Screening of RET gene mutations in multiple endocrine neoplasia type-2 using conformation sensitive gel
Marcelo A C G dos Santos1, Elisangela Pereira de S Quedas, Rodrigo de Almeida Toledo
1Endocrine Genetics Unit (LIM-25), Endocrinology, Department of Internal Medicine, Hospital das Clínicas, University of São Paulo School of Medicine, SP.
Abstract:
Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited tumor syndrome caused by RET proto-oncogene germline mutations (RET). Here we tested the Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for RET hot-spot mutations. Seven MEN2 families were studied by direct sequencing analysis, CSGE and Single Strand Conformational Polymorphism (SSCP). Using CSGE/SSCP, we were able to detect four out of five types of RET mutations verified by sequencing analysis: Cys620Arg, Cys634Arg, Cys634Tyr, and Met918Thr, furthermore a missense substitution at codon 648 (Val648Ile). RET polymorphisms 691 and 769 were also verified. Data obtained using CSGE/SSCP were fully concordant. We conclude that CSGE showed to be a sensitive, fast, low-cost, and simple procedure to detect RET mutations in codons which are reported as the most prevalent RET variants (approximately 95%) in large MEN2 series. As to the Val804Met mutation, this method still needs to be optimized.
Insights
Conformation Sensitive Gel Electrophoresis (CSGE) effectively screens for RET proto-oncogene mutations in Multiple Endocrine Neoplasia type 2 (MEN2). This fast, low-cost method detects most common RET variants, aiding in inherited tumor syndrome diagnosis.
Area of Science:
- Genetics
- Oncology
- Molecular Biology
Background:
- Multiple Endocrine Neoplasia type 2 (MEN2) is an inherited tumor syndrome.
- Germline mutations in the RET proto-oncogene cause MEN2.
- Accurate detection of RET mutations is crucial for diagnosis and management.
Purpose of the Study:
- To evaluate Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for RET proto-oncogene mutations.
- To compare CSGE with direct sequencing and Single Strand Conformational Polymorphism (SSCP) for RET mutation detection.
- To assess the sensitivity and efficiency of CSGE for common MEN2-associated RET variants.
Main Methods:
- Seven MEN2 families were analyzed.
- Direct sequencing was used as the gold standard for mutation verification.
- Conformation Sensitive Gel Electrophoresis (CSGE) and Single Strand Conformational Polymorphism (SSCP) were employed for mutation screening.
Main Results:
- CSGE/SSCP successfully detected four out of five known RET mutations (Cys620Arg, Cys634Arg, Cys634Tyr, Met918Thr) and identified a novel Val648Ile substitution.
- The method also verified known RET polymorphisms at codons 691 and 769.
- CSGE/SSCP data were fully concordant with sequencing analysis for the detected mutations.
Conclusions:
- CSGE is a sensitive, rapid, cost-effective, and simple technique for screening prevalent RET mutations in MEN2 patients.
- The method covers approximately 95% of the most common RET variants.
- Further optimization is needed for detecting specific mutations like Val804Met.
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