Ancestry and self-reported race in Brazilian breast cancer women

SUMMARY OBJECTIVE: This study aimed to evaluate the association between self-reported race/color and ancestry in Brazilian patients with breast cancer. METHODS: This was an observational, transversal, epidemiological study, evaluating race and ancestry in 1,127 patients with breast cancer. For genetic ancestry, a 46-AIM-INDEL panel was used. The ancestral profile was evaluated with the Structure v.2.3.3 software. Descriptive statistics were performed. To assess differences between race and ancestry, an analysis of variance with Bonferoni adjustment was used. RESULTS: The race distribution was 77.7% white, 17.6% brown, 4.1% black, 0.4% yellow, and 0.3% cafuse. The African ancestry proportion was significantly (p<0.001) more evident in black [0.63±0.21 (0.17-0.96)], followed by brown [0.25±0.16 (0.02-0.70)], and less frequent in white skin color. The European ancestry proportion was significantly (p<0.001) higher in white [0.72±0.17 (0.02-0.97)], followed by brown [0.57±0.19 (0.12-0.92)], yellow [0.27±0.31 (0.12-0.620], and black [0.24±0.19 (0.02-0.72)]. The Asiatic ancestry proportion is significantly (p<0.001) higher in yellow [0.48±0.51 (0.04-0.93)]. The Amerindian ancestry proportion frequency was the least frequent in all groups, and cafuse patients did not express differences between all race groups. The brown race group presented differences in African and European ancestry. CONCLUSION: Although we found many similarities between white European ancestry, black African ancestry, and yellow Asian ancestry, there is great miscegenation between patients. Although they can be labeled as having one race, they do present many ancestral genes that would allow their inclusion in another race group.

Detección del polimorfismo p.K952L en pacientes cubanos con la enfermedad de Wilson

RESUMEN Fundamento La enfermedad de Wilson es una entidad rara con patrón de herencia autosómico recesivo, a causa de las mutaciones en el gen ATP7B, lo cual provoca la acumulación de cobre en tejidos y órganos. En la literatura se informan más de 800 polimorfismos. Objetivo identificar el polimorfismo p.K952L en los pacientes cubanos con diagnóstico clínico presuntivo de enfermedad de Wilson. Métodos se realizó un estudio descriptivo, en el Centro Nacional de Genética Médica y el Instituto Nacional de Gastroenterología, que incluyó 35 pacientes con diagnóstico clínico de la enfermedad de Wilson. La extracción del ADN fue por la técnica de precipitación salina; y la amplificación del fragmento de interés, mediante la técnica de Reacción en Cadena de la Polimerasa. Además, se empleó la técnica de Polimorfismo Conformacional de Simple Cadena para la determinación de los cambios conformacionales y la presencia del polimorfismo p.K952L. Resultados en el exón 12 se identificaron los cambios conformacionales denominados b y c, que correspondieron al polimorfismo p.K952L en estado heterocigótico y homocigótico, respectivamente. La frecuencia alélica del polimorfismo p.K952L fue de 38,6 %. Las manifestaciones más frecuentes en los pacientes que presentaron este polimorfismo fueron las hepáticas. Conclusión Se identificó el polimorfismo p.K952L en 21 pacientes cubanos con diagnóstico clínico de enfermedad de Wilson, lo cual posibilita ampliar los estudios moleculares por métodos indirectos.

ABSTRACT Background Wilson's disease is a rare entity with an autosomal recessive inheritance pattern, due to mutations in the ATP7B gene, which causes the accumulation of copper in tissues and organs. More than 800 polymorphisms are reported in the literature. Objective to identify the p.K952L polymorphism in Cuban patients with a presumptive clinical diagnosis of Wilson's disease. Methods a descriptive study was carried out at the Medical Genetics National Center and the Gastroenterology National Institute, which included 35 patients with a Wilson's disease clinical diagnosis. DNA extraction was by saline precipitation technique; and the amplification of the fragment of interest, by means of the Polymerase Chain Reaction technique. In addition, the Simple Chain Conformational Polymorphism technique was used to determine the conformational changes and the presence of the p.K952L polymorphism. Results in exon 12 the conformational changes called b and c were identified, which corresponded to the polymorphism p.K952L in the heterozygous and homozygous state, respectively. The allelic frequency of the p.K952L polymorphism was 38.6%. The most frequent manifestations in patients with this polymorphism were liver. Conclusion The p.K952L polymorphism was identified in 21 Cuban patients with a clinical diagnosis of Wilson's disease, which makes it possible to extend molecular studies by indirect methods.

CRISP3 glycoprotein: a good biomarker for prostate cancer? / Glicoproteína CRISP3: um bom biomarcador para câncer de próstata?

ABSTRACT Introduction: Cysteine-rich secretory protein 3 (CRISP3) is expressed at low levels in normal human prostate but often overexpressed in prostate cancer (PCa). The relevance of this overexpression for the malignancy of PCa is still unclear. The prognostic value of the currently used prostate specific antigen (PSA) test can be misleading under certain circumstances, resulting in overtreatment of indolent tumors. New biomarkers are needed to reduce overtreatment and improve quality of life of men. Objective: Evaluate if CRISP3 expression could be a good biomarker for PCa. Methods: CRISP3 expression was determined by immunohistochemistry in tissue sections of prostate cancer from twenty-five patients subjected to radical prostatectomy. Gleason grading system was used as prognostic indicator and the staging of PCa was defined using the TNM system. Clinical parameters and PSA levels before and after surgery were determined. Results: CRISP3 expression was strong in 14 (56%), moderate in four (16%) and weak in seven (28%) specimens. There was no correlation between the intensity of CRISP3 expression and pre- and post-treatment PSA levels. Fifteen (60%) of PCa biopsies showed extension of the primary tumor pT2. Seven patients (28%) showed Gleason score higher than 7; thirteen (52%) equal to 7, and five (20%) lower than 7. There were no significant statistical differences between Gleason score and CRISP3 expression. Conclusion: CRISP3 is expressed in prostate cancer at different levels. Additional studies are required to better evaluate if CRISP3 could be used as a biomarker.

RESUMEN Introducción: La proteína rica en cisteína secretora 3 (CRISP3) se expresa en bajos niveles en la próstata humana normal, pero está mayormente expresada en el cáncer de próstata (CaP). Todavía, su relevancia en pacientes con CaP aún no está clara. La prueba de antígeno prostático específico (PSA) puede generar interpretaciones erróneas bajo ciertas circumstancias, acarreando sobretratamiento de tumores indolentes. Nuevos biomarcadores son fundamentales para evitar tratamientos innecesarios y mejorar la calidad de vida del paciente. Objetivo: Evaluar se la expresión de CRISP3 podría ser un buen biomarcador de CaP. Métodos: Se determinó la expresión de CRISP3 por inmunohistoquímica en cortes de tejido de CaP de 25 pacientes sometidos a prostatectomía radical. La clasificación Gleason fue utilizada como indicador pronóstico, y la estadificación fue determinada por el sistema TNM. Parámetros clínicos y niveles de PSA antes y después de la cirurgía fueron determinados. Resultados: La expresión de CRISP3 fue fuerte en 14 (56%) muestras; moderada en cuatro (16%) y débil en siete (28%). No hubo relación entre la expresión de CRISP3 y PSA pre y post-tratamiento. Quince (60%) biopsias de CaP tuvieron extensión del tumor primario pT2. Siete pacientes (28%) demostraron escala de Gleason mayor que 7; trece (52%), igual a 7; y cinco (20%), menor que 7. No hubo diferencias estadísticas significativas entre la escala de Gleason y la expresión de CRISP3. Conclusión: CRISP3 se expresa en CaP en diferentes niveles. Se necesitan estudios adicionales para evaluar se CRISP3 puede realmente ser usado como biomarcador.

RESUMO Introdução: A proteína CRISP3 é expressa em baixos níveis na próstata humana normal, mas superexpressa no câncer de próstata (CaP). Contudo, sua relevância em pacientes com CaP ainda não está clara. O teste de antígeno específico da próstata (PSA) pode proporcionar interpretações erradas em determinadas circunstâncias, resultando em excesso de tratamento para tumores indolentes. Novos biomarcadores são fundamentais para evitar tratamentos desnecessários e melhorar a qualidade de vida do paciente. Objetivo: Avaliar se a expressão de CRISP3 poderia ser um bom biomarcador para CaP. Métodos: A expressão de CRISP3 foi determinada por imuno-histoquímica em seções de tecido de CaP de 25 pacientes submetidos a prostatectomia radical. O sistema de classificação Gleason foi utilizado como indicador prognóstico, e o estadiamento foi determinado pelo sistema TNM. Parâmetros clínicos e níveis de PSA antes e pós-cirurgia foram determinados. Resultados: A expressão de CRISP3 foi forte em 14 (56%) amostras; moderada em quatro (16%) e fraca em sete (28%). Não houve correlação entre a expressão de CRISP3 e PSA pré e pós-tratamento. Quinze (60%) biópsias de CaP apresentaram extensão do tumor primário pT2. Sete pacientes (28%) mostraram escore de Gleason maior que 7; treze (52%), igual a 7; e cinco (20%), menor que 7. Não houve diferenças estatísticas significativas entre o escore de Gleason e a expressão de CRISP3. Conclusão: CRISP3 é expresso no CaP em diferentes níveis. Estudos adicionais são necessários para avaliar se CRISP3 realmente pode ser usado como biomarcador.

L1 cell adhesion molecule (L1CAM) in stage IB cervical cancer: distinct expression in squamous cell carcinomas and adenocarcinomas.

AIMS: L1 cell adhesion molecule (L1CAM) has been shown to be correlated with tumour progression, attributed to its possible association with epithelial-mesenchymal transition (EMT), characterised by the expression of vimentin and loss of e-cadherin. Herein, we investigate the associations between L1CAM and clinicopathological parameters, as well as the expression of vimentin and e-cadherin, in carcinomas restricted to the cervix. METHODS: The study was retrospective observational and included 45 squamous cell carcinomas (63.4%) and 26 adenocarcinomas (36.6%) submitted to primary surgical treatment. Patient age, FIGO (International Federation of Gynecology and Obstetrics) stage, tumour size and follow-up were obtained from the medical records. All the slides were revised to evaluate histological differentiation, lymphovascular space invasion, depth of infiltration, disease-free cervical wall thickness, pattern of invasion front, Silva pattern (for adenocarcinomas) and the percentage of tumour-infiltrating lymphocytes. Tissue microarrays were constructed for immunohistochemical staining for L1CAM, e-cadherin and vimentin. RESULTS: Adenocarcinomas were associated with lower disease-free and overall survival. L1CAM and vimentin expressions were more frequent among adenocarcinomas, although loss of e-cadherin expression was more common among squamous carcinomas. L1CAM expression was associated with larger tumours, vimentin expression and lower disease-free survival. No association was observed between the expression of either L1CAM or vimentin and loss of e-cadherin. High levels of tumour-infiltrating lymphocytes were more frequent in squamous cell carcinoma, high-grade tumours, destructive pattern at front of invasion and loss of e-cadherin expression. CONCLUSIONS: Our results confirm the prognostic role of L1CAM in cervical carcinomas, but suggest a role for mechanisms other than EMT.

Detección de la mutación I172N en pacientes cubanos con hiperplasia suprarrenal congénita por insuficiencia de 21 hidroxilasa / Detection of the I172N Mutation in Cuban Patients with Congenital Adrenal Hyperplasia due to 21 Hydroxylase Insufficiency

Resumen Fundamento: la hiperplasia suprarrenal congénita constituye la causa más frecuente de ambigüedad sexual en la infancia y aproximadamente el 95 % de los casos son producto de mutaciones que ocurren en el gen que codifica la enzima 21α-hidroxilasa. El diagnóstico molecular constituye un elemento a considerar para el manejo y asesoramiento genético a pacientes y familiares en riesgo. Objetivo: identificar la mutación I172N, determinar su frecuencia en la población estudiada y su posible relación con los fenotipos clínicos encontrados. Métodos: se realizó un estudio descriptivo, de corte transversal, durante el período 2014-2016 a pacientes cubanos con diagnóstico de hiperplasia suprarrenal congénita por insuficiencia de 21-OH atendidos en el Instituto de Endocrinología de La Habana. El universo de estudio quedó constituido por 32 pacientes. Los resultados se presentaron en tablas o gráficos según fue más factible mostrar la información. Resultados: la mutación I172N fue identificada en individuos de ambos sexos y se estableció su relación con las formas clásicas de la enfermedad. En la población estudiada, se ubicó dentro de las tres mutaciones más frecuentes de las pesquisadas hasta el momento en el Centro Nacional de Genética Médica de la Habana a pacientes con hiperplasia suprarrenal congénita por insuficiencia de 21 OH. Conclusiones: el método estandarizado demostró ser reproducible y confiable para el diagnóstico molecular de los individuos con hiperplasia suprarrenal congénita por insuficiencia de 21-OH. La mutación I172N se ubica dentro de las tres más frecuentes en la población cubana estudiada y se relaciona con las formas clásicas de la enfermedad.

ABSTRACT Background: congenital adrenal hyperplasia is the most frequent cause of sexual ambiguity in childhood. Molecular diagnosis is an element to be considered for the management and genetic counseling of patients and relatives at risk. Objective: to identify the I172N mutation, to determine its frequency in the studied population and its possible relationship with the clinical phenotypes found. Methods: a descriptive, cross-sectional study was conducted during the 2014-2016 period for Cuban patients diagnosed with congenital adrenal hyperplasia due to 21-OH insufficiency treated at the Institute of Endocrinology of Havana. The universe consisted of 32 patients. The variables analyzed were: age, social sex, age at diagnosis, clinical form of hyperplasia, diagnosis by screening program, family history, consanguinity, nonspecific neonatal death, genital crisis of the newborn, previous molecular diagnosis, mutations studied previously, mutation I172N gene CYP21A. The results were presented in tables or graphs as it was more feasible to show the information. Results: the I172N mutation was identified in individuals of both sexes and its relation with the classic forms of the disease was established. In the studied population the three most frequent mutations of the researched ones, so far in the National Center of Medical Genetics of Havana to patients with congenital adrenal hyperplasia due to 21 OH insufficiency. Conclusions: the standardized method proved to be reproducible and reliable for the molecular diagnosis of individuals with congenital adrenal hyperplasia due to 21-OH insufficiency. The I172N mutation is among the three most frequent in the studied Cuban population and is related to the classic forms of the disease.

Diagnóstico molecular de distrofia muscular de Duchenne/Becker en una familia sin antecedentes patológicos de la enfermedad / Molecular Diagnosis of Duchenne/Becker muscular dystrophy in a family with no pathological antecedents of the disease

Fundamento: Las distrofias musculares de Duchenne y de Becker son enfermedades neuromusculares progresivas, con un patrón de herencia recesivo ligado al cromosoma X y causadas por mutaciones en el gen que codifica para la distrofina. El estudio de posibles portadoras en las familias afectadas resulta crucial, ya que genera expectativas y opciones frente al asesoramiento genético.Objetivos: describir el diagnóstico molecular de distrofia muscular de Duchenne/Becker en una familia sin antecedentes patológicos de la enfermedad.Métodos: se realizó un estudio experimental, de las deleciones en el gen distrofia muscular de Duchenne/Becker, en un paciente con diagnóstico clínico de la enfermedad, para lo cual se empleó la técnica de PCR-multiplex siguiendo los métodos descritos por Beggs y Chamberlain. También fueron estudiadas las mujeres de la familia, a través del análisis de marcadores polimórficos mediante repeticiones cortas en tándem de (CA)n.Resultados: fueron identificadas en el paciente deleciones de los exones 47 al 52; así como la procedencia del cromosoma X ligado a la enfermedad (abuelo materno). Se determinó el estado de no portadora en tres mujeres de la familia. No se pudo excluir mosaicismo germinal en la madre del niño.Conclusión: se infirió la ocurrencia de una mutación de novo. El diagnóstico molecular permitió la confirmación diagnóstica de la enfermedad en el niño afectado, además de la posibilidad de brindar un adecuado asesoramiento genético a la familia.

Foundation: Duchenne and  Becker muscular dystrophies are progressive neuromuscular diseases with a pattern of recessive inherited link to chromosome X and caused by mutations in the gene which codifies for dystrophin. The study of possible carriers in affected families is crucial since it generates expectations and options on genetic advisory.Objective: to describe the molecular diagnosis of Duchenne/Becker muscular dystrophy  in a family without pathological antecedents of the disease.Methods: an experimental study was developed about the deletions of Duchenne/Becker gene of muscular dystrophy, in a patient with clinical diagnosis of the disease. It was used multiple PCR technique following the methods described by Beggs and Chamberlain. In addition, the women of the family were studied by the analysis of polymorphic markers through short repetitions in (CA) n tandem.Results: deletions of exons from 47 to 52 were identified in the patient; so as the precedence of the X chromosome related to the disease (maternal grandfather). It was determined the state of non-carrier in three women of the family. It was not possible to exclude germline mosaicism in the child´s mother.Conclusion: the occurrence of a novo mutation was inferred. The molecular diagnosis allowed confirming the diagnosis of the affected child; in addition it was possible to offer adequate genetic advisory to the family.

Desempeño de métodos moleculares para la identificación de subtipos poco comunes del virus de la hepatitis C, genotipo 2 / Performance of molecular methods for identification of unusual subtypes of hepatitis C virus genotype 2

Resumen Introducción. El virus de la hepatitis C (HCV) presenta una gran variabilidad genética, con siete genotipos y numerosos subtipos. La determinación del tipo viral ha sido fundamental para la escogencia y la duración del tratamiento antiviral adecuado. En Venezuela, el genotipo 2 del HCV es relativamente diverso, siendo particularmente prevalente el subtipo 2j. Objetivo. Evaluar el desempeño de las metodologías para la determinación del genotipo del HCV, particularmente para la identificación del subtipo 2j. Materiales y métodos. Se determinaron el genotipo y el subtipo del HCV mediante la técnica de hibridación inversa LiPA (Line Probe Assay) y secuenciación de las regiones genómicas 5'NC y NS5B del virus. Resultados. En 65 muestras analizadas, la metodología basada en la amplificación de la región 5'NC mostró mayor sensibilidad (100 %), en comparación con la técnica LiPA (91 %) y la secuenciación de la región NS5B (77 %). La determinación de genotipo, tomando como método de referencia la secuenciación de NS5B, mostró un alto grado de concordancia para la secuenciación de la región 5´NC y la hibridación inversa LiPA, con 100 % en la asignación de genotipos, comparado con 70 % y 66 % para los subtipos, respectivamente. La secuenciación de la región NS5B permitió identificar los subtipos 2j y 2s, los cuales no fueron detectados por las otras metodologías. No se observó un patrón característico para las muestras subtipo 2j en la hibridación inversa LiPA. Conclusión. Aunque es la metodología con menor sensibilidad, la secuenciación de la región NS5B es una herramienta poderosa para la correcta discriminación de los distintos subtipos circulantes del HCV, lo cual reviste importancia epidemiológica.

Abstract Introduction: Hepatitis C virus (HCV) displays high genetic variability, with seven genotypes and numerous subtypes. The determination of the viral type has been essential for the selection and timing of antiviral treatment. In Venezuela, HCV genotype 2 is relatively diverse, being particularly prevalent subtype 2j. Objective: To evaluate the performance of methodologies for genotyping HCV, particularly for identification of subtype 2j. Materials and methods: HCV genotype and subtype were determined by reverse hybridization technique (LiPA) and sequencing of the HCV 5'UTR and NS5B regions. Results: A total of 65 samples from HCV-infected patients were analyzed. PCR amplifications of the 5'UTR region exhibited the highest sensitivity (100% vs 91% for LiPA and 77% for NS5B). Genotype determination, taking as reference test NS5B, showed 100% concordance with the other methods, and 67% and 59% for subtypes with 5´NC and LiPA, respectively. NS5B sequencing allowed the identification of subtypes 2j and 2s, which were not detected by the other methods. A specific LiPA pattern was not observed for HCV subtype 2j. Conclusion: Although being the methodology with lowest sensitivity for amplification of HCV RNA, sequencing NS5B region remains a powerful tool for correct discrimination of the different HCV subtypes, which is of epidemiological relevance.

Autism spectrum disorders: an updated guide for genetic counseling / Transtornos do espectro autista: um guia atualizado para aconselhamento genético

ABSTRACT Autism spectrum disorder is a complex and genetically heterogeneous disorder, which has hampered the identification of the etiological factors in each patient and, consequently, the genetic counseling for families at risk. However, in the last decades, the remarkable advances in the knowledge of genetic aspects of autism based on genetic and molecular research, as well as the development of new molecular diagnostic tools, have substantially changed this scenario. Nowadays, it is estimated that using the currently available molecular tests, a potential underlying genetic cause can be identified in nearly 25% of cases. Combined with clinical assessment, prenatal history evaluation and investigation of other physiological aspects, an etiological explanation for the disease can be found for approximately 30 to 40% of patients. Therefore, in view of the current knowledge about the genetic architecture of autism spectrum disorder, which has contributed for a more precise genetic counseling, and of the potential benefits that an etiological investigation can bring to patients and families, molecular genetic investigation has become increasingly important. Here, we discuss the current view of the genetic architecture of autism spectrum disorder, and list the main associated genetic alterations, the available molecular tests and the key aspects for the genetic counseling of these families.

RESUMO O transtorno do espectro autista é um distúrbio complexo e geneticamente heterogêneo, o que sempre dificultou a identificação de sua etiologia em cada paciente em particular e, por consequência, o aconselhamento genético das famílias. Porém, nas últimas décadas, o acúmulo crescente de conhecimento oriundo das pesquisas sobre os aspectos genéticos e moleculares desta doença, assim como o desenvolvimento de novas ferramentas de diagnóstico molecular, tem mudado este cenário de forma substancial. Atualmente, estima-se que, por meio de testes moleculares, é possível detectar uma alteração genética potencialmente causal em cerca de 25% dos casos. Considerando-se também a avaliação clínica, a história pré-natal e a investigação de outros aspectos fisiológicos, pode-se atribuir uma etiologia para aproximadamente 30 a 40% dos pacientes. Assim, em vista do conhecimento atual sobre a arquitetura genética do transtorno do espectro autista, que tem tornado o aconselhamento genético cada vez mais preciso, e dos potenciais benefícios que a investigação etiológica pode trazer aos pacientes e familiares, tornam-se cada vez mais importantes os testes genéticos moleculares. Apresentamos aqui uma breve discussão sobre a visão atual da arquitetura genética dos transtornos do espectro autista, listando as principais alterações genéticas associadas, os testes moleculares disponíveis e os principais aspectos a se considerar para o aconselhamento genético destas famílias.

Muir-Torre Syndrome: case report and molecular characterization / Sindrome de Muir-Torre: relato de caso e caracterizacao molecular

CONTEXT: Muir-Torre syndrome is a rare autosomal dominant genodermatosis caused by mutations in the mismatch repair genes. It is characterized by the presence of sebaceous skin tumors and internal malignancies, affecting mainly the colon, rectum and urogenital tract. Awareness of this syndrome among physicians can lead to early diagnosis of these malignancies and a better prognosis. CASE REPORT: We report the case of a Chilean patient who, over the course of several years, had multiple skin lesions, endometrial cancer and colon cancer. The syndrome was diagnosed using molecular techniques such as microsatellite instability analysis, immunohistochemistry and DNA sequencing, which allowed us to find the causative mutation. CONCLUSION: Molecular diagnostics is a highly useful tool, since it allows clinicians to confirm the presence of mutations causing Muir-Torre syndrome. It is complementary to the analysis of the clinical data, such as dermatological presentation, presence of visceral malignancies and family history of colorectal tumors, and it provides important knowledge to help physicians and patients choose between treatment options. .

CONTEXTO: A síndrome de Muir-Torre é uma genodermatose autossômica dominante rara causada por mutações nos genes de reparo de incorreções. Caracteriza-se pela presença de tumores sebáceos da pele e doenças malignas internas, afetando principalmente cólon, reto e trato urogenital. A consciência desta síndrome pelos médicos pode levar ao diagnóstico precoce dessas doenças malignas e a um melhor prognóstico. RELATO DE CASO: Relatamos o caso de uma paciente chilena que, ao longo de vários anos, teve lesões cutâneas múltiplas, câncer de endométrio e câncer de cólon. A síndrome foi diagnosticada com técnicas moleculares, como a análise de instabilidade de microssatélites, imunoistoquímica e sequenciamento de DNA, o que nos permitiu encontrar a mutação causadora. CONCLUSÃO: Diagnóstico molecular é uma ferramenta muito útil, uma vez que permite que os clínicos confirmem a presença de mutações causadoras de síndrome de Muir-Torre. É complementar para a análise dos dados clínicos, tais como a apresentação dermatológica, a presença de doenças malignas viscerais e história familiar de tumores colorrectais, e fornece conhecimentos importantes para ajudar os médicos e os pacientes a escolher entre opções de tratamento. .

Avaliação de estratégia combinada de eletroforese capilar e PCR triplo metilação-específica para diagnóstico molecular da Síndrome do X Frágil / Assessment of a combined strategy of capillary electrophoresis and methylation-specific triple polymerase chain reaction for molecular diagnosis of the Fragile X Syndrome

Introdução: a síndrome do X frágil é a principal causa de retardo mental de natureza familiar. Suas características clínicas pouco marcantes fazem com que um diagnóstico de certeza a partir de testes moleculares seja imprescindível. Objetivo: descrever e avaliar as vantagens e desvantagens de uma estratégia combinada de PCR triplo metilação-específica e eletroforese capilar para o diagnóstico molecular da síndrome do X frágil, visando baixo custo, exequibilidade, reprodutibilidade e sensibilidade. Métodos: foram coletadas 43 amostras de sanguede pacientes com déficit cognitivo e de suas mães, quando indicado. Tais indivíduos possuíam exame citogenético positivo, fenótipo e/ou história familiar sugestivas de síndrome do X frágil. Após extração de DNA, foi realizada eletroforese capilar com marcadores fluorescentes,tratamento com bissulfito de sódio e três reações de PCR metilação-específicas em cada amostra. Resultados: foi possível determinar o genótipo em 29 pacientes: 23 (14 homens e nove mulheres) apresentavam alelos com tamanho normal e seis (todos do sexo masculino) possuíam alelos na faixa de mutação completa. Em outras seis amostras, todas do sexo feminino, foi possível determinar um alelo na faixa normal e outro alelo alterado, entretanto, sem diferenciação entre faixa de pré-mutação ou de mutação completa. Nas demais oito amostras(cinco homens e três mulheres), não se pôde determinar o genótipo. Conclusões: a técnica proposta faz uma triagem de pacientes, mas apresenta desvantagens, como não terem sido obtidos resultados satisfatórios com as reações para alelos metilados e a análise dos rastrosdas PCRs com três primers ter se mostrado difícil e dependente de observador.

Introduction: The fragile X syndrome is the main cause of inherited mental retardation. Its very small remarkable clinical characteristics make that a surely diagnosis from molecular tests be indispensable. Objective: To describe and assess the advantages and disadvantages of a combined strategy of methylation-specific triple polymerase chain reaction (PCR)and capillary electrophoresis for the molecular diagnosis of the fragile X syndrome, seeking low cost, feasibility, reproducibility, and sensibility. Methods: 43 blood samples were collected from patients with cognitive deficit and their mothers, when indicated. Such subjects presented a positive cytogenetic exam, phenotype and/or familiar history suggestive of the fragile X syndrome. After DNA extraction, capillary electrophoresis with fluorescent markers, treatment with sodium bisulfite, and three methylation-specific PCR reactions were performed in each sample. Results: It was possible to determine the genotype in 29 patients: 23 (14 men and 9 women) presented alleles with normal size and six (all male) had them in the complete mutation range. In other six female samples, it was possible to determine an allele in the normal range and another altered; nevertheless, without differentiation between the pre-mutation or complete mutation ranges. In the other eight samples (five men and threewomen), it was not possible to establish the genotype. Conclusions: The suggested technique does a patient?s screening, but it has some disadvantages such as non-satisfying results been seen with the reactions for methylated alleles and analysis of the PCRs tracks with three primers being difficult and dependent on the observer.

Genotipificación del Virus del Papiloma Humano(VPH) en muestras de cepillados cervicales de pacientes de diferentes hospitales de Bogotá y evaluación de la concordancia de dos métodos basados en PCR / Genotyping the human papilloma virus (HPV) in Cytobrush samples taken from patients attending different hospitals in Bogotá (Colombia) and evaluating agreement between two PCR-based methods

Objetivo: genotipificar el VPH en muestras de pacientes con diferentes grados histopatológicos de lesión cervical. Materiales y métodos: estudio de corte transversal en muestras de cepillado cervical de mujeres con diferentes grados de lesiones cervicales histopatológicas en la ciudad de Bogotá en el año 2009. Se utilizó Luminex® xMAP® una técnica altamente sensible, reproducible y automatizada, y Reverse Line Blot (RLB). Resultados: las infecciones por los tipos de VPH de 16 y 18 fueron las más frecuentes en todos los tipos de patología del cérvix. Las infecciones mixtas predominaron con un 78,04% de los casos con patología negativa, 85,7% NIC I, 81,25% NIC II, 78,78% NIC III y 71,42% cáncer cervical. La concordancia entre los dos métodos fue del 67%. Conclusión: las infecciones por los tipos de VPH 16 y 18 predominan en la población estudiada.

Objective: genotyping HPV in samples taken from patients having different histopathological degrees of cervical injury. Materials and methods: this was a cross-sectional study of cervical scrape samples taken from females having different degrees of histopathological cervical injury in Bogotá during 2009. Luminex® xMAP® was used, which is a highly sensitive, reproducible and automated technique, as well as reverse line blot (RLB). Results: infections caused by HPV types 16 and 18 were the most frequently found types of pathology of the cervix. Mixed infections predominated (78.04%); amongst cases having negative pathology, 85.7% was found for cervical intraepithelial neoplasia (CIN) I, 81.25% for CIN II, 78.78% for CIN III and 71.42% for cervical cancer. Agreement between both methods was 67%. Conclusion: infection by VPH types 16 and 18 predominated in the population being studied.