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Several genetic variants act as modifiers of testicular germ cell tumor (TGCT) susceptibility in the 129/Sv mouse model of human pediatric TGCTs. One such modifier, the Steel locus, encodes the transmembrane-bound and soluble ligand of the kit receptor. Some (Sl and SlJ) but not all (Sld) mutations of the Steel locus increase TGCT incidence in heterozygous mutant mice. Because Sl and SlJ are large deletions that affect multiple transcripts and Sld is an intragenic deletion of the kit ligand (Kitl) from which only the soluble protein is produced, it was uncertain whether Kitl or a neighboring gene is a modifier of TGCT susceptibility. We tested the effect of the small Steel grizzle-belly (Slgb) deletion on TGCT susceptibility to determine whether Kitl is a TGCT modifier gene. An increase in TGCT incidence was observed in Slgb/+ heterozygotes and fine-mapping of the deletion breakpoints revealed that Kitl is the only conventional gene deleted by the mutation, suggesting that Kitl is the TGCT modifier gene at the Steel locus. Additionally, we propose that soluble KITL in Sld/+ heterozygous mutant mice complements a dosage effect of transmembrane-associated KITL on TGCT susceptibility and that the kit receptor (Kit) is haplosufficient for primordial germ cell (PGC) development.
Testicular germ cell tumors (TGCTs) are the most common cancer affecting young men (1). Although both environmental and genetic factors contribute to TGCT susceptibility, the genetic component of TGCTs is particularly strong. Statistically, 25% of susceptibility to early-onset TGCTs is attributable to genetic factors, making these tumors the third most heritable form of cancer (2). Despite the strong heritable influence on TGCT incidence and considerable effort to identify susceptibility genes, the genetic basis for TGCTs remains elusive. A recent study concluded that many loci with weak effects control TGCT susceptibility, and to date only one genetic factor, the rare gr/gr Y chromosome deletion, is reproducibly associated with TGCT incidence in humans (3-5).
In mice, spontaneous TGCTs occur at an appreciable frequency only on the 129 inbred background (6). TGCTs in mice are most similar to human pediatric TGCTs and arise during embryogenesis as a result of abnormalities in the development of the primordial germ cell (PGC) lineage, the embryonic precursor of oocytes and sperm (7, 8). As in humans, the genetics of TGCT susceptibility is complex with as many as 6-8 segregating genes controlling susceptibility in mouse crosses (6, 9). In segregating crosses between 129 and other inbred strains the frequency of affected males is less than 0.01% (9). Thus, TGCT susceptibility in mice is too complex for conventional genetic approaches.
Modifier genes are an attractive alternative to the limited power of conventional genetic approaches to dissect the genetic basis of highly complex traits. Several single gene mutations and genetic variants modify TGCT susceptibility when congenic on the 129/Sv background (10). Such modifiers have been useful in defining genetic interactions that control TGCT susceptibility and identifying susceptibility loci in sensitized polygenic trait analyses (11-14).
The Steel (Sl) and White-spotted (W) variants are a group of classic spontaneous mouse mutations affecting the kit ligand (KITL) and its receptor (KIT), respectively (15-19). The KITL/KIT system promotes the survival, proliferation and migration of hematopoietic, melanocyte and germ cell precursors (20). In homozygous Sl and W mice, defects in the development of these progenitor cells causes anemia, loss of pigmentation, and infertility (20). In addition, many homozygous Sl and W embryos die after embryonic day 15.5 (E15.5) (18). Heterozygotes for most of the Sl and W mutations present less severe phenotypes than homozygotes (15). To test whether mutations in the KITL/KIT system increase TGCT susceptibility, various Sl and W mutations were transferred to the 129/Sv background and TGCT incidence was measured in heterozygous mutants and their wild-type siblings (6, 9). Interestingly, some (Sl and SlJ) but not all (Sld) Steel mutations, and none of the tested W mutations, increase TGCT susceptibility in heterozygotes (Table 1).
Variation among the Sl and W alleles was initially puzzling because it was difficult to understand how increased TGCT susceptibility could be limited to only two variants of this ligand-receptor complex. Discovery of the molecular nature of the Steel mutations provided clues. Sl and SlJ are large deletions (~973 kb and ~650 kb, respectively) that remove all of Kitl and a significant amount of flanking sequence (Fig. 1A) (18, 21). By contrast, Sld is a 4kb intragenic deletion of Kitl that retains expression of soluble but not transmembrane KITL at wild-type levels (Fig. 1A) (22, 23). Therefore, two explanations are possible for increased TGCT incidence in Sl and SlJ but not Sld mutant mice: 1) a gene or non-coding regulatory element neighboring Kitl is also deleted in Sl and SlJ mutants and is responsible for increased TGCT susceptibility, or 2) gene dosage effects of soluble Kitl on TGCT susceptibility reduces TGCT incidence to 129/Sv levels in Sld mutants.
The Steel grizzle-belly (Slgb) mutation is a smaller deletion (~120 Kb) than Sl or SlJ, with the 5′ breakpoint approximately 60-40 Kb proximal to exon 1 of Kitl and the distal breakpoint within the 3′UTR of Kitl (Fig. 1A) (18). Preliminary sequence analysis of the Slgb deletion map suggested that Kitl may be the only gene deleted by this mutation. Therefore, the Slgb mutation provides a means to test the influence of a Kitl null deletion on TGCT susceptibility independent of alterations in other genes. In this report, we tested the influence of heterozygosity for the Slgb mutation on spontaneous TGCT incidence in 129/Sv males and fine-mapped the Slgb deletion breakpoints. An increase in TGCT incidence was observed in Slgb/+ heterozygotes. Mapping of the Slgb deletion breakpoints revealed no other genes or obvious functional elements within the deletion interval, suggesting that partial deficiency of Kitl increases TGCT susceptibility. We propose that soluble KITL maintains TGCT susceptibility at 129/Sv baseline frequencies in Sld/+ heterozygous mutant mice and that KIT is haplosufficient for PGC development in mice with the W mutations.
129S1/SvImJ (JR002448) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Heterozygous KitlSlgb/+ mutant mice were obtained from the research colony of M.A. Bedell (24, 25). Prior to the tumor survey, the KitlSlgb mutation was backcrossed for at least 10 generations onto 129S1/SvImJ to establish a 129S1/SvImJ-KitlSlgb/+ (Slgb/+) congenic strain. Homozygous Slgb/Slgb mutant embryos were obtained from timed-matings of heterozygous mutant mice. Embryonic day 0.5 (E0.5) was assumed to be noon of the day the vaginal plug was observed and embryos were then collected at E13.5. Homozygous Slgb/Slgb embryos die after E15.5 (18). Mice were maintained in the CWRU Animal Resource Center on a 12:12 h light:dark cycle and fed Lab Diet 5010 (Richmond, IN). All protocols were approved by the Institutional Animal Care and Use Committee.
A light coat color on the belly and pink tips of the tail and digits on an otherwise normally pigmented mouse distinguished Slgb/+ mice from their wild-type siblings (12, 15). Genomic DNA was isolated from adult tails and E13.5 embryo tissue using the Qiagen DNeasy Blood and Tissue Kit (Valencia, CA). Homozygous mutant embryos were identified using a PCR assay for Kitl. After a 5 minute incubation at 95°C, samples were amplified for 35 cycles: 30 seconds at 95°C, 30 seconds at 58°C, and 1 minute at 72°C, followed by a final extension at 72°C for 5 minutes. Primers were: F: 5′-TCATGGTGCACCGTATCCTA-3′ and R: 5′-CCTTGGCATGTTCTTCCACT-3′ and produce a 170 bp product from wild-type +/+, and Slgb/+ DNA but not homozygous Slgb/Slgb DNA. Sequence information was obtained from the UC Santa Cruz Genome Browser using NCBI Mouse Build 37 (genome.ucsc.edu).
Crosses between 129/Sv and 129/Sv-KitlSlgb/+, in both parental directions, were used for the TGCT surveys. Male offspring were necropsied at 4-6 weeks of age and testes were visually examined for tumors. Chi-square contingency tests were used to test for statistical differences between the number of affected wild-type and Slgb/+ males, and parent-of-origin effects on TGCT susceptibility.
The distal Slgb breakpoint was previously identified to be within the 3′ untranslated region (3′UTR) at base pair position 5287 of the Kitl transcript (18, 26). To identify the proximal breakpoint of the Slgb deletions we used a genotyping and sequencing strategy (21). Primer sets were designed to PCR amplify sequences 60, 50, 40 and 30 kb proximal to the start of Kitl. A PCR product was generated for all primer sets using wild-type adult DNA, but homozygous Slgb/Slgb mutant embryo DNA failed to generate a product with the 30 kb primer set, placing the proximal breakpoint between -40 to -30 kb of Kitl. New primer sets were designed at 1 kb intervals between -40 and -30 kb of Kitl and an additional round of PCR further narrowed the region of the proximal breakpoint to between -38.3 and -37.2 Kb. PCR was then performed on Slgb/Slgb homozygous and wild-type DNA with primers F: 5′-CACAGGTTAAAATATGCACAGCA-3′ (upstream of the 5′ breakpoint) and R: 5′-ATCAAAAGGGTCGGGACATA-3′ (downstream of the 3′ breakpoint) to amplify across the deletion. A 463 bp product was generated from only the Slgb/Slgb homozygous DNA. Sequencing of this PCR product defined the boundaries of the Slgb deletion.
Sequence analysis of the Ensembl (www.ensembl.org), NCBI (www.ncbi.nlm.nih.gov/genome/guide/mouse) and UCSC mouse genome databases was used to identify transcripts and hypothetical proteins within the deletion intervals of the Steel mutations. The Vista human/mouse/rat Genome Browser (pipeline.lbl.gov) and the UCSC Genome Browser Vertebrate Multiz Alignment & Conservation Track were used to identify conserved extragenic sequences within the Slgb deletion interval (27, 28).
If Slgb (~120 kb deletion) increases the number of affected males as do Sl and SlJ (~973 kb and ~650 Kb deletions, respectively) (Table 1), the candidate region that controls TGCT susceptibility in Steel mutant mice would be significantly reduced. We therefore measured the TGCT incidence in Slgb/+ mutant and wild-type male siblings. TGCT incidence was significantly increased (~2-fold) in Slgb/+ mutant males compared to wild-type controls (Table 2). Therefore, our Slgb/+ tumor survey reduces the critical interval for TGCT susceptibility in Steel mutant mice to approximately 120 Kb.
Additionally, we tested the influence of the parent-of-origin of the Slgb mutation on TGCT incidence in Slgb/+ and wild-type offspring. Parental factors did not affect susceptibility in Slgb/+ offspring as TGCT incidence was similar irrespective of the parental origin of the Slgb mutation (Table 3). By contrast, the parent-of-origin of the Slgb mutation influenced TGCT susceptibility in wild-type offspring, with TGCT incidence being significantly lower when the male parent was heterozygous for Slgb (Table 3). A more detailed analysis of this effect will be reported elsewhere.
We mapped and sequenced the breakpoints of the Slgb deletion to identify candidate TGCT susceptibility genes. The Sl mutation spans several genes, including Kitl, and several predicted functional elements, including two putative Ensembl miRNAs (Ensembl gene ID’s: ENSMUSG00000077043 and ENSMUSG00000077034) distal to Kitl and several hypothetical proteins (Fig. 1A) (21). The SlJ mutation also deletes the two putative Ensembl miRNAs and several hypothetical proteins (Fig. 1A) (18). Additionally, the proximity of the deletion breakpoints to neighboring genes may affect their expression. Based on the rough map of the ~120 Kb Slgb deletion, it appeared that Kitl is the only gene affected by the mutation (Fig. 1A) (18).
To fine-map the breakpoints for the Slgb mutation, we used a combination of PCR and sequencing. The Slgb deletion breakpoints were mapped to base pairs 99,440,172 and 99,563,881 of mouse chromosome 10 (Fig. 1B). This 123 Kb deletion starts 38 Kb proximal to exon 1 of Kitl and ends within its 3′UTR. Sequence analysis did not reveal any other genes, transcripts or expressed sequence tags within the Slgb interval in the mouse or other mammalian species. The closest annotated sequences to the proximal end of the Slgb deletion are a hypothetical protein, a ribosomal protein L17-like pseudo-gene (XM_001480394), and citrate synthase-like protein (Csl), -112 Kb and -220 kb from the Slgb breakpoint, respectively.
The VISTA program and UCSC genome browser revealed several cross-species conserved extragenic sequences within the 38 Kb proximal of Kitl. However, due to the distance between the closest neighboring gene and the proximal breakpoint of the Slgb deletion, the cross-species conserved extragenic elements within the Slgb deletion are most likely Kitl transcriptional regulatory elements. In addition, because the Slgb distal breakpoint ends within the Kitl 3′UTR, the mutation most likely does not affect the hypothetical proteins or the putative Ensembl miRNAs distal to Kitl. Furthermore, the known miRNAs on mouse chromosome 10 do not associate with tumor susceptibility loci on this chromosome (29). Together our TGCT survey and sequence analysis of the Slgb mutation suggest that deficiency of Kitl increases TGCT susceptibility in mice.
An increase in TGCT incidence was observed in 129/Sv mice harboring the Slgb deletion of the Steel locus. Furthermore, sequencing of the Slgb deletion breakpoints revealed that Kitl is the only conventional gene deleted by this mutation. These results suggest that Kitl is the TGCT modifier at the Steel locus. Interestingly, only the KITL null deletions (Sl, SlJ and Slgb) influence TGCT susceptibility on the 129/Sv inbred background. The Sld mutation, from which soluble KITL is expressed, does not influence TGCT susceptibility. The contrasting effects of the various Steel mutations on TGCT susceptibility suggest that the transmembrane isoform of KITL has unusual and specific effects on PGC development.
Both KITL isoforms are biologically active but may have distinct functional significance in the survival (transmembrane KITL), proliferation (transmembrane and soluble KITL) and migration (soluble KITL) of stem cells (30-32). Deficiency of KITL in homozygous and heterozygous Steel null mutants results in PGC proliferation, migration and survival defects (18, 21, 25). By contrast, in homozygous Sld/Sld and compound heterozygous Sld/Sl mice, which are transmembrane KITL-deficient, expression of soluble KITL from the Sld locus partially rescues germ cell proliferation and migration (25, 31). Furthermore, PGC development (and TGCT susceptibility) in Sld/+ mice is similar to wild-type siblings. Thus, although soluble KITL cannot fully complement the functions of transmembrane KITL, it appears to compensate for the dosage effect of transmembrane-bound KITL on PGC development and TGCT susceptibility in Sld/+ heterozygous mice.
The parent-of-origin effect of the Slgb mutation on wild-type tumor incidence in our Slgb crosses was unexpected. Recently, we demonstrated that genetic modifiers of TGCTs interact across generations to influence TGCT susceptibility (trans-generational epistasis) (13). These interactions represent a novel mode of epigenetic inheritance and may account for the difficulties in identifying TGCT susceptibility genes in mice and humans. Influences of parental genotype on wild-type TGCT incidence have not, however, been previously reported for single genetic modifiers. These parental effects have either not been tested, reported, or do not occur for most TGCT modifiers. We are currently in the process of replicating the SlJ tumor survey to determine whether the parent-of-origin effect observed in the Slgb survey is a common feature of Steel null mutations.
Most of the pleiotropic effects observed in Steel homozygous and heterozygous mutant mice are also observed in mice with the various W mutations of the KIT receptor. However, unlike the Steel mutations, none of the tested W mutations increased TGCT susceptibility in heterozygous males. PGC development in heterozygous Sl and W mutation mice may explain this difference. Heterozygous Steel mutant mice have reduced numbers of PGCs, due to migration and proliferation deficiencies, and a defect in spermatogenesis in the adult testis (15, 21, 33). By contrast, heterozygous W mutant mice have a statistically similar number of PGCs in the developing genital ridge and type A spermatogonia in the adult testis compared to wild-type littermates (34, 35). Therefore dosage of Kitl but not Kit affects PGC development. Because TGCTs initiate during embryogenesis, it is therefore not surprising that heterozygosity for W mutations does not modify TGCT susceptibility.
The mechanism by which the Steel loss-of-function mutations increase TGCT susceptibility is unknown. It is possible that the PGC migration defect of Steel mutant mice contributes to TGCT formation. Transmembrane-bound KITL is expressed by somatic cells along the route of PGC migration and the genital ridge releases chemo-attractants, such as soluble KITL, to guide PGCs toward the developing gonad (30, 36-38). When migratory signals are disrupted and male PGCs localize to ectopic tissues, they undergo abnormal developmental changes and initiate apoptosis in response to local stimuli (39, 40). It is possible that in heterozygous null mutant males disruption of kit ligand signaling delays germ cell migration into the genital ridge, exposes PGCs to signals outside the developing gonad, and enhance tumorigenic potential after localization into the embryonic testis.
Alternatively, PGCs with altered migration patterns may become apoptotic prior to entering the genital ridge, resulting in decreased PGC numbers within the developing testis, as is observed in Steel mutant mice (15, 21, 33). A decreased ratio of germ cells to somatic cells within the developing testis may increase the availability of growth factors produced by the somatic cells and alter the developmental fate of the PGCs that localized to the genital ridge. Because Steel mutations act as modifiers of a pre-existing susceptibility to TGCTs in 129/Sv inbred mice, it is likely that the effects of delayed migration or altered germ cell to somatic cell ratios are specific to the 129 genetic background.
KIT is one of several candidate TGCT susceptibility genes located within a region of human chromosome 4 (4q12) that is frequently amplified in seminomas, but not nonseminomas (41, 42). Somatic mutations that constitutively activate the KIT receptor are also associated with seminoma initiation, but are extremely rare in nonseminomas and pediatric teratomas (42-45). By contrast, deletions of chromosome 12q22, where Kitl is located, are associated with nonseminomas, but are extremely rare in seminomas (46, 47). In one series of studies, up to 40% of TGCTs showed loss of heterozygosity for a polymorphic marker (D12S7) closely associated with Kitl (47, 48). Interestingly, 4q12 amplifications, 12q22 deletions and activating Kit mutations are rare in TGCT precursor lesions, intratubular germ cell neoplasia (ITGCN) (41, 49). Therefore, modulation of the KIT/KITL signaling cascade is most likely not involved in TGCT initiation, but instead influences the ultimate developmental fate of ITGCN. Because mouse TGCTs are teratomas and teratocarcinomas, our data and those from humans suggest that a decrease in KIT/KITL signaling, through a gene dosage affect of Kitl, supports development of differentiated tumors (teratomas or nonseminomas), where as activation of KIT/KITL signaling supports development of less differentiated seminomas.
We thank L.C. Stevens and D.S. Varnum for sharing their unpublished results and Mary Bedell for providing the Slgb mutant mice. This work was supported by NIH grants CA75056 and CA119623.