Recessive dystrophic epidermolysis bullosa (R-DEB) is caused by mutations in the COL7A1 gene. The most common mutation reported in Mexican families is the c.2470insG mutation, normally detected by DNA sequencing. We report a faster and more economical high-throughput genotyping method to detect the c.2470insG mutation using specific TaqMan probes in a real-time polymerase chain reaction (RT-PCR) that facilitates genotype analysis with allelic discrimination plots. Our new method correctly genotyped 45 samples that had previously been sequenced as 41 wild-type homozygous (-/-), 1 heterozygous (-/G) and three mutant homozygous (G/G) (100% specificity). This new method allows high-throughput screening and furthermore is economical ($3 US/sample), fast (2 h), and sensitive as it requires only 20 ng input DNA. We used the new test to genotype 89 individuals from 32 unrelated Mexican families with R-DEB. The observed genotypic frequencies were 93.3% for the homozygous wild-type and 6.7% for the heterozygous genotype. The homozygous mutant genotype was not found. In conclusion, the allelic discrimination assay by RT-PCR is a sensitive, specific and effective high-throughput test for detecting the c.2470insG mutation.
Moreno-Treviño, M. G., León-Cachón, R. B. R., González-Salazar, F., Aguirre-Garza, M., Cerda-Flores, R. M., Meester, I., & Salas-Alanis, J. C. (2014). Real-time PCR Detection of the Recessive Dystrophic Epidermolysis Bullosa-associated c.2470insG Mutation in Unrelated Mexican Families. Archives of Medical Research, 45(7), 596–599. https://doi.org/10.1016/j.arcmed.2014.09.003