CARD15 Genotyping in Inflammatory Bowel Disease Patients by Multiplex Pyrosequencing

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Abstract
We describe here a method for detection of three common single-nucleotide polymorphisms (SNPs) in the inflammatory bowel disease (IBD)-related CARD15 gene. We used a two-step approach with multiplex PCR and pyrosequencing. Although the three loci are spaced far from one another, the built-in multiplexing capability of pyrosequencing reactions to extend for numerous bases was used with appropriate primers and the design of efficient deoxynucleoside triphosphate (dNTP) dispensation orders to screen for the variants simultaneously. IBDs are chronic relapsing disorders affecting the gastrointestinal tract with a population prevalence in Western countries of 1:1000 (1). The complex trait is subdivided on the basis of clinical and histologic features into two main subtypes: Crohn disease (CD) and ulcerative colitis (UC) (2)(3)(4)(5). Whereas UC is limited to the rectal and colonic mucosal layers, CD can involve any part of the intestine, most frequently the terminal ileum and colon (6)(7)(8)(9). Several candidate loci for IBD have been identified on the basis of whole-genome scans in different populations (10)(11)(12)(13)(14)(15)(16)(17)(18). Of these, the one on chromosome 16 named IBD1 has been confirmed in several independent studies (11)(12)(15)(16)(17)(18). Three SNPs in the CARD15 gene, located in the IBD1 region, have been demonstrated to confer susceptibility to CD, but not to UC, in several populations and to correlate with an ileal site of the disease (6)(7)(8)(9)(10). In this report, using a previously described approach for SNP/mutation analysis (19), we developed and validated a protocol to characterize simultaneously the R702W (SNP8), G908R (SNP12), and L1007fsinsC (SNP13) variants, which are associated with increased risk …