Real-time PCR detection of the recessive dystrophic epidermolysis bullosa-associated c.2470insG mutation in unrelated Mexican families

Abstract

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.

Keywords: Diagnosis; Dystrophic epidermolysis bullosa; Genotyping; Real-time PCR.