Complex interactions of new quantitative trait loci, Sluc1, Sluc2, Sluc3, and Sluc4, that influence the susceptibility to lung cancer in the mouse.

Many complex traits, including susceptibility to lung cancer, are controlled by multiple genes--quantitative trait loci (QTLs). We facilitated the mapping of QTLs by making use of recombinant congenic strains (RCS), a system of mouse inbred strains in which the genetic complexity is reduced, and by applying MQM-mapping (multiple-QTL models or marker-QTL-marker), a multilocus method with an increased power of detecting of individual QTLs and interacting QTLs (epistasis). The mouse strain O20 develops significantly larger N-ethyl-N-nitrosourea induced lung tumours than mice of the RC strain OcB-9 (ref. 5); the latter share approximately 87.5% of their genes with strain O20 and 12.5% with strain B10.O20 (refs 6,7). QTL analysis of 222 (OcB-9 x O20) F2 mice revealed four new loci that influence susceptibility to lung cancer (Sluc genes). They are involved in two significant, partly counteracting interactions which mask their individual main effects: Sluc1 (on chromosome 19) interacts with Sluc2 (chromosome 2), and Sluc3 (chromosome 6) interacts with Sluc4 (chromosome 11). Together with the data of van Wezel et al. in the accompanying report, our results indicate that interactions between tumour susceptibility genes are a common phenomenon which complicates their mapping.

Gene interaction and single gene effects in colon tumour susceptibility in mice.

To dissect the multigenic control of colon tumour susceptibility in the mouse we used the set of 20 CcS/Dem (CcS) recombinant congenic (RC) strains. Each CcS strain carries a unique, random subset of approximately 12.5% of the genome of strain STS/A (STS) on the genetic background of BALB/cHeA (BALB/c). Previously, applying a protocol of 26 injections of 1,2-dimethylhydrazine (DMH), we detected two susceptibility loci, Scc1 and Scc2, on chromosome 2 (refs 4, 5). Using a shorter tumour-induction procedure, combining DMH and N-ethyl-N-nitrosourea (ENU) treatment, we demonstrate that BALB/c, STS and most CcS strains are relatively resistant. The strain CcS-19, however, is susceptible, probably due to a combination of BALB/c and STS alleles at several loci. Analysis of 192 (BALB/c x CcS-19) F2 mice revealed, in addition to the Scc1/Scc2 region, three new susceptibility loci: Scc3 on chromosome 1, Scc4 on chromosome 17 and Scc5 on chromosome 18. Scc4 and Scc5 have no apparent individual effect, but show a strong reciprocal interaction. Their BALB/c and STS alleles are not a priori susceptible or resistant but the genotype at one locus determines the effect of the allele at the second locus and vice versa. These findings and the accompanying paper on lung tumour susceptibility show that interlocus interactions are likely to be an important component of tumour susceptibility.

Mapping a gene for combined hyperlipidaemia in a mutant mouse strain.

Familial combined hyperlipidaemia (FCHL) is a common, multifactorial disorder associated with elevated levels of plasma triglyceride, cholesterol, or both. A characteristic feature is increased secretion of very low density lipoproteins (VLDL) and apolipoprotein B (apoB). Although FCHL is the most common cause of premature coronary artery disease (CAD), accounting for over 10% of cases, its aetiology remains largely unknown. One powerful approach to the dissection of complex genetic traits involves the use of animal models. We have identified a mouse strain, HcB-19/Dem (HcB-19), which exhibits hypertriglyceridaemia, hypercholesterolaemia and elevated levels of plasma apoB. Like FCHL patients, HcB-19 mice also exhibit increased secretion of triglyceride-rich lipoproteins, and their hyperlipidaemia becomes progressively more severe with age. It is likely that the hyperlipidaemia results from a mutation of a novel gene that arose during development of strain HcB-19. We mapped the hyperlipidaemia gene (Hyplip1) to the distal portion of mouse chromosome 3. This region is syntenic to human chromosome 1q21-q23, which has recently been shown to harbour a gene associated with FCHL in families from a Finnish isolate.

Hepatocellular carcinoma in Txnip-deficient mice.

The molecular pathogenesis and the genetic aberrations that lead to the progression of hepatocellular carcinoma (HCC) are largely unknown. Here, we demonstrate that the thioredoxin interacting protein (Txnip) gene is a candidate tumor suppressor gene in vivo. We previously showed that the recombinant inbred congenic strain HcB-19 has a spontaneous mutation of the Txnip gene, and we now show that the strain has dramatically increased incidence of HCC, and that the HCC cosegregates with the Txnip mutation. Approximately 40% of the Txnip-deficient mice developed hepatic tumors with an increased prevalence in male mice. Visible tumors develop as early as 8 months of age. Histological analysis confirmed the morphology of HCC in the Txnip-deficient mice. Molecular markers of HCC, alpha-fetoprotein and p53, were increased in tumors of Txnip-deficient mice. The upregulation of p53 preceded tumor development; however, bromodeoxyuridine (BrdU) labeling of normal hepatic tissue of Txnip-deficient mice did not reveal increased cell proliferation. Finally, microarray analyses of tumor, non-tumor adjacent, and normal tissue of Txnip-deficient mice highlighted the genetic differences leading to the predisposition and onset of HCC. Our findings suggest that Txnip deficiency is sufficient to initiate HCC and suggest novel mechanisms in hepatocarcinogenesis.

Complexity of lung cancer modifiers: mapping of thirty genes and twenty-five interactions in half of the mouse genome.

BACKGROUND: Numerous low-penetrance genes control susceptibility to cancer in experimental animals, but the overall genetic information on this group of genes (i.e., number of loci and their mutual interactions) is missing. We performed a systematic search, scanning roughly half of the mouse genome for lung cancer susceptibility (Sluc) genes affecting tumor size or number by using mouse recombinant congenic (RC) strains. In each RC strain (OcB), approximately 12.5% of the genome is derived from the lung cancer-resistant strain B10.O20, whereas the rest is derived from the lung cancer-susceptible strain O20. METHODS: A total of 730 F2 hybrids from five (OcB x O20) crosses were tested. Pregnant mice were treated on day 18 of gestation with a single dose of N-ethyl-N-nitrosourea. When offspring were 16 weeks old, whole lungs were removed and sectioned semiserially, and the size of all lung tumors (n = 2658) was determined. Analysis of variance was used for detection of linkage, and models (including main effect and two-way interactions) were tested with a statistical program. RESULTS: We detected a total of 30 Sluc loci (16 new plus 14 previously reported) and 25 two-way interactions. Some of these interactions are counteracting (e.g., Sluc17 and Sluc20), resulting in the partial or total masking of the individual independent effect (main effect) of each involved locus. Seven loci (Sluc1, Sluc5, Sluc12, Sluc16, Sluc18, Sluc20, and Sluc26) and two interactions (Sluc5 x Sluc12 and Sluc5 x Sluc26) were detected in more than one RC strain. CONCLUSIONS: The extrapolation of our results to the whole genome suggests approximately 60 Sluc loci (90% confidence intervals = 42 to 78). Despite the genetic complexity of lung cancer, use of appropriate mapping strategies can identify a large number of responsible loci and can reveal their interactions. This study provides an insight into the genetic control of lung tumorigenesis and may serve as a paradigm for investigating the genetics of other cancer types.

H-2-dependent regulation of the high level of expression of ecotropic murine leukemia virus.

Adult B10.Y mice, which are congenic with C57BL/10ScSn ((B10) mice for the H-2 region, expressed a high titer of infectious ecotropic virus in the spleen. F1 hybrids between B10.Y and B10 mice were negative or had very low levels of virus expression. In (B10.Y x B10)F2 segregant mice, the high virus phenotype segregated with the H-2pa haplotype of B10.Y, whereas the virus-negative phenotype was associated with the H-2b haplotype of B10. Molecular hybridization experiments with a selected ecotropic AKR murine leukemia virus cyclic DNA probe indicated that both partner strains possessed ecotropic virus sequences and that the number of sequences present was the same in B10.Y mice as in B10 mice. This finding excluded the possibility that the H-2-related effect might be due to the presence of additional viral structural genes within or close to the H-2 region of B10.Y mice. The level of expression of this endogenous ecotropic virus was therefore affected by regulatory genes of the H-2 region.

Glucocorticoid hormone effect on transplacental carcinogenesis and lung differentiation: influence of histocompatibility-2 complex.

In the mouse, the histocompatibility-2 (H-2) haplotype influences induction of lung and intestinal tumors by N-ethyl-N-nitrosourea (ENU) treatment of fetuses or infant mice. The differentiation of lung and intestinal epithelium is known to be regulated by glucocorticoids. We show that glucocorticoid-induced development of alveolar lung volume is H-2 influenced and that glucocorticoid treatment of fetuses also influences prenatal ENU induction of lung and intestinal tumors. These glucocorticoid effects on tumorigenesis are also H-2 influenced. The number of papillary lung tumors increased in B10 (H-2b) and decreased in B10.A (H-2a) mice. In the intestine, the number of tumors increased in H-2b females and decreased in H-2b males. In H-2a mice, the number of intestinal tumors was unchanged but their location was altered. We propose that the H-2 complex influences tumorigenesis in lung and small intestine by affecting the hormonal regulation of differentiation of target epithelial cells.

A gene for susceptibility to small intestinal cancer, ssic1, maps to the distal part of mouse chromosome 4.

Mouse inbred strains B10.O20 and O20 were used to study the genetics of susceptibility to small intestinal cancer. Strain B10.O20 is susceptible to tumors of the small intestine induced by a transplacental treatment with the carcinogen N-ethyl-N-nitrosourea on day 18 of gestation, whereas strain O20 is completely resistant. F1 hybrid mice are also completely resistant, indicating that homozygosity of the B10.O20 allele of at least one susceptibility gene is required for the development of tumors of the small intestine. To map this gene, we used backcross 2 (N3) mice. We tested 283 [((B10.O20 x O20)F1 x B10.O20) x B10.020] N3 mice, 67 of which developed tumors of the small intestine. Their genotype was determined with the use of more than 85 simple sequence length polymorphisms (SSLPs) spread over the genome. If a SSLP is linked closely to a susceptibility gene, it is expected that almost none of the N3 mice with small intestinal tumors are heterozygous for this marker. We found that very few tumor-bearing N3 mice were heterozygous for SSLP markers on the distal part of chromosome 4 compared with the N3 mice without small intestinal tumors. This difference is highly significant (chi 2 = 31 for D4Mit158). Therefore, one of the genes that influence susceptibility to small intestinal cancer, ssic1, maps to the distal part of chromosome 4.

High frequency of interactions between lung cancer susceptibility genes in the mouse: mapping of Sluc5 to Sluc14.

Although several genes that cause monogenic familial cancer syndromes have been identified, susceptibility to sporadic cancer remains unresolved. Animal experiments have demonstrated multigenic control of tumor susceptibility. Recently, we described four mouse lung cancer susceptibility (Sluc) loci, the main effects of which are masked by their mutual interactions. Because such interactions can considerably affect the strategies for identification of cancer susceptibility genes in humans, it is necessary to establish whether they are common or rare. Here, we report the mapping of 10 additional Sluc loci and show that 13 of the 14 Sluc loci are involved in one or more interactions, demonstrating that interactions of tumor susceptibility genes are frequent and that they probably form complex networks.

Influence of mouse major histocompatibility complex (H-2) on N-ethyl-N-nitrosourea-induced tumor formation in various organs.

The influence of the major histocompatibility complex (MHC) of the mouse (H-2) on carcinogen-induced tumorigenesis was investigated. Mice of five H-2 congenic strains on the C57BL/10 background were treated with the direct-acting carcinogen N-ethyl-N-nitrosourea at the age of 15 days, and examined for tumors when moribund. Significant differences between strains in susceptibility to N-ethyl-N-nitrosourea-induced tumors in lung, small intestine, and liver were found. For lung tumors the strains B10.A and 2R were most susceptible, the strains 4R and B10 were relatively resistant. The strain 5R was intermediate. Susceptibility to small intestine tumors was highest in the strain 2R, intermediate in the strain B10.A, the strains 4R, 5R, and B10 were relatively resistant. The location of the tumors in the intestine was also affected by H-2. In the strain 2R most tumors are located in the proximal part, in 4R in the distal part. Tumorigenesis in the liver was highest in the strain 2R, intermediate in the strains B10.A, 4R, and B10, and lowest in the strain 5R. We conclude that susceptibility to carcinogen-induced tumors in the lung, small intestine, and liver in congenic strains on the C57BL/10 background is H-2 haplotype dependent. Susceptibility to tumors in the lung and intestine has a similar strain distribution, but differs from that for liver tumors. Males were more susceptible than females in the strain B10 (lung tumors) and 4R (small intestine and liver tumors). This indicates haplotype- and organ-specific, sex-related influences on tumor development. The possible mechanism(s) of H-2 effects on chemically induced tumorigenesis are discussed. Apart from the well-known immunological functions of the MHC, the involvement of hormonally related effects of the MHC is considered as well.

Different genetic susceptibility to aberrant crypts and colon adenomas in mice.

Aberrant crypt foci (ACFs) are the earliest identifiable epithelial lesions thought to precede the development of a subset of colon tumors. To assess their predictive value to adenoma development, we have tested in mice whether the development of ACFs and adenomas is controlled by the same genes. Therefore we used the CcS/Dem series of recombinant congenic strains, in which the effect of multiple susceptibility genes might be studied separately. We investigated susceptibility to ACFs in nine CcS/Dem strains and their parental strains, BALB/cHeA and STS/A, 4 weeks after s.c. injection of 1,2-dimethylhydrazine (20 mg/kg body weight). For the strains BALB/cHeA, STS/A, and CcS-19, we also examined the number of ACFs 2, 8, and 12 weeks after treatment. Susceptibility to adenomas was measured as the number of adenomas 6 weeks after 26 weekly s.c. injections of 1,2-dimethylhydrazine (15 mg/kg body weight). The nine CcS/Dem strains, the BALB/ cHeA strain, and the STS/A strain exhibited different patterns of susceptibility to ACFs and adenomas, demonstrating that different subsets of susceptibility genes are involved. Therefore, in evaluating the role of ACFs as a predictive marker for adenoma development, genetic factors must be taken into account.

Four new colon cancer susceptibility loci, Scc6 to Scc9 in the mouse.

Germ-line mutations in APC and mismatch repair genes explain only a small percentage of all colorectal cancer cases. We have used the recombinant congenic strain mouse model to find new loci that are involved in the control of susceptibility to colon cancer. Five different colon cancer susceptibility genes, Scc1-Scc5, have been described previously using the recombinant congenic strains. Two of these loci, Scc4 and Scc5, show a reciprocal, genetic interaction. Here we report the mapping of four new colon tumor susceptibility genes: (a) Scc6 on chromosome 11; (b) Scc7 on chromosome 3; (c) Scc8 on chromosome 8; and (d) Scc9 on chromosome 10. Scc7 and Scc8 show a genetic interaction; Scc7 is only detected by virtue of its interaction with Scc8.

Scc-1, a novel colon cancer susceptibility gene in the mouse: linkage to CD44 (Ly-24, Pgp-1) on chromosome 2.

Mutations of proto-oncogenes and tumor-suppressor genes lead to neoplastic development. Some germline mutations of these genes increase the tumor susceptibility of their carriers, but the relationship between genes controlling tumor susceptibility and the known oncogenes and tumor-suppressor genes remains unelucidated. Moreover, as tumor susceptibility in mouse is controlled by multiple genes, their identification has been virtually impossible. We therefore developed a new system, the recombinant congenic strains (RCS), which separates individual susceptibility genes into different RC strains, thus facilitating their analysis. To map genes controlling the development of colon cancer, we used the Balb/c-c-STS (CcS/Dem) RC strains. Owing to several unidentified genes, Balb/cHeA mice are relatively resistant and STS/A mice highly susceptible to 1,2-dimethylhydrazine-(DMH)-induced colon adenocarcinomas. Each CcS/Dem strain carries a different subset of about 12.5% of genes of the STS strain on the Balb/c background, and individual STS susceptibility genes became segregated into different RC strains. Using CcS-19, one of the highly susceptible RC strains, we mapped a novel colon tumor susceptibility gene, Scc-1, different from the oncogenes and tumor-suppressor genes known to be involved in colon tumorigenesis, in the vicinity of CD44 (Ly-24, Pgp-1) on chromosome 2. The mapping of the Scc-1 gene indicates that the RCS system can be used to map and study the presently unknown genes which control cancer development.

Kras-2 alleles, mutations, and lung tumor susceptibility in the mouse--an evaluation.

Lung Tumor Susceptibility (LTS) in the mouse has been shown to be influenced by loci within the Major Histocompatibility Complex (MHC) and the Kras-2 regions. In crosses between susceptible and resistant strains, one allele of Kras-2 has been linked with a high LTS, whereas the other allele has been linked with a low LTS. Furthermore, these Kras-2 alleles affect differently the occurrence of Kras-2 mutations in lung tumors. In this study we evaluated the relationship between LTS, Kras-2 alleles and Kras-2 mutations, using Recombinant Congenic Strains with identical MHC haplotypes. The Kras-2 region indeed influences the frequency of Kras-2 mutations in N-ethyl-N-nitrosourea-induced lung tumors. These mutations are associated with larger tumors. However, Kras-2 affects LTS only marginally. Several strains with identical Kras-2 alleles and mutation frequency differ widely in LTS, while some strains with different alleles and mutation frequency do not. We conclude that a considerable part of the genetic variability in LTS is caused by LTS-loci other than Kras-2 and MHC.

Modulations of glucocorticoid-induced apoptosis linked to the p53 deletion and to the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1).

We have analysed the effects of p53 and of the apoptosis susceptibility gene Rapop1 (Radiation-induced apoptosis 1) located on chromosome 16 on glucocorticoid- and radiation-induced in vivo apoptosis of thymocytes. For those analyses, we used Rapop1 semicongenic mice heterozygous for the STS and BALB/cHeA alleles in the chromosomal segment containing Rapop1 in the BALB/cHeA background, mice bearing a p53 deficient allele in the BALB/cHeA background and the genetic crosses between these mice. The p53 wild type mice with a STS/A allele at the Rapop1 locus were less susceptible to both radiation- and glucocorticoid-induced apoptosis than those with homozygous BALB/cHeA alleles at this locus. Surprisingly, glucocorticoid-induced apoptosis was enhanced in the p53 hemizygous mice and considerably increased in the p53 nullizygous mice. In contrast, a sizable reduction of radiation-induced apoptosis was seen in the p53 hemizygous mice. The low susceptiblity to glucocortocoid-induced apoptosis linked to the STS allele of Rapop1 was less pronounced in the p53 hemizygous mice and a diminished effect of Rapop1 on radiation-induced apoptosis was seen in these mice. Although it remains to be established whether the genes modulating glucocortocoid-induced apoptosis are identical to p53 and Rapop1, our data suggest that p53 and Rapop1 may participate in glucocorticoid-induced apoptosis of thymocytes.

Haplotype-specific interactions of non-H-2-linked genetic factors controlling the mouse C4 and Slp protein levels.

The influence of non-H-2 linked genes on the plasma levels of the H-2 S-region encoded proteins C4, Slp, and factor B was tested in Recombinant Inbred (RI) strains. The A X B and B X A RI strains exhibit a continuous range of C4 and Slp levels from very high to very low which reach beyond the levels of their parental strains, C57BL/6J and A/J, indicating involvement of several trans-regulatory (non-H-2-linked) genes. Only limited variation in levels of factor B has been found. No linkage relationship could be established for the trans-regulatory genes, because more than one gene is involved. A complex interaction of H-2 haplotype, genetic background, sex, and possibly maternal effect in determining the C4 and Slp protein plasma levels has been observed. The H-2-dependent sex effect is evident, because males have higher C4 levels than females in RI strains with H-2b but not with H-2a haplotype. This sex effect is also background dependent, because it is present in the H-2b congenic strain on A background (A.BY) but not in C57BL/10 and C57BL/6 (both H-2b). Mice from RI strains with H-2b haplotype have in general higher C4 levels than mice with H-2a haplotype.

H-2 antigenic specificities controlled by the translocation chromosome T190.

The H-2 antigens determined by the T(1,17)190Ca translocation chromosome were analyzed serologically. It is shown that this chromosome determines previously unknown specificities H-2.76, H-2.77, H-2.78 and the "private" specificity, H-2.108.

Qualitative and quantitative aspects of anti-H-2Ld sera.

Two anti-H-2Ld sera were analyzed, BALB/c-H-2dm2 anti-BALB/cBy and (C3H X BALB/c-H-2dm2)F1 anti-BALB/cHe, the latter also containing anti-Qa antibodies. Their reaction patterns were compared with an anti-Qa serum (C3H X BALB/cBy)F1 anti-BALB/cHe. Four H-2 specificities could be detected by the anti-H-2Ld sera, two already known (H-2.64, H-2.65) and two new specificities (H-2.81, H-2.82). According to their reaction pattern H-2.64, H-2.81, and H-2.82 can be regarded as members of the H-2.28 family of specificities. A quantitative difference in the expression of these H-2 specificities exists in different haplotypes. The cells of the strain against which the sera were made (BALB/cHe and BALB/cBy, respectively) did not give the highest titers with the antisera and had a relatively low absorbing capacity. The H-2dx haplotype carries two new specificities of the H-2.28 family, namely, H-2.81 and H-2.82. Lysostrip tests showed that the antibodies against those specificities cap the H-2.1-positive H-2Ddx molecules, suggesting that these molecules may react with both anti-H-2.1-like and anti-H-2.28-like antibodies. The H-2 specificities detected by the BALB/c-H-2dm2 anti-BALB/cBy serum were detected also in liver, kidney, spleen, heart, and lung tissue. New information on the strain distribution of Qa-2 was obtained from the experiments and a quantitative difference in Qa-2 antigens between H-2 congenic strains was observed as well. The H-2b strains react with these antibodies with higher titers than the strains carrying the H-2d haplotype.

Susceptibility to tuberculosis as a complex genetic trait: analysis using recombinant congenic strains of mice.

Previous advances in the genetics of infectious diseases derived principally from identification of single genes and their isolated effects on the progression of infection. Modern genetic analysis represents a powerful means of understanding the interplay among different pathways activated in the course of infection, their hierarchy and interactions in terms of the development of an optimal protective strategy. By utilizing both whole-genome scanning of (C3HxC57BL/6)F2 and a set of the recombinant congenic strains, produced by backcrossing B10 onto a C3H background, we demonstrated that susceptibility to tuberculosis is a multigenic trait. We have identified two distinct groups of susceptible mice: one that dies within four to six weeks of infection (supersusceptible) and another that dies within seven to 10 weeks (comparable to the susceptible parental strain). Our preliminary genetic analysis suggests that the susceptibilty of those groups is controlled by different genetic factors. Supersusceptible mice exhibit dramatic lung pathology, not observed in either parental strain, and their survival after infection with virulent Mycobacterium tuberculosis is comparable to that of mice rendered immunodeficient by disruption of essential immune genes. Further genetic and functional analyses of these strains offer possibilities for understanding the control of transmission, preferential growth of the pathogen in the lung, and mechanisms of local and systemic protective immune responses.