Download Different Species Common Arthritis Quantitative Trait Loci in High

High Resolution Mapping of Cia3: A
Common Arthritis Quantitative Trait Loci in
Different Species
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Xinhua Yu, Haidong Teng, Andreia Marques, Farahnaz
Ashgari and Saleh M. Ibrahim
J Immunol 2009; 182:3016-3023; ;
doi: 10.4049/jimmunol.0803005
http://www.jimmunol.org/content/182/5/3016
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Supplementary
Material
The Journal of Immunology
High Resolution Mapping of Cia3: A Common Arthritis
Quantitative Trait Loci in Different Species1
Xinhua Yu, Haidong Teng, Andreia Marques, Farahnaz Ashgari, and Saleh M. Ibrahim2
R
heumatoid arthritis (RA)3 is a chronic inflammatory autoimmune joint disease influenced by genetic and environmental factors (1). The genetic contribution to RA susceptibility is estimated to be as much as 60%, of which the HLA DRB1
locus is thought to account for 30 –50% (2, 3). However, identification
of non-MHC RA susceptibility genes has been challenging due to
genetic heterogeneity and incomplete penetrance, as well as the effect
of environmental factors on RA development. Presently, only a few
genes have been convincingly showed to be associated with RA, including PADI4, PTPN22, and CTLA4 (4, 5, 6). Genetic analysis of
well-defined experimental models of autoimmune arthritis provides
an alternative strategy to study the genetic basis of RA. A good example is identification of NCF1 as a novel susceptibility gene in autoimmune arthritis (7). Animal models of RA have been used to identify susceptibility genes, and multiple quantitative trait loci (QTLs)
have been identified (see Ref. 8, 9).
Despite the many advantages of animal models, identification of
susceptibility genes in animal models is limited by two factors. On
one hand, linkage analysis is not as powerful as case-control association studies. As a consequence, a genomic region containing a susceptibility gene that has very small effect on disease might not reach
the significant threshold of the linkage analysis, and thus will not be
Section of Immunogenetics, University of Rostock, Germany
Received for publication September 10, 2008. Accepted for publication December
24, 2008.
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 study was supported by the EU FP6 contract MRTN-CT-2004-005693
(EURO-RA).
2
Address correspondence and reprint requests to Dr. Saleh M. Ibrahim at the current
address: Genetics Group, Department of Dermatology, University of Lübeck, Ratzeburger
Allee 160, 23538 Lübeck, Germany. E-mail address: [email protected]
3
Abbreviations used in this paper: RA, rheumatoid arthritis; QTL, quantitative
trait loci; CIA, collagen-induced arthritis; AIL, advanced intercross line; chr.,
chromosome; SNP, single nucleotide polymorphism.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803005
defined as a QTL. Therefore, the number of susceptibility genes could
be much larger than the number of the identified QTLs. On the other
hand, identification of susceptibility genes within the QTLs is still a
challenging task, with the exceptions of few genes with very strong
effect on the disease, e.g., NCF1 (7). In most cases, a single quantitative trait gene contributes only mildly or moderately to the outcome
of the complex traits. To accelerate progress in the identification of
susceptibility genes, several complementary approaches have been
suggested, such as identification of polymorphisms in coding or regulatory region, in vitro functional studies, transgenesis, knock-in models, deficiency-complementation testing, mutational analysis, and homology searches (10). In addition, advanced progress in mouse
genetics also accelerates progress in the identification of the quantitative trait genes. For example, genomes of the 16 commonly used
mouse inbred strains have been recently sequenced, and 8.27 million
SNPs have been identified (11). Therefore, polymorphisms within a
QTL identified in those 16 strains or their substrains can be obtained,
which will considerably help in the identification of candidate genes.
Previously we performed a genome-wide linkage analysis in a F2
progeny to identify QTLs controlling collagen-induced arthritis (CIA)
and generated an advanced intercross line (AIL) to refine those QTLs
(12, 13). In the F2 progeny, we identified one QTL with a strong effect
on CIA clinical traits, Cia2, that contributes to only 16% of the phenotype variant (12). This suggests that there could be additional susceptibility gene(s) whose effect is masked by Cia2, and thus failed to
reach a significant threshold. In this study, we investigated six
genomic regions that showed suggestive level of linkage to CIA in F2
mice. We used the AIL to confirm those suggestive QTLs and refine
the positive(s) into a small genomic region. Also, we investigated the
candidate genes for one confirmed and refined QTL by defining differentially expressed genes, identifying the nonsynonymous polymorphisms, and performing comparative genomic mapping.
Materials and Methods
Phenotypic traits of CIA
Two mouse populations used in this study, 290 (DBA/1 ⫻ FVB/N)F2
mice and 308 (DBA/1 ⫻ FVB/N)F11/12 AIL mice, were generated
Downloaded from http://www.jimmunol.org/ by guest on April 29, 2017
Murine collagen induced arthritis (CIA) is a widely used model of rheumatoid arthritis (RA). Identification of CIA susceptibility genes
will aid in the understanding of RA pathogenesis and development of therapeutic targets. This study aims to identify and refine
quantitative trait loci (QTL) controlling CIA. Major CIA clinical traits were evaluated in both (DBA/1ⴛFVB/N) F2 and advanced
intercross line (AIL) mice; QTLs were confirmed and refined in AIL. To search for candidate genes, we applied multiple approaches,
including gene expression profiling, identification of nonsynonymous polymorphism, and comparative genomic mapping. We identified
six suggestive QTLs controlling CIA clinical traits in the F2 progeny; one of these was confirmed and refined in AIL. This QTL is located
on chromosome 6 and overlaps with Cia3, which was identified previously. We refined the 2-log support interval of Cia3 into a 5.6 Mb
genomic region; 15 of 77 genes are differentially expressed or carry nonsynonymous polymorphisms between two parental strains. The
counterpart genomic region of Cia3 on the rat and human genomes are linked to RA. Twenty-nine of 77 genes are located in the
arthritis-linked genomic regions of all three species. Five of those 29 genes are differentially expressed or carry nonsynonymous polymorphisms between parental strains: Timp4, Tmem40, Mbd4, Cacna1c, and Lrtm2. Taken together, we refined Cia3 into a 5.6 Mb
genomic region on mouse chromosome 6 and identified candidate genes. This will aid in the search for susceptibility gene(s) controlling arthritis
development within Cia3 and its counterpart regions in rat and human genomes. The Journal of Immunology, 2009, 182: 3016–3023.
The Journal of Immunology
3017
Table I. List of QTLs controlling clinical traits of CIA
Chr.
Marker
LOD Score
Traits
Susceptible Allele
Overlap with CIA QTLs
2
6
7
10
11
18
19
D2Mit81
D6Mit328
D7Mit248
D10Mit261
D11Mit126
D18Mit222
D19Mit90
10.4ⴱⴱ
2.37ⴱ
2.12ⴱ
2.1ⴱ
2.08ⴱ
2.43ⴱ
2.66ⴱ
severity, onset, susceptibility
severity
severity
severity
severity
susceptibility
onset
DBA/1
FVB/N
FVB/N
DBA/1
FVB/N
FVB/N
FVB/N
Cia2, Cia4
Cia3
Cia8
Cia40
ⴱⴱ, highly significant and ⴱ, suggestive.
previously in our laboratory. Detailed information of mice characteristics and induction of CIA were described previously (12, 13). Three
clinical traits of CIA were used for linkage analysis: severity, onset, and
susceptibility. The CIA severity (maximal score) in the F2 progeny was
reevaluated using the scoring system that was applied in AIL mice (13).
The onset trait of the F2 and AIL mice were calculated previously (12,
13). Susceptibility is a qualitative trait, with a score of 0 and 1 for the
healthy and diseased mice, respectively.
Table II. ANOVA analysis of chromosomes with evidence of linkage in F2 and AIL
AIL mice
a
Chr.
Markers
Position (Mb)
F
p value
Markers
Position (Mb)
F
p value
6
D6Mit67
D6Mit328
97.7
112.7
2.92
5.52
0.053000
0.004400
D6Mit14
D7Mit228
145.6
47.3
1.35
2.69
0.261000
0.070900
D7Mit248
73
5.17
0.006200
D7Mit350
90.7
2.59
0.076000
D10Mit20
66.5
2.94
0.054000
D10Mit261
85.1
4.88
0.008100
D10Mit96
99.1
3.24
0.041000
D11Mit285
89.7
3.21
0.041700
D11Mit126
103.7
5
0.007300
D11Mit214
114.9
4.68
0.009900
D18Mit222
14.7
5.63
0.003900
D18Mit12
36
2.75
0.065000
D19Mit88
D19Mit90
37.3
42.2
5.32
6.58
0.005300
0.001600
D19Mit71
59.6
6.35
0.001900
D6Mit67
D6Mit328
D6MIt10
D6Mit329
D6Mit366
D6Mit115
rs51294806
rs6295683
rs30265977
rs50344715
D6Mit335
D6Mit14
D7Mit228
D7Mit229
D7Mit193
D7Mit83
D7Mit295
D7Mit88
D7Mit248
D7Mit122
D7Mit350
D7Mit183
D7Mit323
D10Mit20
D10Mit32
D10Mit186
D10Mit174
D10Mit132
D10Mit261
D10Mit94
D10Mit161
D10Mit41
D10Mit96
D10Mit70
D11Mit285
D11Mit70
D11Mit289
D11Mit145
D11Mit126
D11Mit58
rs27054829
rs27004424
rs27010185
D11Mit100
D11Mit214
D18Mit67
D18Mit222
D18Mit230
D18Mit22
D18Mit12
D19Mit40
D19Mit46
D19Mit88
D19MIt11
D19Mit38
rs31054271
D19Mit84
rs37383437
D19Mit71
rs46580758
97.7
112.7
113.2
114.1
115.2
116.7
117.6
118.5
119.7
123.5
127.5
145.6
47.3
52.9
57
59.1
63.6
67.3
73
82.4
90.7
101.6
108
66.5
69.1
75.3
75.7
83.6
85.1
87.7
90.2
93.7
99.1
103.5
89.7
93.9
94.7
97.5
103.7
104.4
105.5
106.4
107.4
110
114.9
12.1
14.7
17.8
25.1
36
25.4
32.7
37.3
42
47
53.1
56
57.3
59.6
60.9
0.63
3.56
3.95
3.96
5.96
8.7
6.12
5.77
2.51
1.12
2.78
0.47
0.94
2.13
1.66
1.39
1.52
1.49
1.94
0.76
0.96
0.58
1.26
1.53
1.91
1.08
0.54
0.56
1.47
0.32
0.83
0.12
0.97
0.348
0.47
1.75
0.84
3.2
0.59
0.81
0.86
0.14
2.54
3.41
1.7
0.93
0.91
1.65
1.2
0.6
1.74
1.21
1.62
1.18
0.09
0.94
1.27
1.83
4.57
2.83
0.531
0.029
0.02
0.02
0.00278
0.00021
0.00264
0.00345
0.082
0.327
0.0638
0.624
0.392
0.12
0.191
0.249
0.219
0.226
0.144
0.467
0.382
0.561
0.285
0.217
0.148
0.341
0.582
0.569
0.231
0.722
0.436
0.887
0.378
0.706
0.493
0.175
0.432
0.041
0.551
0.447
0.425
0.873
0.08
0.0344
0.183
0.396
0.404
0.193
0.302
0.548
0.176
0.297
0.197
0.308
0.91
0.389
0.28
0.161
0.011
0.06
7
10
11
18
19
a
Markers showing significant linkage are marked in bold.
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F2 mice
3018
FINE-MAPPING ARTHRITIS QTL
Markers and genotyping
To confirm and refine the suggestive QTLs identified in F2 progeny, we
genotyped the genomic regions containing those QTLs in AIL mice. Because the confidence intervals of QTLs in this AIL range from 4 to 12 Mb
(13, 14), we first selected genetic markers covering those genomic regions
with intermarker distance of ⬃5 Mb. Then, we increased the marker density for the genomic regions showing evidence of linkage ( p ⬍ 0.05).
Genotyping of AIL mice was performed on DNA extracted from tail tips
using PCR amplification for microsatellite markers as described previously
(12) or by a PCR-RFLP method for SNP markers. We genotyped 308 AIL
mice with 38 markers and we included 22 markers genotyped previously
(14). In total, 60 genetic markers (50 microsatellite markers and 10 SNP
markers) in six genomic regions were used for analysis.
Genotypes of all nonsynonymous SNPs in FVB/NJ and DBA/2J
strain and genotypes of part of the nonsynonymous SNPs in DBA/
1J strain were retrieved from the Mouse Phenome Database (http://
phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn ⫽ snps/door). We genotyped the DBA/1J strains with the nonsynonymous SNPs located in the
QTL confidence interval by sequencing. We designed primers to amplify
the genomic fragment comprising a SNP and sequenced the PCR product
directly.
Gene expression profiling
Linkage analysis
All linkage analyses were performed using QTX Map manager software
(18). The physical positions of the loci were obtained from Ensembl (http://
www.ensembl.org). The suggestive and significance linkage threshold values were determined by permutation tests (n ⫽ 500). In accordance with
the suggestion of Lander and Botstein (19), the confidence interval was
defined as the distance between points on each side of the peak of each
QTL at which the LOD score falls by 2.
Comparative genomic mapping
The comparative mapping was performed using HomoloGene orthology
predictions (http://www.ncbi.nlm.nih.gov/projects/homology/maps/) among
mice, rats, and humans. The genomic region of the confidence interval of the
mouse CIA QTL and its counterparts on rat and human genomes were
analyzed. The confidence intervals of rat arthritis QTLs are retrieved from
the original reports, and the confidence intervals of human genomic regions
were artificially defined as 10 Mb at both sides of the microsatellite markers that are linked to RA.
Results
Suggestive QTLs controlling CIA clinical traits in F2 mice
Previously, we performed a genome-wide linkage analysis using
(DBA/1 ⫻ FVB/N) F2 progeny to identify QTLs controlling CIA
(12). A genomic region on chromosome (chr.) 2 was significantly
linked to CIA clinical traits, e.g., severity, onset, and susceptibility.
This genomic region was named Cia2 (12, 20, 21). No other significant QTLs controlling CIA clinical traits were identified in the
F2 progeny; however, the Cia2 contributes only ⬃16% of the clinical variation. This suggests that some susceptibility genes with
relatively small effect on CIA failed to reach the significance linkage threshold due to the masking effect of Cia2. To test this hypothesis, we reanalyzed the F2 mice with suggestive significance
thresholds. Also, we revaluated CIA severity using a more sensi-
FIGURE 1. Cia3 in F2 and AIL mice. Development of CIA in F2 (A)
and in AIL (B). Mice were divided into three groups according to the
genotype of peak markers (D6Mit328 in F2 and D6Mit115 in AIL) of
Cia3, where aa, ab, and bb stand for FVB/N homozygous, heterozygous,
and DBA/1 homozygous alleles. C, Log-likelihood plot showing the
relationship between Cia3 and arthritis clinical traits. The horizontal
line indicates the significant threshold defined by the permutation test.
The filled bar indicates the 2-log confidence interval. The genetic markers used in study are indicated according to their physical positions.
tive scoring system. In addition to Cia2, six suggestive QTLs were
identified on chromosome 6, 7, 10, 11, 18, and 19, with LOD
scores ranging from 2.08 to 2.66. Four of these suggestive QTLs
control CIA severity, and two control CIA onset and susceptibility,
respectively (Table I). Interestingly, three of these QTLs overlap
with previously identified mouse CIA QTLs: they are Cia3 on chr.
6, Cia8 on chr. 10, and Cia40 on chr. 11 (12, 20 –23). These overlaps support the idea that susceptibility genes could be located in
those regions.
Confirming suggestive QTLs in AIL
To confirm the suggestive QTLs, we used an AIL that we generated previously (13). We genotyped 308 AIL mice with 60 genetic
markers covering the six QTLs. We then performed ANOVA analysis and QTL Linkage analysis for each marker. Table II summarizes the results of the ANOVA analysis. Among the six suggestive
Downloaded from http://www.jimmunol.org/ by guest on April 29, 2017
Previously, we detected gene expression profiling in lymph nodes of
DBA/1 and FVB/N mice on day 0 (before immunization), day 10, day 35
(onset phase), as well as day 95 (chronic phase) after CIA induction (15).
In this study, we analyzed the gene expression profiling in joint of DBA/1
and FVB/N mice on days 0 and 35 as well as in the thymus of the two
strains on day 0. Each group contained three mice. Analysis of gene expression was conducted using MOE 430A array (Affymetrix), interrogating
more than 22,000 genes according to procedures described previously (15,
16). The normalized expression values were imported to and analyzed by
Affymetrix dCHIP software (17). Differentially expressed genes were identified by defining the following filtering criteria in the dCHIP software: 1)
The fold change between the group means exceeded 2-fold; 2) The absolute difference between the two groups exceeded 100; and 3) The p-value
threshold of the unpaired t test was 0.05. The false discovery rate was
established with permutation test for each pairwise comparison to estimate
the proportion of false-positive genes.
The Journal of Immunology
3019
Table III. List of nonsynonymous gene polymorphism with Cia3a
Position (Mb)
Allele
Gene
SAP
DBA/2
FVB/N
DBA/1
rs13478988
rs31495179
rs31498257
rs31498687
rs30840549
rs30121304
rs31551252
rs31551918
rs31553514
rs31549850
rs37185903
rs36953372
rs37558791
rs37667924
rs31557206
rs31557210
rs31557212
rs31563226
rs31562616
rs31574016
rs31574018
rs31570654
rs31571560
rs31573792
rs31572531
rs30363420
rs31576356
rs29873127
rs31578998
rs31579689
rs31580309
rs31576214
rs31579954
rs31661638
115.5
115.7
115.7
115.7
115.8
116.4
116.5
116.5
116.5
116.5
116.5
117.4
117.8
117.8
118.4
118.4
118.4
118.4
118.4
118.5
118.5
118.5
118.5
118.5
118.5
118.5
118.5
118.5
118.5
118.5
118.5
118.5
118.6
119.3
C/G
C/T
A/T
A/G
C/T
C/T
A/G
G/T
A/G
G/T
C/T
C/G
C/G
A/C
A/C
C/T
C/G
A/G
A/T
G/T
C/G
C/T
A/G
G/T
C/T
C/T
C/T
A/T
C/G
C/T
C/T
C/G
G/T
G/T
2510049J12Rik
Tmem40
Tmem40
Tmem40
Mbd4
Alox5
Olfr212
Olfr212
Olfr212
Olfr213
Olfr215
LOC100043777
Zfp239
Zfp239
Bms1
Bms1
Bms1
Bms1
Zfp248
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Ankrd26
Cacna1c
Lrtm2
R101G
T118A
Q31H
S3L
N128D
I645V
Q15R
V56L
Q239R
V56L
I213V
F2L
T56S
R127S
G637V
E550G
A520G
A364V
T306S
E1664D
P1657A
Y1509C
A1450V
Q1363H
R848G
T784A
N449S
T425S
H398D
I217M
N113S
H36Q
N1769T
I35L
C
T
A
G
T
T
A
T
A
T
T
C
C
C
A
T
C
G
T
G
C
T
G
G
C
T
C
A
G
C
C
G
G
T
G
C
T
A
C
C
G
G
G
G
C
G
G
A
C
C
G
A
T
T
G
C
A
T
T
C
T
T
C
T
T
C
G
G
C
T
A
G
T
T
A
T
A
T
T
C
C
C
C
C
C
G
A
G
C
T
G
G
C
T
C
A
G
C
C
G
T
T
a
Data obtained from the Mouse Phenome Database.
QTLs, only the QTL on chr. 6 (Cia3) showed significant linkage to
CIA. The peak marker of Cia3 is D6Mit115 (F ⫽ 8.7, p ⫽
0.00021), which is 3.9 Mb away from the peak marker in F2 mice.
The other five genomic regions failed to reach a significant level of
linkage, although the peak markers on chr. 11 and chr. 19 showed
slight linkage ( p ⫽ 0.034 and p ⫽ 0.011, respectively). Therefore,
Cia3 has been confirmed as a CIA QTL in the DBA/1 ⫻ FVB/N
cross.
Fine mapping of Cia3
Cia3 was originally identified in (DBA/1 ⫻ SWR) F2 mice, with
the peak marker of D6Mit10 located at 113.2 Mb on chr. 6 (21).
Our study confirmed this QTL in an independent cross. The peak
markers of Cia3 in our F2 and AIL are D6Mit328 (112.7 Mb) and
D6Mit115 (116.6 Mb), respectively. In both F2 and AIL, the
FVB/N allele enhance the disease in an additive manner (Fig. 1, A
and B). The confidence interval of Cia3 in both (DBA/1 ⫻ SWR)
F2 and (DBA/1 ⫻ FVB/N) F2 mice are ⬎40 Mb. Using AIL, we
refined Cia3 into a 5.6 Mb genomic region with flanking markers
of D6Mit329 and rs30265977 (Fig. 1C).
Nonsynonymous polymorphism within Cia3
A quantitative trait gene polymorphism is either a nonsynonymous polymorphism changing protein structure or a regulatory
sequence variation affecting gene expression. Therefore, identification of the nonsynonymous polymorphisms between parental strains could aid in the search for candidate genes. However, Cia3 is located in a high gene-density genomic region,
containing 77 genes. Sequencing all the coding region of those
genes is time and resource consuming. Fortunately, 16 common
mouse inbred strains have been recently sequenced and 8.4 million SNPs have been identified among them (11). FVB/NJ and
DBA/2J strains are among those 16 strains. DBA/1J and
DBA/2J are substrains, and the confidence interval of Cia3 is an
identical by descent chromosomal region between the two substrains. Therefore, searching for nonsynonymous SNPs between
Table IV. List of differentially expressed genes within Cia3
Thymus
LN
Joint
Gene
Position (Mb)
Probe ID
Day 0
Day 0
Day 10
Day 35
Day 95
Day 0
Day35
Timp4
Cacna1c
115,2
118,5
1423405_at
1421297_a_at
⫺
Down (⫺4.0)
⫺
⫺
⫺
⫺
⫺
⫺
Up (2.7)
⫺
Up (2.1)
⫺
⫺
⫺
⫺, not differentially expressed; Up, with higher expression in DBA/1 strain; Down, with lower expression in DBA/1 strain; ( ), fold change.
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SNP
3020
FINE-MAPPING ARTHRITIS QTL
DBA/2J and FVB/N in this region could help to identify the
polymorphisms between DBA/1J and FVB/N strains. In total,
32 nonsynonymous SNPs were found between DBA/2J and
FVB/NJ strains. Genotyping DBA/1J strain with those SNPs
showed that DBA/1J shared the same alleles with DBA/2J strain
in 30 of 32 SNPs. An additional two nonsynonymous SNPs that
are not polymorphic between FVB/N and DBA/2J strains were
found to be polymorphic between DBA/1 and FVB/N strains.
Taken together, 32 nonsynonymous SNPs in 14 genes were
identified between DBA/1 and FVB/N strains (Table III).
gene expression in the thymus and joints of both strains. In total,
1312 genes were differentially expressed between DBA/1 and
FVB/N strains in one or more tissues (Supplementary Table I).4
Two differentially expressed genes are located within the confidence interval of Cia3. One is tissue inhibitor of metalloproteinases 4 (Timp4), with higher expression in DBA/1 strain than in
FVB/N strain in lymph nodes during chronic phase and in the joint
before immunization. The other gene is voltage-dependent L-type
calcium channel subunit ␣-1C (Cacna1c), with lower expression in
DBA/1J than FVB/NJ in the thymus (Table IV).
Gene expression profiling
Comparative genomic mapping of Cia3
When a quantitative trait gene polymorphism is a sequence variation regulating the expression of a gene, the gene should be differentially expressed between parental strains in disease-related
tissue in a certain phase of the disease. Therefore, defining the
gene expression profile in disease related tissues could aid in the
search for candidate genes. Previously, we performed gene expression profiling on lymph nodes of DBA/1 and FVB/N strains at four
disease phases during the development of CIA (15). To complete
the gene expression profiling in CIA related tissues, we detected
When a genomic region and its counterparts in multiple species are
identified as QTLs controlling diseases, it indicates that a common
susceptibility gene might exist in multiple species. Based on this
hypothesis, comparative mapping among multiple species could be
performed to refine a QTL and to select candidate genes. For the
refined 5.6 Mb confidence interval of mouse Cia3, the counterpart
4
The online version of this article contains supplementary material.
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FIGURE 2. Comparative mapping of genomic regions containing Cia3 among mouse, rat, and human. The comparative maps are calculated using
HomoloGene orthology predictions (http://www.ncbi.nlm.nih.gov/projects/homology/maps/) for the mouse Cia3. The physical positions of the chromosome
are presented in Mb. The confidence intervals of arthritis QTLs and human genomic regions linked to the RA are present as black bars. The confidence
intervals of rat Pia7 and Cia13 are from the original reports, and the confidence intervals of the human genome are artificially defined as 10 Mb at both
side of the microsatellite markers that are linked to RA. Fifty-seven known genes within Cia3 are depicted in the figure according to their physical positions.
The gray areas indicate the homologous region between mouse and rat as well as between mouse and human.
The Journal of Immunology
3021
Table V. List of genes located in arthritis QTLs in mouse, rat, and human
Position (Mb)
Gene
Description
Rat
Position (Mb)
Human
Position (Mb)
Chr. 6
114.1
114.2
114.3
114.6
114.8
115.0
115.1
115.2a
115.4
115.5
115.6
115.6
115.7
115.7
115.8
115.8
115.8
115.9
115.9
115.9
116.0
119.1
118.5
Slc6a11
Slc6a1
Hrh1
Atg7
Vgll4
1500001M20Rik
Syn2
Timp4
Pparg
Tsen2
Mkrn2
Raf1
Tmem40
Cand2
BC060267
Mbd4
Ift122
Rho
H1foo
Plxnd1
Tmcc1
Dcp1b
Cacna1c
Chr. 4
150.1
150.2
150.4
150.8
151.0
151.1
151.3
151.4
151.6
151.7
151.7
151.8
151.9
151.9
151.9
152.0
152.0
152.1
152.1
152.1
152.1
155.5
154.9
Chr. 3
10.8
11.0
11.2
11.3
11.6
11.8
12.0
12.2
12.3
12.5
12.6
12.6
12.8
12.8
130.6
130.6
130.6
130.7
130.7
130.8
130.8
1.9
2.0
119.2
119.3
119.4
119.3
119.4
119.5
Cacna2d4
Adipor2
Wnt5b
Lrtm2
Fbxl14
Erc1
solute carrier family 6, member 11
solute carrier family 6, member 1
histamine receptor H 1
autophagy-related 7 (yeast)
vestigial like 4 (Drosophila)
RIKEN cDNA 1500001M20 gene
synapsin II
tissue inhibitor of metalloproteinase 4
peroxisome proliferator activated receptor gamma
tRNA splicing endonuclease 2 homolog
makorin, ring finger protein, 2
v-raf-leukemia viral oncogene 1
transmembrane protein 40
cullin-associated and neddylation-dissociated 2
cDNA sequence BC060267
methyl-CpG binding domain protein 4
intraflagellar transport 122 homolog
rhodopsin
H1 histone family, member O, oocyte-specific
plexin D1
transmembrane and coiled coil domains 1
DCP1 decapping enzyme homolog b
calcium channel, voltage-dependent, L type,
alpha 1C subunit
calcium channel, voltage-dependent, ␣2/␦ subunit 4
adiponectin receptor 2
wingless-related MMTV integration site 5B
leucine-rich repeats and transmembrane domains 2
F-box and leucine-rich repeat protein 14
ELKS/RAB6-interacting/CAST family member 1
a
Chr. 12
155.5
155.7
155.7
155.6
155.8
155.9
1.8
1.7
1.6
1.8
1.5
1.0
Genes carrying non-synonymous polymorphism(s) or differentially expressed between DBA/1 and FVB/N strains are written in bold letters.
on the rat genome is a 6 Mb (150 –156 Mb) genomic region on
chromosome 4. The peaks of two rat arthritis QTLs, Pia7 and
Cia13, are located in this region (24, 25) (Fig. 2). The counterpart
of Cia3 on the human genome is mapped to three chromosomes:
chromosome 3, 10, and 12. The counterpart genomic regions on
chromosome 3 and 12 have been showed to be linked to RA (26 –
28) (Fig. 2). When comparative mapping was performed, 29 of the
77 genes within the Cia3 confidence interval were presentein
genomic regions linked to arthritis in all three species (Table V).
Among the 29 genes, five genes carry nonsynonymous polymorphism or are differentially expressed between DBA/1 and FVB/N
strains. These are Timp4, Tmem40, Mbd4, Cacna1c, and Lrtm2.
Recently, two genome-wide association studies have been reported
(29, 30), providing an additional novel resource for comparative
genome mapping. In these two published reports, we looked into
the association of SNPs located within counterpart genomic regions of Cia3 on human genome with RA. However, no significant
association was observed.
Discussion
The aim of a significance threshold applied to QTLs is to decrease
the false-positives to a reasonable level, with the drawback of increasing the false negatives (19). Therefore, QTLs identified in a
F2 progeny need to be confirmed in other populations, e.g., congenic strains and AIL. In this study, we used an AIL progeny to
confirm and refine suggestive QTLs identified in F2 progeny. One
of six suggestive QTLs, Cia3, was confirmed in the AIL and refined into a 5.6 Mb genomic region. This is one practical example
of a false negative QTL in F2 mice. Besides Cia3, two other suggestive QTLs, on chr. 11 (Cia40) and on chr. 19, warrant discussion. Although not significant, there is slight linkage with the same
susceptibility alleles in AIL as in F2 (data not shown). This indi-
cates that the two genomic regions might contain small effect CIA
susceptibility genes which remain to be confirmed.
Cia3 was identified in a previous study with an independent
population of (DBA/1 ⫻ SWR) F2 progeny (21). In our study, the
allele from the resistant strain, FVB/N, enhanced susceptibility to
CIA. The susceptible allele of Cia3 in (DBA/1 ⫻ SWR) F2 progeny was not shown. However, SWR mice with Cia2 congenic
fragment from DBA/1 strain showed higher susceptibility to CIA
than DBA/1 mice, indicating that the SWR strain carries CIA susceptibility allele(s) (21). In both F2 and AIL mice, Cia3 affects
CIA severity, onset, and susceptibility. However, the magnitudes
of the effects on individual traits vary between F2 and AIL, with
highest effect on severity in F2 and highest effect on susceptibility
in AIL.
We refined Cia3 into a 5.6 Mb genomic region which contains 77 genes, including 57 known genes and 20 predicted or
hypothetical genes. This relatively small genomic region allowed us to realistically search for the candidate genes. To select the candidate genes, we performed gene expression profiling in disease-related tissues at different phases of the disease.
We also searched for the nonsynonymous polymorphisms in
coding sequence. In total, we identified 15 genes that are either
differentially expressed or carry nonsynonymous polymorphism(s) between the parental strains. These genes could be
considered as candidate genes for further studies. However, this
gene list is not conclusive for two reasons. First, we identified
nonsynonymous polymorphisms between FVB/N and DBA/1
strains by initially comparing FVB/N and DBA/2 strains.
DBA/1 and DBA2 are substrains and the confidence interval of
Cia3 is an identical by descent region. Sequences in the
genomic region of the two substrains should be identical, with
exception of polymorphisms occurring after the separation of
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Mouse
3022
Acknowledgments
We thank Ilona Klamfuss for taking care of the animals.
Disclosures
The authors have no financial conflict of interest.
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