Genetic dissection of the Mss4 locus mediating sex-biased cancer resistance in the rat peripheral nervous system.

The incidence of neural tumors is sexually dimorphic in both, humans and rodents. The identification of genetic determinants contributing to sex-biased tumor development is an essential prerequisite for differential tumor prevention in males and females. F2 hybrids of inbred BDIV and BDIX rats, resistant and susceptible, respectively, to ethylnitrosourea-induced malignant peripheral nerve sheath tumors (MPNST) display a marked sex bias regarding tumor risk. Homozygous BDIV alleles at the Mss4 locus (90.9-111.2 Mb, chromosome 6) mediate MPNST resistance exclusively in female F2 rats according to a genome wide association analysis. This locus was functionally confirmed and fine mapped through MPNST induction in males and females of three congenic rat strains (BDIX.BDIV-Mss4a, b, d). As a consequence, it could be subdivided in Mss4.1 (98.8-99.7 Mb) mediating cancer resistance, and Mss4.2 (99.7-111.2 Mb) enhancing sex specificity. Positional candidate genes were selected through DNA sequencing and expression profiling using RNAs from trigeminal nerve tissue of parental and congenic male and female animals. The transregulatory fingerprint of BDIV or BDIX alleles at Mss4.1 and/or Mss4.2, respectively, provided insight into the processes influencing cancer risk in a sex-biased way. A group of genes, a fraction of which involved in Schwann cell differentiation, showed low, male-biased expression in nerve tissues under the control of BDIX susceptibility alleles, but high, female-biased transcript levels when controlled by BDIV resistance alleles at Mss4. The Esr2 gene located in Mss4.1 constitutes an interesting functional candidate together with a yet unidentified gene/enhancer in Mss4.2.

Ablation of T cell immunity differentially influences tumor risk in inbred BD rat strains.

Inbred rat strains BDIX and BDIV are constitutionally susceptible and resistant, respectively, to the development of malignant peripheral nerve sheath tumors (MPNST) induced by neonatal exposure to N-ethyl-N-nitrosourea (EtNU). They represent a model system for analysis of molecular and cellular processes underlying differential cancer susceptibility. A point mutation in the Neu/ErbB-2 gene is an early marker of Schwann precursor cells at high risk of malignant conversion and is diagnostic of the resulting MPNST predominantly developing in the trigeminal nerves. Initially considerable amounts of Neu/ErbB-2-mutant cells arise in nerve tissue of both rat strains subsequently disappearing in resistant BDIV rats, but persisting and giving rise to MPNST in susceptible BDIX animals. An almost identical cellular immune response-sequentially involving macrophages, T helper- and cytotoxic T lymphocytes-is mounted in the trigeminal nerves of EtNU-treated rats of both strains. In this study, T cell maturation was prevented by neonatal thymectomy following EtNU-exposure. While resistance against MPNST development significantly decreased in BDIV rats MPNST incidence and survival time remained unaltered in thymectomized BDIX rats. Contrary to euthymic animals a number of both thymectomized BDIV and BDIX rats developed MPNST lacking the Neu/ErbB-2-mutation. This suggests that Schwann cells initiated by other genetic alterations can progress to full malignancy in immune-compromised rats only. T cell-dependent resistance against tumorigenesis originating from non-Neu/ErbB-2-mutant Schwann precursors might thus be shared by both strains while BDIV T lymphocytes additionally prevent the development of Neu/ErbB-2-mutant MPNST. Rat strain-specific differences in the interaction of T lymphocytes with (pre)malignant Neu-mutant cells may thus critically contribute to susceptibility and resistance towards EtNU-induced MPNST development.

Gender-specific polygenic control of ethylnitrosourea-induced oncogenesis in the rat peripheral nervous system.

The inbred BD rat strains constitute a model system for analysis of the genetic basis of susceptibility or resistance to the development of neural tumors, as they exhibit distinct strain-specific differences regarding the sensitivity to tumor induction by the alkylating carcinogen N-ethyl-N-nitrosourea (EtNU). Among the different BD strains, BDIX and BDIV rats, respectively, are either highly susceptible or entirely resistant to the development of EtNU-induced malignant schwannomas of the peripheral nervous system (PNS), predominantly of the trigeminal nerves. We have previously mapped one locus associated with susceptibility/resistance to schwannoma induction to the telomeric third of chromosome 10 (Mss1) in segregating (BDIX x BDIV) crosses. We report on the genetic mapping of 6 further loci controlling tumor incidence or survival time on chromosomes 1 (Mss2), 3 (Mss3), 6 (Mss4), 13 (Mss5) and 15 (Mss6) as well as on chromosome 10 (Mss7) close to the centromere. Interestingly, most of these loci mediate gender-specific effects of variable strength ranging from minor influences on tumor development to complete tumor resistance. The gender specificity is reflected by the fact that male (BDIX x BDIV) F2 rats exhibit a 2-fold higher incidence of EtNU-induced schwannomas than females as well as a shorter survival time. A number of human nervous system tumors too arise with a marked gender bias. Genes mediating gender-specific predisposition of developing malignant schwannomas in the rat may be relevant for the human individual risk of developing nervous system tumors.

A 6-Mb contig-based comparative gene and linkage map of the rat schwannoma tumor suppressor region at 10q32.3.

Frequent genetic aberrations of malignant schwannomas induced by the alkylating agent N-ethyl-N-nitrosourea in hybrids from inbred BD rat strains include allelic imbalances of the telomeric 20 Mb of chromosome 5 (Dis-2) and of the telomeric 5 Mb of chromosome 10q32 (Dis-1) in 59 and 94% of the tumors, respectively. The Dis-1 minimal loss of heterozygosity consensus region extends from D10Rat4 to the telomere and harbors a putative tumor suppressor gene(s). We constructed a 6-Mb BAC/PAC contig containing more than 70 known genes, 18 mapped microsatellites, and further ESTs/reference RNAs. A continuous block of strongly conserved synteny with mouse chromosome 11E2 and human chromosome 17q25.3 was found. Combining the sequence information from the rat and closely related syntenic regions of different mammalian species produces nearly complete gene maps as a basis for a positional candidate approach and gives insight into mammalian genomic evolution.

The circling behavior of the deafblind LEW-ci2 rat is linked to a segment of RNO10 containing Myo15 and Kcnj12.

The LEW/Ztm-ci2 rat is an autosomal recessive mutant that displays circling behavior, deafness, progressive retinopathy, locomotor hyperactivity, ataxia, and opisthotonus. We performed a genome-wide scan of a (LEW/Ztm-ci2 x BN/Ztm) F1 x LEW/Ztm-ci2 backcross population with anonymous microsatellite markers to analyze the genetics of this mutant rat. This linkage analysis demonstrated a very strong association of RNO10 SSLP markers to the phenotype with a core region in the central part of the chromosome. The knowledge of genes mapping to this part of the rat genome and their linkage to SSLP markers is still poor. We developed SSLP markers closely linked to genes, which might be responsible for the mutant phenotype by using the growing amount of rat-specific DNA sequences available at World Wide Web databases. Application of this method facilitated the search for candidate genes for the phenotype of the LEW-ci2 rat. We were able to map Myo15 and its neighboring genes, Znf179 and Aldh3a1, to the region of interest and Myo1c to a more distal location on RNO10. Further rat BAC clones were used to create a physical map of the region of interest. This map revealed the position of further genes. Among those is Kcnj12. Owing to their localization on RNO10 and their involvement in a similar pathology in human and mouse, Myo15 and Kcnj12 can be regarded as candidate genes for the deafblind phenotype of the LEW-ci2 rat.

Loss of heterozygosity in malignant rat schwannomas chemically induced in hybrids of inbred rat strains with differential tumor susceptibility.

Rats of the inbred BD strains strongly differ in their susceptibility to the induction of tumors of the central (CNS) and peripheral nervous system (PNS) by N-ethyl-N-nitrosourea (EtNU). Malignant schwannomas induced in (BDIX x BDIV) and (BDIX x BDVI) rat hybrids were analyzed to identify genetic alterations associated with EtNU-induced tumorigenesis in the PNS. EtNU-induced schwannomas exclusively exhibit an A:T T:A transversion mutation of the neu/Erbb-2 gene located on chromosome 10, with subsequent loss of the wild-type neu/Erbb-2 allele at a post-initiation stage. Targeted allelic deletion mapping previously revealed losses of heterozygosity (LOH) at the distal end of chromosome 10 in a large majority of (BDIX x BDIV) schwannomas. The aims of the present study were (i) to scan the whole genome for further LOHs; (ii) to narrow down the consensus regions of frequently occurring allelic deletions using tumors from different crosses of BD rats; and (iii) to determine the sequence of genetic alterations during schwannoma development. A limited number of (BDIX x BDIV) F(1) tumors were initially screened for LOH and microsatellite instability (MI) by amplifying 58 microsatellite markers spanning the whole genome. LOHs on chromosome 5 were detected in 9/17 tumors, with random loss of the parental alleles. Ninety-two schwannomas from different BD rat-crosses were then analyzed to solidify these data and to determine the consensus region of frequent LOHs. The results indicate that LOHs on chromosomes 10 and 5 are required for the development of EtNU-induced malignant schwannomas from immature neu/Erbb-2 mutant glial cells, and that putative tumor suppressor genes are localized on chromosome 10q32.3, corresponding to human chromosome 17q25.3, and the telomeric region of mouse chromosome 11, and on the telomeric quarter of chromosome 5. MI was detected in <0.2% of cases.