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Predisposition to epilepsy in fragile X syndrome: Does the Val66Met polymorphism in the BDNF gene play a role?
Epilepsy & Behavior 22 (2011) 581–583
Contents lists available at SciVerse ScienceDirect
Epilepsy & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ye b e h
Brief Communication
Predisposition to epilepsy in fragile X syndrome: Does the Val66Met polymorphism in the BDNF gene play a role? Mireia Tondo a, Pilar Poo b, Montserrat Naudó a, Teresa Ferrando c, Jordi Genovés a, Marta Molero a, Loreto Martorell a,⁎ a b c
Molecular Genetics Section, Hospital Sant Joan de Déu, Barcelona, Spain Neurology Section, Hospital Sant Joan de Déu, Barcelona, Spain Neuropediatrics Department, Hospital-Quirón, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 9 June 2011 Revised 14 July 2011 Accepted 1 August 2011 Available online 3 September 2011 Keywords: Epilepsy FMR1 gene Polymorphism BDNF gene Fragile X syndrome
a b s t r a c t Epilepsy is detected in about 23% of patients with fragile X syndrome (FXS). Absence or reduced levels of the fragile X mental retardation protein (FMRP), a global regulator of translation in neurons and an important factor in synaptic plasticity, produce the observed epileptic patterns. The brain-derived neurotrophic factor (BDNF) gene is a specific regulator of synaptic plasticity, and disturbances in its function cause dendrite abnormalities similar to those observed in FXS. A putative reciprocal regulation of FMRP and BDNF has been hypothesized. The Val66Met polymorphism in the BDNF gene may be involved in the alteration of normal secretion of the mature peptide and may modulate the epileptic phenotype observed in some patients with FXS. We investigated the relationship of this Met66 allele to the prevalence of epilepsy in 77 patients with FXS. No association was observed between this polymorphism and epilepsy in our group of patients. Therefore, it should not be considered a biomarker for developing epilepsy in patients with FXS. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy affects approximately 0.5 to 2% of the general population, leading to significant clinical and social implications for patients and their families. Fragile X syndrome (FXS) is the most common form of inherited mental retardation affecting 1 in 4000 males and 1 in 6000 females. The disease has a characteristic physical and behavioral phenotype including mental retardation, autism, emotional and psychiatric challenges, and anxiety and mood disorders [1]. Abnormal CGG repeat expansion in the fragile X mental retardation (FMR1) gene causes FXS. Premutation carriers who were previously considered as not affected and therefore healthy (55–200 CGG repeats) have been found to manifest other symptoms, among them epilepsy [1]. The frequency of epilepsy in FXS is greater than in the normal population, ranging from 14 to 50% depending on the study, with 23% the most commonly accepted percentage [2]. Epilepsy in individuals with FXS follows a benign course, disappearing in adolescence [3]. However, in some cases epilepsy is still present in adulthood [4]. The brain-derived neurotrophic factor gene (BDNF) is a member of the neurotrophin family and is implicated in differentiation, maturation, and survival of neurons. Its main function is to regulate the mRNA FMR1 gene in neurons. Some authors suggest it has the
⁎ Corresponding author at: Edifici docent, c/Santa Rosa, 39, Hospital Sant Joan de Déu, 08950 Esplugues, Barcelona, Spain. Fax: + 34 93 6009760. E-mail address: [email protected] (L. Martorell). 1525-5050/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2011.08.003
capacity to induce seizures by indirect reduction of FMR1 protein and self-increase of BDNF expression. The FMR1 protein is present at synaptic sites acting as a protein regulator. Therefore, it plays an important role in physiopathological mechanisms. A large number of polymorphisms have been described for the BDNF gene, among which Val66Met (rs6265) is a frequently studied one. The reason for this is its implication in psychiatric disorders and as a measure of cognitive function. One other polymorphism has also been associated with BDNF and FMRP (rs6784320). However, this SNP is intronic, and no effect on BDNF function could be shown. The authors concluded that its association with epilepsy may reflect strong linkage disequilibrium with functional Val66Met [5]. Concerning allele frequencies, conflicting results have been reported in all studies. Val66 is the most frequent allele, present in about 20% of the Caucasian population [6]. Some studies suggest that although the substitution of a valine for a methionine does not affect the protein itself, it could alter the secretion of the mature peptide, resulting in phenotypical changes [7]. They also suggest that BDNF induces hyperexcitability in the hippocampus and, at the same time, is involved in the epileptogenesis. The major conflicting result concerning involvement of the Val66Met polymorphism in seizures was observed in patients with Rett syndrome. In this syndrome, the Met66 polymorphism has a protective effect against early seizures [8]. However, other authors have observed contrary effects [9]. The aim of the present study was to investigate whether the Met66 polymorphism is associated with epilepsy in FXS and if this
582
M. Tondo et al. / Epilepsy & Behavior 22 (2011) 581–583
clinical form, and genotype), a binary logistic regression test with the Hosmer–Lemeshow goodness-of-fit test correction was applied.
Table 1 Clinical and genetic data of patients and controls. Genotype/clinical form Full (n = 61) Epilepsy Nonepilepsy Premutated (n = 16) Epilepsy Nonepilepsy Total (n = 77) Control group (n = 56)
Met/Met
Met/Val
Val/Val
Total
1M 1F
1M 20 (11 M/9 F)
7 (6 M/1 F) 31 (25 M/6 F)
9 52
0 1F 3 (3.9%) 3 (1 M/2 F) (5.3%)
0 5 (4 M/1 F) 26 (33.8%) 17 (7 M/10 F) (30.3%)
1M 9 (7 M/2 F) 48 (62.3%) 36 (13 M/23 F) (64.3%)
1 15 77 56
3. Results
2. Methods
A total of 77 individuals with a mutation in the FMR1 gene were clinically and molecularly evaluated: 61 patients had the full mutation allele (79.2%) and 16 individuals were premutated carriers (20.8%). Twenty-one were females (27.3%) and 56 were males (72.7%). Ten of 77 patients with FXS (13%) had epilepsy. Clinical and molecular data from patients with FXS (n = 77) and controls (n = 56) are summarized in Table 1. Detailed clinical and genetic data from the 10 patients with epilepsy are summarized in Table 2. The frequency of the Val66 allele in general and in the FXS population was 64.3 and 62.3%, respectively. For the Val66Met allele, the frequency was 30.3 and 33.8%, respectively. The binary logistic regression test with the Hosmer–Lemeshow goodness-of-fit test correction showed no association between epilepsy and the different alleles as none of the variables in the equation was statistically significant (data not shown).
2.1. Patients
4. Discussion
The present study comprises the clinical and genetic evaluation of 77 patients with FXS (age range = 1–65 years, mean = 6 years, SD = 13.2) referred to our genetics unit for molecular testing of the FMR1 gene. All patients belong to families that originate from different regions in Spain. The control group comprised 56 individuals (age range = 2–69 years, mean = 8.3 years, SD = 12.4) without a history of seizures or epilepsy. The study was approved by the ethics committee of our hospital and samples were obtained with the appropriate informed consent. Clinical history of patients was exhaustively reviewed and completed. Genomic DNA was derived from peripheral blood leukocytes using standard procedures [10].
Different studies have speculated on the relationship between the BDNF and FMR1 genes as a cause of epilepsy in FXS. Along these lines, a previous study in a Finnish population described the Met66 allele as a factor predisposing to epilepsy in males with FXS [5]. In our study, the observed frequencies of the Val66Met allele in the control and the FXS populations were practically identical (64.3% and 62.3%, respectively) and did not differ from those described in previous studies for a Caucasian population [6]. In the present study, we found no statistical significance between presence of the Met66 allele and the existence of epilepsy in individuals with FXS. This result is not in agreement with previous results from other groups indicating a positive genetic association [5]. For this reason, the polymorphism of the Met66 allele should not be considered a factor predisposing to seizures in the FXS population. Concerning the data on the Finnish population, our opposing results could be due to the fact that their targeted group contained only 27 patients [5]. They hypothesized that the absence of homozygous individuals with the Met66Met allele in their population was due to the deleterious effect of a Met66Met genotype in FXS. This hypothesis was ruled out in our study as one individual with the full mutation and the Met66Met genotype was found (see Table 2). Moreover, the Met66Met allele was also detected in our control population. In our study, the frequencies of the different alleles did not differ from those described in previous studies for a Caucasian population [6]. Furthermore, the frequencies observed in our group of patients and in our control group were very similar, suggesting no difference in presenting one or another allele and having epilepsy. Both previous studies and the present study found that the relationship between the BDNF and FMR1 genes remains unclear and is more complex than was initially thought. For example, the role that
Note. A binary logistic regression test with Hosmer–Lemeshow goodness-of-fit test correction was applied to search for an association between presenting and not presenting epilepsy and the other variables. No association was observed (P = NS).
polymorphism in the BDNF gene could be a biological marker for a tendency to develop epilepsy in FXS.
2.2. Determination of CGG repeats size in the FMR1 gene Polymerase chain reaction analysis to measure normal alleles and Southern blot for large expansions were carried out as previously described [11]. 2.3. BDNF Val66Met polymorphism analysis The nucleotide change leading to the Val66Met substitution was performed as previously described [8] with a slight variation: the polymerase chain reaction product was analyzed in a 3130 Avant Applied Biosystems sequencer. 2.4. Statistical analysis The SPSS 17.0 program was used for statistical description of the variables. To search for association among the different variables (sex,
Table 2 Clinical and genetic data of patients with seizures. Patient
Clinical form
Sex
Age (years)
BDNF polymorphism
EEG features
Type of seizure
1 2 3 4 5 6 7 8 9 10
Full Full Full Full Full Full Full Full Full premutated
Male Female Male Male Male Male Male Male Female Male
2 3 2 3 4 6 6 12 5 14
Met/Val Val/Val Val/Val Val/Val Val/Val Val/Val Val/Val Met/Met Val/Val Val/Val
Focal paroxysm Normal Multifocal spikes Focal paroxysm Multifocal spikes Focal paroxysm Normal Focal paroxysm Multifocal spikes Normal
Without clinical expression Partial epileptic seizures Myoclonic seizures Without clinical expression Generalized tonic seizures Partial epileptic seizures Generalized tonic–clonic seizures Without clinical expression Partial epileptic seizures Generalized tonic–clonic seizures
M. Tondo et al. / Epilepsy & Behavior 22 (2011) 581–583
these alleles may have in other neurodevelopment disorders such as Rett syndrome is still controversial [8,9]. Furthermore, and as recently speculated, other aspects should be taken into account when studying the relationship between epilepsy and FXS, such as the influence of autoimmunity in mothers of children with FXS and seizures [12]. In conclusion, no relationship was found between the presence of Met66 allele and epilepsy in our fragile X syndrome group. Therefore this polymorphism should not be considered a biomarker for developing epilepsy in patients with fragile X syndrome. Ethical approval We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments We are indebted to the patients for their cooperation in performing this study. We also thank Dr. Sylvie Guiroux, Center de Reserche en Génétique Humanine et Moléculaire, CHUQ, Québec, Canada, for kindly supplying us with the StB12.3 probe.
583
References [1] Van Esch H. The fragile X premutation: new insights and clinical consequences. Eur J Med Genet 2006;49:1–8. [2] Berry-Kravis E. Epilepsy in fragile X syndrome. Dev Med Child Neurol 2002;44: 724–8. [3] Singh R, Sutherland G, Manson J. Partial seizures with focal epileptogenic electroencephalographic patterns in three related female patients with fragile X syndrome. J Child Neurol 1999;14:108–12. [4] Sabaratnam M, Vroegop P, Gangadharan S. Epilepsy and EEG findings in 18 males with fragile X syndrome. Seizure 2001;10:60–3. [5] Louhivuori V, Arvio M, Soronen P, et al. The Val66Met polymorphism in the BDNF gene is associated with epilepsy in fragile X syndrome. Epilepsy Res 2009;85: 114–7. [6] Gratacos M, Gonzalez J, Mercader J, et al. Brain-derived neurotrophic factor Val66Met and psychiatric disorders: meta-analysis of case–control studies confirms association to substance-related disorders, eating disorders, and schizophrenia. Biol Psychiatry 2007;61:911–22. [7] Chen Z, Bath K, McEwen B, et al. Impact of genetic variant BDNF (Val66Met) on brain structure and function. Novartis Found Symp 2008;289:180–95. [8] Nectoux J, Bahi-Buisson N, Guellec I, et al. The p.Val66Met polymorphism in the BDNF gene protects against early seizures in RETT syndrome. Neurology 2008;70: 2145–51. [9] Ben Zeev B, Bebbington A, Ho G, et al. The common BDNF polymorphism may be a modifier of disease severity in Rett syndrome. Neurology 2009;72: 1242–7. [10] Maniatis T, Firtsch EF, Sambrook J. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16: 1215. [11] Fu YH, Kuhl DP, Pizzuti A, et al. Variation of the CGG repeat X site results in genetic instability: resolution of Sherman paradox. Cell 1991;67:1047–58. [12] Chonchaiya W, Tassone F, Ashwood P, et al. Autoinmune disease in mothers with the FMR1 premutation is associated with seizures in their children with fragile X syndrome. Hum Genet 2010;128:539–48.
Contents lists available at SciVerse ScienceDirect
Epilepsy & Behavior j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / ye b e h
Brief Communication
Predisposition to epilepsy in fragile X syndrome: Does the Val66Met polymorphism in the BDNF gene play a role? Mireia Tondo a, Pilar Poo b, Montserrat Naudó a, Teresa Ferrando c, Jordi Genovés a, Marta Molero a, Loreto Martorell a,⁎ a b c
Molecular Genetics Section, Hospital Sant Joan de Déu, Barcelona, Spain Neurology Section, Hospital Sant Joan de Déu, Barcelona, Spain Neuropediatrics Department, Hospital-Quirón, Madrid, Spain
a r t i c l e
i n f o
Article history: Received 9 June 2011 Revised 14 July 2011 Accepted 1 August 2011 Available online 3 September 2011 Keywords: Epilepsy FMR1 gene Polymorphism BDNF gene Fragile X syndrome
a b s t r a c t Epilepsy is detected in about 23% of patients with fragile X syndrome (FXS). Absence or reduced levels of the fragile X mental retardation protein (FMRP), a global regulator of translation in neurons and an important factor in synaptic plasticity, produce the observed epileptic patterns. The brain-derived neurotrophic factor (BDNF) gene is a specific regulator of synaptic plasticity, and disturbances in its function cause dendrite abnormalities similar to those observed in FXS. A putative reciprocal regulation of FMRP and BDNF has been hypothesized. The Val66Met polymorphism in the BDNF gene may be involved in the alteration of normal secretion of the mature peptide and may modulate the epileptic phenotype observed in some patients with FXS. We investigated the relationship of this Met66 allele to the prevalence of epilepsy in 77 patients with FXS. No association was observed between this polymorphism and epilepsy in our group of patients. Therefore, it should not be considered a biomarker for developing epilepsy in patients with FXS. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Epilepsy affects approximately 0.5 to 2% of the general population, leading to significant clinical and social implications for patients and their families. Fragile X syndrome (FXS) is the most common form of inherited mental retardation affecting 1 in 4000 males and 1 in 6000 females. The disease has a characteristic physical and behavioral phenotype including mental retardation, autism, emotional and psychiatric challenges, and anxiety and mood disorders [1]. Abnormal CGG repeat expansion in the fragile X mental retardation (FMR1) gene causes FXS. Premutation carriers who were previously considered as not affected and therefore healthy (55–200 CGG repeats) have been found to manifest other symptoms, among them epilepsy [1]. The frequency of epilepsy in FXS is greater than in the normal population, ranging from 14 to 50% depending on the study, with 23% the most commonly accepted percentage [2]. Epilepsy in individuals with FXS follows a benign course, disappearing in adolescence [3]. However, in some cases epilepsy is still present in adulthood [4]. The brain-derived neurotrophic factor gene (BDNF) is a member of the neurotrophin family and is implicated in differentiation, maturation, and survival of neurons. Its main function is to regulate the mRNA FMR1 gene in neurons. Some authors suggest it has the
⁎ Corresponding author at: Edifici docent, c/Santa Rosa, 39, Hospital Sant Joan de Déu, 08950 Esplugues, Barcelona, Spain. Fax: + 34 93 6009760. E-mail address: [email protected] (L. Martorell). 1525-5050/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2011.08.003
capacity to induce seizures by indirect reduction of FMR1 protein and self-increase of BDNF expression. The FMR1 protein is present at synaptic sites acting as a protein regulator. Therefore, it plays an important role in physiopathological mechanisms. A large number of polymorphisms have been described for the BDNF gene, among which Val66Met (rs6265) is a frequently studied one. The reason for this is its implication in psychiatric disorders and as a measure of cognitive function. One other polymorphism has also been associated with BDNF and FMRP (rs6784320). However, this SNP is intronic, and no effect on BDNF function could be shown. The authors concluded that its association with epilepsy may reflect strong linkage disequilibrium with functional Val66Met [5]. Concerning allele frequencies, conflicting results have been reported in all studies. Val66 is the most frequent allele, present in about 20% of the Caucasian population [6]. Some studies suggest that although the substitution of a valine for a methionine does not affect the protein itself, it could alter the secretion of the mature peptide, resulting in phenotypical changes [7]. They also suggest that BDNF induces hyperexcitability in the hippocampus and, at the same time, is involved in the epileptogenesis. The major conflicting result concerning involvement of the Val66Met polymorphism in seizures was observed in patients with Rett syndrome. In this syndrome, the Met66 polymorphism has a protective effect against early seizures [8]. However, other authors have observed contrary effects [9]. The aim of the present study was to investigate whether the Met66 polymorphism is associated with epilepsy in FXS and if this
582
M. Tondo et al. / Epilepsy & Behavior 22 (2011) 581–583
clinical form, and genotype), a binary logistic regression test with the Hosmer–Lemeshow goodness-of-fit test correction was applied.
Table 1 Clinical and genetic data of patients and controls. Genotype/clinical form Full (n = 61) Epilepsy Nonepilepsy Premutated (n = 16) Epilepsy Nonepilepsy Total (n = 77) Control group (n = 56)
Met/Met
Met/Val
Val/Val
Total
1M 1F
1M 20 (11 M/9 F)
7 (6 M/1 F) 31 (25 M/6 F)
9 52
0 1F 3 (3.9%) 3 (1 M/2 F) (5.3%)
0 5 (4 M/1 F) 26 (33.8%) 17 (7 M/10 F) (30.3%)
1M 9 (7 M/2 F) 48 (62.3%) 36 (13 M/23 F) (64.3%)
1 15 77 56
3. Results
2. Methods
A total of 77 individuals with a mutation in the FMR1 gene were clinically and molecularly evaluated: 61 patients had the full mutation allele (79.2%) and 16 individuals were premutated carriers (20.8%). Twenty-one were females (27.3%) and 56 were males (72.7%). Ten of 77 patients with FXS (13%) had epilepsy. Clinical and molecular data from patients with FXS (n = 77) and controls (n = 56) are summarized in Table 1. Detailed clinical and genetic data from the 10 patients with epilepsy are summarized in Table 2. The frequency of the Val66 allele in general and in the FXS population was 64.3 and 62.3%, respectively. For the Val66Met allele, the frequency was 30.3 and 33.8%, respectively. The binary logistic regression test with the Hosmer–Lemeshow goodness-of-fit test correction showed no association between epilepsy and the different alleles as none of the variables in the equation was statistically significant (data not shown).
2.1. Patients
4. Discussion
The present study comprises the clinical and genetic evaluation of 77 patients with FXS (age range = 1–65 years, mean = 6 years, SD = 13.2) referred to our genetics unit for molecular testing of the FMR1 gene. All patients belong to families that originate from different regions in Spain. The control group comprised 56 individuals (age range = 2–69 years, mean = 8.3 years, SD = 12.4) without a history of seizures or epilepsy. The study was approved by the ethics committee of our hospital and samples were obtained with the appropriate informed consent. Clinical history of patients was exhaustively reviewed and completed. Genomic DNA was derived from peripheral blood leukocytes using standard procedures [10].
Different studies have speculated on the relationship between the BDNF and FMR1 genes as a cause of epilepsy in FXS. Along these lines, a previous study in a Finnish population described the Met66 allele as a factor predisposing to epilepsy in males with FXS [5]. In our study, the observed frequencies of the Val66Met allele in the control and the FXS populations were practically identical (64.3% and 62.3%, respectively) and did not differ from those described in previous studies for a Caucasian population [6]. In the present study, we found no statistical significance between presence of the Met66 allele and the existence of epilepsy in individuals with FXS. This result is not in agreement with previous results from other groups indicating a positive genetic association [5]. For this reason, the polymorphism of the Met66 allele should not be considered a factor predisposing to seizures in the FXS population. Concerning the data on the Finnish population, our opposing results could be due to the fact that their targeted group contained only 27 patients [5]. They hypothesized that the absence of homozygous individuals with the Met66Met allele in their population was due to the deleterious effect of a Met66Met genotype in FXS. This hypothesis was ruled out in our study as one individual with the full mutation and the Met66Met genotype was found (see Table 2). Moreover, the Met66Met allele was also detected in our control population. In our study, the frequencies of the different alleles did not differ from those described in previous studies for a Caucasian population [6]. Furthermore, the frequencies observed in our group of patients and in our control group were very similar, suggesting no difference in presenting one or another allele and having epilepsy. Both previous studies and the present study found that the relationship between the BDNF and FMR1 genes remains unclear and is more complex than was initially thought. For example, the role that
Note. A binary logistic regression test with Hosmer–Lemeshow goodness-of-fit test correction was applied to search for an association between presenting and not presenting epilepsy and the other variables. No association was observed (P = NS).
polymorphism in the BDNF gene could be a biological marker for a tendency to develop epilepsy in FXS.
2.2. Determination of CGG repeats size in the FMR1 gene Polymerase chain reaction analysis to measure normal alleles and Southern blot for large expansions were carried out as previously described [11]. 2.3. BDNF Val66Met polymorphism analysis The nucleotide change leading to the Val66Met substitution was performed as previously described [8] with a slight variation: the polymerase chain reaction product was analyzed in a 3130 Avant Applied Biosystems sequencer. 2.4. Statistical analysis The SPSS 17.0 program was used for statistical description of the variables. To search for association among the different variables (sex,
Table 2 Clinical and genetic data of patients with seizures. Patient
Clinical form
Sex
Age (years)
BDNF polymorphism
EEG features
Type of seizure
1 2 3 4 5 6 7 8 9 10
Full Full Full Full Full Full Full Full Full premutated
Male Female Male Male Male Male Male Male Female Male
2 3 2 3 4 6 6 12 5 14
Met/Val Val/Val Val/Val Val/Val Val/Val Val/Val Val/Val Met/Met Val/Val Val/Val
Focal paroxysm Normal Multifocal spikes Focal paroxysm Multifocal spikes Focal paroxysm Normal Focal paroxysm Multifocal spikes Normal
Without clinical expression Partial epileptic seizures Myoclonic seizures Without clinical expression Generalized tonic seizures Partial epileptic seizures Generalized tonic–clonic seizures Without clinical expression Partial epileptic seizures Generalized tonic–clonic seizures
M. Tondo et al. / Epilepsy & Behavior 22 (2011) 581–583
these alleles may have in other neurodevelopment disorders such as Rett syndrome is still controversial [8,9]. Furthermore, and as recently speculated, other aspects should be taken into account when studying the relationship between epilepsy and FXS, such as the influence of autoimmunity in mothers of children with FXS and seizures [12]. In conclusion, no relationship was found between the presence of Met66 allele and epilepsy in our fragile X syndrome group. Therefore this polymorphism should not be considered a biomarker for developing epilepsy in patients with fragile X syndrome. Ethical approval We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments We are indebted to the patients for their cooperation in performing this study. We also thank Dr. Sylvie Guiroux, Center de Reserche en Génétique Humanine et Moléculaire, CHUQ, Québec, Canada, for kindly supplying us with the StB12.3 probe.
583
References [1] Van Esch H. The fragile X premutation: new insights and clinical consequences. Eur J Med Genet 2006;49:1–8. [2] Berry-Kravis E. Epilepsy in fragile X syndrome. Dev Med Child Neurol 2002;44: 724–8. [3] Singh R, Sutherland G, Manson J. Partial seizures with focal epileptogenic electroencephalographic patterns in three related female patients with fragile X syndrome. J Child Neurol 1999;14:108–12. [4] Sabaratnam M, Vroegop P, Gangadharan S. Epilepsy and EEG findings in 18 males with fragile X syndrome. Seizure 2001;10:60–3. [5] Louhivuori V, Arvio M, Soronen P, et al. The Val66Met polymorphism in the BDNF gene is associated with epilepsy in fragile X syndrome. Epilepsy Res 2009;85: 114–7. [6] Gratacos M, Gonzalez J, Mercader J, et al. Brain-derived neurotrophic factor Val66Met and psychiatric disorders: meta-analysis of case–control studies confirms association to substance-related disorders, eating disorders, and schizophrenia. Biol Psychiatry 2007;61:911–22. [7] Chen Z, Bath K, McEwen B, et al. Impact of genetic variant BDNF (Val66Met) on brain structure and function. Novartis Found Symp 2008;289:180–95. [8] Nectoux J, Bahi-Buisson N, Guellec I, et al. The p.Val66Met polymorphism in the BDNF gene protects against early seizures in RETT syndrome. Neurology 2008;70: 2145–51. [9] Ben Zeev B, Bebbington A, Ho G, et al. The common BDNF polymorphism may be a modifier of disease severity in Rett syndrome. Neurology 2009;72: 1242–7. [10] Maniatis T, Firtsch EF, Sambrook J. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16: 1215. [11] Fu YH, Kuhl DP, Pizzuti A, et al. Variation of the CGG repeat X site results in genetic instability: resolution of Sherman paradox. Cell 1991;67:1047–58. [12] Chonchaiya W, Tassone F, Ashwood P, et al. Autoinmune disease in mothers with the FMR1 premutation is associated with seizures in their children with fragile X syndrome. Hum Genet 2010;128:539–48.