MTHFR C677T Genotype As a Risk Factor for Epilepsy Including Post-Traumatic Epilepsy in a Representative Military Cohort

Abstract

The well-studied C677T variant in the methylenetetrahydrofolate reductase (MTHFR) enzyme is a biologically plausible genetic risk factor for seizures or epilepsy. First, plasma/serum levels of homocysteine, a pro-convulsant, are moderately elevated in individuals with the homozygote TT genotype. Furthermore, the TT genotype has been previously linked with migraine with aura—a comorbid condition—and with alcohol withdrawal seizures. Finally, several small studies have suggested that the TT genotype may be overrepresented in epilepsy patients. In this study, we consider whether the MTHFR C677T or A1298C variants are associated with risk of epilepsy including post-traumatic epilepsy (PTE) in a representative military cohort. Study subjects were selected from the cohort of military personnel on active duty during the years 2003 through 2007 who had archived serum samples at the DoD Serum Repository, essentially all active duty personnel during this time frame. We randomly selected 800 epilepsy patients and 800 matched controls based on ICD-9-CM diagnostic codes. We were able to isolate sufficient genetic material from the archived sera to genotype approximately 85% of our study subjects. The odds of epilepsy were increased in subjects with the TT versus CC genotype (crude OR=1.52 [1.04–2.22], p=0.031; adjusted OR=1.57 [1.07–2.32], p=0.023). In our sensitivity analysis, risk was most evident for patients with repeated rather than single medical encounters for epilepsy (crude OR=1.85 [1.14–2.97], p=0.011, adjusted OR=1.95 [1.19–3.19], p=0.008), and particularly for PTE (crude OR=3.14 [1.41–6.99], p=0.005; adjusted OR=2.55 [1.12–5.80], p=0.026). Our early results suggest a role for the common MTHFR C677T variant as a predisposing factors for epilepsy including PTE. Further exploration of baseline homocysteine and folate levels as predictors of seizure risk following traumatic brain injury is warranted.

Introduction

T he MTHFR gene on chromosome 1p36.3 encodes 5,10-methylenetetrahydrofolate reductase (MTHFR), a key enzyme in the metabolism of the essential amino acid methionine. Two common functional variants in this enzyme, MTHFR C677T and A1298C, have been described in many populations (Botto and Yang, 2000; Esfahani et al., 2003; Schneider et al., 1998). Individuals with the MTHFR 677TT homozygote genotype have approximately 50% reduced enzymatic activity, resulting in moderately increased homocysteine—a sulfur-containing amino acid and by-product of impaired methionine metabolism (de Bree et al., 2003). The effect of the A1298C variant is less certain, but is believed to have a more modest or no effect on homocysteine concentration (Lievers et al., 2003). Although the MTHFR C677T variant is best known for its possible relationship with ischemic vascular disease, it has also been associated with neurological diseases including Alzheimer-type dementia, Parkinson's disease, and migraine with aura (Cronin et al., 2005; Diaz-Arrastia, 2000; Lewis et al., 2005; Mattson and Shea, 2003; Rubino et al., 2007; Schurks et al., 2010).

Several lines of evidence support a role for the MTHFR C677T variant as a predisposing factor for seizures or epilepsy. First, homocysteine lowers seizure thresholds and has been used as a pro-convulsant in animal epilepsy models (Diaz-Arrastia, 2000; Kruman et al., 2000; Kubova et al., 1995; Mares et al., 2002). Second, some studies have suggested that the TT genotype or elevated homocysteine per se may increase the risk of developing alcohol withdrawal seizures in alcohol-dependent subjects (Bleich et al., 2004; Hillemacher et al., 2007; Lutz et al., 2006). Third, the TT genotype has been identified as a risk factor for migraine with aura in several populations (Schurks et al., 2010). Migraine with aura is a comorbid condition (Ludvigsson et al., 2006; Ottman and Lipton, 1994) that may share pathophysiological mechanisms with epilepsy (Bigal et al., 2003).

Finally, some studies have suggested that the TT genotype may be overrepresented in epilepsy patients, including direct evidence from two studies (Dean et al., 2007; Ono et al., 2000), and indirect evidence from other studies that reported genotype frequencies in epilepsy patients and controls for other reasons (e.g., to determine whether the MTHFR C677T genotype might be related to hyperhomocysteinemia in patients receiving anticonvulsants, or whether maternal genotype might be related to risk of congenital malformations in children with in utero anticonvulsant exposure; Apeland et al., 2003; Caccamo et al., 2004; Dean et al., 1999; Kini et al., 2007; Sniezawska et al., 2011; Yoo and Hong, 1999). While these earlier results are suggestive, these findings need to be replicated, as most of the studies are small, are based on somewhat selected populations, and in some studies the source of the control group is either not described or is not comparable to the cases. None of these studies specifically considered a role of MTHFR C677T genotype as it relates to the risk of post-traumatic epilepsy (PTE).

In the present study, we consider the possible role of the MTHFR C677T and A1298C variants as pre-disposing factors for epilepsy including PTE. Study subjects are a randomly selected group of active duty service members with diagnosed epilepsy and age, sex, and race-matched controls. Our source of genetic material is archived serum specimens maintained by the Department of Defense Serum Repository.

Methods

The study protocol was approved by the Institutional Review Board of the Uniformed Services University.

Study population

Since 1985, the United States Department of Defense (DoD) has prospectively stored serum specimens collected from service members in the Armed Forces under direction of the Armed Forces Health Surveillance Center (AFHSC; Rubertone and Brundage, 2002). The DoD Serum Repository (DoDSR) contains over 50 million archived serum specimens kept in perpetual storage at −30°C, the majority comprising residual sera from routine periodic HIV testing collected at intervals of approximately 2 years (Rubertone and Brundage, 2002; Silverberg et al., 2003). The specimens are linked to medical diagnostic data via the Defense Medical Surveillance System (DMSS), through which it is possible to request de-identified specimens from individuals with a history of specific ICD-9-CM diagnoses (Rubertone and Brundage, 2002). Although DoDSR specimens are not collected or stored under conditions generally considered favorable to genetic epidemiology research, our pilot studies have demonstrated the feasibility of recovery of adequate genetic material from a majority of serum specimens.

Cases were randomly selected from eligible service members with one or more medical encounters in 2003 through 2007 indicating a diagnosis of epilepsy (ICD-9-CM code 345.x) occurring while on active duty. As pre-existing seizures would likely be an exclusionary criterion for military service (Headquarters Department of the Army, 2008), our case group is assumed to consist primarily of individuals with adult-onset epilepsy. The most recent available serum specimen collected after January 1, 2003 was selected.

Controls were randomly selected from service members without a documented medical encounter for epilepsy, and on active duty at some time since January 1, 2003. Controls were individually matched to cases for date of birth (±1 year), sex, race (white/black/other), and available specimen collected within±30 days of the matching case. Specimens were retrieved by staff at the DoDSR, thawed according to standard procedures, and one 0.5-cc aliquot was prepared from each specimen.

Diagnostic and demographic data

A de-identified database was created at the DoDSR containing an untraceable and anonymous study ID, linkage to the serum sample, and demographic and diagnostic information. Demographic information included sex, race, year of birth, and date of serum collection. Diagnostic information included details of medical encounters for epilepsy (ICD-9-CM 345.x) and non-fatal traumatic brain injury (TBI) per earlier methodology (Langlois et al., 2006; 800.x-801.x, 803.x-804.x, 850.x-854.1, and 959.01). These medical encounters would include ambulatory or inpatient visits made while on active duty, but would not include medical encounters while deployed to a combat zone. We classified injuries using the International Classification of Diseases Programs for Injury Categorization (ICDPIC; http://ideas.repec.org/c/boc/bocode/s457028.html), a program that maps ICD-9 diagnostic codes into a six-level approximation of the Abbreviated Injury Scale (AIS). We mapped this six-level code into mild, moderate, and severe TBI, per prior methodology (Thurman and Guerrero, 1999).

DNA isolation and genotyping

Processing of sera was done at a commercial laboratory (BioServe Laboratories, Beltsville, MD). More details about the laboratory methods are provided as supplementary data (Supplementary Table 1; see online supplementary material at http://www.liebertonline.com). Genotyping was done twice for the entire 1600 samples (i.e., 100% replication).

Statistical analysis

Primary analyses were planned before the data were collected and we did not look at any other genetic variants. Subjects with unknown or discordant genotype (i.e., the first genotype was different from the replication genotype) were excluded from the analysis. We determined whether control genotype frequencies were in Hardy-Weinberg equilibrium. We compared genotype frequencies between epilepsy cases and controls using unmatched logistic regression as described below. We also considered whether the risk of epilepsy associated with genotype was similar for those with PTE. PTE subjects were defined as those with at least one medical encounter consistent with a TBI as above occurring any time before or proximate to (defined as no more than 30 days after) the first documented medical encounter for epilepsy.

We calculated crude odds ratios (ORs) as an estimate of the relative risk of epilepsy/PTE by MTHFR C677T or A1298C genotype. We calculated crude and adjusted ORs, with adjusted ORs adjusting for age, sex, race (white, black, and other), and DNA concentration (as an additional quality control measure). As epilepsy cases were identified based on ICD-9-CM diagnostic codes without clinical verification, we performed a sensitivity analysis in which we restricted our cases to patients who had at least two medical encounters for epilepsy. All analyses were performed using Stata 11.0.

Results

We identified 1600 individuals, consisting of 800 randomly selected epilepsy cases and 800 matched controls. Study subjects were mostly male (80%) and were a mean/median age of 32/29 years. Ethnicity was white (68%), black (19%), and other races (13%; Table 1). Medical encounters suggestive of TBI were more common in epilepsy cases (137/800=17%) compared to controls (33/800=4%). TBI severity was mostly minor for both cases (69%) and controls (82%). Most of the epilepsy patients who had TBI-related medical encounters (118/137=86%) were classified as PTE, with a median of 96.5 days between the first documented TBI medical encounter and the first documented epilepsy diagnosis.

Table 1.

Description of the Study Subjects

		Controls (n=800)	Cases (I) (n=800)	Cases (II) (n=310)
Age (mean, SD)		32.0 (8.5)	32.0 (8.5)	32.8 (8.3)
Sex	Male	643 (80.4%)	643 (80.4%)	242 (78.1%)
	Female	157 (19.6%)	157 (19.6%)	68 (21.9%)
Race	White	545 (68.1%)	545 (68.1%)	215 (69.4%)
	Black	154 (19.3%	154 (19.3%	57 (18.4%)
	Other	101 (12.6%)	101 (12.6%)	38 (12.3%)
Post-traumatic epilepsy	No	N/A	682 (85%)	266 (86%)
	Yes	N/A	118 (15%)	44 (14%)

Case group I: All epilepsy cases.

Case group II: Limited to cases with two or more medical encounters for epilepsy.

Post-traumatic epilepsy: At least one documented medical encounter for traumatic brain injury occurring before or within 1 month of the first medical encounter for epilepsy.

SD, standard deviation.

A total of 1357 (85%) subjects were successfully genotyped for MTHFR C677T and 1319 (82%) for MTHFR A1298C. The unsuccessfully genotyped subjects for MTHFR C677T (n=243) included 224 individuals not genotyped due to a low quantity of DNA, and an additional 19 individuals with inconsistent genotype at replication. The unsuccessfully genotyped subjects for MTHFR A1298C A1298C (n=281) included 245 individuals not genotyped due to a low quantity of DNA, and an additional 36 individuals with inconsistent genotype at replication. There were no differences in call rates for MTHFR C677T by case status (p=0.14), but call rates were higher in controls for MTHFR A1298C (p=0.01). As expected, the C677T and A1298C variants were in strong linkage disequilibrium (X², p<0.001). None of the study subjects carried the 677TT/1298CC, 677CT/1298CC, or 677TT/1298AC haplotypes.

Genotype frequencies are shown in Table 2. Genotype frequencies for controls were in Hardy-Weinberg equilibrium for both MTHFR C677T (p=0.25) and A1298C (p=0.15). There were the expected (Botto and Yang, 2000; Esfahani et al., 2003; Schneider et al., 1998) differences in genotype frequencies by race (677TT genotype was prevalent in 10% of white, 1% of black, and 7% of other race controls; 1298CC genotype was prevalent in 11% of white, 3% of black, and 7% of other race controls).

Table 2.

MTHFR C677T Genotype and T Allele Frequency by Epilepsy Status

MTHFR C677T genotype
	C/C (n=716)	C/T (n=511)	T/T (n=130)	Total genotyped	T allele frequency ^a	Unknown ^b
Control	366 (54.8%)	249 (37.3%)	53 (7.9%)	668 (100%)	26.6%	132 (16.5%)
Case (I)	350 (50.8%)	262 (38.0%)	77 (11.2%)	689 (100%)	30.2%	111 (13.9%)

	C/C (n=493)	C/T (n=349)	T/T (n=87)	Total genotyped	T allele frequency ^a	Unknown ^b
Control	366 (54.8%)	249 (37.3%)	53 (7.9%)	668 (100%)	26.6%	132 (16.5%)
Case (II)	127 (48.7%)	100 (38.3%)	34 (13.0%)	261 (100%)	32.2%	49 (15.8%)

Allele of genotyped sample.

Allele of total sample.

Case group I: All epilepsy cases.

Case group II: Limited to cases with two or more medical encounters for epilepsy.

For MTHFR C677T, the odds of epilepsy overall were increased in subjects with the TT genotype compared to those with the CC genotype (crude OR=1.52 [1.04–2.22], p=0.031; adjusted OR=1.57 [1.07–2.32], p=0.023; Table 3). Results were somewhat stronger when cases were limited to those with two or more medical encounters for epilepsy (crude OR=1.85 [1.14–2.97], p=0.011, adjusted OR=1.95 [1.19–3.19], p=0.008).

Table 3.

Relative Odds of Epilepsy by MTHFR C677T Genotype

	All epilepsy cases (Case I: 1+medical encounters)				Post-traumatic epilepsy (Case I: 1+medical encounters)
MTHFR C677T	Crude OR	p-value	Adjusted OR	p Value	Crude OR	p Value	Adjusted OR	p Value
CC	1.00 (ref)		1.00 (ref)		1.00 (ref)		1.00 (ref)
CT	1.10 (0.88–1.38)	0.409	1.13 (0.89–1.42)	0.320	0.95 (0.60–1.50)	0.835	0.92 (0.57–1.47)	0.720
TT	1.52 (1.04–2.22)	0.031	1.57 (1.07–2.32)	0.023	1.92 (1.01–3.64)	0.046	1.81 (0.93–3.51)	0.080

	All epilepsy cases (Case II: 2+ medical encounters)				Post-traumatic epilepsy (Case II: 2+ medical encounters)
MTHFR C677T	Crude OR	p Value	Adjusted OR	p Value	Crude OR	p Value	Adjusted OR	p Value
CC	1.00 (ref)		1.00 (ref)		1.00 (ref)		1.00 (ref)
CT	1.16 (0.85–1.57)	0.352	1.17 (0.85–1.61)	0.331	0.60 (0.27–1.33)	0.208	0.52 (0.23–1.16)	0.110
TT	1.85 (1.14–2.97)	0.011	1.95 (1.19–3.19)	0.008	3.14 (1.41–6.99)	0.005	2.55 (1.12–5.80)	0.026

Case group I: All epilepsy cases (also shown separately for cases with post-traumatic epilepsy).

Case group II: Limited to cases with two or more medical encounters for epilepsy (also shown separately for cases with post-traumatic epilepsy).

Odds ratios (ORs) are an estimate of the risk of epilepsy in subjects with the CT or TT genotype relative to those with the CC genotype. Adjusted ORs are adjusted for age, sex, race, and DNA concentration.

Results were similar for PTE (crude OR=1.92 [1.01–3.64], p=0.046; adjusted OR=1.81 [0.93–3.51], p=0.080). Results for PTE again appeared somewhat stronger when cases were limited to those with two or more medical encounters for epilepsy (crude OR=3.14 [1.41–6.99], p=0.005; adjusted OR=2.55 [1.12–5.80], p=0.026).

There was no relationship between A1298C genotype and epilepsy overall, for PTE, or for epilepsy cases with two or more medical encounters (results not shown).

Discussion

Our results suggest that the common and well-studied MTHFR C677T variant is a genetic risk factor for epilepsy in this representative military cohort. While risk was similar for PTE and epilepsy overall, there was the suggestion of a stronger effect for PTE that needs to be confirmed in a study that is adequately powered for this subgroup analysis. Our results are consistent with findings from some earlier small studies, and suggest one possible mechanism for the long-observed epidemiologic link between epilepsy and migraine (Bigal et al., 2003; Ludvigsson et al., 2006; Ottman and Lipton, 1994).

There has been limited success in the identification and replication of genetic variants that increase risk of seizure disorders overall or specifically following head trauma (Cavalleri et al., 2007; Greenberg and Pal, 2007; Helbig et al., 2008; Tan and Berkovic, 2010; Tan et al., 2004). However, some earlier studies have suggested, directly or indirectly, that this variant may be overrepresented in epilepsy patients (summarized in Table 4). Indirect evidence comes from four studies that considered the role of the MTHFR C677T genotype as it relates to homocysteine levels in patients receiving anticonvulsants (Apeland et al., 2003; Caccamo et al., 2004; Sniezawska et al., 2011; Yoo and Hong, 1999), and from two studies considering whether maternal C677T genotype was related to a risk of congenital malformations or fetal anticonvulsant syndrome in their children (Dean et al., 1999; Kini et al., 2007). Two studies looked at the effect of the TT genotype on epilepsy per se (Dean et al., 2007; Ono et al., 2000). In the first study (Ono et al., 2000), the prevalence of the TT genotype in symptomatic/cryptogenic epilepsy patients, idiopathic epilepsy patients, and controls, respectively was 27%, 15%, and 18% (p<0.05 for the difference between controls and symptomatic/cryptogenic epilepsy). In a later study based on 141 women who had been prescribed anti-epilepsy medications during pregnancy and 303 age-matched healthy female blood donor controls, Dean and associates found a higher prevalence of the TT genotype in cases compared to controls (15% versus 10%; p<0.02; Dean et al., 2007). Several other genetic variants that are involved in folate metabolism, including MTHFR A1298C, were not associated with case status in this latter study (Dean et al., 2007). In the aggregate, there is some support for the TT variant being overrepresented in this heterogeneous group of epilepsy patients, with the caveat that individual studies were generally underpowered to detect an OR in the 1.5–2.0 range, and that some studies had additional methodological limitations, particularly non-comparability of cases and controls.

Table 4.

Prior Research Related to MTHFR C677T Genotype and Epilepsy

		Number of subjects		Prevalence of TT Genotype			Source of subjects
Study	Location or ethnicity	Controls	Cases	Controls	Cases	Calculated OR ^a	Controls	Cases
MTHFR 677TT genotype frequencies from studies relating to homocysteine levels in patients on anticonvulsants
1	Korea	103	103	13%	23%	2.7 [1.1–7.0]	Patients receiving health examinations	Epilepsy patients
2	Italy	98	95	18%	20%	1.3 [0.5–3.3]	Healthy drug-free subjects	Epilepsy patients
3	Norway	101	101	8%	7%	0.9 [0.3–3.0]	Hospital employees and blood donors	Epilepsy patients
4	Poland	55	65	4%	11%	2.3 [0.4–24.6]	Unk	Epilepsy patients
MTHFR 677TT genotype frequencies from studies relating to the risk of congenital malformations after exposure to anticonvulsants
5	Northern European	152	36	9%	25%	4.3 [1.3–13.7]	Mixture of matched (to parents) controls and random adults from a primary care registry	Mothers of child affected with fetal anticonvulsant syndrome
6	UK	226	141	11%	12%	1.0 [0.5–2.3]	Mothers without epilepsy at time of antenatal care	Mothers with epilepsy at time of antenatal care
MTHFR 677TT genotype frequencies from gene-association studies
7	Japan	97	92	18%	21%	2.0 [0.8–4.9]^b	Healthy adult controls	Epilepsy patients
8	Scottish	303	170	10%	15%	2.0 [1.1–3.9]	Age-matched female blood donors	Women with epilepsy prescribed anticonvulsants during pregnancy
9	U.S. (present study)	668	689	8%	11%	1.5 [1.04–2.2]	Age, sex, race matched controls	Points with 1+ICD-9-CM medical encounters for epilepsy

Odds of epilepsy for the 677TT versus 677CC genotype calculated based on data in indicated reference.

Odds ratio (OR) significant for symptomatic/cryptogenic epilepsy (OR=2.8) but not for idiopathic epilepsy (OR=1.4)

1 Yoo and Hong, 1999

2 Caccamo et al., 2004

3 Apeland et al., 2003

4 Sniezawska et al., 2011

5 Dean et al., 1999

6 Kini et al., 2007

7 Ono et al., 2000

8 Dean et al., 2007

9 Present Study

This variant is biologically plausible as an epilepsy susceptibility gene due to the pro-convulsant activity of homocysteine in animal models (Diaz-Arrastia, 2000; Kruman et al., 2000; Kubova et al., 1995; Mares et al., 2002), and the observed relationship between the TT genotype or homocysteine levels with susceptibility to alcohol-withdrawal seizures (Bleich et al., 2004; Hillemacher et al., 2007; Lutz et al., 2006). Furthermore, the TT genotype has been identified as a genetic risk factor for migraine with aura, a comorbid condition (Rubino et al., 2007; Schurks et al., 2010). While migraine is prevalent in about 13% of the general population, the prevalence of migraine in individuals with epilepsy is about 24% (Ottman and Lipton, 1994). In a longitudinal study, children with migraine, particularly migraine with aura, were at increased risk of developing an incident unprovoked seizure compared to age- and gender-matched controls (Ludvigsson et al., 2006). Future studies with a detailed and independent assessment of migraine with aura and epilepsy are needed to consider the possible role of this genetic variant as a shared susceptibility gene for these two comorbid conditions.

Strengths of this study include our randomly selected, comparatively large, and multi-ethnic study population. Our control group, which derives from the same well-defined sampling frame as our cases, was carefully matched 1:1 to our case group. Cases and controls were covered under the same healthcare system and thus likely had similar access to medical care and likelihood of diagnosis. Because of the nature of our study population, our results may be specific to epilepsy syndromes with onset in early to middle adulthood, as it is unlikely that individuals with pre-existing seizures would qualify for military service.

Some limitations of these results should also be taken into account. As cases were identified based on ICD-9-CM diagnostic codes without further verification, it is likely that there is some misclassification of case status. This type of misclassification would most likely have attenuated rather than exaggerated our results. We note that a recent study found that ICD9/10 diagnostic codes for epilepsy in two Canadian administrative databases had an almost perfect positive predictive value following physician chart review (Jette et al., 2010). Our identification of PTE cases based on ICD-9-CM diagnostic codes suggestive of TBI may have been an underestimate of the true prevalence of PTE in this cohort. Furthermore, since we did not have information on medical encounters occurring during deployment to a combat zone, we may have incorrectly classified some PTE cases as non-traumatic epilepsy cases. For these reasons the subgroup results reported herein should be confirmed in a larger cohort with a more careful assessment of head trauma.

The source of genetic material was also a limitation to some extent. While specimens stored in the DoDSR may not be an optimal source of DNA, this unique resource made it possible to identify a large number of cases for a condition with low population prevalence, and also to identify a cohort of epilepsy patients with a presumed higher prevalence of PTE than would be seen in a civilian epilepsy population. We note that our observed genotype frequencies overall and by race groups are consistent with other studies (Botto and Yang, 2000; Esfahani et al., 2003). This is the first study to our knowledge to use the DoDSR for a population genetics study.

In conclusion, our and earlier results suggest that the common MTHFR C677T variant is a genetic risk factor for epilepsy including PTE. Additional research is needed to determine whether homocysteine or folate levels are predictive of seizure incidence following TBI, and furthermore, whether lowering of homocysteine levels with folic acid or other interventions might be neuroprotective in populations at high risk of incident seizures or head injury.

Footnotes

Acknowledgments

Funding for this study was provided by the Comprehensive Neuroscience Program of the Henry Jackson Foundation for Military Medicine, and the Intramural Research Program of the Uniformed Services University.

We thank Dr. Christopher Williams for reviewing this manuscript and for his helpful suggestions. We also acknowledge with gratitude the assistance of Dr. Sherman McCall during the pilot phase of this study, and Dr. Angie Eick at the DoDSR.

The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of the Army, Department of Defense, or the U.S. Government.

Author Disclosure Statement

No competing financial interests exist.

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