Vijay S. Ganesh

Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex

Disruption of human neural precursor proliferation can give rise to a small brain (microcephaly), and failure of neurons to migrate properly can lead to an abnormal arrest of cerebral cortical neurons in proliferative zones near the lateral ventricles (periventricular heterotopia). Here we show that an autosomal recessive condition characterized by microcephaly and periventricular heterotopia maps to chromosome 20 and is caused by mutations in the gene ADP-ribosylation factor guanine nucleotide-exchange factor-2 (ARFGEF2). By northern-blot analysis, we found that mouse Arfgef2 mRNA levels are highest during embryonic periods of ongoing neuronal proliferation and migration, and by in situ hybridization, we found that the mRNA is widely distributed throughout the embryonic central nervous system (CNS). ARFGEF2 encodes the large (>200 kDa) brefeldin A (BFA)-inhibited GEF2 protein (BIG2), which is required for vesicle and membrane trafficking from the trans-Golgi network (TGN). Inhibition of BIG2 by BFA, or by a dominant negative ARFGEF2 cDNA, decreases cell proliferation in vitro, suggesting a cell-autonomous regulation of neural expansion. Inhibition of BIG2 also disturbed the intracellular localization of such molecules as E-cadherin and β-catenin by preventing their transport from the Golgi apparatus to the cell surface. Our findings show that vesicle trafficking is an important regulator of proliferation and migration during human cerebral cortical development.

Mutations in PNKP cause microcephaly, seizures and defects in DNA repair

Maintenance of DNA integrity is crucial for all cell types, but neurons are particularly sensitive to mutations in DNA repair genes, which lead to both abnormal development and neurodegeneration. We describe a previously unknown autosomal recessive disease characterized by microcephaly, early-onset, intractable seizures and developmental delay (denoted MCSZ). Using genome-wide linkage analysis in consanguineous families, we mapped the disease locus to chromosome 19q13.33 and identified multiple mutations in PNKP (polynucleotide kinase 3′-phosphatase) that result in severe neurological disease; in contrast, a splicing mutation is associated with more moderate symptoms. Unexpectedly, although the cells of individuals carrying this mutation are sensitive to radiation and other DNA-damaging agents, no such individual has yet developed cancer or immunodeficiency. Unlike other DNA repair defects that affect humans, PNKP mutations universally cause severe seizures. The neurological abnormalities in individuals with MCSZ may reflect a role for PNKP in several DNA repair pathways.

Filamin A mutations cause periventricular heterotopia with Ehlers-Danlos syndrome

Objective: To define the clinical, radiologic, and genetic features of periventricular heterotopia (PH) with Ehlers-Danlos syndrome (EDS). Methods: Exonic sequencing and single stranded conformational polymorphism (SSCP) analysis was performed on affected individuals. Linkage analysis using microsatellite markers on the X-chromosome was performed on a single pedigree. Western blotting evaluated for loss of filamin A (FLNA) protein and Southern blotting assessed for any potential chromosome rearrangement in this region. Results: The authors report two familial cases and nine additional sporadic cases of the EDS-variant form of PH, which is characterized by nodular brain heterotopia, joint hypermobility, and development of aortic dilatation in early adulthood. MRI typically demonstrated bilateral nodular PH, indistinguishable from PH due to FLNA mutations. Exonic sequencing or SSCP analyses of FLNA revealed a 2762 delG single base pair deletion in one affected female. Another affected female harbored a C116 single point mutation, resulting in an A39G change. A third affected female had a 4147 delG single base pair deletion. One pedigree with no detectable exonic mutation demonstrated positive linkage to the FLNA locus Xq28, an affected individual in this family also had no detectable FLNA protein, but no chromosomal rearrangement was detected. Conclusion: These results suggest that the Ehlers-Danlos variant of periventricular heterotopia (PH), in part, represents an overlapping syndrome with X-linked dominant PH due to filamin A mutations.

Autosomal recessive form of periventricular heterotopia

Background: Familial periventricular heterotopia (PH) represents a disorder of neuronal migration resulting in multiple gray matter nodules along the lateral ventricular walls. Prior studies have shown that mutations in the filamin A (FLNA) gene can cause PH through an X-linked dominant inheritance pattern. Objective: To classify cortical malformation syndromes associated with PH. Methods: Analyses using microsatellite markers directed toward genomic regions of FLNA and to a highly homologous autosomal gene, FLNB, were performed on two pedigrees to evaluate for linkage with either filamin gene. Results: Two consanguineous pedigrees with PH that suggest an autosomal recessive inheritance pattern are reported. MRI of the brain revealed periventricular nodules of cerebral gray matter intensity, typical for PH. Seizures or developmental delay appeared to be a common presenting feature. Microsatellite analysis suggested no linkage to FLNA or FLNB. Conclusions: Autosomal recessive PH is another syndromic migrational disorder, distinct from X-linked dominant PH. Further classification of these different syndromes will provide an approach for genetic evaluation.

A novel form of pontocerebellar hypoplasia maps to chromosome 7q11-21

Objective: To describe a novel form of pontocerebellar hypoplasia (PCH) and map its genetic locus. Background: PCH is a heterogeneous group of disorders that are characterized by abnormally small cerebellum and brainstem. Autosomal recessive inheritance has been implied in many cases, but no genetic loci have been mapped to date. Methods: The authors studied a consanguineous family from the Sultanate of Oman with three siblings with a novel form of PCH. The authors performed clinical studies and linkage analysis of this pedigree. Results: The clinical features of the affected children include developmental delay, progressive microcephaly with brachycephaly, seizures during the first year of life, hypotonia with hyperreflexia, short stature, and optic atrophy. Imaging studies showed a small pons and cerebellum, prominent sulci and lateral ventricles, and decreased cerebral white matter volume. A lack of dyskinesias distinguishes this pedigree from PCH type 2. Genetic studies of this family revealed evidence of significant linkage to chromosome 7q11-21 (maximum multipoint lod score 3.23). Conclusions: This pedigree represents a novel form of autosomal recessive PCH, which the authors propose to call cerebellar atrophy with progressive microcephaly (CLAM). This disorder maps to chromosome 7q11-21, and this locus was named CLAM. This report represents the first identification of a genetic locus for PCH.

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.

A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts.

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.

Etiological heterogeneity of familial periventricular heterotopia and hydrocephalus.

Periventricular heterotopia (PH) represents a neuronal migration disorder that results in gray matter nodules along the lateral ventricles beneath an otherwise normal appearing cortex. While prior reports have shown that mutations in the filamin A (FLNA) gene can cause X-linked dominant PH, an increasing number of studies suggest the existence of additional PH syndromes. Further classification of these cortical malformation syndromes associated with PH allows for determination of the causal genes. Here we report three familial cases of PH with hydrocephalus. One pedigree has a known FLNA mutation with hydrocephalus occurring in the setting of valproic acid exposure. Another pedigree demonstrated possible linkage to the Xq28 locus including FLNA, although uncharacteristically a male was affected and sequencing of the FLNA gene in this individual revealed no mutation. However, in the third family with an autosomal mode of inheritance, microsatellite analysis ruled out linkage with the FLNA gene. Routine karyotyping and fluorescent in situ hybridization using BAC probes localized to FLNA also showed no evidence of genomic rearrangement. Western blot analysis of one of the affected individuals demonstrated normal expression of the FLNA protein. Lastly, sequencing of greater than 95% of the FLNA gene in an affected member failed to demonstrate a mutation. In conclusion, these findings demonstrate the etiological heterogeneity of PH with hydrocephalus. Furthermore, there likely exists an autosomal PH gene, distinct from the previously described X-linked and autosomal recessive forms. Affected individuals have severe developmental delay and may have radiographic findings of hydrocephalus.

FLNA genomic rearrangements cause periventricular nodular heterotopia

Objective: To identify copy number variant (CNV) causes of periventricular nodular heterotopia (PNH) in patients for whom FLNA sequencing is negative. Methods: Screening of 35 patients from 33 pedigrees on an Affymetrix 6.0 microarray led to the identification of one individual bearing a CNV that disrupted FLNA. FLNA-disrupting CNVs were also isolated in 2 other individuals by multiplex ligation probe amplification. These 3 cases were further characterized by high-resolution oligo array comparative genomic hybridization (CGH), and the precise junctional breakpoints of the rearrangements were identified by PCR amplification and sequencing. Results: We report 3 cases of PNH caused by nonrecurrent genomic rearrangements that disrupt one copy of FLNA. The first individual carried a 113-kb deletion that removes all but the first exon of FLNA. A second patient harbored a complex rearrangement including a deletion of the 3′ end of FLNA accompanied by a partial duplication event. A third patient bore a 39-kb deletion encompassing all of FLNA and the neighboring gene EMD. High-resolution oligo array CGH of the FLNA locus suggests distinct molecular mechanisms for each of these rearrangements, and implicates nearby low copy repeats in their pathogenesis. Conclusions: These results demonstrate that FLNA is prone to pathogenic rearrangements, and highlight the importance of screening for CNVs in individuals with PNH lacking FLNA point mutations. Neurology® 2012;78:269–278

Rapid Detection of Powassan Virus in a Patient With Encephalitis by Metagenomic Sequencing

Abstract We describe a patient with severe and progressive encephalitis of unknown etiology. We performed rapid metagenomic sequencing from cerebrospinal fluid and identified Powassan virus, an emerging tick-borne flavivirus that has been increasingly detected in the United States.