Interindividual differences in xenobiotic-metabolising enzymes : the human genetic factor
Author: McLellan, Roman A.
Date: 2000-01-21
Location: Rockefeller-salen, Nobels väg 11
Time: 13.00
Department: Institutet för miljömedicin (IMM) / Institute of Enviromental Medicine
Abstract
The vast number of human xenobiotic-metabolising enzymes display
interindividual variability that can alter the disposition of any
compound metabolised by these enzymes, including environmental chemicals
and many clinically used drugs. Both genetic and non-genetic factors
affect the levels and activities of these enzymes. The purpose of this
study was to investigate some of the underlying genetic differences and
also to examine genetic variations among different ethnic groups.
Cytochrome P450 2D6 (CYP2D6) displays both interindividual and
interethnic variation. The CYP2D6 alleles distributed in a Saudi Arabian
population were investigated. We found a high frequency of subjects
carrying duplicated CYP2D6 alleles (21%) and very few defective alleles,
which is in agreement with earlier Saudi Arabian CYP2D6 phenotyping
studies which reported a very low frequency of poor metabolisers. This
suggests that CYP2D6 could be under selective pressure in this region
possibly due to dietary reasons. It has been reported that CYP2D6
expression in easily accessible human peripheral blood mononuclear cells
(PBMCs) can be used to predict whole-body metabolic activity. We examined
if this enzyme is expressed in this cell population and if so, whether
the expression would correlate to gene copy number. However, no correctly
spliced CYP2D6 mRNA or protein was observed in mononuclear cells and the
validity of using PBMCs as a surrogate marker to predict hepatic CYP2D6
activity can be questioned.
The mu class glutathione S-transferase gene, GSTM1, is polymorphic in
humans with roughly half of the population being homozygous deleted for
this gene. In contrast, an earlier Saudi Arabian GSTM1 phenotyping study
revealed that some individuals exhibited ultrarapid enzyme activities,
therefore the genetic basis of this observation was investigated. By
doing restriction fragment length polymorphism (RFLP) analysis and using
a quantitative multiplex PCR method, the existence of a GSTM cluster
containing a duplicated functional GSTM1 gene was revealed. The
generation of the duplicated GSTM1 locus likely involved an unequal
crossover event between homologous regions in the locus. Individuals
carrying extra GSTM1 genes may gain a protective effect against some
chemicals as they are likely to rapidly conjugate and inactivate
carcinogenic compounds.
Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in humans
and can activate procarcinogens. We re-evaluated the genotyping method
for the CYP2A6*3 allele and discovered a gene conversion with CYP2A7 in
the 3'-flanking region of 30-40% of CYP2A6 alleles, causing
misclassification of the CYP2A6 genotype. The CYP2A6*3 allele was not
found using our new genotyping method which questions its existence. The
structure of the CYP2A6 gene deletion was characterised and the
generation of this locus likely involved an unequal crossing-over event
between the 3'-flanking region of the CYP2A6 and CYP2A7 genes. A rapid
PCR-based method was created to detect the allele and it was found at a
frequency of 15% in Chinese but only 1% in Caucasians. Another allele,
CYP2A6*5, was found to encode an enzyme with a G479L substitution that
was very unstable when expressed in yeast and yielded very low protein
levels and coumarin 7-hydroxylase activity. It was shown to be quite rare
with a frequency of only 0-1%.
An inducible enzyme that is expressed in extrahepatic tissues, can
activate procarcinogens, and is a low-Km. 17ß -estradiol 4- and
2-hydroxylase is cytochrome P450 1B1 (CYP1B1). The CYP1B1.2 enzyme
contains the two linked amino acid substitutions R48G and A119S compared
to CYP1B1.1. To functionally characterise the two variants with respect
to kinetic properties and protein stability, the alleles were expressed
in yeast and in mammalian COS-1 cells. It was found that CYP1B1.1. and
CYP1B1.2 share very similar properties with respect to catalytic
activities and stability and that these amino acid substitutions do not
alter CYP1B1 function.
In conclusion, the present study has identified and characterised several
novel alleles for genes encoding some of the human
xenobiotic-metabolising enzymes and examined their distribution along
with other known alleles in different ethnic groups. Genotyping methods
were developed to detect the new alleles that can be used not only in
molecular epidemiological studies but also for helping to design more
efficient and safer drug treatments.
List of papers:
I. McLellan RA, Oscarson M, Seidegård J, Evans DA, Ingelman-Sundberg M (1997). "Frequent occurrence of CYP2D6 gene duplication in Saudi Arabians" Pharmacogenetics 7(3): 187-191
Pubmed
II. McLellan RA, Oscarson M, Dalén P, Ingelman-Sundberg M, Bertilsson L (1970). "Evaluation of human peripheral blood mononuclear cells as a surrogate marker for predicting whole-body cytochrome P450 2D6 metabolic activity" (Manuscript)
III. McLellan RA, Oscarson M, Alexandrie AK, Seidegård J, Evans DA, Rannug A, Ingelman-Sundberg M (1997). "Characterization of a human glutathione S-transferase mu cluster containing a duplicated GSTM1 gene that causes ultrarapid enzyme activity " Mol Pharmacol 52(6): 958-965
Pubmed
IV. Oscarson M, McLellan RA, Gullstén H, Yue QY, Lang MA, Bernal ML, Sinues B, Hirvonen A, Raunio H, Pelkonen O, Ingelman-Sundberg M (1999). "Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population" FEBS Lett 448(1): 105-110
Pubmed
V. Oscarson M, McLellan RA, Gullstén H, Agúndez JA, Benítez J, Rautio A, Raunio H, Pelkonen O, Ingelman-Sundberg M (1999). "Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism" FEBS Lett 460(2): 321-327
Pubmed
VI. McLellan RA, Oscarson M, Hidestrand M, Leidvik B, Jonsson E, Otter C, Ingelman-Sundberg M (1970). "Characterisation and functional analysis of two common human cytochrome P450 1B1 variants" (Submitted)
I. McLellan RA, Oscarson M, Seidegård J, Evans DA, Ingelman-Sundberg M (1997). "Frequent occurrence of CYP2D6 gene duplication in Saudi Arabians" Pharmacogenetics 7(3): 187-191
Pubmed
II. McLellan RA, Oscarson M, Dalén P, Ingelman-Sundberg M, Bertilsson L (1970). "Evaluation of human peripheral blood mononuclear cells as a surrogate marker for predicting whole-body cytochrome P450 2D6 metabolic activity" (Manuscript)
III. McLellan RA, Oscarson M, Alexandrie AK, Seidegård J, Evans DA, Rannug A, Ingelman-Sundberg M (1997). "Characterization of a human glutathione S-transferase mu cluster containing a duplicated GSTM1 gene that causes ultrarapid enzyme activity " Mol Pharmacol 52(6): 958-965
Pubmed
IV. Oscarson M, McLellan RA, Gullstén H, Yue QY, Lang MA, Bernal ML, Sinues B, Hirvonen A, Raunio H, Pelkonen O, Ingelman-Sundberg M (1999). "Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population" FEBS Lett 448(1): 105-110
Pubmed
V. Oscarson M, McLellan RA, Gullstén H, Agúndez JA, Benítez J, Rautio A, Raunio H, Pelkonen O, Ingelman-Sundberg M (1999). "Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism" FEBS Lett 460(2): 321-327
Pubmed
VI. McLellan RA, Oscarson M, Hidestrand M, Leidvik B, Jonsson E, Otter C, Ingelman-Sundberg M (1970). "Characterisation and functional analysis of two common human cytochrome P450 1B1 variants" (Submitted)
Issue date: 1999-12-31
Publication year: 2000
ISBN: 91-628-3924-1
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