Protein accumulation and neurodegeneration in the woozy mutant mouse is caused by disruption of SIL1, a cochaperone of BiP

Endoplasmic reticulum (ER) chaperones and ER stress have been implicated in the pathogenesis of neurodegenerative disorders, such as Alzheimer and Parkinson diseases, but their contribution to neuron death remains uncertain. In this study, we establish a direct in vivo link between ER dysfunction and neurodegeneration. Mice homozygous with respect to the woozy (wz) mutation develop adult-onset ataxia with cerebellar Purkinje cell loss. Affected cells have intracellular protein accumulations reminiscent of protein inclusions in both the ER and the nucleus. In addition, upregulation of the unfolded protein response, suggestive of ER stress, occurs in mutant Purkinje cells. We report that the wz mutation disrupts the gene Sil1 that encodes an adenine nucleotide exchange factor of BiP, a crucial ER chaperone. These findings provide evidence that perturbation of ER chaperone function in terminally differentiated neurons leads to protein accumulation, ER stress and subsequent neurodegeneration.     

A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases

Sialic acid storage diseases (SASD, MIM 269920) are autosomal recessive neurodegenerative disorders that may present as a severe infantile form (ISSD) or a slowly progressive adult form, which is prevalent in Finland (Salla disease). The main symptoms are hypotonia, cerebellar ataxia and mental retardation; visceromegaly and coarse features are also present in infantile cases. Progressive cerebellar atrophy and dysmyelination have been documented by magnetic resonance imaging (ref. 4). Enlarged lysosomes are seen on electron microscopic studies and patients excrete large amounts of free sialic acid in urine. A H+/anionic sugar symporter mechanism for sialic acid and glucuronic acid is impaired in lysosomal membranes from Salla and ISSD patients. The locus for Salla disease was assigned to a region of approximately 200 kb on chromosome 6q14-q15 in a linkage study using Finnish families. Salla disease and ISSD were further shown to be allelic disorders. A physical map with P1 and PAC clones was constructed to cover the 200-kb area flanked by the loci D6S280 and D6S1622, providing the basis for precise physical positioning of the gene. Here we describe a new gene, SLC17A5 (also known as AST), encoding a protein (sialin) with a predicted transport function that belongs to a family of anion/cation symporters (ACS). We found a homozygous SLC17A5 mutation (R39C) in five Finnish patients with Salla disease and six different SLC17A5 mutations in six ISSD patients of different ethnic origins. Our observations suggest that mutations in SLC17A5 are the primary cause of lysosomal sialic acid storage diseases.     

ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating

Stress tolerance of the heart requires high-fidelity metabolic sensing by ATP-sensitive potassium (K(ATP)) channels that adjust membrane potential-dependent functions to match cellular energetic demand. Scanning of genomic DNA from individuals with heart failure and rhythm disturbances due to idiopathic dilated cardiomyopathy identified two mutations in ABCC9, which encodes the regulatory SUR2A subunit of the cardiac K(ATP) channel. These missense and frameshift mutations mapped to evolutionarily conserved domains adjacent to the catalytic ATPase pocket within SUR2A. Mutant SUR2A proteins showed aberrant redistribution of conformations in the intrinsic ATP hydrolytic cycle, translating into abnormal K(ATP) channel phenotypes with compromised metabolic signal decoding. Defective catalysis-mediated pore regulation is thus a mechanism for channel dysfunction and susceptibility to dilated cardiomyopathy.     

First genetic evidence of GABA(A) receptor dysfunction in epilepsy: a mutation in the gamma2-subunit gene

Major advances in the identification of genes implicated in idiopathic epilepsy have been made. Generalized epilepsy with febrile seizures plus (GEFS+), benign familial neonatal convulsions and nocturnal frontal lobe epilepsy, three autosomal dominant idiopathic epilepsies, result from mutations affecting voltage-gated sodium and potassium channels, and nicotinic acetylcholine receptors, respectively. Disruption of GABAergic neurotransmission mediated by gamma-aminobutyric acid (GABA) has been implicated in epilepsy for many decades. We now report a K289M mutation in the GABA(A) receptor gamma2-subunit gene (GABRG2) that segregates in a family with a phenotype closely related to GEFS+ (ref. 8), an autosomal dominant disorder associating febrile seizures and generalized epilepsy previously linked to mutations in sodium channel genes. The K289M mutation affects a highly conserved residue located in the extracellular loop between transmembrane segments M2 and M3. Analysis of the mutated and wild-type alleles in Xenopus laevis oocytes confirmed the predicted effect of the mutation, a decrease in the amplitude of GABA-activated currents. We thus provide the first genetic evidence that a GABA(A) receptor is directly involved in human idiopathic epilepsy.     

Mutations in SYNE1 lead to a newly discovered form of autosomal recessive cerebellar ataxia

The past decade has seen great advances in unraveling the biological basis of hereditary ataxias. Molecular studies of spinocerebellar ataxias (SCA) have extended our understanding of dominant ataxias. Causative genes have been identified for a few autosomal recessive ataxias: Friedreich's ataxia, ataxia with vitamin E deficiency, ataxia telangiectasia, recessive spastic ataxia of Charlevoix-Saguenay and ataxia with oculomotor apraxia type 1 (refs. 6,7) and type 2 (ref. 8). Nonetheless, genes remain unidentified for most recessive ataxias. Additionally, pure cerebellar ataxias, which represent up to 20% of all ataxias, remain poorly studied with only two causative dominant genes being described: CACNA1A (ref. 9) and SPTBN2 (ref. 10). Here, we report a newly discovered form of recessive ataxia in a French-Canadian cohort and show that SYNE1 mutations are causative in all of our kindreds, making SYNE1 the first identified gene responsible for a recessively inherited pure cerebellar ataxia.     

ANG mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis

We recently identified angiogenin (ANG) as a candidate susceptibility gene for amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by adult-onset loss of motor neurons. We now report the finding of seven missense mutations in 15 individuals, of whom four had familial ALS and 11 apparently 'sporadic' ALS. Our findings provide further evidence that variations in hypoxia-inducible genes have an important role in motor neuron degeneration.     

Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2

Ataxia-ocular apraxia 2 (AOA2) was recently identified as a new autosomal recessive ataxia. We have now identified causative mutations in 15 families, which allows us to clinically define this entity by onset between 10 and 22 years, cerebellar atrophy, axonal sensorimotor neuropathy, oculomotor apraxia and elevated alpha-fetoprotein (AFP). Ten of the fifteen mutations cause premature termination of a large DEAxQ-box helicase, the human ortholog of yeast Sen1p, involved in RNA maturation and termination.     

Mutations in SIL1 cause Marinesco-Sjögren syndrome, a cerebellar ataxia with cataract and myopathy

SIL1 (also called BAP) acts as a nucleotide exchange factor for the Hsp70 chaperone BiP (also called GRP78), which is a key regulator of the main functions of the endoplasmic reticulum. We found nine distinct mutations that would disrupt the SIL1 protein in individuals with Marinesco-Sj?gren syndrome, an autosomal recessive cerebellar ataxia complicated by cataracts, developmental delay and myopathy. Identification of SIL1 mutations implicates Marinesco-Sj?gren syndrome as a disease of endoplasmic reticulum dysfunction and suggests a role for this organelle in multisystem disorders.     

Spectrin mutations cause spinocerebellar ataxia type 5

We have discovered that beta-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred descended from President Lincoln's grandparents and two additional families. Two families have separate in-frame deletions of 39 and 15 bp, and a third family has a mutation in the actin/ARP1 binding region. Beta-III spectrin is highly expressed in Purkinje cells and has been shown to stabilize the glutamate transporter EAAT4 at the surface of the plasma membrane. We found marked differences in EAAT4 and GluRdelta2 by protein blot and cell fractionation in SCA5 autopsy tissue. Cell culture studies demonstrate that wild-type but not mutant beta-III spectrin stabilizes EAAT4 at the plasma membrane. Spectrin mutations are a previously unknown cause of ataxia and neurodegenerative disease that affect membrane proteins involved in glutamate signaling.     

Mutations in TTBK2, encoding a kinase implicated in tau phosphorylation, segregate with spinocerebellar ataxia type 11

The microtubule-associated protein tau (encoded by MAPT) and several tau kinases have been implicated in neurodegeneration, but only MAPT has a proven role in disease. We identified mutations in the gene encoding tau tubulin kinase 2 (TTBK2) as the cause of spinocerebellar ataxia type 11. Affected brain tissue showed substantial cerebellar degeneration and tau deposition. These data suggest that TTBK2 is important in the tau cascade and in spinocerebellar degeneration.     

Novel mutations in families with unusual and variable disorders of the skeletal muscle sodium channel.

Mutations in the skeletal muscle sodium channel gene (SCN4A) have been described in paramyotonia congenita (PMC) and hyperkalaemic periodic paralysis (HPP). We have found two mutations in SCN4A which affect regions of the sodium channel not previously associated with a disease phenotype. Furthermore, affected family members display an unusual mixture of clinical features reminiscent of PMC, HPP and of a third disorder, myotonia congenita (MC). The highly variable individual expression of these symptoms, including in some cases apparent non-penetrance, implies the existence of modifying factors. Mutations in SCN4A can produce a broad range of phenotypes in muscle diseases characterized by episodic abnormalities of membrane excitability.     

The gene disrupted in Marinesco-Sjögren syndrome encodes SIL1, an HSPA5 cochaperone

We identified the gene underlying Marinesco-Sj?gren syndrome, which is characterized by cerebellar ataxia, progressive myopathy and cataracts. We identified four disease-associated, predicted loss-of-function mutations in SIL1, which encodes a nucleotide exchange factor for the heat-shock protein 70 (HSP70) chaperone HSPA5. These data, together with the similar spatial and temporal patterns of tissue expression of Sil1 and Hspa5, suggest that disturbed SIL1-HSPA5 interaction and protein folding is the primary pathology in Marinesco-Sj?gren syndrome.     

The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin

The newly recognized ataxia-ocular apraxia 1 (AOA1; MIM 208920) is the most frequent cause of autosomal recessive ataxia in Japan and is second only to Friedreich ataxia in Portugal. It shares several neurological features with ataxia-telangiectasia, including early onset ataxia, oculomotor apraxia and cerebellar atrophy, but does not share its extraneurological features (immune deficiency, chromosomal instability and hypersensitivity to X-rays). AOA1 is also characterized by axonal motor neuropathy and the later decrease of serum albumin levels and elevation of total cholesterol. We have identified the gene causing AOA1 and the major Portuguese and Japanese mutations. This gene encodes a new, ubiquitously expressed protein that we named aprataxin. This protein is composed of three domains that share distant homology with the amino-terminal domain of polynucleotide kinase 3'- phosphatase (PNKP), with histidine-triad (HIT) proteins and with DNA-binding C2H2 zinc-finger proteins, respectively. PNKP is involved in DNA single-strand break repair (SSBR) following exposure to ionizing radiation and reactive oxygen species. Fragile-HIT proteins (FHIT) cleave diadenosine tetraphosphate, which is potentially produced during activation of the SSBR complex. The results suggest that aprataxin is a nuclear protein with a role in DNA repair reminiscent of the function of the protein defective in ataxia-telangiectasia, but that would cause a phenotype restricted to neurological signs when mutant.     

Frequent somatic mutations in MAP3K5 and MAP3K9 in metastatic melanoma identified by exome sequencing

We sequenced eight melanoma exomes to identify new somatic mutations in metastatic melanoma. Focusing on the mitogen-activated protein (MAP) kinase kinase kinase (MAP3K) family, we found that 24% of melanoma cell lines have mutations in the protein-coding regions of either MAP3K5 or MAP3K9. Structural modeling predicted that mutations in the kinase domain may affect the activity and regulation of these protein kinases. The position of the mutations and the loss of heterozygosity of MAP3K5 and MAP3K9 in 85% and 67% of melanoma samples, respectively, together suggest that the mutations are likely to be inactivating. In in vitro kinase assays, MAP3K5 I780F and MAP3K9 W333* variants had reduced kinase activity. Overexpression of MAP3K5 or MAP3K9 mutants in HEK293T cells reduced the phosphorylation of downstream MAP kinases. Attenuation of MAP3K9 function in melanoma cells using siRNA led to increased cell viability after temozolomide treatment, suggesting that decreased MAP3K pathway activity can lead to chemoresistance in melanoma.     

An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8)

Myotonic dystrophy (DM) is the only disease reported to be caused by a CTG expansion. We now report that a non-coding CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). This expansion, located on chromosome 13q21, was isolated directly from the genomic DNA of an ataxia patient by RAPID cloning. SCA8 patients have expansions similar in size (107-127 CTG repeats) to those found among adult-onset DM patients. SCA8 is the first example of a dominant SCA not caused by a CAG expansion translated as a polyglutamine tract.     

Intragenic deletion in the gene encoding ubiquitin carboxy-terminal hydrolase in gad mice.

The gracile axonal dystrophy (gad) mouse is an autosomal recessive mutant that shows sensory ataxia at an early stage, followed by motor ataxia at a later stage. Pathologically, the mutant is characterized by 'dying-back' type axonal degeneration and formation of spheroid bodies in nerve terminals. Recent pathological observations have associated brain ageing and neurodegenerative diseases with progressive accumulation of ubiquitinated protein conjugates. In gad mice, accumulation of amyloid beta-protein and ubiquitin-positive deposits occur retrogradely along the sensory and motor nervous systems. We previously reported that the gad mutation was transmitted by a gene on chromosome 5 (refs 10,11). Here we find that the gad mutation is caused by an in-frame deletion including exons 7 and 8 of Uchl1, encoding the ubiquitin carboxy-terminal hydrolase (UCH) isozyme (Uch-l1) selectively expressed in the nervous system and testis. The gad allele encodes a truncated Uch-l1 lacking a segment of 42 amino acids containing a catalytic residue. As Uch-l1 is thought to stimulate protein degradation by generating free monomeric ubiquitin, the gad mutation appears to affect protein turnover. Our data suggest that altered function of the ubiquitin system directly causes neurodegeneration. The gad mouse provides a useful model for investigating human neurodegenerative disorders.     

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa (RP). So far, six loci (USH1A-USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A for USH1B and the gene encoding harmonin for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G-->C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, Delta M1281 and IVS51+5G-->A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.     

Dominant missense mutations in ABCC9 cause Cantú syndrome

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (K(ATP)) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the K(ATP) channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.     

Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca2(+)-permeable cation channels which are blocked by extracellular Mg2(+) in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2A(N615K) (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg2(+) block and a decrease in Ca2(+) permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.     

Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions.

Progressive external ophthalmoplegias (PEO) characterized by accumulation of large-scale mitochondrial DNA (mtDNA) deletions are rare human diseases. We mapped a new locus for dominant PEO at 15q22-q26 in a Belgian pedigree and identified a heterozygous mutation (Y955C) in the polymerase motif B of the mtDNA polymerase gamma (POLG). We identified three additional POLG missense mutations compatible with recessive PEO In two nuclear families. POLG is the only DNA polymerase responsible for mtDNA replication.