Genetic Factors on Mouse Chromosome 18 Affecting Susceptibility to Testicular Germ Cell Tumors and Permissiveness to Embryonic Stem Cell Derivation Philip D

Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-3342

Molecular Biology, Pathobiology, and Genetics

Genetic Factors on Mouse Chromosome 18 Affecting Susceptibility to Testicular Germ Cell Tumors and Permissiveness to Embryonic Stem Cell Derivation Philip D. Anderson,1 Vicki R. Nelson,1 Paul J. Tesar,1,2 and Joseph H. Nadeau1,3

1Department of Genetics, 2Center for Stem Cell and Regenerative Medicine, and 3Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, Ohio

Abstract susceptibility have also been described in mice (12, 13). Despite Despite strong heritability, little is known about the genetic these advances, the genetic factors that contribute to the disease control of susceptibility to testicular germ cell tumors (TGCT) remain poorly understood. The difficulty of finding TGCT genes in in humans or mice. Although the mouse model of spontane- humans makes a mouse model relevant to characterizing the ge- ous TGCTs has been extensively studied, conventional linkage netic control of susceptibility. analysis has failed to locate the factors that control terato- Strain 129 males develop spontaneous TGCTs at a rate of 1% to carcinogenesis in the susceptible 129 family of inbred strains. 8%, depending on the substrain (14, 15). These TGCTs are an es- As an alternative approach, we used both chromosome sub- tablished model of spontaneous testicular teratomas and terato- stitution strains (CSS) to identify individual chromosomes carcinomas that occur in human infants (14, 15). In both infants that harbor susceptibility genes and a panel of congenic and mice, TGCTs arise from primordial germ cells (PGCs; ref. 12) strains derived from a selected CSS to determine the num- and are thought to lack both carcinoma in situ and characteristic ber and location of susceptibility variants on the substi- karyotypic abnormalities such as isochromosome 12p that are tuted chromosome. We showed that 129-Chr 18MOLF males found in adult TGCTs in humans (16). As in humans, genetic con- are resistant to spontaneous TGCTs and that at least four trol of TGCT tumorigenesis in mice is complex, and linkage studies genetic variants control susceptibility in males with this have met with only modest success. The first linkage study failed to substituted chromosome. In addition, early embryonic cells uncover any statistically significant linkages, although the best in- from this strain fail to establish embryonic stem cell lines as volved distal chromosome 19 (17). A separate cross implicated a ∼ efficiently as those from the parental 129/Sv strain. For the 23 cM quantitative trait locus (QTL) on chromosome 13 (18). This QTL contains >400 genes, which represents almost half of first time, 129-derived genetic variants that control TGCT 4 susceptibility and fundamental aspects of embryonic stem all the genes on chromosome 13. cell biology have been localized in a genetic context in Chromosome substitution strains (CSS) are a powerful alterna- which the genes can be identified and functionally charac- tive to segregating crosses for discovering genes involved in TGCT terized. [Cancer Res 2009;69(23):9112–7] susceptibility. CSSs, which are also known as consomic strains, are inbred strains in which an entire chromosome is replaced by its homologue from a different inbred strain. CSSs have several logis- Introduction tical and statistical advantages (19, 20). Fine mapping is accom- Testicular germ cell tumors (TGCT) are the most common solid plished either with crosses or preferably with panels of congenic tumors in men 15 to 34 years of age, accounting for >1% of all male strains in which the substituted chromosome is partitioned into cancers (1, 2). More than 90% of testicular cancers are TGCTs (3). segments of various lengths on the inbred host background (21–24). Family history is a significant risk factor, with a frequency in affect- Based on the weak linkage to chromosome 19 (17), Matin and ed males that is 10 to 13 times higher among brothers, 4 times colleagues made the first autosomal CSS in mice to test whether higher among sons of affected fathers (4–7), and a 75-fold in- chromosome 19 had QTLs involved in TGCTs (25). MOLF is an creased risk for monozygotic twins (8). Unfortunately, linkage inbred strain that is derived from Mus musculus molossinus and and association studies to identify genes that control susceptibility is genetically distant from 129/Sv (26–28). Among 129-Chr 19MOLF have been difficult because of a shortage of multigenerational ped- males, 82% developed at least one TGCT, showing that MOLF-19 igrees with a sufficient number of affected individuals, the sterility had at least one TGCT-promoting QTL (25). A panel of 13 con- that often results from treatment, and the genetic complexity of genic strains was then made to map the QTLs (21). Analysis of the disease. Using a candidate gene approach, Nathanson and col- TGCT prevalence in the congenic strains showed that at least five leagues identified a 1.6-Mb deletion (gr/gr) on Yq11 that confers TGCT QTLs were located on this substituted chromosome (21). susceptibility to TGCTs (9). Recently, a genome-wide association Moreover, closely linked QTLs are readily resolved in a panel of study implicated Kitl, a component of the c-kit signal transduction congenic strains, which is difficult to accomplish in segregating pathway (10, 11). Mutations in the Kitl gene that enhance TGCT crosses because too few closely linked crossovers occur to provide strong evidence for independence. In addition, the boundaries of these candidate intervals are genetically, rather than statistically, Note: Supplementary data for this article are available at Cancer Research Online defined (cf. refs. 21–24). (http://cancerres.aacrjournals.org/). Requests for reprints: Joseph H. Nadeau, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106. Phone: 216-368-0581; Fax: 216-368-3832; E-mail: [email protected]. ©2009 American Association for Cancer Research. 4 University of California Santa Cruz Table Browser. http://genome.ucsc.edu/cgi- doi:10.1158/0008-5472.CAN-09-3342 bin/hgTables. August 12, 2009.

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To identify other 129-derived TGCT factors that must exist to all calculations was set at P < 0.05 after Bonferroni correction for multiple account for the complex patterns of inheritance, we began by test- testing. ing for the action of QTLs with dominant effects on susceptibility Testing for QTLs with dominant effects on TGCT prevalence. We in a panel of incipient CSSs and found statistically significant evi- used the following method to identify donor chromosomes that harbor QTLs that confer dominant effects on TGCT prevalence: dence for QTLs suppressing TGCTs in two CSSs, 129-Chr 2MOLF and 129-Chr 18MOLF. An incipient CSS is one that has not yet been Total % affected ¼ mð% affected with QTLÞ backcrossed 10 generations (19, 29). Then, with a panel of congenic þð1 − mÞð% affected without QTLÞ strains that together span the length of chromosome 18, we identified four TGCT QTLs on this substituted chromosome. Finally, we where “total % affected” is the fraction of all males that had at least one MOLF showed that early embryonic cells from 129-Chr 18 are less TGCT, m is the fraction of backcrossed mice that inherited the MOLF allele likely to yield pluripotent cell lines in vitro than those from 129/Sv. (their genotype is designated M1), “% affected with QTL” is the percentage of males that are heterozygous for the MOLF allele and affected with at least one TGCT, 1 − m is the fraction of males that inherited two copies “ Materials and Methods of the 129-derived allele (their genotype is designated 11), and % affected without a QTL” is the percentage of all males that are homozygous for the Mice. MOLF/Ei (MOLF) and 129S1/SvImJ (129/Sv) mice were purchased 129 allele and affected with at least one TGCT. from The Jackson Laboratory. MOLF mice were maintained on LabDiets The m and (1 − m) terms, which represent the actual segregation ratios 7960 rodent chow (PMI Nutrition International). 129/Sv and backcrossed for the alternative alleles (cf. Table 1), were included to avoid an assump- mice were maintained on 5010 rodent chow (PMI Nutrition International). tion of normal (1:1) Mendelian segregation, i.e., m = 0.5. If heterozygosity for Mice were housed in the Case Western Reserve University Animal Resource MOLF-derived alleles does not affect susceptibility, then the overall fraction Center and maintained on a 12:12-h light/dark cycle. Mice were provided of affected males is equivalent to the baseline rate in 129/Sv males. This water and diet ad libitum. The mouse work was approved by the Case fraction will be higher (or lower) than the baseline rate if heterozygous Western Reserve University Institutional Animal Care and Use Committee. males have enhancer (or suppressor) effects. CSS construction. To make the 129-ChrMOLF panel, MOLF mice were The total percentage of affected males (term 1) was estimated by survey- mated with 129/Sv and the progenies were backcrossed to 129/Sv. N2 mice ing backcrossed males for TGCTs. The percentage of affected males with- were genotyped and mice with nonrecombinant chromosomes of interest out the putative MOLF-derived QTL (term 3) was 5% (Table 1), which is the were backcrossed to the host strains. This step was repeated until at least baseline TGCT prevalence in 129/Sv males in this survey and is consistent the N10 generation. Nonrecombinant N10+ mice were brother-sister mated with published estimates (14, 15, 21, 30–32). Because the value for two of to make the substituted chromosome homozygous. the three terms was known, we were able to solve for the unknown term, Genotyping. Mice were genotyped with either SNaPshot single base ex- i.e., the percentage of affected males with a putative QTL for each CSS. tension (Applied Biosystems) or microsatellite markers. For single base ex- Because of the modes of inheritance of the Y chromosome and the mi- tension, SNaPshot was performed as described by the manufacturer's tochondria, this method was not needed for CSSs 129-Chr YMOLF and protocol with the following changes: 1 unit of shrimp alkaline phosphatase 129-Chr MitoMOLF. (USB Corporation) and 0.2 units of Exonuclease I (USB Corporation) each Construction of congenic strains from 129-Chr 18MOLF. N12 mice were added to 15 μL of PCR product to remove unused deoxynucleotide that were heterosomic for MOLF-derived chromosome 18 were back- triphosphates and PCR primers. The custom single base extension oligonu- crossed to 129/Sv and N13 progeny were typed for six microsatellite mark- cleotide, which anneals immediately upstream of the SNP, was used at a ers. Recombinant N13 mice were backcrossed to 129/Sv and N14 working concentration of 0.2 to 1.0 μmol/L, depending on the efficiency heterozygous progeny were intercrossed to produce homozygous congenic of each primer. Reactions were multiplexed at least four and at most six mice. Each congenic strain was then maintained with brother-sister mat- times. After primer extension, the reactions were subjected to capillary ing. The identities of the microsatellite markers used for genotyping are electrophoresis on ABI DNA Analyzers (Applied Biosystems) in the Case listed in Supplementary Table S3. Western Reserve University Genomics Core. For SNP genotyping, at least Recombination breakpoint mapping. We sequenced portions of chro- four and at most six SNPs were typed per chromosome. Two SNPs were as mosome 18 to discover additional SNPs that could be used to map the re- close to each of the ends of each chromosome as possible, and two to four combination breakpoints in selected congenic strains. The identities of the SNPs were evenly spaced in between. There were no gaps >23 cM. The iden- PCR primers used for breakpoint mapping are listed in Supplementary tities of the SNPs and the primers used for genotyping are listed in Supple- Table S4. mentary Table S1. Histology. Testes were fixed in 10% phosphate-buffered formalin (Fisher For microsatellite marker genotyping, polymorphic MIT markers were Scientific SF100-4) at 4°C for at least 48 h, rinsed once in 1× PBS at room identified (Center for Inherited Disease Research). DNA for PCR genotyping temperature, and equilibrated in 30% sucrose in 1× PBS at 4°C for at least was isolated from mouse tail as previously described (18) and resuspended 2 d before embedding and freezing in optimal cutting temperature com- in 10 mmol/L of Tris. PCR amplification was performed in a 96-well PTC- pound (Sakura Finetek USA 4583). Embedded gonads were sectioned at 225 tetrad thermal cycler (MJ Research). PCR conditions were as follows: 5to10μm with a Leica CM3050 cryostat and slides were dried in the dark 94°C for 2 min followed by 39 cycles of 94°C for 60 s, 55°C for 35 s, and 72°C at room temperature for 1 h before staining with H&E. for 45 s. A final extension of 72°C for 5 min was performed. PCR products Derivation of mouse embryonic stem cells. Mouse embryonic stem were resolved in 4% agarose gels (Invitrogen 15510-027) in 1× TAE and vi- (ES) cell lines were derived as described previously (33). In brief, blastocysts sualized with ethidium bromide. Genotypes were inferred by length poly- were flushed from the uterus at embryonic day 3.5 (E3.5) into HEPES-buffered morphism. Four MIT markers were typed per chromosome. Two markers mKSOM (34). Individual blastocysts were explanted onto γ-irradiated mouse were as close to the ends of each chromosome as possible, and two were embryo fibroblasts in mouse ES cell medium that consisted of knockout evenly spaced in between. There were no gaps >34 cM. The identities of the DMEM (Invitrogen) supplemented with 15% knockout serum replacement, microsatellite markers are listed in Supplementary Table S2. 5% ES cell–qualified fetal bovine serum (Invitrogen), 103 units/mL of recom- TGCT survey. Three- to 5-wk-old males (a minority were >5 wk) were binant mouse leukemia inhibitory factor (ESGRO, Millipore), 2 mmol/L euthanized with CO2 inhalation or cervical dislocation. Incisions were of L-glutamine, 1× nonessential amino acids (both from Invitrogen), and made in the abdomen to expose the testes. TGCTs are identifiable with 0.1 mmol/L of 2-mercaptoethanol (Sigma). After 5 to 6 d, the inner cell masses macroscopic inspection and scored for laterality (14). TGCT prevalence had reached a suitable size and were picked from the plate with a pulled glass was analyzed using the χ2 goodness-of-fit test or Fisher's exact test and pipette and incubated for 5 min at room temperature in 0.25% trypsin-EDTA. compared with the host TGCT prevalence. The significance threshold for Trypsin was neutralized with serum-containing medium and the inner cell www.aacrjournals.org 9113 Cancer Res 2009; 69: (23). December 1, 2009

Table 1. Estimated QTL effects in 129/Sv control and nine incipient 129-ChrMOLF CSSs

Strain Generations nnaffected Total M Affected χ2 P surveyed affected (%) with QTL (%)

Control 129/Sv N/A 140 7 5.0 N/A N/A N/A N/A 129-ChrMOLF CSSs MOLF-2 N3-N10 227 5 2.2 0.61 0.4 10.16 0.013 MOLF-3 N3-N5 59 2 3.4 0.53 1.9 1.16 ns MOLF-7 N3-N11 144 6 4.2 0.62 3.7 0.55 ns MOLF-11 N3-N8 94 6 6.4 0.47 7.9 1.71 ns MOLF-16 N4-N9 122 6 4.9 0.48 4.8 0.01 ns MOLF-18 N5-N13 245 5 2.0 0.64 0.4 11.12 0.008 MOLF-X N3-N5 125 7 5.6 0.48 6.3 0.41 ns MOLF-Y N3-N16 363 13 3.6 N/A N/A 1.54 ns MOLF-M N5-N21 537 29 5.4 N/A N/A 0.18 ns

NOTE: Control and incipient CSS males were surveyed at the backcross generations indicated. n affected and total affected (%) gives the total number and percentage of males with TGCT. All TGCT cases were unilateral. M is the proportion of mice that inherited one copy of the putative MOLF QTL and was calculated separately for each strain except 129-Chr YMOLF and 129-Chr MMOLF. The percentage affected with QTL is the TGCT prevalence among incipient CSS males with one copy of a putative dominant QTL. We tested whether the TGCT prevalence in males with the QTL deviated significantly from the control 129/Sv TGCT prevalence using the χ2 goodness-of-fit test. The significance threshold was set at P < 0.05 after the correction for nine multiple tests. P values in boldface indicate significant reductions from the control. No incipient CSSs had significantly elevated TGCT prevalence relative to control. N/A, not applicable; ns, not significant.

masses were partially dissociated individually with the pulled glass pipette. dominance effects, 129-Chr YMOLF and 129-Chr MitoMOLF. For those The partial dissociates were plated individually onto a feeder layer of irra- two CSSs, we simply tested whether the total percentage of affected 2 diated mouse embryo fibroblasts in a single well of a 24-well plate (1.9 cm males differed significantly from the percentage in 129/Sv controls. surface area) and cultured with mouse ES cell medium. After 5 d, individual We examined 140 control 129/Sv males and a total of 1,916 post- wells were trypsinized and passaged into a single well of a six-well plate (9.6 N2 129-ChrMOLF backcross males for TGCTs among the nine incip- cm2 surface area). ES cell colonies became readily evident between days 2 ient CSSs (Table 1). Only two incipient CSSs, 129-Chr 2MOLF and and 7. Colonies were individually picked and dissociated for further expan- MOLF sion. Dissociated colonies that gave rise to ES cell lines were expanded and 129-Chr 18 , provided evidence for QTLs with dominant effects cryopreserved. To minimize variability, comparison of ES cell line deriva- on the baseline rate of susceptibility in 129/Sv males; both showed tion efficiencies between strain 129/Sv and 129-Chr 18MOLF was performed a significantly reduced percentage of affected males (Table 1). during an overlapping time period and all reagents used were identical. ES These results are consistent with substitution of at least one cell derivation efficiency was analyzed using the χ2 goodness-of-fit test and 129-derived susceptibility gene or at least one MOLF-derived TGCT compared with the standard value for strain 129 (30%; see refs. 33, 35–37). suppressor on each chromosome. The significance threshold for all calculations was set at P < 0.05 after Yates' We prioritized the development of these CSSs and surveyed 365 correction for continuity. homosomic 129-Chr 18MOLF males for TGCTs. Remarkably, none of Mouse embryo fibroblasts were maintained with DMEM supplemented the 365 adult 129-Chr 18MOLF males examined were affected with a with 10% fetal bovine serum, 2 mmol/L of L-glutamine, and 0.1 mmol/L of −6 2-mercaptoethanol. Mouse embryo fibroblasts were irradiated with 30 to 60 TGCT (P <10 ; Fig. 1). These results validate the evidence from Gy and seeded as feeders at a density of 5.2 × 104 cells/cm2 for mouse ES the incipient CSSs for at least one TGCT QTL on chromosome MOLF cell derivation and maintenance cultures. All cells were grown on Nunclon 18. 129-Chr 2 was only recently made homozygous, the prev- Δ–treated dishes or multiwell plates (Fisher Scientific) and coated for 2 h alence of affected males is being assessed, and results will be re- at 37°C with 0.1% (w/v) gelatin (Sigma). Antibiotics at concentrations of ported elsewhere. 50 units/mL and 50 μg/mL for penicillin and streptomycin, respectively, Evidence for at least four TGCT QTLs on chromosome 18. were used only for primary explants of blastocysts. To determine the number and location of TGCT QTLs on chromosome 18, we constructed a panel of eight congenic strains from MOLF Results 129-Chr 18 (Fig. 1). In total, we surveyed 1,979 congenic males for TGCTs. All but four of the TGCTs were histologically verified. CSS survey. We selected nine MOLF chromosomes to substitute All TGCT cases were unilateral. onto the 129 inbred background, i.e., chromosomes 2, 3, 7, 11, 16, 18, We analyzed TGCT prevalence in the congenic strains using a X, Y, and the mitochondria. These chromosomes were selected method involving sequential comparisons between pairs of con- based on prior association with TGCTs (7, 18, X, Y; see refs. 9, 17, genic strains (22–24).5 First, the strains with the smallest MOLF- 38) or for diversity in chromosome length and architecture (2, 3, derived segments (C1 and C6) were compared with 129/Sv. Then, 11, 16, and mitochondria). At each backcross generation, males were each of these two strains was compared with the congenic strains surveyed for TGCTs (Table 1). For the autosomal CSSs and 129-Chr containing the next longer, overlapping MOLF-derived segments XMOLF, we tested whether the frequency of affected males with the putative, dominantly acting QTL differed significantly from the control 129/Sv rate. Two CSSs entail donor chromosomes that segregate but do not undergo genetic recombination and are not subject to 5 Shao et al., submitted for publication.

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(e.g., C1 versus C3 and separately C6 versus C7). This process of Length of the congenic segments may influence TGCT prev- sequential comparisons was continued until each strain had been alence. Youngren and colleagues provided evidence that they in- tested once and only once. Significant differences in TGCT preva- terpreted as epigenetic control of the 129-Chr 19MOLF CSS (21). In a lence between 129/Sv and congenic strains indicate the presence of panel of congenic strains made from 129-Chr 19MOLF, the length of QTLs affecting TGCT prevalence. the congenic segments, rather than their locations on the chromo- This systematic process tested whether the unique MOLF- some, predicted TGCT prevalence (21). The only exceptions were derived segment in each pairwise congenic comparison harbors two congenic strains containing the MOLF-derived telomeric seg- a QTL that affects that rate of affected males in the comparison ment, suggesting that the proposed epigenetic effect involved the strain. The TGCT prevalences in C1 and C6 were both significantly centromeric and central portions but not the distal portion of reduced relative to the 129/Sv control (Fig. 1), suggesting the pres- chromosome 19 (21). Using the panel of eight congenic strains ence of TGCT suppressor QTLs in both of these MOLF-derived made from 129-Chr 18MOLF, we tested whether length rather than congenic intervals. The reduced frequency of affected C1 males location predicted TGCT prevalence in a manner similar to the (0.87%) defines a small (2 cM) QTL near the centromere, an inter- 129-Chr 19MOLF congenic strains. The correlation (r = −0.53) be- val labeled region 1 (Fig. 1). Similarly, C6 males were affected at a tween the length of the congenic segments and TGCT prevalence significantly reduced rate (1.20%), and although C6 lacks region 1 was not statistically significant (Supplementary Fig. S1), arguing near the centromere, it has a ∼6 cM MOLF-derived chromosome against a chromosome-wide epigenetic defect and supporting the segment at the telomere (region 2; Fig. 1). The simplest explanation existence of QTLs located at discrete sites on the chromosome. for these results is that two independently acting TGCT QTLs, Do TGCTs and ES cell derivations share genetic determi- which respectively confer a 4-fold to 6-fold reduction in suscepti- nants? 129 is the most permissive mouse strain for ES cell deriva- bility, are located at opposite ends of chromosome 18. tion (39), which interestingly is the only strain to show an The TGCT prevalence in C7 was enhanced compared with C6 appreciable frequency of spontaneous TGCTs (14, 15, 39). The ge- (3.85% versus 1.20%, P = 0.0008; Fig. 1), which is evidence for a netic elements of strain 129 that result in high-frequency repro- TGCT enhancer in the portion of C7 that does not overlap the con- gramming of PGCs to a tumorigenic, pluripotent state could also genic segment in the C6 strain. This ∼19 cM interval defines the facilitate the unique ability to efficiently derive pluripotent cell location of a third TGCT QTL (region 3; Fig. 1). lines from strain 129 early embryos. We therefore compared the Finally, with a single exception in the C4 congenic strain, four of frequency of ES cell derivation in 129-Chr 18MOLF and control the eight congenic strains (C3, C4, C5, and C9) lacked TGCTs as 129/Sv mice. We obtained four 129/Sv ES cell lines from 18 blas- adults, recapitulating the absence of TGCTs in the 129-Chr 18MOLF tocysts (22.2%), which is not significantly different from the accept- parent strain. The four strains share a ∼4 cM region of chromo- ed and reproducible average for this strain (30%; P = 0.391; Table some 18 between markers M21 (21 cM) and M25 (25 cM), defining 2). By contrast, the ES cell derivation efficiency of the 129-Chr the location of a fourth TGCT QTL (region 4; Fig. 1). 18MOLF strain was significantly reduced (4.8%; P = 0.008) as we only

Figure 1. TGCT data from adult parental strains and congenic strains. The sizes of the congenic intervals are shown relative to the two parent strains. M (MOLF/MOLF) and 1 (129/129) denote genotypes at each marker. The identities of the markers are listed in Supplementary Tables S3 and S4. The number of adult males surveyed and the number and percentage of males affected with TGCTs is shown. The TGCT prevalences in the congenic strains were compared sequentially using the χ2 goodness-of-fit test or Fisher's exact test. P values have been corrected for eight tests and those with significant differences from control are listed. *, not genotyped at this marker; **, for the comparisons in which the expected TGCT rate was 0%, Fisher exact test was substituted for the χ2 test and only the P-value is reported. www.aacrjournals.org 9115 Cancer Res 2009; 69: (23). December 1, 2009

Table 2. Efficiency of ES cell derivation from 129-Chr 18MOLF and 129/Sv

Strain Blastocysts (n) Hatched and attached to feeders (n) ES cell lines (n) Derivation efficiency (%)

129-Chr 18MOLF (test) 24 21 1 4.8 129/Sv (control) 19 18 4 22.2

obtained one ES cell line from a total of 21 blastocysts (Table 2). with human TGCT susceptibility in two linkage studies (maximum These results suggest that the genetic elements contributing to the heterogeneity LOD score = 1.81 and 1.44, respectively; ref. 40). formation of TGCTs in vivo may also influence the ability of early Region 3 is a large ∼19 cM segment containing ∼148 genes. embryo cells to form pluripotent ES cell lines in vitro. Unlike regions 1 or 2, region 3 contains at least one factor that increases TGCT prevalence. Region 3 belongs to a conserved synteny with 5q, 18p, and 18q, with 18q having been weakly associated with Discussion human TGCTs (40), and 5q being significantly associated with human TGCT susceptibility (11). Because the locations of the TGCTs are the most common type of testicular cancer as well as recombination breakpoints of C5 and C6 are only roughly mapped, the most common tumor in men ages 15 to 34 years (1–3). Despite the boundaries of region 3 are uncertain. Additional mapping their prevalence, the genetic control of susceptibility is poorly un- with subcongenic strains should reduce the number of candi- derstood. Family history suggests a strong genetic component, but date genes and facilitate identification of the TGCT enhancer studies have found a small number of genetic variants that each at this QTL. account for a relatively small fraction of the overall inherited risk Region 4 is a ∼4 cM segment containing ∼48 genes. Region 4 (9–11). The 129/Sv inbred strain is an established model in which belongs to a conserved synteny with 5q in humans, which has been to investigate the genetic components of TGCT susceptibility (14, associated with significant TGCT risk (11). Region 4 contains Eif1a, 15, 30–32). A previous study showed that QTLs affecting TGCT a translation factor that is functionally related to Eif2s2,whose prevalence could be mapped using chromosome substitution misexpression is linked to TGCT suppression in 129/Sv males het- strains (25). We therefore constructed and screened a panel of in- erozygous for the agouti yellow mutation (Ay) and those that have cipient 129-ChrMOLF CSSs to test whether other MOLF chromo- reduced expression of Eif2s2 (46). There are no known nonsynon- somes had QTLs affecting TGCT prevalence. We identified two ymous differences in coding sequences between the 129/Sv and CSSs (129-Chr 2MOLF and 129-Chr 18MOLF)withdominantQTLs. MOLF alleles of Eif1a.6 To map these QTLs on chromosome 18, we constructed and sur- Interestingly, none of the four regions contain the dead-end veyed a panel of eight congenic strains derived from 129-Chr (Dnd1) gene, an allele of which, Ter, is a known modifier of 18MOLF. The results of our study show that four regions of chromo- TGCT susceptibility (47, 48). Similarly, none of the regions con- some 18 contain QTLs affecting TGCT prevalence. tain sprouty (Spry4), the mouse orthologue of a human gene as- The key to predicting how the four regions on chromosome 18 sociated with TGCTs in genome-wide association studies (10). affect TGCT prevalence is knowing which genes are in each QTL. Therefore, the genes and factors in the four regions on chromo- Region 1 is a ∼2 cM segment containing ∼27 genes. Region 1 be- some 18 have not been previously associated with TGCT suscep- longs to a conserved synteny involving 10p in humans, a region that tibility. Nonetheless, both Dnd1 and Spry4 map close to the has not been previously associated with TGCTs (40). One of the proximal boundary of region 4. If region 4 contained a long- genes in region 1, Map3k8, is a mitogen-activated protein kinase range transcriptional regulator, then either gene could account and a known proto-oncogene; overexpression of Map3k8 in various for the modifier effect of region 4. Unfortunately, the transcrip- cell lines promotes proliferation and transformation (41, 42). Inter- tional control of both genes is poorly described. estingly, Spry4 is a negative regulator of mitogen-activated protein Although ES cells are a widely used research tool, the mechan- kinase activity (43), and SNPs in Spry4 were recently associated isms that permit their derivation from early embryos remain with human TGCT in a genome-wide association study (10). Spry4 unclear. The genetic contribution to the ability to derive ES cell is located on mouse chromosome 18, but outside the candidate re- lines is often overlooked and poorly understood as the over- gions, and coding nonsynonymous nucleotide differences are not whelming majority of ES cell lines in use today are derived from known between the 129/Sv and MOLF alleles of Map3k8 or Spry4.6 strain 129. Using standard techniques, the majority of other Perhaps the most interesting candidate gene in region 2 is strains have proven refractory to ES cell derivation. In the limited Fbxo15, one of the few known targets of stem cell pluripotency fac- number of other strains in which ES cells can be derived, the ef- tor Oct3/4 (44). Oct3/4 is aberrantly expressed in human TGCTs ficiencies are consistently much lower than that of strain 129. For (45), which may link Fbxo15 to TGCT susceptibility. The 129/Sv example, despite the attempts of multiple groups, the nonobese and MOLF alleles of Fbxo15 differ in a single G→Asubstitution diabetic strain has not yielded ES cell lines. Interestingly, in exon 5, which leads to a valine to isoleucine (V→I) shift at amino NOD129F1 blastocysts yield ES cell lines at a frequency nearly acid position 188.6 Interestingly, region 2 belongs to a conserved equivalent to that of strain 129 itself (49). Furthermore, genetic synteny with 18q22 in humans, which has been weakly associated manipulation of the nonobese diabetic strain by constitutive expression of Myc or Klf4 results in the ability to derive ES cells from this classically nonpermissive strain (50). These results show 6 Mouse Genome Informatics: mouse strains, SNPs, and polymorphisms. http://www. that heritable genetic or epigenetic factors influence the ability to informatics.jax.org/strains_SNPs.shtml. August 12, 2009. derive ES cell lines from early embryos.

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Strikingly, strain 129 is also the only mouse strain to show an understanding of these factors may have a direct effect on the clin- appreciable frequency of TGCTs. We therefore investigated ES cell ical treatment of TGCTs. derivation efficiency in 129-Chr 18MOLF, which is resistant to TGCTs despite having all but one chromosome derived from 129. Disclosure of Potential Conflicts of Interest We found that the ES cell derivation efficiency in 129-Chr 18MOLF was significantly reduced as we could only derive one ES cell line No potential conflicts of interest were disclosed. from 21 blastocysts, suggesting that the genetic elements contributing to the formation of TGCTs from primordial germ cells might Acknowledgments also contribute to the derivation of ES cell lines from early embryos. Further analysis of the ES cell derivation efficiency and TGCT Received 9/9/09; revised 9/29/09; accepted 10/1/09; published OnlineFirst 11/24/09. MOLF Grant support: National Cancer Institute grant CA75056, National Center for Re- prevalence in 129-Chr 18 subcongenic strains should allow for search Resources grant RR12305, and Case Comprehensive Cancer Center and the the precise mapping, and ultimately, the identification of the fac- Center for Stem Cell and Regenerative Medicine. The costs of publication of this article were defrayed in part by the payment tors responsible for the acquisition and maintenance of the plurip- of page charges. This article must therefore be hereby marked advertisement in otent state whether in vitro for ES cells or in vivo for TGCTs. An accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Genetic Factors on Mouse Chromosome 18 Affecting Susceptibility to Testicular Germ Cell Tumors and Permissiveness to Embryonic Stem Cell Derivation

Philip D. Anderson, Vicki R. Nelson, Paul J. Tesar, et al.

Cancer Res 2009;69:9112-9117. Published OnlineFirst November 24, 2009.

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