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Master's Dissertation
DOI
10.11606/D.5.2007.tde-06062007-170334
Document
Author
Full name
Marcelo Augusto Cortina Gonçalves dos Santos
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2007
Supervisor
Committee
Toledo, Sergio Pereira de Almeida (President)
Billerbeck, Ana Elisa Correia
Marui, Suemi
Title in Portuguese
Detecção e rastreamento de mutações no proto-oncogene RET em pacientes com neoplasia endócrina múltipla tipo 2 por meio de eletroforese em gel sensível à conformação
Keywords in Portuguese
Análise mutacional de DNA/métodos Reação em cadeia por polimerase/métodos
Carcinoma medular/genética
Eletroforese/métodos
Mutação/genética
Neoplasia endócrina múltipla tipo 2A/genética
Neoplasia endócrina múltipla tipo 2B/genética
Neoplasias da tireóide/diagnóstico
Proto-oncogenes/genética
Abstract in Portuguese
A neoplasia endócrina múltipla tipo 2 (NEM-2) é uma síndrome tumoral herdada por mutações germinativas no proto-oncogene RET (RET) e transmitida por herança autossômica dominante. Atualmente, a indicação de tireoidectomia total preventiva é recomendada a indivíduos portadores de mutações no RET. Analisamos a aplicação do método Eletroforese em Gel Sensível à Conformação (CSGE) no rastreamento de mutações hot-spots do RET. Sete famílias com NEM-2 foram rastreadas pelo CSGE, seqüenciamento gênico e análise do Polimorfismo Conformacional de Cadeia Simples (SSCP). Usando o CSGE e SSCP, identificamos cinco das seis (83,3%) mutações verificadas pelo seqüenciamento: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile e Met918Thr. Foram analisados 128 amplicons englobando mutações hot-spots do RET e 116 dentre 128 (90.6%) concordaram com o seqüenciamento genético. Os polimorfismos 691 e 769 também foram documentados pelo CSGE e SSCP. Os dados obtidos por CSGE e SSCP foram totalmente (100%) concordantes. O CSGE revelou ser metodologia sensível, rápida, fácil de ser executada e de baixo custo na detecção de mutações nos códons 620, 634, 648, e 918, as quais constituem grande maioria (~95%) dos pacientes com NEM-2. Quanto à mutação Val804Met (prevalência na população inferior a 3%), o método necessita ser otimizado. Concluímos que o CSGE é uma metodologia efetiva para o rastreamento de mutações que mais freqüentemente ocorrem no RET como causadora de NEM-2.
Title in English
RET proto-oncogene mutations screening and detection in patients with multiple endocrine neoplasia type 2 using conformation sensitive gel electrophoresis
Keywords in English
DNA mutational analyses/methods
Electrophoresis/methods
Medullary carcinoma/genetic
Multiple endocrine neoplasia type 2A/genetic
Multiple endocrine neoplasia type 2B/genetic
Mutation/genetic
Polymerase chain reaction/methods
Proto-oncogene/genetic
Thyroid neoplasia/diagnosis
Abstract in English
Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited tumor syndrome caused by activating germline mutations in RET proto-oncogene (RET). Presently, the prophylactic total thyroidectomy is recommended to all RET mutations carriers. Here we tested the Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for the RET hot-spot mutations. Seven MEN2 families were studied by CSGE, as well as by Single Strand Conformational Polymorphism (SSCP) and direct sequencing analysis. Using CSGE and SSCP, we were able to detect five out of the six (83.3%) RET mutations verified by direct sequencing analysis: Cys620Arg, Cys634Arg, Cys634Tyr, Val648Ile and Met918Thr. RET polymorphisms 691 and 769 were verified by CSGE and SSCP. In our sample, data obtained using CSGE were fully concordant (100%) with SSCP findings. Thus, CSGE showed to be a sensitive, fast, low-cost, and ease procedure to detect RET mutations in codons 620, 634, 648, and 918 which are reported as the most prevalent RET variants (~95%) in large MEN2 series. As to the Val804Met mutation (prevalence in the population lower than 3%), this method still needs to be optimized. We concluded that CSGE is an effective screening method for the most frequent RET hot-spot disease-causing mutations.
 
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Publishing Date
2007-07-03
 
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