A novel multiplex analysis of filaggrin polymorphisms: A universally applicable method for genotyping

Background: The filaggrin protein is expressed as profilaggrin mainly in stratum granulosum cells of the epidermis. The proffiaggrin gene codes for 10-12 filaggrin repeats. The filaggrin protein is important for skin barrier function. Filaggrin deficiency due to functional null-polymorphisms affects 8-10% of the people in Northern Europe and is a strong risk factor for several diseases. Here, we describe a novel method for efficient, multiplexed genotyping of variations in the profilaggrin gene. Methods: Five known techniques were combined: i) allele-specific PCR, ii) PCR with tagged primers, iii) asymmetric PCR, iv) multiplex PCR, and v) hybridization of single-stranded PCR products to spectrally coded microbeads carrying tag sequences as capture probes. Asymmetry of PCR was accomplished by having the tagged and allele-specific forward primers present in limiting concentrations. Asymmetry ensured that the later PCR cycles generated only single-stranded reverse-strand products. This greatly improved the assay sensitivity and allowed for simple optimization. Results: The specificity of the tags was verified with single PCR in wildtype and homozygous samples. Only the PCR products with the appropriate anti-tag hybridized to the corresponding beads, demonstrating the specificity of the signal. The hybridization signal is strongly dependent on single-stranded PCR products. After 46 PCR cycles, double-stranded products are clearly present, but only the single-stranded products generated in later cycles hybridize to the beads and elicit the strong signals that allow for unambiguous genotyping. Conclusions: We have tested 17,000 samples for three filaggrin polymorphisms using this method, with a call rate exceeding 99% and a reagent cost of US $ 0.75 per sample. The method is universally applicable for multiplex genotyping of e.g. hereditary hemochromatosis, lactose intolerance, or cystic fibrosis. (C) 2012 Elsevier B.V. All rights reserved.