Subcortical Laminar Heterotopia Research Articles

ILAE neuroimaging task force highlight: Subcortical laminar heterotopia.

The ILAE Neuroimaging Task Force publishes educational case reports that highlight basic aspects of neuroimaging in epilepsy consistent with the ILAE's educational mission. Subcortical laminar heterotopia, also known as subcortical band heterotopia (SBH) or "double cortex," is an intriguing and rare congenital malformation of cortical development. SBH lesions are part of a continuum best designated as agyria-pachygyria-band-spectrum. The malformation is associated with epilepsy that is often refractory, as well as variable degrees of developmental delay. Moreover, in an increasing proportion of cases, a distinct molecular-genetic background can be found. Diagnosing SBH can be a major challenge for many reasons, including more subtle lesions, and "non-classic" or unusual MRI-appearances. By presenting an illustrative case, we address the challenges and needs of diagnosing and treating SBH patients in epilepsy, especially the value of high-resolution imaging and specialized MRI-protocols.

The late diagnosis of double cortex syndrome in a 36-year-old woman with resistant atonic seizures

Subcortical laminar heterotopia (double cortex syndrome) is an orphan disease with an incidence of 1 to 200,000 people in the population. The cause of the disease is mutation of the gene DCX (synonyms: DBCN, XLIS) in chromosome Xq22.3-q23. The type of inheritance is X-linked dominant. Correct diagnosis requires a high degree of skills of a neurologist/epileptologist and a radiologist. The paper describes a clinical case of the late diagnosis of double cortex syndrome in a 36-year-old woman with a long history of resistant atonic seizures and mental retardation.

Analysis of DCX gene mutation in a patient featuring X-linked subcortical laminar heterotopia and epilepsy

To detect potential mutation of Doublecortin (DCX) gene in a patient featuring X-linked subcortical laminar heterotopia (X-SCLH) and epilepsy. Mutation of the DCX gene was screened by PCR and direct sequencing. Pathogenicity of the mutation was analyzed with a PolyPhen-2 software. A de novo missense mutation c.971T>C (p.Phe324Ser) was discovered. A diagnostic method for X-SCLH has been established, which may facilitate diagnosis and genetic counseling of patients featuring this disease.

Clinical Features of Cerebral Cortex Malformations in Children: A Study in Upper Egypt

Malformation of Cortical Development (MCD) corresponds to a broad spectrum of cerebral lesions resulting from cortical development abnormalities during embryogenesis. MCD are increasingly recognized as an important cause of epilepsy, especially refractory ones. Aims: Evaluation of pattern and clinical spectrum of MCD. Patients and methods: The data of children presented to the Pediatric Department, or Pediatric Neurology Clinic, Sohag University Hospital whom Computed Tomography (CT) or Magnetic Resonance Imaging (MRI) brain showing MCD were collected retrospectively regarding their history, examinations, and developmental parameters, Electroencephalography (EEG), Intelligence Quotient (IQ). Results: during the study periods we reviewed the data of 50 Child with MCD, the mean age of the study group was 3.7 years with age range from 4 days to 15 years. The majority of the patients (50%) belonged to the age group 1-5 years old. Convulsions were the main complaint in the majority of children 21 (42%) out of 50 child, followed by global developmental delay in 20 out of 50 child. Seizures generally were present in (82%). Concerning type of seizures, multiple seizure types were found in 16 (39%) out of 41 child, partial seizures found in 13 (31.7%), generalized tonic in 6 (14.6%), infantile spasm in 4 (9.8%), and generalized tonic-clonic in 2 (4.8%). Types of malformation of cortical development in the studied children as defined by neuro-imaging, (42%) had lissencephaly, (16%) had bilateral Schizencephaly, (12%) had unilateral schizencephaly, (12%) had polymicrogyria, (10%) had focal cortical dysplasia, (6%) had periventricular heterotopia, and one child with subcortical laminar heterotopia. Conclusion: The common cortical malformations found in Sohag University Hospital were lissencephaly, bilateral Schizencephaly, unilateral schizencephaly. Also the most common clinical features of MCDs in our community are refractory seizures and/or global developmental delay, and the most valuable tool in diagnosis is brain MRI. Recommendations: Informing pediatricians to raise their clinical suspicion to MCDs in children presented by frequent, intractable, early onset, and partial or multiple types’ seizures and/or global developmental delay, and that MRI brain must be done then.

JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration

Doublecortin (DCX) is expressed in young neurons and functions as a microtubule-associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migration. Previous in vitro phosphorylation experiments revealed that cyclin-dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5-/- mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP-DCX-WT, or GFP-DCX-S332A and analyzed neurite extension and migration. Introduction of GFP-DCX-WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP-DCX-S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5-/- brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function.

Coexistence of mitochondrial and nuclear DNA mutations in a woman with mitochondrial encephalomyopathy and double cortex

We describe a 16-year-old girl with mental retardation, myoclonic epilepsy, ataxia, mitochondrial myopathy, sensorineural hearing loss, lactic acidosis, and MRI evidence of diffuse subcortical laminar heterotopia and agyria/pachygyria. Restriction fragment length polymorphism (RFLP) and DNA sequence analyses revealed two pathogenic mutations: a heteroplasmic m.3243A > G in muscle and blood, and a new heterozygous insertion at nt697 in the doublecortin gene (DCX), resulting in a frameshift after amino acid residue 232, with a premature stop codon at amino acid residue 244. This is yet another example of genetic “double trouble” resulting in a complex phenotype.

JNK phosphorylates Ser332 of doublecortin and regulates its function in neurite extension and neuronal migration

Doublecortin (DCX) is expressed in young neurons and functions as a microtubule-associated protein. DCX is essential for neuronal migration because humans with mutations in the DCX gene exhibit cortical lamination defects known as lissencephaly in males and subcortical laminar heterotopia (or double cortex syndrome) in females. Phosphorylation of DCX alters its affinity for tubulin and may modulate neurite extension and neuronal migration. Previous in vitro phosphorylation experiments revealed that cyclin-dependent kinase 5 (Cdk5) phosphorylates multiple sites of DCX, including Ser332, (S332). However, phosphorylation at only Ser297 has been shown in vivo. In the present study, we examined phosphorylation of S332 of DCX in the Cdk5-/- mouse brain and results found, unexpectedly, indicate an increased DCX phosphorylation at S332. We found that JNK, not Cdk5, phosphorylates DCX at S332 in vivo. To examine the physiological significance of S332 phosphorylation of DCX in neuronal cells, we transfected cells with either GFP, GFP-DCX-WT, or GFP-DCX-S332A and analyzed neurite extension and migration. Introduction of GFP-DCX-WT enhanced neurite extension and migration. These effects of DCX introduction were suppressed when we used GFP-DCX-S332A. Treatment of neurons with JNK inhibitor increased the amount of DCX that bound to tubulin. Interestingly, amount of DCX that bound to tubulin decreased in Cdk5-/- brain homogenates, which indicates that phosphorylation of DCX by JNK is critical for the regulation of DCX binding to tubulin. These results suggest the physiological importance of phosphorylation of DCX for its function.

Spinophilin Facilitates Dephosphorylation of Doublecortin by PP1 to Mediate Microtubule Bundling at the Axonal Wrist

The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. We find that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the "wrist" of growing axons, leading to activation. Loss of activity at the "wrist" is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton.

Doublecortin interacts with the ubiquitin protease DFFRX, which associates with microtubules in neuronal processes

Doublecortin (DCX) is a microtubule-associated protein involved in neuronal migration, which causes X-linked lissencephaly and subcortical laminar heterotopia (SCLH) when mutated. Here we show that DCX interacts with the ubiquitin-specific protease Drosophila fat facets related on X chromosome (DFFRX). This interaction was confirmed by targeted mutagenesis, colocalization, and immunoprecipitation studies. DFFRX is thought to deubiquitinate specific substrates including β-catenin, preventing their degradation by the proteasome. Interestingly, unlike β-catenin, no ubiquitinated forms of DCX could be detected, and indeed we show that DCX interacts with a novel recognition domain in DFFRX, located outside of its catalytic site. We also show that DFFRX associates with microtubules at specific subcellular compartments, including those enriched in DCX. These results thus suggest that in addition to vesicular trafficking, DCX may play a role in the regulation of cell adhesion via its interaction with DFFRX in migrating and differentiating neurons.

CNS malformations: gene locations of known human mutations

aMalformationInheritanceLocusSymbol: gene or transcription productKey referencesAgenesis corpus callosum with neuropathyARSLC12A6 for transporter protein KCC35Cerebro-hepato-renal syndrome (Zellweger)aARXq22.3-q23DCX6Coffin–Lowry syndromeXRXp22.2RSK27,8Congenital muscular dystrophy with cerebral and cerebellar dysplasiaARFKRP (Fukutin)9HemimegalencephalybARXq28L1-CAM10HoloprosencephalycAD; AR7q36-qterSHH11–13HoloprosencephalyAR; sporadic13q32ZIC214HoloprosencephalyAR; sporadic2q21SIX315HoloprosencephalyAD; sporadic18p11.3TGIF16HoloprosencephalyAR; sporadic9q22.3PTCH (SHH receptor)17HoloprosencephalyAR; sporadic10q11.2DKK (head inducer)18HoloprosencephalyAR; sporadicDhcr7 (SHH related)19Kallmann syndromeaXRXp22.3KAL1; EMX220–22Lissencephaly I (isolated and Miller–Dieker syndrome)AR17p13.3LIS123–25Lissencephaly II with cerebellar hypoplasia #AR7q22RELN26Lissencephaly II, muscle–eye–brain diseaseAR1p32POMGnT127Microcephaly, primaryAR1pq25-q32MCPH54,28Midbrain agenesis and cerebellar hypoplasia?AR; sporadic7q36EN229Periventricular nodular heterotopiaXDXq28FLN-A30,31Periventricular nodular heterotopiaAD??32Periventricular nodular heterotopia and posterior pituitary ectopiaARHESX133Pituitary aplasia, ectopia (neurohypophysis)ARHESX133–35Pituitary aplasia (adenohypophysis)Pitx236Rett syndromeXDXq28MECP237Sacral agenesisdAD7q36.1-qter, 1q41-q42.1SHH, HLXB938–40SchizencephalyAR10q26.1EMX241,42Septo-optic-pituitary dysplasiaAR; sporadic3p21.1-p21.2HESX1, PAX31,2,34,35,43,44Subcortical laminar heterotopia (band heterotopia; double cortex)XDXq22.3-q23DCX3,45,46,56Tuberous sclerosisAD9q34.3, 16p13.3TSC1; hamartin, TSC2; tuberin47–52X-linked hydrocephalus (X-linked aqueductal stenosis and pachygyria)XRXq28L1-CAM53–55aThe DCX (doublecortin) mutation is primary in subcortical laminar heterotopia, but also is described in Zellweger syndrome, though it is likely only a secondary defect in this lysosomal disease associated with major neuroblast migratory defects; DCX is localized on the X-chromosome and Zellweger is an AR trait. DCX also is a secondary genetic defect in Kallmann syndrome (anosmia due to agenesis or defective migration of olfactory bulb neurons and hypogonadotropic hypogonadism, the hypothalamic secretory cells having the same origin as the olfactory neurons).bThe role of L1-CAM in hemimegalencephaly is not certain and is more likely a secondary defect and not the primary genetic mutation.cHoloprosencephaly is associated with many chromosomal defects in addition to those here listed, but the gene products associated with the others have not yet been identified. Only 20% of genetically studied cases have one of the six genetic mutations demonstrated.aThe DCX (doublecortin) mutation is primary in subcortical laminar heterotopia, but also is described in Zellweger syndrome, though it is likely only a secondary defect in this lysosomal disease associated with major neuroblast migratory defects; DCX is localized on the X-chromosome and Zellweger is an AR trait. DCX also is a secondary genetic defect in Kallmann syndrome (anosmia due to agenesis or defective migration of olfactory bulb neurons and hypogonadotropic hypogonadism, the hypothalamic secretory cells having the same origin as the olfactory neurons).dSacral agenesis (AD form) maps to the same locus at 7q36 as one form of holoprosencephaly and also is associated with defective SHH expression, the same genetic defect expressed at opposite ends of the neural tube. Both sacral agenesis and holoprosencephaly also occur with a high incidence in infants born to mothers with diabetes mellitus. Agenesis of more than two vertebral bodies is generally associated with dysplasia of the spinal cord in that region during fetal development: fusion of ventral horns; deformed central canal with heterotopic ependyma, consistent with defective neural induction. A second gene with a locus at 1q41-q42.1 is also identified as another cause of autosomal dominantly transmitted sacral agenesis.

Identification of a novel human doublecortin-domain-containing gene (DCDC1) expressed mainly in testis.

Mutations in the X-linked gene doublecortex (DCX) result in lissencephaly in males or subcortical laminar heterotopia (double cortex) in females. Recently, an evolutionarily conserved doublecortin (DC) domain important for microtubule binding and microtubule polymerization was defined according to detailed sequence analysis of DCX and DCX-like proteins. Subsequently we cloned a novel human cDNA that contained a DC domain during large-scale DNA sequencing of the human fetal brain cDNA library, and termed it doublecortin-domain-containing 1 (DCDC1). According to a search against the human genome database, DCDC1 was mapped to 11p13. Expression analysis showed that DCDC1 was mainly expressed in adult testis. Furthermore, the expression level of DCDC1 in fetal brain was much higher than in adult brain.

Magnetoencephalographic studies of two cases of diffuse subcortical laminar heterotopia or so-called double cortex

Two cases (a young male and a girl, suffering intractable epilepsy) of diffuse subcortical laminar heterotopia, or so-called double cortex (DC) have been investigated using magnetoencephalography (MEG). MEG confirmed involvement of both cortices (hetero- and normocortex) in the genesis of interictal spikes, and, according to the heterogeneity of DC syndrome, some differences were observed: spike initiation in the normocortex and latter involvement of the heterotopic cortex in the man, and rather a cancellation in both cortices in the girl. In addition, participation of heterotopic cortex in physiological activities could be demonstrated in the man.

Doublecortin functions at the extremities of growing neuronal processes.

Type I lissencephaly is a cortical malformation disorder characterized by disorganized cortical layers and gyral abnormalities and associated with severe cognitive impairment and epilepsy. The exact pathophysiological mechanisms underlying the epilepsy and mental retardation in this and related disorders remain unknown. Two genes, LIS1 and doublecortin, have both been shown to be mutated in a large proportion of cases of type I lissencephaly and a milder allelic disorder, subcortical laminar heterotopia (SCLH). Studying the protein products of these genes and the biochemical pathways in which they belong is likely to yield important information concerning both normal and abnormal cortical development. The relationships between the LIS1 and Doublecortin proteins are not yet well defined, but both are believed to play a critical role in cortical neuronal migration. Lis1 is expressed from very early development in the mouse and in both proliferating cells and post-mitotic neurons of the cortex. This protein is likely to have multiple functions since it is a subunit of the enzyme platelet-activating factor acetylhydrolase, which degrades platelet activating factor, and has also been shown to be involved in microtubule dynamics, potentially influencing nuclear migration through its interaction with the dynein motor protein complex. Doublecortin on the other hand is exclusively expressed in post-mitotic neurons and is developmentally regulated. In young developing neurons Doublecortin has a specific subcellular localization at the ends of neuritic and leading processes. This localization, combined with our previous data showing that it is a microtubule-associated protein and that it interacts with adapter complexes involved in vesicle trafficking, suggests a role in the growth of neuronal processes, downstream of directional or guidance signals. The observations summarized here favor the suggestion that whereas LIS1 may play a role in nuclear migration, Doublecortin is instead restricted to functions at the leading edge of the cell.

Molecular pathology of human cerebral malformations.

Molecular pathogenesis of human cerebral malformations is briefly reviewed from a neuropathologic viewpoint, with emphasis on holoprosencephaly and neuronal migration disorders. Immunopathologic approaches are useful in elucidating the essential pathomechanism of these anomalies. In alobar holoprosencephaly, for instance, immunostaining for glial fibrillary acidic protein clarifies the pathologic significance of the leptomeningeal glioneuronal heterotopia along the ventral prosencephalic surface. In type 1 lissencephaly and subcortical laminar heterotopia, immunohistochemistry for the causative gene products revealed the temporal and spatial pattern of their localization in the normally developing cerebrum, as well as their reduction in these disorders.

Molecular pathology of human cerebral malformations

Molecular pathogenesis of human cerebral malformations is briefly reviewed from a neuropathologic viewpoint, with emphasis on holoprosencephaly and neuronal migration disorders. Immunopathologic approaches are useful in elucidating the essential pathomechanism of these anomalies. In alobar holoprosencephaly, for instance, immunostaining for glial fibrillary acidic protein clarifies the pathologic significance of the leptomeningeal glioneuronal heterotopia along the ventral prosencephalic surface. In type 1 lissencephaly and subcortical laminar heterotopia, immunohistochemistry for the causative gene products revealed the temporal and spatial pattern of their localization in the normally developing cerebrum, as well as their reduction in these disorders.

Neuroimaging characteristics of pseudosubcortical laminar heterotopia.

Subcortical laminar heterotopia (SLH) is a subtype of malformation of cortical development characterized by laminar gray matter between the cortex and ventricles, which can vary in thickness and may be continuous or discontinuous. The objective of this study is to describe a normal finding of high-resolution magnetic resonance imaging that may simulate an SLH. SLH is isointense to cortex on both T1- and T2-weighted/FLAIR images, usually both anteriorly and posteriorly in location. Conversely, pseudo-SLH is a normal variant present only at the posterior aspect of the brain, and with dark signal on both T1- and T2-weighted/FLAIR images.

Magneto-encephalographic features of two cases of diffuse subcortical laminar heterotopia: a study of epileptic spikes generation and somatosensory evoked activities organisation

Magneto-encephalographic features of two cases of diffuse subcortical laminar heterotopia: a study of epileptic spikes generation and somatosensory evoked activities organisation

Genetic alteration of the DCX gene in Japanese patients with subcortical laminar heterotopia or isolated lissencephaly sequence

We examined mutations of the doublecortin (DCX) gene, which is responsible for X-linked subcortical laminar heterotopia (SCLH) and lissencephaly, in eight unrelated Japanese patients, four with SCLH and four with isolated lissencephaly sequence (ILS). Polymerase chain reaction (PCR) disclosed a deletion of part of the DCX gene in one male ILS patient. Single-strand conformational polymorphism analysis and subsequent sequence analysis were carried out in the remaining seven patients. One male ILS patient had a nonsense mutation in exon V, which would result in premature termination of the gene product. One female SCLH patient had a missense mutation in exon IV. Our results indicate that in the Japanese, as has been seen elsewhere, abnormality of the DCX gene is the common cause of SCLH and ILS.

Colocalization of doublecortin with the microtubules: An ex vivo colocalization study of mutant doublecortin

Doublecortin (DCX) plays an important role in neuronal migration and development, and the participation of DCX in neuronal migration has been demonstrated by intensive mutational analysis for patients with X-linked or sporadic lissencephaly, and/or subcortical laminar heterotopia. Although a previous search for protein similarity showed that DCX has a region homologous to the putative Ca(2+)/calmodulin-dependent protein kinase, the function of the DCX gene (DCX) has remained unknown. We show here that mouse DCX colocalizes with the microtubules and provide evidence that its conformational structure is important for its subcellular localization by means of mutant doublecortin expression study. The results of our study may suggest that the cytoskeleton involving DCX mediates the neuronal migration during brain development.

Doublecortin mutations cluster in evolutionarily conserved functional domains.

Mutations in the X-linked gene doublecortin ( DCX ) result in lissencephaly in males or subcortical laminar heterotopia ('double cortex') in females. Various types of mutation were identified and the sequence differences included nonsense, splice site and missense mutations throughout the gene. Recently, we and others have demonstrated that DCX interacts and stabilizes microtubules. Here, we performed a detailed sequence analysis of DCX and DCX-like proteins from various organisms and defined an evolutionarily conserved Doublecortin (DC) domain. The domain typically appears in the N-terminus of proteins and consists of two tandemly repeated 80 amino acid regions. In the large majority of patients, missense mutations in DCX fall within the conserved regions. We hypothesized that these repeats may be important for microtubule binding. We expressed DCX or DCLK (KIAA0369) repeats in vitro and in vivo. Our results suggest that the first repeat binds tubulin but not microtubules and enhances microtubule polymerization. To study the functional consequences of DCX mutations, we overexpressed seven of the reported mutations in COS7 cells and examined their effect on the microtubule cytoskeleton. The results demonstrate that some of the mutations disrupt microtubules. The most severe effect was observed with a tyrosine to histidine mutation at amino acid 125 (Y125H). Produced as a recombinant protein, this mutation disrupts microtubules in vitro at high molar concentration. The positions of the different mutations are discussed according to the evolutionarily defined DC-repeat motif. The results from this study emphasize the importance of DCX-microtubule interaction during normal and abnormal brain development.