Eriko F. Jimbo

Circadian-relevant genes are highly polymorphic in autism spectrum disorder patients.

BACKGROUND The genetic background of autism spectrum disorder (ASD) is considered a multi-genetic disorder with high heritability. Autistic children present with a higher prevalence of sleep disorders than has been observed in children with normal development. Some circadian-relevant genes have been associated with ASD (e.g., PER1, PER2, NPAS2, MTNR1A, and MTNR1B). METHODS We analyzed 28 ASD patients (14 with sleep disorders and 14 without) and 23 control subjects of Japanese descent. The coding regions of 18 canonical clock genes and clock-controlled genes were sequenced. Detected mutations were verified by direct sequencing analysis, and additional control individuals were screened. RESULTS Thirty-six base changes with amino acid changes were detected in 11 genes. Six missense changes were detected only in individuals with ASD with sleep disturbance: p.F498S in TIMELESS, p.S20R in NR1D1, p.R493C in PER3, p.H542R in CLOCK, p.L473S in ARNTL2, and p.A325V in MTNR1B. Six missense changes were detected only in individuals with ASD without sleep disturbance: p.S1241N in PER1, p.A325T in TIMELESS, p.S13T in ARNTL, p.G24E in MTNR1B, p.G24E in PER2, and p.T1177A in PER3. The p.R493C mutation in PER3 was detected in both groups. One missense change, p.P932L in PER2, was detected only in the control group. Mutations in NR1D1, CLOCK, and ARNTL2 were detected only in individuals with ASD with sleep disorder. The prevalence of the mutations detected only single time differed significantly among all ASD patients and controls (p=0.003). Two kinds of mutations detected only in individuals with ASD with sleep disorder, p.F498S in TIMELESS and p.R366Q in PER3, were considered to affect gene function by three different methods: PolyPhen-2, scale-invariant feature transform (SIFT) prediction, and Mutation Taster (www.mutationtaster.org). The mutations p.S20R in NR1D1, p.H542R in CLOCK, p.L473S in ARNTL2, p.A325T in TIMELESS, p.S13T in ARNTL, and p.G24E in PER2 were diagnosed to negatively affect gene function by more than one of these methods. CONCLUSION Mutations in circadian-relevant genes affecting gene function are more frequent in patients with ASD than in controls. Circadian-relevant genes may be involved in the psychopathology of ASD.

MAOA/B deletion syndrome in male siblings with severe developmental delay and sudden loss of muscle tonus

Deletion of the monoamine oxidase (MAO)-A and MAO-B was detected in two male siblings and in their mother. The approximately 800-kb deletion, extending from about 43.0MB to 43.8MB, was detected by array comparative genomic hybridization analysis. The MAOA and MAOB genes were included in the deletion, but the adjacent Norrie disease gene, NDP, was not deleted. The boys had short stature, hypotonia, severe developmental delays, episodes of sudden loss of muscle tone, exiting behavior, lip-smacking and autistic features. The serotonin levels in their cerebrospinal fluid were extremely elevated. Another set of siblings with this deletion was reported previously. We propose recognition of MAOA/B deletion syndrome as a distinct disorder.

Role of an adaptor protein Lin-7B in brain development: possible involvement in autism spectrum disorders

Using comparative genomic hybridization analysis for an autism spectrum disorder (ASD) patient, a 73‐Kb duplication at 19q13.33 (nt. 49 562 755–49 635 956) including LIN7B and 5 other genes was detected. We then identified a novel frameshift mutation in LIN7B in another ASD patient. Since LIN7B encodes a scaffold protein essential for neuronal function, we analyzed the role of Lin‐7B in the development of cerebral cortex. Acute knockdown of Lin‐7B with in utero electroporation caused a delay in neuronal migration during corticogenesis. When Lin‐7B was knocked down in cortical neurons in one hemisphere, their axons failed to extend efficiently into the contralateral hemisphere after leaving the corpus callosum. Meanwhile, enhanced expression of Lin‐7B had no effects on both cortical neuron migration and axon growth. Notably, silencing of Lin‐7B did not affect the proliferation of neuronal progenitors and stem cells. Taken together, Lin‐7B was found to play a pivotal role in corticogenesis through the regulation of excitatory neuron migration and interhemispheric axon growth, while further analyses are required to directly link functional defects of Lin‐7B to ASD pathophysiology.

LIN7A Depletion Disrupts Cerebral Cortex Development, Contributing to Intellectual Disability in 12q21-Deletion Syndrome

Interstitial deletion of 12q21 has been reported in four cases, which share several common clinical features, including intellectual disability (ID), low-set ears, and minor cardiac abnormalities. Comparative genomic hybridization (CGH) analysis using the Agilent Human Genome CGH 180K array was performed with the genomic DNA from a two-year-old Japanese boy with these symptoms, as well as hypoplasia of the corpus callosum. Consequently, a 14 Mb deletion at 12q21.2-q21.33 (nt. 77 203 574–91 264 613 bp), which includes 72 genes, was detected. Of these, we focused on LIN7A, which encodes a scaffold protein that is important for synaptic function, as a possible responsible gene for ID, and we analyzed its role in cerebral cortex development. Western blotting analyses revealed that Lin-7A is expressed on embryonic day (E) 13.5, and gradually increases in the mouse brain during the embryonic stage. Biochemical fractionation resulted in the enrichment of Lin-7A in the presynaptic fraction. Suppression of Lin-7A expression by RNAi, using in utero electroporation on E14.5, delayed neuronal migration on postnatal day (P) 2, and Lin-7A-deficient neurons remained in the lower zone of the cortical plate and the intermediate zone. In addition, when Lin-7A was silenced in cortical neurons in one hemisphere, axonal growth in the contralateral hemisphere was delayed; development of these neurons was disrupted such that one half did not extend into the contralateral hemisphere after leaving the corpus callosum. Taken together, LIN7A is a candidate gene responsible for 12q21-deletion syndrome, and abnormal neuronal migration and interhemispheric axon development may contribute to ID and corpus callosum hypoplasia, respectively.

Role of a circadian-relevant gene NR1D1 in brain development: possible involvement in the pathophysiology of autism spectrum disorders

In our previous study, we screened autism spectrum disorder (ASD) patients with and without sleep disorders for mutations in the coding regions of circadian-relevant genes, and detected mutations in several clock genes including NR1D1. Here, we further screened ASD patients for NR1D1 mutations and identified three novel mutations including a de novo heterozygous one c.1499 G > A (p.R500H). We then analyzed the role of Nr1d1 in the development of the cerebral cortex in mice. Acute knockdown of mouse Nr1d1 with in utero electroporation caused abnormal positioning of cortical neurons during corticogenesis. This aberrant phenotype was rescued by wild type Nr1d1, but not by the c.1499 G > A mutant. Time-lapse imaging revealed characteristic abnormal migration phenotypes in Nr1d1-deficient cortical neurons. When Nr1d1 was knocked down, axon extension and dendritic arbor formation of cortical neurons were also suppressed while proliferation of neuronal progenitors and stem cells at the ventricular zone was not affected. Taken together, Nr1d1 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation. These results suggest that functional defects in NR1D1 may be related to ASD etiology and pathophysiology.

An Xp22.12 microduplication including RPS6KA3 identified in a family with variably affected intellectual and behavioral disabilities

The ribosomal protein S6 kinase, 90 kb, polypeptide 3 gene (RPS6KA3) is responsible for Coffin–Lowry syndrome (CLS), which is characterized by intellectual disability (ID) and facial and bony abnormalities. This gene also affects nonsyndromic X-linked ID and nonsyndromic X-linked ID without bony abnormalities. Two families have been previously reported to have genetic microduplication including RPS6KA3. In the present study, we used array-comparative genomic hybridization (CGH) analysis with Agilent Human genome CGH 180K and detected a 584-kb microduplication spanning 19.92–20.50 Mb of Xp22.12 (including RPS6KA3) in the members of one family, including three brothers, two sisters, and their mother. The 15-year-old male proband and one of his brothers had mild ID and localization-related epilepsy, whereas his other brother presented borderline intelligence quotient (IQ) and attention-deficit-hyperactivity disorder (ADHD). One sister presented pervasive development disorder (PDD). Analysis of this family suggests that RPS6KA3 duplication is responsible for mild ID, ADHD, and localization-related epilepsy, and possibly for PDD.

Gene therapy for a mouse model of glucose transporter-1 deficiency syndrome

Objective We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene was expressed under the synapsin I promoter (AAV-hSLC2A1) and examined if AAV-hSLC2A1 administration can lead to functional improvement in GLUT1-deficient mice. Methods AAV-hSLC2A1 was injected into heterozygous knock-out murine Glut1 (GLUT1+/−) mice intraperitoneally (systemic; 1.85 × 1011 vg/mouse) or intra-cerebroventricularly (local; 1.85 × 1010 vg/mouse). We analyzed GLUT1 mRNA and protein expression, motor function using rota-rod and footprint tests, and blood and cerebrospinal fluid (CSF) glucose levels. Results Vector-derived RNA was detected in the cerebrum for both injection routes. In the intra-cerebroventricular injection group, exogenous GLUT1 protein was strongly expressed in the cerebral cortex and hippocampus near the injection site. In the intraperitoneal injection group, exogenous GLUT1 protein was mildly expressed in neural cells throughout the entire central nervous system. The motor function test and CSF/blood glucose ratio were significantly improved following intra-cerebroventricular injection. Conclusions AAV-hSLC2A1 administration produced exogenous GLUT1 in neural cells and improved CSF glucose levels and motor function of heterozygous knock-out murine Glut1 mice.

Role of Class III phosphoinositide 3‐kinase in the brain development: possible involvement in specific learning disorders

Class III phosphoinositide 3‐kinase (PIK3C3 or mammalian vacuolar protein sorting 34 homolog, Vps34) regulates vesicular trafficking, autophagy, and nutrient sensing. Recently, we reported that PIK3C3 is expressed in mouse cerebral cortex throughout the developmental process, especially at early embryonic stage. We thus examined the role of PIK3C3 in the development of the mouse cerebral cortex. Acute silencing of PIK3C3 with in utero electroporation method caused positional defects of excitatory neurons during corticogenesis. Time‐lapse imaging revealed that the abnormal positioning was at least partially because of the reduced migration velocity. When PIK3C3 was silenced in cortical neurons in one hemisphere, axon extension to the contralateral hemisphere was also delayed. These aberrant phenotypes were rescued by RNAi‐resistant PIK3C3. Notably, knockdown of PIK3C3 did not affect the cell cycle of neuronal progenitors and stem cells at the ventricular zone. Taken together, PIK3C3 was thought to play a crucial role in corticogenesis through the regulation of excitatory neuron migration and axon extension. Meanwhile, when we performed comparative genomic hybridization on a patient with specific learning disorders, a 107 Kb‐deletion was identified on 18q12.3 (nt. 39554147–39661206) that encompasses exons 5–23 of PIK3C3. Notably, the above aberrant migration and axon growth phenotypes were not rescued by the disease‐related truncation mutant (172 amino acids) lacking the C‐terminal kinase domain. Thus, functional defects of PIK3C3 might impair corticogenesis and relate to the pathophysiology of specific learning disorders and other neurodevelopmental disorders.

Gene therapy for Glut1-deficient mouse using an adeno-associated virus vector with the human intrinsic GLUT1 promoter

We generated an adeno‐associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV‐GLUT1). We examined whether AAV‐GLUT1 administration could lead to functional improvement in GLUT1‐deficient mice.

Mutational and functional analysis of Glucose transporter I deficiency syndrome

OBJECTIVE We investigated a correlation between a mutation in the SLC2A1 gene and functional disorders in Glucose transporter I deficiency syndrome (GLUT1DS). METHODS We performed direct sequence analysis of SLC2A1 in a severe GLUT1DS patient and identified a novel frame shift mutation, c.906_907insG, p.V303fs. We created a plasmid vector carrying the c.906_907insG mutation, as well as A405D or R333W in the SLC2A1, which are found in patients with mild and moderate GLUT1DS severity, respectively. We transiently expressed these mutants and wild type SLC2A1 plasmids in a human embryonic kidney cell line (HEK293), and performed immunoblotting, immunofluorescence, and enzymatic photometric 2-deoxyglucose (2DG) uptake assays. RESULTS GLUT1 was not detected after transient expression of the SLC2A1 plasmid carrying c.906_907insG by either immunoblotting or immunofluorescence. The degree of glucose transport reduction as determined by enzymatic photometric 2DG assay uptake correlated with disease severity. CONCLUSIONS Enzymatic photometric 2DG uptake study appears to be a suitable functional assay to predict the effect of SLC2A1 mutations on GLUT1 transport.