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Genes and Immunity (2006) 7, 592–599
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ORIGINAL ARTICLE
Identification of chromosome intervals from 129 and
C57BL/6 mouse strains linked to the development
of systemic lupus erythematosus
Y Heidari1,3, AE Bygrave1,3, RJ Rigby1, KL Rose1, MJ Walport1,4, HT Cook2, TJ Vyse1 and M Botto1
Molecular Genetics and Rheumatology Section, Faculty of Medicine, Imperial College, Hammersmith Campus, London, UK and
Department of Histopathology, Faculty of Medicine, Imperial College, Hammersmith Campus, London, UK
1
2
Systemic lupus erythematosus is an autoimmune disease in which complex interactions between genes and environmental
factors determine the disease phenotype. We have shown that genes from the non-autoimmune strains 129 and C57BL/6 (B6),
commonly used for generating gene-targeted animals, can induce a lupus-like disease. Here, we conducted a genome-wide
scan analysis of a cohort of (129 B6)F2 C1q-deficient mice to identify loci outside the C1qa locus contributing to the
autoimmune phenotype described in these mice. The results were then confirmed in a larger dataset obtained by combining the
data from the C1q-deficient mice with data from previously reported wild-type mice. Both analyses showed that a 129-derived
interval on distal chromosome 1 is strongly linked to autoantibody production. The B6 genome contributed to anti-nuclear
autoantibody production with an interval on chromosome 3. Two regions were linked to glomerulonephritis: a 129 interval on
proximal chromosome 7 and a B6 interval on chromosome 13. These findings demonstrate that interacting loci between 129
and B6 mice can cause the expression of an autoimmune phenotype in gene-targeted animals in the absence of any disrupted
gene. They also indicate that some susceptibility genes can be inherited from the genome of non-autoimmune parental strains.
Genes and Immunity (2006) 7, 592–599. doi:10.1038/sj.gene.6364335; published online 31 August 2006
Keywords: systemic lupus erythematosus; autoantibodies; rodent; gene-targeting
Introduction
Systemic lupus erythematosus (SLE) is a systemic
autoimmune disease characterized by the production of
autoantibodies against a variety of nuclear and cell
surface antigens, resulting in immune-complex mediated
damage to vascular, dermatological, renal, neurological
and rheumatological tissues. The aetiology of SLE is
complex, with a combination of multiple genes and
environmental factors determining both susceptibility
and disease phenotype.
Spontaneous murine models of SLE, such as the New
Zealand, BXSB and MRL mouse strains, have been widely
used to dissect the complex genetic component of SLE.
Comparison of the many linkage studies performed in
these lupus-prone strains illustrates the complexity of this
disease. Numerous disease susceptibility loci have been
identified, with some loci, such as distal chromosome 1,
mid to distal chromosome 4, proximal chromosome 7 and
proximal chromosome 17 (the H2 complex) being linked
to disease in different murine models.1 However, it is also
Correspondence: Professor M Botto, Molecular Genetics and
Rheumatology Section, Faculty of Medicine, Imperial College,
Hammersmith Campus, Du Cane Road, London W12 0NN, UK.
E-mail: [email protected]
3
These authors equally contributed to the work.
4
Current address: The Wellcome Trust, London, UK.
Received 5 July 2006; accepted 31 July 2006; published online 31
August 2006
clear that several intervals are strain-specific, confirming
the genetic complexity of the disease and indicating the
presence of extensive heterogeneity in the genes contributing to SLE pathogenesis.
More recently, gene-targeting technology has allowed
researchers to investigate the impact of a single gene on
murine physiology. There is, however, accumulating
evidence that genetic factors other than the actual
disrupted gene can influence the resulting phenotype
of the knockout mouse. In this regard, it is of note that
the majority of the gene-targeted strains are initially
developed on a hybrid genetic background between 129
and C57BL/6 (B6) mice, which has been shown to be
spontaneously predisposed to development of humoral
autoimmunity with low levels of glomerulonephritis.2–5
The initial knockout strain is then usually backcrossed
onto B6 in order to remove as much 129 genome as
possible. However, despite 10 or more generations of
backcrossing, a considerable 129-derived genome interval, flanking the targeted gene, will remain. We have
previously shown that a number of genetic loci, derived
from both 129 and B6, are linked to the development of
disease in the (129 B6) mice, with the most statistically
significant of these being a 129-derived interval on distal
chromosome 1 linked to increased autoantibody production.6 This region has been consistently linked to
autoimmune traits in a number of lupus-prone strains,
including NZB (Nba2),7,8 NZM2410 (Sle1)9 and BXSB
(Bxs3).10,11 A congenic strain, comprising the 129-derived
locus on distal chromosome 1 on a B6 background, a
Autoimmunity in 129 and C57BL/6 mice
Y Heidari et al
combination commonly created by backcrossing onto B6
a knockout strain in which the gene located in that region
has been inactivated in 129 embryonic stem cells, also
developed an autoimmune phenotype. The humoral
autoimmunity in this congenic strain was indistinguishable to that observed in a mouse carrying a deletion of
the Apcs gene, located within the lupus-linked genomic
region on distal chromosome 1 and considered as a
candidate gene for murine SLE.6 Therefore, backgroundderived genes can significantly contribute to the phenotype observed in knockout strains even when the mice
have been extensively backcrossed onto the B6 strain,
greatly complicating the interpretation of the phenotypic
analysis of gene-targeted animals.
The influence of background genes on the development or modification of spontaneous autoimmune
disease is well known, especially with respect to the lpr
and Yaa disease-susceptibility genes.12–14 Not surprisingly important effects of the genetic background on the
expression of autoimmunity have also been reported in
gene-targeted mice.5,15,16 For example, C1q deficiency, a
condition that in humans is strongly associated with the
development of a lupus-like disease,17 in mice appears to
have a disease-accelerating effect only in lupus-prone
strains, including the (129 B6) genetic background.3,16
Likewise, inactivation of the FcgRIIB gene results in the
development of a lupus-like phenotype in the context of
the B6 genomic background, but not the BALB/c
genomic background.15 In this example, it is of note that
two recessive B6-derived loci outside the targeted region
were linked to the development of the disease phenotype.18 Thus, SLE exists as a complex-trait disorder in
which specific combinations of susceptibility alleles are
required for the expression of the full phenotype.
In order to identify loci outside the C1qa locus that
modify the autoimmunity observed in (129 B6).C1qa/
mice we carried out a linkage analysis to both autoantibody production and nephritis in a new cohort of
(129 B6)F2.C1qa/ mice. In addition, we combined
the data from this cohort with data from a previously
published cohort of (129 B6)F2 wild-type mice,6 in
order to expand the sample size and confirm our
observations. We show here that a number of genetic
loci outside the C1q locus on distal chromosome 4 are
linked to autoimmunity, both confirming previously
described loci and identifying novel regions in this strain
combination.
Results
Mapping of loci predisposing to lupus in the
(129 B6).C1qa/ mice
The observation that 129.C1qa/ and B6.C1qa/ mice
did not develop any autoimmune traits16 while the
(129 B6).C1qa/ mice developed a lupus-like disease3
suggest that the disease-modifying loci may arise as a
result of interaction between specific combinations of
alleles inherited from both the 129 and B6 parental
strains. In order to investigate the genetic contribution of
129 and B6 genes to the lupus-like disease observed in
the (129 B6).C1qa/ mice, we generated a new cohort
of (129 B6)F2.C1qa/ animals and monitored these for
a year. In this cohort of C1q-deficient mice, anti-nuclear
antibodies (ANA) were detected in 38% (median 0,
range 0–10240), anti-chromatin antibodies (Abs) in 58%
(median: 2.23, range 0–4.0), anti-double-stranded DNA
(anti-dsDNA) Abs in 18% (median 0; range 0–2560) and
anti-single-stranded DNA (anti-ssDNA) Abs in 85% of
the mice. Histological evidence of glomerulonephritis
(above grade I) was found in 32% of the mice.
Interval mapping demonstrated significant linkage to
ANA (LOD ¼ 4.0, P ¼ 9.9 105, Figure 1a), anti-dsDNA
Abs (LOD ¼ 5.3, P ¼ 4.9 106, Figure 1b) and antissDNA Abs (LOD ¼ 5.5, P ¼ 3.1 106, Figure 1c) at
a locus approximately 95 cM from the centromere of
chromosome 1 in the (129 B6)F2.C1qa/ cohort. Antichromatin Abs were also linked to this chromosome 1
region, but at a more distal locus and with a lower level
of significance (LOD ¼ 2.2, P ¼ 6.3 104, Figure 1d). All
these loci were derived from 129.
In addition to the linkage observed on distal chromosome 1, ANA titres were linked to a locus on mid-distal
chromosome 3, albeit with a reduced degree of significance (LOD ¼ 2.3, P ¼ 5 104, Figure 2). This locus
was derived from the B6 background. In this context,
it is of note that QTL analysis of the mid-distal region
of chromosome 4 could not be applied to the
(129 B6)F2.C1qa/ mice as this region was of fixed
129 origin.
Owing to the reduced variability in the glomerulonephritis (GN) data compared to the autoantibody titre
data, the linkage to GN was determined using two
methods – as a regular quantitative trait, and in an
analysis of extremes, where only mice that were clearly
negative or positive were included. Both these analyses
showed that GN was linked to a 129-derived region of
proximal chromosome 7 in the (129 B6)F2.C1qa/
cohort. The QTL analysis demonstrated suggestive
linkage (LOD ¼ 2.2, P ¼ 6.3 104) to a 22 cM region
between D7Mit246 (15 cM) and D7Mit30 (37 cM), and
the analysis of extremes showed linkage to D7Mit230
at 26.3 cM, with a w2 of 8.84.
593
Confirmation of linkage analysis data in a combined cohort of
(129 B6)F2.C1qa/ and (129 B6)F2 mice
Overall the study of the (129 B6)F2.C1qa/ cohort
confirmed a number of the 129 and B6 loci previously
described as being associated with SLE traits, but failed
to support others.6 In order to increase the power of our
study, we repeated the QTL analysis in a larger data set,
obtained by combining data from the (129 B6)F2.C1qa/
mice presented in this study with data from a previously reported (129 B6)F2 intercross,6 resulting in 297
female mice. In this analysis, the medial and distal
regions of chromosome 4 were not included. The results
from the IgG anti-ssDNA ELISA and IgG anti-chromatin
ELISA assays were ranked, as described in the Materials
and methods section, in order to compare the data from
the two cohorts. The ranking method was verified by
comparing the patterns of genome-wide linkage to both
ranked and unranked ELISA data in a single cohort, and
confirming that the linkage patterns were comparable in
both position and magnitude of linkage.
The combined cohort of (129 B6)F2.C1qa/ and
(129 B6)F2 mice confirmed the linkage of ANA
(LOD ¼ 6.3, P ¼ 5 107, Figure 3a), IgG anti-dsDNA
Abs (LOD ¼ 7.6, P ¼ 2.5 108, Figure 3b), IgG antissDNA Abs (LOD ¼ 8.3, P ¼ 4.9 109, Figure 3c) and
IgG anti-chromatin Abs (LOD ¼ 4.7, P ¼ 2 105,
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Figure 1 Interval maps showing QTLs on chromosome 1 with ANA (a), anti-dsDNA Abs (b), anti-ssDNA Abs (c) and anti-chromatin Abs
(d) in (129 B6)F2.C1qa/ mice. Centimorgan positions were deduced by interval mapping, anchoring marker locations according to data
from www.informatics.jax.org. Intermittent dashed lines indicate threshold over which linkage is considered suggestive; dashed lines
indicate threshold over which linkage is considered significant; dotted lines indicate threshold over which linkage is considered highly
significant (see Materials and methods). LOD scores were generated with Map Manager QTb29.
Figure 2 Linkage of ANA to a B6-derived region of Chromosome 3
in (129 B6)F2.C1qa/ mice. Threshold for suggestive linkage
(intermittent dashed line), as determined by 1000 cross- and traitspecific permutation tests, is indicated (LOD ¼ 2.3, P ¼ 5 104).
Figure 3d) to a locus on distal chromosome 1 with a peak
of around 95 cM. All of the above disease traits were
linked to the distal chromosome 1 region with a higher
degree of significance in the combined than the
Genes and Immunity
individual cohorts. It is of note that the linkage to IgG
anti-chromatin Abs mapped to a more distal region of
chromosome 1 than the other autoantibodies in the
(129 B6)F2.C1qa/ cohort, whereas in the combined
cohort analysis it mapped to the same locus as the other
autoantibodies (B95 cM from the centromere). Akin to
the linkage on chromosome 1, the ANA linkage to middistal chromosome 3 was observed in the combined
cohort of (129 B6)F2.C1qa/ and (129 B6)F2 mice.
ANA titre was linked to a locus of around 50 cM from the
centromere, again with an increased degree of significance in the combined cohort (LOD ¼ 8.0, P ¼ 9 109,
Figure 4).
To investigate any potential interactions between the
B6-derived gene(s) on chromosome 3 and the 129derived gene(s) on chromosome 1, we grouped the mice
accordingly to their genotype at these two loci and
compared ANA titres. We selected a marker at the peak
of the linkage to ANA ((D1Mit206 (95.8 cM) on chromosome 1 and D3Mit103 (51.1 cM) on chromosome 3) to
define the genotype of the mice. As illustrated in Figure 5,
ANA levels were significantly higher in the mice
carrying two B6 alleles on chromosome 3 in combination
with one or two 129 alleles on chromosome 1 compared
to all the other genetic combinations. Of note the ANA
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Figure 3 Interval maps showing QTLs on chromosome 1 with ANA (a), anti-dsDNA Abs (b), anti-ssDNA Abs (c) and anti-chromatin Abs
(d) in a combined analyses of (129 B6)F2.C1qa/ and wild-type (129 B6)F2 mice. Centimorgan positions were deduced by interval
mapping, anchoring marker locations according to data from www.informatics.jax.org. Intermittent dashed lines indicate threshold over
which linkage is considered suggestive; dashed lines indicate threshold over which linkage is considered significant; dotted lines indicate
threshold over which linkage is considered highly significant. Thresholds were determined by 1000 cross- and trait-specific permutation tests.
Figure 4 Linkage of ANA to a B6-derived region of Chromosome 3
in a combined analyses of (129 B6)F2.C1qa/ and wild-type
(129 B6)F2 mice. Centimorgan positions were deduced by interval
mapping, anchoring marker locations according to data from
www.informatics.jax.org. Intermittent dashed lines indicate threshold over which linkage is considered suggestive; dashed lines
indicate threshold over which linkage is considered significant;
dotted lines indicate threshold over which linkage is considered
highly significant (LOD ¼ 8.0, P ¼ 9 109).
titres were not significantly different between the
animals homozygous or heterozygous for the 129derived segment on chromosome 1, when in combination with B6 homozygosity on chromosome 3 suggesting
that the presence of a single 129-derived allele on
chromosome 1 was sufficient to drive loss of tolerance
to nuclear antigens. This analysis provided further
support to the hypothesis that the B6-derived loci on
chromosome 3 tend to operate in a recessive manner
while the 129-derived loci on chromosome 1 operate in a
dominant fashion.
As in the (129 B6)F2.C1qa/ cohort, the linkage
analysis of GN in the combined cohort was carried out
using both a quantitative trait analysis and analysis of
extremes. Using the quantitative trait analysis method,
linkage of GN to a 129-derived locus on proximal
chromosome 7 was confirmed with an increased significance (LOD ¼ 3.8, P ¼ 1.6 105, Figure 6a). This was
confirmed by the analysis of extremes which showed a
linkage to D7Mit230 (26.3 cM), with a w2 of 11.72,
P ¼ 0.0028 (Table 1). Interestingly, a further linkage to
GN, this time derived from a B6 locus, was observed in
the combined cohort. Located on proximal chromosome
13, this locus reached the cutoff for suggestive linkage
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Figure 5 ANA titres of various chromosome 1 and chromosome 3
genotype combinations in a pooled cohort of (129 B6)F2.C1qa/
and wild-type (129 B6)F2 mice. Small symbols represent one
mouse; large symbols a variable number of animals as indicated in
parentheses. The genotype was established using a single marker at
the peak linkages on chromosome 1 (D1Mit206) and chromosome 3
(D3Mit103). The mice carrying two B6-derived alleles on chromosome 3 and one or two 129-derived alleles on chromosome 1 had
significantly higher levels of ANA compared to all the other genetic
combinations (Po0.001). The most relevant comparisons are shown.
One way ANOVA with Bonferonni’s multiple comparison tests
were applied.
(LOD ¼ 2.4, P ¼ 4 104, Figure 6b) in the combined
cohort, but not in the individual (129 B6)F2.C1qa/ or
(129 B6)F2 cohorts. Like the locus on chromosome 7,
this linkage was also observed in an analysis of extremes,
with GN linked to D13Mit117 at B19 cM, with a w2 of
15.3, P ¼ 4.77 104 (Table 1).
Discussion
Multiple genetic loci are known to contribute to the
development and pathogenesis of SLE in mice and in
humans. In this study, we have provided further
evidence that epistatic interactions between 129 and B6
mice, even though autoimmunity has not been reported
in either of the strains, can lead to a spontaneous lupuslike phenotype. In particular, we confirmed that a 129derived region of chromosome 1, when expressed in
context of the B6 genome, is strongly linked to autoantibody production. Consistent with this, the B6 genome
contributed to the autoimmune phenotype with an
interval on chromosome 3, displaying a highly significant linkage to anti-nuclear autoantibodies. Interestingly
glomerulonephritis was linked to different chromosomes: a 129 region on proximal chromosome 7 and a
B6 interval on chromosome 13 (Figure 7).
A genome-wide scan of the (129 B6)F2.C1qa/ mice
demonstrated linkage of lupus serological markers
(ANA, anti-dsDNA Abs, anti-ssDNA Abs and antichromatin Abs) to a 129-derived locus on distal chromosome 1 – recently named Sle 16 (http://www.informatics.
jax.org). This is in agreement with our previous study,6 in
which SLE traits were mapped in (129 C57BL/6)F2
wild-type and (129 C57BL/6)F2.Apcs/ mice. The
linkages were markedly increased when we enlarged
the sample size by combining data from the
Genes and Immunity
Figure 6 Interval maps showing QTLs on chromosome 7 (a) and
chromosome 13 (b) with GN in a combined analysis of
(129 B6)F2.C1qa/ and wild-type (129 B6)F2 mice. Centimorgan
positions were deduced by interval mapping, anchoring marker
locations according to data from www.informatics.jax.org. Intermittent dashed lines indicate threshold over which linkage is
considered suggestive; dashed lines indicate threshold over which
linkage is considered significant. (LOD ¼ 3.8, P ¼ 1.6 105 and
LOD ¼ 2.4, P ¼ 4 104, respectively).
Table 1 Linkage of chromosomes 7 and 13 to GN in the combined
cohort of (129 B6)F2.C1qa/ and wild-type (129 B6)F2 mice
Marker
Position (cM)
Origin
w2
P
D7Mit178
D7Mit246
D7Mit158
D7Mit230
D7Mit30
D7Mit253
0.5
15.0
23.0
26.3
37.0
52.8
129
129
129
129
129
129
4.14
11.04
10.05
11.72
7.57
6.13
1.26E-01
4.01E-03
6.58E-03
2.86E-03
2.27E-02
4.66E-02
D13Mit135
D13Mit117
D13Mit64
D13Mit248
D13Mit193
D13Mit30
D13Mit262
10.0
19.0
30.0
34.0
43.0
52.0
68.0
B6
B6
B6
B6
B6
B6
B6
12.53
15.29
5.81
3.54
2.51
0.63
1.61
1.90E-03
4.77E-04
5.47E-02
1.70E-01
2.85E-01
7.31E-01
4.48E-01
Centimorgan positions were deduced by interval mapping, anchoring marker locations according to data from www.informatics.jax.org. w2-values are calculated with a standard (2 2) contingency
table with 2 degree of freedom. Suggestive linkages, defined as
described in the material and method section, are underlined.
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1
3
7
Sles3*
Bxs5
1,2
1
Sle1
1,2,4
Nba2
1,2
Bxs3
1
Lbw7
ANA
ANA
anti-ssDNA
anti-dsDNA
anti-chromatin
13
1,2
1,2
Sle3
3
Lbw5
2
Nba3
GN
GN
1,2
Yaa1
Figure 7 A summary of the lupus susceptibility loci mapped in this study on chromosomes 1, 3, 7 and 13. Regions previously linked to SLE
in the other lupus models are also indicated. Linkage to SLE traits from this study are shown as open boxes ¼ 129, filled boxes ¼ B6. 1ANA
(any), 2GN, 3mortality, 4gp70/gp70 immune complexes, *disease suppressor locus.
(129 B6)F2.C1qa/ mice with data from a previously
reported (129 B6)F2 intercross, indicating that this 129
region is the main locus capable of initiating the humoral
autoimmune response to nuclear antigens upon interaction with the B6 genome. The distal chromosome 1
region is associated with a number of potent SLE
susceptibility loci including Sle1,9 Nba219 and Bxs3,10 all
of which are strongly associated with the production of
autoantibodies to nuclear antigens. The significance of
this chromosome 1 locus has also been confirmed by
several congenic dissection analyses.1,20 Furthermore,
recent genomic characterization of the Sle1b locus
(located between 171.8 and 173.1 Mbp) has identified a
highly polymorphic cluster of Slam/Cd2 family genes,
encoding key regulators of lymphocyte function, as the
strongest candidate genes for mediating the Sle1b
autoimmune phenotype.21 The autoimmune-associated
haplotype of the lupus-prone NZM2410 (NZW) strain,
named the Slam/Cd2 haplotype 2, is also present in the
129/SvJ mice,21 indicating that these two strains may
share the same pathways leading to loss of peripheral
tolerance when in combination with one or more
polymorphic genes in the B6 genome. These observations
give compelling evidence for the presence of a 129 locus
influencing systemic autoimmunity on telomeric chromosome 1 as revealed in the context of B6 genome.
Whether this 129 lupus locus can contribute to the
development of autoimmunity on other genetic backgrounds is still unknown.
In common with the distal chromosome 1 region,
numerous studies have demonstrated linkage of SLE
traits to proximal chromosome 7. In this study, we
confirm that there is a 129-derived locus on proximal
chromosome 7 linked to GN.6 Furthermore, this locus
colocalizes with a number of loci from various lupusprone mouse strains, including Sle3 (NZM2401 locus
linked to GN and ANA);20,22 Lbw5 (NZW locus linked to
mortality),23 Lmb3 (MRL locus linked to splenomegaly,
lymphadenopathy, anti-dsDNA Abs),24 Nba3 (NZB locus
linked to GN)25 and Nba5.26 As this is an analogous
situation to that on distal chromosome 1, a similar
haplotype-based candidate gene identification strategy
could be applied, especially as murine single nucleotide
polymorphism (SNP) databases continue to increase in
both SNP density and the number of typed strains. The
presence of a 129-derived lupus locus on proximal
chromosome 7 also has impact on the phenotype of
any targeted gene in this region – it is conceivable that
the 129 gene(s) that predispose to GN in the (129 B6)F2
strain could modify or augment the GN observed in a
knockout strain. Recently a knockout of the Bcl-2
associated protein (Bax) gene, located on proximal
chromosome 7, was reported to have GN as one of its
resulting phenotypes.27 In light of the data presented
herein, one cannot exclude the possibility that the
observed GN phenotype may have been due to the
surrounding 129 genome, and not the inactive Bax gene.
A previously described locus of B6 origin on chromosome 3,6 was here confirmed to be linked to ANA
production in both the (129 B6)F2.C1qa/ mice and
combined cohorts. Unlike the loci on chromosomes 1 and
7, this locus has not been consistently linked to lupus
disease traits, the SLE-associated loci Sles3 and Bxs5
being proximal (between 20 and 40 cM) to our observed
linkage peak. However, they intriguingly both involve
non-autoimmune strains; in the case of Sles3,
NZWxC57BL/6 heterozygosity,1 and in the case of
Bxs5, C57BL/10 homozygosity.11 The confirmation of a
B6-derived locus on chromosome 3 that modifies ANA
titres (in combination with 129-derived genes) highlights
the likelihood of epistatic interactions influencing the
phenotype of a knockout strain, in spite of extensive
backcrossing to B6.
The possibility of B6 genes influencing the phenotype
of a knockout mouse was further underlined by the
observation of B6-derived linkage to GN on proximal
chromosome 13. This linkage was only observed in
the combined analysis of (129 B6)F2.C1qa/ and
(129 B6)F2 mice, and had thus a less penetrant effect
than the loci on chromosomes 1, 3 and 7. Nonetheless,
such minor loci may together have a significant
cumulative influence on the phenotype of a knockout
strain bred onto the B6 background. Hence, it is
important to identify both the genomic position and
the resulting phenotypes of these loci. In this context, it is
of note that we had previously reported a B6-derived
suggestive linkage to anti-dsDNA Abs on the mid-distal
region of chromosome 4.6 However, this observation
could not be validated in the current study as this region
was of fixed 129-origin in the (129 B6)F2.C1qa/ mice.
Hence, we cannot exclude the potential effect of a minor
B6-derived locus on distal chromosome 4.
In conclusion, we have identified linkage of lupus
disease traits to loci present on chromosomes 1, 3, 13 and
7 in the 129 B6 mice (Figure 7). As this is the strain
combination of choice for gene-targeted mice, knowledge
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of the influence of parental genetic loci on the disease
phenotype is critical if we are to avoid attributing false
positive phenotypes to knockout strains.
Materials and methods
Mice
The C1q-deficient mice, C1qa/ were generated as
previously reported3 and the (129 B6)F1.C1qa/ mice
were generated by crossing 129.C1qa/ mice with
B6.C1qa/ mice that had been backcrossed onto B6 for
10 generations. (129 B6)F2.C1qa/ were obtained by
intercrossing the (129 B6)F1.C1qa/ mice. A total of 156
(129 B6)F2.C1qa/ female mice were produced and
monitored for 1 year. The strain- and sex-matched wildtype (129 B6)F2 cohort was as previously described.6
Mice were maintained in specific pathogen-free conditions, and all procedures were in accordance with
institutional guidelines.
Serological analyses
Serum was collected at 12 months of age and assayed for
autoantibodies. IgG ANA and IgG anti-dsDNA Abs were
measured by indirect immunofluorescence using Hep-2
cell and Crithidia luciliae slides (The Binding Site,
Birmingham, UK) respectively.16 Serum samples were
screened at a 1:80 (ANA) or 1:20 (anti-dsDNA Abs)
dilution and the positive samples titrated to end point.
IgG anti-ssDNA Abs and anti-chromatin Abs were
measured by ELISA as described previously.28 For
determination of anti-chromatin antibodies ELISA plates
(Nunc-Immuno MaxiSorp, NUNC, Denmark) were
coated with 50 ml of PBS Thimerosal (0.1 g/l) containing
0.5 mg/ml nucleohistones from calf thymus (Lorne
laboratories Ltd, Reading, UK) at 41C overnight, then
blocked with PBS 5% milk powder for 1 h at 371C. Sera
diluted 1/100 in PBS 2% BSA 0.05% Tween20 were
incubated for 1 h at 371C. For measuring anti-ssDNA Abs
plates were coated with 50 ml of 10 mg/ml ssDNA (Sigma
Chemical Co., Poole, UK) in sodium carbonate buffer pH
9.6 at 41C overnight and then blocked with 100 ml PBS
0.5% BSA. Samples were screened at a 1/100 dilution in
PBS 2% BSA 0.05% Tween20 for 1 h at 371C with 50 ml/
well. Bound antibodies were detected with alkaline
phosphatase (AP)-conjugated goat anti-mouse IgG
(g-chain specific) (Sigma-Aldrich, Dorset, UK). The plates
were developed using the substrate p-nitrophenyl phosphate (Sigma Chemical Co., Poole, UK). The OD of the
reaction mixture at 405 nm wavelength was measured
using an ELISA plate reader (Titertek Labsystems,
Basingstoke, UK). Samples were tested in duplicate with
a non-specific binding control and the results were
expressed in arbitrary ELISA units (AEU) relative to
serial dilutions of a standard positive sample derived
from pooled autoimmune MRL/Mp.lpr/lpr serum. Serum
samples were considered positive if above the mean
72s.d. of the blank. The intra-assay coefficient of
variation was between 5 and 8%.
Histological analysis
All the mice were killed at 1 year of age. Kidney tissue
was fixed in Bouin’s solution for at least 2 h, transferred
into 70% ethanol, and then processed into paraffin.
Sections were cut, mounted, stained with periodic acidGenes and Immunity
Schiff reagent and scored for GN. Glomerular histology
was graded as follows: grade 0 – normal, grade I – focal
hypercellularity in 10–25% of the glomeruli, II –
hypercellularity involving 450% of the glomerular tuft
in 25–50% of glomeruli, grade III – hypercellularity
involving 450% of the glomerular tuft in 50–75% of
glomeruli, grade IV – glomerular hypercellularity in
475% or crescents in 425% of glomeruli. Histological
analysis was performed in a blinded fashion and 50
glomeruli per section were analysed.
Genotypic analysis
Genotyping of the (129 B6)F2.C1qa/ cohort was
carried out using polymorphic microsatellite markers, a
standard polymerase chain reaction and either 4%
MetaPhor agarose (Cambrex Bioscience Rockland, Rockland, ME, USA) or 16% polyacrylamide gels stained with
ethidium bromide. The average marker density was one
marker per 10 centimorgans (cM) across the autosomes,
the positions and sequences of which were determined
from the Mouse Genome Informatics (MGI) database
(http://www.informatics.jax.org). The list of markers
used is available on request.
Statistical analyses
All linkage analyses and interval mapping were conducted using MapManager QTb29 (ftp://mcbio.med.buffalo.edu/pub/MapMgr/).29 Marker maps were
generated to determine the accuracy of genotyping, with
all markers mapping to within 3 cM of the marker
position in the MGI database. All centimorgans positions
of markers referred to in this study are from the MGI
database.
Anti-ssDNA and anti-chromatin ELISA data were log
transformed prior to linkage analysis as this resulted in a
more normalized distribution. As the ELISA assays for
these two autoantibodies in the two cohorts of mice had
been carried on separate occasions and using a different
MRL/Mp.lpr/lpr standard positive sample, for the
combined analysis of the two sets of data the samples
in each group were ranked using uncategorized (continuous) arbitrary values and the ranked values were
subsequently used for the quantitative trait locus (QTL)
analysis.
Thresholds for suggestive, significant and highly
significant linkages were determined using cohort- and
trait-specific permutation tests, based on 1000 permutations of the data, in Map Manager QTb29.29 A logarithm
of odds ratio (LOD) of X2.0 (Pp9.9 104), of X3.6
(Pp2.5 105) and of X5.7 (Pp2.0 106) was indicative of suggestive, significant and highly significant
linkage, respectively, in the (129 B6)F2.C1qa/ cohort
of mice. In the combined cohort of C1q-deficient
and wild-type F2 mice, the threshold for suggestive,
significant and highly significant linkages were
LOD X2.1 (Pp7.9 104), X3.5 (Pp3.1 105) and
X5.3 (Pp4.9 106), respectively. The calculated thresholds for suggestive, significant and highly significant
linkages were similar across the different traits.
Glomerulonephritis score was analysed as both a
quantitative trait in Map Manager and in an analysis of
extremes, in which mice with a GN score of II or above
were considered as positive for glomerulonephritis, and
mice with a GN score of 0 were considered negative.
All mice with grade I GN were excluded. Linkage to
Autoimmunity in 129 and C57BL/6 mice
Y Heidari et al
microsatellite markers was determined using a Chi
square (w2) test in an Excel (2003) spreadsheet. w2-values
of over 12.9 (P ¼ 0.0016) was considered to be indicative
of suggestive linkage.30
Non-parametric data are presented as median, with
range of values in parentheses unless otherwise stated.
Statistics were calculated using GraphPad Prism version
3.0 (GraphPad Software, San Diego, CA, USA). One way
ANOVA with Bonferonni;s multiple comparison tests
were applied for analysis of multiple groups.
Abbreviations
Abs, antibodies; AEU, arbitrary ELISA units; ANA,
antinuclear antibody; AP, alkaline phosphatase; antidsDNA, anti-double stranded DNA; anti-ssDNA,
anti-single stranded DNA; B6, C57BL/6; GN, glomerulonephritis; QTL, quantitative trait locus; SLE, Systemic
lupus erythematosus.
Acknowledgements
We thank Mrs Margarita Lewis for technical assistance
with the processing of tissue for histological studies and
the staff of the Biological Services Unit at our institution
for the care of the animals involved in this study. This
work was supported by the Wellcome Trust (Grant
number 071467).
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Genes and Immunity