Rapid detection of the SPINK5 polymorphism Glu420Lys by real-time PCR technology.
BACKGROUND
The SPINK5 gene, encoding the serine protease inhibitor LEKTI maps to chromosome 5q32, and has been suggested to be a locus predisposing to atopic diseases in general. The Glu420Lys variant showed significant association with atopy, asthma and atopic dermatitis in recent studies.


OBJECTIVE
Development of a high throughput assay to analyse the polymorphism G1258A (Glu420Lys) in exon 14 of the SPINK5 gene followed by the validation using samples of 235 latex-allergic health care workers (HCWs) with (N=63) and without asthma (N=172), and 80 non-atopic controls.


METHODS
Twenty DNA samples were first analysed by a polymerase chain restriction fragment analysis (RFLP) using Hph I to generate defined control DNAs which were used for the development of the assay suitable for the detection of the Glu420Lys variant by LightCycler technology.


CONCLUSIONS
315 samples were successfully screened with this new assay. The temperatures in the melting analysis of the SPINK5 exon 14 PCR product were characteristic to the probes hybridised to the mutant (AA) at 51.5 degrees C and to the wild-type (GG) at 59.5 degrees C. The fast and reliable mutation detection in the tested samples makes this high-speed method suitable for larger epidemiological studies.
Rihs
Hans-Peter
HP
Research Institute of Occupational Medicine of the Berufsgenossenschaften (BGFA), Ruhr University Bochum, Germany. rihs@bgfa.de
Kowal
Anna
A
Raulf-Heimsoth
Monika
M
Degens
Paul-Otto
PO
Landt
Olfert
O
Brüning
Thomas
T
D000328:Adult; D000368:Aged; D000483:Alleles; D001249:Asthma / Q000235:genetics; D002352:Carrier Proteins / Q000032:analysis* / Q000235:genetics*; D005260:Female; D005838:Genotype; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000235:genetics; D008297:Male; D008875:Middle Aged; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D053491:Proteinase Inhibitory Proteins, Secretory; D015203:Reproducibility of Results; D000074163:Serine Peptidase Inhibitor Kazal-Type 5; D013696:Temperature
D002352:Carrier Proteins; D053491:Proteinase Inhibitory Proteins, Secretory; C120650:SPINK5 protein, human; D000074163:Serine Peptidase Inhibitor Kazal-Type 5