LINE-1 methylation is inherited in familial testicular cancer kindreds

Testicular germ cell tumors (TGCT) are the most commonly diagnosed cancer among young American men aged 20-35 years, and their incidence has doubled over the last 40 years [1]. Family history of TGCT is one of the strongest and most consistent risk factors for this tumor. Brothers of affected cases have an 8-to 10-fold increased relative risk compared with the general population and fathers/sons a 4-and 6-fold higher risk [2, 3]. These high familial risks suggest that inherited susceptibility and/or environmental factors that cluster in families may account for a significant portion of TGCT cases. Testicular microlithiasis (TM), a condition characterized by the presence of calcium deposits within the seminiferous tubules, aggregates in families and has been associated with testicular malignancy [4, 5]. Linkage and candidate gene studies [6–10] have identified several genomic regions of interest, including Xq27, 2p23, 3p12, 3q26, 12p13-q21 and 18q21-q23. Candidate gene studies have identified two loci of interest, i.e., the Y-chromosome gr/gr deletion [9] and the PDE11A gene [8], while recent GWAS analyses identified KIT-ligand, SPRY4 and BAK1) [6, 7]. However, a specific high-penetrance susceptibility gene has yet to be proven, suggesting that the combined contribution of multiple common genetic variants of lower penetrance may account for the inherited component of TGCT susceptibility [10].

Epigenetic changes in the genome, such as aberrant DNA methylation, are an increasingly recognized contributor to cancer development. TGCTs have distinct DNA methylation profiles, and parallels have been observed between the epigenetic properties of TGCTs and embryogenesis (e.g., DNA methylation increases with differentiation) [11]. The seminoma genome is essentially devoid of DNA methylation, while the nonseminoma genome is less extensively hypomethylated and has variable CpG island hypermethylation levels [12, 13]. A few tumor suppressor genes are inactivated by DNA promoter hypermethylation in a small proportion of TGCTs, while DNA hypomethylation of testis-and cancer-associated genes and unmethylated XIST are frequently observed in TGCTs (Reviewed in [11]). Imprinting defects have also been observed in TGCT somatic tissues, suggesting that epigenetic defects may be present. In the aggregate, the data suggest that methylation may provide an alternate genetic mechanism for TGCT susceptibility.

Transgenerational epigenetic inheritance has been well studied and documented in many eukaryotic organisms (e.g., maize, yeast, Drosophila, mice) [14–16], and recent human studies suggest that epigenetic aberrations that influence cancer risk can be inherited through the germline from parent to child [17–20]. Epigenetic patterns have been observed to segregate in both Mendelian and non-Mendelian patterns, as well as in a pattern consistent with environmental exposure. Non-Mendelian and environmentally-induced transgenerational inheritance of epimutations is supported by model organism data [21–27]. There are several potential modes of transgenerational epigenetic inheritance, including paramutations, aberrant gene imprinting, and dsRNA processes (Reviewed in [28]). Retrotransposons are thought to be resistant to epigenetic reprogramming during embryogenesis in mice [29], and thus may play a role in epigenetic heritability (e.g., if inserted in or near the affected gene). A recent mouse model study found that transgenerational epigenetic inheritance through the germline controls susceptibility to TGCTs [21].

Global DNA hypomethylation, characterized by a global loss of 5-methylcytosine (5-mC), contributes to malignant transformation by activating oncogenes or latent retrotransposons, such as LINE-1, and by loss of imprinting [30]. LINE-1 retrotransposons are hypomethylated in many cancers and their lack of repression may contribute to genome disorganization, expression changes, and chromosomal instability [31]. The nucleotide patterns of LINE-1 repeats have been shown to both segregate in human pedigrees, and to provide an individual-specific fingerprint [32]. LINE-1 insertion dimorphisms may reflect DNA mutations or methylation changes [32]. Methylation levels at LINE-1 loci have been shown to differ among different loci in normal tissues, suggesting that cis-elements near each LINE-1 may influence their epigenetics [33]. In addition, locus-specific LINE-1 methylation has been shown to be differentially influenced by carcinogenic processes, depending on where the LINE-1s are located in the genome [33]. LINE-1 retrotransposons are strongly expressed in TGCTs [31, 34, 35], and it was hypothesized that active LINE-1 retrotransposon events are involved in primordial germ cell differentiation, and the consequential genome instability contributes to malignant transformation [35].

In recent case-control studies, hypomethylation of peripheral blood DNA was found to be associated with increased risks of bladder and head/neck cancers, suggesting that global demethylation in genomic DNA is a potential biomarker of cancer susceptibility [36, 37]. We performed a pilot analysis of global DNA methylation levels in peripheral blood among family members from 101 multiple-case familiar testicular cancer families. Methylation at LINE-1 regions using pyrosequencing was used as a proxy for measuring global methylation levels. We examined the heritability of the global genomic methylation phenotype in our families, as well as the association between global (LINE-1) methylation levels and testicular cancer.

We found that males had significantly higher global (LINE-1) methylation levels than females, consistent with prior reports [37–39]. This difference has been variably attributed to gender-related dietary differences, reduced circulating levels of folate due to female menstruation, and/or the presence of an additional X chromosome in female cells [37, 39]. We found no association between LINE-1 methylation levels and age [36, 37, 40], smoking [36, 38], or alcohol usage [36, 37], also consistent with prior studies.

LINE-1 methylation levels of offspring were found to be significantly positively correlated with parental levels, particularly between mother-daughter, father-daughter, and affected father-affected son pairs. There are limited data investigating epigenetic inheritance in humans, although it has been suggested that epimutations may be maintained more frequently during oogenesis [41, 42] than during spermatogenesis [43]. Differences in epigenetic reprogramming between maternal and paternal genomes have been observed in mouse and drosophila studies [22, 23, 25–27]. In mice, epigenetic inheritance at a locus involved in fur color demonstrated that there was incomplete erasure of an epigenetic modification at a retrotransposon when passed through the female germ line [23].

However, others have shown that exposure to an endocrine disruptor during embryonic gonadal sex determination in rats induced permanent DNA methylation imprinting of the male germline (i.e., sperm) that was transmitted to subsequent generations [25–27]. This epigenetic alteration was associated with transgenerational disease states (approximately 85% frequency), including abnormalities in testis function, male infertility, and tumor development. This observation illustrates how an environmental exposure can epigenetically reprogram the germline and promote adult-onset defects in the germline, providing one possible mechanism by which environmental exposures may promote TGCTs [44].

Transgenerational epigenetic interactions have been shown to control susceptibility to TGCTs in mice with Dead-end homologue 1 (Dnd1) mutations [21]. It was suggested that these epigenetic modifiers may mark the germline, and offspring that also carry a genetic susceptibility factor will have an increased risk of TGCTs; or, environmental factors may influence genes to act as mediators and to monitor cellular conditions that then induce epigenetic alterations [28]. Lam et al. [21] suggested that, in humans, transgenerational epigenetics may explain the 2-3 fold risk difference in brothers and sons of TGCT affected individuals. The strong correlation we observed between LINE-1 methylation levels among affected father-affected son pairs may reflect an inherited or germline LINE-1 hypomethylation abnormality. Such an inherited epigenetic event could be associated either with an environmental factor or genetic susceptibility.

We observed an association between TGCT and global hypomethylation that was marginally significant when considering LINE-1 methylation as a continuous variable after controlling for potential confounders and testicular cancer risk factors (age and microlithiasis). Our study had ≥80% statistical power to detect ORs of 1.6 and 2.4 with LINE-1 methylation in the second and first tertiles, respectively, assuming a type I error of 0.05. Other studies have found that global hypomethylation in DNA from blood was associated with bladder and head/neck cancers [36, 37]. Genomic global (LINE-1) methylation may be an independent risk factor or a phenotypic marker of cancer risk associated with genetic instability, aberrant epigenetic regulation, or other factors.

Our small sample size with attendant reduced statistical power to detect differences in LINE-1 methylation levels is a limitation of this study, although ours is the largest cohort of multiple-case families available for analyses of this kind. The family-based design of this study also limits our exploring the association between TGCT and global hypomethylation levels, since we have observed that this may be an heritable phenotype; a non-family based matched control group would be a useful follow-up. However, the unique multiple-case family design of this study is a strength for investigating inheritance, which allowed us to study multiple individuals per family with and without cancer, and to eliminate potential confounding due to ethnic background or population stratification.

LINE-1 retrotransposons are highly-activated in TGCTs and may lead to insertional mutations, transcriptional deregulation, DNA breaks, and an increased frequency of recombinational events [31, 34, 35]. Little is known regarding the mechanisms underlying hypomethylation changes of LINE-1 sequences; however, available data suggest that LINE-1 retrotransposon events may contribute to genome instability and malignant transformation. This is the first study to show that global LINE-1 methylation levels may be inherited from parent to child in humans, and that hypomethylation is associated with testicular cancer risk. More research is needed to help explain differences in maternal/paternal effects and mechanisms of transgenerational epigenetic inheritance.

The results of this study suggest that LINE-1 methylation is heritable in humans, and that transgenerational inheritance of LINE-1 methylation levels may be associated with testicular cancer risk. The incomplete penetrance and non-Mendelian inheritance of epigenetic variation are consistent with complex disease phenotypes, which familial testicular cancer displays, and thus it may provide some insight into the basis of this complex disease. It has proved difficult to identify high-penetrance TGCT susceptibility genes in humans, despite a high familial risk and likely inherited susceptibility. Transgenerational epigenetic mechanisms could help explain the difficulty of finding classical TGCT susceptibility genes in humans. Larger studies are warranted to further explore the association between genomic global hypomethylation and TGCT risk, and to investigate the hypothesis that LINE-1 epigenetic inheritance is associated with TGCT susceptibility.