F1 Backcross Mice Research Articles

Abstract 1368: Variable penetrance of mammary tumors in mouse models of Li-Fraumeni syndrome is linked to replication-associated repair

Abstract Background: The p53 tumor suppressor gene plays a key role in sporadic and inherited breast cancers. Spontaneous mammary tumors develop in ~55% of female BALB/c-Trp53+/- mice providing a model for breast cancer in Li-Fraumeni Syndrome. In contrast, C57BL/6J-Trp53+/- females are devoid of mammary tumors. This strain difference offers a dramatic example of variable penetrance and tools to define genes contributing to the risk of mammary tumors. Methods: Genome-wide linkage analysis of the mammary tumor incidence was performed in F1 and N2 backcross mice. The C57BL/6J alleles of SM1 locus were introgressed into the BALB/c genetic background through 10 backcrosses to generate SM1-Trp53+/- mice. Radiation sensitivity, DNA double-strand break repair, processitivity of DNA replication and expression of DNA repair genes in SM1-Trp53+/- mice were compared with BALB/c-Trp53+/- and C57BL/6J-Trp53+/-. Results: Genome-wide linkage analysis identified a major modifier locus on chromosome 7 (designated SM1) and another on chromosome 2 with an overall LOD score of 6.1 using a multigenic model of inheritance. The SM1 locus does not undergo loss of heterozygosity in mammary tumors consistent with dominant-acting risk alleles from BALB/c mice. Mammary epithelial cells (MMEC) of SM1 mice, having C57BL/6 alleles of SM1 in BALB/c background, were sensitive to radiation as were BALB/c MMEC. Functional assays of DNA double-strand break repair (1) demonstrate error-prone repair through the single strand annealing (SSA) pathway level in SM1 embryo fibroblasts (MEF) was similar to that in C57BL/6 MEF, which was 2.8-fold higher in BALB/c-Trp53+/- MEF and 2.5-fold higher in [BALB.B6]F1-Trp53+/-. Similarly, DNA fiber assays showed that processivity of DNA replication was significantly decreased in BALB/c and F1 MEF with median fork length (MFL) 6.4µM but increased in C57BL/6 and complemented in SM1 MEF (MFL ~8.2µM). In addition, gene expression profile of at least 5 DNA repair proteins (Ercc5, Rev1, Rev3l, Trex2, Rdm1) that are differentially expressed in C57BL/6 and BALB/c were complemented by SM1 locus. Conclusions: Gene(s) within the 20Mb SM1 locus impair movement of replications forks. Stalling of replication may precipitate fork collapse and DNA double strand breaks resulting in the loss of heterozygosity for Trp53 through recombination observed in 90% of mammary tumors.

Congenic mapping and allele-specific alteration analysis of Stmm1 locus conferring resistance to early-stage chemically induced skin papillomas.

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.

Independent genetic control of early and late stages of chemically induced skin tumors in a cross of a Japanese wild-derived inbred mouse strain, MSM/Ms

MSM/Ms is an inbred mouse strain derived from a Japanese wild mouse, Mus musculus molossinus. In this study, we showed that MSM/Ms mice exhibit dominant resistance when crossed with susceptible FVB/N mice and subjected to the two-stage skin carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA)/ 12-O-tetradecanoylphorbol-13-acetate (TPA). A series of F1 backcross mice were generated by crossing p53(+/+) or p53(+/-) F1 (FVB/N × MSM/Ms) males with FVB/N female mice. These generated 228 backcross animals, approximately half of which were p53(+/-), enabling us to search for p53-dependent skin tumor modifier genes. Highly significant linkage for papilloma multiplicity was found on chromosomes 6 and 7 and suggestive linkage was found on chromosomes 3, 5 and 12. Furthermore, in order to identify stage-dependent linkage loci we classified tumors into three categories (<2mm, 2-6mm and >6mm), and did linkage analysis. The same locus on chromosome 7 showed strong linkage in groups with <2mm or 2-6mm papillomas. No linkage was detected on chromosome 7 to papillomas >6mm, but a different locus on chromosome 4 showed strong linkage both to papillomas >6mm and to carcinomas. This locus, which maps near the Cdkn2a/p19(Arf) gene, was entirely p53-dependent, and was not seen in p53 (+/-) backcross animals. Suggestive linkage conferring susceptibility to carcinoma was also found on chromosome 5. These results clearly suggest distinct loci regulate each stage of tumorigenesis, some of which are p53-dependent.

M-cadherin, a candidate gene for type 2 diabetes and related phenotypes in a KK/Ta mouse model

The KK/Ta strain serves as a suitable polygenic mouse model for type 2 diabetes associated with fasting hyperglycaemia, glucose intolerance, hyperinsulinaemia, mild obesity and dyslipidaemia. Recently, we reported the susceptibility loci contributing to type 2 diabetes and related phenotypes in KK/Ta mice. In the present study, to identify susceptibility genes for type 2 diabetes and related disorders, GeneChip Expression Analysis was employed to survey the gene expression profile in the liver of KK/Ta and BALB/c mice. M-cadherin, a calciumdependent intercellular adhesion molecule, showed increased expression in the liver of KK/Ta mice, and sequence analysis revealed three missense mutations. The relationship between these polymorphisms and various phenotypes in 208 KK/Ta x (BALB/c x KK/Ta) F1 backcross mice was analysed. Statistical analysis revealed that M-cadherin exhibits linkage to levels of triglyceride and insulin in sera, glucose tolerance and body weight. Although it has been postulated that M-cadherin may be important for the regulation of morphogenesis of skeletal muscle cells, these results suggest that M-cadherin may influence hypertriglyceridaemia, glucose intolerance, hyperinsulinaemia and obesity in KK/Ta mice.

Minor gene effect of leptin receptor variant on the body weight in KK/Ta mice

Leptin is an adipocyte-derived hormone involved in body weight regulation that acts through the leptin receptor. Previous studies exploring potential association between the leptin receptor (Lepr) variant and obesity have reported conflicting results. The objectives of the present study are to evaluate (1) whether the Lepr variant contributes to type 2 diabetes and its related disorders such as obesity and (2) whether the gene interaction between Lepr and Zn-alpha(2) glycoprotein1 (Azgp1) genes is recognized using genetically homogeneous type 2 diabetic KK/Ta mice. The levels of leptin (Lep) and Lepr mRNA in adipose tissues and brain were measured by relative quantitative RT-PCR. The levels of leptin protein in sera were measured by enzyme-linked immunosorbent assay. Genotyping of backcross mice was performed using a mismatch primer. Leptin protein and its mRNA levels were increased in KK/Ta mice. Lepr mRNA levels of KK/Ta mice did not differ from those of BALB/c mice. Sequence analysis revealed that the coding region of Lep in KK/Ta mice was identical to that in BALB/c mice. Six nucleotide polymorphisms were observed in the coding region of Lepr. In KK/Ta x (BALB/c x KK/Ta) F1 backcross mice, the Lepr variant of KK/Ta mice failed to alter any of the variables of obesity except for body weight at 20 weeks of age. However, it enhanced the effect of Azgp1 on body weight. It is concluded that the Lepr variant contributes to obesity to some degree in KK/Ta mice.

Genetic variants ofTgfb1act as context-dependent modifiers of mouse skin tumor susceptibility

The human TGFB1 gene is polymorphic, and genetic variants are associated with altered cancer risk. However, human genetic association studies have had variable outcomes because TGFbeta1 action is context-dependent. We used the murine skin model of chemical carcinogenesis in genetic linkage analysis of three independent Mus musculus NIH/Ola x (Mus spretus x M. musculus NIH/Ola)F1 backcrosses, to identify a skin tumor susceptibility locus, Skts14, on proximal chromosome 7. Tgfb1 maps at the peak of linkage. The mouse Tgfb1 gene is polymorphic, resulting in cis-regulated differential allelic mRNA expression between M. spretus and M. musculus in F1 mouse skin. This phenomenon is reflected in differential phospho-SMAD2 levels, downstream of TGFbeta signaling, between these two mouse species. In normal F1 mouse skin, the Tgfb1SPR allele is expressed at higher levels than the Tgfb1NIH allele, and this differential is accentuated by phorbol 12-myristate 13-acetate treatment. In benign F1 papillomas, this imbalance is reversed, possibly by selection against expression of a hyperactive Tgfb1SPR allele in TGFbeta growth-responsive tumors. We demonstrate that skin tumor susceptibility is altered by Tgfb1 gene dosage, but that manifestation of Tgfb1-linked skin tumor susceptibility in M. musculus NIH/Ola x (M. spretus x M. musculus NIH/Ola)F1 backcross mice depends on interactions with another unlinked tumor modifying locus, Skts15, that overlaps Tgfbm3 on chromosome 12. These findings illustrate the power of complex genetic interactions in determining disease outcome and have major implications to the assessment of disease risk in individuals harboring variant TGFB1 alleles.

Identification of epistatic interaction involved in obesity using the KK/Ta mouse as a Type 2 diabetes model: is Zn-alpha2 glycoprotein-1 a candidate gene for obesity?

The KK/Ta strain serves as a suitable polygenic mouse model for the common form of type 2 diabetes associated with obesity in humans. Recently, we reported the susceptibility loci contributing to type 2 diabetes and related phenotypes in KK/Ta mice. In this study, we focused on expression in the kidneys and liver of KK/Ta and BALB/c mice using differential display (DD) PCR. Zn-alpha(2) glycoprotein-1 (Azgp1) mRNA levels were increased in the kidneys and liver in KK/Ta mice, and sequence analysis revealed a missense mutation. We analyzed the relationship between this polymorphism and various phenotypes in 208 KK/Ta x (BALB/c x KK/Ta) F1 backcross mice. Statistical analysis revealed that Azgp1 and D17Mit218 exhibit a suggestive linkage to body weight (8 weeks) (logarithm of odds 2.3 and 2.9, respectively). Moderate gene-gene interactions were observed at these loci. Adiponectin mRNA levels in 3T3-L1 cells transfected with the expression pcDNA 3.1 vector containing Azgp1 coding sequence of KK/Ta mice were significantly higher than those of BALB/c mice. These results suggest that Azgp1 is a possible candidate gene for regulation of body weight, elucidation of polygenic inheritance, and age-dependent changes in the genetic control of obesity.

Myeloid, B and T lymphoid and mixed lineage thymic lymphomas in the irradiated mouse.

Thymic lymphoma is a very common spontaneous and/or induced malignancy in both inbred mice and in transgenic mouse models of human cancer. Although a thymic lymphoma is defined as thymus-dependent T-cell malignancy, diagnostic criteria vary between studies and considerable heterogeneity has been reported. To define and classify the thymic lymphomas that arose in our study of X-irradiated (CBA/HxC57BL/6)F1, F1 backcross and F1 intercross mice, 66 thymic lymphomas were immunogenotyped for immunoglobulin heavy chain (IgH) and T-cell receptor beta (TCRbeta) gene rearrangements, and/or analysed for expression of lineage-specific markers and allelic loss on chromosome 4. The data indicate that 33% of the thymic lymphomas are very similar to mouse radiation-induced acute myeloid (AML) and mixed lineage (IgH(R), TCRbeta(G)) pre-B lympho-myeloid (L-MLs) leukaemias, 33% are mixed lineage (IgH(R), TCRbeta(R)) B/T lymphoid and <33% can be described as single lineage (IgH(G), TCRbeta(R)) T-cell malignancies. As the myeloid and L-ML leukaemias are not thymus-dependent this suggests that a malignant myeloid or pre-B lympho-myeloid cell can colonize the spleen to give an AML or L-ML leukaemia, or can colonize the thymus where TCRbeta gene rearrangement(s) may be induced to give the mixed lineage thymic lymphomas. Thus, assuming the single lineage T-cell thymic lymphomas fulfil the criteria of a thymus-dependent T-cell malignancy, thymic lymphomas are comprised of at least three distinct malignancies.

Characterisation of the mouse diabetes susceptibilty locus Nidd/SJL: islet cell destruction, interaction with the obesity QTL Nob1, and effect of dietary fat.

The diabetes susceptibility locus Nidd/SJL was identified in an outcross of New Zealand obese (NZO) and lean Swiss/Jackson Laboratory mouse strain (SJL) mice. Here we characterise its effects in a NZO x F1(SJLxNZO) backcross population raised on high-fat or standard diet, and describe its interaction with the obesity quantitative trait locus (QTL) Nob1. NZO x F1(SJLxNZO) backcross mice were raised on a normal or high fat diet and were monitored (body weight, blood glucose, serum insulin) for 22 weeks. Genotypes of polymorphic markers were determined by PCR, and linkage analysis was done. Pancreas morphology was assessed by conventional staining and immunohistochemistry of insulin. In backcross mice raised on a high-fat diet, Nidd/SJL produced hyperglycaemia (maximum likelihood of the odds (LOD) score 9.9), hypoinsulinaemia, reduction of islet-cell volume, and loss of beta cells. No effect was observed on body weight and serum insulin concentrations before the onset of hyperglycaemia. The development of diabetes in carriers of Nidd/SJL was markedly accelerated and aggravated by the obesity/hyperinsulinaemia QTL Nob1; together, these loci were responsible for approximately 90% of the diabetes observed in the backcross population. When raised on a standard diet, Nidd/SJL carriers exhibited a fivefold higher prevalence of diabetes, but Nob1 failed to enhance the effect of Nidd/SJL. Diabetes in this obese mouse model is the result of an interaction of genes responsible for obesity/insulin resistance (e.g. Nob1) and islet cell failure ( Nidd/SJL). The combined diabetogenic effects of Nidd/SJL and Nob1 were markedly enhanced by a high-fat diet, whereas that of Nidd/SJL alone was independent of the dietary fat content.

The complement factor 5a receptor gene maps to murine chromosome 7

Complement factor 5a (C5a) promotes local inflammation and is a potent chemoattractant for neutrophils and macrophages. We had an interest in C5a and its receptor, C5r1, because we previously identified C5a as a positional candidate gene for the quantitative trait locus Abhr2, which determines allergen-induced bronchial hyperresponsiveness in our murine model of asthma. To study the significance of C5r1 in our asthma model we first had to determine its genomic map location in mice. Genomic sequence surrounding murine C5r1 was analyzed for polymorphisms and two variable microsatellites were identified. These microsatellites were genotyped in A/J × (C3H/HeJ × A/J)F1 backcross mice (n = 355) and mapped in a panel of 164 markers spaced at approximately 10 cM intervals throughout the genome. Multipoint linkage analysis placed C5r1 on murine chromosome 7, 3.9 cM from the top of the linkage group. This map location has been previously identified as containing an additional quantitative trait locus for allergen-induced airway hyperresponsiveness, Abhr3, in this population of mice.

Genetic contributions of nonautoimmune SWR mice toward lupus nephritis.

(SWR x New Zealand Black (NZB))F(1) (or SNF(1)) mice succumb to lupus nephritis. Although several NZB lupus susceptibility loci have been identified in other crosses, the potential genetic contributions of SWR to lupus remain unknown. To ascertain this, a panel of 86 NZB x F(1) backcross mice was immunophenotyped and genome scanned. Linkage analysis revealed four dominant SWR susceptibility loci (H2, Swrl-1, Swrl-2, and Swrl-3) and a recessive NZB locus, Nba1. Early mortality was most strongly linked to the H2 locus on chromosome (Chr) 17 (log likelihood of the odds (LOD) = 4.59 - 5.38). Susceptibility to glomerulonephritis was linked to H2 (Chr 17, LOD = 2.37 - 2.70), Swrl-2 (Chr 14, 36 cM, LOD = 2.48 - 2.71), and Nba1 (Chr 4, 75 cM, LOD = 2.15 - 2.23). IgG antinuclear autoantibody development was linked to H2 (Chr 17, LOD = 4.92 - 5.48), Swrl-1 (Chr 1, 86 cM, colocalizing with Sle1 and Nba2, LOD = 2.89 - 2.91), and Swrl-3 (Chr 18, 14 cM, LOD = 2.07 - 2.13). For each phenotype, epistatic interaction of two to three susceptibility loci was required to attain the high penetrance levels seen in the SNF(1) strain. Although the SWR contributions H2, Swrl-1, and Swrl-2 map to loci previously mapped in other strains, often linked to very similar phenotypes, Swrl-3 appears to be a novel locus. In conclusion, lupus in the SNF(1) strain is truly polygenic, with at least four dominant contributions from the SWR strain. The immunological functions and molecular identities of these loci await elucidation.

Allelic Loss Mapping and Physical Delineation of a Region Harboring a Thymic Lymphoma Suppressor Gene on Mouse Chromosome 16

Our previous mapping of allelic loss in γ-ray induced thymic lymphomas in F1 hybrid and backcross mice between BALB/c and MSM strains identified three regions with high frequencies of allelic loss which probably harbor a tumor suppressor gene. One region, Tlsr7, exists near the D16 Mit122 locus on chromosome 16. This study has further localized Tlsr7 by constructing a physical map and scanning a total of 587 thymic lymphomas. The map consists of 13 overlapping BAC clones and isolation of BAC-derived polymorphic probes leads to fine mapping of allelic losses. Eleven lymphomas show informative breakpoints of allelic loss regions relative to the flanking markers on the map. Pulsed-field gel electrophoresis of NotI digests of the clones shows that the commonly lost region is localized within an approximately 300 kb interval near D16Mit192. This map is invaluable to facilitate the identification of genes in the Tlsr7 region.

Quantitative trait loci for obesity and insulin resistance (Nob1, Nob2) and their interaction with the leptin receptor allele (LeprA720T/T1044I) in New Zealand obese mice.

To locate genes responsible for obesity and insulin resistance, a backcross model of New Zealand obese (NZO) mice with the lean Swiss/Jackson Laboratory (SJL) strain was stablished. In female NZO x F1 backcross mice, two major quantitative trait loci for variables of obesity (body weight, body mass index, total body fat) and insulin resistance (hyperinsulinaemia) were identified on chromosomes 5 (Nob1) and 19 (Nob2) close to the markers D5Mit392 and D19Mit91. The aberrant alleles have presumably contributed by the NZO genome. Whereas Nob1 contributed mainly to higher body weight, Nob2 seemed to mainly aggravate insulin resistance independent of obesity. The leptin receptor variant of NZO (LeprA720T/T1044I) failed to alter any of the variables of obesity. It seemed, however, to enhance the effect of Nob1 on body weight and that of Nob2 on serum insulin concentration. When expressed in COS-7 cells, LeprA720T/T10441 produced a normal basal and maximum activation with a minor increase in the EC50 of leptin. The data identify two new quantitative trait loci that are responsible for a major part of obesity and hyperinsulinaemia as produced by recessive genes in NZO mice. LeprA720T/T1044I alone cannot produce obesity, but may enhance the effects of other obesity/insulin resistance genes in this mouse model.

Type 2 diabetes-like hyperglycemia in a backcross model of NZO and SJL mice: characterization of a susceptibility locus on chromosome 4 and its relation with obesity.

A backcross model of New Zealand obese mice (NZO) with the lean, atherosclerosis-resistant SJL strain was established to locate genes responsible for obesity, insulin resistance, and type 2 diabetes-like hyperglycemia. In male NZO x F1 backcross mice, a major susceptibility locus for the development of hyperglycemia and hypoinsulinemia (Nidd/SJL) was identified on chromosome 4 between the markers D4Mit278 and D4Mit232, 10-28 cM distal of the previously described Nidd1 locus. The diabetogenic allele has presumably been contributed by the SJL genome, and it appeared to be responsible for approximately 60% of the total prevalence of hyperglycemia. The presence of Nidd/SJL did not alter body weight or weight gain by week 12. Thereafter, it was associated with reduced weight gain or weight loss, presumably as a consequence of decompensated hyperglycemia. In all male backcross mice, the prevalence of hyperglycemia at week 22 increased with the body weight at week 12, suggesting that the development of hyperglycemia was dependent on the degree of obesity. In the absence of Nidd/SJL, mice weighing <50 g at week 12 did not develop hyperglycemia by week 22. In contrast, in animals carrying the diabetogenic allele, the prevalence of hyperglycemia was 20 and 64% when the 12-week weight was <45 and 45-50 g, respectively. These data are consistent with the conclusion that Nidd/SJL represents a diabetes gene that lowers the obesity threshold for the development of hyperglycemia and hypoinsulinemia.

Genome analysis of collagen disease in MRL/ lpr mice: polygenic inheritance resulting in the complex pathological manifestations

MRL/MpJ- lpr/ lpr (MRL/lpr) mice develop collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren’s syndrome, respectively. In the previous studies, we observed genetic segregation of these complex pathological manifestations throughout the genome recombination with a C57Bl/6- lpr/ lpr or a C3H/HeJ- lpr/ lpr (C3H/lpr) strain of mice which rarely develops such lesions, indicating that development of collagen disease is dependent on an MRL host genetic background. To clarify the mode of inheritance and the gene loci affecting four types of the lesions in MRL/lpr mice; vasculitis, glomerulonephritis, arthritis and sialoadenitis, a genetic dissection of the lesions was carried out by using MRL/lpr, C3H/lpr, (MRL/lpr×C3H/lpr) F1 intercross, and MRL/lpr×(MRL/lpr×C3H/lpr) F1 backcross mice. Definition of each lesion was performed by histopathology under light microscopy, and genomic DNA of the backcross mice were subjected to association studies by chi-square analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. We observed that gene loci recessively associated with each lesion were mapped on different chromosomal positions. We conclude that each of four types of the lesions in MRL/lpr mice is under the control of different set of genes, suggesting the complex pathological manifestations of collagen disease result from polygenic inheritance.

Genetic basis of autoimmune sialadenitis in MRL/lpr lupus-prone mice: additive and hierarchical properties of polygenic inheritance.

To clarify the mode of inheritance of autoimmune sialadenitis in MRL/MpJ-lpr/lpr (MRL/lpr) lupus-prone mice and identify the susceptibility loci. MRL/lpr, C3H/HeJ-lpr/lpr (C3H/lpr), (MRL/lpr x C3H/lpr)F1 intercross, and MRL/lpr x (MRL/lpr x C3H/lpr)F1 backcross mice were prepared, and sialadenitis in individual mice was analyzed by histopathologic grading. The genomic DNA of the backcross mice was examined by simple sequence-length polymorphism analysis, and the highly associated polymorphic microsatellite markers with sialadenitis were determined as sialadenitis susceptibility loci. Four susceptible gene loci recessively associated with sialadenitis were mapped on chromosomes 10, 18, 4, and 1, respectively. These loci manifested additive and hierarchical properties in the development of sialadenitis. The results indicate that sialadenitis in MRL/lpr mice is under the control of polygenic inheritance, possibly involving allelic polymorphism.

Genetic dissection of the complex pathological manifestations of collagen disease in MRL/lpr mice.

An MRL strain of mice bearing a Fas-deletion mutant gene, lpr, MRL/MpJ-lpr/lpr (MRL/lpr) develops collagen disease involving vasculitis, glomerulonephritis, arthritis and sialoadenitis, each of which has been studied as a model for polyarteritis, lupus nephritis, rheumatoid arthritis and Sjögren's syndrome, respectively. Development of such lesions seems dependent on host genetic background since the congenic C3H/HeJ-lpr/lpr (C3H/lpr) mice rarely develop them. To identify the gene loci affecting each lesion, a genetic dissection of these complex pathological manifestations was carried out. First, histopathological features in MRL/lpr, C3H/lpr, (MRL/lpr x C3H/lpr) F1 intercross, and MRL/lpr x (MRL/lpr x C3H/lpr) F1 backcross mice were analyzed. Genomic DNA of the backcross mice were subjected to association studies by Chi-squared analysis for determining which polymorphic microsatellite locus occurs at higher frequency among affected compared to unaffected individuals for each lesion. As a result, gene loci recessively associated with each lesion were mapped on different chromosomal positions. We concluded that each of these lesions in MRL/lpr mice is under the control of a different set of genes, suggesting that the complex pathological manifestations of collagen disease result from polygenic inheritance.

Allelic loss mapping and physical delineation of a region harboring a putative thymic lymphoma suppressor gene on mouse chromosome 12.

Our previous allelic loss analysis of gamma-ray induced thymic lymphomas in F1 hybrid and backcross mice between BALB/c and MSM strains mapped the Tlsr4 region exhibiting a high frequency of allelic loss (62%) to a 2.9 cM interval between the markers D12Mit53 and D12Mit279 on mouse chromosome 12. To narrow further the interval harboring a putative tumor suppressor gene, a high-density scan has been carried out for informative 361 thymic lymphomas. Construction of a physical map of Tlsr4 with 3 YAC and 15 BAC clones and isolation of YAC- and BAC-derived polymorphic probes lead to fine allelic loss mapping. Three successive polymorphic sites within one BAC exhibit the retention of both alleles in seven, one and four lymphomas, suggesting that a common region of allelic loss for Tlsr4 exists within the BAC region. Pulsed-field gel electrophoresis of NotI digests of this and other clones determines that the commonly lost region is a 35 kb interval with a NotI site. NotI sites are frequently associated with coding regions, and our preliminary sequencing has identified ESTs in the region. Thus, the present study facilitates the identification of genes in the Tlsr4 region that would lead to isolation of a novel tumor suppressor gene.

Specificity of loss of heterozygosity in radiation-induced mouse myeloid and lymphoid leukaemias

Purpose : To determine whether loss of heterozygosity (LOH) at specific chromosomal loci in radiation-induced leukaemias, arising in a similar genetic background, is leukaemia-type specific (myeloid versus lymphoid) or common to both. Materials and methods : Leukaemias that arose in 3Gy X-irradiated (CBA/H C57BL/6)F1 intercross and backcross mice were diagnosed as acute myeloid leukaemia (AML) or thymic lymphoma (TL). LOH was determined using 28 polymorphic microsatellite markers distributed over seven chromosomes using control and leukaemic DNA from individual mice. Results : LOH incidences of 0-20% were observed at most loci in both leukaemia types. Specific LOH incidences of 38-76% were observed for myeloid (chromosome 2) and lymphoid (chromosomes 11 and 14) leukaemias. Chromosome 4 LOH was frequently (38-50%) observed in both types, although the commonly deleted regions differed. LOH was detected at either chromosome 2 or 4 in AML and either chromosome 4 or 11 in TL. Conclusions : LOH incidences of 38-76% suggest a causal role of particular loci which is mainly, but not exclusively, dependent on leukaemia type. LOH incidences of 0-20% at other loci in both leukaemias suggest that many genetic deletions are noncausal and incidental in radiation-leukaemogenesis.

Vasculitis-susceptible genes in mice with a deficit in Fas-mediated apoptosis

Autoimmune diseases show complex pathological manifestations, which frequently involve systemic vasculitis. This complication is understood to be a manifestation of advanced disease, or to represent distinct entities, restricted by genetic and/or environmental factors. An MRL/Mp strain of mice bearing the Fas deletion mutant gene, lpr (MRL/lpr), spontaneously develop systemic vasculitis coincidentally with glomerulonephritis, arthritis and sialoadenitis, but a C3H/HeJ- lpr/lpr (C3H/lpr) strain does not. Thus, this is a suitable model for analyzing the genetic basis of vasculitis in autoimmune diseases. To genetically dissect these complex pathological manifestations, a linkage analysis of each lesion with polymorphic microsatellite markers was performed by using MRL/lpr×(MRL/lpr×C3H/lpr)F1 backcross mice. Vasculitis-susceptible gene loci were mapped on chromosomes 3 and 4, which were not associated with glomerulonephritis, arthritis and sialoadenitis. These results indicate that systemic vasculitis in MRL/lpr mice may be under the control of host genes which are different from those for other autoimmune diseases.