title: 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.
authors.affiliation: División Ciencias de la Salud, Departamento de Ciencias Básicas, Universidad de Monterrey, San Pedro Garza García, Nuevo León, México.
authors.forename: María G
authors.initials: MG
authors.lastname: Moreno-Treviño
authors.affiliation: División Ciencias de la Salud, Departamento de Ciencias Básicas, Universidad de Monterrey, San Pedro Garza García, Nuevo León, México. Electronic address: rafael.reyesleon@udem.edu.
authors.forename: Rafael B R
authors.initials: RB
authors.lastname: León-Cachón
authors.affiliation: División Ciencias de la Salud, Departamento de Ciencias Básicas, Universidad de Monterrey, San Pedro Garza García, Nuevo León, México; Centro de Investigaciones Biomedicas del Noreste, Monterrey, Nuevo León, México.
authors.forename: Francisco
authors.initials: F
authors.lastname: González-Salazar
authors.affiliation: División Ciencias de la Salud, Departamento de Ciencias Básicas, Universidad de Monterrey, San Pedro Garza García, Nuevo León, México.
authors.forename: Marcelino
authors.initials: M
authors.lastname: Aguirre-Garza
authors.affiliation: Facultad de Enfermeria, Universidad Autonoma de Nuevo León, Monterrey, Nuevo León, México.
authors.forename: Ricardo M
authors.initials: RM
authors.lastname: Cerda-Flores
authors.affiliation: División Ciencias de la Salud, Departamento de Ciencias Básicas, Universidad de Monterrey, San Pedro Garza García, Nuevo León, México.
authors.forename: Irene
authors.initials: I
authors.lastname: Meester
authors.affiliation: División Ciencias de la Salud, Departamento de Ciencias Básicas, Universidad de Monterrey, San Pedro Garza García, Nuevo León, México; DebRA México A.C., Guadalupe, Nuevo León, México.
authors.forename: Julio C
authors.initials: JC
authors.lastname: Salas-Alanis
mesh_terms: D000328:Adult; D000483:Alleles; D001483:Base Sequence; D002648:Child; D024143:Collagen Type VII / Q000235:genetics*; D004247:DNA / Q000235:genetics; D016108:Epidermolysis Bullosa Dystrophica / Q000175:diagnosis* / Q000235:genetics; D005260:Female; D005838:Genotype; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006720:Homozygote; D006801:Humans; D008297:Male; D008800:Mexico; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: C569053:COL7A1 protein, human; D024143:Collagen Type VII; D004247:DNA
keywords: Diagnosis; Dystrophic epidermolysis bullosa; Genotyping; Real-time PCR