Allele and genotype frequencies were calculated using SPSS v20 (

Allele and genotype frequencies were calculated using SPSS v.20 (IBM Corp., Armonk, NY). Allele and genotype frequencies were calculated

for a) the studied population as a whole, b) the R-DEB patient group, and c) 13 selected families in which, apart from the patient, both parents could be genotyped. We improved the detection efficiency of the c.2470insG mutation by being able to screen 96 samples in 2 h with 100% sensitivity and specificity. Forty-five samples of R-DEB patients and their relatives that had been genotyped previously by DNA sequencing and included representatives of all genotype options (−/−,−/G, and G/G) (6) were genotyped identically by our real-time allelic discrimination method (Figure 1). The 89 subjects who participated in this study come from 32 unrelated Mexican families in which at least one find more member suffered from R-DEB. There were 39 (40%) R-DEB patients (36 children, two fathers and one mother) and 50 healthy individuals (24 mothers, 18 fathers, and eight siblings). In the studied population of 89 subjects, the G allele was contributed only by heterozygous genotypes. PD-166866 nmr The frequency of the G allele was 3.4%. The observed genotype frequencies were 93.3% for the homozygous wild-type (−/−) and 6.7% for the heterozygous genotype

(−/G). The homozygous mutant genotype (G/G) was not found (Table 1). Among subjects suffering from R-DEB (n = 39), the mutant G allele frequency was 3.8%; a 0.4% increase as compared to the studied population (n = 89). The genotype frequencies among patients suffering R-DEB were 92.3% for the −/− and 7.7% for −/G genotypes, respectively ( Table 1). The genotype distribution in the 13 selected families was as follows: the homozygous cAMP wild-type (−/−) was found in 13 healthy mothers, 11 healthy fathers and 16 offspring, of which three were healthy but 13 were affected. The heterozygous genotype (−/G) corresponded to two healthy fathers and four offspring, of which three were affected and one was healthy. The nucleotide sequencing method is considered a

gold standard for mutation detection (11). However, its low sensitivity may lead to inadequate analysis and false negatives in samples with a low DNA concentration (13). Furthermore, sequencing is more laborious, time-consuming, and expensive as compared to real-time PCR genotyping (12). We succeeded in developing a real-time PCR genotyping assay that detects the c.2470insG mutation with a 100% concordance as compared to the standard method of nucleotide sequencing. Advantages of the real-time PCR genotyping assay are that a) less input DNA input is required (20 ng), b) high-throughput option facilitates the parallel analysis of a large number of patient samples (96), c) results are obtained rapidly (2 h), d) analysis is simplified as genotypes can be read from allelic discrimination plots (Figure 1), and e) low cost per test ($3 US). The allele frequency of the c.2470insG mutation in our R-DEB population in the current study (3.