Institutionen för medicinsk biokemi och biofysik (MBB) / Department of Medical Biochemistry and Biophysics
Abstract
There is a pronounced interindividual variability in the levels and
activity of many drug metabolising enzymes, which might cause differences
in the sensitivity to and the toxicity of many clinically used drugs as
well as environmental compounds such as nicotine and precarcinogens. In
the present investigation the molecular genetic basis for some of the
differences in the CYP2A6, CYP2D6, CYP2E1 and GSTM1 enzymes have been
elucidated.
Cytochrome P450 2D6 (CYP2D6) exhibits a marked interindividual and
interethnic variability, with many Asian and black African populations
have a generally reduced CYP2D6 activity compared to Caucasians. In
Chinese, who are known to having reduced capacity for metabolism of
CYP2D6 substrates, we have identified and characterised the CYP2D6*10
allele. This allele was shown to carry two missense mutations, P34S and
S486T, and heterologous expression in COS-1 cells revealed that the P34S
substitution yields an unstable enzyme that IS rapidly degraded. Indeed
CYP2D6*10 was found to be the most common CYP2D6 allele among Chinese.
Among black African populations, it has been shown that the presence of
the CYP2D6*17 allele correlates well to reduced in vivo activity. The
three missense mutations found in this allele, T107I, R296C and S486T,
were introduced in all eight possible combinations into a CYP2D6 cDNA and
were expressed in yeast. This revealed that a combination of the T107I
and R296C substitutions yielded an enzyme with a 5-fold higher apparent
Km for the substrates, whereas no effect was seen when the amino acid
substitutions were introduced separately. In a Saudi Arabian population
we found a low frequency of these reduced activity alleles and inactive
alleles but a Egli frequency of the CYP2D6 gene duplication which is
consistent with earlier phenotyping studies in this population.
The expression of ethanol-inducible P450 2E1 (CYP2E1) is also known to
exhibit striking interindividual variability. In order to elucidate
possible genetic causes for this variation, we screened all exons for
mutations using genomic DNA from almost 200 unrelated individuals. Two
novel alleles were found, yielding enzymes with the amino add
substitutions R76H and V3891, respectively. These alleles were however
very rare, implicating a selective pressure for active CYP2E1 enzyme,
most likely because of an important endogenous function. Sequencing and
characterisation of the 5' flanking region of the CYP2E1 gene revealed
however the presence of a polymorphic repeat region, with an allele
frequency of 23 % in a Chinese population, but only 1 % in Swedes. This
polymorphism might be of importance for interindividual differences in
the regulation of the CYP2E1 gene.
Cytochrome P450 2A6 (CYP2A6) is the major human nicotine C-oxidase. In
fact, data has been presented where a correlation was seen between the
number of defective CYP2A6*2 and CYP2A6*3 alleles in an individual and
the risk of becoming a smoker as well as the number of cigarettes being
smoked. The method used for CYP2A6 genotyping has, however, been found to
give erroneous results with respect to the coumarin hydroxylase
phenotype, a probe drug for the CYP2A6 enzyme. Improved genotyping
methods were developed which could correctly predict the CYP2A6
phenotype. We could not, however, detect any CYP2A6*3 alleles in contrast
to the 2-28 % previously reported and propose that a previously unknown
common gene conversion event in the 3' flanking region of the CYP2A6 gene
is the reason for this discrepancy. Furthermore, we found that the CYP2A6
gene deletion was due to an unequal cross-over event between the CYP2A6
gene and the related CYP2A7 gene. A PCR-based genotyping method was
developed which showed a frequency of the deletion allele to be 15 % in a
Chinese population, but only 1 % in Europeans.
Finally, the molecular mechanism behind the ultrarapid glutathione
S-transferase M1 (GSTM1) activity found in certain individuals was
studied. Southern blotting and PCR analysis revealed a novel GSTM1 allele
with a duplication of the active GSTM1 gene, which most likely occurred
through an unequal cross-over event. A quantitative PCR method was
developed which accurately quantified the number of GSTM1 genes.
In summary, several novel cytochrome P450 and GSTM1 alleles have been
identified and characterised. These are of putative importance for
explanation of impaired drug metabolism and possibly for smoking
behaviour and genetically determined sensitivity to carcinogens. These
findings form a basis for future molecular epidemiological studies and
for use in patients as a tool for individualised drug therapy resulting
in less side effects and better efficacy