Diagnostic method validation: High resolution melting (HRM) of small amplicons genotyping for the most common variants in the MTHFR gene.
OBJECTIVE
According to OECD guidelines methods implemented in a diagnostic laboratory should be properly validated prior their implementation. For this purpose we selected genotyping by High Resolution Melting (HRM) of small amplicons using common variants in MTHFR as a model.


METHODS
We selected previously typed samples on which selected analytical validation-related parameters relevant to DNA diagnostics - specificity, sensitivity, precision, robustness and ability to perform reliable calls were evaluated.


RESULTS
Correct genotype was assigned in 375/381 (98.4%) for c.677 C>T (rs1801133: C>T; p.A222 V) and in 102/104 (98.1%) for c.1298 A>C (rs1801131: A>C; p.E429A) of all cases. Low analytical failure rate and very high specificity/sensitivity were achieved. Similarly, precision and robustness were consistent.


CONCLUSIONS
We have successfully validated HRM of small amplicons using common MTHFR variants as a model. We proved that this technique is highly reliable for routine diagnostics and our diagnostic validation strategy can serve as a model for other applications.
Norambuena
Patricia A
PA
Department of Biology and Medical Genetics, Charles University 2nd Medical School and University Hospital Motol, Praha, Czech Republic. patynorambuena@gmail.com
Copeland
Joshua A
JA
Krenková
Petra
P
Stambergová
Alexandra
A
Macek
Milan
M
D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D042965:Methylenetetrahydrofolate Reductase (NADPH2) / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D042965:Methylenetetrahydrofolate Reductase (NADPH2)