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Keywords:

  • Epilepsy;
  • Gowers’ dictum;
  • idiopathic;
  • inheritance;
  • mapping;
  • QTL;
  • symptomatic

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

Mice

The inbred EL/Suz (EL) mice were originally obtained from J. Suzuki (Tokyo Institute of Psychiatry) and are now available from The Jackson Laboratory, Bar Harbor, ME. The inbred non-epileptic ABP/LeJ (ABP) strain was purchased from the Jackson Laboratory. Mice were housed in plastic cages with Sani-chip bedding that were changed once per week. The mice were kept on a 12-h light/dark cycle with food (Agway Prolab Rat/Mouse/Hamster 3000; Richmond, IN, USA) and water provided ad libitum. All mice used were maintained in the Boston College Animal Care Facility, and the procedures for their use were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care Committee.

EL and ABP mice were crossed to produce reciprocal F1 hybrids. The female mouse is presented first in a cross, e.g. the (ABP × EL) F1 hybrid is derived from crossing an ABP female with an EL male. F1 hybrids were crossed to produce (ABP × EL) F2 generation mice. Reciprocal crosses were used to produce both F1 and F2 generation mice. All mice were weaned at 30 days of age, and a tail clip was obtained after weaning for genotyping.

Seizure testing

Mice were tested for seizures, and the severity of seizures was graded as previously described (Todorova et al. 1999). Briefly, the test included two phases (I and II) that were separated by 1 week. Each phase involved two handling trials that were separated by 30 min. In each trial, a mouse was held by the tail for 30 seconds about 10–15 cm above the bedding of its home cage. The mouse was then placed in a cage containing fresh bedding for 2 min. The mouse was held again for 15 seconds and then returned to the home cage. The test was repeated after 1 week (phase II). Mice were undisturbed (no cage changing) for 1 week prior to testing, and all tests were performed between 1300 h and 1800 h. During each testing phase, a numerical score, depending on the seizure severity, was assigned to each animal. The final test score was calculated as a mean from the scores in phases I and II. Seizure severity was graded in the parental EL and ABP and the F1, and F2 hybrid mice according to the following scores: 1, squeaking; 2, immobility, blinking and mild facial clonus; 3, catatonic posture with Straub tail; 4, forelimb clonus with rearing and 5, generalized tonic convulsions with ventroflexion or dorsoflexion of the neck.

Seizure testing environments

Seizure susceptibility was determined for the EL, ABP, F1 and F2 mice in three different environments as described in Fig. 1. The three environments included:

image

Figure 1. Seizure testing environments used to characterize the influence of genetic and environmental factors on epilepsy in EL mice. Test is the handling-induced seizure test as described in Materials and methods. For each environment, seizure susceptibility data for the genetic analysis in environments I, II and III were at 141, 150 and 261 days of age, respectively.

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Environment I: mice were tested for seizures four times with 30-day intervals between the consecutive tests starting at 30 days of age. The test scores from the fourth test were used for the genetic analysis.

Environment II: naïve mice, previously untested, were tested for seizures once at 150 days of age. The test scores from the first test were used for the genetic analysis.

Environment III: mice were tested for seizures four times with 30-day intervals between the consecutive tests starting at 150 days of age. The test scores from the fourth test were used for the genetic analysis.

Genetic marker analysis

Genomic DNA was isolated from mouse tails using a modified DNA isolation procedure as we previously described (Allen & Seyfried 1994; Hoffman & Winston 1987). Simple sequence-length polymorphism (SSLP) markers were used to identify genetic loci associated with seizure susceptibility (Frankel et al. 1995a). SSLPs were characterized by the polymerase chain reaction (PCR) using oligonucleotide primers purchased from Research Genetics (Invitrogen, Carlsbad, CA, USA). PCR products were separated by electrophoresis on 6% denaturing polyacrylamide gels. The gels were exposed to X-ray film overnight at −80 °C. At least two of the authors manually read the films and recorded the genotype information.

Statistical analysis

Genotype and quantitative trait data were stored and sorted by Excel (Microsoft Inc., Redmond, WA, USA). The seizure susceptibilities of animals in the different groups were analyzed by the two-tailed t-test. The position and the effect of the QTLs were determined as logarithm of the odds (LOD) scores through an interval mapping procedure using Mapmaker/EXP and Mapmaker/QTL software (Lander et al. 1987). Associations between seizure susceptibility and genetic markers were also analyzed using the two-tailed t-test.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

The seizure susceptibility phenotype in EL mice is complex, and there has been controversy on the number and the location of the genes involved. To address the influence of seizure testing environment, we evaluated seizure susceptibility in crosses between epileptic EL mice and non-epileptic ABP mice in three environments that differed with respect to age (young or old) and the number of seizure tests administered (one or four) (Fig. 1). Environments I and III involved repetitive seizure testing at 30-day intervals beginning at either 30 days of age (young) or 150 days of age (old), respectively. Environment II involved a single seizure at 150 days of age.

Seizure phenotypes

We previously showed that the young EL brain is less susceptible to seizures than the old EL brain, as the percentage of EL mice experiencing seizures at 30 days of age after a single seizure test was zero, whereas 75% of EL mice experienced seizures after a single seizure test at 180 days of age (Todorova et al. 1999). The seizure susceptibility scores of the EL and ABP parental strains, the F1 hybrids and the F2 generation mice were determined for each of the three environments (Fig. 2). Although the mice tested in environments I and II were of similar age (141–150 days, respectively), the mean seizure severity scores for each genetic group were significantly higher in environment I than in environment II (P < 0.001, two-tailed t-test) (Fig. 2a,b). The mean seizure severity scores of the EL and the F1 hybrid mice were similar to each other and significantly higher (P < 0.001, two-tailed t-test) than those of the ABP mice in environments I and III simulating dominant inheritance for seizure susceptibility (Fig. 2a,c). On the other hand, the mean seizure severity scores of the ABP and the F1 hybrid mice were similar to each other and significantly lower (P < 0.001, two-tailed t-test) than those of the EL mice in environment II simulating recessive inheritance (Fig. 2b). The variance in the F2 generation mice was higher than that in the F1 generation mice in all environments (Fig. 2). Thus, the heritability of epilepsy in each of the three environments was high and varied between 0.65 and 0.77. These findings indicate that environmental and genetic factors influence seizure susceptibility in EL mice.

image

Figure 2. Mean seizure severity score (SSS) and heritability (h2) estimates for seizure susceptibility in the three different testing environments: a, environment I; b, environment II; c, environment III. The h2 estimates were calculated by subtracting the (ABP × EL) F1 (environmental) variance from the (ABP × EL) F2 (genetic and environmental) variance and dividing by the (ABP × EL) F2 (genetic and environmental) variance. Reciprocal F1 hybrids had similar variance in environment I. Only (EL × ABP) F1 hybrids were used for the estimation of environmental variance in environments II and III. The test and age of the animals are as described in Fig. 1 and Materials and methods. For environment III, naïve EL mice between 286 and 295 days of age were used to determine seizure susceptibility. Although these EL mice were about 1 month older than the ABP, F1 or F2 mice, seizure susceptibility is similar in all EL mice older than 200 days of age, and repetitive seizure testing does not influence their already maximum seizure susceptibility; n, number of mice tested; v, variance.

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Seizure susceptibility QTL

To determine the effect of previously identified QTL on the seizure susceptibility in the three different environments, F2 mice were genotyped for genetic markers on chromosomes 2, 9 and 10 that were previously associated with seizures (Frankel et al. 1995a; Legare et al. 2000; Poderycki et al. 1998; Rise et al. 1991). Thirteen SSLPs were tested for association with seizures in the F2 mice in each environment. In environment I, a region on chromosome 9 containing El4 (near D9Mit22) was strongly associated with seizure susceptibility, whereas no significant association was found with the region containing El1 (near D9Mit35) (Fig. 3a). The LOD score peak on chromosome 9 was located between markers D9Mit22 and D9Mit32 (LOD = 4.35, with 20.1% of the total variance explained) (Fig. 3a; Table 1). The analysis of chromosome 2 markers (associated with El2) revealed only a weak association with seizure susceptibility (LOD = 2.16, with 10.2% of the total variance explained) located between D2Mit30 and D2Mit21 (Fig. 3b). However, this region had a gender-specific effect and was strongly associated with seizure susceptibility in females (LOD = 3.72, accounting for 35.2% of the total variance), but not associated in males (Fig. 3b, inset; Table 1). Furthermore, the gender effect for El2 was observed only in environment I. No association was found between seizure susceptibility and El3 located on chromosome 10 near D10Mit42 (Fig. 3c).

image

Figure 3. Genetic maps indicating interval logarithm of the odds (LOD) scores for linkage to loci controlling seizure susceptibility in environment I: a, chromosome 9; b, chromosome 2 (females and males, inset); c, chromosome 10. LOD scores were determined with Mapmaker/QTL using seizure scores as the trait measure.

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Table 1.  Quantitative trait loci (QTL) for seizure susceptibility in different environments *
EnvironmentQTLGender specificityPositionLOD scoreTotal variance (%)
  • *

    Mapmaker/QTL was used for the interval mapping analysis.

  • The QTL position corresponds to the maximal logarithm of the odds (LOD) score in an interval between neighboring markers.

  • Total variance was obtained from Mapmaker/QTL analysis.

IEl4Male/femaleD9Mit22 + 12 cM4.3520.1
El2FemaleD2Mit30 + 6 cM3.7235.2
IIEl-NMale/femaleD9Mit188 + 6 cM3.469.2
El3Male/femaleD10Mit42 + 4 cM2.295.6
IIIEl-NMale/femaleD9Mit188 + 6 cM6.2915.7
El4Male/femaleD9Mit22 + 8 cM4.2812.7

In environment II, no significant associations were found between seizure susceptibility and the previously identified QTL, El1 or El2 (Fig. 4a,b). A weak association was found for El3 (peak LOD score of 2.29 broadly positioned between markers D10Mit42 and D10Mit134) that accounted for 5.6% of the total variance (Fig. 4c; Table 1). However, a new (previously unknown) QTL on proximal chromosome 9 was found that influenced seizure susceptibility in environment II (Fig. 4a). The new locus was named El-N (for Epilepsy-Naive quantitative trait locus). The peak LOD score (LOD = 3.46) was positioned between D9Mit188 and D9Mit2 and accounted for 9.2% of the total variance (Fig. 4a; Table 1).

image

Figure 4. Genetic maps indicating interval logarithm of the odds (LOD) scores for linkage to loci controlling seizure susceptibility in environment II: a, chromosome 9; b, chromosome 2; c, chromosome 10. LOD scores were determined as in Fig. 3.

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In environment III, two regions on chromosome 9 containing El4 and El-N were associated with seizures (Fig. 5a). The peak LOD score (LOD = 6.29) for El-N was positioned between D9Mit188 and D9Mit2 and accounted for 15.7% of the total variance (Fig. 5a; Table 1). As in environment I, the peak LOD score (LOD = 4.28) for El4 was located between D9Mit22 and D9Mit32 and accounted for 12.7% of the total variance (Fig. 5a; Table 1). In contrast to environments I and II, no significant associations were observed between genetic markers and seizure susceptibility QTL on chromosome 2 or 10 in environment III (Fig. 5b,c). The results were similar for the association between seizure susceptibility and the epilepsy QTL in the three environments when the data were also analyzed using the t-test analysis (Table 2). Viewed together, these findings indicate that both internal (age and gender) and external (repetitive testing) environmental risk factors determine the genetic architecture for seizure susceptibility in EL mice.

image

Figure 5. Genetic maps indicating interval logarithm of the odds (LOD) scores for linkage to loci controlling seizure susceptibility in environment III: a, chromosome 9; b, chromosome 2; c, chromosome 10. LOD scores were determined as in Fig. 3.

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Table 2.  Influence of environment on epilepsy quantitative trait locus (QTL) detection in (ABP × EL) F2 mice*
 Mean SSS 
EnvironmentEpilepsy QTLE alleleA allelet-Value§dfP-value**
  • *

    Data were analyzed using the two-tailed Student s t-test, and the mean seizure severity score (SSS) of all F2 mice homozygous for the E allele at a given epilepsy QTL was compared with that of mice homozygous for the A allele.

  • Epilepsy QTL in EL mouse with associated microsatellite marker in parentheses.

  • Numbers in parentheses indicate the number of mice homozygous for the E allele or the A allele.

  • §

    The t-value for unequal variances was used, when the Levene’s Test for Equality of variances was rejected.

  • df, degrees of freedom.

  • **

    The probability of Type I error.

IEl4 (D9Mit32)4.16 (28)2.59 (34)−3.602600.001
El-N (D9Mit2)4.10 (24)2.95 (22)−2.185360.036
El2 F (D2Mit30)4.40 (10)2.39 (14)−3.356210.003
El2 M (D2Mit30)4.05 (19)3.22 (16)−1.286330.207
El3 (D10Mit134)3.78 (28)3.50 (31)−0.600570.551
IIEl4 (D9Mit32)0.95 (42)0.45 (47)−2.067680.042
El-N (D9Mit2)1.44 (46)0.47 (45)−3.485680.001
El2 F (D2Mit30)0.58 (24)0.57 (22)−0.053440.958
El2 M (D2Mit30)1.03 (18)0.57 (21)−1.008250.323
El3 (D10Mit134)1.31 (39)0.53 (63)−2.525530.015
IIIEl4 (D9Mit32)3.97 (41)2.56 (44)−4.046830.0001
El-N (D9Mit2)4.36 (44)2.55 (43)−5.682850.0001
El2 F (D2Mit30)3.37 (23)3.17 (21)−0.413420.682
El2 M (D2Mit30)3.29 (18)2.70 (20)−0.913360.368
El3 (D10Mit134)3.29 (38)3.06 (61)−0.569970.570

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

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.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments
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Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. References
  7. Acknowledgments

This research was supported by the Boston College Research Fund and NIH grants (NS23355) and (HD39722). We also thank Michael Kiebish and Dr Richard McGowan, SJ, for technical help.