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

Clin Chim Acta. 2012 Oct 9;413(19-20):1488-92. doi: 10.1016/j.cca.2012.06.014. Epub 2012 Jun 14.


Background: The filaggrin protein is expressed as profilaggrin mainly in stratum granulosum cells of the epidermis. The profilaggrin 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.

MeSH terms

  • Alleles
  • Biotinylation
  • DNA Primers
  • DNA, Single-Stranded
  • Filaggrin Proteins
  • Genotyping Techniques*
  • Humans
  • Intermediate Filament Proteins / genetics*
  • Microspheres
  • Multiplex Polymerase Chain Reaction / methods*
  • Nucleic Acid Hybridization
  • Polymorphism, Single Nucleotide
  • Protein Isoforms / genetics
  • Sensitivity and Specificity


  • DNA Primers
  • DNA, Single-Stranded
  • FLG protein, human
  • Filaggrin Proteins
  • Intermediate Filament Proteins
  • Protein Isoforms