Novel pharmacogenetic markers for treatment outcome in azathioprine-treated inflammatory bowel disease

M.A. Smith; A.M. Marinaki; M. Arenas; M. Shobowale-Bakre; C. M. Lewis; A. Ansari; J. Duley; J.D.  Sanderson

Disclosures

Aliment Pharmacol Ther. 2009;30(4):375-384. 

In This Article

Methods

Patients and Samples

Patients were recruited as part of a prospective multi-centre study of AZA for the treatment of IBD undertaken by members of the London IBD Forum.[41] The study was initially designed to assess the impact of pre-treatment TPMT genotype and activity and subsequent red cell TGN levels on clinical outcome. All patients were aged between 16 and 80 years of age and had IBD diagnosed by standard criteria. After excluding any patients with zero TPMT activity, all patients received 2 mg/kg of AZA. Complete response was defined as the achievement of each patient's indication for treatment and stated treatment goal. Steroid withdrawal was defined as complete withdrawal of corticosteroids by 3 months and remaining off corticosteroids for further 3 months. Maintenance of remission was defined as no active disease for 6 months. Remission of active disease was defined by disease activity criteria (Harvey-Bradshaw indices, Truelove and Witts criteria). Requirement for surgery, an alternative immunomodulator or a biologic were considered treatment failure. Adverse drug effects were carefully defined and included only those resulting in AZA withdrawal. The full protocol of this study is reported elsewhere.[41]

Pharmacogenetic Analysis

Known SNPs in XDH, MOCOS and AOX1 were selected using the Applied Biosystems International database and the online SNP registry: http://www.ncbi.nlm.nih.gov/SNP. Only coding region SNPs with a minor allele frequency greater than 0.02 in the Caucasian population were selected. TaqMan SNP genotyping assays were obtained from Applied Biosystems (Warrington, UK) and details are shown in Table 1 .

Patients were genotyped by real-time PCR using a Biorad Miniopticon (Hemel Hempstead, UK). 1.8 μL of DNA was added to Absolute QPCR Mix (Abgene, Epsom, UK) and Taqman SNP genotyping assay and diluted up to a reaction volume of 10 μL with DNA-free water, according to the manufacturers' instructions. PCR conditions were 15 min enzyme activation at 95 °C, then 42 cycles of: denaturation (15 s at 95 °C) and anneal/extension (1 min at 60 °C).

Sequencing

Due to the highly significant association discovered between one AOX1 SNP and clinical outcome, the coding region of AOX1 was then sequenced in 10 nonresponders (5 with and 5 without the SNP).

Primers for each exon were designed using the web-based tool primer3 (http://frodo.wi.mit.edu/) and synthesized by MWG Biotech, (Ebersberg Germany). The primer sequences are shown in the supplemental information (Table S1) available online. PCR conditions were 1 min denaturation at 94 °C then 35 cycles of (45 s denaturation at 94 °C, then 30 s annealing at 54 °C and 50 s extension at 72 °C) then 5 min extension at 72 °C. PCR product was then purified using QIAquick DNA extraction kits according to the manufacturer's instructions (Qiagen Ltd. Crawley, UK). PCR products were sequenced using Beckman Coulter Dye Terminator Sequencing Kit, (High Wycombe, UK), according to the manufacturer's instructions. The sequences obtained were compared with the published sequences obtained from the NCBI website cDNA:BC117179, genomic DNA: AC007163 (http://www.ncbi.nlm.nih.gov/sites/entrez?db=Nucleotide).

Statistical Analysis

The frequency of each SNP was compared with the published frequencies for Caucasian individuals in dbSNP and all SNPs tested for departure from Hardy–Weinberg equilibrium. Association tests were performed under a dominant model, testing for association between presence of the SNP minor allele and clinical outcome (both response and occurrence of side effects). Chi-square tests were used, unless cells contained values less than 5 in which instance a Fisher's Exact test was performed. Analysis was performed using Instat version 3.0a for Macintosh, (Graphpad Software, San Diego, CA, USA http://www.graphpad.com). Chi-square test for trend was used for 2 × 2 × 2 contingency tables. Difference in means was analysed using unpaired Student's t-test. No correction for multiple testing has been applied. Haplotype analysis was performed in XDH and MOCOS using UNPHASED.[42]

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