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Genetic Complementation in Female (BXSB ×
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J Immunol 2003; 171:6442-6447; ;
doi: 10.4049/jimmunol.171.12.6442
http://www.jimmunol.org/content/171/12/6442
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References
Dwight H. Kono, Miyo S. Park and Argyrios N.
Theofilopoulos
The Journal of Immunology
Genetic Complementation in Female (BXSB ⴛ NZW)F2 Mice1
Dwight H. Kono,2 Miyo S. Park, and Argyrios N. Theofilopoulos
S
ystemic lupus erythematosus is a genetically heterogeneous disease inherited as a polygenic threshold trait with
manifestations dependent on the number and specific combinations of predisposing alleles (reviewed in Refs. 1–3). Due to
the complexity of defining this type of inheritance in humans, inbred mouse strains that spontaneously develop lupus-like disease
are being studied to determine the nature of the genetic contributions to disease induction, severity, and the diversity of manifestations. This approach has enabled the identification of several
susceptibility genes, including Fas (Tnfrsf6; lpr and lprcg mutations), Fasl (Tnfsf6; gld mutation), Src homology domain 2-containing tyrosine phosphatase-1 (Hcph; me and mev mutations) and
a putative gene that has not yet been cloned, the Y chromosome
accelerator of autoimmunity and lymphoproliferation (Yaa)3 (1).
These genes had mutations with specific phenotypes that provided
the basis for their identification. Because most lupus susceptibility
alleles do not exhibit such distinguishing phenotypes, however,
genome-wide searches have been performed as the initial step toward identifying genes based on chromosomal location.
Several genomic intervals from lupus-prone and nonautoimmune backgrounds have been linked to a variety of lupus traits
with varying degrees of confidence. Among the lupus-prone
strains, five quantitative trait loci (QTL) have been identified for
the New Zealand Black (NZB) (chromosomes 1, 4, 5, 11, and 17)
(3–5), eight for the New Zealand White (NZW) (chromosomes 1,
6, 7, 13, 16, 17, 18, and 19) (3– 6), eight for the MRL-Faslpr
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
Received for publication August 1, 2003. Accepted for publication October 15, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grants AR42242,
ES08666, and AR39555. This is Scripps Research Institute Publication 12241-IMM.
2
Address correspondence and reprint requests to Dr. Dwight H. Kono, Department of
Immunology, The Scripps Research Institute-IMM3, 10550 North Torrey Pines Road,
La Jolla, CA 92037. E-mail address: [email protected]
3
Abbreviations used in this paper: Yaa, Y chromosome accelerator of autoimmunity
and lymphoproliferation; QTL, quantitative trait locus; GN, glomerulonephritis; PAS,
periodic acid-Schiff; DVD, degenerative vascular disease; MI, myocardial infarction.
Copyright © 2003 by The American Association of Immunologists, Inc.
(chromosomes 2, 4, 5, 7, 10, 11, 12, and 16) (7–9), and at least 11
loci for the Yaa⫹ BXSB (possibly four regions on chromosome 1,
possibly two on chromosome 4, and a single locus each on chromosomes 3, 7, 8, 10, 13, 14, and 17) (10 –12). Although some of
the overlapping loci from different strains may represent the same
gene, it is evident from this and other studies of gene knockout
animals (reviewed in Ref. 1) that a substantial number of genes can
contribute to the induction of systemic autoimmunity. Little, however, is known about the relative strength and interaction of these
loci to the autoimmune process, which will be important for determining the significance of these loci.
F1 complementation studies have shown that acceleration of disease occurs not only in the BWF1 hybrid, but also in male and
female F1 hybrids of the BXSB with either the NZB or NZW
strains (13). In contrast, minimal complementation is observed between these strains and the MRL-Faslpr, with the sole exception of
the male, but not female, (MRL-Faslpr ⫻ BXSB)F1 hybrid, which
develops accelerated disease because of the Yaa gene (13). These
findings raise the possibility of shared susceptibility genes or
mechanisms between the BXSB and NZ strains that may not apply
to the MRL background. Because the NZB and NZW strains have
different sets of susceptibility genes (1–3), complementation of
either strain by the BXSB background suggests that at least some
of the BXSB QTL responsible for complementation are unique.
Furthermore, because F1 hybrids of autoimmune strains with nonautoimmune strains develop less severe to no autoimmune disease
(3, 4, 13), it is likely that complementation is from predisposing
genes contributing positively to disease rather than recessive suppressor genes that are negated by the F1 heterozygosity.
The BXSB is a recombinant inbred strain generated from crossing C57BL/6 and SB/Le strains that develops a severe form of
systemic autoimmunity (1, 13). Male BXSB mice develop severe
lupus-like disease by 5 mo of age characterized by lymphoproliferation, high levels of Mac1-positive peripheral blood cells, high
titers of autoantibodies, glomerulonephritis (GN) and vasculitis.
Genetic susceptibility, however, is highly dependent on the Yaa
gene because female BXSB mice develop only very mild disease.
The Yaa gene alone can promote disease in other lupus backgrounds. Consomic NZB.Yaa and NZW.Yaa that differ from the
0022-1767/03/$02.00
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F1 hybrids among New Zealand Black (NZB), New Zealand White (NZW), and BXSB lupus-prone strains develop accelerated
autoimmunity in both sexes regardless of the specific combination. To identify BXSB susceptibility loci in the absence of the Y
chromosome accelerator of autoimmunity (Yaa) and to study the genetics of this complementation, genome-wide quantitative trait
locus (QTL) mapping was performed on female (BXSB ⴛ NZW)F2 mice. Six QTL were identified on chromosomes 1, 4, 5, 6, 7,
and 17. Survival mapped to chromosomes 5 and 17, anti-chromatin Ab to chromosomes 4 and 17, glomerulonephritis to chromosomes 6 and 17, and splenomegaly to chromosomes 1, 7, and 17. QTL on chromosomes 4 and 6 were new and designated as
Lxw1 and -2, respectively. Two non-MHC QTL (chromosomes 1 and 4) were inherited from the BXSB and the rest were NZWderived, including two similar to previously defined loci. Only two of 11 previously defined non-MHC BXSB QTL using male
(Yaaⴙ) crosses were implicated, suggesting that some male-defined BXSB QTL may require coexpression of the Yaa. Findings from
this and other studies indicate that BXSB and NZB backgrounds contribute completely different sets of genes to complement NZW
mice. Identification of susceptibility genes and complementing genes in several lupus-prone strain combinations will be important
for defining the epistatic effects and background influences on the heterogeneous genetic factors responsible for lupus
induction. The Journal of Immunology, 2003, 171: 6442– 6447.
The Journal of Immunology
6443
Materials and Methods
Mice
NZW/LacScr, BXSB/Scr, (BXSB ⫻ NZW)F1 (XWF1), and (BXSB ⫻
NZW)F2 (XWF2) intercross mice were bred and maintained in The Scripps
Research Institute animal facility (La Jolla, CA). Female mice were used in
this study. Mice were autopsied at 1 year of age or earlier if moribund.
Lupus phenotypes
Mice were bled at monthly intervals from 5 mo of age. Autopsies and
histologic procedures were performed as previously described (5, 7).
Briefly, tissue sections were fixed in Bouin’s solution and stained with
periodic acid-Schiff (PAS) reagent. Severity of GN was graded blindly on
a 0 to 4 scale as previously described (5), with scores ⬎2 considered
pathologic. Severity of degenerative vascular disease of coronary vessels,
myocardial infarction, and necrotizing polyarteritis was graded on a 0 –3
scale as previously described (20). Briefly, for degenerative vascular disease, grade 1 contained minimal PAS-positive deposits along and within
one coronary blood vessel wall, grade 2 had PAS-positive deposits with
narrowing of the lumen in two or three vessels, and grade 3 involved four
or more affected vessels. For myocardial infarction, the size of myocardial
necrosis was 1–2 mm for grade 1, 2–3 mm for grade 2, and ⬎3 mm for
grade 3. Arteritis was graded on the number of affected small and mediumsized muscular arteries and the number of organs involved. Only arteries
with necrotizing and exudative inflammation of the intima and media were
included. Grade 1 was limited to one organ, grade 2 required involvement
of three vessels in at least two different organs, and grade 3 involved
vessels in at least three different organs. The ELISA for serum antichromatin Abs was performed as described (5).
Microsatellite analysis
Genome-wide microsatellite scanning was performed by PCR of tail DNA
using 132 simple sequence length polymorphisms (list available on request) selected from 361 microsatellite markers (Research Genetics, Huntsville, AL). PCRs used standard reagents containing 1.5 mM MgCl2 and 0.4
␮M primers under the following conditions: 40 cycles of 92°C for 20 s,
42°C to 60°C (depending on primers) for 1.5 min and 72°C for 2 min.
Products were visualized on agarose gels stained with ethidium bromide.
Statistics and linkage analysis
Survival was analyzed by the Kaplan-Meier statistic. Comparisons of traits
between parental strains and crosses were performed with the two-tailed
unpaired t test or ANOVA. Associations between quantitative traits in F2
mice were determined by regression coefficients with p values derived from
Fisher’s transformation.
The linkage map for the (BXSB ⫻ NZW)F2 cross was created with Mapmaker3 (http://waldo.wi.mit.edu/ftp/distribution/software/mapmaker3) (21).
QTL were identified using QTL Cartographer version 1.17 (http://statgen.ncsu.edu/qtlcart). Likelihood ratios (LR) were calculated using the LRmapqtl
program. Composite interval mapping was performed using model 6 of the
Zmapqtl program with options set at 2-cM intervals, 10-cM window size, and
five background parameters. The experiment-wise significance level for each
trait was determined by analyzing 1000 random shuffling permutations of the
actual phenotype data. Log transformations of quantitative traits were used
when they resulted in more normalized distributions. GN scores were normalized by regrouping into five categories as previously described for QTL (7):
GN scores ⱕ1 were scored as 1 (n ⫽ 11), between 1 and ⬍2 as 2 (n ⫽ 65),
2 to ⬍2.5 as 3 (n ⫽ 122), 2.5 to ⬍3 as 4 (n ⫽ 42), and ⱖ3 as 5 (n ⫽ 24). New
loci were designated Lxw for lupus BXSB ⫻ NZW.
Results
Disease traits in female BXSB, NZW, F1, and F2 intercross mice
The incidence and severity of major disease manifestations for
female BXSB, NZW, XWF1, and XWF2 mice are summarized on
Table I and Fig. 1. Traits examined included those previously
tested in a BWF2 linkage study (survival, GN, anti-chromatin Ab
levels, and spleen weight) (5), as well as others that have high
incidence in XWF1 hybrids, including myocardial infarction (MI),
degenerative vascular disease (DVD), arteritis, and thymic atrophy
(13). For all traits, disease was significantly worse in XWF1 hybrid
mice than in one or both parental strains.
The 1-year survival for female BXSB mice was 100% (14 mice)
and 89% (8 of 9) for NZW mice, but only 31% (5 of 16) for female
XWF1 mice ( p ⬍ 0.01). The female XWF2 mice had an intermediate survival of 66% (81 of 123) consistent with multigenic inheritance of this trait. The average IgG anti-chromatin Ab levels in
BXSB mice and XWF1 hybrids were elevated, whereas, average
levels in NZW mice were nearly normal throughout the 1-year
observation period (Fig. 1). The average anti-chromatin Ab levels
Table I. Summary of lupus manifestations in female BXSB, NZW, XWF1, and XWF2 micea
Strain
Survival
at 12 mo
(%)
Spleen Weight
GN Score
MIb
DVDb
Arteritis
Thymus Atrophy
BXSB
NZW
XWF1
XWF2
100c
89c
31
66
129 ⫾ 12d,e
134 ⫾ 16e
340 ⫾ 81
188 ⫾ 16
0.8 ⫾ 0.1 (0)f
1.4 ⫾ 0.4 (22)c
2.7 ⫾ 0.2 (54)
1.9 ⫾ 0.1 (39)
0c
0.11 ⫾ 0.11 (11)
0.75 ⫾ 0.27 (58)
0.29 ⫾ 0.06 (22)
0.11 ⫾ 0.11 (11)
0.58 ⫾ 0.18 (58)
0.38 ⫾ 0.07 (32)
0c
0e
0.40 ⫾ 0.16 (40)
0.21 ⫾ 0.05 (15)
0.21 ⫾ 0.10 (14)f
0.83 ⫾ 0.12 (78)
0.88 ⫾ 0.08 (85)
0.57 ⫾ 0.05 (61)
a
Number of mice in each group: 14 BXSB, 9 NZW, 10 –16 XWF1, 107–123 XWF2.
Scoring criteria are described in the Methods section.
A value of p ⬍ 0.01 for the Kaplan-Meier log-rank test (survival only) or t tests of parental and XWF1 mice.
d
Mean ⫾ SE (% incidence).
e
A value of p ⬍ 0.05 for parental and XWF1 mice.
f
A value of p ⬍ 0.0001 for t tests of parental and XWF1 mice.
b
c
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parental strains by only the BXSB Y chromosome develop accelerated autoimmune disease similar to Yaa containing male F1
crosses of NZB or NZW to BXSB mice (14). The Yaa gene, however, requires other background genes for disease because C57BL/
6.Yaa or CBA/J.Yaa mice are largely unaffected (13–15). The Yaa
gene has a greater effect on mice with mild lupus than in those with
severe disease (15). Expression of Yaa on T cells is not required
for disease acceleration (16, 17), and double bone marrow chimera
experiments using mixtures of Yaa⫹ and Yaa⫺ cells demonstrated
selective production of anti-DNA and hypergammaglobulinemia
by Yaa⫹ B cells (18). The Ab promoting effect of the Yaa gene was
observed not only for self Ags, but also for foreign Ags, particularly those that elicit low T cell-dependent Ab responses (19).
Thus, weak autoimmune promoting genes might be expected to be
most affected by the Yaa gene.
Thus far, BXSB QTL have been mapped only in Yaa⫹ mice
(10 –12) and therefore, the extent to which these loci are dependent
on the Yaa is not known. In this study, a genome-wide QTL scan
of female (BXSB ⫻ NZW)F2 (XWF2) intercross mice was performed to identify BXSB (H-2b) susceptibility loci in the absence
of the Yaa gene and to determine whether complementation of
NZW (H-2z) to BXSB or to NZB (H-2d) involves similar or diverse QTL. Multiple QTL from both parental strains were found to
predispose to lupus-like disease, including two new loci on chromosomes 4 and 6. Furthermore, BXSB QTL that complemented
the NZW genome to promote lupus were completely different from
the NZW-complementing NZB QTL.
LUPUS SUSCEPTIBILITY LOCI IN (BXSB ⫻ NZW)F2 MICE
6444
FIGURE 1. Cumulative survival and anti-chromatin
Ab levels for female BXSB, NZW, and crosses. A, Percent cumulative mortality (p ⬍ 0.006 for XWF1 and
NZW, p ⬍ 0.0002 for XWF1 and BXSB). B, Mean and
SE ELISA OD units for anti-chromatin Ab levels. A
value of p ⬍ 0.05 for BXSB vs NZW (at 9–12 mo of
age, by unpaired two-tailed t test), NZW vs XWF1
(8 –12 mo), BXSB vs XWF1 (12 mo), BXSB vs XWF2
(11 and 12 mo), NZW vs XWF2 (9–12 mo). The number of mice in each group was 14 BXSB, 8–9 NZW,
7–16 XWF1, and 93–123 XWF2. Mortality of NZW
mice was reported previously (5).
genically related, e.g., survival, GN, and anti-chromatin Ab production, suggests that to some extent, independent immunopathologic or additional pathways may be involved.
Loci with linkage to survival
Two loci associated with survival were identified on chromosomes
5 (D5Mit55, p ⬍ 0.007) and 17 (Tnf, p ⬍ 0.0016) (Table II). The
locus on chromosome 5 accounts for 18.0% of the variance and the
chromosome 17 locus for 6.8%. The chromosome 5 locus maps to
the proximal-mid portion of the chromosome (Fig. 2) and the Tnf
marker on chromosome 17 is located within the MHC complex ⬍1
cM from the class II genes and will be considered equivalent to the
MHC. The predisposing allele for the chromosome 5 locus was
inherited from NZW strain, whereas, for the MHC, the heterozygous genotype (H-2b/z) conferred somewhat higher susceptibility
than the homozygous BXSB genotype (H-2b/b), while the NZW
genotype (H-2z/z) was resistant (Table II). The contributions of the
two loci were additive ( p ⬍ 0.0005), and when all combinations of
alleles were examined, appeared dependent on specific combinations (epistasis) (Fig. 3). Thus, the locus on chromosome 5 had a
strong effect on survival when the MHC (Tnf) was heterozygous
(70, 43, and 25%, when D5Mit55 was of the X, F, and W genotypes, respectively, Fig. 2); however, there was minimal effect
when the MHC was of the least susceptible NZW genotype (83–
100% survival for all D5Mit55 genotypes).
Table II. Susceptibility loci for (BXSB ⫻ NZW)F2 mice
Marker
Survival
D5Mit55
Tnf
Chr(cM)a
NZW
BXSB
F1
pb
Allelec
5 (28)
17 (19)
9.4 ⫾ 1.1d
11.9 ⫾ 1.0
11.4 ⫾ 1.0
10.6 ⫾ 1.0
10.8 ⫾ 1.0
10.1 ⫾ 1.0
⬍0.001
⬍0.003e
W
XW
35 ⫾ 19
44 ⫾ 21
236 ⫾ 96
570 ⫾ 159
389 ⫾ 97
238 ⫾ 73
0.0002
⬍0.001
X
X
6 (26)
17 (19)
3.7 ⫾ 0.2
2.1 ⫾ 0.2
2.8 ⫾ 0.4
3.6 ⫾ 0.2
2.7 ⫾ 0.2
3.2 ⫾ 0.2
0.0038
0.0001
W
X
1 (43)
7 (1)
17 (19)
151 ⫾ 41
335 ⫾ 70
126 ⫾ 8
231 ⫾ 30
159 ⫾ 20
287 ⫾ 39
183 ⫾ 20
155 ⫾ 13
179 ⫾ 26
0.003
0.001
⬍0.0001
X
W
X
1 (106)
6 (61)
17 (47)
0.52 ⫾ 0.15
0.57 ⫾ 0.17
0.07 ⫾ 0.07
0.000
0.04 ⫾ 0.04
0.47 ⫾ 0.13
0.14 ⫾ 0.06
0.15 ⫾ 0.06
0.09 ⫾ 0.04
0.0004
0.001
0.0016
W
W
X
Anti-chromatin autoantibody levels
D4Mit2
4 (7)
Tnf
17 (19)
Glomerulonephritis
D6Mit33
Tnf
Splenomegaly
D1Mit46
D7Mit152
Tnf
Arteritis
D1Mit17
D6Mit256
D17Mit42
a
Chromosome and (cM location) from http://www.informatics.jax.org/searches/marker_form.shtml.
Values of p from the Kaplan-Meier log-rank test (survival only) or ANOVA, logarithm transformed data were used for anti-chromatin Ab and splenomegaly.
X ⫽ BXSB; W ⫽ NZW.
d
Mean ⫾ SE for survival, anti-chromatin ELISA (OD units) at 6 mo, GN score, spleen weight, and arteritis score.
e
p ⫽ 0.0006 for F vs W (Tnf; survival).
b
c
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in XWF1 mice at older ages (11 and 12 mo) were likely reduced
as a result of death or renal insufficiency of the most severely
affected individuals. The XWF2 female mice had slightly higher
anti-chromatin Ab levels than the BXSB and XWF1 early in disease and lower levels at later time points, consistent with additive
polygenic inheritance. Spleen weights were in the normal range for
both BXSB and NZW mice, but were more than 2-fold greater in
XWF1 mice (Table I). Interestingly, while there was no evidence
of GN in BXSB mice, 22% (2 of 9) of NZW mice developed GN
(score ⬎2.0) despite the much lower anti-chromatin Ab levels. The
XWF1 hybrid had significantly more GN than the NZW ( p ⬍
0.01) and BXSB ( p ⬍ 0.0001) strains. MI and DVD occurred
more frequently in the XWF1 hybrid (58%) than the BXSB (none)
and NZW (11%) mice. Thymic atrophy was present in all groups,
but to a significantly lower extent in BXSB mice ( p ⬍ 0.01 compared with XWF1 hybrids). Arteritis was present in 40% of XWF1
mice, but in none of the parental strains ( p ⬍ 0.05). Overall, the
findings suggest that NZW mice may be more susceptible than
BXSB mice to most of the traits including mortality, GN, MI,
DVD, arteritis, and thymic atrophy, although BXSB mice are
clearly more susceptible to anti-chromatin Ab production.
Among the XWF2 mice, significant correlation (r ⬎0.65) between traits was observed only for DVD and MI (r ⫽ 0.85, p ⬍
0.0001) consistent with DVD as the major cause of MI. The lack
of correlation between some of the other traits considered patho-
The Journal of Immunology
Mapping of QTL predisposing to anti-chromatin Ab production
The MHC (Tnf, p ⫽ 5.1 ⫻ 10⫺6) and a QTL on proximal chromosome 4 (D4Mit2, p ⫽ 0.0002) were linked to IgG antichromatin Ab production at 6 mo (Table II, Fig. 2). The MHC
accounted for 29.7% and the chromosome 4 locus for 13.0%. In
both cases, the susceptibility allele was inherited from the BXSB
strain. When analysis was performed for anti-chromatin Ab production at later time points (10 and 11 mo), there was linkage to
the MHC and a NZW allele on chromosome 1 (D1Mit54, p ⫽
4.2 ⫻ 10⫺4), but not to the chromosome 4 QTL. This is consistent
with the early anti-chromatin Ab production observed in the
BXSB, but not NZW strain, and may have been affected by the
earlier sacrifice of more severely diseased XWF2 mice (⬃20% of
mice at 10 mo).
QTL predisposing to GN
Two QTL were identified for GN, one was the MHC (Tnf, p ⫽
4.3 ⫻ 10⫺5) and the other a proximal interval on chromosome 6
(D6Mit33, p ⫽ 0.004) (Table II, Fig. 2). The susceptible allele on
the chromosome 6 QTL was recessively inherited from the NZW,
whereas the H-2b/b haplotype was associated with worse disease.
Chromosome 6 QTL appears to be novel. The chromosome 6 locus
accounted for 16.0% of the variance and the MHC for 25.7%.
QTL predisposing to splenomegaly
Three QTL predisposing to splenomegaly were identified on midproximal chromosome 1 (D1Mit46, p ⫽ 0.03), the acrocentric end
FIGURE 3. Cumulative survival of XWF2 mice segregated by the chromosome 5 QTL and MHC genotypes. Cumulative survival of all combinations of the chromosome 5 QTL (probably Sle6) and MHC (chromosome
17) genotypes from 4 to 12 mo (left panel). Percent survival at 12 mo is
shown on the right panel. First letter of genotype is the chromosome 5 QTL
and second is the MHC; F ⫽ F1, X ⫽ BXSB, and W ⫽ NZW. The genotype of the chromosome 5 locus was defined at D5Mit55 (28 cM from
centromere) and the MHC at Tnf (19 cM from centromere).
of chromosome 7 (D7Mit152, p ⫽ 5.5 ⫻ 10⫺5) and the MHC (Tnf,
p ⫽ 1.1 ⫻ 10⫺3) (Table II, Fig. 2). These loci accounted for 19.0,
15.5, and 21.5% of variance, respectively. The susceptible locus on
chromosome 1 was inherited from the BXSB strain and appeared
to be additive. The susceptible allele for the chromosome 7 QTL
was recessively inherited from the NZW. The H-2b/b haplotype
was again associated with the greatest severity. The chromosome
7 QTL overlaps and is probably identical to Lbw5 and Sle3/Sle5,
which were previously identified in BWF2 intercross (5) and
NZM/Aeg2410 ⫻ (NZM/Aeg2410 ⫻ C57BL/6)F1 backcross (4)
studies, respectively.
QTL predisposing to other traits
Three QTL were identified for arteritis on chromosomes 1
(D1Mit54, p ⫽ 2.9 ⫻ 10⫺4), 6 (D6Mit256, p ⫽ 7 ⫻ 10⫺4), and 17
(D17Mit42, p ⫽ 1.2 ⫻ 10⫺3), however, none of these reached the
0.1 level of genome-wide significance. The susceptibility alleles
for QTL on chromosomes 1 and 6 were from the NZW strain and
for the chromosome 17 QTL from the BXSB. Genome-wide
searches of DVD and MI failed to identify any QTL. This may be
due to the contribution of a large number of genes and a relatively
low incidence of DVD and MI in the XWF2 intercrosses in this
study (Table I).
Discussion
Herein, lupus-predisposing QTL for NZW and BXSB backgrounds
were identified in the absence of the Yaa gene by analyzing female
XWF2 mice. Six significant QTL linked to one or more traits were
found on chromosomes 1, 4, 5, 6, 7, and 17, including two potentially new QTL and four previously defined loci. Non-MHC QTL
from the NZW strain included loci on chromosomes 5, 6, and 7,
while there were two non-MHC QTL on chromosomes 1 and 4
from the BXSB background. The BXSB-derived QTL on chromosome 1 (at ⬃43 cM), was linked to splenomegaly and mapped
between two previously reported loci, Bxs1 (at 32.8 cM) and Bxs2
(at 63 cM). Both of these QTL were identified using male BXSB ⫻
B10 crosses and interestingly both were linked to splenomegaly,
although at a suggestive level of significance (10, 12). Both loci
were also linked to GN and autoantibody production. Thus, it is
likely that the chromosome 1 locus identified in this study represents the same locus. This locus mapped centromeric to previously
described NZB (Lbw7/Nba2) (3, 5) and NZW (Sle1) (4) chromosome 1 QTL and is likely distinct from these.
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FIGURE 2. QTL scans for chromosomes 1, 4, 5, 6, 7, and 17. Composite interval maps of LR test (LRT) are shown for the following traits:
chromosome (Chr) 1: spleen weight; Chr 4: anti-chromatin Ab; Chr 5:
survival; Chr 6: GN; Chr 7: spleen weight; Chr 17: GN, anti-chromatin Ab;
and spleen weight. For chromosomes 1–7, horizontal lines are the experiment-wise significance levels ␣ ⫽ 0.1 (dotted line) and 0.05 (solid line)
obtained by analyzing 1000 random permutations of the experimental data
(QTL Cartographer). For Chr 17, QTL scans for the three different traits
are: spleen weight (thin line), GN (bold line), and anti-chromatin Ab (dotted line), along with corresponding horizontal lines for the experiment-wise
significance levels ␣ ⫽ 0.05. Œ, Positions of the following markers in order
from the acromere. Chr 1: D1Mit294, D1Nds4, D1Mit212, D1mit302,
D1Mit46, D1Mit10, D1Mit54, D1Mit105, D1Mit115, D1Mit17; Chr 4:
D4Mit1, D4Mit2, D4Mit53, D4Mit9, D4mit28, D4Mit258, D4Mit54,
D4Mit14, D4Mit48; Chr 5: D5Mit48, D5Mit348, D5Mit55, D5Mit254,
D5Nds2, D5Mit312, D5Mit371, D18Mit7, D5Mit31, D5Mit43; Chr 6:
D6Mit138, D6Mit33, D6Mit70, D6Mit10, D6Mit256, D6Mit291, D6Mit14;
Chr 7: D7Mit152, D7Mit56, D7Mit294, cd22, D7Mit55, D7Mit227,
D7Nds5, D7Mit211, D7Mit84, D7Mit281, D7Mit12, D7Nds4, D7Mit259;
Chr 17: D17Mit97, Tnf, D17Mit10, D17Mit42, D17Mit129.
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LUPUS SUSCEPTIBILITY LOCI IN (BXSB ⫻ NZW)F2 MICE
The BXSB-inherited chromosome 4 locus, designated Lxw1 (for
lupus BXSB ⫻ NZW), was linked to early anti-chromatin production consistent with the predilection of female BXSB, but not female NZW, mice to this trait (Fig. 1). Lxw1 maps proximal to
another BXSB locus (Acla-2) that was linked to the production of
anti-cardiolipin IgG Ab (11) and a more distal BXSB chromosome
4 locus that was linked to lymphadenopathy (10). This raises the
possibility that Lxw1 and Acla-2, although appearing to be distinct because of their locations, may represent a single BXSB
QTL that broadly enhances autoantibody production even in the
absence of the Yaa gene, but this will need to be determined.
Lxw1, because of its proximal location, does not overlap the
chromosome 4 NZB locus, Nba1/Sle2/Lbw2 (4, 5, 22), and
clearly represents a distinct QTL.
The chromosome 5 locus was linked to mortality and appeared
strongly dependent on the MHC haplotype (Fig. 3). This locus
likely represents the NZW chromosome 5 QTL, Sle6 (23). The
chromosome 6 locus linked to GN is novel and will be designated
Lxw2. The NZW QTL on chromosome 7 overlaps and is probably
the same as a NZW locus previously defined as Sle3/5 or Lbw5 (4,
5). This QTL has been verified in C57BL/6 mice congenic for the
Sle3/Sle5 region of NZM/Aeg2410 (24) and in NZB backgrounds
congenic for the Lbw5 fragment of NZW (our unpublished observations). The fact that this locus can contribute to some disease
manifestations in both autoimmune and nonautoimmune backgrounds (Refs. 4, 5, and this study) suggests its involvement in a
common or fundamental mechanism in the autoimmune process.
Several BXSB QTL were previously defined in mapping studies
of male Yaa⫹ crosses. Hogarth et al. (10, 12) using BXSB ⫻
(B10 ⫻ BXSB)F1 and B10 ⫻ (B10 ⫻ BXSB)F1 backcross mice,
identified four regions on chromosome 1 with significant linkage
to nephritis or anti-dsDNA Ab (Bxs1– 4) and another to ANA/antissDNA on chromosome 3 (Bxs5). In addition, other QTLs of suggestive linkage were also described, including a region on distalmid chromosome 4 to lymphadenopathy, proximal-mid
chromosome 10 to anti-dsDNA Ab and chromosome 13 to antissDNA Ab. In another study, Ida et al. (11), analyzing NZW ⫻
(NZW ⫻ BXSB)F1 backcross mice identified BXSB QTL in mid
chromosome 4 (Acla-2) with linkage to anti-cardiolipin Ab, distal
chromosome 7 (Myo-1) to MI, proximal chromosome 8 (Pbat-2) to
anti-platelet Ab and thrombocytopenia, mid chromosome 14
(Myo-2) to MI, and the MHC region to anti-cardiolipin (Acla-1)
and anti-platelet (Pbat-1) Abs. Strikingly, of these 11 or more
potential BXSB QTL identified in male (Yaa⫹) mice, only the
MHC and a chromosome 1 locus were linked to disease in this
study of female XWF2 mice. Some of these loci may not have
been identified because they were mapped to different traits,
while others may be dependent on the Yaa mutation. Other sexrelated factors may also play a role although predisposition of
male BXSB mice to lupus is dependent on the Yaa gene and not
sex hormones (13).
Previous studies using (NZW ⫻ BXSB)F1 (H-2z/b), (NZW.H-2d
⫻ BXSB) F1 (H-2d/b), and NZW ⫻ (NZW ⫻ BXSB) F1 backcross
mice have shown that H-2b/z confers increased autoimmune susceptibility compared with the z/d, z/z, or d/b haplotypes (11, 25).
In addition to two of these haplotypes (b/z and z/z), the current
XWF2 analysis examined linkage of the homozygous b/b haplotype to disease severity. Strikingly, H-2b/b was linked to all
mapable traits except for arteritis and was the haplotype associated
with worse disease in all cases except for survival in which case
the heterozygous H-2b/z haplotype was associated with a slightly
worse outcome. This suggests that expression of the H-2b/b haplotype on the NZW background might result in significant disease
acceleration in a manner similar to the autoimmune-enhancing effect of H-2bm12 in NZB mice (26).
Without exception, the development of lupus-like disease in
predisposed mouse models requires the additive contributions
of multiple genetic defects that vary in strength, associated
traits, and dependence on other genes. Although identification
of susceptibility genes and definition of their effects on the immune system are paramount, understanding the overall contribution of these genes to disease in a variety of genetic backgrounds is also important. Classification of lupus-predisposing
genes in such a manner should also aid in the selection of potential gene targets for diagnosis and intervention. Further studies of complementation among lupus-prone strains will be important for defining relative contributions and epistatic
interactions of susceptibility alleles.
Acknowledgments
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