Pharmacogenetics of drug metabolizing enzymes with special emphasis on Ethiopians

Sammanfattning: Pharmacogenetics of drug metabolising enzymes has been well studied in Caucasians and Asians, but relatively little in Africans. The aim of this thesis was to investigate the molecular genetics of drug metabolising enzymes, in particular CYP2D6, CYP2C19, CYP2C9, CYP1B1, CYP1A2 and XO, with special emphasis on the Ethiopian population. Two different Ethiopian populations were investigated, healthy unrelated subjects living in Ethiopia (n=122) and Ethiopians living in Sweden (n=73). In the first group, it was found that only 1.8% were PMs for debrisoquine, whereas 29 % carried duplicated or multiduplicated CYP2D6 genes, having higher enzyme activity. We identified new haplotypes carrying 4 or 5 CYP2D6'2 gene copies on one allele. We found a significantly lower rate of CYP2D6 but not of CYP2C19-catalyzed reactions in Ethiopians living in Ethiopia as compared to Ethiopians living in Sweden of the same genotypes. We suggest that the dietary habits in Ethiopia have had an inhibitory effect on CYP2D6 and have contributed to dietary related selection for CYP2D6 duplication in Ethiopians. We screened for new SNPs in the CYP1B1 gene and detected 3 novel SNPs one of which (4360C>G in exon 3) was present at a frequency of 7 %, and causes an Ala443Gly (m5) amino acid substitution. In total, 5 missense SNPs were found yielding in total 32 possible CYP1B1 haplotypes. However, we could only find 7 of them carrying 1-4 different missense SNPs. Functional characterization of these revealed that CYP1B1.6 and CYP1B1.7, having the Arg48Gly, Ala119Ser and Leu432Val mutations in common, had significantly decreased Vmax/Km for both the 2- and 4-hydroxylation of 17 b-estradiol whereas the other were similar to the CYP1B1.1 enzyme. It is emphasized that haplotypes rather than single SNPs are to be considered in e.g. molecular epidemiological studies. Ethiopians living in Ethiopia (n=115) or in Sweden (n=73) were phenotyped with caffeine to evaluate any influence of environmental and gender related differences on XO and CYP1A2 activities. XO activity was significantly higher in Ethiopians living in Ethiopia compared with those living in Sweden. We suggest that the dietary habits in Ethiopia might have caused the elevated XO-expression. Genomic DNA sequencing of CYP1A2 gene from 12 subjects revealed the presence of a novel intron 1 SNP, -730C>T. Genotyping and molecular haplotyping methods for the intron 1 SNPs were developed and 4 different haplotypes were identified: CYP1A2'1A (wt for all SNPs); CYP1A2'1F (-164A); CYP1A2'1J (-740G and -164A) and CYP1A2'1K (-730T, -740G and -164A) with frequencies of 39.9, 49.6, 7.5 and 3.0 %, respectively. Subjects with CYP1A2'1K had significantly decreased CYP1A2 activity in vivo (p<0.02) and reporter constructs with this haplotype revealed significantly less inducibility with TCDD in human B16A2 hepatoma cells. EMSA analysis using nuclear extracts from B16A2 cells indicated that the -730 C>T mutation abolishes a binding site for a transcription factor of the Ets family. It is concluded that this polymorphism might be of great importance for individual sensitivity to carcinogen activation and rate of drug metabolism. In conclusion unique novel alleles have been identified in the Ethiopian population and has given light to mechanisms behind their evolution and putative importance in carcinogen and drug metabolism.