Características otorrinolaringológicas en niños con trisomía 21: un estudio de 171 pacientes en el Hospital Infantil de México Federico Gómez / Otolaryngological features in children with trisomy 21: a study of 171 patients at the Hospital Infantil de México Federico Gómez

Resumen Introducción: Los niños con trisomía 21 enfrentan una amplia gama de problemas en la región de la cabeza y el cuello, por lo cual es importante reconocer las manifestaciones otorrinolaringológicas que presentan, así como su apropiado manejo. Métodos: Estudio de serie de casos retrospectivo de pacientes pediátricos con trisomía 21. De cada caso se analizó el espectro de manifestaciones otorrinolaringológicas, el manejo establecido y los resultados. Resultados: Se incluyeron 171 niños. La edad media de la primera valoración por otorrinolaringología en la institución fue de 7.2 ± 4.2 años. Las manifestaciones otológicas más frecuentes fueron la estenosis del conducto auditivo externo y la disfunción de la trompa de Eustaquio. Más de la mitad de los pacientes (63 %) presentaron hipoacusia, principalmente de tipo conductivo bilateral, y hasta el 75 % de los pacientes con afectación otológica requirieron algún procedimiento quirúrgico. Las manifestaciones rinológicas más comunes fueron la rinosinusitis crónica y la rinitis alérgica. La apnea obstructiva del sueño estuvo presente en el 30% de los pacientes. El tratamiento principal fue la amigdalectomía, seguida del tratamiento con dispositivos de presión positiva de la vía aérea. Menos del 5 % de los pacientes presentaron un compromiso laríngeo. Conclusiones: Los pacientes pediátricos con trisomía 21 deben ser remitidos sistemáticamente a una evaluación otorrinolaringológica periódica, debido a la alta incidencia de manifestaciones en esta región. Se deben ofrecer tratamientos oportunos para mejorar su salud y calidad de vida.

Abstract Introduction: Children with trisomy 21 face a wide range of conditions in the head and neck region, for which it is important that physicians are aware and have a strong understanding of the ear, nose, and throat (ENT) disorders, and their management as well. Methods: Retrospective case series of pediatric patients with trisomy 21. The spectrum of otolaryngological manifestations, their management, and outcomes of each case were analysed. Results: One hundred and seventeen pediatric patients were included. The mean age was 7.2 ± 4.2 years. More than half of the patients (63 %) had hearing loss (HL). The most frequent presentation was conductive HL, predominating the mild and bilateral type. The most common otological manifestations found were external ear canal stenosis and Eustachian tube dysfunction. Up to 75 % of the patients with otologic involvement required some surgical procedure. The most common rhinological manifestations were chronic rhinosinusitis and allergic rhinitis. Obstructive sleep apnea (OSA) was present in 30% of all patients, which main treatment was tonsillectomy, followed by continuous positive and biphasic positive airway pressure treatments. Less than 5 % of the patients presented a laryngeal compromise. Conclusions: Pediatric patients with trisomy 21 systematically should be referred to periodic ENT assessment due to the high incidence of manifestations in this region. Timely treatments should be offered in order to improve the health and the quality of life of the patient.

Pre-analytical parameters associated with unsuccessful karyotyping in myeloid neoplasm: a study of 421 samples

Cytogenetics is essential in myeloid neoplasms (MN) and pre-analytical variables are important for karyotyping. We assessed the relationship between pre-analytical variables (time from collection to sample processing, material type, sample cellularity, and diagnosis) and failures of karyotyping. Bone marrow (BM, n=352) and peripheral blood (PB, n=69) samples were analyzed from acute myeloid leukemia (n=113), myelodysplastic syndromes (n=73), myelodysplastic syndromes/myeloproliferative neoplasms (n=17), myeloproliferative neoplasms (n=137), and other with conclusive diagnosis (n=6), and reactive disorders/no conclusive diagnosis (n=75). The rate of unsuccessful karyotyping was 18.5% and was associated with the use of PB and a low number of nucleated cells (≤7×103/µL) in the sample. High and low cellularity in BM and high and low cellularity in PB samples showed no metaphases in 3.9, 39.7, 41.9, and 84.6% of cases, respectively. Collecting a good BM sample is the key for the success of karyotyping in MN and avoids the use of expensive molecular techniques.

Genetic Testing and Pregnancy Outcome Analysis of 362 Fetuses with Congenital Heart Disease Identified by Prenatal Ultrasound / Análise de Teste Genético e de Desfecho da Gestação de 362 Fetos com Cardiopatia Congênita Identificados por Ultrassom Pré-Natal

Abstract Background: Congenital heart defects (CHD), as the most common congenital anomaly, have been reported to be associated with chromosomal abnormalities. Currently, patients with CHD are routinely offered karyotyping and chromosomal microarray (CMA) testing, but the genotype-phenotype relationship has not yet been fully established. Objective: To determine the type and frequency of chromosomal abnormalities in fetuses with CHD and to analyze pregnancy outcomes of fetuses with heart abnormalities caused by different genetic factors. Methods: A total of 362 cases of CHD were enrolled from 2009 to 2016. Detailed ultrasound and laboratory examinations, including karyotyping and CMA, were performed. Outcome was obtained from discharge summaries. Results: Of the 362 fetuses, 220 were found with an isolated CHD, and 142 had CHD with extracardiac anomaly. Among these 362 fetuses, 140 were identified with a genetic cause, including 111 cases with aneuploidy, 10 cases with abnormality of chromosomal structure by karyotyping and 19 cases with pathogenic or likely pathogenic copy-number variations (CNVs) by CMA. The detection rate is close to 38.7%. Only one (identified as trisomy 18 syndrome) in 140 positive cases resulted in perinatal death, with the others being induced. The remaining 222 cases had negative results for both genetic testing and of these cases, 56 resulted in induced labor, and 77 had natural childbirth or caesarean births. The pregnancy outcome of the remaining 89 cases was uncertain. Conclusions: Karyotyping and CMA are effective and accurate prenatal genetic techniques for identifying fetal chromosomal abnormalities associated with cardiac defects, and this can assist clinical doctors to perform appropriate genetic counselling with regard to the etiology and outcome of CHD.

Resumo Fundamento: As cardiopatias congênitas (CCs) são as anomalias congênitas mais comuns, e têm sido associadas a anormalidades cromossômicas. Atualmente, a cariotipagem e a análise cromossômica por microarray (CMA) são oferecidas rotineiramente aos pacientes, mas a relação genótipo-fenótipo ainda não foi totalmente estabelecida. Objetivo: Determinar o tipo e a frequência das anomalias cromossômicas em fetos com CC e analisar os desfechos da gestação de fetos com anormalidades cardíacas causadas por diferentes fatores genéticos. Métodos: No total, foram admitidos 362 casos de CC entre 2009 e 2016. Ultrassonografia e exames laboratoriais detalhados foram realizados, incluindo cariotipagem e CMA. O resultado foi obtido a partir das folhas de epicrise. Resultados: Dos 362 fetos, 220 apresentaram doença coronariana isolada e 142 apresentaram doença coronariana com anomalia extracardíaca. Entre esses 362 fetos, foram identificados 140 com causa genética, incluindo 111 casos com aneuploidia, 10 casos com anormalidade da estrutura cromossômica por cariotipagem e 19 casos com variações no número de cópias (CNVs) patogênicas ou provavelmente patogênicas por CMA. A taxa de detecção é de aproximadamente 38,7%. Apenas um (identificado como síndrome da trissomia do cromossomo 18) em 140 casos positivos resultou em morte perinatal, com as demais sendo induzidas. Os 222 casos restantes tiveram resultados negativos para ambos os testes genéticos e, destes, 56 resultaram em trabalho de parto induzido e 77 tiveram partos naturais ou cesarianas. O desfecho da gravidez dos 89 casos restantes foi incerto. Conclusões: A cariotipagem e a CMA são técnicas genéticas pré-natais eficazes e precisas para a identificação de anomalias cromossômicas fetais associadas a defeitos cardíacos, e isso pode ajudar os médicos a realizar aconselhamento genético adequado com relação à etiologia e ao desfecho das cardiopatias congênitas.

Un caso de deleción parcial 1p36. 1 y trisomía parcial 6p diagnosticadas por cariotipo / A case of partial 1p36. 1 deletion and partial trisomy 6p diagnosed by karyotype

La deleción de la región cromosómica 1p36 es una de las anomalías subteloméricas más frecuentes y causa rasgos dismórficos distintivos. Por otro lado, la trisomía distal del brazo corto del cromosoma 6 es una anormalidad cromosómica poco frecuente de fenotipo variable. Objetivo: Presentar el caso de un paciente con ambas alteraciones cromosómicas, y resaltar la vigencia e importancia del cariotipo como herramienta diagnóstica en dismorfología. Caso clínico: Lactante de 2 meses de edad con múltiples anomalías craneofaciales, hemangioma en la nuca, fosita sacra, acortamiento rizomélico, pies y manos pequeños, criptorquidia unilateral izquierda e hipotonía. Además, antecedente de restricción del crecimiento intrauterino. Producto del octavo embarazo de una mujer G8A7C1 de 28 años. Con estos hallazgos inespecíficos en el fenotipo se solicitó cariotipo que mostró una deleción parcial de 1p36.1 y una trisomía parcial de cromosoma 6p. Conclusión: El cariotipo convencional sigue siendo una herramienta importante para el etiológico en pacientes con anomalías congénitas (múltiples), mostrando en este caso una deleción parcial de 1p36.1 y una trisomía parcial de cromosoma 6p, alteraciones cromosómicas estructurales.

The deletion of chromosomal region 1p36 is one of the most common sub-telomeric microdeletion syndromes and has distinctive dysmorphic features. On the other hand, partial trisomy of the short arm of chromosome 6 is a rare chromosomal abnormality with a variable phenotype. Objective: To report a case with both chromosome abnormalities, and to highlight the importance of the karyotype as a diagnostic tool in dysmorphology. Clinical case: The case of is presented of a two month-old infant with several craniofacial anomalies, neck haemangioma, sacral pit, rhizomelic shortening, small hands and feet, left unilateral cryptorchidism, and hypotonia. The infant also suffered intrauterine growth restriction and is the product of the eighth pregnancy of a 28 years old woman. Due to the unspecific findings in phenotype, a karyotype was requested, which showed a partial deletion of 1p36.1 and a partial trisomy of chromosome 6. Conclusion: The development of new techniques in molecular biology has improved diagnostic possibilities in medical genetics. However, the traditional karyotype remains as an important diagnostic tool in patients with multiple congenital anomalies.

Karyotypic diversity among three species of the genus Astyanax (Characiformes: Characidae) / Diversidade Cariotípica entre três espécies do gênero Astyanax (Characiformes, Characidae)

Abstract The group Incertae sedis within the Characidae family currently includes 88 genera, previously included in the subfamily Tetragonopterinae. Among them is the genus Astyanax comprising a group of species with similar morphology and widely distributed in the Neotropics. Thus, the present study aimed to analyze the karyotype diversity in Astyanax species from different watersheds by conventional Giemsa staining, C-banding and fluorescence in situ hybridization (FISH rDNA 18S) probe.specimens of Astyanax aff. paranae belonging to the “scabripinnis complex”, Astyanax asunsionensis and Astyanax aff. bimaculatus were analyzed”. Two sympatric karyomorphs were observed in Astyanax.aff paranae, one of them having2n=48andthe other one with 2n=50 chromosomes. Other population of this same species also presented 2n=50 chromosomes, but differing in the karyotype formula and with macro supernumerary chromosome found in 100% of the cells in about 80%of females analyzed. Two population of A. asuncionensis and one population of Astyanax. aff. bimaculatus, also showed a diploid number of 50 chromosomes, but also differing in their karyotype formulas. Therefore, A. asuncionensis was also characterized by intraspecific chromosome diversity. The C-banding analysis was able to demonstrate a distinctable to demonstrate a distinct pattern of heterochromatin differing A. asuncionensis from Astyanax aff. paranae and Astyanax aff. bimaculatus. The supernumerary chromosome of Astyanax aff. paranae proved completely heterochromatic. Only Astyanax.aff. bimaculatus multiple showed multiple sites of nucleolar organizing regions. The other species were characterized by having a simple system of NOR. These data contributes to the know ledge of the existing biodiversity in our fish fauna, here highlighted by the inter- and intraspecific chromosomal diversity in the genus Astyanax.

Resumo O grupo Incertae sedis, dentro da família Characidae inclui atualmente 88 gêneros, anteriormente incluídos na subfamília Tetragonopterinae. Dentre eles encontra-se o gênero Astyanax que compreende um grupo de espécies com morfologia similar e com ampla distribuição na região Neotropical. Assim, o presente estudo teve como objetivo analisar a diversidade cariotípica em espécies de Astyanax de diferentes bacias hidrográficas, através da coloração convencional com Giemsa, bandeamento C e hibridização fluorescente in situ (FISH com rDNA 18S). Exemplares de Astyanax aff. paranae, pertencentes ao “complexo scabripinnis”; Astyanax asunsionensise Astyanax aff. bimaculatus foram analisados. Dois cariomorfos foram observados em A. aff. paranae, um deles com 2n=48 cromossomos e outro com 2n=50 cromossomos. Outra população apresentou 2n=50 cromossomos, ambas diferindo na fórmula cariotípica e um cromossomo supranumerário encontrado em 100% das células, em aproximadamente 80% das fêmeas analisadas. Populações de A.asunsionensis e uma população de Astyanax aff. Bimaculatus também mostraram número diplóide de 50 cromossomos, mas diferindo em suas fórmulas cariotípicas. Portanto, A. asuncionensis foi também caracterizado por uma diversidade cariotípica intraespecífica. As análises de bandeamento C foi capaz de demonstrar um padrão distinto de heterocromatina, diferindo A. asuncionensis de A.aff. paranae e A. aff. bimaculatus. O cromossomo supranumerário de Astyanax aff. paranae mostrou-se completamente heterocromático. Apenas Astyanax aff. bimaculatus mostrou múltiplos sítios de regiões organizadoras de nucléolo(NORs). As outras espécies foram caraterizadas por apresentar um sistema simples de NOR. Estes dados contribuem para o conhecimento da existência de biodiversidade em nossa fauna de peixes, aqui em destaque pela diversidade cromossômica inter e intraespecífica no gênero Astyanax.

Retrospective karyotype study in mentally retarded patients / Estudo retrospectivo de cariótipo em pacientes com retardo mental

SUMMARY Objective: To describe the chromosomal alterations in patients with mental retardation (MR) using G-banding karyotype analysis. Method: A retrospective study of the results G-banding karyotype analysis of 369 patients investigated for MR was performed. Based on the structural rearrangements found, the authors searched all chromosomal regions related with breakpoints, and these were compared with the literature on MR and databases. Results: 338 (91.6%) normal cases, and 31 (8.4%) with some type of chromosomal abnormality were identified. Among the altered cases, 21 patients (67.8%) were identified with structural chromosomal alterations, nine (29%) with numerical alterations, and one (3.2%) with numerical and structural alterations. Conclusion: Structural chromosomal abnormalities were observed more frequently in this study. G-banding karyotyping contributes to the investigation of the causes of MR, showing that this technique can be useful for initial screening of patients. However, higher resolution techniques such as array based comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MPLA) can detect submicroscopic alterations commonly associated with MR.

RESUMO Objetivo: descrever as alterações cromossômicas em pacientes com retardo mental (RM) pela análise do cariótipo com bandas G. Método: foi realizado um estudo retrospectivo dos resultados de cariótipo com bandas G de 369 pacientes em investigação de RM. A partir dos rearranjos estruturais encontrados, foram levantadas todas as regiões cromossômicas envolvidas nos pontos de quebra e elas foram comparadas com a literatura para RM e bancos de dados. Resultados: foram identificados 338 (91,6%) casos normais e 31 (8,4%) com algum tipo de alteração cromossômica. Dentre os casos alterados, 21 pacientes (67,8%) foram identificados com alterações cromossômicas estruturais, 9 (29%) com alterações numéricas e 1 (3,2%) com alteração numérica e estrutural. Conclusão: as alterações cromossômicas estruturais foram aquelas observadas com maior frequência. O cariótipo com bandas G contribui para a investigação das causas de RM, mostrando que essa técnica pode ser útil como uma primeira triagem dos pacientes. No entanto, técnicas mais resolutivas como o array based comparative genomic hibridization (aCGH) e o multiplex ligation dependent probe amplification (MLPA) permitem detectar alterações submicroscópicas comumente associadas ao RM.

Homogeneidade cromossômica em diferentes populações de Panstrongylus megistus do Brasil / Chromosome homogeneity in different populations of Panstrongylus megistus of Brazil

Os triatomíneos são insetos vetores do protozoário Trypanosoma cruzi, agente etiológico da doença de Chagas. Existem seis espécies de maior importância vetorial, a saber, Triatoma infestans, Panstrongylus megistus, T. brasiliensis, T. sordida, T. pseudomaculata e Rhodnius neglectus. P. megistus apresenta grande distribuição geográfica e já foi coletada em 22 Estados brasileiros. Variações cromossômicas intraespecíficas já foram detectadas na subfamília Triatominae. Assim, o presente trabalho teve como objetivo analisar as características cromossômicas de P. megistus provenientes de sete diferentes Estados brasileiros (Rio Grande do Norte, Rio Grande do Sul, Paraná, São Paulo, Minas Gerais, Bahia e Santa Catarina), por meio de técnicas citogenéticas clássicas. Todos os insetos analisados apresentaram as mesmas características cromossômicas: 21 cromossomos, sistema de determinação do sexo do tipo X1X2Y, tamanho relativo dos autossomos com pequena variação, tamanho relativo dos cromossomos sexuais (Y>X1>X2), cromocentro na prófase meiótica formado apenas pelos cromossomos sexuais e presença de heterocromatina apenas no cromossomo sexual Y. Sendo assim, o presente trabalho apresenta dados evolutivos sobre as populações de P. megistus do Brasil, demonstrando que as populações de diferentes Estados brasileiros apresentam homogeneidade cromossômica.(AU)

The triatomines are insects vectors of the protozoan Trypanosoma cruzi, etiologic agent of Chagas disease. There are six species of greatest importance vector, out more, Triatoma infestans, Panstrongylus megistus, T. brasiliensis, T. sordida, Rhodnius neglectus and T. pseudomaculata. P. megistus presents great geographic distribution and has been collected in 22 Brazilian states. Intraspecific chromosomal variations have been detected in Triatominae. Thus, the present study aimed to analyze the chromosomal characteristics of P. megistusfrom seven different Brazilian states (Rio Grande do Norte, Rio Grande do Sul, Paraná, São Paulo, Minas Gerais, Bahia and Santa Catarina) by means of classical cytogenetic techniques. All insects analyzed showed the same chromosomal characteristics: 21 chromosomes, sex determination system the type X1X2Y, relative size of autosomes with little variation, relative size of the sex chromosomes (Y>X1>X2), chromocenter in meiotic prophase formed only by sex chromosomes and the presence of heterochromatin only in sex chromosome Y. Thus, this paper presents evolutionary data on the populations of P. megistus of Brazil demonstrating that populations from different Brazilian states have chromosomal homogeneity.(AU)

Deoxyribonucleic acid damage study in primary amenorrhea by comet assay and karyotyping.

AIM: This study aims at evaluating the chromosomal abnormalities and deoxyribonucleic acid (DNA) damage in cases with primary amenorrhea by karyotyping and comet assay. STUDY DESIGN: A total of 30 cases of primary amenorrhea were recruited. Secondary sexual characters were assessed by Tanner staging. Chromosomal analysis was performed by conventional phytohemagglutinin stimulated lymphocyte cell culture technique. Alkaline version of comet assay was used to evaluate DNA damage. RESULTS: The chromosomal pattern of 20 subjects (66.7%) was found to be normal (46,XX). Two subjects had 46,XY pattern and eight subjects had Turner syndrome (45,X or 45,X/46,XX). The comet parameters were found to be increased among subjects with 45,X monosomy, when compared to the rest of the study group and also in subjects with Tanner stage 1 when compared to stage 2. CONCLUSION: Comet assay revealed increased DNA damage in cases with 45,X monosomy, compared with subjects with 46,XX and 46,XY karyotype, which correlated with clinical features.

The application of an in situ karyotyping technique for mesenchymal stromal cells: a validation and comparison study with classical G-banding

The cytogenetic analysis of mesenchymal stromal cells (MSCs) is essential for verifying the safety and stability of MSCs. An in situ technique, which uses cells grown on coverslips for karyotyping and minimizes cell manipulation, is the standard protocol for the chromosome analysis of amniotic fluids. Therefore, we applied the in situ karyotyping technique in MSCs and compared the quality of metaphases and karyotyping results with classical G-banding and chromosomal abnormalities with fluorescence in situ hybridization (FISH). Human adipose- and umbilical cord-derived MSC cell lines (American Type Culture Collection PCS-500-011, PCS-500-010) were used for evaluation. The quality of metaphases was assessed by analyzing the chromosome numbers in each metaphase, the overlaps of chromosomes and the mean length of chromosome 1. FISH was performed in the interphase nuclei of MSCs for 6q, 7q and 17q abnormalities and for the enumeration of chromosomes via oligo-FISH in adipose-derived MSCs. The number of chromosomes in each metaphase was more variable in classical G-banding. The overlap of chromosomes and the mean length of chromosome 1 as observed via in situ karyotyping were comparable to those of classical G-banding (P=0.218 and 0.674, respectively). Classical G-banding and in situ karyotyping by two personnel showed normal karyotypes for both cell lines in five passages. No numerical or structural chromosomal abnormalities were found by the interphase-FISH. In situ karyotyping showed equivalent karyotype results, and the quality of the metaphases was not inferior to classical G-banding. Thus, in situ karyotyping with minimized cell manipulation and the use of less cells would be useful for karyotyping MSCs.

Análise dos resultados maternos e fetais dos procedimentos invasivos genéticos fetais: um estudo exploratório em Hospital Universitário / Analysis of fetal and maternal results from fetal genetic invasive procedures: an exploratory study at a University Hospital

OBJETIVO: Caracterizar as indicações das gestantes que procuraram o serviço de Medicina Fetal do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo para realização de procedimentos invasivos diagnósticos e avaliar os resultados dos cariótipos fetais e de suas gestações. MÉTODOS: Estudo observacional retrospectivo das gestantes que realizaram biópsia de vilo corial (BVC), amniocentese e cordocentese no período de fevereiro de 2005 a dezembro de 2009. Não foram incluídos outros procedimentos diagnósticos ou procedimentos terapêuticos. O resultado da gestação foi obtido através de consulta de prontuário eletrônico e/ou físico e/ou contato telefônico. RESULTADOS: Foram realizados 713 procedimentos (113 BVC, 340 amniocenteses e 260 cordocenteses). A principal indicação para a realização dos procedimentos invasivos foi a presença de alterações estruturais nos fetos, seguido por valores aumentados da translucência nucal e pela idade materna avançada. O cariótipo fetal esteve alterado em 186 casos (26,1%). A trissomia do cromossomo 18 foi a aneuploidia mais comum, seguida pela trissomia do 21, a monossomia do X e a trissomia do cromossomo 13. Ocorreram 4,9% de abortamento, 25,7% de natimortos e 13% de neomortos. Oito gestantes optaram pela interrupção judicial, e 99% das gestantes cujos fetos não apresentavam malformação e que apresentavam cariótipo fetal normal tiveram nativivos.

OBJECTIVE: To characterize the indications of pregnant women who sought the Fetal Medicine Services of the Hospital das Clínicas, at the Medical School of the Universidade de São Paulo for performing invasive diagnostic procedures, and to evaluate the results of fetal karyotypes and their pregnancies. METHODS: A retrospective and observational study on pregnant women who underwent chorionic villus sampling (CVS), amniocentesis, and cordocentesis in the period from February, 2005 to December, 2009. Other diagnostic or therapeutic procedures were not included. The result of pregnancy was obtained by consulting patient electronic records, medical records, and/or telephone call. RESULTS: 713 procedures were performed (113 CVS, 340 amniocenteses, and 260 cordocenteses). The main indication for performing invasive procedures was the presence of structural changes in fetuses, followed by increased values of nuchal translucency, and advanced maternal age. Fetal karyotype was altered in 186 cases (26.1%). The 18 trisomy was the commonest aneuploidy followed by the 21 trisomy, X monosomy, and 13 trisomy. There were 4.9% cases of miscarriage, 25.7% cases of stillborn infants, and 13% cases of neonatal deaths. Eight pregnant women opted for legally induced abortion. 99% of pregnant women whose fetuses did not present abnormalities and presented normal fetal karyotype had infants who were born alive.

Philadelphia chromosome detection in chronic myeloid leukemia: Utility of phytohemagglutinin-stimulated peripheral blood culture.

Background: The conventional cytogenetic approach to demonstrate Philadelphia (Ph) chromosome at times does not yield enough number of metaphases or are of suboptimal quality. Further, the rapid molecular tests have completely pushed this simple technique into disrepute. Aims: This study aimed to evaluate usefulness of phytohemagglutinin (PHA)-stimulated peripheral blood culture for detection of Ph chromosome in chronic myeloid leukemia (CML) patients. Materials and Methods: Fifty-six patients, including 11 newly diagnosed cases of CML and 45 patients of CML on imatinib therapy showing the presence of Ph chromosome in unstimulated samples, were included in the study. Cytogenetic analysis was done on unstimulated samples, i.e. bone marrow aspirate, 24- and 48-h peripheral blood culture, and compared with PHA-stimulated 72-h peripheral blood culture. Results: The preparations from PHA-stimulated peripheral blood culture samples in all 56 patients yielded high number of good-quality metaphases. All the 11 (100%) newly diagnosed patients and 39/45 (87%) of the patients on imatinib therapy showed the presence of Ph chromosome in PHA-stimulated samples. Addition of PHA-stimulated 72-h peripheral blood culture preparation can be of use for increasing the diagnostic yield in cases of CML with suboptimal results on conventional cytogenetics from bone marrow aspirate sample.

Karyotypic findings in chronic myeloid leukemia cases undergoing treatment.

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative expansion of primitive hematopoietic progenitor cells. MATERIALS AND METHODS: In the present study, CML samples were collected from various hospitals in Amritsar, Jalandhar and Ludhiana. RESULTS: Chromosomal alterations seen in peripheral blood lymphocytes of these treated and untreated cases of CML were satellite associations, double minutes, random loss, gain of C group chromosomes and presence of marker chromosome. No aberrations were observed in control samples. Karyotypic abnormalities have also been noted in the Ph-negative cells of some patients in disease remission. CONCLUSION: This is a novel phenomenon whose prognostic implications require thorough and systematic evaluation.

Octaploidy in idiopathic thrombocytopenic purpura.

We report a case of an elderly 68-year-old male who presented in our hospital with chief complaints of petechial rashes and ecchymosis over extremities and bleeding from the oral cavity since 3–4 days prior to hospitalization. He saw a physician before coming to our hospital and received one dose of IV methylprednisolone and oral wysolone. He had come to our hospital for further management. Bone marrow karyotyping was done and chromosomal analysis revealed two cell lines. Eighty percent of the cells analyzed revealed apparently normal male karyotype. However, 20% cells analyzed revealed a total of 184 chromosomes, suggesting octaploidy.

Detection of PML/RARA Rearrangement by Reverse Transcriptase-PCR and Sequencing in a Case of Microgranular Acute Promyelocytic Leukemia Lacking t(15;17) on Karyotype and FISH / 대한진단검사의학회지

We report a case of morphologically microgranular acute promyelocytic leukemia with PML/RARA fusion transcripts demonstrated by reverse transcriptase-PCR and cDNA sequencing, and no PML/RARA fusion detected by karyotype and FISH analyses. Karyotype was 47,XX,+8[19]/46,XX[1]. Although the newer FISH probes provide more accurate detections of t(15;17), it would be necessary to perform other molecular tests to further identify the masked PML/RARA fusions.

Caracterización citogenética del pez neotropical Brycon henni (Pisces: Characidae)

Cytogenetic characterization of the neotropical fish Brycon henni (Pisces: Characidae). Brycon henni, is a native fish from Western Colombia is important as food and in sport fishing, and could be cultured in waters between 18 and 28ºC. A previous cytogenetic study in branchial cells indicated different chromosomal complements. Total blood samples were seeded in 4 ml Ham-F12 culture medium, supplemented with 0.5 ml Bovine Fetal Serum and 0.7 ml Phyitohemaglutinin M (Gibco®) during 96 hr at 28ºC; an antimitotic factor (100µl Colcemid® 10%) was added. Cells were incubated in 10 ml KCl hypotonic solution (0.56%) for 24 min at 37ºC, fixed in methanol; acetic acid (3: 1) three times, separated by centrifugations, extended drip, and the chromosomes slides were stained with 5% Giemsa (5%). The best metaphases (6 males and 8 females) were photographed. There was a diploid number of 50 chromosomes: Metacentrics (26M), Submetacentrics (16SM) and Subtelocentrics (8ST). The fundamental number is 100 arms. The evolutionary tendency is type B with no supernumerary chromosomes; a first pair of big metacentric chromosomes is characteristic for Brycon, while no chromosomic sexual heteromorfism was detected. Rev. Biol. Trop. 56 (4): 1619-1628. Epub 2008 December 12.

La sabaleta Brycon henni, es un pez nativo del occidente colombiano importante para la seguridad alimentaria de las poblaciones ribereñas y para la pesca deportiva por sus característica combativas. El desarrollarse entre los 18 y 28ºC la convierte en una especie de cultivo promisoria. Un estudio citogenético a partir de células branquiales indicó diferentes complementos cromosómicos. Este trabajo aplicó la técnica de cultivo de linfocitos a muestras de sangre de adultos. Las muestras de sangre total fueron sembradas en 4 ml de medio de cultivo Ham-F12, suplementado con 0.5 ml de Suero Fetal Bovino y 0.7 ml del mitógeno Fitohemaglutinina M (Gibco®) durante 96 hr a 28ºC; 1.5 hr antes de la cosecha agregamos 100µl de Colcemid al 10% como factor antimitótico. Cada cultivo se incubó con 10 ml de solución hipotónica KCl 0.56% por 24 min a 37ºC, para continuar con tres fijaciones sucesivas, centrifugaciones y tinción Giemsa al 5%. Las mejores metafases fueron fotografiadas en microscopio, correspondientes a 6 machos y 8 hembras, indicando un número diploide de 50 cromosomas, clasificados en Metacéntricos (26M), Submetacéntricos (16SM) y Subtelocéntricos (8ST), para un número fundamental (NF) de 100 brazos. La tendencia evolutiva hallada fue de tipo B; no se encontraron cromosomas supernumerarios pero sí un primer par de cromosomas metacéntricos grandes, considerado marcador para el género Brycon, no determinante de heteromorfismo sexual. Estos resultados coinciden con los demás bricónidos investigados, en donde se podría considerar un ancestro común con un número cromosómico básico y cariotipos simétricos.

Condiciones óptimas de cultivo de linfocitos y análisis parcial del cariotipo de la tortuga cabezona, Caretta caretta (Testudines: Cheloniidae) en Santa Marta, Caribe Colombiano

Lymphocyte culture and partial karyotype of the marine turtle Caretta caretta (Testudines: Cheloniidae) in Santa Marta, Colombian Caribbean. Over the past few years an important reduction in the number of nesting marine turtle Caretta caretta individuals has been registered in the Colombian Caribbean, raising the question of a possible extinction in the medium term. A conservation plan is needed. We studied the culture requirements for C. caretta lymphocytes and preliminary karyotype analysis for cytogenetic identification, immunological study and toxicology without the need to kill individuals. Peripheral blood samples were obtained from 47 individuals in Santa Marta, Colombia and tests were made until optimal conditions were established for lymphocyte culture. The karyotype had 56 chromosomes, 32 macrochromosomes and 24 micro-chromosomes. An ideogram showed that C. caretta has four groups of chromosomes. Sexual chromosomes were not observed. These results do not coincide with the karyotype described from the Pacific (Japan). The present study is the first to include a complete description of the chromosome morphology of turtles from the Atlantic Ocean. it is possible that one of the adaptive strategies of this species is genetic interchange with other species of the family, producing viable hybrids. individuals in this study might be viable hybrids of C. caretta and further molecular studies are needed. Rev. Biol. Trop. 56 (3): 1459-1469. Epub 2008 September 30.

En los últimos años se ha registrado una disminución importante en el número de individuos de la tortuga marina Caretta caretta anidantes en el Caribe colombiano, situación que pone en evidencia la posibilidad de su extinción a mediano plazo. Por esto, es necesario implementar planes para su manejo y conservación. En este estudio se determinaron los requerimientos del cultivo de linfocitos de Caretta caretta para la obtención de cariotipos que permitan la identificación citogenética, el estudio inmunológico y toxicológico de individuos sin necesidad de sacrificarlos. Se muestrearon 47 individuos de C. caretta de Santa Marta, Colombia obteniendo sangre periférica con la que se realizaron ensayos de las diferentes variables hasta obtener las condiciones óptimas para el cultivo convencional de linfocitos. El cariotipo obtenido presentó 56 cromosomas: 32 macrocromosomas y 24 microcromosomas. El ideograma mostró que C. caretta tiene cuatro grupos de cromosomas: el grupo A compuesto por doce (12) pares de cromosomas de mayor tamaño. El Grupo B compuesto por cuatro (4) pares de cromosomas medianos y pequeños y el Grupo C conformado por 12 pares de microcromosomas. No se observaron cromosomas sexuales. Estos resultados están en desacuerdo con el cariotipo descrito por Kamesaki (1989), debido posiblemente a que las muestras analizadas en ese estudio fueron colectadas en el Océano Pacifico (Japón). El presente estudio es el primero realizado con tortugas del Océano Atlántico que cuenta con la descripción completa de la morfología cromosómica. Es posible, que una de las estrategias adaptativas de esta especie sea el intercambio genético con otras especies de la familia, que produce individuos híbridos viables. En este aspecto se ha descrito la hibridación de tortugas Caretta caretta con Eretmochelys imbricata, Chelonia mydas y Lepidochelys kempii, esto sugiere la posibilidad que los individuos caracterizados en este estudio sean híbridos viables de C. caretta, por lo tanto, se hace necesario realizar estudios a nivel molecular.

Risk factors for hypospadias: a case control study

Despite being one of the most common congenital defects in boys, the etiology of hypospadias remains largely unknown. In this study we evaluated a spectrum of potential risk factors for hypospadias in which we focused on both paternal and maternal factors and chromosomal aberrations. Cases were selected from the Genetic Clinic, Medical Research Institute, University of Alexandria. A total of 176 cases with hypospadias were included in this study, and a matching control group ofnormal 300 boys for the association study. All cases were subjected to detailed family, pregnancy, genetic histories, clinical examination, and pedigree study. Chromosome analysis was performed using peripheral blood lymphocyte cultures by trypsin G-banding technique. Hormonal assays, abdominal and pelvic ultrasound were carried out according to case presentation. Both parents of cases and the control group completed written questionnaires. Abnormal karyotyes were detected in 23 cases [13.07%] associated with other anomalies, sex chromosome abnormalities were present in 69.56% and autosomal aberrations in 30.43%. Patients with chromosomal abnormalities were excluded from the association study. Logistic regression analysis was used to assess the independent contribution of different factors to the risk of hypospadias. Our data did not support an association with increased parental age. The most profound result was the increased risk of hypospadias for boys with positive family history [n=23; OR=26.36; 95%Cl: 5.90-164.23]. Strong indications for an increased risk of hypospadias were also found with low birth weight [n=45; OR=1 3.47; 95%Cl=6.09-30.70], preterm birth [n=6; OR=1 2.20; 95%Cl=1.45-271.47], twin or triplet pregnancy [n=4; OR=8.03; 95%Cl=0.84-190.23], and when mothers had preeclampsia [n=16; OR=11.56; 95%Cl=3.11-50.77]. Associations with pregnancy achieved with fertility treatment, and mother used iron supplements were also found. In conclusion, routine karyotype screening permits the diagnosis of chromosomal anomalies especially in those with the most severe forms of hypospadias and additional anomalies. Several risk factors have been identified for hypospadias which support the idea that genetic predisposition, placental insufficiency, and substances that interfere with natural hormones before conception or during fetal development play a role in the etiology of hypospadias

Cariotipos de los caracoles de tinte Plicopurpura pansa y Plicopurpura columellaris (Gastropoda: Muricidae)

Karyotypes of the purple snails Plicopurpura pansa and Plicopurpura columellaris (Gastropoda: Muricidae). The karyotypes of the purple snails Plicopurpura pansa (Gould, 1853) and P. columellaris (Lamarck, 1816) were established from 17 and 13 adults, respectively; and from eight capsules with embryos of P. pansa. In P. pansa were counted 59 mitotic fields in the adults and 127 in embryos; and 118 fields in P. columellaris. Chromosome numbers from 30 to 42 were observed in both species. Such a variation was notorious in each sample and there was no evidence of any relationship with tissue (gill, muscle and stomach). Both species has a typical modal number of 2n=36 chromosomes. Five good quality chromosome spreads were selected from adults of each species to assemble the karyotype. Classic cytogenetics statistics like relative lengths, arm ratio, centromeric index and the difference between long and short arms are presented. There were three pairs of metacentric and fifteen pairs of telocentric chromosomes in both species. This classification was not strong enough, so the chromosome complement by species was divided in four groups ("a", "b", "c" and "d") on the basis of relative lengths (p+q). A comparison of p+q in each chromosome pair was estimated within and between species by two ways analysis of variance and Tukey tests (P<0.05). Significant differences were identified among chromosome groups in each species; the differences between species were given by the first three pairs of chromosomes (group "a" biarmed) and the last two pairs (group "d" uniarmed). Deviations in chromosome number and relative lengths probably are given by chromosome rearrangements, related with chromosome polymorphism and presence of the atypical microchromosome "B". The fundamental number in both species was characterized by 42 chromosome arms. No sex chromosomes were identified. Rev. Biol. Trop. 55 (3-4): 853-866. Epub 2007 December, 28.

El cariotipo de Plicopurpura pansa y P. columellaris fue determinado a partir de 17 y 13 especímenes adultos respectivamente. Adicionalmente, se utilizaron ocho cápsulas de P. pansa. Contamos 186 campos mitóticos en P. pansa: 59 en los adultos y 127 en los embriones; y 118 campos en P. columellaris. En ambas especies se observaron números cromosómicos desde 30 hasta 42. Las variaciones en número cromosómico fueron identificadas en cada individuo, no habiendo ninguna relación con los tejidos (branquias, músculo y estómago) empleados. El número modal diploide típico fue de 2n=36 cromosomas en ambas especies. En los especímenes adultos seleccionamos cinco de las mejores dispersiones cromosómicas para armar el cariotipo. Calculamos los estadísticos citogenéticos clásicos, longitudes relativas, proporción de brazos, índice centromérico y la diferencia entre brazos. Identificamos en ambas especies tres pares de cromosomas metacéntricos y quince pares de cromosomas telocéntricos. Esta clasificación no fue suficientemente robusta, por lo que dividimos el complemento cromosómico de cada especie en cuatro grupos ("a", "b", "c" y "d") utilizando como criterio las longitudes relativas (p+q). Hubo diferencias significativas entre grupos cromosómicos por especie y entre especies, los tres primeros pares de cromosomas (grupo "a" birrámeos) y los dos últimos pares (grupo "d" monorrámeos menores) (análisis de varianza de dos vías, p<0.05). Las desviaciones en número cromosómico y en las longitudes relativas, posiblemente se deban a reorganizaciones cromosómicos asociadas con el polimorfismo cromosómico y presencia de microcromosomas tipo "B". El número fundamental en ambas especies se caracterizó por 42 brazos cromosómicos. No identificamos cromosomas sexuales.

Can fetal echocardiogram and karyotyping be fairly offered for fetal umbilical cord abnormalities?

A prospective study to determine if fetal echocardiography and karyotyping by amniocentesis are strongly indicated in fetus with single umbilical artery [SUA]? During the period from March 2002-September 2006 all cases of fetal SUA, from Security Forces Hospital, Riyadh, Kingdom of Saudi Arabia, in a routine obstetric population were studied. Targeted sonograms and perinatal follow up were obtained. Sixteen thousand women completed the study, 162 cases of SUA were diagnosed on the basis of prenatal sonograms, however 169 cases [53 non isolated SUA and 116 isolated SUA] confirmed postnatafly. The sensitivity, specificity, positive predictive value and negative predictive value of prenatal ultrasonographic diagnosis of SUA were 90.5%, 99.9%, 94.4% and 99.8% respectively. Aneuploidy and cardiac abnormalities were present in 8 out of the 53 [15%] and 17/53 [32%] In none isolated SUA respectively. fetuses with SUA should get further evaluation at centers that handle high-risk cases and parent should strongly counseled for fetal echocardiogram and fetal karyotyping In case of none isolated SUA