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 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.
Department of Clinical Biochemistry, Gentofte Hospital, University of Copenhagen, Denmark. michael.meldgaard@regionh.dk
Michael
M
Meldgaard
Pal B
PB
Szecsi
Berit C
BC
Carlsen
Jacob P
JP
Thyssen
Jeanne D
JD
Johansen
Torkil
T
Menné
Steen
S
Stender
D000483:Alleles; D019921:Biotinylation; D017931:DNA Primers; D004277:DNA, Single-Stranded; D000091344:Filaggrin Proteins; D060005:Genotyping Techniques; D006801:Humans; D007381:Intermediate Filament Proteins / Q000235:genetics*; D008863:Microspheres; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009693:Nucleic Acid Hybridization; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000235:genetics; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004277:DNA, Single-Stranded; C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D020033:Protein Isoforms