Aim: Epilepsy is a neurological disorder with a great impact on the quality of life and potential disability of the affected patients. In the present study, we have investigated chromosomal aberrations (CA), methylenetetrahydrofolate reductase (MTHFR)-C677T genotypes and homocysteine levels in a group of epileptic patients (EP) from South India.
Methods: Blood samples were collected from a total of 29 patients (17 females; 12 males) and subjected to chromosomal and genotypic analyses.
Results: Chromosomal alterations, such as deletions, duplications, and ring chromosomes (20 and 14), were found in epileptic patients, with an average of 12.76 ± 1.96 chromosomal defects per patient (1.53 ± 0.83 in controls; P < 0.001). The frequency of the MTHRF-C/T genotype was higher in EP, whereas the MTHFR-T/T genotype was lower than in control subjects. Strikingly, the levels of homocysteine were found to be significantly higher in EP (14.57 ± 2.97 µmol/L) than in controls (8.83 ± 2.39 µmol/L; P < 0.001). Pharmacotherapy in EP led to increases in homocysteine levels. Individuals with the MTHFR-C/T genotype tended to have a greater predisposition to an increase in homocysteine levels during pharmacological treatment.
Conclusion: Accumulation of CA may contribute to the epileptic phenotype, and diverse genetic factors may be associated with antiepileptic drug-related homocysteine disturbances.
Keywords: Chromosome aberrations, cytogenetics, epilepsy, homocysteine, MTHFR genotyping, karyotyping
Epilepsy has largely been defined as a condition that disrupts the functioning of the brain and is characterized by abnormal, recurrent and disorganized electrical impulses. An estimated global impact of epilepsy is observed to affect nearly 50 million people in areas of the world which have limited medical treatments.[1,2] Interestingly the World Health Organization had released the statistics of epilepsy to be similar to breast cancer in women or lung cancer among men. Community-based surveys in India over the recent years have shown the prevalence rate to be five per 1,000 with an incidence of nearly 50 per 100,000; much higher than developed nations. Epileptic subjects are associated with nearly a threefold increase in mortality when compared to the general population.
The neurophysiology of epilepsy can be specifically characterized by the pattern of thinking difficulties, frequency and severity of seizures, duration of seizures, anti-epileptic medications and causative feature of seizures. Unlike other conditions of the brain, epilepsy has been identified with over forty neurological conditions among which convulsions and seizures are the common symptoms. Further, idiopathic and familial conditions are recognized to constitute the major percentage of epileptic variants. Other conditions include inherent hyperexcitability due to brain tumors, trauma, developmental lesions and seizures due to sensory stimuli.
Although the studies regarding the mode of transmission of epilepsy have been challenged on many levels, the involvement of the associated genetic component has clearly been identified. The complexity of inheritance and clinical heterogeneity further contributed to favor the role of genetics in epilepsy. Among researchers, epilepsy represents a diverse group of neurological entities in which genetic factors and non-genetic causative factors may be involved. This has increased the importance of evaluation and management of epilepsy by genetic measures among all developed and developing countries.
The relevance of the field of genetics has been widely recognized by researchers who have clarified the distinct role of de novo mutations, genetic heterogeneity (one disorder having multiple genetic causes), pleiotropy (a single gene associated with different phenotypes) and somatic postzygotic mutations in unraveling epilepsy. Major advances in neurogenetics have recently helped us in better understanding the genetic basis of epilepsy, including several chromosomal abnormalities and multiple loci associated with different types of epilepsy and these advances have also made it necessary to reclassify the current classification of epilepsies and epileptic syndromes.
The aim of the present study was three-fold: (i) to investigate chromosomal abnormalities of epileptic patients by cytogenetic analysis, using the trypsin G-banding method; (ii) to determine the frequency of methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphisms in patients and controls by using polymerase chain reaction (PCR)-restriction fragment length polymorphism; and (iii) to analyze the plasma levels of total homocysteine (tHcy) in epileptic patients and control subjects from South India.
Experimental samples were selected from the hospitals at Coimbatore City, Tamilnadu, South India. A total of 44 subjects were selected, including 29 experimental subjects (age: 11.53 ± 3.64 years; 17 males, age: 12.89 ± 4.42 years; and 12 females, age: 10.66 ± 3.02 years) and 15 controls (age: 11.73 ± 3.26 years; 8 males, age: 11.87 ± 2.69 years; and 7 females, age: 11.57 ± 3.26 years). The controls selected were mentally normal and physically healthy, and resided in the same area. A detailed questionnaire was administered to both experimental and control subjects, registering data on respiration, skin, reproduction, birth order, parental age, marital status, family history and clinical features. The work was carried out in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki, and written informed consent was obtained from the individuals. Healthy controls of similar ethnicity were selected.
Blood samples of about 5 mL were collected from all subjects to assess the biochemical profiles, while the separated sera were assayed for hematological parameters. The samples were transported to the culture laboratory in airtight ice-packed containers to carry out the chromosomal and genotypic analysis.
Chemical reagents were procured from Sigma Chemicals (St. Louis, MO) and Gibco Laboratory (Grand Island, NY). The blood samples were prepared to establish cell cultures, according to the standard procedures of our laboratory. The author added 0.5 mL of whole blood to 4.5 mL RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 1% streptomycin-penicillin antibiotics and 0.2 mL reagent-grade phytohemagglutinin and incubated at 37 ˚C for 71 h after which 0.1 µg/mL of colcemid was added to block cells in mitosis. The lymphocytes were harvested at 72 h by centrifugation (800-1,000 g, 7 min) and followed by the addition of hypotonic solution (KCl 0.075 mol/L) at room temperature for 20 min. The cells were then treated twice with methanol and acetic acid [3:1 vol/vol]. Cytological preparations were made by placing two to three drops of the concentrated cell suspension onto slides moistened with ice-cold acetic acid (60%), and carefully dried on a hot plate (56 ˚C for 2 min). For identification of numerical and structural chromosomal aberrations (CA), 100 complete metaphase cells of the first cell cycle were evaluated under a microscope (×100) according to the International System for Human Cytogenetic Nomenclature. Data were registered on master tables and later transferred to a computer file.
The tHcy biochemical ranges were analyzed using an Hcy Elisa kit. The acceptable normal reference range was 5-15 µmol/L.
Whole blood was collected in ethylenediaminetetraacetic acid-coated collection tubes to avoid clotting. The extraction procedure was by lysis of the red blood cells followed by lysis of the white blood cells and nuclei. Finally, genomic DNA was concentrated and desalted by alcohol precipitation using a descending alcohol series. Agarose gel electrophoresis was carried out using 1% gel and triterpenic acid-enriched fraction buffer to confirm the presence of PCR-grade DNA. Further, the purity of DNA extracted was estimated by the ratio of absorbance at 260 nm and 280 nm.
The primers 677 F (5’ CTC GCC TTG AAC AGG TGG AG 3’) and 677 R (5’ CTG GAT GGG AAA GAT CCC GG 3’) (Bangalore Genei, India) were used for amplifying genomic DNA. 4 µL of 400 ng template DNA, 1 µL each of forward and reverse primers and 12.5 µL of 2X PCR Master Mix were used in a total reaction volume of 25 µL by following an initial denaturation step at 95 °C (10 min) followed by 30 cycles of denaturation step of 95 °C (30 s), annealing step of 58 °C (30 s), extension step of 72 °C (1 min) and a final elongation step of 72 °C (10 min).
The allelic variants were identified by the use of restriction enzymes that differentiate between alleles. The MTHFR gene (GenBank Accession No. EF026975.1) [gene names, gene symbols, and accession numbers are from GenBank (http://www.ncbi.nlm)] was amplified and then digested with HinfI enzyme using the previously described primers and conditions. For digestion of PCR product with HinfI restriction enzyme, 10 µL of PCR reaction product, 18 µL nuclease-free water, 2 µL enzyme buffer (10X) and 2 µL of HinfI restriction enzyme were mixed gently and incubated at 37 °C for 8 h to 12 h. The digestion products were visualized on 4% metaphor agarose gel containing ethidium bromide.
All statistical analyses were performed using the SPSS software (version 17) to assess the group statistics for subjects and controls as mean ± SD. For statistical data inference, the t-test for independent variables and ANOVA were used to compare mean values of the quantitative variables. The comparison and deviation of genotype frequencies from the expected were examined by the chi-square test. Multiple regression analysis was carried out to determine the correlation of continuous variables, by using them as dependent variables, with several independent variables. A significance level of 0.05 was adopted; all the levels were compared and analyzed for the statistical difference. Analysis of variance was carried out to find the significance.
Chromosomal alterations, such as deletion, duplication, and ring chromosomes, were found in epileptic patients [Figure 1]. Ring chromosome-20 was found in 5 patients; ring chromosome-14 was observed in 7 patients; ring chromosome-5 was noted in 2 patients; and dup (5p), del (1q-), del (2p-), del (3p-), del (4p-) were also observed in the patients. Table 1 shows total CA (CSA: chromosomal type aberration; CTA: chromatid type aberrations) and tHcy levels in epileptic patients, and Table 2 shows the same in control subjects [Figure 1]. CA are significantly more frequent in epileptic patients (12.76 ± 1.96) than in controls (1.53 ± 0.83; P < 0.001) [Table 2; Figure 1]. tHcy levels were also higher in epileptic patients (14.59 ± 2.97 µmol/L) than in controls (8.83 ± 2.39 µmol/L) [Table 2; Figure 2] in both sexes [Figure 2]. Furthermore, tHcy levels showed a clear age-dependent profile in epileptic children (P < 0.0001), but not in healthy children [Figure 3]. Anti-epileptic drug (AED) users exhibit a higher frequency of CA (7.28 ± 2.49) compared to non-AED users (6.16 ± 1.32), whereas chromatid aberrations were more frequent in non-AED users (6.5 ± 1.64) as compared to AED users (6.14 ± 2.67). High levels of tHcy (16.02 ± 2.54 µmol/L) were also observed in AED users [Table 3].
Table 1: Chromosomal aberrations and homocysteine levels in patients with epilepsyClick here to view
Table 2: Chromosomal aberrations and homocysteine levels in control subjectsClick here to view
Table 3: Number of chromosomal aberrations and homocysteine levels in epileptic patients and control subjectsClick here to view
Figure 1: Chromosomal aberrations in patients with epilepsyClick here to view
Figure 2: Homocysteine levels in patients with epilepsyClick here to view
Figure 3: Age-related homocysteine levels in patients with epilepsy and age-matched healthy control subjects. HCY: homocysteineClick here to view
The frequencies of the MTHFR genotypes (CC, CT, and TT) among epileptic patients (EP) were 23.07%, 61.53%, and 15.38%, respectively; and in controls, 26.6%, 40%, and 33.3%, respectively [Table 4]. Combining the heterozygous and homozygous MTHFR variant genotypes (CT + TT), a significant increase in the prevalence of the C677T mutation was found in EP. The prevalence of the CT heterozygous variant genotype alone was significantly higher in EP than in controls.
Table 4: Frequency of MTHFR (C677T) variants in epileptic patients and controlsClick here to view
Epileptic cases in India have been seen with a perspective emphasis in prevention, management, and understanding along with sociodemographic transition. For this purpose, the epidemiology of this condition in national populations is mandatory to develop a response for control of epilepsy among men, women, and children in India.
Rural and urban epidemiology of epilepsy
In countries with health care integrated with a good recording system, data sources and software-based techniques have been used to understand the epidemiology of epilepsy. Additionally, the doctor’s records of the neurological evaluation have also been added into the methodology. Sources of data such as national and regional registers, medical records, and practice records, a mandated record filing system with linkage system and prescription database can also be used to identify the cases under study. But the limited availability of different data sources restrict such applications and community-based derivations alone must be depended on.
Community-based studies have also shown the prevalence of epilepsy in India among the rural and urban populations. The pattern observed shows a higher susceptibility to men than women in both urban and rural populations with a slightly higher prevalence rate among urban men and women. Pedigree analysis by Satishchandra et al. described the factor of hereditary epilepsy from relatives in 13.7% of the cases. Mathai showed a higher percentage of epilepsy in family members (24.4%). A higher percentage of mental retardation was observed in earlier studies by Sharma et al. and Kumari et al. than in those reported by Das and Sanyal. This may be due to the use of antiepileptic drugs.
For the care of epileptic patients, proper knowledge, care and no secondary infections by bacteria, viruses or other parasites are of importance. Further, the attitude of the entire community towards epileptic subjects and their families is also mandatory. Within communities, the rise in discrimination impedes care and increases the stigma leading to limited diagnosis. Despite significant advancements in diagnosis and treatment in India, lack of knowledge and other beliefs raise a widely different approach among the urban and rural populations. A stark contrast can be observed among EP in rural and urban populations. In India, over 70% of cases were confirmed to be epileptic without receiving any treatment, as reported by Das and Sanyal. In contrast, the same authors revealed that only 6% of the cases were not receiving any treatment for epilepsy in urban populations. Further, 17% were not on medication on the assessment day. Similar observations have been reported by Sharma et al. on studies centered in the Northwestern region of India.
Chromosomal regions with aberrations such as anomalies and deletions are quite evident among populations that are affected by seizures. These regions can act as useful targets for geneticists to identify specific epileptic syndromes. Several abnormal chromosomal regions have been associated with epilepsy among which anomalies in the distal end of Chromosome 1, deletions in band p36.3 and delineated del(1) (p36) syndrome are the most common. The 4p-deletion syndrome (Wolf-Hirsch horn Syndrome, WHS) is well known and is strongly associated with epilepsy. Febrile seizures were observed in a study in 1990 in a case that showed terminal deletion of the long arm of Chromosome 6. On comparisons with the genetic study of chromosomes, 74% of the cases that showed CA have been associated with seizures and spasms. In our study, there is a clear accumulation of CA in patients with epilepsy. We observed ring (20) in 2 patients, ring (14) in 3 patients, ring (5) in 1 patient. According to Ieshima et al. Ring Chr-14 is the only anomaly of chromosome 14 with a striking association with epilepsy. These genomic segments with structural alterations in chromosomes and chromatids might be responsible for the heterogeneous expression of different epileptic genotypes in cases with idiopathic epilepsies.
Homocysteine and epilepsy
The interest in the homocysteine molecule by the medical and the research community has mainly been due to its association to atherosclerosis and cardiovascular diseases. Further, its elevated levels in the plasma of the population of interest has concluded it to an independent risk factor. Its association to mental disorders has primarily been in an associated relation with B-vitamins.[17-19]
Although, the conditions of hyperhomocysteinemia have been observed to yield convulsions in animals, direct relation between homocysteine levels and epilepsy remains unclear. The entire mechanism of this action has still not been demonstrated; however, it is well-known that tHcy may act as an agonist of N-methyl-D-aspartate-type glutamate receptor that plays a role in epilepsy. The methionine load test appears to be important in order to identify abnormal tHcy metabolism. Furthermore, as a consequence of the use of antiepileptic drugs, an increase in the levels of Hcy has been identified as a risk factor for neurodegenerative disorders and other neurological conditions such as epilepsy. Antiepileptic drugs can induce Hcy through the following mechanisms: inhibition of vitamin absorption, Hcy metabolism dysfunction, accelerated vitamin catabolism, and modulation of renal function. High tHcy levels have also been associated with congenital malformations among children whose mothers have received anticonvulsants during the first three months of pregnancy. Hyperhomocysteinemia is easily modifiable through folate supplementation. Therefore, in the present study, we investigated both fasting and progressive multifocal leukoencephalopathy levels of plasma total homocysteine in patients receiving different AEDs.
In a population, there is a gradually increased risk of stroke or coronary events as the tHcy concentrations rise from the lower quartile to the upper quartile. Our study shows that Hcy levels are significantly different in epileptic patients and controls, with a clear age-dependent pattern in epileptic patients, but not in controls. However, the comparison of Hcy, folic acid and vitamin B12 serum levels in patients receiving AEDs, non-AED users and controls showed results similar to those reported in previous studies,[23,24] with some differences probably due to the characteristics and age of the selected population, as well as the type of drug used for the treatment of epileptic seizures.
MTHFR gene in epilepsy
Different mutations in several genes distributed across the human genome may play causative or susceptibility roles in diverse forms of epilepsy. We have studied the potential contribution of the MTHFR gene, encoding the enzyme 5, 10-MTHFR, in EP. Severe deficiency of MTHFR among infants with convulsions or seizures deserves diagnostic consideration. High Hcy levels are more frequent in MTHFR-C677T homozygotes than in heterozygotes or in patients with no apparent mutations. However, Hcy levels showed no significant differences between patients who were receiving carbamazepine or valproic acid, in line with studies reported by Apeland et al. and Vurucu et al. In contrast, Gidal et al. reported a statistically significant decline in plasma Hcy concentration among patients receiving treatment. About 90% of the patients older than 10 years of age showed Hcy levels over 10 µmol/L, whereas an average of 6 µmol/L was found in patients younger than 10 years of age. This result is in agreement with findings reported by Eldeen et al.
Our study suggests a potential association between MTHFR 677C > T and epilepsy, although our sample is very small. The role of MTHFR in brain development and functioning has been classically proposed, with reports suggesting a more sensitive role of MTHFR in developing embryos. In addition, the potential implication of MTHFR 677C > T variants in susceptibility to epilepsy could be redirected at studies that deal with its association with increased malformations among children that carry pathogenic variants via maternal linage.
In conclusion, there is a clear accumulation of CA in patients with epilepsy. These genomic alterations may contribute to defining the heterogeneous phenotype of idiopathic epilepsy. tHcy levels are significantly elevated in epileptic patients, either treated with conventional anti-epileptic drugs or in those who do not receive any treatment. In addition, tHcy levels exhibit an age-dependent profile in epileptic patients, which is absent in healthy children. An over-representation of the MTHFR-C677T variant in epileptic patients may indicate that mutations in the MTHFR gene might contribute to increase the risk of epilepsy in selected populations. These three findings (CA, MTHFR variation, hyperhomocysteinemia) might be important in the pathogenesis of epilepsy and in the pharmacogenetics of anti-epileptic drugs.
Helping in concept, design and definition of intellectual content: K. Sasikala, M. Arun, R. Cacabelos
Bibliographic research: R.K.M. Kumar, K.B. Haripriya
Performing clinical studies: B. Balamuralikrishnan
Performing cytogenetic studies: S. Sureshkumar, A.K. Kumar
Contributing with data acquisition, data analysis, and statistical analysis: R.N. Meenakshi, E. Murugesh, R. Suganthi, P. Shanmughavel
Manuscript preparation: M. Arun, R.N. Meenakshi
Manuscript editing and review: R. Cacabelos, K. Sasikala
The authors thank the Management of Bharathiar University for providing infrastructure facilities for this research work and the subjects who volunteered to take part in this study.
Financial support and sponsorship
This work was supported by Dr. Ramón Cacabelos, EuroEspes Biomedical Research Center, Institute of Medical Science and Genomic Medicine, 15165 Bergondo, Corunna.
Conflicts of interest
There are no conflicts of interest.
Patients were selected and taken from hospitals in Coimbatore and Chennai (Tamil Nadu), and written informed consent was obtained from individuals. Healthy controls of similar ethnicity were selected.
The work was carried out in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.
- 1. World Health Organization. Atlas: epilepsy care in the world. Geneva: World Health Organization; 2005. p. 91.
- 2. Nei M, Hays R. Sudden unexpected death in epilepsy. Curr Neurol Neurosci Rep 2010;10:319-26.
- 3. Murray CJL, Kreuser J, Whang W. Cost-effectiveness analysis, and policy choices: investing in health systems. In: Murray CJL, Lopez AD, editors. Global comparative assessments in the health sector: disease burden, expenditures, and intervention packages. Geneva: World Health Organization; 1994. p. 181-92.
- 4. Singh R, Gardner RJ, Crossland KM, Scheffer IE, Berkovic SF. Chromosomal abnormalities and epilepsy: a review for clinicians and gene hunters. Epilepsia 2002;43:127-40.
- 5. 52nd WMA General Assembly. Ethical Principles for Medical Research Involving Human Subjects. World Medical Association Declaration of Helsinki. Edinburgh: Bulletin of the World Health Organization; 2001. p. 79.
- 6. Moorhead PS, Novell PC, Mellman WJ, Battips DM, Hungerford DA. Chromosome preparation of leukocyte culture from peripheral blood. Exp Cell Res 1960;20:613-6.
- 7. Amudhan S, Gururaj G, Satishchandra P. Epilepsy in India I: epidemiology and public health. Ann Indian Acad Neurol 2015;18:263-77.
- 8. Satishchandra P, Ulla GR, Sinha A, Shankar SK. Pathophysiology and genetics of hot-water epilepsy. In: Berkovic SF, Genton P, Hirsch E, Picard F, editors. Genetics of focal epilepsies: clinical aspects and molecular biology. London: John Libbey & Company Ltd; 1999. p. 169-76.
- 9. Mathai KV. Epilepsy-some epidemiological experimental and surgical aspects. Neurol India 1986;14:299-314.
- 10. Sharma S, Raina SK, Bhardwaj AK, Chaudhary S, Kashyap V, Chander V. Socio demography of mental retardation: A community-based study from a goitre zone in rural sub-Himalayan India. J Neurosci Rural Pract 2015;6:165-9.
- 11. Kumari S, Mishra SN, Chaudhury S, Singh AR, Verma AN, Kumari S. An experience of community mental health program in rural areas of Jharkhand. Ind Psychiatry J 2009;18:47-50.
- 12. Das SK, Sanyal K. Neuroepidemiology of major neurological disorders in rural Bengal. Neurol India 1996;44:47.
- 13. Sharma S, Raina SK, Bhardwaj AK, Chaudhary S, Kashyap V, Chander V. Prevalence of mental retardation in urban and rural populations of the goiter zone in Northwest India. Indian J Public Health 2016;60:131-7.
- 14. Knight-Jones E, Knight S, Heussler H, Regan R, Flint J, Martin K. Neurodevelopmental profile of a new dysmorphic syndrome associated with submicroscopic partial deletion of 1p36.3. Dev Med Child Neurol 2000;42:201-6.
- 15. Francis GL, Flannery DB, Byrd JR. An apparent de novo terminal deletion of chromosome 2 (pter----p24:). J Med Genet 1990;27:137-8.
- 16. Ieshima A, Takeshita K, Yamamoto K. Ring 14 syndrome with decreased bone mineral content in two pubertal girls. Jinrui Idengaku Zasshi 1983;28:35-43.
- 17. Bonaa KH, Njolstad I, Ueland PM, Stott DJ, MacIntosh G, Lowe GD, Gussekloo J. Homocysteine, B vitamins, and cardiovascular disease. N Engl J Med 2006;2006:205-11.
- 18. Lonn E, Yusuf S, Arnold MJ, Sheridan P, Pogue J, Micks M, McQueen MJ, Probstfield J, Fodor G, Held C, Genest J Jr; Heart Outcomes Prevention Evaluation (HOPE) 2 Investigators. Homocysteine lowering with folic acid and B vitamins in vascular disease. N Engl J Med 2006;354:1567-77.
- 19. Coppola G, Ingrosso D, Operto FF, Signoriello G, Lattanzio F, Barone E, Matera S, Verrotti A. Role of folic acid depletion on homocysteine serum level in children and adolescents with epilepsy and different MTHFR C677T genotypes. Seizure 2012;21:340-3.
- 20. Belcastro V, Gaetano G, Italiano D, Oteri G, Caccamo D, Pisani LR, Striano P, Striano S, Ientile R, Pisani F. Antiepileptic drugs and MTHFR polymorphisms influence hyper-homocysteinemia recurrence in epileptic patients. Epilepsia 2007;48:1990-4.
- 21. Graham IM, Daly LE, Refsum HM, Robinson K, Brattström LE, Ueland PM, Palma-Reis RJ, Boers GH, Sheahan RG, Israelsson B, Uiterwaal CS, Meleady R, McMaster D, Verhoef P, Witteman J, Rubba P, Bellet H, Wautrecht JC, de Valk HW, Sales Lúis AC, Parrot-Rouland FM, Tan KS, Higgins I, Garcon D, Medrano MJ, Candito M, Evans AE, Andria G. Plasma homocysteine as a risk factor for vascular disease. The European Concerted Action Project. JAMA 1997;277:1775-81.
- 22. Schwaninger M, Ringleb P, Winter R, Kohl B, Fiehn W, Rieser PA, Walter-Sack I. Elevated plasma concentrations of homocysteine in antiepileptic drug treatment. Epilepsia 1999;40:345-50.
- 23. Sener U, Zorlu Y, Karaguzel O, Ozdamar O, Coker I, Topbas M. Effects of common anti-epileptic drug monotherapy on serum levels of homocysteine, vitamin B12, folic acid and vitamin B6. Seizure 2006;15:79-85.
- 24. Kurul S, Unalp A, Yis U. Homocysteine levels in epileptic children receiving antiepileptic drugs. J Child Neurol 2007;22:1389-92.
- 25. Loo KW, Griffiths LR, Gan SH. A novel multiplex PCR-RFLP method for simultaneous detection of the MTHFR 677 C > T, eNOS +894 G > T and - eNOS -786 T > C variants among Malaysian Malays. BMC Med Genet 2012;13:34.
- 26. Apeland T, Mansoor MA, Strandjord RE, Kristensen O. Plasma homocysteine and serum folate in patients with epilepsy on carbamazepine or valproate monotheraphy. Carbamazepine and valproate have different effects on folate and homocysteine metabolism. Acta Neurol Scand 2000;101:352.
- 27. Vurucu S, Demirkaya E, Kul M, Unay B, Gul D, Akin R, Gokçay E. Evaluation of the relationship between c677t variants of methylenetetrahydrofolate reductase gene and hyperhomocy-steinemia in children receiving antiepileptic drug therapy. Prog Neuropsychopharmacol Biol Psychiatry 2008;32:844-8.
- 28. Gidal BE, Tamura T, Hammer A, Vuong A. Blood homocysteine, folate and Vitamin B-1 concentrations in patients with epilepsy receiving lamotrigine or sodium valproate for initial monotherapy. Epilepsy Res 2005;64:161-6.
- 29. Eldeen ON, Abd Eldayem SM, Shatla RH, Omara NA, Elgammal SS. Homocysteine, folic acid and vitamin B12 levels in serum of epileptic children. Egypt J Med Hum Genet 2012;13:275-80.