Rapid detection of common CARD15 variants in patients with inflammatory bowel disease.
BACKGROUND
Three mutations (R702W, G908R, and 1007fs) within the CARD15 gene have been identified as independent risk factors for the development of Crohn's disease (CD). Virtually all studies investigating the occurrence of these mutations in patients with CD have used separate PCR-based methods to screen patient DNA, here we describe a novel multiplex amplification refractory mutation system (ARMS) assay that allows the simultaneous detection of R702W, G908R, and 1007fs, and a fourth CARD15 variant, P268S, at a fraction of the cost of the pre-existing genotyping assays.


METHODS
Allele-specific primer sets were designed for each CARD15 variant, optimized separately for annealing temperature and MgCl2 and then multiplexed. The mutant- and wild-type-specific primers were split across two tubes so that each multiplex reaction was internally controlled for amplification failure. An additional primer pair specific to beta2-microglobulin was included as an independent control for DNA quality. The specificity of each primer set was tested using positive controls that had been validated by sequencing, and the robustness of the final ARMS assay was assessed by genotyping 111 Caucasian patients with inflammatory bowel disease (IBD).


RESULTS
The specificity of each primer set was confirmed using a sequence validated positive control for each of the four CARD15 variants. Of the 111 DNA samples screened with our ARMS assay, a clear CARD15 genotype was obtained for 109 patients.


CONCLUSIONS
Given the potential predictive value of R702W, G980R, and 1007fs, a robust genotyping method for these variants would be of considerable value both in diagnostic and research settings. Our ARMS assay only takes 3-4 hours to perform once DNA has been extracted and requires only 1U of Taq DNA polymerase, making it a rapid, reliable, and cost-effective alternative to current CARD15 genotyping methods.
Roberts
Rebecca L
RL
Department of Pathology, Christchurch School of Medicine and Health Sciences, University of Otago, New Zealand. rebecca.roberts@chmeds.ac.nz
Gearry
Richard B
RB
Barclay
Murray L
ML
Kennedy
Martin A
MA
D004252:DNA Mutational Analysis / Q000379:methods*; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease / Q000235:genetics; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000175:diagnosis* / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D053473:Nod2 Signaling Adaptor Protein; D016133:Polymerase Chain Reaction / Q000379:methods*
D047908:Intracellular Signaling Peptides and Proteins; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein