DCX Gene Research Articles

Phenotypic Variability in Novel Doublecortin Gene Variants Associated with Subcortical Band Heterotopia.

Doublecortin, encoded by the DCX gene, plays a crucial role in the neuronal migration process during brain development. Pathogenic variants of the DCX gene are the major causes of the "lissencephaly (LIS) spectrum", which comprehends a milder phenotype like Subcortical Band Heterotopia (SBH) in heterozygous female subjects. We performed targeted sequencing in three unrelated female cases with SBH. We identified three DCX-related variants: a novel missense (c.601A>G: p.Lys201Glu), a novel nonsense (c.210C>G: p.Tyr70*), and a previously identified nonsense (c.907C>T: p.Arg303*) variant. The novel c.601A>G: p.Lys201Glu variant shows a mother-daughter transmission pattern across four generations. The proband exhibits focal epilepsy and achieved seizure freedom with a combination of oxcarbazepine and levetiracetam. All other affected members have no history of epileptic seizures. Brain MRIs of the affected members shows predominant fronto-central SBH with mixed pachygyria on the overlying cortex. The two nonsense variants were identified in two unrelated probands with SBH, severe drug-resistant epilepsy and intellectual disability. These novel DCX variants further expand the genotypic-phenotypic correlations of lissencephaly spectrum disorders. Our documented phenotypic descriptions of three unrelated families provide valuable insights and stimulate further discussions on DCX-SBH cases.

Lissencephaly, Pachygyrias, Band Heterotopias, RELN Pathway, and ARX Mutations (Incomplete Neuron Migration)

AbstractLissencephaly (LIS) is a group of malformations of cortical development consisting of a defective neuronal migration that results in lack of formation of the normal cerebral convolutions. It includes a spectrum of defect with varying degrees of severity, from agyria and pachygyria to subcortical band heterotopia. The etiopathogenesis of LIS includes both genetic and environmental factors. Although nongenetic forms of LIS have been reported, genetic causes are certainly more frequent and to date 19 LIS-SBH-associated genes have been identified. Most common mutations involve LIS1, DCX, ARX, and RELN genes. Clinically affected individuals present with early hypotonia, which can progress to limb spasticity, seizures, and psychomotor retardation. Convulsive episodes usually appear early (first months of life) and include infantile spasms, akinetic or myoclonic seizures, up to the development of complex epileptic syndromes, including atypical absences, myoclonia, and partial or tonic–clonic seizures. Several clinical entities are associated with classical LIS, including the following: isolated lissencephaly sequence (ILS); Miller–Dieker syndrome (MDS; OMIM 247200); subcortical band heterotopia (OMIM 300067); X-linked LIS with abnormal genitalia; and LIS with cerebellar hypoplasia. Diagnosis primarily depends on genetic and neuroimaging. Magnetic resonance imaging (MRI) is the gold standard, and it detects the presence of thick cortical cortex, its location, and the layers' architecture. Based on neuroimaging, it is possible to distinguish six subtypes of gyral malformations. Clinical and therapeutic management of these patients is challenging, considering the necessity to face drug-resistant epilepsy, intellectual disability, spasticity, and dysphagia and feeding problems. At the present moment, no gene-specific treatment for LIS is available.

Generation of an induced pluripotent stem cell line (SDQLCHi067-A) from a patient with subcortical band heterotopia harboring a heterozygous mutation in DCX gene

Subcortical band heterotopia (SHB) is a rare severe brain developmental malformation caused by deficient neuronal migration during the development of cerebral cortex. Here, a human induced pluripotent stem cell (iPSCs) line was established from a 4-year-1-month-old girl with SHB carrying a heterozygous mutation (c.568A > G, p.K190E) in DCX. The generated iPSC line showed the ability to differentiate into three lineages in vitro and was confirmed by pluripotency markers and the original gene mutation.

Hypothalamic molecular correlates of photoperiod-induced spring migration in intact and castrated male redheaded buntings

This study investigated the molecular changes associated with neural plasticity in photoperiodic induction of spring migration in intact and castrated redheaded bunting, Emberiza bruniceps. We measured the hypothalamic mRNA expression of genes in birds that were photostimulated into winter non-migratory and spring (vernal) migratory phenotypes under short and long photoperiods, respectively. These included genes associated with the appetitive phase of reproduction (spring migration drive, th and ddc genes encoding for tyrosine hydroxylase and dopamine decarboxylase enzymes, respectively), sleep/awake state (pmch gene encoding for pro-melanin concentrating hormone; hcrt and hcrtr2 encoding for the hypocretin/orexin and its receptor, respectively) and neurogenesis (dcx and neuN coding for doublecortin and neuronal nuclear proteins, respectively). Higher th mRNA levels suggested an upregulated dopamine synthesis in the hypothalamus of spring migrants. Similarly, elevated hcrt and hcrtr2 mRNA levels suggested an increased wakefulness, and those of dcx and neuN genes suggested an enhanced neurogenesis during the spring migration state. Further, compared to intact birds, the lower th and pmch, and higher hcrtr2 and neuN mRNA levels in castrates suggested a role of testicular steroids in modulation of the appetitive phase of reproduction, sleep and awake states, and neurogenesis during spring migration period. These results provide insights into molecular changes linked with important hypothalamic molecular pathways and steroidal influence in the photoperiodic induction of spring migration in obligate migratory songbirds.

Effect of Haloperidol and Olanzapine on Hippocampal Cells' Proliferation in Animal Model of Schizophrenia.

Aberrant neurogenesis in the subventricular zone (SVZ) and hippocampus (HIP) contributes to schizophrenia pathogenesis. Haloperidol (HAL) and olanzapine (OLA), commonly prescribed antipsychotics for schizophrenia treatment, affect neurogenesis too. The effect of HAL and OLA on an mHippoE-2 cell line was studied in vitro where we measured the cell number and projection length. In vivo, we studied the gene expression of DCX, Sox2, BDNF, and NeuN in the SVZ and HIP in an MK-801-induced animal schizophrenia model. Cells were incubated with HAL, OLA, and MK-801 for 24, 48, and 72 h. Animals were injected for 6 days with saline or MK801 (0.5 mg/kg), and from the 7th day with either vehicle HAL (1 mg/kg) or OLA (2 mg/kg), for the next 7 days. In vitro, HAL and OLA dose/time-dependently suppressed cells’ proliferation and shortened their projection length. HAL/OLA co-treatment with MK-801 for 24 h reversed HAL’s/OLA’s inhibitory effect. In vivo, HAL and OLA suppressed DCX and NeuN genes’ expression in the HIP and SVZ. MK-801 decreased DCX and NeuN genes’ expression in the HIP and OLA prevented this effect. The data suggest that subchronic HAL/OLA treatment can inhibit DCX and NeuN expression. In an MK-801 schizophrenia model, OLA reversed the MK-801 inhibitory effect on DCX and NeuN and HAL reversed the effect on DCX expression; however, only in the HIP.

Novel DCX pathogenic variant in a girl with subcortical band heterotopia

Abstract Subcortical band heterotopia (SBH), is a brain malformation defined by symmetrical and bilateral heterotopic gray matter bands localized deep within the white matter, between the cortex and lateral ventricles. SBH is the result of abnormal neuronal migration, with improper positioning of the cortical neurons. DCX gene (doublecortin), a microtubule-associated protein with essential roles in neuronal migration and differentiation during brain development, is one of the main contributors to the X-linked Lissencephaly spectrum pathogenesis (OMIM #300067). DCX variants are responsible for SBH in females and isolated lissencephaly in males. Herein, we present a 7-year-old girl with a de novo frameshift variant in DCX gene, unreported by date. The patient has focal complex seizures with onset at 23 months of age, fully controlled with medication, mild tremor and coordination impairment of fine movements and some learning difficulties, otherwise with normal development. The brain magnetic resonance imaging revealed the presence of thick SBH. Direct sequencing of DCX gene revealed a pathogenic heterozygous cytosine duplication in exon 3; this frameshift variant leads to a premature stop codon in position 164 (p.Gln160Profs*5). The variant type and its predicted consequence at protein level correlates with the severity of radiological findings. The clinical presentation of our patient is, however, milder than expected. Our research expands the mutational spectrum of DCX gene in SBH females and provides a detailed clinical and imagistic description of the patient. This paper highlights the utility of single gene sequencing as a first-tier diagnostic test of patients with gene-specific phenotypic features.

Expression of neuroblastoma-related genes in bone marrow at end of high-risk neuroblastoma therapy.

Minimal disease quantification may predict event-free survival (EFS) and overall survival (OS). We evaluated mRNA expression of five neuroblastoma-associated genes (NB5 assay) in bone marrows (BM) of patients with newly diagnosed high-risk neuroblastoma who received consistent immunotherapy. mRNA expression of CHGA, DCX, DDC, PHOX2B, and TH genes in BM of 479 patients enrolled on the immunotherapy arm of Children's Oncology Group trials ANBL0032 and ANBL0931 was evaluated using real-time polymerase chain reaction (PCR)-based TaqMan low-density array. Results from end-consolidation and end-therapy were analyzed for association with five-year EFS/OS and patient and tumor characteristics. Tests of statistical significance were two-sided. NB5 assay detected neuroblastoma-related mRNA in 222 of 286 (77.6%) of BMs obtained at end-consolidation and 188 of 304 (61.8%) at end-therapy. Any mRNA level detected in end-therapy BM correlated with significantly worse EFS (57% [49.6%-63.7%] vs 73.0% [63.5%-80.4%]; P=0.005), but not OS. Analysis limited to patients in complete response at end-therapy still found a significant difference in EFS with detectable versus not detectable NB5 assay results (58.9% [49.5%-67.1%] vs 76.6% [66.1%-84.2%]; P=0.01). End-consolidation results did not correlate with EFS or OS. Multivariable analysis determined end-therapy NB5 assay BM results (P=0.02), age at diagnosis (P=0.002), and preconsolidation response (P=0.02) were significantly associated with EFS independent of other clinical and biological parameters evaluated, including end-therapy response. If further validated in additional patient cohorts, the NB5 assay's ability to independently predict EFS from end-therapy could improve patient stratification for novel maintenance therapy trials after current end-therapy to improve outcome.

Genotype-phenotype correlations for the type 1 lissencephaly of eight Tunisian children

Background: Lissencephaly represents a rare subgroup of genetically distinct neurological disorders of neuronal migration characterized by a paucity or absence of cerebral gyration. The most common form of lissencephaly has been isolated and referred to as classic or type 1 lissencephaly. It is frequently related to abnormalities within LIS1 or DCX genes, with abnormalities ranging from single base pair substitutions to contiguous gene deletions. Methods: In this study, we report, for the first time, a clinical and genetic characterization of eight unrelated Tunisian children presenting type 1 lissencephaly. We screened LIS1 and DCX abnormalities thanks to a combination of molecular cytogenetic methods and next generation sequencing. Results: One deletion of DCX and three deletions of LIS1 were observed. One of these deletions was inherited from a maternal reciprocal translocation and estimated to approximately 2,9 Mb length. In addition, a 26 Kb LIS1 deletion was detected and refined between exon 3 up to exon 11 by target capture and sequencing. The last LIS1 rearrangement was a mutation (c.779T>A, p.V260E). Finally, two novel DCX mutations were found out (c.910G>C, p.G304R/c.436T>C, p.F146L). Conclusions: Our data confirm the individuality and originality of type 1 lissencephaly on both the phenotypic and the genetic levels. Furthermore, our data confirm again that LIS1 and DCX are the most genes associated with type 1 lissencephaly and spotlight the usefulness of developing approaches and methods for detecting a large number of known causative gene mutations.

Dcx expression defines a subpopulation of Gdf5+ cells with chondrogenic potentials in E12.5 mouse embryonic limbs

Growth differentiation factor 5 (Gdf5) and doublecortin (Dcx) genes are both expressed in joint interzone cells during synovial joint development. In this study, we re-analyzed the single cell RNA-sequencing data (Gene Expression Omnibus GSE151985) generated from Gdf5+ cells of mouse knee joints at embryonic stages of E12.5, E13.5, E14.5, and E15.5, with a new focus on Dcx. We found that Dcx expression was enriched in clusters of Gdf5+ cells, with high expression levels of pro-chondrogenic genes including sex determining region Y-box transcription factor 5 (Sox5), Sox6, Sox9, Gdf5, versican, matrilin 4, collagen type II α 1 chain (Col2a1), Col9a1, Col9a2, and Col9a3 at E12.5. Dcx+ and Dcx- cells had differential gene expression profiles. The up-regulated genes in Dcx+ vs. Dcx- cells at E12.5 and E13.5 were enriched in chondrocyte differentiation and cartilage development, whereas those genes up-regulated at E14.5 and E15.5 were enriched in RNA splicing, protein stability, cell proliferation, and cell growth. Gene expression profiles in Dcx+ cells showed rapid daily changes from E12.5 to E15.5, with limited number of genes shared across the time period. Expression of Gdf5, Sox5, Sox6, melanoma inhibitory activity, noggin, odd-skipped related transcription factor 2, matrilin 4, and versican was positively correlated with Dcx expression. Our results demonstrate that Dcx expression defines a subpopulation of Gdf5+ cells with chondrogenic potentials in E12.5 mouse embryonic limbs.

Subcortical Band Heterotopia Presented With Refractory Epilepsy and Reversible Aphasia

Subcortical band heterotopia (SBH) is a rare neurodevelopmental disorder due to mutation in the DCX or LIS1 gene. It is predominantly a disease of females. Its presentation varied widely, ranging from mild epilepsy and mental retardation to refractory epilepsy and severe mental retardation. Here, a case of a 22-year-old lady with refractory seizure is reported. She also had expressive aphasia which had reversed after adjustment of the anti-epileptic drugs and control of the seizure. Her MRI of the brain revealed a band of complete gray matter deep to the pachygyric cortex and an electroencephalogram (EEG) revealed bi-frontal slow waves.

Clinical and genetic characteristics of type 3 lissencephaly caused by a mutation in the TUBA1A gene (OMIM: 611603)

Background. Lissencephaly (LIS) is a spectrum of malformations of the cerebral cortex that occur as a result of impaired migration of neuronal precursors to the cortical plate and the formation of furrows and convolutions in the post‑migration period of embryonic development. In recent years, a significant role of hereditary factors in the occurrence of LIS has been shown due to the improvement of methods of molecular genetic diagnostics. Today, 13 genetic variants have been identified in the LIS group, six of which are inherited autosomal recessively, five are autosomal dominant, and two are linked to the X chromosome. It is shown that 80 % of cases of hereditary LIS is caused by mutations in two genes: PAFAH1B1 , which is responsible for the occurrence of the LIS 1 type with an au‑ tosomal dominant type of inheritance, and in the DCX gene, localized on the X chromosome. The rest of the genetic variants account for from 1 % to 5 % of cases of defects accompanied by dysgenesis of the cerebral cortex. In recent years, the number of works devoted to the analysis of clinical and genetic characteristics of monogenic variants of LIS has increased. The results of such studies will allow us to improve our understanding not only of the pathogenetic mechanisms of this group of diseases, but also of the molecular basis for the formation of brain structures in the normal embryonic period. Objective: to describe the clinical and genetic characteristics of three Russian patients with autosomal dominant LIS type 3 (OMIM: 611603) caused by mutations in the TUBA1A gene. Materials and methods. All patients were under observation in the consultative and diagnostic department of Research Centre for Medical Genetics. The diagnosis was established on the basis of clinical data, genealogical anamnesis, results of brain MRI, EEG night video monitoring and exome sequencing by NGS. The validation of the identified nucleotide substitutions and analysis of the disease segregation were performed using the Sanger direct automatic sequencing method. Results. The clinical and genetic characteristics of three patients with newly identified and previously described mutation in the TUBA1A gene were analyzed. Possible effects of new missense mutations in the gene on the function of the protein product of the gene are discussed. Conclusions. The results of the analysis of the clinical and genetic characteristics of the patients we observed contribute to the study of the polymorphism of clinical manifestations resulting from mutations in the TUBA1A gene. The previously stated assumption about a wide range of malformations of the brain in patients with mutations in this gene was confirmed, which should be taken into account when making a diagnosis.

In vitro investigation of growth factors including MGF and IGF-1 in neural stem cell activation, proliferation, and migration

Neurogenesis is mainly activated after damage in adult tissues. This destruction activates the neural stem cells (NSCs) by exiting from a quiescent state and initiating proliferation, differentiation, and migration towards the damaged area. Although studies have investigated to clarify the process of NSC biology and neurogenesis, there are still significant artifacts in understanding the primary mechanism. It is known that only a small percentage of NSC become neurons and integrate into the brain tissue after this process. The significant proportion differentiates to become either astrocytes or oligodendrocytes. Furthermore, the quiescent stem cells in the niche are mainly activated by the stimuli affect. In recent years, many studies have been conducted with varying hormones, some of which might provide neuro-stimulation effect and/or involved in the regeneration of the brain tissue and/or neuroprotection from traumatic or ischemic pathologies, including Insulin-like growth factor 1 (IGF-1), Mechano Growth Factor (MGF), Basic Fibroblast Growth Factor (FGF-2), Erythropoietin (EPO), Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF).In this study, we examined the effects of FGF-2, MGF, IGF-1, EPO, EGF, NGF, and BDNF alone or with various combinations on rat hippocampal NSC by tracking the changes in the expression of Nestin, GFAP, TUBB3, and DCX genes during 24 h (h), 72 h and 168 h time frame. The apoptosis analysis revealed that FGF-2 and FGF-2 coupled growth factors effectively protect NSCs against apoptosis, whereas MGF coupled growth factors failed in this protection. The cell cycle analysis demonstrated that these growth factors had accumulated the NSCs exit from the quiescent phase to the Mitosis phase, mostly without being long in the Synthesis Phase. Neurosphere sizes were increased with MGF, signifying MGF being effective in neural progenitor cells. The combined use of MGF with FGF-2 was more effective in postmitotic neurons than MGF alone.We have comparatively demonstrated the effect of cytokines alone and combined administration on activation, proliferation, and migration of NSCs. Although many issues are still waiting to be investigated in adult neurogenesis, neural regeneration, and adult neural stem cell biology, the results provide vital resources to the researchers that are interested in the varying effect of growth factor on NSC.

Long-term treadmill exercise upregulated hippocampal learning-related genes without improving cognitive behaviour in socially isolated rats.

Some environment enrichments such as exercise has been reported to improve the diminished cognitive functions and related gene expression. Therefore, we aimed to investigate the effects of prolonged treadmill exercise on long-term learning and hippocampal gene expression, which involves learning and plasticity. Male Wistar rats (n = 32) randomly assigned into four groups: control (C), social isolation (SI), exercised (E), social isolation + exercise (SE) during postnatal days (PNDs) 21-34. Social isolation protocol was applied during 14 days by placing the rat alone in a cage. Rats were exercised daily, 5 days per week, for overall 4 weeks. Finally, learning performance was evaluated by the novel object recognition test. At the end of learning test, the rats were decapitated to isolate hippocampus tissues for learning related gene expression such as N-methyl-d-aspartate receptor (NMDAR) subunit genes (Grin1, Grin2a, Grin2b) and cyclin dependent kinase 5 (Cdk5), Cdk5 regulatory subunit p35 (Cdk5r), activity-regulated, cytoskeletal-associated protein (Arc), the immediate early gene (c-Fos, a marker of neuronal activation), doublecortin (DCX), achaetescute homolog 1 (ASCL1), brain-derived neurotrophic factor (BDNF) by real-time polymerase chain reaction (RT-PCR). Grin1, NMDAR subunit gene expression was increased significantly in E group compared to other groups. Grin2b, NMDAR subunit gene expression was increased in E group compared to the SI group. Cdk5 level increased in E group compared to the SE group. The ASCL1 gene expression increased in E group compare to the SE group. The DCX gene expression increasing in C group compared to SI and SE groups. Taken together these findings may point out that long-term social isolation down-regulated learning-related genes. However, treadmill exercise together with social isolation did not restore this down-regulation although treadmill exercise increased learning-related genes without improving cognitive behaviour.

Analysis of 17 genes detects mutations in 81% of 811 patients with lissencephaly

PurposeTo estimate diagnostic yield and genotype-phenotype correlations in a cohort of 811 patients with lissencephaly or subcortical band heterotopia. MethodsWe collected DNA from 756 children with lissencephaly over 30 years. Many were tested for deletion 17p13.3 and mutations of LIS1, DCX, and ARX, but few other genes. Among those tested, 216 remained unsolved and were tested by a targeted panel of 17 genes (ACTB, ACTG1, ARX, CRADD, DCX, LIS1, TUBA1A, TUBA8, TUBB2B, TUBB, TUBB3, TUBG1, KIF2A, KIF5C, DYNC1H1, RELN, and VLDLR) or by whole-exome sequencing. Fifty-five patients studied at another institution were added as a validation cohort. ResultsThe overall mutation frequency in the entire cohort was 81%. LIS1 accounted for 40% of patients, followed by DCX (23%), TUBA1A (5%), and DYNC1H1 (3%). Other genes accounted for 1% or less of patients. Nineteen percent remained unsolved, which suggests that several additional genes remain to be discovered. The majority of unsolved patients had posterior pachygyria, subcortical band heterotopia, or mild frontal pachygyria. ConclusionThe brain-imaging pattern correlates with mutations in single lissencephaly-associated genes, as well as in biological pathways. We propose the first LIS classification system based on the underlying molecular mechanisms.

Bezafibrate Upregulates Mitochondrial Biogenesis and Influence Neural Differentiation of Human-Induced Pluripotent Stem Cells

Bezafibrate (BZ) regulates mitochondrial biogenesis by activation of PPAR’s receptors and enhancing the level of PGC-1α coactivator. In this report, we investigated the effect of BZ on the expression of genes (1) that are linked to different pathways involved in mitochondrial biogenesis, e.g., regulated by PPAR’s receptors or PGC-1α coactivator, and (2) involved in neuronal or astroglial fate, during neural differentiation of hiPSC. The tested cell populations included hiPSC-derived neural stem cells (NSC), early neural progenitors (eNP), and neural progenitors (NP). RNA-seq analysis showed the expression of PPARA, PPARD receptors and excluded PPARG in all tested populations. The expression of PPARGC1A encoding PGC-1α was dependent on the stage of differentiation: NSC, eNP, and NP differed significantly as compared to hiPSC. In addition, BZ-evoked upregulation of PPARGC1A, GFAP, S100B, and DCX genes coexist with downregulation of MAP2 gene only at the eNP stage of differentiation. In the second task, we investigated the cell sensitivity and mitochondrial biogenesis upon BZ treatment. BZ influenced the cell viability, ROS level, mitochondrial membrane potential, and total cell number in concentration- and stage of differentiation-dependent manner. Induction of mitochondrial biogenesis evoked by BZ determined by the changes in the level of SDHA and COX-1 protein, and mtDNA copy number, as well as the expression of NRF1, PPARGC1A, and TFAM genes, was detected only at NP stage for all tested markers. Thus, developmental stage-specific sensitivity to BZ of neurally differentiating hiPSC can be linked to mitochondrial biogenesis, while fate commitment decisions to PGC-1α (encoded by PPARGC1A) pathway.

Germline and somatic mutations in cortical malformations: Molecular defects in Argentinean patients with neuronal migration disorders.

Neuronal migration disorders are a clinically and genetically heterogeneous group of malformations of cortical development, frequently responsible for severe disability. Despite the increasing knowledge of the molecular mechanisms underlying this group of diseases, their genetic diagnosis remains unattainable in a high proportion of cases. Here, we present the results of 38 patients with lissencephaly, periventricular heterotopia and subcortical band heterotopia from Argentina. We performed Sanger and Next Generation Sequencing (NGS) of DCX, FLNA and ARX and searched for copy number variations by MLPA in PAFAH1B1, DCX, POMT1, and POMGNT1. Additionally, somatic mosaicism at 5% or higher was investigated by means of targeted high coverage NGS of DCX, ARX, and PAFAH1B1. Our approach had a diagnostic yield of 36%. Pathogenic or likely pathogenic variants were identified in 14 patients, including 10 germline (five novel) and 4 somatic mutations in FLNA, DCX, ARX and PAFAH1B1 genes. This study represents the largest series of patients comprehensively characterized in our population. Our findings reinforce the importance of somatic mutations in the pathophysiology and diagnosis of neuronal migration disorders and contribute to expand their phenotype-genotype correlations.

Novel Mutation chrX:110644366 C>A of the DCX Gene in 4-year-old Girl with Sporadic Double Cortex Syndrome

Novel Mutation chrX:110644366 C>A of the DCX Gene in 4-year-old Girl with Sporadic Double Cortex Syndrome

P.2.a.018 - The effects of ketamine on behaviour, plasma corticosterone and neurogenesis in socially isolated rats

To study the genetics and functional alteration of a family with X-linked lissencephaly and subcortical band heterotopia.Five affected patients (one male with lissencephaly, four female with subcortical band heterotopia) and their relatives were studied. Sanger sequencing of DCX gene, allele specific PCR and molecular inversion probe technique were performed. Mutant and wild type of the gene products, namely doublecortin, were expressed in cells followed by immunostaining to explore the localization of doublecortin and microtubules in cells. In vitro microtubule-binding protein spin-down assay was performed to quantify the binding ability of doublecortin to microtubules.We identified a novel DCX mutation c.785A > G, p.Asp262Gly that segregated with the affected members of the family. Allele specific PCR and molecular inversion probe technique demonstrated that the asymptomatic female carrier had an 8% mutant allele fraction in DNA derived from peripheral leukocytes. This mother had 7 children, 4 of whom were affected and all four affected siblings carried the mutation. Functional study showed that the mutant doublecortin protein had a significant reduction of its ability to bind microtubules.Low level mosaicism could be a cause of inherited risk from asymptomatic parents for DCX related lissencephaly-subcortical band heterotopia spectrum. This is particularly important in terms of genetic counselling for recurrent risk of future pregnancies. The reduced binding affinity of mutant doublecortin may contribute to developmental malformation of the cerebral cortex.

The use of proteomic analysis to study trafficking defects in axons.

Mutations in microtubule subunits and microtubule-associated proteins are the causes of many neurological disorders. These human conditions are usually associated with axonal tract defects or degeneration. The molecular mechanisms of these axonal dysfunction are still largely unknown. Conventional methods may not yield a complete analysis of downstream molecules related to axonal dysfunctions. Therefore, we devised a simple unbiased method to screen molecular motors and axonal molecules, which might be involved in axonal defects. We performed our analysis in the mouse with a targeted deletion in the doublecortin (Dcx) gene. Dcx is a microtubule-associated protein with direct effects on microtubule motors. Furthermore, the knockout of Dcx and its functionally redundant structurally similar paralog, doublecortin-like kinase 1 (Dclk1), in mouse results in thinner or absent axon tracts, including the corpus callosum and anterior commissures. We compared protein profiles of corpus callosum from Dcx knockout and wild-type mouse of P0-P2 using mass spectrometry. This strategy allowed us to identify novel candidates downstream of Dcx involved in axon transport.

Doublecortin marks a new population of transiently amplifying muscle progenitor cells and is required for myofiber maturation during skeletal muscle regeneration.

Muscle satellite cells are indispensable for muscle regeneration, but the functional diversity of their daughter cells is unknown. Here, we show that many Pax7(+)MyoD(-) cells locate both beneath and outside the basal lamina during myofiber maturation. A large majority of these Pax7(+)MyoD(-) cells are not self-renewed satellite cells, but have different potentials for both proliferation and differentiation from Pax7(+)MyoD(+) myoblasts (classical daughter cells), and are specifically marked by expression of the doublecortin (Dcx) gene. Transplantation and lineage-tracing experiments demonstrated that Dcx-expressing cells originate from quiescent satellite cells and that the microenvironment induces Dcx in myoblasts. Expression of Dcx seems to be necessary for myofiber maturation because Dcx-deficient mice exhibited impaired myofiber maturation resulting from a decrease in the number of myonuclei. Furthermore, in vitro and in vivo studies suggest that one function of Dcx in myogenic cells is acceleration of cell motility. These results indicate that Dcx is a new marker for the Pax7(+)MyoD(-) subpopulation, which contributes to myofiber maturation during muscle regeneration.