Genes and Immunity (2000) 1, 251–259
 2000 Macmillan Publishers Ltd All rights reserved 1466-4879/00 $15.00
www.nature.com/gene
Genetic control of susceptibility to UV-induced
immunosuppression by interacting quantitative trait loci
KE Clemens1, G Churchill2, N Bhatt1, K Richardson1 and FP Noonan1
1
Laboratory of Photobiology and Photoimmunology, Department of Dermatology, The George Washington University Medical
Center, Washington DC 20037, USA; 2The Jackson Laboratories, Bar Harbor, ME, USA
Ultraviolet B radiation (290–320 nm) initiates a dose and wavelength dependent down-regulation of cell-mediated immunity
which is critical in experimental ultraviolet radiation (UV) carcinogenesis, preventing immune attack on highly antigenic
UV-induced tumors. UV-induced immunosuppression has been demonstrated in humans and may be a risk factor for skin
cancer. In this study, we have investigated genetic linkage of the autosomal loci controlling this trait. Previously, we had
derived a model describing control of susceptibility to UV-induced immunosuppression in inbred mice by unlinked
interacting autosomal and X-linked loci. A genome-wide scan using MIT microsatellite markers was carried out on 100
backcross {BALB/c × (BALB/c × C57BL/6) F1} mice derived from the inbred strains BALB/c (low susceptibility) and
C57BL/6 (high susceptibility) and tested for systemic UV-induced immunosuppression of a contact hypersensitivity
response. The values for % suppression for each animal and the genotype data were used to investigate genetic linkage
by multiple regression analysis. Significance was assessed using the permutation test. Both main effects and interactive
effects were investigated, first with each genotype marker singly, and secondly, in a novel approach using markers
pairwise. A joint model was derived in which all loci and pairs of loci identified were included simultaneously in a multiple
regression model. This model indicates four quantitative trait loci (QTLs) with significant main effects, one on chromosome
10 which decreased susceptibility to UV-induced immunosuppression and QTLs on chromosomes 6, 17 and 1 which
increased susceptibility. Additionally, loci on chromosomes 14 and 19 showed significant interaction with the locus on
chromosome 1. Further investigation indicated a potential three-way interaction involving the loci on chromosomes 1,14
and 19. Genes and Immunity (2000) 1, 251–259.
Keywords: ultraviolet radiation; immunosuppression; epistasis
Introduction
A body of experimental evidence has resulted in a model
for skin cancer formation in which ultraviolet B radiation
(290–320 nm) (UVB) initiates two separate events, the
neoplastic transformation of skin cells and a systemic
down-regulation of cell mediated immune function.
There is considerable evidence from experimental systems that this form of immunosuppression is a critical
step in UV carcinogenesis, preventing immunologic
destruction of highly antigenic UV-induced skin cancers.1,2 UVB-induced immune suppression may be a fundamental regulatory mechanism, controlling interaction
between mammals and potentially deleterious environmental radiation.3
We previously derived a genetic model for control of
systemic susceptibility to UV-induced immunosuppression in inbred strains of mice.4,5 We described a six-fold
difference in UV dose required for generating 50%
immunosuppression of contact hypersensitivity between mouse
Correspondence: FP Noonan, Laboratory of Photobiology and Photoimmunology, Ross Hall, Room 113, The George Washington University Medical Center, Washington DC 20037, USA. Tel: 202 994 3970, Fax: 202
994 0409, E-mail: drmfpn얀gwumc.edu
These studies were funded by NIH RO1CA53765 to FPN.
Received 18 October 1999; revised and accepted 29 November 1999
strains which was independent of pigmentation and
independent of the innate ability to be contact sensitized
in unirradiated animals. Using parental backcross and F2
generation animals, derived from the parental strains
BALB/c (low susceptibility to UV immunosuppression)
and C57BL/6 (high susceptibility to UV immunosuppression), we demonstrated the action of three interacting but unlinked loci, two autosomal loci and an Xlinked locus. In the current study we have investigated
genetic linkage of the autosomal loci, using a backcross
model in which the X-chromosome did not segregate.
With loci identified by a novel method of statistical analysis, we have derived a model describing main and interacting effects controlling susceptibility to UV immunosuppression.
Results
Experimental strategy
Animals were derived from a backcross, BC1(C), between
parental BALB/c female mice and (BALB/c × C57BL/6)
F1 male mice. In this backcross the X chromosome did
not segregate, being fixed as the BALB/c allele; thus linkage of autosomal loci was investigated. Mice were tested
for quantitative susceptibility to UV immunosuppression
as previously described (Material and methods). All animals phenotyped in four separate experiments were
QTLs for UV-induced immunosuppression
KE Clemens et al
252
used. Each experiment contained a number of internal
controls to ensure that the expected parental levels of UV
immunosuppression were achieved (Materials and
methods). The statistical analysis employed controlled for
inter-experimental variation that was not accounted for
by these internal controls. In order to derive as complete
a model as possible for genetic control of this trait, data
was analyzed three ways. Firstly, linkage analysis was
carried out for each marker singly. Secondly, an analysis,
which could detect interactions between loci, was used
in which markers were investigated pairwise. Finally, all
loci and pairs of loci identified in the genome-wide scans
were included simultaneously in a joint model derived
by regression analysis.
Quantitation of UV-induced immunosuppression
The distributions of values for % suppression of contact
hypersensitivity in parental and backcross animals are
shown in Figure 1. Values for backcross animals were
distributed over the entire range of both sets of parental
values. The data in backcross mice was obtained using
53 female and 47 male mice. Gender was not a significant
variable, in agreement with our previous observations.4,5
The data was obtained in four separate experiments in
which parental animals were also UV irradiated. In each
of the four experiments, values for % suppression for parental BALB/c females and CB6F1 males were within the
range shown in Figure 1. For determination of linkage, in
order to stabilize the variance and obtain approximate
normality, values for % suppression for backcross ani-
mals were transformed by arcsin.sqrt (Materials and
methods), a standard variance stabilizing transformation6
(Figure 1). Since variation across experiments was statistically significant, experiment number was included as a
blocking factor in all subsequent analyses. This was done
by permuting the transformed value for % suppression
together with experiment number in the permutation test
for significance.
The means and distributions of positive control values
for the contact hypersensitivity response (Mean ± s.d of
ear swelling in cm × 10−3) in unirradiated animals within
this dataset were similar in BALB/c parental mice
(19.6 ± 6.4, n = 28) and in BC1 (C) mice (20.0 ± 5.6, n = 58),
indicating that segregation of ability to be contact sensitized in the absence of UV radiation was not a
confounding factor, in agreement with our previous
findings.4,5
Single marker analysis: A genome-wide scan was carried
out by regression of each marker on the transformed
value for the phenotype. The regression included a
‘blocking factor’ for experiments one through four to
account for experiment to experiment variation. No significant associations for gender were noted and gender
was not included in the regression. The genome-wide significance of the regression was assessed by a permutation
test in which the phenotype and the experiment number
were permuted together and regressed against the genotype data for each marker.7
The two highest peaks detected were on chromosomes
Figure 1 Distribution of values for % suppression for parental BALB/c female (n = 123) and CB6F1 male (n = 107) and parental backcross
BC1(C) male and female (n = 100) mice and arcsin.sqrt transformation of values for % suppression for BC1(C) animals. y-axis represents
number of animals.
Genes and Immunity
QTLs for UV-induced immunosuppression
KE Clemens et al
10 and 17, at markers D10Mit170, with an F statistic of
12.87 and P = 0.00053 and at D17Mit49, F = 10.18 and
P = 0.0019 (Figure 2). D10Mit170 is associated with a
decrease in susceptibility to UV-induced immunosuppression and D17Mit49 with an increased susceptibility. Using the stringent criteria of genome-wide significance8 which takes into account the error due to
multiple testing, D10Mit170 was significant at P ⬍ 0.05
and the peak for D17Mit49 falls just below the P = 0.1
level.
Pairwise marker analysis: Single marker analysis revealed
two quantitative trait loci (QTLs) with opposing effects.
These two QTLs would therefore be predicted to cancel
each other out, indicating that further QTLs, undetected
in the single marker analysis, were contributing to the
high susceptibility phenotype. In order, therefore, to
derive a more complete model, including any possible
interactive effects, a genome-wide scan was carried out
for all markers considered pairwise (see Materials and
methods) controlling for experiment number as a variable. Figure 3 shows the results of the pairwise scan
graphically, with color-coded significance levels for each
pair of markers considered overall (lower right half of
Figure 3) or significance levels of interaction between
each pair of markers (upper left half of Figure 3). Table 1
lists F statistics and P values both overall (Foverall) and
for interactions (Finteraction) for all marker pairs for which
overall significance was P ⬍ 0.001 (Foverall ⬎ 5.91). These
results should be viewed as suggestive. None of the
marker pairs exceeded the genome-wide threshold at
P = 0.05 (Foverall ⬎ 8.55) as determined by permutation
analysis of the pairwise search. Failure to achieve genome-wide significance is not surprising given the stringency of the criteria which accounts for more than 2000
tests carried out on a sample of 100 mice.
The pair D10Mit170 and D6Mit389 had the highest
pairwise score (Foverall = 7.76) but there was only weak
evidence of interaction (P = 0.107). There was a cluster of
high scoring locus pairs between D10Mit170 on chromosome 10 and a number of loci on chromosome 17, with
a peak at the pair D10Mit170 and D17Mit49
(Foverall = 7.61) with no evidence of interaction (P = 0.97).
There were two pairs of loci on chromosomes 10 and 14
with weak evidence for interaction (P = 0.02 and 0.055).
There are two pairs of loci noted between chromosome
1 and 14 with significant interaction (P ⬍ 0.001). Another
pair of loci between chromosomes 1 and 19 also shows
significant interaction (P ⬍ 0.001). The final significant
locus pair is D1Mit411 with D17Mit49, each of which
occurs in other significant pair combinations and which
shows no suggestion of interaction (P = 0.32). Although
all these locus pairs fail to meet the stringent genomewide permutation thresholds, (F = 8.55 for P = 0.05) they
are nominally significant as indicated above at a stringent
level (P ⬍ 0.001). Furthermore they form a consistent set
in which each locus (with the exception of D6Mit389)
occurs in combination with at least two other loci. This
observation led us to consider a joint model for these loci
in which all effects are included simultaneously in a multiple regression model.
253
Joint modeling: We used the results of the genome-wide
scans described above to build a multiple regression
model for the trait using the loci and interactions
detected. A joint model with significant main effects on
chromosomes 1, 6, 10 and 17 and interaction effects
between chromosomes 1 and 14 and 1 and 19 was
determined to provide the best overall fit to the data
(Table 2, Figures 4 and 5). All individual terms were significant with the exception of chromosomes 14 and 19 as
main effects, these loci being significant only as a result
Figure 2 Genome-wide scan for susceptibility to UV-induced immunosuppression derived from single marker analysis. Horizontal lines
represent F values for genome-wide significance of P = 0.05 and P = 0.1.
Genes and Immunity
QTLs for UV-induced immunosuppression
KE Clemens et al
254
Figure 3 Pairwise scan for linkage of susceptibility to UV-induced immunosuppression. The grid is a 64 × 64 plot, each unit representing
an MIT marker which are presented in a sequence according to chromosome number and cM position on the chromosome. The white
boxes represent chromosomes. Bar shows color-coded negative log10 P values from blue, P = 1 (100) to red P = 0.0001 (10−4). Each square
represents the significance (color-coded) of a pair of markers; the lower right half of the graph represents the significance of each pair of
markers considered overall and the upper left half of the graph represents the significance of interaction between each pair of markers.
For example, the red square at the intersection of chromosomes 6 and 10 (lower right half of graph) represents the significance of the third
marker on chromosome 6 (D6Mit389) with the second marker on chromosome 10 (D10Mit170). Inspection of the intersection of these two
markers in the upper half of the plot reveals no significant interaction (blue square). Pairs of markers having overall significance P ⬍ 0.001
are listed in Table 1.
Table 1 Marker pairs significant in pairwise scan for linkage to susceptibility to UV-induced immunosuppression
Foveralla
Marker Pair
D6Mit389
D10Mit170
D10Mit170
D10Mit170
D10Mit170
D10Mit170
D10Mit170
D1Mit411
D1Mit411
D1Mit411
D1Mit411
D10Mit170
D17Mit49
D17Mit123
D17Mit187
D17Mit100
D14Mit185
D14Mit165
D14Mit260
D14Mit185
D19Mit19
D17Mit49
7.76
7.61
7.03
6.24
6.06
6.98
6.48
6.91
6.90
6.77
6.43
P
0.00012
0.00013
0.00026
0.00067
0.0008
0.00027
0.00049
0.0003
0.0003
0.00035
0.00052
Finteractionb
2.63
0.00
0.01
0.04
0.01
5.38
3.78
12.87
12.31
12.03
0.98
Table 2 Joint model of genetic control of susceptibility to UVinduced immunosuppression using loci and interactions detected
in the genome-wide scans: analysis of variance table
P
0.107
0.97
0.91
0.85
0.91
0.02
0.055
0.00053
0.00069
0.00079
0.32
Source
df
Adj SSa
F statistic
P valueb
D10Mit170
D17Mit49
D6Mit389
D1Mit411
D14Mit260
D19Mit19
D1Mit411:D14Mit260
D1Mit411:D19Mit19
Experiment No
Error
Total
1
1
1
1
1
1
1
1
3
90
99
0.706
0.575
0.540
0.365
0.143
0.079
0.813
0.571
2.19
7.267
13.751
9.41
7.66
7.19
4.87
1.9
1.06
10.84
7.61
9.72
0.0029
0.0069
0.0088
0.03
0.172
0.306
0.0014
0.0071
0.000013
a
Foverall compares the full model with two loci and an interaction
effect to the null model of genetic effects. It has 3 and 42 degrees
of freedom.
b
Finteraction compares the full model to an additive model, ie two
markers with no interaction. It has 1 and 92 degrees of freedom.
of their interaction with the locus on chromosome 1. In
this joint model, each of the loci was significant at
P ⬍ 0.01 either alone or in combination with another
locus. Reduced significance of individual terms in a multiple regression is typical. There is no obvious approach
Genes and Immunity
a
Adjusted sum of squares, Type III.
Nominal P values.
b
to establishing a significance for inclusion in such a
model. The level of P ⬍ 0.01 is reasonably stringent. This
model explains approximately 52% of the total variance
in the data, with the genetic effects explaining 36% of
the variance.
A number of alternative models were compared in this
process. We determined that, on chromosome 14, the
QTLs for UV-induced immunosuppression
KE Clemens et al
255
Figure 4 Significant main effects in the joint model for genetic control of susceptibility to UV-induced immunosuppression. For significance
levels see Table 2. Shown are the mean ± s.e.m. of values for % suppression when the locus indicated is O (homozygous ie, two BALB/c
alleles) and E (heterozygous one BALB/c and one C57BL/6 allele). The main effects on chromosomes 1, 6 and 17 significantly increase
susceptibility and the main effect on chromosome 10 significantly decreases susceptibility to UV-induced immunosuppression when the
locus is heterozygous (E).
marker D14Mit260 provided the strongest evidence for
an effect in the joint model. Even after accounting for the
effects at D14Mit260, however, the marker at D14Mit185
still appeared to have a significant effect on the trait.
Because D14Mit260 (19.5 cM) and D14Mit185 (54 cM) are
distant, there may be multiple loci on chromosome 14.
However with the sample size available, this conclusion
is speculative and it is not possible to resolve these two
loci. Adding D14Mit185 and its interaction with
D10Mit170 to the model was significant (P = 0.029), but
was less well supported than other effects.
Interactive effects: The pairwise scan (Figure 3 and Table 1)
showed significant interaction between D1Mit411 and
both D14Mit260 and D19Mit19. In the joint model
(Table 2, Figures 4 and 5), these interactions are strong
effects, the D1Mit411: D14Mit260 interaction being the
strongest term and the D1Mit411: D19Mit19 the fourth
strongest genetic term in the model. These interactions
are shown in Figure 5. Further investigation revealed evidence for a three-way interaction among these loci. There
are several ways to vizualize a three-way interaction. In
Figure 5 we show that the genotype of D19Mit19 alters
the D1Mit411: D14Mit260 interaction. In Figure 5 and
Table 3 we can see that when both D14Mit260 and
D19Mit19 were of the opposite genotype to D1Mit411,
susceptibility to UV-immunosuppression was reduced
compared to other combinations of genotypes for these
loci. An F-test comparing the transformed UV suppression across the eight genotype categories defined by
these marker genotypes (Table 3) with adjustment for
experiment number is highly significant (F7,89 = 6.84;
P = 1.5 × 10−6). The posthoc comparison of categories OEE
and EOO for markers on chromosomes 1, 14 and 19
respectively (Table 3) versus the remaining six genotypes
is also highly significant (F1,95 = 40.02, P = 8.2 × 10−9).
Discussion
Irradiation with UVB at biologically relevant doses
causes a dose and wavelength dependent immunosuppression of cell-mediated immunity in inbred mice3
and in man.9,10 Experimental evidence indicates a critical
role for UVB-induced immunosuppression in UV carcinogenesis. Thus genetically determined susceptibility to
UV-induced immunosuppression may represent a risk
factor for skin cancer. We have found evidence for six
QTLs controlling susceptibility to UV-induced immunosuppression of contact hypersensitivity in the mouse.
Four of these QTLs, on chromosomes 10, 17, 6 and 1 are
main effects and two additional QTLs on chromosomes
14 and 19 were detected via interaction with the QTL on
Genes and Immunity
QTLs for UV-induced immunosuppression
KE Clemens et al
256
Figure 5 Interactive effects in genetic control of susceptibility to UV-induced immunosuppression. The genotype of D14Mit260 or D19Mit19
affects % suppression associated with D1Mit411 (upper panels). A three-way interaction between these loci is shown in the lower panels.
Upper left panel: Lines connect points for mean ± SEM of values for % suppression for D14Mit260 as O (line with negative slope) or D14Mit
260 as E (line with positive slope) associated with D1Mit411 as O or E (x axis). Upper right panel: comparable data with D19Mit19 and
D1Mit411. Altering genotype of either D14Mit260 or D19Mit19 alters % suppression associated with D1Mit411 and changes the slope of
the line. Lower two panels: Data for D14Mit260 and D1Mit411 is shown with D19Mit19 as O (left lower panel) or D19Mit19 as E (right lower
panel). The genotype of D19Mit19 affects the interaction between D1Mit411 and D14Mit260. In the left panel (D19Mit19 = O), interaction is
marked and the lines have opposing slopes. In the right panel (D19Mit19 = E), interaction is less marked and lines have slopes of the same
sign. Note that there is no direct interaction between D14Mit260 and D19Mit19 (Figure 3).
Table 3 Evidence for a three-way interaction between loci on chromosomes 1, 14 and 19
Genotypea of QTL on chromosome
1
14
19
O
O
O
O
E
E
E
E
O
O
E
E
O
O
E
E
O
E
O
E
O
E
O
E
% UV Suppression of CHSb
Mean ± s.e.m.
71.0 ± 4.3
56.3 ± 5.8
54.7 ± 9.5
c
26.5 ± 7.9
c
34.5 ± 8.0
68.7 ± 7.9
78.0 ± 7.4
61.4 ± 6.0
a
O represents homozygous ie, with two BALB/c alleles; E represents heterozygous ie, with one BALB/c and one C57BL/6 allele.
Value of phenotype (see Materials and methods and Figure 1).
An F test comparing the transformed UV suppression across the
eight genotype categories was highly significant (see text).
c
Post-hoc comparison of categories OEE and EOO vs the remaining
six categories was highly significant (see text).
b
chromosome 1. In a joint model, considering the effect of
all these loci simultaneously, all were significant at
P ⬍ 0.01. In this study, it was not experimentally possible
to test all animals for phenotype at one time and,
although internal controls were included within each
experiment, inter-experimental variation remained a significant contribution to the variance, indicating environGenes and Immunity
mental factors play a significant role in this phenotype.
In our analysis, the effect of inter-experimental variability
was controlled for by using experiment number as a
blocking factor.
The statistical analysis used in this study is based on
multiple linear regression6 using microsatellite MIT markers as covariates. Our approach was to identify key loci
using genome scans on single markers and pairs of markers. We then estimated the effects of the loci and the interactions identified in a joint model, which provided a complete description of how the multiple genetic factors
simultaneously influenced the trait. We note that using
markers as surrogates for the unobserved QTLs can result
in a slight downward bias in estimated effect sizes and
significance levels. In this study, the genome-wide scan
using markers one at a time revealed two QTLs with
opposing effects on UV immunosuppression on chromosomes 10 and 17. A more complete model, which
included interactive effects, was detected using a novel
methodology in which pairs of markers were used as
covariates. This approach identified two further loci on
chromosomes 6 and 1 which increased susceptibility and
also enabled the detection of interactive effects involving
loci on chromosomes 14 and 19 and chromosome 1. A
significant three-way interaction was observed between
the loci on chromosomes 1, 14 and 19. The interactive
effects detected were among the strongest terms in the
joint model, indicating that this type of pairwise analysis
is essential to obtaining an accurate description of the
genetic control of susceptibility to UV immunosuppres-
QTLs for UV-induced immunosuppression
KE Clemens et al
sion. Although the additional QTLs detected by this
methodology did not reach the stringent level of genomewide significance (P = 0.05) and thus are suggestive in
this sample of 100 animals, all showed a high level of
nominal significance in the pairwise scan at P ⬍ 0.001
and thus indicate regions for further detailed mapping.
Selection of candidate genes for these QTLs requires
fine mapping of the regions identified and derivation of
marker-assisted congenics for these chromosomal
regions. Nevertheless, there are some interesting genes
located near the QTLs. D1Mit411 is located at 18.5 cM on
chromosome 1, near the protein tyrosine kinase Zap70
(20 cM) which acts in signal transduction in T lymphocytes, and in the region of the genes Stat1 and Stat4
(28 cM) which code for two of the STAT transcription factors which are involved in signal transduction in
response to cytokines. D14Mit260, which interacts with
D1Mit411, is located at 19.5 cM near the T cell receptor
␣ and ␦ chains (19–20 cM), suggesting possible
Tcr/Zap70 or Tcr/STAT interactions. D19Mit19 which
affects the D1Mit411:D14Mit260 interaction is located at
26 cM close to Jak2 (24 cM), known to interact with the
STAT transcription factors. We have recently demonstrated that the number of dermal mast cells is a critical
determinant of susceptibility to UV-induced immunosuppression.11 D10Mit170 (29 cM), a marker for the QTL
which down-regulates susceptibility to UV immunosuppression, is located near the gene Gp49b (32 cM) for
gp49B, a type I membrane glycoprotein which inhibits
mast cell signaling.12 D17Mit49 (23.2 cM) is located just
distal to the MHC (18–20 cM). Our previous studies,
however, using major histocompatibility complex (MHC)
congenics4 do not support a role for the MHC in controlling this trait, suggesting other loci in this region are
responsible for this QTL. This is in contrast to recent
studies using a different model of UV immunosuppression in which, using MHC congenics, a UVB
susceptibility region was mapped within the Bat5 and H2D segment of mouse chromosome 17.13 Finally, D6Mit
389 (48.2 cM) is located near the histamine H1 receptor
Hrh1 (49 cM) Histamine has been implicated in UV
immunosuppression.14 D6Mit 389 also maps in the region
of Tnfrsf1a (57.1 cM) which codes for the ␣ chain of the
tumour necrosis factor (TNF) receptor. Mice deficient in
TNF receptors have recently been demonstrated to show
impaired susceptibility to UV-induced immunosuppression associated with a decrease in dermal mast
cell number.15
Linkage for susceptibility to UV-induced immunosuppression was not detected to the chromosomal
regions of a number of genes implicated in UV-induced
immunosuppression, most notably to cytokines,16 including the cytokine cluster on chromosome 11 and to cytokine receptors, with the exception of the ␣ chain of the
IL-1 receptor which maps near D1Mit411.
As far as we are aware, QTLs for susceptibility to UVinduced immunosuppression have not previously been
reported. Association of susceptibility to UV-induced
immunosuppression in man with IL-10 or TNF polymorphisms was investigated but no such association could
be detected.17 It is anticipated that further investigation
of these QTLs and ultimate identification of the genes
responsible and their interactions will be significant in
understanding mechanisms of UV immunosuppression.
257
Materials and methods
Mice
BALB/cAnNCr female and F1 (BALB/cAnNCr
female × C57BL/6NCr male) (CB6F1) male mice were
purchased from Frederick Cancer Research Facility
(supplier, Charles River). BALB/c female × CB6F1 male
{BC1 (C)} parental backcross mice were bred in our
facility and tested for susceptibility to UV immunosuppression between 9 and 30 weeks old. Age at test was
not a significant variable (P = 0.19, correlation test). For
this study, backcross animals derived from first and
second litters only were used. All experiments were carried out according to NIH guidelines.
Phenotyping
Phenotype for UV-induced immunosuppression was
established, as previously published,4,5 by quantitation of
the systemic decrease in contact hypersensitivity
response (CHS) after treatment with a standard dose of
UV radiation. Briefly, backcross mice had their backs
shaved with electric clippers, the ears covered with black
electrical tape and were anesthetized with Nembutal
(50 mg/kg) i/p and UV irradiated with a dose of
4.5 kJ/m2 (15 min exposure) of UV from a bank of FS40
sunlamps. Three days after UV irradiation, animals were
sensitized on the shaved abdomen with 1 mg of trinitrochlorobenzene (TNCB) in acetone. Backcross control
animals were treated identically except that either no UV
treatment was given (positive controls) or no UV was
given and no sensitizer applied (negative controls). Five
to 7 days after sensitization, all mice were anesthetized
with halothane, ear thickness measured with a spring
loaded micrometer (mean of three readings per ear) and
5 ␮l of 1% TNCB applied to each ear surface. Twentyfour hours later ear thickness was again measured and
ear swelling computed for each ear of each animal. Percentage suppression for individual UV irradiated animals
was derived as described previously as:
%suppression = 100 ×
ear swelling of test mouse − ear swelling of negative control
1−
ear swelling of positive control − ear swelling of negative control
冋
册
As an internal control, parental BALB/c (low susceptibility phenotype) females and CB6F1 (high susceptibility
phenotype) males were also UV irradiated and appropriate controls of each parental strain included in each
experiment to establish that values for % suppression
were within the predicted range.
Genotyping
PCR primer pairs for mapped microsatellite markers of
the MIT series18 were obtained from Research Genetics
(Huntsville, AL, USA). DNA was prepared from tail tips
by salt extraction followed by phenol/chloroform extraction and ethanol precipitation. PCR was performed using
a ‘touchdown’ procedure19 in which the first annealing
temperature was set at 60°C and reduced by 0.5°C each
cycle for 30 cycles. PCR products were separated using
3 to 4% agarose (EEO; Fisher Scientific) gel electrophoresis in 0.5X TBE buffer and visualized by ethidium
bromide staining. Parental DNA was amplified for each
primer set used per PCR session and a 100 bp DNA ladder included in the gel to confirm identification of parental PCR bands based on expected size. All genotypes
Genes and Immunity
QTLs for UV-induced immunosuppression
KE Clemens et al
258
were read by two independent observers. DNA from any
discordant or ambiguous genotyping was subjected to
triplicate PCR reactions for confirmation. Crossovers
were confirmed by a minimum of duplicate PCR reactions. Data were scanned for the occurrence of double
recombinants and genotyping repeated for confirmation.
Genotyping was spot-checked by extracting fresh DNA
from frozen kidney tissue from random samples of
backcross animals and re-testing.
We used the Chromosome Committee mapped positions in the Mouse Genome Database (www.jax.org) to
choose the markers:
D1Mit411, D1Mit365, D1Mit403, D2Mit365, D2Mit272,
D2Mit456, D3Mit62, D3Mit241, D3Mit147, D4Mit236,
D4Mit15, D4Mit16, D4Mit284, D5Mit386, D5Mit239,
D5Mit372, D5Mit31, D6Mit159, D6Mit223, D6Mit389,
D6Mit373, D7Mit145, D7Mit238, D7Mit362, D8Mit4,
D8Mit106, D8Mit14, D9Mit297, D9Mit75, D9Mit18,
D10Mit213, D10Mit170, D10Mit10, D11Mit53, D11Mit339,
D11Mit333, D12Mit9, D12Mit114, D12Mit132, D13Mit3,
D13Mit179,
D13Mit151,
D14Mit121,
D14Mit260,
D14Mit165,
D14Mit185,
D14Mit266,
D15Mit226,
D15Mit71, D15Mit79, D16Mit131, D16Mit5, D16Mit106,
D17Mit100,
D17Mit49,
D17Mit187,
D17Mit123,
D18Mit110, D18Mit202, D18Mit49, D18Mit4, D19Mit68,
D19Mit19, D19Mit34.
Statistical analysis
The phenotype % suppression was converted to a fraction (% suppression/100) and transformed by arcsin.sqrt
to stabilize variance and to obtain approximate normality
(Figure 1). A multiple regression program (Minitab) was
used to fit a multiple regression model to the response
using genetic markers as covariates. The markers acted
as a surrogate for the actual QTL state.
Main effects-single marker analysis
A genome-wide scan was carried out by marker
regression (for each marker) on the phenotype. The
regression included a dummy variable ‘blocking factor’
for experiments one through four to account for experiment to experiment variation. No significant associations
for gender were noted. The significance of the regression
was assessed by the permutation test in which the phenotype and the experiment number were permuted together
and regressed against the genotype data for each
marker.7,20
Pairwise genome scans
Since some loci can affect a phenotype primarily through
interaction effects, we have developed an approach and
implemented software in Matlab (Mathworks Inc, Natick,
MA, USA) to conduct a simultaneous search for pairs of
interacting loci. The method of simultaneous search21
examined all pairs of marker loci for association with the
trait in a two-dimensional genome scan. In our procedure, the likelihood under the full regression model
(with two main effects and an interaction) was compared
to that under the null model of no genetic effects. This
comparison generated an F-statistic (with 3 and 96
degrees of freedom) for every marker pair, which was
the primary screen for identifying interesting locus pairs.
Significance of this F-statistic was assessed by permutation analysis, which accounted for the large number of
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pairs tested in the genome-wide search. We carried out
several additional tests to determine if the locus pair represented a genuine interaction or two additive main
effects, or was merely an artifact resulting from a strong
association with only one of the two loci. A second Fstatistic (with 1 and 96 degrees of freedom) was computed which compared the full model (with interaction)
to an additive model with two main effects but no interaction. If this test was significant (P ⬍ 0.001), we concluded that the loci interacted and stopped. If this second
F-statistic was not significant (P ⬎ 0.001), we concluded
that there was not strong evidence for interaction and
proceeded to the next level of testing. Nominal P-values
are appropriate for the test of interactive effects because
we have already selected the locus pair using stringent
genome-wide criteria. The choice of a nominal level at
P = 0.001 for significance is stringent but somewhat arbitrary.
The joint model was derived by including all loci and
interactions detected by the genome scans in a multiple
regression model. All terms were checked for individual
significance and choices between alternative linked markers were made, based on the overall significance of the
regression.
Acknowledgements
The authors thank Drs G Morahan and EC De Fabo for
helpful discussions.
References
1 De Fabo EC, Kripke ML. Wavelength dependence and dose-rate
independence of UV radiation induced suppression of immunologic unresponsiveness of mice to a UV-induced fibrosarcoma.
Photochem Photobiol 1980; 32: 183–188.
2 Fisher M, Kripke M. Systemic alteration induced in mice by
ultraviolet irradiation and its relationship to ultraviolet carcinogenesis. Proc Natl Acad Sci (USA) 1977; 74: 1688–1692.
3 Noonan FP, De Fabo EC. Immunosuppression by ultraviolet B
radiation: initiation by urocanic acid. Immunology Today 1992; 13:
250–254.
4 Noonan FP, Hoffman HA. Control of UVB immunosuppression
in the mouse by autosomal and sex-linked genes. Immunogenetics 1994; 40: 247–256.
5 Noonan FP, Hoffman HA. Susceptibility to immunosuppression
by ultraviolet B radiation in the mouse. Immunogenetics 1994; 39:
29–39.
6 Snedecor GW, Cochran WG. Statistical Methods. 8th edn. Ames
IA: Iowa State University Press, 1989, p 289.
7 Doerge RW, Churchill GA. Permutation tests for multiple loci
affecting a quantitative character. Genetics 1996; 142: 285–294.
8 Lander E, Kruglyak L. Genetic dissection of complex traits:
guidelines for interpreting and reporting linkage results. Nat
Genet 1995; 11: 241–247.
9 Skov L, Hansen H, Dittmar HC, Barker JN, Simon JC,
Baadsgaard O. Susceptibility to effects of UVB irradiation on
induction of contact sensitivity, relevance of number and function of Langerhans cells and epidermal macrophages. Photochem
Photobiol 1998; 67: 714–719.
10 Duthie MS, Kimber I, Norval M. The effects of ultraviolet radiation on the human immune system. Br J Dermatol 1999; 140:
995–1009.
11 Hart PH, Grimbaldeston MA, Swift G, Jaksic A, Noonan FP,
Finlay-Jones JJ. Dermal mast cells determine susceptibility to
UVB-induced systemic suppression of contact hypersensitivity
responses in mice. J Exp Med 1998; 187: 2045–2053.
12 Kuroiwa A, Yamashita Y, Inui M et al. Association of tyrosine
QTLs for UV-induced immunosuppression
KE Clemens et al
13
14
15
16
phosphatases SHP-1 and SHP-2, inositol 5-phosphatase SHIP
with gp49B1, and chromosomal assignment of the gene. J Biol
Chem 1998; 273: 1070–1074.
Handel-Fernandez ME, Kurimoto I, Streilein JW, Vincek V. Genetic mapping and physical cloning of UVB susceptibility region
in mice. J Invest Dermatol 1999; 113: 224–229.
Hart PH, Jaksic A, Swift G, Norval M, el Ghorr AA, Finlay Jones
JJ. Histamine involvement in UVB- and cis-urocanic acidinduced systemic suppression of contact hypersensitivity
responses. Immunology 1997; 91: 601–608.
Hart PH, Grimbaldeston MA, Swift GJ, Sedgwick JD, Korner H,
Finlay-Jones JJ. TNF modulates susceptibility to ultraviolet Binduced systemic immunomodulation in mice by effects on dermal mast cell prevalence. Eur J Immunol 1998; 28: 2893–2901.
Ullrich SE. The role of epidermal cytokines in the generation of
cutaneous immune reactions and ultraviolet radiation-induced
immune suppression. Photochem Photobiol 1995; 62: 389–401.
17 Allen MH, Skov L, Barber R et al. Ultraviolet B induced suppression of induction of contact sensitivity in human skin is not
associated with tumour necrosis factor-alpha-308 or interleukin10 genetic polymorphisms. Br J Dermatol 1998; 139: 225–229.
18 Dietrich W, Miller J, Steen R et al. A genetic map of the mouse
with 4,006 simple sequence length polymorphisms. Nat Genet
1994; 7: 220–245.
19 Hecker KH, Roux KH. High and low annealing temperatures
increase both specificity and yield in touchdown and stepdown
PCR. Biotechniques 1996; 20: 478–485.
20 Churchill GA, Doerge RW. Empirical threshold values for quantitative trait mapping. Genetics 1994; 138: 963–971.
21 Dupuis J, Brown PO, Siegmund D. Statistical methods for linkage analysis of complex traits from high-resolution maps of
identity by descent. Genetics 1995; 140: 843–856.
259
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