Modulation of multiple experimental arthritis models by collagen-induced arthritis quantitative trait loci isolated in congenic rat lines: different effects of non-major histocompatibility complex quantitative trait loci in males and females

OBJECTIVE: Collagen-induced arthritis (CIA) is a model of inflammatory arthritis with many similarities to rheumatoid arthritis (RA). We previously mapped in F(2) offspring of CIA-susceptible DA and CIA-resistant F344 rats, 5 quantitative trait loci (QTLs) for which F344 alleles were associated with reduced CIA severity. In the present study, we sought to characterize the independent arthritis-modulating effects of these 5 QTLs. METHODS: CIA-regulatory regions were transferred from the F344 genome to the DA background or vice versa by repeated backcrossing. The arthritis-modulating effects of the transferred alleles were determined by comparing the severity of experimentally induced arthritis in congenic rats with that in DA rats. RESULTS: Congenic lines with either the F344 major histocompatibility complex (MHC) on the DA background or the DA MHC on the F344 background were resistant to CIA, confirming both MHC and non-MHC contributions to the genetic regulation of CIA. F344 alleles at the Cia3 and Cia5 regions of chromosomes 4 and 10 reduced CIA severity relative to that observed in DA rats. F344 Cia4 and Cia6 regions of chromosomes 7 and 8 failed to significantly alter CIA severity. Arthritis-modifying effects of Cia4 and Cia6 were, however, detected in pristane-induced and/or Freund's incomplete adjuvant oil-induced arthritis. The arthritis-modifying effects of the non-MHC CIA-regulatory loci differed in males and females. CONCLUSION: These congenic lines confirmed the existence and location of genes that regulate the severity of experimental arthritis in rats. Mechanisms responsible for the sex-specificity of individual arthritis-regulatory loci may explain some of the sex differences observed in RA and other autoimmune diseases in humans.     

A major quantitative trait locus on chromosome 3 controls colitis severity in IL-10-deficient mice

Colitic lesions are much more severe in C3H/HeJBir (C3H) than C57BL/6J (B6) mice after 10 backcrosses of a disrupted interleukin-10 (Il10) gene. This study identified cytokine deficiency-induced colitis susceptibility (Cdcs) modifiers by using quantitative trait locus (QTL) analysis. A segregating F(2) population (n = 408) of IL-10-deficient mice was genotyped and necropsied at 6 weeks of age. A major C3H-derived colitogenic QTL (Cdcs1) on chromosome (Chr.) 3 contributed to lesions in both cecum [logarithm of odds ratio (LOD) = 14.6)] and colon (LOD = 26.5) as well as colitis-related phenotypes such as spleen/body weight ratio, mesenteric lymph node/body weight ratio, and secretory IgA levels. Evidence for other C3H QTL on Chr. 1 (Cdcs2) and Chr. 2 (Cdcs3) was obtained. Cdcs1 interacted epistatically or contributed additively with loci on other chromosomes. The resistant B6 background also contributed colitogenic QTL: Cdcs4 (Chr. 8), Cdcs5 (Chr. 17, MHC), and Cdcs6 (Chr. 18). Epistatic interactions between B6 QTL on Chr. 8 and 18 contributing to cecum hyperplasia were particularly striking. In conclusion, a colitogenic susceptibility QTL on Chr. 3 has been shown to exacerbate colitis in combination with modifiers contributed from both parental genomes. The complex nature of interactions among loci in this mouse model system, coupled with separate deleterious contributions from both parental strains, illustrates why detection of human inflammatory bowel disease linkages has proven to be so difficult. A human ortholog of the Chr. 3 QTL, if one exists, would map to Chr. 4q or 1p.     

Identification of two novel female-specific non-major histocompatibility complex loci regulating collagen-induced arthritis severity and chronicity, and evidence of epistasis

OBJECTIVE: To identify additional sex-specific and epistatic quantitative trait loci (QTL) regulating collagen-induced arthritis (CIA) severity overall, as well as within different stages during the disease course, in an intercross between major histocompatibility complex-identical inbred rat strains DA/Bkl (susceptible) and ACI/Hsd (resistant). METHODS: Arthritic male (DA x ACI)F2 intercross offspring (n = 143) were analyzed separately from the females (n = 184). Phenotypic extremes (maximum arthritis scores [MAS]) were genotyped and used for QTL analysis. All 327 rats were genotyped with the simple sequence-length polymorphism (SSLP) markers closest to the peak of Cia7 and Cia10, the major loci previously identified in this intercross, and with SSLPs covering chromosomes 12 and 18. Phenotypes studied were disease onset, arthritis severity scores on days 14-39, MAS, mean and cumulative arthritis scores, delayed-type hypersensitivity, and antibody responses to rat type II collagen. RESULTS: A new female-specific arthritis-severity recessive locus was identified on rat chromosome 12 (Cia25), with a maximum effect observed on day 28 (logarithm of odds [LOD] 4.7). The homozygous DA genotype at Cia25 was associated with a 45% higher median arthritis score in females. Sequencing analyses of the Cia25 candidate gene Ncf1 revealed polymorphisms between DA and ACI. The previously identified locus, Cia10, was found to be male-specific. A 2-locus interaction model analysis identified a novel recessive chromosome 18 QTL, Cia26, which was dependent on Cia7, with its maximum effect observed at later stages during the disease course (peak LOD score of 3.6 for arthritis scores on day 39). CONCLUSION: This study identified 2 novel female-specific loci, and 1 male-specific locus. Cia25 regulates MAS and disease severity during the mid-to-late stages of the disease course and may be accounted for by Ncf1 polymorphisms. Cia26 is in epistasis with Cia7 and regulates later stages of disease, suggesting an involvement in disease perpetuation and/or chronicity.     

Identification of four new quantitative trait loci regulating arthritis severity and one new quantitative trait locus regulating autoantibody production in rats with collagen-induced arthritis

OBJECTIVE: Collagen-induced arthritis (CIA) is a polygenic model of experimentally induced autoimmunity and chronic joint inflammation. This study maps genetic loci that regulate CIA susceptibility in DA/Bkl (DA) and BN/SsNHsd (BN) rats. METHODS: Genome scans covering chromosomes 1-20 and interval mapping techniques using 159 simple sequence-length polymorphism markers were used to identify quantitative trait loci (QTLs) that regulate CIA in (DA x BN)F2 hybrids. Serum antibody titers to type II collagen were determined by enzyme-linked immunosorbent assay. RESULTS: DA rats were high responders to porcine type II collagen (PII) and developed severe CIA (100%). BN rats were low responders to PII and resistant to CIA (0%). BN genes strongly repressed PII-induced CIA. Only 12% of (DA x BN)F1 rats (7 of 60) and 31% of (DA x BN)F2 rats (307 of 1,004) developed CIA. Three new QTLs (Cia11, Cia12, and Cia13) with significant logarithm of odds (LOD) scores of 5.6, 4.6, and 4.5, respectively, plus a suggestive QTL (Cia14*, LOD 3.0) regulating arthritis severity were identified on chromosomes 3, 12, 4, and 19. A new QTL, Ciaa3, associating with anticollagen antibody titer (antibody to PII LOD 6.5; antibody to rat type II collagen LOD 5.2) mapped to chromosome 9. Of 10 CIA QTLs previously identified in (DA x F344) and (DA x ACI) rats, only Cia1 in the major histocompatibility complex and a region coincident to Cia5 on chromosome 10 (LOD >8.0) influenced CIA severity in (DA x BN)F2 rats. CONCLUSION: Since CIA exhibits many of the pathologic features of rheumatoid arthritis, the data indicate that the variety of genetic elements regulating human autoimmune and rheumatic diseases may be much larger and more varied than originally envisioned.     

Susceptibility to adjuvant arthritis in DA and F344 rats. A dominant trait controlled by an autosomal gene locus linked to the major histocompatibility complex

The hereditary of adjuvant-induced arthritis in rats of the DA and Fisher 344 strains was examined. Development of arthritic lesions in response to a single injection of supplemented Freund's complete adjuvant has been found to be controlled by an autosomal, dominant gene locus, Ar. The capacity to respond was present in DA rats and absent in our Fisher rats. Ar has been found to be linked to the major histocompatibility complex genes with which it exhibits a high rate of recombination.     

Modulation of multiple experimental arthritis models by collagen‐induced arthritis quantitative trait loci isolated in congenic rat lines: Different effects of non–major histocompatibility complex quantitative trait loci in males and females

Objective

Collagen‐induced arthritis (CIA) is a model of inflammatory arthritis with many similarities to rheumatoid arthritis (RA). We previously mapped in F₂ offspring of CIA‐susceptible DA and CIA‐resistant F344 rats, 5 quantitative trait loci (QTLs) for which F344 alleles were associated with reduced CIA severity. In the present study, we sought to characterize the independent arthritis‐modulating effects of these 5 QTLs.

Methods

CIA‐regulatory regions were transferred from the F344 genome to the DA background or vice versa by repeated backcrossing. The arthritis‐modulating effects of the transferred alleles were determined by comparing the severity of experimentally induced arthritis in congenic rats with that in DA rats.

Results

Congenic lines with either the F344 major histocompatibility complex (MHC) on the DA background or the DA MHC on the F344 background were resistant to CIA, confirming both MHC and non‐MHC contributions to the genetic regulation of CIA. F344 alleles at the Cia3 and Cia5 regions of chromosomes 4 and 10 reduced CIA severity relative to that observed in DA rats. F344 Cia4 and Cia6 regions of chromosomes 7 and 8 failed to significantly alter CIA severity. Arthritis‐modifying effects of Cia4 and Cia6 were, however, detected in pristane‐induced and/or Freund's incomplete adjuvant oil–induced arthritis. The arthritis‐modifying effects of the non‐MHC CIA‐regulatory loci differed in males and females.

Conclusion

These congenic lines confirmed the existence and location of genes that regulate the severity of experimental arthritis in rats. Mechanisms responsible for the sex‐specificity of individual arthritis‐regulatory loci may explain some of the sex differences observed in RA and other autoimmune diseases in humans.

     

Confirmation and genetic dissection of a major quantitative trait locus for alcohol preference drinking

OBJECTIVE: In previous work, we created congenic strains that carry the DBA/2IBG (D2) region for alcohol preference on chromosome 2, on an otherwise C57BL/6IBG (B6) background. Here, we report construction and testing of interval-specific congenic recombinant strains (ISCRSs) for the purpose of narrowing the quantitative trait loci (QTL) interval. METHODS: ISCRSs were derived by identifying mice that carry recombination events in the D2 interval, during the backcrossing for congenics. Recombinant mice were backcrossed to B6, and progeny that carry the reduced chromosome 2 region were tested for its effect on the alcohol preference phenotype. RESULTS: We developed multiple ISCR strains, which spanned the QTL interval. Three of these showed the D2 phenotype of reduced alcohol consumption. The overlap of two of these strains reduced the QTL interval from 66.8 to 3.5 Mb. A third positive ISCRS suggests the possibility of a second, linked QTL. CONCLUSIONS: Use of ISCRSs can narrow a QTL region to a few Mb. This reduced interval size will facilitate identification of candidate genes, through bioinformatics, gene expression, and DNA sequencing strategies. Potential difficulties, including reduced power as a result of variable phenotypes or small effect size, are discussed.     

The major histocompatibility complex gene region and sarcoidosis susceptibility in African Americans

Investigators have intensively evaluated the major histocompatibility (MHC) complex for sarcoidosis susceptibility genes with the majority of reports implicating the human leukocyte antigen (HLA)-DRB1 gene. Because most studies have been performed in white and Asian populations, we sought to determine which MHC genes might be risk factors for sarcoidosis in African Americans. We genotyped six microsatellite markers spanning 11.6 megabases that overlapped the MHC region on chromosome 6p21-22 in 225 nuclear families ascertained by African American probands with a history of sarcoidosis. Using a family-based association methods approach, we performed multiallelic tests of association between each marker and sarcoidosis. A statistically significant association was detected between sarcoidosis and the DQCAR marker (p = 0.002) less than two kilobases telomeric from the HLA-DQB1 gene. Typing two additional markers in this region revealed that DQCAR-G51152 haplotypes, spanning a 38-kilobase region across the HLA-DQB1 gene, were associated with sarcoidosis on a global level (p = 0.022). Analysis of individual DQCAR and G51152 alleles showed that the DQCAR 178 (expected = 21.0; observed = 10; p = 0.0005) and G51152 217 (expected = 25.6; observed = 14; p = 0.0009) alleles were transmitted to affected offspring less often than expected; whereas the DQCAR 182 allele was transmitted more often than expected (expected = 52.6; observed = 66; p = 0.002). Our results indicate that HLA-DQB1 and not HLA-DRB1 plays an important role in sarcoidosis susceptibility in African Americans. Identification of the specific HLA-DQB1 alleles that influence sarcoidosis susceptibility in African Americans and the study of their antigenic-binding properties may reveal why African Americans suffer disproportionately from this disease.     

Molecular identification of a major quantitative trait locus, qLTG3-1, controlling low-temperature germinability in rice

Tolerance to abiotic stress is an important agronomic trait in crops and is controlled by many genes, which are called quantitative trait loci (QTLs). Identification of these QTLs will contribute not only to the understanding of plant biology but also for plant breeding, to achieve stable crop production around the world. Previously, we mapped three QTLs controlling low-temperature tolerance at the germination stage (called low-temperature germinability). To understand the molecular basis of one of these QTLs, qLTG3–1 (quantitative trait locus for low-temperature germinability on chromosome 3), map-based cloning was performed, and this QTL was shown to be encoded by a protein of unknown function. The QTL qLTG3–1 is strongly expressed in the embryo during seed germination. Before and during seed germination, specific localization of beta-glucuronidase staining in the tissues covering the embryo, which involved the epiblast covering the coleoptile and the aleurone layer of the seed coat, was observed. Expression of qLTG3–1 was tightly associated with vacuolation of the tissues covering the embryo. This may cause tissue weakening, resulting in reduction of the mechanical resistance to the growth potential of the coleoptile. These phenomena are quite similar to the model system of seed germination presented by dicot plants, suggesting that this model may be conserved in both dicot and monocot plants.     

Evaluation of quantitative trait loci regulating severity of mycobacterial adjuvant-induced arthritis in monocongenic and polycongenic rats: identification of a new regulatory locus on rat chromosome 10 and evidence of overlap with rheumatoid arthritis susceptibility loci

OBJECTIVE: To evaluate the regulatory potential of genetic loci controlling Mycobacterium butyricum adjuvant-induced arthritis (Mbt-AIA) using mono- and polycongenic rats. METHODS: Of 4 quantitative trait loci (QTLs) that regulate Mbt-AIA, F344 alleles at 3 of these loci, Aia1, Aia2, and Aia3, are associated with lower arthritis severity, whereas F344 alleles at Aia4 are associated with greater arthritis severity. In this study, we constructed congenic lines by transferring 1 or more of the F344 genomic segments containing Aia1, Aia2, and Aia3 onto the DA genome. We comparatively evaluated their responses to Mbt-AIA with the responses of parental DA and F344 rats. RESULTS: Aia1, encompassing the rat major histocompatibility complex, reduced arthritis severity in monocongenic rats of both sexes. The arthritis-lowering effects of Aia2 and Aia3 were sex-influenced and were therefore observed in only males and only females, respectively. Polycongenic rats containing F344 genomic regions at Aia1, Aia2, and Aia3 developed Mbt-AIA of relatively greater severity than did F344 rats, implying that in DA and F344 rats, there could be other Mbt-AIA loci in addition to Aia1, Aia2, Aia3, and Aia4. To test the possibility that some of these Mbt-AIA-regulatory loci may colocalize with other arthritis QTLs, we evaluated Mbt-AIA in DA.F344 monocongenic rats containing collagen-induced arthritis QTLs. Cia5 (the QTL region on chromosome 10), but not Cia5a, Cia4, or Cia6, also regulated Mbt-AIA, and was named Aia5. CONCLUSION: F344 genomic regions at Aia1, Aia2, and Aia3 and the newly identified Aia5 contain genes that reduce Mbt-AIA severity in DA rats. These Mbt-AIA-regulatory loci overlap rheumatoid arthritis-susceptibility loci in humans.     

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.     

Evaluation of quantitative trait loci regulating severity of mycobacterial adjuvant‐induced arthritis in monocongenic and polycongenic rats: Identification of a new regulatory locus on rat chromosome 10 and evidence of overlap with rheumatoid arthritis susceptibility loci

Objective

To evaluate the regulatory potential of genetic loci controlling Mycobacterium butyricum adjuvant‐induced arthritis (Mbt‐AIA) using mono‐ and polycongenic rats.

Methods

Of 4 quantitative trait loci (QTLs) that regulate Mbt‐AIA, F344 alleles at 3 of these loci, Aia1, Aia2, and Aia3, are associated with lower arthritis severity, whereas F344 alleles at Aia4 are associated with greater arthritis severity. In this study, we constructed congenic lines by transferring 1 or more of the F344 genomic segments containing Aia1, Aia2, and Aia3 onto the DA genome. We comparatively evaluated their responses to Mbt‐AIA with the responses of parental DA and F344 rats.

Results

Aia1, encompassing the rat major histocompatibility complex, reduced arthritis severity in monocongenic rats of both sexes. The arthritis‐lowering effects of Aia2 and Aia3 were sex‐influenced and were therefore observed in only males and only females, respectively. Polycongenic rats containing F344 genomic regions at Aia1, Aia2, and Aia3 developed Mbt‐AIA of relatively greater severity than did F344 rats, implying that in DA and F344 rats, there could be other Mbt‐AIA loci in addition to Aia1, Aia2, Aia3, and Aia4. To test the possibility that some of these Mbt‐AIA–regulatory loci may colocalize with other arthritis QTLs, we evaluated Mbt‐AIA in DA.F344 monocongenic rats containing collagen‐induced arthritis QTLs. Cia5 (the QTL region on chromosome 10), but not Cia5a, Cia4, or Cia6, also regulated Mbt‐AIA, and was named Aia5.

Conclusion

F344 genomic regions at Aia1, Aia2, and Aia3 and the newly identified Aia5 contain genes that reduce Mbt‐AIA severity in DA rats. These Mbt‐AIA–regulatory loci overlap rheumatoid arthritis–susceptibility loci in humans.

     

Functional and inducible expression of a transfected murine class II major histocompatibility complex gene.

Using the spheroplast fusion technique, we have introduced the cloned E beta b gene into two d haplotype cell lines, the B lymphoma line A20-2J and the macrophage tumor line P388D1. Analysis with a monoclonal antibody indicates that the product of the transfected E beta b gene associates with the endogenous E alpha chain to form an E alpha dE beta b complex. While expression of E alpha dE beta b is constitutive in A20-2J cells transfected with the E beta b gene, surface expression of E alpha dE beta b is detected in transfected macrophage cells only after treatment of cells with culture supernatants from concanavalin A (Con A)-stimulated T cells. Transfected B lymphoma cells and transfected Con A supernatant-treated macrophage cells have acquired the ability to present antigen to E alpha dE beta b-restricted T-cell hybridomas. The observed inducible expression of the transfected gene in the macrophage host indicates that sequences responsible for regulated expression of the E beta b gene may be associated with the transfected gene. In combination with directed mutagenesis, the system described here provides a means to study (i) E beta b sequences that are important in determining the restriction specificity of the E molecule and (ii) sequences associated with the E beta gene that may be important in the regulation of E beta chain expression.