzada determina una alta tasa de falsos positivos. Por lo que este marcador, unido a la evaluación de los marcadores ecográficos del primer trimestre para recalcular el riesgo individual, puede aumentar la efectividad en el diagnóstico y disminuir el número de pruebas invasivas. Conclusiones: La estimación de riesgo de síndrome de Down basada únicamente en la edad materna avanzada determina una alta tasa de falsos positivos. Por lo que este marcador, unido a la evaluación de los marcadores ecográficos del primer trimestre para recalcular el riesgo individual, puede aumentar la efectividad en el diagnóstico y disminuir el número de pruebas invasivas(AU)

ABSTRACT Introduction: The prenatal investigation of chromosomal abnormalities through the use of epidemiological and echographic markers on the first trimester, allows to identify pregnant women with an increased risk of Down syndrome. Objectives: To analyze maternal age, nuchal translucency, venous ductus and nasal bone, during the first trimester screening, in pregnant women who underwent prenatal cytogenetic diagnosis, in order to evaluate effectiveness in early detection of Down syndrome and the value for reducing the number of invasive tests. Methods: A descriptive retrospective cross-sectional study was carried out and a sample of 3439 pregnant women was studied. The cytogenetic study ordered at Havana Provincial Center for Medical Genetics was carried out from January 3, 2006 to December 30, 2008. Results: Advanced maternal age showed 87 percent sensitivity and 99 percent of false positive rate. Nuchal translucency accounted 10 percent of sensitivity. The nasal bone showed no association with positive karyotypes for Down syndrome. A statistical association of the venous ductus presence could not be established since the search was not systematically. Conclusions: The estimation of Down syndrome risk based solely on advanced maternal age determines high false positive rate. Therefore, this marker, together with the evaluation of the first trimester ultrasound markers for recalculating the individual risk, can increase the diagnostic effectiveness and decrease the number of invasive tests(AU)

Cytogenetic markers as tools in delimiting species of the highly diverse Neotropical fish Bryconamericus (Characiformes: Characidae)

Bryconamericus is a highly diverse group of characid fishes, being cytogenetic a valuable tool for the delimitation of species. Bryconamericus aff. iheringii (Upper Uruguay/Lower Paraná), B. coeruleus (Upper Paraná), B. cf. ecai e B. cf. eigenmanni (Upper Uruguay) were studied cytogenetically, and presented 2n=52 chromosomes, with interpopulational/interspecific variation of karyotype and fundamental number. Heterochromatin was evidenced in pericentromeric, telomeric and interstitial regions, and it was shown to be an important cytogenetic marker. Single nucleolar organizing regions (NORs) were found in B. cf. eigenmanni, B. cf. ecai and B. aff. iheringii (Lower Paraná), and multiple in B. aff. iheringii (Upper Uruguay) and B. coeruleus, with occurrence of two patterns for the first species, and three for the second. The 5S/18S rDNA-FISH confirmed the location of the NORs and showed single 5S rDNA cistrons only in B. aff. iheringii (Lower Paraná), evidencing the dispersion of both genes, often co-located, in the karyotype of the others species. The data of this work contribute for the delimitation of the species of the genus. Co-localization of ribosomal genes may represent a plesiomorphic condition for the group, and their dispersion suggest the occurrence of duplication, pseudogeneization and transposition events mediated by mobile genetic elements.(AU)

Bryconamericus é um grupo altamente diverso de caracídeos, sendo a citogenética uma valiosa ferramenta para a delimitação de espécies. Bryconamericus aff. iheringii (Alto Uruguai/Baixo Paraná), B. coeruleus (Alto Paraná), B. cf. ecai e B. cf. eigenmanni (Alto Uruguai) foram estudados citogeneticamente, e apresentaram 2n=52 cromossomos, com variação interpopulacional/interespecífica de cariótipo e número fundamental (NF). Heterocromatinas foram evidenciadas nas regiões pericentromérica, telomérica e intersticial, e mostrou-se um importante marcador citogenético. Regiões organizadores de nuclcéolos (RONs) simples foram encontradas em B. cf. eigenmanni, B. cf. ecai e B. aff. iheringii (Baixo Paraná), e múltiplas em B. aff. iheringii (Alto Uruguai) e em B. coeruleus, com a ocorrência de dois padrões de localização para a primeira espécie, e três para a segunda. A FISH-DNAr 5S/18S confirmou a localização das RONs e mostrou cístrons simples de DNAr 5S apenas em B. aff. iheringii (Baixo Paraná), evidenciando a dispersão de ambos os genes, muitas vezes co-localizados, no cariótipo das demais espécies. Os dados deste trabalho contribuem para a delimitação das espécies do gênero. A co-localização dos genes ribossomais pode representar uma condição plesiomórfica para o grupo, e sua dispersão sugere a ocorrência de eventos de duplicação, pseudogenização e transposição mediada por elementos genéticos móveis.(AU)

Síndrome de Turner en una adolescente / Turner syndrome in an adolescent

Se presenta el caso clínico de una adolescente ecuatoriana de 17 años de edad, blanca, quien nació producto de un embarazo normal y parto eutócico. Posterior al nacimiento se le realizó estudio genético que mostró la presencia de un cariotipo X0, por lo cual se le diagnosticó síndrome de Turner. A los 3 años fue operada de hipoplasia del arco aórtico con una evolución favorable. En la actualidad la paciente tiene baja talla con orejas de baja implantación, así como implantación alta del cabello en la nuca, tórax plano en forma de escudo, escaso bello pubiano y ausencia de menstruación

The case report of a 17 years Ecuadorian white adolescent is presented who was born from a normal pregnancy and eutocic delivery. After birth, a genetic study that showed the presence of a X0 Karyotype was carried out, reason why she was diagnosed Turner syndrome. She was operated of hypoplastic aortic arch when she was 3 years with a favorable clinical course. At the present time the patient has short height with ears of low implantation, as well as high implantation of the hair in the back of the neck, flat thorax in shield form, scarce pubic hair and amenorrhea

Espermiogênese como ferramenta citotaxonômica para diferenciar Triatoma guazu e T. williami, espécies vetoras da doença de Chagas / Spermiogenesis as cytotaxonomic tool to differentiate Triatoma guazu and T. williami, vector species of Chagas disease

Os triatomíneos pertencem à ordem Hemiptera, subordem Heteroptera, família Reduviidae e subfamília Triatominae. Todas as 148 espécies são hematófagas estritas e potenciais vetoras do protozoário Trypanosoma cruzi, agente etiológico da doença de Chagas. Algumas espécies são extremamente semelhantes do ponto de vista morfológico, o que pode dificultar o trabalho dos programas de controle de vetores. Triatoma guazu e T. williami são consideradas como espécies irmãs e não podem ser diferenciadas por diferentes abordagens, como análises morfométricas, isoenzimáticas e cromossômicas. Assim, o presente trabalho analisou as células haplóides durante a espermiogênese, com o objetivo de auxiliar na diferenciação desses vetores. A análise das espermátides de T. guazu e T. williami permitiu diferenciá-los, pois T. williami apresentou apenas um filamento heteropicnótico nas espermátides iniciais que foi parcialmente mantido durante o alongamento celular e T. guazu apresentou dois filamentos heteropicnóticos periféricos que se uniram durante o alongamento celular, dando origem a uma espermátide alongada totalmente heteropicnótica. Assim, ressaltando a importância dessa ferramenta na diferenciação de espécies relacionadas. No entanto, sugerimos que cruzamentos híbridos experimentais devem ser realizados entre esses triatomíneos, com o intuito de confirmar o status específico desses insetos vetores da doença de Chagas.(AU)

Triatomines belong to the Hemiptera order, Heteroptera suborder, Reduviidae family and Triatominae subfamily. All the 148 species are haematophagous strict and potential vectors of the protozoan Trypanosoma cruzi, etiologic agent of Chagas disease. Some species are very similar from a point of view morphological, what can difficult the work of vector control programs. Triatoma guazu and T. williami are considered sister species and cannot be differentiated by many approaches such as morphological, isoenzyme and chromosome analysis. Thus, the present study examined the haploid cells during spermiogenesis, aiming to assist the differentiation of these vectors. The analysis of spermatids of T. guazu and T. williami allowed to differentiate them because T. williami presented only one heteropyknotic filament in the early spermatids which was partially maintained during cell elongation and T. guazu presented two peripheral heteropycnotic filaments that have joined during cell elongation, leading to a elongated spermatid fully heteropycnotic. Thus highlighting the importance of this tool in the differentiation of related species. However, we suggest that experimental hybrid crosses should be made between these triatomines, in order to confirm the specific status of these insect vectors of Chagas disease.(AU)

Pattern of chromosome involvement in childhood hyperdiploid pre-B-cell acute lymhoblastic leukemia cases from India.

BACKGROUND: Hyperdiploid pre‑B‑cell acute lymhoblastic leukemia (pre‑B‑ALL) is a common form of childhood leukemia with very good prognosis with present day chemotherapy. However, the chromosomal composition of the hyperdiploidy has not been extensively studied and possible mechanism for this pathology remains so far conjectural. OBJECTIVE: To analyze the pattern of chromosome involvement in a cohort of childhood hyperdiploid pre‑B‑ALL from India and from this pattern to develop an understanding on the causation of such pathology. Whether such patients also carry translocations and FLT3 mutations in addition to their hyperdiploid karyotype. MATERIALS AND METHODS: One hundred and twenty‑six childhood pre‑B‑ALL patients were studied. Bone marrow aspirate of these patients were evacuated for morphology, FAB classification, immunophenotyping and both conventional and molecular cytogenetics. RESULTS: Of 126 patients with pre‑B‑ALL (age 2-15 years), 90 patients with abnormal karyotype showed 50 with hyperdiploid karyotype (50/90 i.e. 55.5%). Chromosomes 9, 10, 14, 17, 18, 20 and 21 were more often involved in hyperdiploidy. Chromosome 21 duplication was present in 92% of the cases. Chromosomes 5, 15, 16, 17 and Y were less often involved (18-20%) in hyperdiploidy. About 44% of patients with hyperdiploidy had additional karyotypic abnormality of which TEL‑AML1 was predominant (24%). Chromosome loss was rare and accounted for 20% of the cases only. We did not find any FLT3 mutation in our patients. CONCLUSION: In this study, the pattern of chromosome involvement in hyperdiploid karyotype of ALL patients is same as other studies except some chromosomes like 1, 6, 11, 12, 19 and 22 have some more frequent involvement than other studies. This study also showed the occurrence of TEL/AML1 fusion is more (19.8%) than other reports from India.

Real time polymerase chain reaction in diagnosis of chronic myeloid leukemia

To compare the sensitivity and specificity of Real Time Polymerase Chain Reaction [RT-PCR] with conventional cytogenetics in diagnosis of chronic myeloid leukemia. A cross-sectional, analytical study. The Armed Forces Institute of Pathology [AFIP], Rawalpindi, from December 2010 to January 2012. A total number of 40 patients were studied, in which all were diagnosed as CML on peripheral blood and bone marrow aspiration. The subjects were tested for the presence of Philadelphia [Ph] chromosome by cytogenetics and BCR-ABL fusion gene by RT-PCR. 2-3 ml of venous blood was collected, half in sodium heparin [anti-coagulant] for cytogenetics and half in EDTA for PCR. For cytogenetics, cells were cultured for 72 hours in RPMI 1640 medium and examined by arresting in metaphase using Colchicine to identify Philadelphia chromosome. For PCR, RNA extraction was done by Tri Reagent LS [MRC, USA] and cDNA was synthesized using reverse transcriptase and gene specific primer. RT- PCR was done on ABI-7500. The positive samples were identified when fluorescence exceeded threshold limit. Results of cytogenetics and RT PCR were compared. Out of the 40 patients, PCR showed 37 [92.5%] were positive and 3 [7.5%] were negative for BCR-ABL fusion gene, whereas in cytogenetics 28 [70%] were positive for Ph chromosome and 12 [30%] were negative for Ph chromosome. Sensitivity and specificity of cytogenetics was 75.6% and 100% respectively. Real time PCR as compared to cytogenetics is less tedious, gives quick results, does not require multiple sampling due to culture failure and can be done on peripheral blood

Cytogenetic studies of Brazilian pediatric myelodysplastic syndrome cases: challenges and difficulties in a large and emerging country

Myelodysplastic syndromes (MDS) and juvenile myelomonocytic leukemia (JMML) are rare hematopoietic stem cell diseases affecting children. Cytogenetics plays an important role in the diagnosis of these diseases. We report here the experience of the Cytogenetic Subcommittee of the Brazilian Cooperative Group on Pediatric Myelodysplastic Syndromes (BCG-MDS-PED). We analyzed 168 cytogenetic studies performed in 23 different cytogenetic centers; 84 of these studies were performed in patients with confirmed MDS (primary MDS, secondary MDS, JMML, and acute myeloid leukemia/MDS+Down syndrome). Clonal abnormalities were found in 36.9% of the MDS cases and cytogenetic studies were important for the detection of constitutional diseases and for differential diagnosis with other myeloid neoplasms. These data show the importance of the Cooperative Group for continuing education in order to avoid a late or wrong diagnosis.

Trisomy 8 in leukemia: A GCRI experience.

Trisomy of chromosome 8 is frequently reported in myeloid lineage disorders and also detected in lymphoid neoplasms as well as solid tumors suggesting its role in neoplastic progression in general. It is likely to be a disease-modulating secondary event with underlying cryptic aberrations as it has been frequently reported in addition to known abnormalities contributing to clinical heterogeneity and modifying prognosis. Here, we share our findings of trisomy 8 in leukemia patients referred for diagnostic and prognostic cytogenetic assessment. Total 60 cases of trisomy 8, as a sole anomaly or in addition to other chromosomal aberrations, were reported (January 2005–September 2008). Unstimulated bone marrow or blood samples were cultured, followed by GTG banding and karyotyping as per the ISCN 2005. Patients with +8 were chronic myeloid leukemia (CML) (36), acute myeloid leukemia (AML) (17), and acute lymphoblastic leukemia (ALL) (7). In 7 patients, trisomy 8 was the sole anomaly, whereas in 6 patients +8 was in addition to normal clone, in 47 patients, the +8 was in addition to t(9;22), t(15;17), and others, including 3 with tetrasomy 8. Only one patient showed constitutional +8. The present study will form the basis of further cumulative studies to correlate potential differential effects of various karyotypic anomalies on disease progression and survival following a therapeutic regime. To unravel the role of extra 8 chromosome, constitutional chromosomal analysis and uniparental disomy will be considered.

Acute promyelocytic leukemia with unusual karyotype.

Acute myeloid leukemia (AML-M3) is associated with the translocation t(15;17)(q22;q12-21) which disrupts the retinoic acid receptor alpha (RARA) gene on chromosome 17 and the PML gene on chromosome 15. We report a two-year-old patient with AML-M3 without the usual translocation t(15;17). Cytogenetic studies demonstrated normal appearance of chromosome 15 while the abnormal 17 homologue was apparently a derivative 17, der(17)(17qter-cen-q21:), the rearrangement distinctly shows deletion at 17q21 band and the morphology corresponding to an iso chromosome i(17q-). This case report is a rare cytogenetic presentation of acute promyelocytic leukemia (APML).

Cytogenetic and comorbidity profile of Down syndrome in Mansoura University Children's Hospital, Egypt.

BACKGROUND: Down syndrome (DS) is the most common chromosomal disorder. It has three chromosomal patterns. AIM: To determine the cytogenetic and comorbidity profiles of DS in the Genetic Unit of Mansoura University Children's Hospital, Mansoura, Egypt. MATERIALS AND METHODS: A retrospective analysis was performed on the case records of 712 cytogenetically diagnosed cases of DS at the Genetic Unit of Mansoura University Children's Hospital, Egypt, during a 10-year period. RESULTS: About 19% of the cases had one or more cardiac anomalies and about 8% were hypothyroid. Nondisjunction was the most common type of abnormality, followed by translocation and lastly mosaic: 96.1, 3.1, and 0.8%, respectively. Hypothyroidism was significantly more common in translocation and mosaic karyotypes than in the nondisjunction karyotypes. First and second birth orders were significantly higher in the translocation and mosaic groups than in the nondisjunction group. Mothers are significantly older at the index pregnancy in the nondisjunction group than in the other two groups. We compared our findings with those of previous studies. CONCLUSION: Knowing karyotype of DS will help in genetic counseling of the parents. Wide-scale national community-based survey with DS registry could help in estimating the size of the problem.

Assessment of fibrosis and vascularization of bone marrow stroma of chronic myeloid Leukemia patients treated with imatinib mesylate and their relationship with the cytogenetic response / Avaliação da fibrose e vascularização do estroma da medula óssea de pacientes com leucemia mielóide crônica tratados com mesilato de imatinibe e sua relação com a resposta citogenética

Chronic Myeloid Leukemia (CML) is a myeloproliferative disease characterized by the presence of the Philadelphia chromosome (translocation between chromosomes 9 and 22), resulting in the formation of the hybrid BCR-ABL protein. Currently, the treatment of CML patients is performed with imatinib mesylate (IM), which promotes the elimination of leukemic cells by inhibiting the kinase activity of BCR-ABL. This study evaluated the effectiveness of IM by monitoring 22 CML patients in a chronic phase treated at the CEPON/SC with IM for a minimum follow-up period of two years. Cytogenetic Response (CR) and bone marrow biopsies (BMB) were evaluated before and after IM treatment. BMB were evaluated by detection of reticulin degree and vascularization. The results were correlated to the CR. Mean time to achieve CR was 9 months and was attained by 77.27 percent of the patients. The results from the initial BMB analysis showed that 59.09 percent presented reticulin of between 2+ and 4+ whereas after treatment, only 27.17 percent presented this degree. With regard to vascularization of the initial sample, 90.91 percent were graded between II and IV, whereas after treatment, 40.91 percent had this degree. The results suggest a positive correlation of degree of reticulin and vascularization with CR.

A Leucemia Mielóide Crônica (LMC) é uma doença mieloproliferativa caracterizada pela presença do cromossomo Filadélfia (translocação entre os cromossomos 9 e 22), que resulta na formação da proteína híbrida BCR-ABL. Atualmente o tratamento de pacientes com LMC é realizado com mesilato de imatinibe (MI), o qual promove a eliminação das células leucêmicas pela inibição da atividade quinase de BCR-ABL. O presente estudo avaliou a eficácia do MI por meio do acompanhamento de pacientes portadores de LMC em fase crônica, atendidos no CEPON/SC tratados com MI pelo tempo mínimo de dois anos. Foram avaliadas a Resposta Citogenética (RC) e as biópsias de medula óssea (BMO) antes e após o tratamento com MI. As BMO foram avaliadas quanto ao grau de reticulina e vascularização. Os resultados correlacionaram-se com a RC cujo tempo médio para obtenção da RC foi de 9 meses, sendo atingida por 77.27 por cento dos pacientes. Na primeira BMO, 59.09 por cento dos pacientes apresentaram grau de reticulina entre 2+ e 4+ e após o tratamento, apenas 27.17 por cento apresentaram esta graduação. Quanto à vascularização da primeira amostra, 90.91 por cento foram graduadas entre II e IV e após o tratamento, 40.91 por cento apresentaram esta graduação. Os resultados sugerem uma correlação diretamente proporcional entre os graus de reticulina e vascularização com a RC.

Karyotype variability in neotropical catfishes of the family pimelodidae (Teleostei: Siluriformes)

Dados cariotípicos são apresentados para quatro espécies da família Pimelodidae. Todas apresentaram o mesmo número diploide, 2n = 56 cromossomos, com diferenças nas fórmulas cariotípicas. As espécies aqui analisadas mostraram pouca quantidade de heterocromatina localizada preferencialmente na região centromérica e telomérica de alguns cromossosmos do complemento cariotípico. As regiões organizadoras de nucléolo (Ag-RONs) e a localização dos genes ribossomais pela hibridização in situ fluorescente (FISH), com sondas 18S e 5S, evidenciaram somente um par cromossômico marcado portador de genes ribossomais, à exceção de Pimelodus britskii que apresentou NORs múltiplas e localização sintênica das sondas 18S e 5S. Eventos não-Robertsonianos, como inversão pericêntrica e duplicação das NORs são requeridos para explicar a diversificação cariotípica em Pseudoplatystoma do rio Paraguai (MS), Pimelodus do rio Iguaçu (PR), Sorubim do rio Paraguai (MS) e Steindachneridion do rio Paraíba do Sul (SP). Os dados obtidos para a macroestrutura cariotípica destas espécies corrobora um padrão conservado observado na família Pimelodidae. Por outro lado, evidências de variações interespecíficas pelos marcadores de citogenética molecular empregados possibilitam inferências citotaxonômicas e diferenciação das espécies aqui analisadas.

Karyotypic data are presented for four species of fish belonging to the Pimelodidae family. These species show a conserved diploid number, 2n = 56 chromosomes, with different karyotypic formulae. The analyzed species showed little amount of heterochromatin located preferentially in the centromeric and telomeric regions of some chromosomes. The nucleolus organizer regions activity (Ag-NORs) and the chromosomal location of ribosomal genes by fluorescent in situ hybridization (FISH), with 18S and 5S probes, showing only one chromosome pair marked bearer of ribosomal genes, the only exception was Pimelodus britskii that presented multiple NORs and syntenic location of the 18S and 5S probes. Non-Robertsonian events, as pericentric inversion and NORs duplication are requested to explain the karyotype diversification in Pseudoplatystoma from the rio Paraguay (MS), Pimelodus from the rio Iguaçu (PR), Sorubim from the rio Paraguay (MS) and Steindachneridion from the rio Paraíba do Sul (SP). The obtained data for the karyotype macrostructure of these species corroborates a conserved pattern observed in Pimelodidae. On the other hand, interspecific variations detected by molecular cytogenetics markers made possible cytotaxonomic inferences and differentiation of the species here analyzed.

Chromosomal analyses in Megalonema platanum (Siluriformes: Pimelodidae), an endangered species from South American rivers

Este estudo apresenta dados cromossômicos de Megalonema platanum do rio Tibagi, Paraná, Brasil e do rio Paraná, Argentina. O número diploide foi igual 54 com composição cariotípica de 24m+16sm+2st+12a em ambas populações. Os sítios AgNORs foram detectados na posição terminal de um par submetacêntrico das duas populações analisadas, coincidindo com constrição secundária no braço curto do par 15. CMA3 e FISH com sonda de DNAr 18S exibiram sinais fluorescentes que correspondem aos sítios AgNORs e à constrição secundária. A presença de um pequeno cromossomo supranumerário acrocêntrico foi observado em M. platanum do rio Tibagi, com heterocromatina centromérica. Outros blocos heterocromáticos foram evidenciados na posição terminal de alguns cromossomos e um par cromossômico submetacêntrico grande, provavelmente o primeiro par, mostrou heterocromatina intersticial. Na população do rio Paraná foram observados ainda blocos heterocromáticos em ambas regiões terminais em alguns cromossomos. Este trabalho mostra pela primeira vez dados citogenéticos de M. platanum, que é uma espécie muito rara na bacia do rio Paraná e pode estar ameaçada de extinção.

This study presents chromosomal data of Megalonema platanum from rio Tibagi, Paraná, Brazil and from rio Paraná, Argentina. The diploid number was equal 54 with karyotype composition of 24m+16sm+2st+12a in both populations. The AgNOR siteswere detected in the terminal position of a submetacentric pair of the two analyzed populations, coinciding with secondary constrictions on the short arm of pair 15. CMA3 and FISH with 18S rDNA probe displayed fluorescent signals that correspond to the AgNOR sites and secondary constriction. The presence of a small acrocentric supernumerary chromosome can be observed in M. platanum from rio Tibagi, with centromeric heterochromatin. Others heterochromatic blocks were evidenced in the terminal position of some chromosome and one metacentric large chromosome pair, probably the first pair, showed an interstitial heterochromatin. In the population of the rio Paraná were still observed heterochromatic blocks in both ends in some chromosomes. This work brings for the first time cytogenetic date of M. platanum, which is a very rare species in the rio Paraná basin and may be endangered.

Investigation of chromosomal aberrations in Egyptian hepatocellular carcinoma patients by fluorescence in situ hybridization.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a very common and highly malignant tumor, associated mainly with chronic viral hepatitis, cirrhosis of any cause, aflatoxin exposure and ethanol consumption. Cytogenetic analysis on HCC has been limited because of poor hepatocyte growth in vitro. Conventional cytogenetic studies have demonstrated frequent abnormalities of specific chromosomes in HCC. Molecular cytogenetic approaches have been applied only rarely in the characterization of HCC. The main aim of this study was to evaluate genetic aberrations of different chromosomes in HCC. The study included 35 patients with HCC, who have been diagnosed and treated at National Cancer Institute, Cairo University, Egypt. The clinico-pathologic features of the studied patient were collected from patient’s files. MATERIALS AND METHODS: Interphase cytogenetics by fluorescence in situ hybridization with the use of a panel of centromere-associated DNA probes for chromosomes 1, 4, 8, 9, 13, 17, 20 and Y were performed on paraffin-embedded HCC specimens. RESULTS: The most common chromosomal aberrations detected were gain of chromosomes 8 in 12 cases (34.28%), 17 in 6 cases (17.14%). Loss of chromosome Y was detected in 6 of male cases (30%). Monosomy 4 was also detected in 5 cases (14.28%). Negative correlation could be detected only between chromosome 4 and 8. (r = -0.381, P < 0.05). Correlations between gain or loss of chromosomes and the different clinicopathologic parameters in the patients investigated, indicated negative correlation between: chromosome Y and age and chromosome 1 and cirrhosis. CONCLUSION: Gains and losses of DNA found in this study probably involve oncogenes and tumor suppressor genes that play a role in the puzzle of hepatocarcinogenesis.

Estudio citogenético en sangre periférica de pacientes con melanoma / Cytogenetic study in peripheral blood of melanoma patients

En Colombia el melanoma es la principal causa de muerte por enfermedades dermatológicas (40%) y representa el 1% del total de muertes por cáncer. El rápido incremento en la incidencia del melanoma hace necesaria la realización de estudios que permitan entender mejor los mecanismos implicados en su génesis y progresión. En este estudio se determinaron anomalías cromosómicas en sangre periférica de 30 pacientes con melanoma y en 23 individuos control mediante Citogenética Convencional (Bandeo G), observándose alta incidencia de anomalías numéricas y baja incidencia de rearreglos estructurales recurrentes, siendo las pérdidas cromosómicas las alteraciones prevalentes en todos los estadíos tumorales estudiados. El análisis citogenético de los pacientes mostró que, los cromosomas X, 9 y 17 fueron los más frecuentemente afectados. De las anomalías numéricas las monosomías de los cromosomas X y 17 y la trisomía formada por un cromosoma marcador fueron las más frecuentes, en estadíos tempranos y tardíos de la enfermedad. Deleciones y translocaciones se presentaron como anomalías únicas. En el grupo control ningún tipo de anomalía fue identificada, y se observó bajo porcentaje de fragilidades en comparación con el grupo de pacientes. En comparación con los controles se observó alta frecuencia de anomalías cromosómicas en los pacientes, lo que sugiere la existencia de heterogeneidad y predisposición genética en el desarrollo de la enfermedad, que con investigaciones adicionales deben ser analizadas y validadas como posibles fuentes de marcadores moleculares, útiles para el diagnóstico temprano, tratamiento y seguimiento de la enfermedad.

Among all the skin diseases, melanoma is the main cause of death in Colombia (40%) and it represents 1% of all deaths by cancer. Due to the fast increase in the incidence of melanoma, it is necessary to carry out research on the mechanisms involved in its genesis and progression. This study determined chromosomal anomalies from peripheral blood samples on 30 patients with melanoma and on 23 control subjects using conventional cytogenetics (G Banded), where a high incidence in numerical anomalies and a low incidence in recurrent structural rearrangements were observed. Chromosomic losses were prevalent in all the tumor stages studied. The analysis showed that the chromosomes X, 9 and 17 were mainly affected. Among the numerical anomalies, monosomies in X and 17 chromosomes, as well as trisomies formed by a marker chromosome, were the most common in both early and late stages of the disease. Deletions and chromosomal crossovers appeared to be as isolated anomalies. In the control group no anomaly was identified, and a low percentage of fragility was observed when compared with the patients group. A high frequency in chromosomal anomalies was observed in patients, in contrast with the control subjects. This suggests the existence of heterogeneity and genetic predisposition during the illness development. To further research, these must be analyzed and validated as possible sources of molecular markers, which could be of use for the early diagnosis, treatment and follow up of the disease.

Cytogenetic analysis of Astyanax laticeps (Cope, 1894) (Ostariophysi: Characidae) from the laguna dos Patos system

The genus Astyanax is prominent among Characiformes, due to the large number of species found and its wide geographic distribution. In this work, Astyanax laticeps specimens from the laguna dos Patos system were cytogenetically analyzed. A diploid number of 2n = 50 chromosomes distributed into 6m+16sm+16st+12a (FN = 88) was found, without differences between males and females. A few small heterochromatin blocks were observed, besides three more conspicuous C-bands, corresponding to NORs, as confirmed by silver nitrate and CMA3 staining, FISH, and DAPI negative staining. These regions were located in a medium-sized subtelocentric and in a large subtelocentric chromosomal pair, probably because of a deletion of this region in one homologous chromosome, or due to a transposition event between them.(AU)

O gênero Astyanax é destacado entre os Characiformes, pelo grande número de espécies encontradas e a ampla distribuição geográfica. Neste trabalho, foram analisados citogeneticamente espécimes de Astyanax laticeps do sistema da laguna do Patos. O número diplóide observado foi de 2n = 50 cromossomos distribuídos em 6m+16sm+16st+12a (NF= 88), sem diferenças entre machos e fêmeas. Foram observados poucos blocos de heterocromatina, além de três bandas-C mais conspícuas, correspondentes às NORs, confirmado pela coloração com nitrado de prata,CMA3, FISH, e coloração negativa ao DAPI. Estas regiões foram localizadas em um cromossomo subtelocêntrico de tamanho médio e em um par subtelocêntrico grande, provavelmente devido a deleção desta região em um dos cromossomos homólogos, ou por eventos de transposição entre eles.(AU)

Biogeography and comparative cytogenetics between two populations of Hoplias malabaricus (Bloch, 1794) (Ostariophysi: Erythrinidae) from coastal basins in the State of Bahia, Brazil

The species Hoplias malabaricus is a predator fish found in nearly all cis-Andean basins. From a cytogenetic point of view, this species comprises, at least, seven differentiated karyomorphs. Several localities have been formerly analyzed in Brazil, however, some regions, such as Bahia State, remain underrepresented. Recently, the Brazilian Environment Ministry classified both Itapicuru and Contas river basins (entirely located within Bahia territory) as priority conservation areas, whose biodiversity status lacks enough information. Therefore, the goal of the present work was to characterize, cytogenetically, populations of H. malabaricus from both basins, by using conventional staining, Ag-NOR and C-banding techniques. All specimens presented a diploid number of 2n = 40 with metacentric/submetacentric chromosomes, without differences between sexes, thereby representing the so-called "karyomorph F". The first metacentric pair presented a remarkably larger size in relation to the other pairs. The NORs were multiple, comprising the terminal region on long arms of two chromosomal pairs in both populations. However, the C-banding pattern was somewhat distinguishable between samples. Although sharing heterochromatic blocks at centromeric region of all chromosomes, the population from Itapicuru River basin appeared to have some more conspicuous blocks than those observed in the population from Contas River basin. The similar karyotype observed in both populations suggests a common geological history between them. The present results represent an advance in the knowledge about the cytogenetic pattern of H. malabaricus populations from poorly studied basins.(AU)

A espécie Hoplias malabaricus é um predador que ocorre em praticamente todas as bacias cis-andinas. Sob o ponto de vista citogenético, ela compreende, pelo menos, sete cariomorfos diferenciáveis. Várias localidades já foram previamente analisadas no Brasil, porém, algumas regiões, como o Estado da Bahia, permanecem pouco amostradas. Recentemente, o Ministério de Meio Ambiente classificou as bacias do rio Itapicuru e Contas (inteiramente localizadas na Bahia), como áreas prioritárias de conservação, cuja biodiversidade carece de informações suficientes. Neste sentido, o objetivo do presente trabalho foi caracterizar citogeneticamente populações de H. malabaricus nessas bacias, por meio de técnicas de coloração convencional, Ag-RON e bandamento C. Todos os espécimes e populações analisadas apresentaram número diploide 2n = 40 com cromossomos metacêntricos/submetacêntricos, sem diferenças entre os sexos, representando assim o denominado "cariomorfo F". O primeiro par metacêntrico apresentou tamanho notavelmente maior que os demais pares. As RONs foram múltiplas, ocupando a região terminal do braço longo de dois pares cromossômicos em ambas populações. Entretanto, os padrões de heterocromatina foram relativamente diferenciáveis entre as bacias hidrográficas estudadas. Apesar de compartilharem blocos heterocromáticos na região centromérica de todos os cromossomos, a população da bacia do Itapicuru apresentou alguns blocos mais conspícuos em relação aos da bacia do rio de Contas. O cariótipo similar encontrado em ambas as populações parece indicar uma história geológica em comum. Os dados obtidos representam um avanço no conhecimento dos padrões citogenéticos de populações de H. malabaricus provenientes de bacias pouco estudadas(AU)

Study of the cytogenetic effects of occupational exposure to pesticides on sanitation workersin Belo Horizonte, Brazil

Sanitation workers handling pesticides in the control of disease vectors constitute an occupationally exposed population to genotoxic substances. The aim of the present study was to investigate the relation between the occupational exposure to various pesticides and the presence of cytogenetic damage. Fifty-nine men were selected (29 sanitation workers and 30 control individuals) with ages varying between 18-57 years who lived and worked in the same area in Belo Horizonte (Brazil). The following parameters were determined for all individuals using the cytokinesis-block micronucleus (MN) assay in peripheral blood lymphocytes: MN/1000 binucleated cells (BC), BC with MN (BCMN)/1000 BC, nucleoplasmic bridges (NB)/1000 BC, apoptotic and necrotic cells/500 cells and nuclear division index. The analysis of covariance showed significantly higher (p < 0.05) mean frequencies of MN (15.81 ± 1.31 vs 4.71 ± 0.42), BCMN (15.10 ± 1.22 vs 4.62 ± 0.44), NB (4.59 ± 0.76 vs 1.00 ± 0.34), and necrotic cells (12.07 ± 1.45 vs 5.17 ± 0.70) in the exposed group when compared to the control group. There was no significant difference in the apoptotic cell frequency between the two groups, while the nuclear division index was significantly lower (1.49 ± 0.02 vs 1.61 ± 0.02) in the control group. Neither the time of exposure nor the smoking or alcohol drinking habit influenced the cytogenetic parameters examined. According to these results, occupational exposure to pesticides induced genotoxic and cytotoxic effects in sanitation workers.