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Gene identification has progressed rapidly for monogenic epilepsies, but complex gene–environmental interactions have hindered progress in gene identification for multifactorial epilepsies. We analyzed the role of environmental risk factors in the inheritance of multifactorial idiopathic generalized epilepsy in the EL mouse. Seizure susceptibility was evaluated in the EL (E) and seizure-resistant ABP/LeJ (A) parental mouse strains and in their AEF1 and AEF2 hybrid offspring using a handling-induced seizure test. The seizure test was administered in three environments (environments I, II and III) that differed with respect to the number of seizure tests administered (one test or four tests) and the age of the mice when tested (young or old). The inheritance of seizure susceptibility appeared dominant after repetitive seizure testing in young or old mice, but recessive after a single test in old mice. Heritability was high (0.67–0.77) in each environment. Significant quantitative trait loci (QTL) that were associated with environments I and III (repetitive testing) were found on chromosomes 2 and 9 and colocalized with previously mapped El2 and El4, respectively. The El2 QTL found in environment I associated only with female susceptibility. A novel QTL, El-N, for age-dependent predisposition to seizures was found on proximal chromosome 9 only in environment II. The findings indicate that environmental risk factors determine the genetic architecture of seizure susceptibility in EL mice and suggest that QTL for complex epilepsies should be defined in terms of the environment in which they are expressed.
Epilepsy is a significant health problem in human and with the exception of stroke is the most prevalent neurological affliction. The number of persons with epilepsy in the United States is estimated at 2.5 million or about 1% of the population. Many persons with epilepsy manifest generalized or partial seizures without signs of organic brain disorder, i.e. idiopathic epilepsy (Marini et al. 2004; Wolf 1994; Wolf 2005). This contrasts with symptomatic epilepsy, where seizures arise from brain injury, disease or neurostructural abnormality (Baumann 1982; Hauser 1982; Wolf 1991). Although many idiopathic generalized epilepsies (IGEs) have a genetic predisposition, the genes for only a few have been identified (Marini et al. 2004). Genetic heterogeneity, variable age of onset and multifactorial inheritance have hindered progress in identifying the genetic and biochemical defects responsible for the most common forms of human IGE (Tan et al. 2004).
Multifactorial disorders involve the action of more than one gene together with environmental factors (Poderycki et al. 1998; Todorova et al. 1999). As a result, these disorders do not usually follow simple Mendelian modes of inheritance. A failure to identify and measure the environmental risk factors for complex diseases will hinder the resolution of genetic architecture (Woolf 1952). The environmental factors that influence multifactorial disorders can be both external and internal (Strickberger 1985; Wright 1934). In the case of epilepsy, external environmental factors can include temperature, light, sound, nutrition, maternal effects, infectious agents and brain trauma. Internal environmental factors, on the other hand, can include gender, circadian rhythms, hormones, seizure history and age (Todorova et al. 1999). In humans, it is difficult to identify subtle environmental factors that influence seizure susceptibility and gene mapping. Hence, an animal model such as the EL mouse, having IGE with multifactorial etiology, is ideally suited for characterizing the mechanisms by which genetic and environmental risk factors interact to influence seizure susceptibility.
The epilepsy (EL) mouse, discovered in Japan in 1954, expresses complex partial seizures with secondary generalization and is a model of idiopathic epilepsy (Kurokawa et al. 1966; Naruse & Kurokawa 1992; Suzuki 2004; Todorova et al. 1999). The seizures in EL mice normally occur during routine handling associated with cage changing at about 70–90 days of age but can be induced in younger mice (45–50 days) by rhythmic vestibular stimulation, e.g. tossing, rotation, rocking or handling (Frankel et al. 1995b; Fueta et al. 1983; Poderycki et al. 1998; Seyfried et al. 1992; Suzuki 2004; Todorova et al. 1999). Seizure onset is earlier in males than in females, but the gender effect dissipates with age (Todorova et al. 1999). The seizures originate in or near the parietal lobe, secondarily generalize to the hippocampus and other brain regions and produce abnormal plasticity (Ishida et al. 1993; Kasamo et al. 1992; Suzuki et al. 1991). Electroencephalographic abnormalities also accompany the seizures in addition to vocalization (squeaking), incontinence, loss of postural equilibrium, excessive salivation as well as head, limb and chewing automatisms (Kurokawa et al. 1966; Seyfried et al. 1999; Suzuki 2004; Suzuki & Nakamoto 1977; Todorova et al. 1999; Uchibori et al. 2002). Although the seizures in EL mice occur without gross signs of organic brain disorder or cell loss, a reactive gliosis involving astrocytes and microglia is associated with seizure progression (Brigande et al. 1992; Drage et al. 2002; Murashima et al. 2005). Hence, the seizure disorder in EL mice is best characterized as IGE.
As occurs with the inheritance of seizures in many persons with IGE, the inheritance of seizure susceptibility in EL mice is complex (Frankel et al. 1995a, 1995b; Fueta et al. 1986; Legare et al. 2000; Marini et al. 2004; Poderycki et al. 1998; Rise et al. 1991; Tan et al. 2004). Several quantitative trait loci (QTL) were found that influenced susceptibility to tossing-induced seizures. The detection of these ‘El’ epilepsy genes, however, depended on genetic background (the strain to which EL was crossed) and on the type of cross (intercross or backcross). El1 (chromosome 9) and El2 (chromosome 2), for example, accounted for much of the phenotypic variation in seizure frequency in a backcross to the non-epileptic ABP strain, whereas El3 (chromosome 10) and El4 (chromosome 9) had the largest effects in the intercross. In crosses between EL and DDY, a seizure-resistant mouse strain related to EL, El5 (chromosome 14) was the major susceptibility QTL detected in the intercross but was undetectable in the backcross to DDY. A recessive seizure enhancer, El6 (chromosome 11), was also found in the seizure-resistant DDY mice. It was concluded that no single El locus is essential for high seizure frequency and that the extreme genetic complexity of EL seizure susceptibility resulted from complex epistatic or additive interactions between many non-allelic El epilepsy genes (Frankel et al. 1995b). It is important to note that these studies employed repeated mouse tossing in mechanical shakers (250–280 tosses/min, three times a week for many weeks) for seizure induction (Frankel et al. 1995a; Poderycki et al. 1998; Rise et al. 1991). Because the seizure phenotype was identical in young EL mice that were repeatedly tossed and in older EL mice that were handled, little consideration was given to the possibility that the seizure test itself might complicate the inheritance of seizure susceptibility. In addition to epistatic and additive genetic interactions, environmental risk factors also contribute to the complex inheritance of seizures in EL mice (Todorova et al. 1999).
We and others found that repeated mouse tossing produces phenocopies, the most extreme form of environmental influence (Muraguchi & Serikawa 1995; Poderycki et al. 1998). Furthermore, the strength of associations between genetic markers and seizure frequency was strongly dependent on the number of tossing tests that the animals received. For example, the association between seizures and El1 decreased with the increasing number of tossing tests, whereas the opposite was the case for El2. These findings indicate that the seizure frequency phenotype is not independent of the seizure test and that the mapping of ‘El’ epilepsy genes can be influenced by test-associated complications. Hence, it is unclear whether the previously mapped El epilepsy genes influence susceptibility to idiopathic epilepsy, to symptomatic epilepsy caused by repeated tossing or to some combination of these.
To reduce test-associated complications, we developed a new procedure for seizure induction in EL mice that involves gentle handling. The mice are simply held by the tail for short consecutive intervals (Todorova et al. 1999). The procedure simulates the situation that normally occurs during routine handling associated with cage changing. Most EL mice tested using this procedure beginning at 30 days develop seizures by 50–60 days of age. In contrast to previous testing procedures involving repeated tossing, our handling test for seizure induction does not increase risk for brain damage and does not induce seizures (false positives) in non-epileptic control strains. Furthermore, the handling paradigm simulates procedures used for inducing emotional stress in rodents (Balcombe et al. 2004; Beck & Gavin 1976; Drage & Heinrichs 2005; Misslin et al. 1982). This is important because emotional stress has long been considered a trigger for the induction of seizures in humans with idiopathic epilepsy (Gowers 1901; Nakken et al. 2005; Todorova et al. 1999). This new testing procedure best approximates stress-related conditions that naturally precipitate epileptic seizures in these mice. In this study, we used this new seizure testing method to map genes involved in the predisposition to IGE and to analyze gene–environmental interactions influencing the expression of the seizure phenotype in EL mice.
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Recent advances in molecular biology and genetics have facilitated the discovery of genetic defects for several rare epilepsy syndromes with simple Mendelian mode of inheritance (Anderson et al. 2002; Steinlein 2004). However, most common human epilepsies are multifactorial disorders where more than one gene and environmental risk factors contribute to the seizure phenotype (Doose & Maurer 1997; Jimenez et al. 1996; Marini et al. 2004; Zifkin et al. 2005). In these epilepsies, large single families are rarely identified, and genetic heterogeneity is common. Moreover, little consideration is given to the nature or types of environmental risk factors that might influence the seizure phenotype. According to Woolf (1952), whenever a character is influenced by environment, the genetic analysis cannot be considered adequate in either practice or theory unless it clearly specifies the precise features of the environment that affect the character and their quantitative importance in relationship to different genotypes. In the absence of such information, it is not surprising that genetic linkage studies for the common epilepsies and other complex behavioral disorders have been difficult to replicate (Marini et al. 2004; Plomin & McGuffin 2003; Tan et al. 2004).
In this study, we used the EL mouse, an animal model of IGE, to investigate the role of gene–environmental interactions in the development of seizure susceptibility. As environmental risk factors contribute significantly to seizure susceptibility in this animal model (Poderycki et al. 1998; Todorova et al. 1999), the EL mouse is well suited for the evaluation of the environmental influences on genetic predisposition to multifactorial IGE. We examined the genes controlling epilepsy in EL mice by mapping seizure susceptibility QTL in three different testing environments. In environment I, mice were exposed to stressful stimuli earlier in life, and their seizure susceptibility was significantly higher than that of naïve, age-matched animals (environment II). These findings suggest that repetitive testing and seizures may kindle the nervous system, thereby lowering the seizure threshold to later tests. Similar kindling-like phenomena were reported in epileptic gerbils and in rats that were repeatedly exposed to stressful environmental stimuli and hyperthermia (Klauenberg & Sparber 1984; Scotti et al. 1998). These effects are related to Gowers’ dictum that seizure begets seizures (Todorova et al. 1999). Furthermore, the etiology of epilepsy in environment I may be related to that of symptomatic epilepsy and may highlight genetic factors influencing the predisposition to seizures after emotional and physical trauma. We previously indicated that repeated tossing of mice over several weeks obscures the boundaries between idiopathic and symptomatic etiologies and will produce a seizure phenotype that is extremely complex (Poderycki et al. 1998).
In environment II, the mice were allowed to age without exposure to repetitive emotional stress or seizures (repetitive testing). The QTL associated with seizure susceptibility in this environment likely determine age-dependent predisposition to idiopathic epilepsy. The conditions for environments I and II are combined in environment III, where mice were allowed to age without exposure to a stressful environment and were then repetitively tested. Because different factors precipitated seizures in environments I and II, we hypothesized that QTL controlling the expression of phenotypically similar seizures would be different in these two environments. This hypothesis was supported by our findings that QTL identified in environments I and II were indeed different and that the QTL seen in environment III, which is a combination of environments I and II, were also seen separately in environments I and II. Moreover, the identified QTL in environment I were previously reported in studies where repetitive shaking and pentylenetetrazole (PTZ) were used to induce seizures, supporting the hypothesis that these loci are important for stress/trauma-induced epilepsy (Legare et al. 2000). In addition, the studies in environment I confirmed a gender-specific QTL on chromosome 2 (El2) that was previously identified in studies in which seizures were induced by shaking and PTZ, suggesting the importance of gender-specific genes controlling the predisposition to symptomatic epilepsy (Legare et al. 2000). Our findings in EL mice support previous suggestions that the estimates of QTL positions and effects are relevant only to the environment and sex in which the phenotype is assessed and may not replicate across sexes or environments (Mackay 2001).
Our studies also identified a novel susceptibility factor, El-N, on chromosome 9 between D9Mit188 and D9Mit2. These findings also showed that El-N is distinct from the previously identified El1 and El4 also located more distally on this chromosome (Frankel et al. 1995b; Rise et al. 1991). This region of chromosome 9 had not been previously associated with seizures in other animal epilepsy models, and no epilepsy locus has been mapped on human chromosome 11, which is syntenic to the region on mouse chromosome 9 containing El-N. Although the glutamate receptor subunit 4 gene, GluR4, maps to this region of chromosome 9, no seizure-associated polymorphism was detected in a preliminary screen of this gene (unpublished observation). We do not, however, exclude the possibility that GluR4 expression might be influenced by the different seizure testing environments (Naka et al. 2005).
El-N was identified in naïve adult mice that aged without exposure to environmental stress, suggesting that this locus determines the predisposition to age-dependent idiopathic epilepsy. El-N was not detected in previous studies, because it potentially played only a minor role in developing symptomatic epilepsy. Our data from environment III suggest that El-N influenced seizure susceptibility in aging adult animals that received repetitive stressful stimuli. The identification of El-N also illustrates the potential influence of age and repetitive seizures (Gowers’ dictum) on epilepsy gene mapping (Todorova et al. 1999). These results support the hypothesis of Lennox that the division between idiopathic and symptomatic epilepsy is not clear and that environmental and genetic factors determine seizure susceptibility in every epileptic patient (Lennox 1951).
Previous studies of multifactorial traits in experimental animals have emphasized the role of genotype–environment interactions in the determination of phenotype (Falconer & Mackay 1996; Mackay 2001; Wahlsten et al. 2003). The existence of such interactions indicates that a single genotype can have different performances in different environments (Cooper & Zubek 1958; Crabbe et al. 1999; Henry 1967; Schridde & van Luijtelaar 2004). Falconer (1960) also suggested that a character measured in two different environments should be regarded as two characters rather than as one and proposed that different genes may control the character in different environments. This was supported further from recent findings in Drosophila melanogaster that the expression of QTLs controlling longevity is strongly dependent on temperature and food availability in the environment and that no single QTL was present in all environmental conditions (Vieira et al. 2000). Similarly, the survival and fertility of Caenorhabditis elegans are also determined by QTL whose expression depends on the type of media used (Shook & Johnson 1999). Although the heritability estimates for seizure susceptibility were generally similar in the three environments, our results indicate that the underlying genetic factors responsible for the estimates differ in each environment.
In summary, we have defined how different seizure testing environments influence the inheritance and genetic architecture for seizure susceptibility in epileptic EL mice. Moreover, our findings suggest that QTL for complex epilepsies should be defined in terms of the environment in which they are expressed. These findings in mice should be instructive for those studies attempting to define the genetic architecture of the common epilepsies in humans where significant environmental effects may influence seizure predisposition.