Development of an allele-specific real-time PCR assay for discrimination and quantification of p63 R279H mutation in EEC syndrome

The ectrodactyly-ectodermal dysplasiaclefting syndrome is a rare autosomal dominant disorder caused by heterozygous mutations in the p63 gene, a transcription factor belonging to the p53 family. The majority of cases of ectrodactyly-ectodermal dysplasia syndrome are caused by de novo mutations and are therefore sporadic in approximately 60% of patients. The substitution of arginine to histidine (R279H), due to a c.836G>A mutation in exon 7 of the p63 gene, represents 55% of the identified mutations and is considered a mutational hot spot. A quantitative and sensitive real-time PCR was performed to quantify both wild-type and R279H alleles in DNA extracted from peripheral blood and RNA from cultured epithelial cells. Standard curves were constructed for both wild-type and mutant probes. The sensitivity of the assay was determined by generating serial dilutions of the DNA isolated from heterozygous patients (50% of alleles mutated) with wild-type DNA, thus obtaining decreasing percentages of p63 R279H mutant allele (50%, 37.5%, 25%, 12.5%, 10%, 7.5%, 5%, 2.5%, and 0.0%). The assay detected up to 1% of the mutant p63. The high sensitivity of the assay is of particular relevance to prenatal diagnosis and counseling and to detect therapeutic effects of drug treatment or gene therapy aimed at reducing the amount of mutated p63. © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.