Jijun Wan

C-terminal truncations in human 3'-5' DNA exonuclease TREX1 cause autosomal dominant retinal vasculopathy with cerebral leukodystrophy

Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3′-5′ exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.

Mutations in the RNA exosome component gene EXOSC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration

RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.

Episodic Ataxia Associated With EAAT1 Mutation C186S Affecting Glutamate Reuptake

BACKGROUND Episodic ataxia (EA) is variably associated with additional neurologic symptoms. At least 4 genes have been implicated. Recently, a mutation in the SLC1A3 gene encoding the glutamate transporter EAAT1 was identified in a patient with severe episodic and progressive ataxia, seizures, alternating hemiplegia, and migraine headache. The mutant EAAT1 showed severely reduced uptake of glutamate. The syndrome was designated EA6 and shares overlapping clinical features with EA2, which is caused by mutations in CACNA1A. OBJECTIVE To test the role of the SLC1A3 gene in EA. DESIGN Genetic and functional studies. We analyzed the coding region of the SLC1A3 gene by direct sequencing. SETTING Academic research. PATIENTS DNA samples from 20 patients with EA (with or without interictal nystagmus) negative for CACNA1A mutations were analyzed. MAIN OUTCOME MEASURES We identified 1 novel EAAT1 mutation in a family with EA and studied the functional consequences of this mutation using glutamate uptake assay. RESULTS We identified a missense C186S mutation that segregated with EA in 3 family members. The mutant EAAT1 showed a modest but significant reduction of glutamate uptake. CONCLUSIONS We broadened the clinical spectrum associated with SLC1A3 mutations to include milder manifestations of EA without seizures or alternating hemiplegia. The severity of EA6 symptoms appears to be correlated with the extent of glutamate transporter dysfunction.

CACNA1A mutations causing episodic and progressive ataxia alter channel trafficking and kinetics

Background: CACNA1A encodes CaV2.1, the pore-forming subunit of P/Q-type voltage-gated calcium channel complexes. Mutations in CACNA1A cause a wide range of neurologic disturbances variably associated with cerebellar degeneration. Functional studies to date focus on electrophysiologic defects that do not adequately explain the phenotypic findings. Objective: To investigate whether some missense mutations might interfere with protein folding and trafficking, eventually leading to protein aggregation and neuronal injury. Methods: The authors studied the functional consequences of two pore missense mutations, C287Y and G293R, in two families with EA2, one newly discovered and the other previously reported. Both mutations caused episodic and interictal ataxia. The biophysical properties of mutant and wild type calcium channels were examined by whole-cell patch-clamp recordings in transfected COS-7 cells. The plasma membrane targeting was visualized by confocal fluorescence imaging on CaV2.1 tagged with green fluorescent protein. Results: The mutant channels exhibited a marked reduction in current expression and deficiencies in plasma membrane targeting. Conclusions: In addition to altered channel function, the deficiency in protein misfolding and trafficking associated with the C287Y and G293R mutants may contribute to the slowly progressive cerebellar ataxia.

Presynaptic failure of neuromuscular transmission and synaptic remodeling in EA2

Objective: To further investigate the basis of abnormal neuromuscular transmission in two patients with congenital myasthenic syndrome associated with episodic ataxia type 2 (EA2) using stimulated single fiber EMG (SFEMG) and in vitro microelectrode studies. Methods: Two patients with genetically characterized EA2 previously shown to have abnormal neuromuscular transmission by voluntary SFEMG were studied with stimulated SFEMG and anconeus muscle biopsy with microelectrode studies and electron microscopy of the neuromuscular junction. Results: In vivo stimulated SFEMG showed signs of presynaptic failure, with jitter and blocking that improved with increased stimulation frequency. Additional evidence of presynaptic failure was provided by the in vitro microelectrode studies, which showed marked reduction of the end plate potential quantal content in both patients. Of note, the end plate potentials showed high sensitivity to N-type blockade with ω-conotoxin not seen in controls. The ultrastructural studies revealed some evidence of small nerve terminals apposed to normal or mildly overdeveloped postsynaptic membranes, suggesting an ongoing degenerative process. Conclusions: The authors demonstrated presynaptic failure of neurotransmission in patients with heterozygous nonsense mutations in CACNA1A. The contribution of non-P-type calcium channels to the process of neurotransmitter release in these patients likely represents a compensatory mechanism, which is insufficient to restore normal neuromuscular transmission.

Large genomic deletions in CACNA1A cause episodic ataxia type 2

Episodic ataxia (EA) syndromes are heritable diseases characterized by dramatic episodes of imbalance and incoordination. EA type 2 (EA2), the most common and the best characterized subtype, is caused by mostly nonsense, splice site, small indel, and sometimes missense mutations in CACNA1A. Direct sequencing of CACNA1A fails to identify mutations in some patients with EA2-like features, possibly due to incomplete interrogation of CACNA1A or defects in other EA genes not yet defined. Previous reports described genomic deletions between 4 and 40 kb in EA2. In 47 subjects with EA (26 with EA2-like features) who tested negative for mutations in the known EA genes, we used multiplex ligation-dependent probe amplification to analyze CACNA1A for exonic copy number variations. Breakpoints were further defined by long-range PCR. We identified distinct multi-exonic deletions in three probands with classic EA2-like features: episodes of prolonged vertigo and ataxia triggered by stress and fatigue, interictal nystagmus, with onset during infancy or early childhood. The breakpoints in all three probands are located in Alu sequences, indicating errors in homologous recombination of Alu sequences as the underlying mechanism. The smallest deletion spanned exons 39 and 40, while the largest deletion spanned 200 kb, missing all but the first three exons. One deletion involving exons 39 through 47 arose spontaneously. The search for mutations in CACNA1A appears most fruitful in EA patients with interictal nystagmus and onset early in life. The finding of large heterozygous deletions suggests haploinsufficiency as a possible pathomechanism of EA2.

Nonconsensus intronic mutations cause episodic ataxia

We discovered intronic mutations in two episodic ataxia type 2 (EA2) families: a four‐nucleotide GAGT deletion at IVS41+(3–6) and a single nucleotide insertion (insT) at IVS24+3. We expressed minigenes harboring the mutations in cell lines to demonstrate exon skipping from the deletion mutation and the activation of a cryptic splice donor site from the insertion mutation. The identification of these disease‐causing mutations expands the spectrum of EA2 mutations and emphasizes the importance of intronic sequences in regulating gene expression. Ann Neurol 2005;57:131–135

Loss of function of SLC25A46 causes lethal congenital pontocerebellar hypoplasia.

Disturbed mitochondrial fusion and fission have been linked to various neurodegenerative disorders. In siblings from two unrelated families who died soon after birth with a profound neurodevelopmental disorder characterized by pontocerebellar hypoplasia and apnoea, we discovered a missense mutation and an exonic deletion in the SLC25A46 gene encoding a mitochondrial protein recently implicated in optic atrophy spectrum disorder. We performed functional studies that confirmed the mitochondrial localization and pro-fission properties of SLC25A46. Knockdown of slc24a46 expression in zebrafish embryos caused brain malformation, spinal motor neuron loss, and poor motility. At the cellular level, we observed abnormally elongated mitochondria, which was rescued by co-injection of the wild-type but not the mutant slc25a46 mRNA. Conversely, overexpression of the wild-type protein led to mitochondrial fragmentation and disruption of the mitochondrial network. In contrast to mutations causing non-lethal optic atrophy, missense mutations causing lethal congenital pontocerebellar hypoplasia markedly destabilize the protein. Indeed, the clinical severity appears inversely correlated with the relative stability of the mutant protein. This genotype-phenotype correlation underscores the importance of SLC25A46 and fine tuning of mitochondrial fission and fusion in pontocerebellar hypoplasia and central neurodevelopment in addition to optic and peripheral neuropathy across the life span.

Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria

SCL25A46 is a mitochondrial carrier protein that localizes to the outer membrane. Mutation L341P causes rapid degradation of SLC25A46 by the ubiquitin-proteasome system, independent of activated stress pathways, including mitophagy and apoptosis. SLC25A46 regulates oligomerization of MFN1/2 and mitochondrial dynamics.