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P365. Genetic alterations of innate immune response in IBD: Planning and setting up a polymorphisms analysis method by multiplex PCR and minisequencing

A. Crudeli, G. Ferraguti, D. Guagnozzi, S. Bruno, M. Lucarelli, P. Vernia

“Sapienza” University, Rome, Italy

Introduction and Objectives: The genetic background is involved in the pathogenesis of IBD. High-throughput SNP analysis is one of the most promising areas for clinical applications of genetic research. Here we describe a rapid, inexpensive and reliable assay to simultaneously investigate 10 nucleotidic variations by MULTIPLEX PCR and MINISEQUENCING in IBD patients. Minisequencing consist in a single base extension of a primer, using dye terminator nucleotides, that anneals next to the variant nucleotide followed by a fragment analysis performed in a capillary electrophoresis system.

Aims and Methods: To set up and validate 10 polymorphisms panel [NOD2 (SNP8, SNP12, SNP13), TLR4 (Asp299Gly), TLR2 (R753G), CD14 (260C>T) e TLR9 (1237;2848) associated to IBDs and TOLLIP (−526 C/G; Ala222Ser) not previously studied in IBD patients] using MULTIPLEX PCR and MINISEQUENCING techniques. To evaluate allelic and genotypic frequency of sequence variations and seek associations between genotype and phenotype.

DNA was extracted from venous blood of 40 IBD patients and 24 healthy blood donors, using the QIAamp DNA BLOOD MIDI KIT (Qiagen).

DNA amplification was performed by MULTIPLEX PCR (two PCR of 5 amplicons plus one negative control each). The PCR products were run on agarose gel and after purified to remove unused primers and dNTPs. The purified template was used for Minisequencing reaction followed by a second purification to remove unused fluorescent ddNTPs.

Capillary electrophoresis was performed on an ABI PRISM 3100 Avant; for each patient an electropherogram was generated after Genotyper software analysis of raw data. Genotypes were represented by peaks distributed as expected in 4 different colours (representing fluorescent bases). Any electropherogram differing from the wild type reference can be related to a polymorphic base in heterozygosis or in homozygosis.

Results: Validation of Minisequencing results was performed by sequencing analysis method of some obtained genotypes. The genotypic and allelic frequencies obtained in our small population are in keeping with those described in the literature for the already studied SNPs. Tollip gene variations showed the same frequencies in IBD patients and controls.

Conclusion: In the present study we developed a reliable, rapid, and cost effective Minisequencing assay that may represent an useful tool for the identification of subgroups of IBD patients, helping to predict the course of the disease and to individualize therapy.