The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rigby, R. J.
Right arrow Articles by Vyse, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rigby, R. J.
Right arrow Articles by Vyse, T. J.
Right arrowPubmed/NCBI databases
*Gene
Medline Plus Health Information
*Lupus
The Journal of Immunology, 2004, 172: 4609-4617.
Copyright © 2004 by The American Association of Immunologists

New Loci from New Zealand Black and New Zealand White on Chromosomes 4 and 12 Contribute to Lupus-Like Disease in the Context of BALB/c1

Robert J. Rigby2,*, Stephen J. Rozzo2,{ddagger}, Joseph J. Boyle{dagger}, Margarita Lewis*, Brian L. Kotzin{ddagger} and Timothy J. Vyse3,*

* Rheumatology Section and {dagger} Department of Histopathology, Imperial College Faculty of Medicine, Hammersmith Campus, London, United Kingdom; and {ddagger} Departments of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
New Zealand Black (NZB) and New Zealand White (NZW) mice are genetically predisposed to a lupus-like autoimmune syndrome. To further define the loci linked to disease traits in NZB and NZW mice in the context of the BALB/c genetic background, linkage analyses were conducted in two crosses: (NZW x BALB/c.H2z)F1 x NZB and (NZB x BALB/c)F2. Novel loci linked to autoantibody production and glomerulonephritis, present in both NZB and NZW mice, were identified on proximal chromosomes 12 and 4. The chromosome 12 locus showed the strongest linkage to anti-nuclear Ab production. Additionally, a number of other novel loci linked to lupus traits derived from both the New Zealand and non-autoimmune BALB/c genomes were identified. Furthermore, we confirm the linkage of disease to a number of previously described lupus-associated loci, demonstrating that they are relatively background independent. These data provide a number of additional candidate gene regions in murine lupus, and highlight the powerful effect the non-autoimmune background strain has in influencing the genetic loci linked to disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE)4 is a clinically heterogeneous autoimmune disease characterized by the production of autoantibodies to a number of nuclear and cell surface Ags. Susceptibility to, and progression of, SLE is believed to result from a combination of genetic, environmental, and stochastic factors. The New Zealand Black (NZB), New Zealand White (NZW), (NZB x NZW)F1 hybrid, and NZM2410 (a recombinant inbred strain derived from an (NZB x NZW)F2 intercross) are the most widely studied murine models of SLE. All of the above mice, to varying degrees, exhibit anti-nuclear Ab production and immune complex glomerulonephritis (GN), with disease severity biased toward females. The New Zealand model develops spontaneous disease and hence is an excellent model to study the genetic contribution to human SLE. A number of linkage analyses have been conducted in both the New Zealand mice and other murine models of SLE. These studies have uncovered multiple genetic loci linked with expression or suppression of various SLE traits ( 1, 2). The same loci have been identified in a number of independent crosses, and some regions, such as loci on chromosomes 1, 4, 7, and 17 seem to be common between lupus-prone strains ( 3).

It is evident from many studies in both humans and mice that the phenotype of a disease-causing gene can depend on the genetic background ( 4). The effects of background-specific modifier genes are often observed in single-gene-modified mice, such as knockouts or transgenics. Studies in MRL/lpr mice crossed with various background strains have demonstrated that the linkage of disease in a complex trait, murine SLE, can be affected by the background strain ( 5, 6, 7, 8). Additionally, work by Rozzo et al. ( 9) studying the effect of genetic background on the contribution of NZB loci to autoimmune lupus nephritis clearly demonstrated disparate background-specific linkage patterns between crosses of NZB and C57BL/6 or BALB/c. In this paper, we investigate the influence of the BALB/c genetic background on the development or inhibition of mouse SLE, and how the background alters the identity of lupus susceptibility loci from New Zealand mice. Linkage analysis to SLE traits in two cohorts of mice, an (NZW x BALB/c.H2z)F1 x NZB backcross (W-BC) and a (NZB x BALB/c)F2 intercross (B-F2) was conducted. We present data to confirm that the BALB/c genetic background can both alter the New Zealand loci associated with murine SLE and directly contribute to disease pathogenesis, and describe several novel loci associated with SLE disease traits.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

(NZB x BALB/c)F2 cohort. NZB/BINJ (NZB) and BALB/cByJ (BALB/c) mice were purchased from Harlan Olac (Bicester, Oxfordshire, U.K.) and maintained in the Biological Services Unit of Imperial College Faculty of Medicine (London, U.K.). These mice were crossed, and the resulting F1 progeny intercrossed to produce the (NZB x BALB/c)F2 cohort (B-F2; n = 222 female mice). Additionally, control NZB, NZW, and BALB/c female mice were obtained from the same source and studied in parallel to the B-F2 cohort.

(NZW x BALB/c.H2z)F2 x NZB cohort. NZB/BINJ, NZW/LacJ, and BALB/cByJ mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the animal care facilities at the National Jewish Center for Research and Medicine and University of Colorado Health Sciences Center (Denver, CO). BALB/cByJ mice congenic for the NZW H2z locus were obtained from a cohort maintained as above. Briefly, BALB/cJ mice were crossed with NZW and backcrossed to BALB/cJ for 12 generations, with the inheritance of the H2z locus determined by both the presence of a simple sequence length polymorphism in the TNF-{alpha} gene and expression of I-Az ( 9). These mice were crossed to produce a backcross cohort, (NZW x BALB/c.H2z)F1 x NZB (W-BC; n = 126 female mice).

Genetic mapping using simple sequence length polymorphisms

Oligonucleotides flanking microsatellite repeat regions polymorphic between NZB and BALB/c (B-F2) or NZW and BALB/c (W-BC) were used to amplify genomic DNA in a standard 35-cycle PCR. The resulting PCR product was electrophoresed on polyacrylamide gels (MiniProtean II electrophoresis system; Bio-Rad, Hemel Hempstead, U.K.) at 2.25 V/mm for 90–120 min, stained with ethidium bromide solution, viewed under UV light, and digitally photographed.

An autosome-wide map, at an average marker distance of <20 cM, was created for both cohorts. Marker positions were based on data from the Mouse Genome Informatics (MGI) database (www.informatics.jax.org).

Sample collection and phenotyping

The B-F2 cohort was bled from the tail every 2 mo, from 6 mo until 14 mo of age. The W-BC cohort was bled from the tail at 7 and 9 mo of age. Blood was incubated at 37°C for 30 min and centrifuged for 10 min at 13,500 rpm at room temperature, and the serum fraction was removed. Samples were stored at -70°C. Urine was tested for proteinuria with Combur3 urine dipsticks (Roche Diagnostics, Lewes, U.K.) every month between 6 and 14 mo of age in the B-F2 cohort and every month between 6 and 12 mo of age in the W-BC cohort. Mice were sacrificed at 14 mo of age (B-F2) or 12 mo of age (W-BC), when a proteinuria level of >=5 g/L (3+) at one time point, or a proteinuria level of 1g/L (2+) for two consecutive time points occurred.

ELISA for serum autoantibody and total Ig levels

Abs to ssDNA and chromatin were assayed as previously described ( 10). Briefly, Nunc Maxisorb plates (VWR, Poole, U.K.) were incubated at 4°C overnight with calf thymus ssDNA at 10 ng/µl (Sigma-Aldrich, Poole, U.K.) or calf thymus chromatin at 500 ng/µl (Lorne Laboratories, Reading, U.K.) and postcoated with PBS-gelatin (0.8 mg/ml). Serum samples were assayed at 1:300 or 1:600, along with a descending log2 range of seven standards (pooled (NZB x NZW)F1 serum). Following washing, bound Ab was detected by incubation with anti-IgG HRP, anti-IgG1-HRP, or anti-IgG2a-HRP (Serotec, Oxford, U.K.) at room temperature for 1 h. ABTS (Sigma-Aldrich) was added postwashing, and the absorbance at 405 nm was assayed after ~1 h. Concentrations of Ab were determined by assigning the standard curve samples nominal values (100–1.56) and plotting absorbance vs value with a third-order polynomial equation. Unknown values were then calculated from the standard curve.

Abs to dsDNA were assayed as previously described ( 11). Briefly, Nunc Immunosorb plates were coated with 1 µg/ml streptavidin (Sigma-Aldrich), incubated at 4°C overnight, and postcoated with 0.8 mg/ml gelatin. {phi}X174 double-stranded plasmid DNA (Promega, Southampton, U.K.) was biotinylated with Photoprobe biotin (Vector Laboratories, Peterborough, U.K.) and added to the streptavidin plate at 200 ng/ml and incubated overnight at 4°C. The plates were postcoated with 0.8 mg/ml gelatin, and serum samples were assayed as above but in triplicate, with one well per sample containing no dsDNA to allow determination of any nonspecific binding to streptavidin. Analysis was conducted as above. Serum IgM or IgG was assayed using a capture ELISA. Nunc Immunosorb plates were coated with anti-mouse IgM or IgG (DAKO, Ely, U.K.), postcoated with 0.8 mg/ml gelatin, and incubated with serum at 1:10,000 along with a standard curve of IgM or IgG (Serotec) at log2 dilutions from 4 µg/ml. Following washing, bound IgM or IgG was detected by adding anti-Ig-HRP (DAKO) for 1 h at room temperature. ABTS was added postwashing, and the absorbance at 405 nm was assayed after ~1 h, and concentrations of IgM or IgG were determined by plotting absorbance vs IgM or IgG concentration with a third-order polynomial equation. Unknown values were then calculated from the standard curve.

All genome mapping and serological assays on mice maintained at the University of Colorado Health Sciences Center were identical with those conducted at Imperial College, with reagents being obtained from the equivalent U.S. suppliers.

Histology

Kidneys from the B-F2, NZB, NZW, and BALB/c cohorts were fixed in Bouin’s solution (three parts saturated picric acid to one part 40% formaldehyde and 5% glacial acetic acid; all VWR) for 2–4 h, transferred to 70% ethanol, processed overnight, and embedded in paraffin wax. Two-micrometer sections were cut and stained with H&E. Kidneys were assessed by J.J.B. using the criteria shown in Table I.


View this table:
[in this window]
[in a new window]
  		Table I. Criteria for assessment of kidneys

 
The trait total histology was two times the glomerular cell score plus the scores for the other traits. Kidney sections from the W-BC cohort were unavailable for study.

Statistical analysis

All linkage analyses and interval maps were conducted using Map Manager QTb29 (Ref. 12 ; http://mapmgr.roswellpark.org). Marker maps were generated to determine the accuracy of genotyping, typically the markers mapping to within ±1–3 cM of the published marker position (MGI; www.informatics.jax.org). All centimorgan positions of markers referred to in this study are those published by MGI. Thresholds for suggestive, significant, and highly significant linkage were based on the results of 1000 permutations for a given trait and cohort, again calculated using Map Manager QTb29. Typically, a log of the odds ratio (LOD) of >=2.1 was indicative of suggestive linkage, LOD of >=3.5 was indicative of significant linkage, and LOD of >=5.2 was indicative of highly significant linkage in the B-F2 cohort; and LOD of >=1.3 was indicative of suggestive linkage, LOD of >=2.5 was indicative of significant linkage, and LOD of >=4.0 was indicative of highly significant linkage in the W-BC cohort. A {chi}2 analysis was used to analyze the linkage of proteinuria in the W-BC cohort.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
NZB and NZW mice are genetically predisposed to the development of a spontaneous SLE-like autoimmune syndrome. To determine disease-modifying loci in NZB and NZW mice in the context of a BALB/c background both (NZB x BALB/c)F2 intercross (B-F2) and (NZW x BALB/c.H2z)F1 x NZB backcross (W-BC) mice were studied.

Linkage to a novel NZB and NZW region on chromosome 12

Interval mapping demonstrated the linkage of serological and histological SLE traits to regions on chromosome 12 in both the B-F2 and W-BC cohorts (Table II and Fig. 1). In the B-F2 cohort;T2,F1> serum IgG anti-chromatin Abs (p = 1.0 x 10-4), IgG1 anti-chromatin Abs (p = 1.7 x 10-4), IgG2a anti-chromatin Abs (p = 1.x10-5), IgG anti-dsDNA Abs (p = 2.9 x 10-3), IgG anti-ssDNA Abs (p = 1.8 x 10-5), and total serum IgG (p = 1.7 x 10-3) were linked to a region on proximal chromosome 12 with a peak linkage between 2.3 and 4.8 cM from the centromere. Interestingly, all of these traits, except IgG1 anti-chromatin Abs, demonstrated a bimodal linkage pattern, with an additional peak at a more distal region of chromosome 12. This more distal peak varied in both position and significance between traits, but was in a region between 27 and 42 cM from the centromere.


View this table:
[in this window]
[in a new window]
  		Table II. Linkage to SLE disease traits on chromosome 12 in the B-F2 cohort

 


View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. Linkage of IgG anti-chromatin Abs (solid line) and histological GN (dotted line) to chromosome 12 in B-F2 mice. Threshold for significant (dashed line) linkage, as determined by 1000 cross- and trait-specific permutation tests, is indicated (LOD = 3.6).

 
In the W-BC cohort, IgG anti-chromatin (p = 5.9 x 10-4), IgG2a anti-chromatin (p = 1.5 x 10-3), IgG anti-ssDNA (p = 1.1 x 10-3), and IgG anti-dsDNA (p = 7.3 x 10-3) were all linked to proximal chromosome 12, albeit at more variable positions than in the F2 cohort (peak linkage at 19, 19, 6–6.5, and 10–11.5 cM, respectively). The variation in position of these linkage data probably reflects the lower numbers and recombinations in the W-BC. None of the serology traits in the W-BC exhibited a bimodal linkage pattern.

Histological evidence of renal damage was also linked to proximal chromosome 12 in the B-F2 cohort (Table II and Fig. 1). Total renal histopathology (p = 4.5 x 10-5), glomerular cell damage (p = 6.8 x 10-5), and tubulointerstitium matrix damage (p = 7.8 x 10-3) were all linked to a region 3.5 cM from the centromere, with a bimodal linkage pattern observed in linkage to glomerular cell damage, the second peak being around 28.5 cM. In the W-BC mice, severe proteinuria was linked to a marker (D12Nds11) at a position of 6 cM from the centromere (p = 5.5 x 10-4). No histological data were available for this cross. However, we have observed a correlation between the development of severe proteinuria and histological GN in the B-F2 cohort (rs = 0.44; p < 1.0 x 10-4) and in previous investigations (T. J. Vyse and B. L. Kotzin, unpublished observations).

Linkage to a novel NZB and NZW region on proximal chromosome 4

A region on proximal chromosome 4 was linked to total serum IgM (p = 9.6 x 10-3) and IgG (p = 1.1 x 10-3) in the B-F2 cohort at ~9.3 and 15.6 cM from the centromere, respectively. A trend for linkage in the B-F2 cohort of IgG2a anti-chromatin Abs and IgG anti-ssDNA Abs to proximal chromosome 4 was also observed, with the p values just outside than those indicating suggestive linkage (p = 1.6 x 10-2 and 7.8 x 10-2, respectively). Histological evidence of renal damage in the B-F2 cohort was also linked to proximal chromosome 4. The peak linkage on proximal chromosome 4 to total histopathology was at 21.9 cM (p = 6.8 x 10-5), the peak linkage to glomerular cell damage was at 20.9 cM (p = 6.8 x 10-5), and the peak linkage to glomerular matrix damage was at 19.8 cM (p = 4.5 x 10-5) (Fig. 2, Table III).



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 2. Linkage of total histopathology in B-F2 (A), total serum IgG in B-F2 (B), and IgG anti-dsDNA Abs in W-BC (C) to chromosome 4. Thresholds for suggestive (dotted line) and significant (dashed line) linkage, as determined by 1000 cross- and trait-specific permutation tests, are indicated. LOD >= suggestive linkage in A and B = 2.1; LOD >= significant linkage in A = 3.5. LOD >= suggestive linkage in C = 1.3; LOD >= significant linkage in C = 2.5.

 

View this table:
[in this window]
[in a new window]
  		Table III. Linkage of SLE traits to additional novel New Zealand loci

 
Linkage to proximal chromosome 4 was also observed in the W-BC cohort, with IgG anti-dsDNA (p = 4.5 x 10-4), IgG anti-ssDNA (p = 1.1 x 10-3), and IgG2a anti-chromatin (p = 8.2 x 10-3) autoantibodies all being linked to a region peaking at 2–3 cM from the centromere (Fig. 2, Table III).

Linkage to a novel BALB/c-derived region on distal chromosome 9

Linkage of both serological and histological SLE traits was observed on chromosome 9 (Table IV). This linkage was only observed in the B-F2 cohort, and was consistently associated with BALB/c homozygosity and was observed only in the B-F2 cohort. IgG anti-chromatin Abs (p = 3.9 x 10-3), IgG1 anti-chromatin Abs (p = 3.9 x 10-3), IgG2a anti-chromatin Abs (p = 8.2 x 10-3), IgG anti-ssDNA (p = 3.2 x 10-5), IgG anti-dsDNA (p = 1.3 x 10-4), and total serum IgM (p = 3.5 x 10-3) were all linked to an area on distal chromosome 9, in a region around 48–59 cM from the centromere. Of the serological traits, only IgG2a anti-chromatin exhibited a bimodal linkage pattern, with an additional peak at a region around 31 cM (p = 9.6 x 10-4) from the centromere.


View this table:
[in this window]
[in a new window]
  		Table IV. Linkage of SLE disease traits to BALB/c-derived regions on chromosome 9 in B-F2 mice

 
Histological evidence of renal damage was also linked to chromosome 9 in the B-F2 cohort and again consistently associated with BALB/c homozygosity (Table IV). There is a clear disparity of linkage between glomerular and tubulointerstitial traits. Glomerular cell and matrix damage was linked to a region spanning 15–40 cM from the centromere (Fig. 3A), whereas tubulointerstitial cell and matrix damage was linked to a more distal region, at around 58–61 cM from the centromere (B). This is within the same region associated with the serological traits described above. No linkage to chromosome 9 was observed in the W-BC cohort; however, it is emphasized that the design of the W-BC cohort did not allow for BALB/c homozygosity.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. Linkage to BALB/c-derived regions on chromosome 9 to tubulointerstitium cell (solid line) and matrix (dashed line) damage (A) and glomerular cell (solid line) and matrix (dashed line) damage (B). Thresholds for suggestive (dotted line) linkage, as determined by 1000 cross- and trait-specific permutation tests, are indicated (LOD = 2.1).

 
Linkage to additional BALB/c alleles

In addition to those on chromosome 9, other transgressive loci were linked to SLE traits (Table V). Two regions on chromosome 8 were mapped in the B-F2 cohort, one at around 16–18 cM linked with anti-dsDNA IgG (p = 7.1 x 10-4) and anti-ssDNA IgG (p = 5.8 x 10-4) autoantibodies, and another, more distal, locus linked to total serum IgG levels (p = 2.6 x 10-3). These loci are recessive and dominant, respectively, and therefore may represent two separate loci. Furthermore, BALB/c alleles on proximal chromosome 16 were associated with serum IgG levels in both the B-F2 and W-BC cohorts. The peaks of linkage in the two crosses are around 5 cM apart, and may be due to the same gene or genes. Interestingly, a locus (Lprm5) that is linked to anti-dsDNA Ab levels has been mapped to this region in MRL mice ( 8).


View this table:
[in this window]
[in a new window]
  		Table V. Linkage of SLE traits to BALB/c-derived alleles on chromosomes 8 and 16

 
Additional new SLE susceptibility loci in New Zealand mice

A number of other loci were linked with SLE traits in the B-F2 and/or W-BC cohorts apparent in New Zealand mice in the context of the BALB/c genetic background and not observed in previous crosses on the C57BL/6 background (Table III). A region on distal chromosome 4, with a linkage peak at 79 cM, was strongly associated with total IgG levels in the W-BC. This locus is around 10 cM distal from the previously mapped cluster of SLE susceptibility loci on distal chromosome 4 ( 10, 13, 14, 15, 16, 17). Linkage was also observed on medial chromosome 6 to anti-dsDNA IgG in the W-BC and distal chromosome 7 to anti-dsDNA and ssDNA IgG in the B-F2 cohort.

Linkage to genomic regions previously reported to harbor SLE susceptibility loci

Our current study also confirmed a number of NZB and NZW loci previously described as being associated with SLE traits, suggesting that they are background independent. All of these regions are detailed and referenced in Table VI, and include loci mapped in other lupus-prone mice, such as MRL and BXSB. We confirmed the linkage of a distal chromosome 1 region colocalizing with Nba2, Sle1, Lbw7, and Bxs3, to serum IgM in the B-F2 cohort and IgG anti-dsDNA and anti-chromatin Abs in the W-BC cohort. Additionally, IgG anti-dsDNA and anti-chromatin Abs showed a weak trend for linkage to this region in the B-F2 cohort, with p values close to the threshold for suggestive linkage (p = 3.5 x 10-2 and 1.5 x 10-2, respectively).


View this table:
[in this window]
[in a new window]
  		Table VI. Linkage to SLE traits in B-F2 and W-BC cohorts in the vicinity of previously described SLE-modifying regions

 
Mid-chromosome 3 was significantly linked to total serum IgM in the W-BC, with a weak trend for linkage to this region in the B-F2 cohort (p = 3.3 x 10-2). This region colocalizes with the SLE-modifying regions Sles3 and Bxs5.

Mid-chromosome 4, colocalizing with Nba1, Sle2, Adaz1, and Lbw2, was strongly linked with both histological GN traits (Fig. 2) and total serum IgM (p = 1.0 x 10-9) in the B-F2 cohort. Additionally, weaker linkage to IgG anti-ssDNA Abs (p = 3.0 x 10-3) and IgG anti-dsDNA (p = 7.1 x 10-3) was observed to this region; the peak p value for all traits was centered 50–55 cM from the centromere. No strong linkage was observed in the W-BC cohort to this region; however, total serum IgM showed a weak trend for linkage in the region of Sle2 (p = 2.0 x 10-2).

Mid-chromosome 5 was linked to IgG and IgG2a anti-chromatin Abs in the B-F2 cohort, the region identified in this study mapping around 10 cM distal of the NZM2410 disease locus Sle6. Distal chromosome 5 was linked to both IgG anti-ssDNA and dsDNA Abs in the W-BC cohort. This linkage region colocalized with the locus Lbw3, an NZW locus linked to mortality in (NZB x NZW)F2 mice.

A region of proximal chromosome 7 rich in SLE-associated loci (Lrdm1, Yaail, Sle3, Lwb5, and Lmb3) was linked to total serum IgM in the B-F2 cohort and IgG anti-ssDNA Abs in the W-BC. Distal chromosome 10 (Swrl4, Lmb4, and Sle12) was associated with both total serum IgG and IgG anti-ssDNA Abs in the B-F2 cohort, and mid-chromosome 11 (Lbw8, Sle13) was linked to total serum IgM in the B-F2 cohort. A locus on distal chromosome 17, linked to anti-chromatin IgG1, was observed in the B-F2 cohort. This colocalizes with the locus Agnz2, although the traits linked are quite disparate ( 15).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have investigated the genetic basis of SLE disease traits in New Zealand mice in the context of the BALB/c genome. These data are summarized in Fig. 4. A region on proximal chromosome 12 derived from both the NZB and NZW genomes and inherited in a recessive manner (in NZB at least) was significantly linked with IgG, IgG1, and IgG2a anti-chromatin Abs, IgG anti-dsDNA Abs, and IgG anti-ssDNA Abs. Based on the above data, we propose to name this locus Nbwa1, New Zealand Black and White autoimmunity 1. This region was also linked to total serum IgG levels. Additionally, we have linked this region with serum gp70 and gp70-anti-gp70 immune complexes (gp70IC) ( 32) and anti-erythrocyte IgM autoantibodies (manuscript in preparation). In combination, these data suggest that the gene/s on proximal chromosome 12 are influencing B cell responses to a wide range of autoantigens. In this study, we have also linked GN to the same region on proximal chromosome 12. Because GN in New Zealand mice is believed to be immune complex mediated, it is likely that the linkage to GN is related to the enhancement of autoantibody production, not with gene/s at the same locus that influence glomerular damage by other mechanisms.



View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 4. Autosomal linkage of lupus traits mapped in this study with > suggestive linkage. Regions previously linked with lupus in New Zealand mice are indicated. GN1, Glomerular GN traits; GN2, tubulointerstitial GN traits; ANA, anti-nuclear Abs; IgM, total serum IgM; IgG, total serum IgG. {blacksquare}, NZB linkage region. {square}, NZW linkage region. , Both NZB and NZW linkage region. , BALB/c linkage region. Autosomes demonstrating no linkage in this study are omitted ( 2 13 14 15 18 19 ). *, >= Significant linkage.

 
Work by Bolland et al. ( 18), in which SLE traits were mapped in a (C57BL/6.Fc{gamma}RIIB-/- x BALB/c.Fc{gamma}RIIB-/-)F2 intercross showed linkage of anti-nuclear Abs to a locus on proximal chromosome 12, named by the authors as Asbb2. In combination with the work described in this study, this gives compelling evidence for the presence of a locus influencing systemic autoimmunity on proximal chromosome 12 that is revealed only in the context of the BALB/c genome. Furthermore, no linkage to this region has been shown in crosses of NZB, NZW, and NZM mice when crossed with C57BL/6 despite multiple independent investigations ( 13, 19, 20, 21, 22, 23, 24). Together, these data suggest permissive alleles on chromosome 12 in C57BL/6 and New Zealand strains that are absent on BALB/c chromosome 12. Both autoantibodies and histological GN traits exhibit a bimodal linkage pattern on chromosome 12, with a more distal peak at a region of either 27–29 or 40–42 cM from the centromere. This raises the intriguing possibility that there are two loci on chromosome 12 influencing SLE disease traits. A congenic mouse line, carrying a 0- to 34-cM region of NZW chromosome 12 on a BALB/c background, is currently being investigated to explore the above linkage data.

A region on proximal chromosome 4, also derived from the New Zealand genome and inherited in a recessive manner, was associated with both serological and histological traits. Peak linkage to serological traits was observed within a region 1–15 cM from the centromere, and peak linkage to histologic GN at around 20 cM from the centromere. This region has not been identified in crosses between New Zealand mice and C57BL/6 ( 13, 19, 20, 21, 22, 23, 24) and therefore represents a New Zealand-derived disease-associated region that is revealed only in the presence of the BALB/c genome. The proximal chromosome 4 region mapped in this study is, at a minimum, 25 cM more proximal from the closest New Zealand-associated disease locus (Sle2) in the cluster of SLE susceptibility loci on mid- to distal chromosome 4. Thus, we propose to name this locus Nbwa2, New Zealand Black and White Autoimmunity 2.

The proximal region has been described in a study of MRL/lpr-/- x C3H/lpr-/- mice and was associated with renal vasculitis, named by the authors as Arvm1 ( 25). Strong linkage to histological GN traits was mapped in the B-F2 cohort to a region on mid-chromosome 4, with a peak linkage at around 40–50 cM from the centromere. This colocalizes with the GN-associated loci Sle2 (at 45 cM), Lbw2 (at 55.6 cM), and Nba1 (at 60–70 cM). The rate of decay of the linkage to GN on mid-chromosome 4 gives convincing evidence that there is a genetic contribution from both mid- and proximal chromosome 4. Considering the trait total histology on chromosome 4, the rate of decay to the distal end of chromosome 4 is around a halving of LOD score over a 15-cM interval from the peak of linkage. However, this rate is not mirrored proximal of the mid-chromosome 4 linkage peak, with the LOD score reaching half the value over a 45-cM interval. Thus, the proximal linkage region is not the result of a wide single-locus linkage pattern (Fig. 2A).

A number of BALB/c alleles were associated with SLE traits in this study. The strongest linkage was observed at a region of distal chromosome 9 around 50–60 cM from the centromere. Both serological traits and renal tubulointerstitial damage mapped to this region, and are consistently associated with the presence of BALB/c alleles acting in a recessive manner. Therefore, in line with previous nomenclature, we propose to name this locus Baa2, BALB/c autoimmunity 2.

Three loci on proximal chromosome 9 have previously been linked to SLE traits: Sles4 at 4 cM ( 23), Baa1 at 28 cM ( 26), and Asbb1 at 17 cM ( 18). In this study, we mapped a number of traits to Baa1, namely anti-chromatin IgG2a (as part of a bimodal linkage pattern), glomerular cell damage, and glomerular matrix damage. These traits were all consistently associated with the presence of BALB/c alleles acting in a recessive manner. No linkage to chromosome 9 was observed in the W-BC cohorts, giving further evidence that the BALB/c disease modifying alleles on chromosome 9 are acting in a recessive manner.

The linkage of renal histopathology showed an interesting pattern—linkage was clearly split into two regions depending on whether the trait was glomerular or tubulointerstitial. Tubulointerstitial damage is known to have prognostic value when considering the outcome of human lupus nephritis ( 27). Our data indicate a separate genetic influence on the development of tubulointerstitial vs glomerular pathology, suggesting that the tubulointerstitial disease is not simply a secondary consequence of glomerular damage. This conclusion is further reinforced by the linkage of a broad range of anti-nuclear Abs to the same locus on distal chromosome 9 that is linked with tubulointerstitial pathology. It is perhaps surprising that, considering the immune complex-mediated pathogenesis of GN in New Zealand mice, the glomerular traits link to the more proximal region of chromosome 9, whereas the majority of autoantibodies link to the distal region.

IgG anti-dsDNA and IgG anti-ssDNA Abs were also linked to a BALB/c-derived region on proximal chromosome 8. Total serum IgG was linked to a transgressive region on proximal chromosome 16 in both the B-F2 and W-BC cohorts. A viable candidate for this locus is the cluster of Ig {lambda} chain genes, situated at 13 cM from the centromere.

A number of other loci, derived from the New Zealand genome, but revealed only in the context of the BALB/c background, were associated with SLE traits. A region on distal chromosome 4 was strongly associated with total serum IgG levels in the W-BC cohort. This region is distal of the cluster of SLE-associated loci on chromosome 4, which map to a region of around 45–65 cM from the centromere, and thus may represent a novel locus. Additionally, a locus on mid-chromosome 6 was linked, in the W-BC cohort, to anti-dsDNA Abs. A region on distal chromosome 7, at least 20 cM from the most distal of the SLE-associated gene cluster on proximal chromosome 7, was linked to both anti-ssDNA and anti-dsDNA autoantibodies in the B-F2 cohort.

As would perhaps be expected, a number of SLE-associated loci appear to be background independent (at least between C57BL/6 and BALB/c) and in some cases present in other lupus-prone mouse strains. Autoantibodies and total IgM were linked to distal chromosome 1, a region previously linked to SLE traits in NZB, NZM2410 (NZW region), and BXSB mice ( 13, 16, 21, 22, 28, 29). Strong linkage of serum IgM was observed on distal chromosome 4 in the region of the locus Imh1, previously described as being associated with hypergammaglobulinemia in NZB mice ( 30). The locus Sle2, a disease region identified in NZM2410 mice as influencing (among other traits) total IgM levels, was also within the region of greater than suggestive linkage to total IgM. Distal chromosome 4 was also linked to autoantibody production in this study, mirroring the phenotypes of the loci Adaz1 (in NZM2328 mice ( 15)) and Lbw2 (in NZB mice (16)). However, Sle2 is more proximal than the region of greater than suggestive linkage to autoantibodies mapped in this study ( 13). Linkage to histologic GN spanned both Sle2 and Nba1, seemingly distinct regions that are associated with the development of GN in NZM2410 and NZB mice, respectively ( 10, 13).

Two areas of linkage to chromosome 5 were seen in this study, both of which are in the region of previously described SLE-associated loci. IgG and IgG2a anti-chromatin Abs were linked in the B-F2 to proximal chromosome 5, around 10 cM distal of the locus Sle6 ( 23), with IgG anti-dsDNA and -ssDNA Abs being linked to distal chromosome 5 in the W-BC cohort, close to the locus Lbw3, linked by Kono et al. ( 16) to mortality and derived from the NZW genome.

Two traits, namely total IgM and IgG anti-ssDNA Abs, were mapped in this study to a region on proximal chromosome 7 abundant with SLE loci. However, the most phenotypically similar locus in this region is Sle3, associated by Morel et al. ( 13, 21) to anti-nuclear Abs, ssDNA, and GN in NZM2410 mice. Linkage on distal chromosome 10 to total IgG, anti-ssDNA IgG, and anti-chromatin IgG2a was observed in the B-F2 cohort. The locus Sle12 was linked to GN, with a possible, more proximal, linkage region to anti-dsDNA Abs in NZM2410 mice, and is a probable candidate for the traits mapped to distal chromosome 10 in this study ( 21).

A number of other linkage analyses in lupus-prone mice identified the H2 complex, on proximal chromosome 17, as being associated with disease traits. In this study, the H2 type was fixed in both the B-F2 and W-BC cohorts, H2d and H2d/z, respectively. The lack of linkage to the H2-containing region in the B-F2 cohort suggests that the H2 complex itself, and not other genes in the same region, underlies the linkage to proximal chromosome 17 identified in other studies. The data herein highlight a number of novel genetic regions associated with SLE traits in New Zealand mice. As well as providing new regions to investigate for genes that cause or modify SLE traits in New Zealand mice, this study highlights the effect of the non-autoimmune background strain, both in determining the New Zealand-derived loci that can be detected using linkage analysis and directly influencing the disease phenotype.


   Acknowledgments
 
We thank Dr. Bernard Morley for critical reading of the manuscript and Ms. Nikki Lee for technical assistance.


   Footnotes
 
1 This study was supported by a Wellcome Trust Senior Fellowship grant (to T.J.V.) and National Institutes of Health Grant AR37070 (to B.L.K.). Back

2 R.J.R. and S.J.R. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Timothy J. Vyse, Rheumatology Section, Imperial College Faculty of Medicine, Hammersmith Campus, London, W12 0NN, U.K. E-mail address: t.vyse{at}imperial.ac.uk Back

4 Abbreviations used in this paper: SLE, systemic lupus erythematosus; GN, glomerulonephritis; LOD, log of the odds ratio. Back

Received for publication October 29, 2003. Accepted for publication January 23, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Vyse, T. J., B. L. Kotzin. 1998. Genetic susceptibility to systemic lupus erythematosus. Annu. Rev. Immunol. 16:261.[Medline]
  2. Wakeland, E. K., K. Liu, R. R. Graham, T. W. Behrens. 2001. Delineating the genetic basis of systemic lupus erythematosus. Immunity 15:397.[Medline]
  3. Nguyen, C., N. Limaye, E. K. Wakeland. 2002. Susceptibility genes in the pathogenesis of murine lupus. Arthritis Res. 4:(Suppl. 3):S255.
  4. Nadeau, J. H.. 2001. Modifier genes in mice and humans. Nat. Rev. Genet. 2:165.[Medline]
  5. Watson, M. L., J. K. Rao, G. S. Gilkeson, P. Ruiz, E. M. Eicher, D. S. Pisetsky, A. Matsuzawa, J. M. Rochelle, M. F. Seldin. 1992. Genetic analysis of MRL-lpr mice: relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci. J. Exp. Med. 176:1645.[Abstract/Free Full Text]
  6. Vidal, S., D. H. Kono, A. N. Theofilopoulos. 1998. Loci predisposing to autoimmunity in MRL-Faslpr and C57BL/6-Faslpr mice. J. Clin. Invest. 101:696.[Medline]
  7. Gu, L., A. Weinreb, X. P. Wang, D. J. Zack, J. H. Qiao, R. Weisbart, A. J. Lusis. 1998. Genetic determinants of autoimmune disease and coronary vasculitis in the MRL-lpr/lpr mouse model of systemic lupus erythematosus. J. Immunol. 161:6999.[Abstract/Free Full Text]
  8. Wang, Y., M. Nose, T. Kamoto, M. Nishimura, H. Hiai. 1997. Host modifier genes affect mouse autoimmunity induced by the lpr gene. Am. J. Pathol. 151:1791.[Abstract]
  9. Rozzo, S. J., T. J. Vyse, C. G. Drake, B. L. Kotzin. 1996. Effect of genetic background on the contribution of New Zealand Black loci to autoimmune lupus nephritis. Proc. Natl. Acad. Sci. USA 93:15164.[Abstract/Free Full Text]
  10. Vyse, T. J., C. G. Drake, S. J. Rozzo, E. Roper, S. Izui, B. L. Kotzin. 1996. Genetic linkage of IgG autoantibody production in relation to lupus nephritis in New Zealand hybrid mice. J. Clin. Invest. 98:1762.[Medline]
  11. Emlen, W., P. Jarusiripipat, G. Burdick. 1990. A new ELISA for the detection of double-stranded DNA antibodies. J. Immunol. Methods 132:91.[Medline]
  12. Manly, K. F., J. M. Olson. 1999. Overview of QTL mapping software and introduction to map manager QT. Mamm. Genome 10:327.[Medline]
  13. Morel, L., U. H. Rudofsky, J. A. Longmate, J. Schiffenbauer, E. K. Wakeland. 1994. Polygenic control of susceptibility to murine systemic lupus erythematosus. Immunity 1:219.[Medline]
  14. Mohan, C., L. Morel, P. Yang, E. K. Wakeland. 1997. Genetic dissection of systemic lupus erythematosus pathogenesis: Sle2 on murine chromosome 4 leads to B cell hyperactivity. J. Immunol. 159:454.[Abstract]
  15. Waters, S. T., S. M. Fu, F. Gaskin, U. S. Deshmukh, S. S. Sung, C. C. Kannapell, K. S. Tung, S. B. McEwen, M. McDuffie. 2001. NZM2328: a new mouse model of systemic lupus erythematosus with unique genetic susceptibility loci. Clin. Immunol. 100:372.[Medline]
  16. Kono, D. H., R. W. Burlingame, D. G. Owens, A. Kuramochi, R. S. Balderas, D. Balomenos, A. N. Theofilopoulos. 1994. Lupus susceptibility loci in New Zealand mice. Proc. Natl. Acad. Sci. USA 91:10168.[Abstract/Free Full Text]
  17. Drake, C. G., S. K. Babcock, E. Palmer, B. L. Kotzin. 1994. Genetic analysis of the NZB contribution to lupus-like autoimmune disease in (NZB x NZW)F1 mice. Proc. Natl. Acad. Sci. USA 91:4062.[Abstract/Free Full Text]
  18. Bolland, S., Y. S. Yim, K. Tus, E. K. Wakeland, J. V. Ravetch. 2002. Genetic modifiers of systemic lupus erythematosus in Fc{gamma}RIIB-/- mice. J. Exp. Med. 195:1167.[Abstract/Free Full Text]
  19. Tucker, R. M., T. J. Vyse, S. Rozzo, C. L. Roark, S. Izui, B. L. Kotzin. 2000. Genetic control of glycoprotein 70 autoantigen production and its influence on immune complex levels and nephritis in murine lupus. J. Immunol. 165:1665.[Abstract/Free Full Text]
  20. Rozzo, S. J., T. J. Vyse, K. Menze, S. Izui, B. L. Kotzin. 2000. Enhanced susceptibility to lupus contributed from the nonautoimmune C57BL/10, but not C57BL/6, genome. J. Immunol. 164:5515.[Abstract/Free Full Text]
  21. Morel, L., C. Mohan, Y. Yu, J. Schiffenbauer, U. H. Rudofsky, N. Tian, J. A. Longmate, E. K. Wakeland. 1999. Multiplex inheritance of component phenotypes in a murine model of lupus. Mamm. Genome 10:176.[Medline]
  22. Vyse, T. J., S. J. Rozzo, C. G. Drake, S. Izui, B. L. Kotzin. 1997. Control of multiple autoantibodies linked with a lupus nephritis susceptibility locus in New Zealand Black mice. J. Immunol. 158:5566.[Abstract]
  23. Morel, L., X. H. Tian, B. P. Croker, E. K. Wakeland. 1999. Epistatic modifiers of autoimmunity in a murine model of lupus nephritis. Immunity 11:131.[Medline]
  24. Santiago, M. L., C. Mary, D. Parzy, C. Jacquet, X. Montagutelli, R. M. Parkhouse, R. Lemoine, S. Izui, L. Reininger. 1998. Linkage of a major quantitative trait locus to Yaa gene-induced lupus-like nephritis in (NZW x C57BL/6)F1 mice. Eur. J. Immunol. 28:4257.[Medline]
  25. Qu, W. M., T. Miyazaki, M. Terada, L. M. Lu, M. Nishihara, A. Yamada, S. Mori, Y. Nakamura, H. Ogasawara, C. Yazawa, et al 2000. Genetic dissection of vasculitis in MRL/lpr lupus mice: a novel susceptibility locus involving the CD72c allele. Eur. J. Immunol. 30:2027.[Medline]
  26. Vyse, T. J., L. Morel, F. J. Tanner, E. K. Wakeland, B. L. Kotzin. 1996. Backcross analysis of genes linked to autoantibody production in New Zealand White mice. J. Immunol. 157:2719.[Abstract]
  27. Austin, H. A., D. T. III, E. M. Vaughan Boumpas, J. E. Balow. 1994. Predicting renal outcomes in severe lupus nephritis: contributions of clinical and histologic data. Kidney Int. 45:544.[Medline]
  28. Morel, L., K. R. Blenman, B. P. Croker, E. K. Wakeland. 2001. The major murine systemic lupus erythematosus susceptibility locus, Sle1, is a cluster of functionally related genes. Proc. Natl. Acad. Sci. USA 98:1787.[Abstract/Free Full Text]
  29. Haywood, M. E., M. B. Hogarth, J. H. Slingsby, S. J. Rose, P. J. Allen, E. M. Thompson, M. A. Maibaum, P. Chandler, K. A. Davies, E. Simpson, et al 2000. Identification of intervals on chromosomes 1, 3, and 13 linked to the development of lupus in BXSB mice. Arthritis Rheum. 43:349.[Medline]
  30. Hirose, S., H. Tsurui, H. Nishimura, Y. Jiang, T. Shirai. 1994. Mapping of a gene for hypergammaglobulinemia to the distal region on chromosome 4 in NZB mice and its contribution to systemic lupus erythematosus in (NZB x NZW)F1 mice. Int. Immunol. 6:1857.[Abstract/Free Full Text]
  31. Xie, S., S. H. Chang, P. Sedrak, A. Kaliyaperumal, S. K. Datta, C. Mohan. 2002. Dominant NZB contributions to lupus in the (SWR x NZB)F1 model. Genes Immun. 3:(Suppl. 1):S13.
  32. Rigby R. J., S. J. Rozzo, H. Gill, T. Fernandez-Hart, B. J. Morley, S. Izui, B. L. Kotzin, and T. J. Vyse. A novel locus regulates both retroviral glycoprotein 70 and anti-glycoprotein 70 antibody production in New Zealand mice when crossedwith BALB/c. J. Immunol. In press.



This article has been cited by other articles:


Home page
 Int ImmunolHome page
A. Nijnik, H. Ferry, G. Lewis, E. Rapsomaniki, J. C. H. Leung, A. Daser, T. Lambe, C. C. Goodnow, and R. J. Cornall
Spontaneous B cell hyperactivity in autoimmune-prone MRL mice
Int. Immunol., July 1, 2006; 18(7): 1127 - 1137.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rigby, R. J.
Right arrow Articles by Vyse, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rigby, R. J.
Right arrow Articles by Vyse, T. J.
Right arrowPubmed/NCBI databases
*Gene
Medline Plus Health Information
*Lupus

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS