Mol Neurobiol DOI 10.1007/s12035-013-8589-1

Genetics, Molecular Biology, and Phenotypes of X-Linked Epilepsy Hao Deng & Wen Zheng & Zhi Song

Received: 25 August 2013 / Accepted: 5 November 2013 # Springer Science+Business Media New York 2013

Abstract Epilepsy is a common and diverse set of chronic neurological disorders characterized by spontaneous, unprovoked, and recurrent epileptic seizures. Environmental factors and acquired disposition are proposed to play a role to the pathogenesis of epilepsy. Genetic factors are important contributors as well. Comparing to the phenotype of epilepsy caused by mutation of single gene on an autosome, the phenotype of X-linked epilepsy is more complex. X-linked epilepsy usually manifests as part of a syndrome or epileptic encephalopathy, and the variability of clinical manifestations of X-linked epilepsy may be attributed to several factors including the type of genetic mutation, methylation, X chromosome random inactivation, and mosaic distribution. As a result, it is difficult to establish the genotype–phenotype correlation, diagnostic tests, and genetic counseling. In this review, we provide an overview of the X-linked epilepsy including responsible loci and genes, the molecular biology, the associated complex phenotypes, and the interference factors. This information may provide us a better understanding of the pathogenesis of X-linked epilepsy and may contribute to clinical diagnosis and therapy of epilepsy.

Keywords Genetics . Molecular biology . Phenotypes . Seizure . X-linked epilepsy

Abbreviations CDKL5 ARX ATP6AP2 SYN1 ARHGEF9 PCDH19 SRPX2 SLC9A6 MeCP2 RAB39B DCX ISSX RTT XLAG HYD-AG XLMR PRTS XMESID EIEE XLSCLH/LIS

Hao Deng and Wen Zheng contributed equally to this work. H. Deng (*) : W. Zheng Center for Experimental Medicine, The Third Xiangya Hospital, Central South University, Tongzipo Road 138, Changsha, Hunan 410013, China e-mail: [email protected] H. Deng : W. Zheng : Z. Song Department of Neurology, the Third Xiangya Hospital, Central South University, Changsha, Hunan, China

The cyclin-dependent kinase-like 5 gene The aristaless-related homeobox X-linked gene The ATPase H+ transporting lysosomal accessory peotein 2 gene The synapsin 1 gene The Rho guanine nucleotide exchange factor 9 gene The protocadherin 19 gene The sushi-repeat protein X-linked 2 gene The solute carrier family 9 member 6 gene The methyl-CpG-binding protein 2 gene The Ras-associated protein gene The doublecortin gene X-linked infantile spasm Rett syndrome X-linked lissencephaly with ambiguous genitalia Hydranencephaly with abnormal genitalia X-linked mental retardation Partington syndrome X-linked myoclonic epilepsy with spasticity and intellectual development Early infantile epileptic encephalopathy X-linked subcortical laminar heterotopia and lissencephaly syndrome

Introduction Epilepsy is a common and diverse set of neurologic disorders associated with transient, hypersynchronous neuronal discharges and characterized by spontaneous, unprovoked, recurrent epileptic seizures. The etiology of epilepsy is complex.

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Multiple genetic factors have been identified to be involved in epilepsy. These genetic factor can be divided in two broad categories: (1) loci and genes identified in primary epilepsy syndromes, in which the epileptic seizures is the predominant clinical features; (2) loci and genes identified in brain developmental disorders presenting with epileptic seizures and other clinical characteristics, such as epileptic encephalopathy, a heterogenous category of devastating epileptic disorders characterized by frequent severe seizures, prominent interictal epileptiform discharges on the EEG, and progressive cognitive and neuropsychological deterioration [1]. Progress has been made in genetic study of epilepsy in the past 18 years and many disease-causing genes for epilepsy and epileptic encephalopathy have been identified. The pathogenic role of the genetic mutations involves the synaptogenesis, pruning, neuronal migration and differentiation, neurotransmitter synthesis and release, and structures and functions of membrane receptors and transporters [2, 3]. The phenotypes associated with some genetic mutations are sex-limited expression pattern and restricted to female or male patients, such as the cyclindependent kinase-like 5 gene (CDKL5), the aristaless-related homeobox X-linked gene (ARX), the ATPase H+ transporting lysosomal accessory peotein 2 gene (ATP6AP2), the synapsin 1 gene (SYN1), the Rho guanine nucleotide exchange factor 9 (ARHGEF9), the protocadherin 19 gene (PCDH19), the sushirepeat protein X-linked 2 gene (SRPX2), the doublecortin gene (DCX), the solute carrier family 9 member 6 gene (SLC9A6), the methyl-CpG-binding protein 2 gene (MeCP2), the Rasassociated protein (RAB39B), etc., which encompass many common syndromes of diverse age at onset, types of epileptic seizures, severity, prognosis, and specific management approaches. Moreover, different epileptic syndromes sometimes link to the same genetic mutation and mutations in different genes result in similar phenotype. All these increase complexities of the genotype–phenotype correlation and make accurate clinical diagnosis more difficult. In this review, we provide an overview of the X-linked loci and genes responsible for some epilepsy and their associated phenotypes. This information may provide us a better understanding of the pathogenesis of epilepsy and may contribute to clinical diagnosis and therapy of epilepsy.

Loci with Disease-Causing Genes Xp22.13 and the CDKL5 Gene Early infantile epileptic encephalopathy 2 (EIEE2), also known as X-linked infantile spasm (ISSX), is a disorder characterized by frequent tonic seizures or spasms beginning in infancy, hypsarrhythmia, and mental retardation (MR). In 2003, Vera Kalscheuer reported that two unrelated female patients with EIEE2 were associated with de novo-balanced

X, autosome translocations, and both disrupted serine threonine kinase 9 gene (STK9) [4]. STK9 gene was mapped to Xp22 and was eventually renamed the CDKL5 gene because of similarity to some cell division protein kinases [5]. The human CDKL5 gene, mapped to Xp22, spans approximately 228 kb. It contains 24 exons and encodes a protein of 1,030 amino acids. The first three exons (exons 1, 1a, and 1b) are not translated. Two splice variants with distinct 5′ untranslated regions (UTRs) have been identified. Isoform I, containing exon 1, is transcribed in a wide range of tissues, whereas isoform II, including exons 1a and 1b, is expressed in the testis and fetal brain [5, 6]. To date, approximately 90 patients with the CDKL5 gene mutations have been reported. Pathogenic mutations in CDKL5 gene include missense, nonsense, deletion, frameshift, and aberrant splicing. Most of these mutations are sporadic [7]. More than 50 % of these mutations reside in the catalytic domain [7]. The CDKL5 protein is unique in kinase family. It has an unusually long tail consisting of more than 600 amino acids. This long tail resides in the C-terminus and has no obvious similarity to any other protein domains. There is a high degree of conservation in the N-terminal region among different CDKL5 orthologs that differ only in the most extreme Cterminus [5]. CDKL5 signaling cascades are reported to be involved in synaptic plasticity and learning, affecting spines, dendritic branching, and actin cytoskeleton formation in the cytoplasm [5]. The CDKL5 gene mutations residing in the catalytic domain encode defective CDKL5 proteins and influence the enzymatic activity of the CDKL5 protein. As a result, there may be impairment in autophosphorylation of CDKL5 protein and phosphorylation of its target proteins, and alteration of the trafficking of CDKL5 protein between the nucleus and the cytoplasm (Fig. 1) [8, 9]. The conserved catalytic domain is also necessary to stabilize the interaction of CDKL5 protein with other nuclear proteins, including the MeCP2 protein, a member of splicing regulatory protein family [10, 11]. MeCP2 is associated with epileptic encephalopathy as well. The spatial and temporal overlapping between expression profiles of MeCP2 and CDKL5 genes have also been documented in embryonic and postnatal mouse brain [7, 11]. Additionally, CDKL5 deficiency in human cells decreased the expression of mitogen-activated protein kinase kinase kinase 5 (MAP3K5) in both non-neuronal and neuronal cells [12]. MAP3K5 −/− mice display complete elimination of recognition memory and slight impairment of fear memory [13]. Recently, CDKL5 protein is suggested to alter RNA processing and splicing of several genes that play important roles in the maturation, function, or survival of neurons in the brain [14]. The CDKL5 gene mutations may cause severe mental retardation, early-onset seizures, or infantile spasms [4, 15, 16]. Additional phenotypes include stereotypic hand movements, severe hypotonia, severe visual impairment, feeding

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Fig. 1 Neuronal location and proposed function of gene products associated with epilepsy. CDKL5 protein usually locates in nucleus and mediates the phosphorylation of the MeCP2 protein. Defective CDKL5 protein influences phosphorylation of MECP2 and the trafficking of itself between the nucleus and the cytoplasm. The ARX protein is a transcription factor and locates in nucleus. Loss of ARX protein increases or decreases the expression of many other proteins. The mutations of the ATP6AP2 gene cause truncated renin receptor, which locates in cytomembrane and results in a modest and reproducible impairment of ERK1/ 2 activation. The synapsin I protein encoded by the SYN1 gene locates in synapse associated with the membranes of small synaptic vesicles and regulates the neurotransmitter release from adult nerve terminals. The collybistin protein encoded by the ARHGEF9 gene locates in synapse and is a brain-specific guanine nucleotide exchange factor, which takes part in the formation of postsynaptic glycine and gamma-aminobutyric acid receptor clusters, affects the plasticity and polarity of the eukaryotic actin cytoskeleton and alters cell signaling transduction pathways. The

mutations of PCDH19 gene cause loss of function of the protocadherin 19 protein, which locates in cytomembrane and synaptic membrane, and affects cell adhesion, signal transduction at the synaptic membrane, and specific synaptic connection. The SRPX2 protein, a ligand for uPAR, locates in cytoplasm and takes part in the extracellular proteolysis by binding uPAR. The doublecortin protein, encoded by the DCX gene and located in cytoplasm, directs neuronal migration by regulating the organization and stability of microtubules. NHE6 encoded by the SLC9A6 gene was found in the membranes of intracellular organelles including mitochondria and endosome. This protein controls of intracellular pH, maintenance of cellular volume. The MeCP2 protein locates in nucleus and as a transcriptional repressor acts as a link between DNA methylation and chromatin remodeling. The RAB39B protein locates to the Golgi compartment and plays a pivotal role in synapse formation, possibly by mediating an intracellular pathway connecting the plasma membrane, endosomes and trans Golgi network, and possibly in the organization of neuronal circuits

difficulties, and impaired psychomotor development [17, 18]. The frequency of CDKL5 gene mutations is estimated to account for about 8.6 % of female cases of early-onset seizures [17], 14 % of cases of epileptic encephalopathy [19], and 28 % of female cases of early-onset seizures and infantile spasms [17]. Mutations of CDKL5 gene in males are thought to be lethal [16]. However, some mutations of the CDKL5 gene have been reported in up to 5 % of boys with epileptic encephalopathy [19]. CDKL5-related epilepsy may be divided into three phases: (1) frequent early-onset seizures starting before 3 months of the age, (2) epileptic encephalopathy, and (3) refractory myoclonic epilepsy [20]. In general, seizures are highly polymorphic, including complex partial seizures, infantile spasms, myoclonic seizures, generalized tonic–clonic seizures, atypical absence, and tonic seizures [5]. One patient can have many different seizure types changing with time. Frequent myoclonic jerks are an important component of the

CDKL5 gene mutation-related epileptic seizures [21]. The severity of epilepsy varies among different types of CDKL5 gene mutations. Mutations affecting the N-terminal domain of CDKL5 are thought to result in a more severe phenotype in infancy [16]. Clinical and EEG data do not recognize any exact pattern, and no specific abnormalities have been detected by MRI [4, 19]. Patients treated with antiepileptic drugs (AEDs) usually experience an initial seizure-free honeymoon period, followed by relapses [5]. Early drug-resistant epilepsy starting from 10 days to 6 months of life is the primary clinical feature differentiating patient with CDKL5 gene mutations from patients with MeCP2 gene mutations [7]. Xp22.13 and the ARX Gene ISSX, characterized by infantile spasms, hypsarrhythmia, and mental retardation, is a classical form of epilepsy. It occurs in

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early infancy and is etiologically heterogeneous. In 1999, Bruyere et al. mapped the disease gene locus of a multigeneration Canada family with ISSX to a 7.0 cM interval between markers DXS1226 and the adrenal hypoplasia locus on Xp21.3-Xp22.1 [22]. Mutations of the ARX gene, located in this region, have been found to be one of the major contributors to X-linked mental retardation (XLMR) and epilepsy comprising ISSX in several families [23]. The human ARX gene, mapped to Xp22.13, spans approximately 12 kb. It contains 5 exons and encodes a protein of 562 amino acids. To date, more than 100 ARX gene mutations, including a 24-bp duplication in Exon 2(c.428_451dup), expansion in the first or second poly-alanine tract, frameshift, and deletion and point mutation within and outside of the homeodomain, have been identified [24, 25]. The c.428_451dup is the most frequent mutation and represents an ARX mutation hotspot [26]. More than 5 % of male patients with infantile spasms are due to expansion in the first or second polyalanine tract of ARX gene [27]. Expansion of the first polyalanine tract is more likely related to infantile spasms compared to that of the second poly-alanine tract [28]. Truncation mutations and missense mutations in the ARX homeodomain result in not only loss of DNA binding activity but also loss of transcriptional repression activity and cause severe brain malformation phenotypes [24, 25, 29], whereas missense mutations outside of the homeodomain and expansion of the poly-alanine tracts are associated with nonmalformation phenotypes [29, 30]. Some degree of genotype–phenotype correlation existing with other factors, including familial modifiers, X inactivation, and environmental mediators, results in phenotypic variability in patients, even from the same family [31]. Some cases with ARX mutations, with or without clear brain malformations, have also been reported to have cysts within the basal ganglia [32, 33], which may be a typical character for ARX patients. The ARX protein belongs to the Aristaless-related subset of the paired class of homeodomain proteins. It has several conserved domains, including the octapeptide domain, three nuclear localization sequences, four poly-alanine tracts, the DNA-binding homeodomain, and the Aristaless domain [23, 34]. The presence of four alanine repeats in the protein is a unique feature. Most proteins containing poly-alanine tracts either are transcription factors or have nuclear localization signals [35]. ARX protein is localized to the nucleus and is expressed predominantly in fetal and adult brain (Fig. 1) including the developing hypothalamus, thalamus, basal ganglia, and cerebral cortex beginning at embryonic day 8 [36, 37]. ARX protein acts as a regulator of proliferation and differentiation of neuronal progenitors, and plays an important role in ventral forebrain development [30], likely for basal ganglia formation; cholinergic neuronal specification; and interneuron production, migration, and specification [31, 34, 38–40]. In addition, ARX is a direct target of transcription factor Dlx1/2. ARX primarily functions as a transcriptional

repressor [41, 42], but it can also act as an activator [43]. Loss of ARX increases the expression of the gastrulation brain homeobox 1 gene (Gbx1), the Lim homeobox gene 6 gene (Lhx6), the distal-less homeobox 2 gene (Dlx2 ), the Nk2 homeobox gene (Nkx2 ), the mage-like 2 gene (Magel2 ), and the Lim domain only 3 gene (Lmo3 ) in ventral regions [39, 40, 42], but decreases the expression of the chemokine CXC motif receptor 4 gene (Cxcr4 ) and the bone morphogenetic protein-binding endothelial regulator protein gene (Bmper ) [40]. These neurological disorders caused by ARX gene mutations can be divided into two categories based on presence or absence of brain malformation, all with epilepsy as a major symptom [31]. Brain malformation is present in X-linked lissencephaly with ambiguous genitalia (XLAG) [32, 34], Proud syndrome, and hydranencephaly with abnormal genitalia (HYD-AG) [32], and is absent in XLMR [23, 36], West Syndrome/ISSX [23, 34], Partington syndrome (PRTS) [23], X-linked myoclonic epilepsy with spasticity and intellectual development (XMESID) [23], Ohtahara syndrome, early infantile epileptic encephalopathy (EIEE) [44], and other cases with epilepsy, intellectual disability, and dystonia/spasticity as part of the syndrome. A number of ARX mouse models have been constructed, leading to different insights into the disorder [33, 34, 45–47]. The first ARX knockout mouse model produced an Arx allele with a premature stop codon and resulted in a truncated protein. The phenotype of knockout mice is similar to that of human XLAG, including a poorly formed small cortex, absent corpus collosum, abnormal tangential migration, and differentiation of interneurons and abnormalities in the male genitalia. Knockout mice died shortly after birth, limiting its usefulness for studying the role of Arx in adulthood [34]. Collombat et al. created another mouse model with targeted knockout of exons 1 and 2 of the Arx gene. The mice produced a mutant protein with the loss of the first 360 Nterminal amino acids and died during perinatal period [47]. To prove that interneuron dysfunction is essential to the epilepsy phenotype in various brain malformations, the conditional Arx knockout mice was created by using the Cre-Lox system to stop expression of Arx in cells expressing Dlx5/6. Knockout mice displayed spontaneous seizures by postnatal day 14, abnormal EEG patterns, and loss of calbindin-positive interneurons [33]. Recently, two knock-in Arx mouse models with expansions in the first polyalanine tract were established. The models reproduced the human 7-alanine expansion and resulted in abnormality of Arx expressing interneurons. These knock-in mice have either normal life span or shortened life span of 5 months with the phenotype resembling human ISSX. The knock-in models are valuable to study the pathophysiology and potential future treatment strategies of ARX diseases [45, 46].

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Xp11.4 and the ATP6AP2 Gene In 2002, seven male patients from a kindred with mental retardation and epilepsy were reported, and the disease gene locus was mapped to Xp21.1-p11.4, spanning 7.7cM between DXS1049 and DXS8054 [48]. In 2005, a silent mutation (c.321C>T, p.D107D) in the ATP6AP2 gene residing in a putative exonic splicing enhancer site was identified in this kindred [49]. The mutation resulted in inefficient inclusion of exon 4 in 50 % of renin receptor mRNA and produced truncated proteins in the patients. The human ATP6AP2 gene, mapped to Xp11.4, spans approximately 26 kb. It contains 9 exons and encodes a protein of 350 amino acids. Similar to the wild-type receptor, the mutated renin receptor could bind renin and increase renin catalytic activity, but resulted in a modest and reproducible impairment of extracellular signal-regulated kinase1/2 (ERK1/2) activation (Fig. 1). The silent mutation in the kindred did not affect the downstream angiotensin system [49]. Xp11.23 and the SYN1 Gene In 2004, ten male patients from a four-generation family with X-linked epilepsy disorder characterized by epilepsy, learning difficulties, macrocephaly, and aggressive behavior was reported. A W356X mutation in the SYN1 gene was found in all male patients and in obligate female carriers [50]. The human SYN1 gene, mapped to Xp11.23, spans approximately 48 kb. It contains 13 exons and encodes synapsin I of 705 amino acids [51]. Synapsin I is a neuronal phosphoprotein associated with the membranes of small synaptic vesicles and takes part in the regulation of synaptogenesis and neurotransmitter release from adult nerve terminals (Fig. 1). Synapsin I stimulates the Rab3a cycle by increasing GTP binding, GTPase activity, and recruitment of Rab3a to the synaptic vesicle membrane; conversely, Rab3a inhibits the actin binding and synapsin I-induced synaptic vesicle clustering activity [52]. In synapsin I-deficient embryonic mice generated by homologous recombination, synapse formation and outgrowth of predendritic neurites and axons were markedly retarded in the mutated neurons [53]. In addition, the organization of synaptic vesicles at presynaptic terminals was significantly altered. Synapsin I-deficient mice has a strikingly increased response to electrical stimulation [54]. Xq11.1-q11.2 and the ARHGEF9 Gene In 2004, a G55A missense mutation in the ARHGEF9 gene, mapped to Xq11.1-q11.2, was found in a patient with clinical symptoms of both hyperekplexia and EIEE [55]. A loss-offunction mutation, Q2X, in the ARHGEF9 gene was reported in another patient with refractory seizures, right frontal polymicrogyria, and severe psychomotor retardation [56].

XLMR associated with epilepsy is a common phenotype of the ARHGEF9 gene mutation [56]. The human ARHGEF9 gene, mapped to Xq11.1-q11.2, spans approximately 150 kb. It contains 12 exons and encodes collybistin of 526 amino acids [55]. Collybistin is a brainspecific guanine nucleotide exchange factor, and belongs to a family of Rho-like GTPases [56]. Collybistin plays a critical role in the formation of postsynaptic glycine and gammaaminobutyric acid receptor clusters (Fig. 1) [57]. Moreover, it affects the plasticity and polarity of the eukaryotic actin cytoskeleton and alters cell signaling transduction pathways [56]. Xq22.1 and the PCDH19 Gene Early infantile epileptic encephalopathy 9 (EIEE9), also known as epilepsy female-restricted with mental retardation, is a disorder characterized by seizures and mental retardation. Ryan et al. assigned the EIEE9 disease locus to Xq22 in 1997 [58]. The PCDH19 gene mutations were first identified in seven families with EIEE9 by systematic sequencing of 737 genes on X-chromosome in 2008 [59], and it was confirmed by subsequent studies [60–63]. The human PCDH19 gene, mapped to Xq22.1, spans approximately 119 kb. It contains 6 exons and encodes a protein of 1,148 amino acids. The first exon is unusually large and most mutations identified to date were clustered in it [59–61]. Usually, patients inherit the PCDH19 gene mutations from their unaffected carrier fathers or affected mothers, and sporadic cases, overwhelmingly more frequent, are caused by de novo mutations [60–63]. The mutations include nonsense, frameshift, and missense mutation, which would probably cause loss-of-function of the mutated allele [60]. The mRNA containing a truncating mutation is highly unstable, and can be degraded by nonsense-mediated mRNA decay, an evolutionarily conserved process ensuring the degradation of transcripts carrying premature termination codon [59]. The PCDH19 gene mutations usually spare transmitting males and affect only females. Male PCDH19 gene mutation carriers with moderate mental retardation and the mother of female patients as heterozygous PCDH19 gene mutation carrier without any clinical features have also been reported [58–60, 64, 65]. One attractive hypothesis for this phenotypic variability is due to cellular interference, which bases on the random or skewed X inactivation. This mechanism is likely due to the existence of compensatory factors in patients with the mutated PCDH19 gene, resulting in a functional mosaicism and the co-existence of PCDH19 -positive and PCDH19-negative cells [59, 60]. The severity of the epilepsy is correlated with the relative amount of inactivated neurons for each chromosome in the brain [65]. The PCDH19 protein, belonging to the cadherin family of calcium-dependent cell–cell adhesion molecules, contains a

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signal sequence, six extracellular cadherin repeats, a transmembrane domain, and a cytoplasmic region with conserved cytoplasmic domains 1 and 2 [59]. Members of protocadherin family are highly expressed in the central nervous system [66, 67]. In the postnatal brain, protocadherins may be involved in modulating signal transduction at the synaptic membrane and establishing specific synaptic connection (Fig. 1) [68]. The main phenotype of the PCDH19 mutations is EIEE9. But mutations in this gene can also be detected in about 30 % of the SCN1A gene mutation negative females with Dravetlike symptoms [69]. Patients usually have normal neuroimaging [69]. Even in monozygotic twins [63], the clinical phenotype of the PCDH19 gene mutation, including seizure severity and degree of cognitive impairment, varies from mild to severe [70]. Seizures begin in infancy or early childhood (6– 36 months) [65]. The severity and frequency of seizures have been reported to be the worst during infancy and early childhood, and improve with age [62, 65]. Seizures are sensitive to fever in most patients, and usually are resistant to antiepileptic therapies [63]. Xq22.1 and the SRPX2 Gene In 2006, p.N327S and p.Y72S in the SRPX2 gene were respectively identified in patients from two unrelated families with rolandic seizure, speech dyspraxia, and mental retardation [71]. The human SRPX2 gene, mapped to Xq22.1, spans approximately 27 kb. It contains 11 exons and encodes a protein of 465 amino acids. SRPX2 protein has an N-terminal signal peptide and 3 consensus sushi repeats, each of which spans approximately 60 amino acids and contains 6 conserved cysteines [72]. Proteins with sushi domains usually participate in important functions in the brain, probably by regulating protein interactions with a variety of cell adhesion molecules [73]. The SRPX2 protein is a ligand for urokinase-type plasminogen activator receptor (uPAR) as well as for other members of the extracellular proteolysis system (Fig. 1), and p.Y72S mutant protein displays increased affinity for uPAR [73]. The SRPX2 protein has been reported to be directly associated with altered speech production and abnormalities in brain speech areas, especially in the rolandic and perisylvian regions [73]. Xq23 and the DCX Gene X-linked subcortical laminar heterotopia and lissencephaly syndrome (XLSCLH/LIS) is a severe neuronal migration disorder caused by migrational arrest of cortical neurons short of their normal destination. It mainly presents with epilepsy and mental retardation of variable severity [74]. In 1997, potential disease intervals in Xq22.3-q23 or Xq27 were found by linkage analysis in three unrelated families

in which males were affected with lissencephaly and females with subcortical laminar heterotopia (SCLH) [74]. In 1998, the breakpoint interrupting the DCX gene on Xq22.3-Xq23 was isolated in an X-linked lissencephaly (XLIS) patient with a balanced chromosomal translocation [75]. Subsequently, the association between the DCX mutations and XLIS or SCLH has been further confirmed in other cases [76, 77]. The human DCX gene, mapped to Xq23, spans approximately 118 kb. It contains 9 exons and encodes doublecortin of 446 amino acids. The doublecortin domain of the protein consists of two evolutionarily conserved repeats, namely NDC from amino acids 42–150 and C-DC from amino acids 171–275 [77]. Only 16 % of the DCX gene's sequence is coding and the 3′ UTR, which contains only one exon, is 7.9 kb long [78]. The mutations reported to date include missense, nonsense, deletion, splice, and insertion mutations [76]. The majority of missense mutations are located in the two evolutionarily conserved domains [79]. Hemizygous males are usually associated with lissencephaly and present with agyria or pachygyria with global developmental delay, severe mental retardation, and seizures. Heterozygous females present with a milder phenotype known as SCLH [80]. Xinactivation patterns in neuronal precursor cells are likely to contribute to the variable clinical phenotype in females [81]. Equivalently, somatic mosaicism is likely to account for the milder phenotype of this disorder in males [82]. In adult human, the doublecortin protein is expressed exclusively at a very high level in the frontal lobe [83], which directs neuronal migration by regulating the organization and stability of microtubules (Fig. 1) [84]. Doublecortin interacts with lissencephaly 1 protein, another lissencephaly-associated protein, to enhance tubulin polymerization in an additive fashion. This interaction is important for proper microtubule function in the developing cerebral cortex [85].

Xq26.3 and the SLC9A6 Gene Christianson syndrome is an XLMR syndrome characterized by severe global developmental delay or regression, earlyonset seizures of variable types, hypotonia, microcephaly, and abnormal ocular movement. This syndrome mainly affects males, and female carriers may be mildly affected [86]. In 1999, Christianson et al. mapped the disease gene locus of a five-generation South African family with this syndrome between markers DXS424 and DXS548, with a maximum twopoint Lod score of 3.10 [87]. In 2008, mutations of the SLC9A6 gene, localized to Xq26.3, were identified in three XLMR families with epilepsy and ataxia, including the family with Christianson syndrome [88]. Subsequently, additional mutations resulting in Christianson type XLMR and epilepsy were further identified by other studies [86, 89].

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The human SLC9A6 gene, mapped to Xq26.3, spans approximately 62 kb. It contains 16 exons and encodes the sodiumselective sodium/hydrogen exchanger 6 (NHE6) of 701 amino acids. NHE6 expresses as two splice variant isoforms: NHE6.0 and NHE6.1 [90, 91]. Both isoforms have 12 putative membrane-spanning segments within the N-terminal region and a hydrophilic C terminus, similar to the topologies predicted for other NHEs. In addition, the SLC9A6 gene has a putative mitochondrial inner membrane targeting signal at its N terminus [90]. NHE6 expresses ubiquitously with the most abundant expression in mitochondrion-rich tissues, including brain, skeletal muscle, and heart. NHE6 is also found in the membranes of intracellular organelles, including mitochondria and endosomes (Fig. 1) [90]. NHE6 was regulated by receptor for activated C kinase-1 in distribution between endosomes and the plasma membrane [92]. NHEs take part in a wide array of essential cellular processes, including control of intracellular pH, maintenance of cellular volume, and reabsorption of sodium across renal, intestinal, and other epithelia [91]. Knockdown and overexpression of NHE6.1 respectively reduces and elevates endosomal pH, which inhibits the maintenance of apical, bile canalicular plasma membranes [91]. Xq28 and the MeCP2 Gene Rett syndrome (RTT) is characterized by a nonsymptomatic phase for the first 6–18 months of age followed by regression of acquired skills, loss of speech, microcephaly, gait abnormalities, stereotypic hand movements, and mental retardation [93]. Many of these patients also have seizures. Linkage analysis mapped the locus to Xq28 [94, 95]. In 1999, five de novo mutations including three missense mutations, a frameshift mutation, and a nonsense mutation in the MeCP2 gene, located in Xq28 region, were identified in five sporadic patients with RTT [96]. Since then, more than 600 pathogenic MeCP2 gene mutations, including missense, nonsense, frameshift, duplication, and deletion mutations have been identified [97], which account for 80–96 % of the classical RTT cases and 40–50 % of atypical RTT manifestations [98, 99]. More than 95 % of the MeCP2 mutations are de novo, and they usually occur on the paternal X chromosome [100, 101]. Eight recurrent missense or nonsense mutations within the methyl-CpG-binding domain (MBD) (R106W, R133C, T158M, and R168X) or transcription repression domain (TRD; R255X, R270X, R294X, and R306C) are responsible for 65 % of MeCP2 RTT cases [93]. The human MeCP2gene, mapped to Xq28, spans approximately 76 kb. It contains four exons and encodes the MeCP2 protein. The protein expresses as two major splice variant isoforms, MeCP2_e1 and MeCP2_e2. The 498 amino-acid MeCP2_e1 encompasses exons 1, 3, and 4, and 486 aminoacid MeCP2_e2 residues comprises exons 2, 3, and 4 [102, 103]. Both isoforms contain an MBD, a TRD, two nuclear localization signals, and a C-terminal domain [104], and only

differ in their N-terminal sequences [93]. MeCP2_e2 is expressed in various tissues, including fetal and adult brain, where the expression is ten times higher than that of MeCP2_e1. MeCP2_e1 is expressed highly in placenta, liver, and skeletal muscle [103]. The MeCP2 expression is lower in immature neurons, while its expression is highest in postmitotic neurons [105]. The elevated level of MeCP2 expression in mature neurons is maintained throughout adulthood, indicating its important role in postmitotic neuronal function. The MBD domain promotes binding to methylated CpG dinucleotides and the preference for adjacent A/T-rich motifs [106]. The MBD domain can also bind to nonmethylated DNA sequences, such as the four-way DNA junctions [107]. But the role of MeCP2 as a transcriptional repressor is mostly mediated through its TRD domain. The TRD domain interacts with the transcription silencer co-repressor mammalian Sin3 yeast homolog of A, further recruiting histone deacetylase 1 (HDAC1) and histone deacetylase 2, and thereby acting as a link between DNA methylation and chromatin remodeling [108]. The TRD domain further facilitates interaction between MeCP2 and other partners including c-SKI [109], YY1, and YB1 [110, 111]. The nuclear localization signals guide the MeCP2 protein into the nucleus, and the C-terminal domain promotes binding of MeCP2 protein to the nucleosome core [112, 113]. Transgenic mice lacking MeCP2 C-terminal domain display RTT phenotypes [114]. Depending on its interacting protein partners and target genes, MeCP2 can act either as an activator or as a repressor [115]. By deacetylation of histones and chromatin condensation, MeCP2 protein represses transcription and silences the specific target genes [108]. Loss of function of MeCP2 in cells, especially in differentiated postmitotic neurons, may lead to overexpression of a number of other genes, with potentially damaging effect during central nervous system maturation [116]. The MeCP2 expression in glial cells is identified and significantly lower compared to neurons [117, 118]. MeCP2deficient astrocytes are unable to support proper neuronal growth while exert a noncell autonomous effect on neuronal properties, probably as a result of aberrant secretion of soluble factor(s) [117, 118]. The deficiency of MeCP2 in microglia leads to elevated secretion of glutamate and contributes to neuronal abnormalities in RTT [119]. Glia is an integral component of the neuropathology of RTT, which targeting as a strategy for improving the associated symptoms [120], although MeCP2 deficiency in neurons is sufficient to cause RTT-like neurological phenotypes in mouse [121]. RTT is the main phenotype of the MeCP2 gene mutation. The MeCP2 gene mutations have also been reported in a few patients with Prader–Willi syndrome [122], Angelman syndrome [123], nonsyndromic mental retardation [124], or autistic disorder [125]. Boys inheriting a mutant MeCP2 allele present with fatal infantile encephalopathy and usually do not survive infancy [93, 97].

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Overexpression of MeCP2 by duplication of the MeCP2 gene also leads to a variety of neurological symptoms [126, 127]. The cardinal features of the MeCP2 gene duplication syndrome include early infantile hypotonia, delayed psychomotor development resulting in profound intellectual disability, absent or limited speech, abnormal gait, epilepsy, spasticity, and recurrent infections. Seizures are observed in 54 % of patients. The onset and the severity of these seizures often correlate with neurological deterioration, and these seizures are refractory to treatment in some patients. Male mouse embryonic stem cells in which Mecp2 is disrupted are unable to support development, consistent with the male lethality of RTT. In contrast, chimeric mice, in which a small proportion of cells are derived from Mecp2-deficient embryonic stem cells, are viable [128]. Mice with a truncating mutation of the Mecp2 gene (Mecp2 308/y) appear normal and exhibit normal motor function for about 6 weeks, but then develop a progressive neurological disease that includes many features of RTT. Most Mecp2 308/y mice survive to at least 1 year of age [114], which is much longer than conditional knockout mice. A twofold increase in Mecp2 expression in transgenic mice results in enhanced motor and contextual learning and enhanced synaptic plasticity in the hippocampus beginning around 10 weeks of age. But after 20 weeks of age, mice develop seizures, hypoactivity, and spasticity, and 30 % of mice die by 1 year of age [129]. In 2011, an adult-onset model of RTT was developed by crossing mice harboring a floxed Mecp2 allele and tamoxifen-inducible CreER allele to delete Mecp2 when mice were fully mature [130].

Xq28 and the RAB39B Gene In 2000, the disease gene of a four-generation Sardinian family with XLMR was mapped to Xq28 with the maximum lod score of 2.71. Three patients in the family had seizures during childhood [131]. In 2010, a truncated mutation of Y7X and a splice site mutation of c.215+1G>A in the RAB39B gene were found in two unrelated families affected by XLMR. Some of the male patients primarily presented with mental retardation, autism spectrum disorder, epileptic seizures, and macrocephaly [132]. The human RAB39B gene, mapped to Xq28, spans approximately 6 kb. It contains 2 exons and encodes a protein of 213 amino acids. The RAB39B has four domains involved in GTP/GDP binding, five RabF domains predicted to interact with regulatory proteins, and a C-terminal prenylation motif. The protein is highly expressed in neurons and seems to play a pivotal role in synapse formation (Fig. 1), possibly by mediating an intracellular pathway connecting the plasma membrane, endosomes and trans Golgi network, and possibly in the organization of neuronal circuits [132].

Conclusions In this article, we summarized the genetics (gene structure, mutation types, and mutation frequencies of causing genes), functional biology (the roles of proteins, expression distribution, and signal pathway), and phenotypes (onset age, severity, and gender distribution difference) of X-linked epilepsy. Monogenic disease is defined as a disease that arises from one dominant or two recessive allelic mutations in the causative gene, whereas polygenic disorder is associated with more than one gene in combination with lifestyles and environmental factors. Since the identification of the cholinergic receptor neuronal nicotinic alpha polypeptide 4 gene (CHRNA4) as the disease-causing gene of an idiopathic partial epilepsy syndrome in 1995, a series of genes involved in ion channel function on autosome have been discovered to be responsible for a group of monogenic epilepsy disorders. Comparing to epilepsy caused by mutated single gene on autosome, the phenotype of X-linked epilepsy is more complex, and usually manifests as a part of a syndrome or epileptic encephalopathy with other psychoneurological abnormalities such as MR, delayed psychomotor development, autism, absent speech, spasticity, oral and speech dyspraxia (OSD). This phenomenon may be caused by several factors including multiple intelligence-related genes on the X chromosome, important role of random X chromosome inactivation, or imprint. The variability of clinical manifestations of X-linked epilepsy may be attributed to several factors, such as the type of gene mutation, methylation, X chromosome random inactivation, and mosaic distribution. The pathogenic role of these genetic mutations usually involve in the synaptogenesis, pruning, neuronal migration and differentiation, neurotransmitter synthesis and release, structures and functions of membrane receptors, and transporters. The difference in gender distribution is a character of Xliked epilepsy. Among these genes located on the X chromosome, the ARX, RAB39B, SYN1, ATP6AP2, SLC9A6 gene mutations and the duplication of MeCP2 gene are responsible for epileptic disorders predominantly affecting male. The CDKL5, PCDH19 gene mutations, and other type mutations of MeCP2 in sporadic cases are more common in female patients (Table 1). There is no obvious difference in gender distribution in the patients with epileptic disorder associated to the ARHGEF9 , SRPX2 , and DCX gene mutations, or in familial patients with the MeCP2 mutations. Epilepsy cannot be simply categorized as a monogenic disease. Many epileptic disorders may comply with the theoretical model of polygenic inheritance. The heterogeneity of gene mutations and complexity of the etiology hinder the development of simple diagnostic tests and genetic consulting. Application of new molecular tools, including genome wide association studies, whole genome sequencing, and exome sequencing, may detect subtle gene variations associated

228

12

26 48 150

119 27 118

62 76

6

CDKL5

ARX

ATP6AP2 SYN1 ARHGEF9

PCDH19

SRPX2

DCX

SLC9A6

MeCP2

RAB39B

Xp22.13

Xp22.13

Xp11.4 Xp11.23 Xq11.1-q11.2

Xq22.1

Xq22.1

Xq23

Xq26.3

Xq28

Xq28

2

4

16

9

11

6

9 13 12

5

24

213

498 or 486

701

446

465

1,148

350 705 526

562

1,030

Exons Amino acids

Females except 6 males

719 sporadic cases

11 cases from two families

23 males, 23 females

46 cases from 20 families

Males

Males

42 males, 102 females

Males

Females except a mosaic male 5 males, 9 females

Males Males 3 males, 2 females, a case not reported

Males

Females except 5 males

Gender tendency

63 sporadic cases, 7 families 14 cases from two families 61 familial cases, 80 sporadic cases, 3 cases not reported 36 cases from 7 families 80 sporadic cases

106 cases from 38 families, 16 sporadic cases, 11 families without case number 7 cases from a family 10 cases from a family 6 sporadic cases

73 sporadic cases, 2 families

Patients reported

RS rolandic seizure, MR mental retardation, OSD oral and speech dyspraxia

Gene size (kb)

Causative gene

Chromosome position

Missense, splice, frameshift, nonsense, deletion Missense, splice, frameshift, nonsense, deletion Nonsense, nucleotide substitution

Missense, splice, insertion, deletion, nonsense Deletion, Splice, truncation, nonsense Duplication

c.321C>T, p.D107D W356X Nonsense, deletion, missense, chromosomal disruptions Missense, deletion nonsense, frameshift p.N327S, p.Y72S

Missense, splice, frameshift, nonsense, deletion Duplication 24 bp, deletions, indels, splice, nonsense, missense

Mutation type

MR, seizures, autism

Classical or atypical RTT

MR, seizures, absent speech, hypotonia Classical or atypical RTT, MR

Christianson syndrome

XLIS, SCLH

EIEE9, Dravet-like symptom RS, OSD and MR

XLMR, epilepsy Epileptic disorder EIEE8, MR, seizures, hyperekplexia

EIEE2, RTT-like phenotype, autism, ISSX, EIEE1, ISSX, XLAG, PRTS, XLMR, HYD-AG, XMESID, Proud syndrome

Main phenotype

[132]

[96, 113, 123, 173]

[96, 171–173]

[126, 162–170]

[86–89]

[80]

[71]

[59–63, 65, 158–161]

[48, 49] [50] [55, 56, 155–157]

[23, 32, 34, 36, 145–154]

[12, 15, 21, 133–144]

Reference

Table 1 Loci and genes identified associated with X-linked epilepsy disorders. This table describes the numbers of case or family, gender tendency, mutation types and main phenotypes of the gene mutations reported before July 2013 associated with X-linked epilepsy disorders

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Mol Neurobiol

with epilepsy. Additionally, combination of data from genetic variants, gene expression, and protein interaction may shed a new light on the identification of mechanism of epileptic syndromes. Furthermore, functional studies of these mutations, especially construction of animal models with genetic deficiency, may contribute to the understanding of genotype– phenotype relations and development of novel antiepileptic drugs. These AEDs may help to reach the goal of personalized and targeted medicine. All these may have significant impact on both the clinical diagnosis and the treatment of epileptic syndromes in the future. Acknowledgments This work was funded by the National Natural Science Foundation of China [81271921, 81101339 and 30971534]; the Sheng Hua Scholars Program of Central South University, China (H.D.); the Research Fund for the Doctoral Program of Higher Education of China [20110162110026]; and the Natural Science Foundation of Hunan Province, China (10JJ5029). Conflict of Interest The authors declare that they have no conflict of interest.

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55.

56.

57. 58.

59.

60.

61.

62.

63.

64.

65.

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