II-69Short invited lectures – abstracts
GENETIC AND EXPRESSION PROFILES AS PHARMACOGENOMIC
MARKERS IN COMPLEX DISEASES
Uroš Potočnik1, 2, Damjan Glavač3, Ivan Ferkolj4, Silvo Koder5, Michael Dean6
1 Center for Human Molecular Genetics and Pharmacogenomics, Medical Faculty
2 Laboratory for Biochemistry, Molecular Biology and Genomics, Faculty for Chemistry and Chemical
Engeneering, University of Maribor, Slovenia
3 Laboratory of Molecular Genetics, Institute of Pathology, Medical Faculty
4 University Medical Centre, Division of Internal Medicine, Department of Gastroenterology; Ljubljana,
Slovenia
5 Teaching Hospital Maribor, Slovenia
6 Laboratory of Genomic Diversity, National Cancer Institute, Frederick, Maryland, USA;
uros.potocnik@uni-mb.si
Introduction The aim of our study is identification of single nucleotide polymorphisms (SNPs), haplo-
types and gene expression profiles of disease susceptibility, drug metabolizing and drug
transporter genes associated with treatment outcome in complex diseases including, In-
flammatory bowel diseases (IBD), asthma, aspirin intolerance, Chronic obstructive pul-
monary disease (COPD), cardiovascular diseases, cancer, psychiatric diseases. In this re-
port we focus on the pharmacogenomics of human inflammatory bowel diseases (IBD),
usually classified into Crohn’s disease (CD) and ulcerative colitis (UC). Accumulating phar-
macogenetic data in IBD strongly support that several genes are important in drug absorp-
tion, disposition, metabolism and resulting side effects and treatment outcome in IBD pa-
tients for instance mutations in gene coding for thiopurin methyl transferase (TPMT) re-
sult in altered metabolism of azathioprine (AZA), drug commonly used in IBD treatment,
and are associated with bone marrow suppression and hepatotoxicity. The expression of
Multidrug resistance 1 gene (MDR1/ABCB1) coding for ABC transmembrane transporter
was higher in peripheral blood lymphocytes in CD patients who required bowel resection
for failed medical therapy as compared to controls. Polymorphism in FCG3A gene was
associated with response to Inflaximab treatment in CD patients. In this report we provide
our pharmacogenomic data on IBD patients resistant to conventional therapy including
corticosteroids, antibiotics, and immunosuppressant. We also correlate genetic and gene
expression data to efficiency of treatment with novel biological therapeutics Infliximab
(antibody against TNF-α) and recently approved biologic drug HUMIRA (adalimumab).
Patients and The DNA was isolated from blood and from paraffin embedded biopsy sections from 355
methods Slovenian patients with verified chronic inflammation including 139 patients with Crohn
disease, 144 patients with ulcerative colitis, including 39 patients with inflammation re-
stricted to rectum (proctitis), and 30 unclassified IBD patients.1 Informed consent was
obtained from all patients enrolled in the study. Therapy and therapeutic outcome was
monitored and recorded for all patients for the last 10 years. Twenty four patients with
refractory Crohn’s disease that failed standard therapy with antibiotics, corticoids and
immunosuppressant were enrolled in treatment with anti-TNF alpha (Inflaximab) accord-
ing to strictly defined criteria. Inclusion criteria for active luminal disease comprised: mod-
erately to severely active CD (11 patients) not adequately responding to prior convention-
al treatment with 5-aminosalicylates 4.5 g/day given for at least 2 months, antibiotics (met-
ronidazole 1.2 g/day and/or ciprofloxacine 1 g/day) given for at least 1 month, 6-methyl-
prednisolone 42 mg/day given for at least 2 months, and azathioprine (AZA) 2.5mg/kg/
day given for at least 6 months.
We use combination of different approaches in our disease association and pharmacoge-
nomic studies including haplotype analysis, single nucleotide polymorphism (SNP) analy-
sis in candidate genes, disease pathway analysis and microarray analysis in Slovenian
IBD patients. For genotyping we used the TaqMan allele discrimination assay (Applied
Byosystems). Haplotypes were estimated using the Expectation-Maximization algorithm.
Results We have genotyped 22 SNPs from 4 different genes, including some IBD candidate genes,
genes coding for drug transporters, drug metabolizing enzymes and randomly selected
II-70 Zdrav Vestn 2007; 76: SUPPL II
genes in IBD patients and controls. We have identified novel association between multi-
drug resistance 1 (MDR/ABCB1) gene polymorphisms in intron 13 (rs2235035) and in
intron 16 (rs1922242) and haplotypes defined by SNPs in exons 12 (1236 C > A), 21(A893S)
and 26 (3435 C > T), and UC patients as well as refractory Crohn disease patients, who do
not respond to standard therapy with glucocorticoids, including patients that develop fistu-
las.
Conclusions We report here novel correlation of SNPs and haplotypes in MDR1/ABCB1 multidrug trans-
porter with IBD patients not responding to standard treatment with corticoids. We suggest
patients not responding to treatment with corticoids for IBD and other autoimmune dis-
ease including rheumatoid arthritis should be analyzed for MDR1/ABCB1 polymorphisms.
Our results also suggest advantage of haplotype analysis versus single SNP analysis. Our
results also suggest the importance of non coding SNPs in pharmacogenetic and disease
association studies.
References
1. Potocnik U, Ferkolj I, Glavac D, Dean M. Polymorphisms in mul-
tidrug resistance 1 (MDR1) gene are associated with refractory
Crohn disease and ulcerative colitis. Genes Immun 2004; 5: 530–
9.