Rapid detection of the SPINK5 polymorphism Glu420Lys by real-time PCR technology
Hans-Peter Rihs1, Anna Kowal, Monika Raulf-Heimsoth
1Research Institute of Occupational Medicine of the Berufsgenossenschaften (BGFA), Ruhr University Bochum, Germany. rihs@bgfa.de
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.
Aims Of The Study:
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.
Results And 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.
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