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Human Molecular Genetics Advance Access originally published online on July 5, 2007
Human Molecular Genetics 2007 16(18):2233-2240; doi:10.1093/hmg/ddm175
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Trans-generational epistasis between Dnd1Ter and other modifier genes controls susceptibility to testicular germ cell tumors

Man-Yee J. Lam, Jason D. Heaney, Kirsten K. Youngren, Jean H. Kawasoe and Joseph H. Nadeau*

Department of Genetics, BRB731, Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA

* To whom correspondence should be addressed. Tel: +1 2163680581; Fax: +1 2163683832; Email: jhn4{at}case.edu/ jhn4{at}po.cwry.edu

Received May 2, 2007; Revised June 19, 2007; Accepted June 27, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENATRY MATERIAL
 REFERENCES
 
The genetic basis for susceptibility to testicular germ cell tumors (TGCTs) has been remarkably elusive. Although TGCTs are the most common cancer in young men and have an unusually strong familial risk, only one low-frequency susceptibility gene has been identified for this highly multigenic trait. In tests to determine whether pairs of genetic variants act epistatically to modulate susceptibility in the 129/Sv mouse model of spontaneous TGCTs, we discovered an unusual mode of inheritance that involved interactions between different genes in different generations. Any of six genetic variants, in either the female or male parent interacted with the Dnd1Ter mutation in male offspring to significantly increase both the frequency of affected Ter/+ males and the proportion of bilateral cases. Trans-generational epistasis is a novel mode of epigenetic inheritance that could account for the difficulty of finding TGCT susceptibility genes in humans and might represent a mechanism for transmitting information about genetic and environmental conditions from parents to offspring through the germline.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENATRY MATERIAL
 REFERENCES
 
Both environmental and genetic factors contribute to susceptibility to testicular germ cell tumors (TGCTs), the most common cancer affecting young men (1,2). Estrogenic-like compounds and other environmental agents have been proposed to account for the doubling in incidence of TGCTs over the last 40 years (3–6), especially in northern and eastern Europe (5,6). However, the specific factors and their mode of action remain to be identified (4,5). Considerable evidence also implicates familial factors. For example, TGCTs show the second highest familial index among all cancers (7). In addition, individuals have an 8–10-fold increased risk if they have an affected brother and a 4–6-fold increased risk with an affected father (8,9). A recent survey concluded that many loci with weak effects control inherited susceptibility (10). The only validated genetic factor is a rare deletion called gr/gr on the Y chromosome (11). This deletion removes two of four copies of the DAZ gene and occurs in 3% of affected males with a family history of TGCTs, 2% of males who do not have a family history of TCGTs, but only 1% of unaffected males. Despite the strong genetic component to inherited susceptibility, linkage, association and gene discovery studies have proven unexpectedly difficult.

The 129 family of inbred strains of laboratory mice is an established model for certain kinds of spontaneous TGCTs (12). However, in crosses between 129 and other inbred strains, the frequency of affected males is less than 10–4 (13). Thus, in both humans and mice, multigenic control and, perhaps, low penetrance and epistasis complicate the use of conventional genetic approaches for discovering TGCT susceptibility genes.

Modifier genes are an alternative to the limited power of conventional approaches for dissecting the genetic basis for highly complex traits (14). Acting as genetic modifiers, various single-gene mutations, a chromosome substitution strain (CSS), and congenic strains that were derived from a CSS modulate TGCT susceptibility only on the 129 background (Table 1). Several linkages for TGCT susceptibility genes were discovered by using Dnd1Ter (Ter) and Trp53null as TGCT modifiers to sensitize mapping studies (15,16).


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  		Table 1. Genes and loci that affect TGCT susceptibility in 129/Sv mice

 
Epistasis between modifiers can also be tested to identify interacting genetic variants that modulate susceptibility. These tests avoid the complications of the low frequency of affected mice in segregating crosses by taking advantage of the uniform background of the genetically predisposed 129/Sv inbred strain. Recent studies based on this paradigm showed that KitlSlJ (SlJ) and Trp53null interact genetically to suppress susceptibility in double-modifier 129/Sv mice, and separately that SlJ and the 129-Chr 19MOLF CSS (M19) interact to enhance susceptibility (17). With the identification of Dnd1 as the gene that is mutated in Ter mice (18), we tested interactions between Dnd1Ter and six TGCT modifiers (SlJ, Trp53null, Ay, CSS M19, M19-A2 and M19-C2 (Fig. 1). (M19-A2 and M19-C2 are congenic strains that were derived from M19.) A brief review of the genetics and biology of TGCT modifiers that were used in this study is provided in Box 1. These tests, which involved a total of 1458 males (Table 2), were designed so that segregation of single-modifier and wild-type males provided controls within each test cross (Fig. 1).


Figure 1
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  		Figure 1. Crosses, segregants and frequencies of affected males in modifier interaction tests, showing expected and observed results for controls and test groups in modifier interaction tests. ‘m’ designates any TGCT modifier gene except Ter. Because differences were not found in reciprocal crosses, gender is not shown. Expected frequencies were based on published results and on control crosses (cf. Table 2), whereas the observed frequencies were shown as either being consistent with expectations (+/+ +/+ and m/+ +/+) or 2- or 3-fold higher than the expected frequency (Ter/+ +/+ and Ter/+ m/+). See Youngren et al. (22,37) and Heaney and Nadeau (37) for details about the analytical methods.

 


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  		Table 2. Comparison of the observed and expected numbers of affected males in modifier interaction tests

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENATRY MATERIAL
 REFERENCES
 
Interactions tests revealed consistent evidence for epistasis between Ter and the TGCT modifiers, and an unexpected and striking effect on the frequency of affected Ter/+ control males in the interaction tests, but not in the independent control crosses. Among the Ter/+ m/+ double-modifier test males, we found a 3-fold increase in frequency for affected Ter/+ SlJ/+ males and a ~2-fold increase for double-modifier males in the Trp53null, Ay, M19, M19-A2 and M19-C2 tests (Table 2), suggesting strong interactions between Ter and TGCT susceptibility genes. (m denotes one of the six modifiers other than Ter.) Surprisingly, one of the control classes in the interaction tests also showed evidence for interactions. The frequencies of affected Ter/+ +/+ males were significantly elevated to a remarkably consistent level—37% in the Ter-Ay test, 36% in the Ter-SlJ test and 35% in the Ter-Trp53null test (Table 3), each of which was significantly higher than the expected frequency of 17% (Table 2). In contrast, the two other control segregants in the interaction test (m/+ and +/+) showed the expected frequencies of affected males. (By design, the M19, M19-A2 and M19-C2 tests did not include a Ter/+ control.) In interaction tests such as these, a departure from expectations in double-modifier versus single-modifier and wild-type controls usually suggests strong genotype-specific interactions. However, the higher than expected frequency of affected Ter/+ +/+ control males, together with the expected frequencies of affected m/+ and +/+ control males, suggests a more complicated and perhaps more interesting interpretation.


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  		Table 3. Frequency of affected hybrids and mutant males in control tests and crosses

 
Three kinds of control studies were undertaken to test conventional explanations for the unusual pattern of TGCT inheritance. First, three control crosses were made to test whether the frequency of affected Ter/+ males in our 129T1/SvJ colony was consistent with published rates, whether genetic differences between the 129T1/SvJ and 129S1/SvJ substrains affected the baseline frequency of affected wild-type (+/+) males, and whether these substrain differences affected the frequency of affected Ter/+ males (Table 3). A total of 358 male offspring were examined in these crosses. In each test, the frequency of males with at least one TGCT was consistent with expectations, suggesting that colony-specific effects and substrain differences did not significantly affect susceptibility. Second, the SlJ and Trp53null interaction tests included single-modifier (SlJ /+ and Trp53null/+) and wild-type (+/+) males as controls (Table 2). The frequency of affected males in these tests did not differ significantly from published frequencies (13,19), demonstrating that the unexpected interaction effects were specific to Ter/+ males. Third, reciprocal crosses were made for the Ter-SlJ and separately the Ter-Trp53null tests to determine whether factors such as maternal nutrition or growth factors, X, Y or mitochondrial inheritance, imprinting, or other gender-specific effects affected susceptibility (Supplementary Material, Table S1). Comparable results for these reciprocal crosses suggest that factors associated with parental gender or germ lineage (oogenesis or spermatogenesis) in the parents did not influence the results significantly.


Box 1. Characteristics of TGCT modifier genes.

Dnd1Ter causes germ cell deficiency in homozygous males and females on all genetic backgrounds that have been tested as well as a high incidence of spontaneous TGCT on the 129/Sv-Ter background (19,39). Reduced numbers of PGCs are first apparent at embryonic day 8 (E8) in Ter/Ter homozygous mutants, whereas PGCs in wild-type males are proliferating from ~10 cells on E7.5 to 25 000–30 000 cells on E13.5 (ref. 18). Homozygous males have a tumor frequency of ~94%, whereas heterozygous males have a tumor frequency of 17%. The Dnd1*Ter mutation arose on the 129T1/SvJ background (19,39). Positional cloning studies identified a point mutation in Ter mice that introduces a termination codon in the mouse ortholog (Dnd1) of the zebrafish dead-end (dnd) gene (31). The protein sequence shares significant similarity with apobec complementation factors, which encode the RNA-binding subunit of the DNA- and RNA-editing enzyme complex that converts specific cytidines to uridines in the apolipoprotein B transcript and other RNAs (30–32) (hereafter abbreviated as Ter).

KitlSlJ encodes the ligand for the KIT cell-surface receptor and is the only gene deleted in SlJ mutant mice (J.H. Nadeau, unpublished data). The SlJ mutation arose on another inbred background and was made congenic on the 129S1/SvJ background. 29-SlJ/+ heterozygous males have a TGCT incidence of 14%, which is 2-fold higher than the rate in 129/Sv wild-type mice; homozygous mutants are embryonic lethal (13,17). Somatic mutations in KIT, the receptor for the ligand encoded by the Kitl gene, have been reported in TGCTs in humans and occur frequently in cases of bilateral TGCTs (24,40) (hereafter abbreviated as SlJ).

Trp53 is one of the most frequently mutated tumor suppressor genes in human cancers (41). Although somatic Trp53 mutations are rare in TGCTs in humans (42), individuals with Li–Fraumeni syndrome, which results from heritable Trp53 mutations, show increased susceptibility to TGCTs (43). Mice with an engineered deficiency of TRP53 are susceptible to various tumors including TGCTs (44), with the specific tumor spectrum and the incidence of TGCTs dependent on genetic background (45,46). The engineered mutation used in these studies was made on the 129S1/SvJ background (45,46). On the 129/Sv genetic background, 35% of the Trp53-deficient mice develop testicular tumors (16,17,45,46) (hereafter abbreviated as Trp53null).

Ay. The agouti-yellow (Ay) mutation increases the incidence of liver, pulmonary, skin and mammary tumors (16,47). Interestingly, Ay on the 129/Sv inbred genetic background decreases the TGCT incidence to one-tenth the incidence in 129/Sv wild-type males (13). Ay is a ~175 kb deletion on chromosome 2 that deletes the coding region of the Raly gene and the entire Eif2s2 gene and places the Agouti gene under the transcriptional control of the Raly promoter (48,49). The Ay mutation arose on another inbred background and was made congenic on the 129S1/SvJ background (hereafter abbreviated as Ay).

129-Chr 19MOLF. A sensitized genome survey suggested that TGCT susceptibility genes were located on chromosome 19 in the MOLF/Ei strain (15). This suggestion was verified with the first autosomal CSS (36). Males that are homosomic for MOLF-derived chromosome 19 on the 129/Sv inbred background have a TGCT incidence of ~80% and many of these males develop bilateral tumors, whereas heterosomic males have a tumor frequency of ~20% and few develop bilateral tumors (36. Analysis of congenic strains derived from this CSS showed that at least five susceptibility loci, some of which act alone and some in combination, control susceptibility to TGCTs (22). A congenic strain derived from M19 has the proximal portion of chromosome 19 from MOLF/Ei homozygous on the 129S1/SvJ background. The proximal boundary presumably includes the centromere and the distal boundary is located between D19Mit132 and D19Mit5/D19Mit133 (22). Another congenic strain that was also derived from M19 has its distal boundary at the telomere of the long arm of chromosome 19 and its proximal boundary between the same markers as M19-A2. These congenic strains overlap only for the short chromosome segment that contains the markers D19Mit46, D19Mit64 and D19Mit81. Genetic variants in this overlapping segments does not affect the incidence of males with TGCTs, but instead interacts with several other linked genes to dramatically increase susceptibility (22) (these strains are hereafter abbreviated as M19, M19-A2 and M19-C2).

 

Given these unexpected results, we re-examined results for the Ter-SlJ and Ter-Trp53null tests to determine whether the increased frequency of affected Ter/+ males accounted for the increased frequency of affected Ter/+ m/+ males. We used the observed frequency of affected Ter/+ +/+ male segregants in the interaction tests to calculate a revised expected frequency of affected double-modifier males. These revised expected frequencies were 41% (instead of 22%) and 43% (instead of 25%) in the SlJ and Trp53null tests, respectively (Table 2). If the Ter/+ effect fully accounted for the double-modifier results, tests based on the revised frequencies should no longer be statistically significant. The frequency of affected Ter/+Trp53null/+ males in the Ter-Trp53null test was consistent with these revised expectations ({chi}2=1.30, P>0.05). In contrast, a significant excess of affected Ter/+ SlJ/+ males remained in the Ter-SlJ test after taking into account the observed frequency of affected Ter/+ +/+ males in this interaction test ({chi}2=21.42, P<10–4). Thus, interactions that led to the higher than expected frequency of affected Ter/+ +/+ males fully accounted for the increased frequency of affected Ter/+ Trp53null/+ segregants, but only partly for the frequency of affected Ter/+ SlJ/+ segregants, suggesting that additional epistatic effects are involved in Ter-SlJ double-modifier males.

Tests to determine whether modifier interactions affected the relative numbers of unaffected, unilateral and bilateral cases revealed a highly significant increase in the proportion of bilateral cases. In these tests, the expected numbers were based on the frequencies of cases with a TGCT in the left (fL) and the right testis (fR) such that, for example, the expected frequency of bilateral cases (fbil) was the product (fL)x(fR) (Eq. (1)). Similar calculations were used to estimate the expected frequency of unaffected and unilateral cases (Eqs. (2) and (3)). A strong and consistent excess of bilateral cases was found in Ter segregants in every interaction test but not in any of the Ter control crosses or in any of the crosses that did not involve Ter (Table 4 and see Supplementary Material, Table S2 for additional results). In the seven tests involving Ter, the frequency of bilateral cases was on average nearly 6-fold higher than expected (range 3–7-fold) and involved both Ter/+ +/+ as well as Ter/+ m/+ males (Table 4). In contrast, 21 of the 24 classes of segregants that did not involve Ter had the expected proportion of bilateral cases. The three exceptions were a 6-fold increase in +/+ M19/+ segregants from the Ter-M19 interaction test and a 13-fold and a 14-fold increase in SlJ/+ M19/+ and +/+ M19/+ segregants in the SlJ-M19 interaction tests (Supplementary Material, Table S2), suggesting that M19 might also affect the proportion of bilateral cases. In addition, modifier interactions did not affect the strong 3:1 left–right laterality bias that characterizes TGCTs in 129 males (13). Thus, the increased frequency of affected Ter/+ males in the six interaction tests resulted largely from a dramatic 6-fold increase in the proportion of affected males with bilateral TGCTs.


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  		Table 4. Observed and expected classes of TGCT cases in Ter–SlJ and Ter–Trp53null cases

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENATRY MATERIAL
 REFERENCES
 
Interaction tests revealed an unusual mode of inheritance for TGCT susceptibility. When one parent had Ter and the other parent had a different modifier, their Ter/+ male offspring showed 2–3-fold enhanced susceptibility and a 6-fold increase in the proportion of bilateral cases, regardless of whether other modifiers were present in these offspring. Direct molecular interactions between products of the modifier genes were not involved because modifiers were never present in the same parent and because both Ter/+ +/+ and Ter/+ m/+ male offspring were affected similarly. In humans, trans-generational epistatic effects could account for the unexpected 2–3-fold difference in risk for brothers (8–10-fold) and sons (4–6-fold) of affected individuals (8,9). Interactions between different genetic variants in different generations complicate studies of heritable traits in humans and model organisms because contributing variants are not necessarily present in affected individuals.

The high frequency of bilateral cases in Ter interaction tests has implications about the time, location and nature of events that initiate the transformation of primordial germ cells (PGCs—the TGCT stem cell) to TGCTs. In both humans and mice, bilateral TGCTs are infrequent and usually involve less than 5% of all cases (9,13,17,20–22). Moreover, the relative numbers of unilateral, bilateral and unaffected cases are consistent with a model of independent occurrence of TGCTs in each testis (22). In contrast, in every Ter interaction test, bilateral cases were frequent and were found more often than expected for the ‘independent occurrence’ model (Table 4) (22). Several developmental aspects of the PGC lineage could influence laterality. Between embryonic days 7.5 and 12.5, PGCs arise near the base of the allantois and migrate through the mesentery of the gut to the urogenital ridges, where the fetal testes will subsequently develop (23). During this time, PGCs proliferate from ~30 cells to ~25 000 cells. TGCT development might therefore involve distinct periods of vulnerability depending on whether transforming events occur in PGCs or in neighboring somatic cells and in part on whether these events occur during PGC migration or after their arrival in the urogenital ridges. Clonality of TGCTs with Kit mutations in many bilateral cases in humans supports events in PGCs before they arrive in the ridges (24).

TGCT modifiers in the parents provide clues to the signaling pathways that transduce information about environmental conditions through the epigenome to subsequent generations. The six TGCT modifiers were selected for these studies because they modulate susceptibility by acting directly or indirectly on PGCs in m/+ males (Table 1 and Box 1) and may at the same time mark the germline in ways that result in increased susceptibility in the next generation. These marks do not usually have phenotypic effects unless they are transmitted to a predisposed genetic background, such as Ter/+ males. Several hormonal and dietary factors modify the epigenetic code in the germline with adverse immunologic, neoplastic and reproductive consequences in subsequent generations (25–29). These environmental factors probably do not affect the epigenetic code directly, but rather are monitored by the organism and their inputs translated into changes in DNA methylation and various histone modifications. Perhaps in the same way organisms interpret the action of TGCT modifiers in parents as genetic analogs of these environmental perturbations and modify their epigenetic code in response to both genetic and environmental perturbations. Alternatively, environmental factors probably do not act directly on the epigenetic code, but instead molecular mechanisms, acting as intermediaries, monitor conditions and then modify the code.

The Dnd1Ter TGCT modifier provides clues to the complex interplay between the genome and epigenome in the dynamic control of gene expression during development. Dnd1 is closely related to apobec complementation factors (Acf1, Acf2 and Acf3) (30). These factors cooperate with the apobec family of cytidine deaminases to mediate cytidine-to-uridine (C-to-U) editing of repeated DNA elements, double-stranded RNAs and mRNA transcripts (31,32). Whether Dnd1 is a mediator or a target of the epigenetic code and how the Ter mutation affects these processes remains to be determined. We propose that epigenetic marks on the germline modulate the timing and level of expression of genes such as miRNAs and other RNAs that are essential for PGC biology and contribute to TGCT susceptibility (33–35) and that anomalies in these processes result in increased susceptibility to TGCTs in individuals with the Dnd1Ter mutation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENATRY MATERIAL
 REFERENCES
 
Mice
129S1/SvImJ (JR002448, previously known as 129/Sv, 129/SvJ and 129S3/SvImJ), 129S1/Sv-p+Tyr+KitlSlJ/+ (JR000090), 129-Trp53tm1Tyj (JR002080) and 129T1/Sv- p+Tyrc-ch Dnd1Ter/J (JR000091) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The 129-Chr 19MOLF CSS was described previously (36) and was obtained from our research colony. The 129.B6-Ay congenic strain was described previously (17). Mice were maintained in the CWRU Animal Resource Center on a 12:12 h light:dark cycle and fed Lab Diet 5010 (Richmond, IN, USA). All protocols were approved by the Institutional Animal Care and Use Committee.

Genotyping
DNA for PCR genotyping was extracted from tail tissue (19).

Dnd1Ter. The nucleotide substitution in the Ter mutant results in the creation of a Dde1 site that was used for genotyping (37). KitlSlJ. The SlJ mutation can be identified by a light coat color on the belly and pink tips of the tail and digits on an otherwise pigmented mouse (17,38).

Trp53. A three primer PCR assay was used to distinguish wild-type from Trp53null heterozygous males (17). In addition, the sequence for primer X7 in ref. 17 is incorrect; the correct sequence is 5'-tatactcagagccggcct-3'.

Ay. Mice that were heterozygous for this mutation were identified by their yellow coat color (38).

Necropsy
Males were necropsied at approximately 4 weeks of age and testes were visually examined for tumors.

Expected frequency of affected males in interaction tests
The null hypothesis was that TGCT modifiers act in an additive and independent manner, and therefore that the expected frequency of affected double-mutant males can be estimated from the frequencies of affected single-mutant and wild-type males. We used the frequency of affected single mutant (fm) and control males (fc) to calculate the expected frequency of affected double-mutant males (fm1m2=fm1+fm2–fc) in each interaction test, where m designates the mutant, CSS or congenic in the interaction test. Chi-square goodness-of-fit tests were then used to compare the observed and expected numbers of affected males in each genotypic class. Significance levels were adjusted to account for the 14 {chi}2-tests among the three interaction tests (SlJ—4 tests, Trp53null—4 tests, and M19–3 tests).

Expected frequencies of unaffected, unilateral and bilateral tgct cases
We hypothesized that bilateral cases result from the coincidental and independent occurrence of TGCTs in the left and right testes, and that the relative proportions of bilateral, unilateral and unaffected cases can be predicted simply from the frequency of left testes with a TGCT and from the frequency of right testes with a TGCT. The analytical methods to test this hypothesis were described previously (22,37). Briefly, for each genotypic class in each interaction test, we calculated the observed frequency of males with a TGCT in the left testis (fL) and separately the observed frequency of males with a TGCT in the right testis (fR), regardless of presence of a TGCT in the contralateral testis. To calculate the expected frequencies of unaffected males, males with a unilateral TGCT (either left or right), and males with bilateral TGCTs, we made the following calculations:

  1. expected frequency of unaffected males


    Formula 175M1

    		(1)

  2. expected frequency of males with a unilateral TGCT


    Formula 175M2

    		(2)

  3. expected frequency of males with bilateral TGCTs


    Formula 175M3

    		(3)

The expected number of affected males were then obtained from the product of each of these expected frequencies and the sample size (n) for each genotypic class in each test group. Significance levels were adjusted to account for the multiple statistical tests.


   SUPPLEMENATRY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENATRY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Drs. Ron Conlon, Angabin Matin, Kathy Molyneaux and Peter Scacheri for critically reading a draft of this manuscript. This work was supported by NIH grant CA75056.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENATRY MATERIAL
 REFERENCES
 

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