Análise comparativa entre as metodologias de PCR metilação-específica (MSP), southern blot (SB) e FISH utilizadas no diagnóstico genético molecular de pacientes com suspeita clínica das síndromes de Prader-Willi ou Angelman / Comparative analysis of methylation-specific PCR (MSP), southern blot (SB) and FISH in molecular genetic diagnosis of patients with clinical picture suggestive of Prader-Willi or Angelman syndromes

Introdução: Prader-Willi (SPW) e Angelman (SA) são síndromes clinicamente distintas, causadas pela perda de expressão de genes na região cromossômica 15q11.2-q13, de origem paterna ou materna, respectivamente. Ambas compartilham os mesmos métodos diagnósticos. Nossos objetivos foram: a) analisar por PCR metilação-específica (MSP) pacientes com suspeita clínica de SPW/SA; b) comparar resultados de diferentes metodologias de diagnóstico molecular; c) aplicar a técnica MSP na rotina assistencial de pacientes encaminhados ao Serviço de Genética Médica/Hospital de Clínicas de Porto Alegre (SGM/HCPA). Métodos: Foram analisados 123 pacientes com suspeita clínica de SPW (n = 71) ou SA (n = 52) por MSP. Desses, 79 possuíam análise prévia por hibridação in situ fluorescente (FISH) e/ou Southern blot (SB). Resultados: Foram detectados 21 casos positivos – 15 de SPW (12,19%) e 6 de SA (4,88%). Nove pacientes tiveram etiologia molecular determinada, sendo sete com diagnóstico de SPW (quatro dissomias uniparentais – UPD15 materna – e três deleções na região 15q11-13) e dois com diagnóstico de SA (um com UPD15 paterna e um com deleção na região 15q11-13). Foram observados resultados equivalentes entre MSP e SB e resultados discrepantes entre MSP e FISH (n = 4). Foram padronizados dois protocolos de MSP para confirmação dos resultados e controle interno de qualidade. Conclusão: O perfil de detecção de cada técnica varia de acordo com o mecanismo etiológico presente. A análise por MSP detecta alterações no padrão de metilação geradas por deleção, UPD e defeitos de imprinting, sem identificar o mecanismo etiológico responsável...

Introduction: Prader-Willi (PWS) and Angelman (AS) are clinically different syndromes caused by loss of expression of genes located on the chromosome 15q11.2-q13, of paternal or maternal origin, respectively. Both syndromes have the same diagnostic methods. The aims of the present study were: a) to perform a molecular analysis of 123 patients with clinical findings suggestive of PWS or AS using methylation-specific PCR (MSP); b) to compare the results obtained using different molecular diagnostic methodologies; c) to standardize MSP to be used in the routine care of patients at Medical Genetics Service/Hospital de Clínicas de Porto Alegre (SGM/HCPA). Methods: 123 patients with clinical findings suggestive of PWS (n = 71) or AS (n = 52) were analyzed by MSP. 79 had undergone previous laboratory analysis by fluorescence in situ hybridization (FISH) and/or Southern blot (SB). Results: MSP detected 21 positive cases – 15 PWS (12,19%) and 6 AS (4,88%). Molecular etiology was determined in 9 patients only – 7 were diagnosed with PWS (4 had uniparental disomy – maternal UPD15 – and 3 had deletions at 15q11-13) and 2 were diagnosed with AS (1 of paternal UPD15 and 1 deletion at 15q11-13). Comparing both methodologies, it was possible to observe concordant results between MSP and SB and discordant results between MSP and FISH (n = 4). We standardized two MSP methods in order to confirm the results and for internal quality control. Conclusion: The resulting profile of each technique varies according to the existing etiological mechanism. The methylation analysis by MSP technique detects changes on methylation pattern caused by deletion, UPD and imprinting defects, but it does not identify the responsible etiologic mechanism...

Régimen de movilización para prevenir hipomovilidad mandibular en pacientes irradiados: análisis y comparación de dos técnicas / A mobilization regimen to prevent mandibular hypomobility in irradiated patients: an analysis and comparison of two techniques

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Radiotherapy, when used in head and neck cancer treatment, can produce side effects in the patients, such as decreased salivary production, xerostomia, opportunistic infections, radiation caries, dysphagia, local discomfort and the limitation of mouth opening. The aim of this study was to evaluate the amplitude of mouth opening in patients before and immediatelyafter the completion of radiotherapy, comparing the effectiveness of two physiotherapy exercises. The irradiated sites included the masticatory muscles. The results demonstrated that there were no statistically significant differences between the two instituted exercises; however there was a trend towards better clinical results in group 2. The amplitude of mouth opening showed a trend towards reduction, but this was not statistically significant. When the pterygoid and sternocleidomatoid muscles were included in the irriated field, patients were observed to have more morbidity. This indicates the great importance of these muscles in mouth opening. Based on the results obtained within this study, it is not possible to conclude that physiotherapy exercies are efficacious in preventing trismus. Future longitudinal studies are required to verify the onset of trismus in radiotherapy patients

A mobilization regimen to prevent mandibular hypomobility in irradiated patients: an analysis and comparison of two techniques.

Radiotherapy, when used in head and neck cancer treatment, can produce side effects in the patients, such as decreased salivary production, xerostomia, opportunistic infections, radiation caries, dysphagia, local discomfort and the limitation of mouth opening. The aim of this study was to evaluate the amplitude of mouth opening in patients before and immediately after the completion of radiotherapy, comparing the effectiveness of two physiotherapy exercises. The irradiated sites included the masticatory muscles. The results demonstrated that there were no statistically significant differences between the two instituted exercises; however there was a trend towards better clinical results in group 2. The amplitude of mouth opening showed a trend towards reduction, but this was not statistically significant. When the pterygoid and sternocleidomatoid muscles were included in the irriated field, patients were observed to have more morbidity. This indicates the great importance of these muscles in mouth opening. Based on the results obtained within this study, it is not possible to conclude that physiotherapy exercies are efficacious in preventing trismus. Future longitudinal studies are required to verify the onset of trismus in radiotherapy patients.

CFTR gene: molecular analysis in patients from South Brazil.

Cystic fibrosis (CF) is the most common genetic disease among Caucasians. The CF gene, named cystic fibrosis transmembrane conductance regulator (CFTR), codifies a protein that acts as a channel through the epithelial membrane. The present work aimed (1) to detect sequence alterations in the nucleotide binding regions and at the membrane spanning domain of the CFTR gene and (2) to detect the following frequent mutations R347P, R347H, R334W, and Q359K (located in exon 7), DeltaF508 (located in exon 10), G542X, G551D, R553X, and S549N (located in exon 11), W1282X (located in exon 20), and N1303K (located in exon 21). Seventy-seven unrelated CF patients were analyzed, who were previously diagnosed and currently under treatment at the Pneumology Service of our hospital. Regions of interest were amplified by PCR using specific primers. Each sample was analyzed by a non-radioactive single-stranded conformational polymorphism (SSCP) analysis technique and restriction enzyme digestion. The DeltaF508 mutation was found in 48.7% of the alleles. Frequencies of G542X, R334W, R553X, and W1282X mutations in our population were 3.25, 1.3, 0.65, and 0.65%, respectively. No alleles were found to carry mutations G551D, R334W, R347P, R347H, Q359K, S549N, and N1303K, which were included in the screening protocol. This study allowed the characterization of 84 out of 154 CF mutant alleles (54.5%). The incidence of main CF mutations analyzed was similar to that of the south European population. Mutation data presented here will be useful for designing new DNA testing strategies for CF in South Brazil.

Detecþao e tipagem molecular de papilomavírus humano (HPV) em 460 amostras de colo uterino: estudo comparativo com exames citopatológico e colposcópico / Detection and typing of human papillomavirus (HPV) in 460 positives samples: comparative study with colposcopy and cytology

A reaþao em cadeira da polimerase (PCR-Polymerase Chain Reaction) é uma técnica de amplificaþao enzimática de seq³Ûncias específicas de ácidos nucleicos. Essa técnica tem sido amplamente descrita para detecþao e tipagem do Papilomavírus Humano (HPV - Human Papilomavirus). Neste trabalho, 460 amostras de colo uterino foran avaliadas para a presenþa do DNA do HPV pela técnica de PCR. Amostras positivas foram subseq³entemente tipadas por RFLP (Restriction Fragment Lenght Polymorphism). Os resultados de PCR-RFLP foram comparados com os exames colposcópico e citopatológico (Papanicolou). O PCR-RFLP demonstrou ser uma técnica eficaz na detecþao e tipagem virais, apresentando maior sensibilidade do que os exames citopatológico e colposcópico (AU)

Detecçao e tipagem molecular de papilomavírus humano (HPV) em 460 amostras de colo uterino: estudo comparativo com exames citopatológico e colposcópico / Detection and typing of human papillomavirus (HPV) in 460 positives samples: comparative study with colposcopy and cytology

A reaçao em cadeira da polimerase (PCR-Polymerase Chain Reaction) é uma técnica de amplificaçao enzimática de seqüências específicas de ácidos nucleicos. Essa técnica tem sido amplamente descrita para detecçao e tipagem do Papilomavírus Humano (HPV - Human Papilomavirus). Neste trabalho, 460 amostras de colo uterino foran avaliadas para a presença do DNA do HPV pela técnica de PCR. Amostras positivas foram subseqüentemente tipadas por RFLP (Restriction Fragment Lenght Polymorphism). Os resultados de PCR-RFLP foram comparados com os exames colposcópico e citopatológico (Papanicolou). O PCR-RFLP demonstrou ser uma técnica eficaz na detecçao e tipagem virais, apresentando maior sensibilidade do que os exames citopatológico e colposcópico

Detecção e tipagem molecular de Papilomavírus Humano (HPV) em 460 amostras e colo uterino: estudo comparativo com exaes citopatológico e colposcópico / Human Papillomavirus (HPV) detection and molecular typing from 460 cervical samples: comparative study between colposcopy and citology (Papanicolaou smears)

A Reação em Cadeia da Polimerase (PCR – Polymerase Chain Reaction) é uma técnica de amplificação enzimática de sequências específicas de ácidos nucléicos. Essa técnica tem sido amplamente descrita para detecção e tipagem do Papilomavírus Humano (HPV – Human Papillomavirus). Neste trabalho, 460 amostras de colo uterino foram avaliadas para a presença do DNA do HPV pela técnica de PCR. Amostras positivas foram subsequentemente tipadas por RFLP (Restriction Fragment Polymorphism). Os resultados de PCR-RFLP foram comparados com os exames colposcópico e citopatológico (Papanicolaou). O PCR-RFLP demonstrou ser uma técnica eficaz na detecção e tipagem virais, apresentando maior sensibilidade do que os exames citopatológico e colposcópico.