Identification of a minimal congenic interval containing a modifier locus
affecting drug metabolism as a complex trait in mice
Carolina
12
Ogrodowczyk ,
Carole
2
LeBlanc-Westwood ,
2
Nowakowska ,
Margeryta
Bozena
and William L Casley2
2
Jaentschke ,
Russell
12
Taylor ,
Michèle
2
Lemieux ,
37
33
1 Department
of Biology, University of Ottawa
2 Center for Biologics Research, Biologics and Genetic Therapies Directorate, Health Canada
Introduction
The metabolism of drugs by the body is quite an important and complex process that usually
requires many factors, particularly those that deal with excretion of the compound. Humans, by the
nature of their genetic makeup, may respond differently to the same drug and this field of study is
called pharmacogenomics (1). There are polymorphisms found between individuals and some of them
have been identified for genes encoding drug metabolizing enzymes (DMEs), but much less is known
about modifier loci affecting DME expression. One of these enzymes is CYP1A2, part of the
cytochrome P450 family of enzymes dealing with phase I drug metabolism. It is quite an important
enzyme, because it has been found to be involved in metabolism of a number of drugs, as well be
involved in metabolic activation of several environmental procarcinogens (2).
CYP1A2 is the principal enzyme of caffeine metabolism in mice (3), which is how its activity has
been studied (4). However, even a single step in this metabolism, the rate of the 3-demethylation of
caffeine to paraxanthine (Figure 1), is a genetically complex trait governed by more than one genetic
locus. In an intercross between a Swiss Webster-derived APN mouse strain (5) and C3H/HeJ mice,
three quantitative trait loci (QTLs) were discovered and located on chromosome 1, chromosome 9 (colocalizing with the Cyp1a2 locus) and chromosome 4 (6).
CYP1A2
Figure 1: Caffeine 3-demethylation. Caffeine, also known as 1,3,7-trimethylxanthine, gets metabolized by
CYP1A2 to paraxanthine, also known as 1,3-dimethylxanthine. Figure modified from Miners and Birkett, 1996
(8).
Congenic mouse strains were established, containing the C3H/HeJ chromosomal segment for a
minimum of 2 LOD interval around the QTL on an APN background, for each of the QTLs (C1, C9
and C4) using marker-assisted selection. This study deals with the chromosome 4 congenic line which
contains a 30 cM C3H/HeJ interval (Figure 2).
Mouse #
APN
C1
C3H
C4
C9
Figure 3: Cyp1a2 gene expression as quantified through real-time PCR by looking at mRNA
expression. Plot of CYP1A2 expression normalized to beta-2-microglobulin and calibrated against APN
males. Each group corresponds to the particular mouse strain and each group is the mean of 4 technical
replicates of a sample consisting of pooled liver RNA from 5 males. The error bars represent the 95 %
confidence interval. C1, C4 and C9 respectively represent chromosome 1, 4 and 9 congenic strains. C3H
represents the C3H/HeJ genotype. Result courtesy of Russell Taylor.
D4Mit199
D4Mit334
D4Mit204
D4Mit251
D4Mit232
0
1
2
3
4
LOD
Figure 2: LOD plot for linkage to the caffeine 3-demethylation phenotype with an unconstrained genetic
model. In an F2 intercross between APN and C3H/HeJ mice, a QTL was colocalized to chromosome 4. On the
left hand side, the markers used for the genetic analysis are found and the yellow line indicates a 1-LOD interval
from the peak value. This corresponds to a region greater than 30 cM. Figure modified from Casley et al, 1999.
77
14
A
Methods
Mouse Lines
 The original C4 congenic strain was lost due to lack of fertility, thus C4C9 double congenic
mice had to be backcrossed to the APN strain in order to re-establish the C4 line.
 The F1 generation chromosome 4 rescue (C4R) mice were selected through marker-assisted
selective breeding to be APN/C3H/HeJ heterozygous at chromosome 4 and APN homozygous at
chromosome 9. Unfortunately, the C9 APN homozygosity was not adequately confirmed
resulting in the carryover of the C3H/HeJ allele at the Cyp1a2 locus into the C4 lines used for
these experiments.
 The F1 generation was selfed in order to obtain an F2 generation with varying congenic
regions. This F2 generation was then setup into ten breeding groups as seen in Figure 4 in order
to obtain an F3 generation.
Marker
cM
D4-196
16.4
D4-214
21.9
D4-17
29.5
D4-26
35.0
D4-9 / D4166
39.3
D4-255
45.9
D4-155
48.1
D4-199
52.5
D4-203
60.1
D4-204
61.2
D4-251
66.7
D4-170
D4-232
69.9
49
5
68
78
APN/APN
1
4
6
C3H/C3H
Alleles
Homozygous C3H/HeJ
Homozygous APN
Heterozygous APN/C3H/HeJ
71.0
Real-time PCR (Quantitative PCR – Q-PCR)
A 20 mg piece of liver sample was used to extract RNA using the RNeasy Mini Kit from
Qiagen. The RNA was then reverse-transcribed using the High Capacity cDNA Archive Kit
from Applied Biosystems and the cDNA used in a real-time reaction with TaqMan assays for
CYP1A2 and beta-2-microglobulin as an endogenous control on the 7500 Fast Real-Time
PCR System.
Paraxanthine/Caffeine Index Phenotyping
Mice at 7 weeks of age were dosed with caffeine at a concentration of 10 mg/kg body weight
by oral gavage. Serum was collected after two hours and analyzed by HPLC for paraxanthine
and caffeine. The paraxanthine to caffeine ratio was calculated.
B
0.3
*
0.5
*
The purpose of this project is to use marker-assisted selective breeding in order to create
congenic mouse sub-lines that contain different regions of the original C3H/H3J congenic
interval and use them in CYP1A2 mRNA, as well as, paraxanthine to caffeine ratio analysis. By
correlating a particular genetic region with the observed phenotype, one should be able to
reduce the 30 cM QTL found on chromosome 4 and thus aid in the identification of the modifier
locus.
Furthermore, these genetic regions would then be subject to functional and
bioinformatical analysis to ultimately identify the candidate gene.
Genotyping
Genomic DNA was extracted from mouse tail tips and used in a PCR reaction with genetic
markers based on short tandem repeats (STRs) labelled with fluorescent dyes. These reactions
were then diluted in water, followed by formamide and mixed with a GS400HD size standard.
The samples were run on the ABI 3100 Genetic Analyzer and size peaks analyzed using the
GeneScan software.
D4Mit26
D4Mit255
D4Mit155
69
Figure 5: Cyp1a2 gene expression as quantified through real-time PCR by looking at mRNA
expression. Plot of CYP1A2 expression normalized to beta-2-microglobulin and calibrated against one
APN/APN male (# 68). Each bar represents a male C4R F2 mouse corresponding to the particular
chromosome 4 genotype as indicated. Means of 3 technical replicates are displayed with error bars
representing the 95 % confidence interval. C3H represents the C3H/HeJ genotype.
D4Mit214
D4Mit17
2
APN/C3H
Figure 4: The C4 rescue (C4R) F2 generation breeding set up. The F2 generation was divided into ten
different breeding groups corresponding to different regions of C3H/HeJ. Groups 1, 3, 4, and 5 consisted of
2 breeding pairs each; group 2 consisted of 5 breeding pairs, while groups 6-10 consisted of a single
breeding pair. On the left are the genetic markers used and their corresponding centiMorgan (cM) locations
on chromosome 4.
D4Mit1
Results
Paraxanthine/Caffeine Ratio
Caffeine 3-demethylation, the metabolism of caffeine to paraxanthine, is a genetically complex
trait mediated by the cytochrome P450 enzyme 1A2 (CYP1A2). A genome–wide scan of an F2 intercross
between APN, an in-house strain with low CYP1A2 expression, and C3H/HeJ, a laboratory strain
expressing normal CYP1A2 levels, determined that this phenotype is mediated by three quantitative
trait loci (QTL) localized to chromosomes 1, 4 and 9, as previously reported. The QTL on chromosome
9 co-localized with the Cyp1a2 gene, an obvious candidate for the gene underlying that QTL. Congenic
mice were bred in which a segment of chromosome 4 from the C3H/HeJ mouse encompassing the QTL
was introgressed onto an APN genetic background. The congenic interval on chromosome 4 spanned a
30 centiMorgan (cM) distance which is too great to identify a possible candidate gene that acts as a
modifier locus affecting CYP1A2 expression. Thus the purpose of this study was to reduce the congenic
region to a much smaller genetic distance. By using marker-assisted selective breeding, mouse sub-lines
that contained different regions of the original C3H/HeJ congenic interval were derived from the
congenic founder. However, upon a closer genotypic analysis of chromosome 9 markers in the F3
generation, it was discovered that the C3H/HeJ allele of Cyp1a2 had been retained in some of the
founders used to generate these sub-lines, thus confounding attempts to attribute changes in CYP1A2
expression to the chromosome 4 congenic content. Previous reports of male and female phenotypic
differences were confirmed by looking at the paraxanthine to caffeine ratio (p=0.001). Additionally,
predicted phenotypic differences between the Cyp1a2 C3H/HeJ homozygous and APN homozygous mice
were also confirmed by looking at the male population (p=0.004). With the identification of residual
CYP1A2 activity, as well as discovering that the APN allele of Cyp1a2 appears to act in a recessive
fashion, further breeding will be required to remove the C3H/HeJ Cyp1a2 allele and allow analysis of
the chromosome 4 congenic sub-lines against a uniform genetic background. These new sub-lines should
be valuable in permitting the use of CYP1A2 mRNA levels as a phenotype for identifying a minimum
congenic interval for the QTL acting as a modifier of CYP1A2 expression and ultimately identify a
possible candidate gene through functional and bioinformatics means.
In the lab, it has been discovered by Q-PCR that the C4 line shows an elevated CYP1A2
mRNA expression relative to the background strain, indicating that the QTL in the C4 segment
may act as a modifier locus for the gene (Figure 3) (7). In order to more finely map this locus, it is
necessary to reduce the congenic region as much as possible. Previous studies of the different
models during linkage analysis suggested that the APN allele at chromosome 4 QTL might act in
a dominant manner. If it does, one should be able to map the locus based on Q-PCR analysis.
Paraxanthine/caffeine ratio
Abstract
0.25
0.2
0.15
0.1
0.05
0.45
*
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
0
Males
Females
Gender
APN/C3H
APN/APN
C3H/C3H
Chromosome 9 Genotype
Figure 6: Caffeine 3-demethylation phenotypic differences as determined by the
paraxanthine/caffeine ratio. C4R F3 mice were injected with caffeine at a concentration of 10 mg/kg
body weight by oral gavage. Serum was collected after two hours, analyzed by HPLC and the
paraxanthine/caffeine ratio calculated. (A) Phenotypic differences between males and females. The
means (±SEM) of n = 25 for male mice and n = 19 for female mice are plotted. Student t-test for
significance was applied to the data (p=0.001). (B) Phenotypic differences between APN/C3H,
APN/APN, and C3H/C3H chromosome 9 genotype male mice. The means (±SEM) of n = 26 for
chromosome 9 heterozygous APN/C3H mice, n = 4 for chromosome 9 homozygous C3H mice and n =
25 for chromosome 9 homozygous APN mice are plotted. Student t-test for significance was applied to
the data. A p-value of <0.001 was obtained for a comparison between the APN/C3H and APN/APN mice.
A p-value of 0.004 was obtained for a comparison between the APN/APN and C3H/C3H mice. A p-value
of 0.330 was obtained for a comparison between the APN/C3H and C3H/C3H mice. C3H represents the
C3H/HeJ genotype.
Conclusions
 The apparent lack of phenotypic division between the C3H/HeJ homozygous, APN
homozygous, and the APN/C3H/HeJ heterozygous mice is due to a residual C3H/HeJ allele
of Cyp1a2
 Nonetheless, congenic sub-lines have been established that contain defined chromosome 4
congenic regions
 Previous reports of male and female phenotypic differences in the caffeine 3-demethylation
(5) were confirmed by looking at the paraxanthine to caffeine ratio (p=0.001)
 No significant phenotypic differences in the caffeine 3-demethylation were observed
between the APN/C3H/HeJ heterozygous and the C3H/HeJ homozygous male mice
(p=0.330)
 Furthermore, a comparison between APN homozygous and C3H/HeJ homozygous at
chromosome 9 male mice, as well as, APN homozygous and APN/C3H/HeJ heterozygous at
chromosome 9 male mice rendered these groups significantly different, p-values of 0.004
and <0.001 respectively, suggesting that the APN allele on chromosome 9 acts in a recessive
fashion
Future Studies
 Established congenic chromosome 4 sub-lines will have to undergo further breeding in
order to remove the C3H/HeJ allele from the Cyp1a2 locus and allow for chromosome 9
APN homozygosity
 The F3 generation will have to be examined for CYP1A2 mRNA expression within groups
normalized for the Cyp1a2 allele
 The F4 generation obtained from proper APN chromosome 9 founders will be used to
explore whether or not a particular genetic marker correlates to an up-regulated CYP1A2
mRNA expression and/or paraxanthine to caffeine ratio
 By having established congenic mouse lines with a reduced congenic interval of the
chromosome 4 QTL, functional and bioinformatical analysis can take place in order to
identify the modifier locus and ultimately a candidate gene
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