Establishing high resolution melting analysis: method validation and evaluation for c-RET proto-oncogene mutation...

Area of Science:

• Molecular diagnostics
• Genetic screening
• Oncology

Background:

• Accurate primary screening of mutation carriers is crucial for diagnostics.
• Germline point mutations in the c-RET proto-oncogene, linked to multiple endocrine neoplasia type 2 (MEN2), are suitable for analysis.
• Early identification of mutation carriers impacts survival due to medullary thyroid carcinoma (MTC) and treatment resistance.

Purpose of the Study:

• To validate and optimize high-resolution melting (HRM) analysis for routine primary mutation screening.
• To evaluate HRM's effectiveness for detecting c-RET gene mutations.

Main Methods:

• Validation assays were performed following International Conference on Harmonization (ICH) guidelines.
• Optimization experiments were conducted.
• HRM analysis was used for primary screening of 28 pathogenic c-RET mutations across nine exons.

Main Results:

• Validation confirmed HRM's repeatability, robustness, accuracy, and reproducibility.
• All pathogenic c-RET gene variants were successfully detected.
• No false-positive or false-negative results were observed.

Conclusions:

• HRM analysis is a powerful and reliable tool for primary screening of genetic variants.
• The method effectively distinguishes heterozygous point mutation carriers from wild-type carriers.
• HRM can serve as a first-line diagnostic tool for rapid, cost-effective screening of mutations like those in the c-RET gene.