Novel SETX variants in a patient with ataxia, neuropathy, and oculomotor apraxia are associated with normal sensitivity to oxidative DNA damaging agents.

BACKGROUND: Homozygous and compound heterozygous mutations in SETX are associated with AOA2 disease, a recessive form of ataxia with oculomotor apraxia and neuropathy with onset of ataxia between the first and second decade of life. The majority of the AOA2 mutated cell lines tested show hypersensitivity to oxidative DNA damaging agents, with one exception. RESULTS: We describe a patient presenting with early-onset progressive ataxia, oculomotor apraxia, axonal sensory-motor neuropathy, optic atrophy, delayed psychomotor development, and a behavior disorder. The patient carries two novel missense variants in the SETX gene. Based on the hypothesis that the patient's clinical phenotype may represent an atypical form of the AOA2 disease, we tested the patient-derived cell line for hypersensitivity to oxidative DNA damaging agents, with negative results. CONCLUSIONS: The lack of hypersensitivity we observed may be explained either by considering the atypical clinical picture of the patient analyzed or, alternatively, by hypothesizing that the variants detected are not the cause of the observed phenotype. Consistent with the first hypothesis of an atypical AOA2 form and based on the multiple functions of senataxin reported so far, it is likely that different sets of SETX mutations/variants may have variable functional effects that still need to be functionally characterized. The possibility that the severe and complicated clinical picture presented by the patient described here represents a clinical entity differing from the known recessive ataxias should be considered as well.

Defective autophagy in spastizin mutated patients with hereditary spastic paraparesis type 15.

Hereditary spastic paraparesis type 15 is a recessive complicated form of the disease clinically characterized by slowly progressive spastic paraparesis and mental deterioration with onset between the first and second decade of life. Thinning of corpus callosum is the neuroradiological distinctive sign frequently associated with white matter abnormalities. The causative gene, ZFYVE26, encodes a large protein of 2539 amino acid residues, termed spastizin, containing three recognizable domains: a zinc finger, a leucine zipper and a FYVE domain. Spastizin protein has a diffuse cytoplasmic distribution and co-localizes partially with early endosomes, the endoplasmic reticulum, microtubules and vesicles involved in protein trafficking. In addition, spastizin localizes to the mid-body during the final step of mitosis and contributes to successful cytokinesis. Spastizin interacts with Beclin 1, a protein required for cytokinesis and autophagy, which is the major lysosome-mediated degradation process in the cell. In view of the Beclin 1-spastizin interaction, we investigated the possible role of spastizin in autophagy. We carried out this analysis by using lymphoblast and fibroblast cells derived from four different spastizin mutated patients (p.I508N, p.L243P, p.R1209fsX, p.S1312X) and from control subjects. Of note, the truncating p.R1209fsX and p.S1312X mutations lead to loss of spastizin protein. The results obtained indicate that spastizin interacts with the autophagy related Beclin 1-UVRAG-Rubicon multiprotein complex and is required for autophagosome maturation. In cells lacking spastizin or with mutated forms of the protein, spastizin interaction with Beclin 1 is lost although the formation of the Beclin 1-UVRAG-Rubicon complex can still be observed. However, in these cells we demonstrate an impairment of autophagosome maturation and an accumulation of immature autophagosomes. Autophagy defects with autophagosome accumulation can be observed also in neuronal cells upon spastizin silencing. These results indicate that autophagy is a central process in the pathogenesis of complicated forms of hereditary spastic paraparesis with thin corpus callosum.

Senataxin modulates neurite growth through fibroblast growth factor 8 signalling.

Senataxin is encoded by the SETX gene and is mainly involved in two different neurodegenerative diseases, the dominant juvenile form of amyotrophic lateral sclerosis type 4 and a recessive form of ataxia with oculomotor apraxia type 2. Based on protein homology, senataxin is predicted to be a putative DNA/RNA helicase, while senataxin interactors from patients' lymphoblast cell lines suggest a possible involvement of the protein in different aspects of RNA metabolism. Except for an increased sensitivity to oxidative DNA damaging agents shown by some ataxia with neuropathy patients' cell lines, no data are available about possible functional consequences of dominant SETX mutations and no studies address the function of senataxin in neurons. To start elucidating the physiological role of senataxin in neurons and how disease-causing mutations in this protein lead to neurodegeneration, we analysed the effect of senataxin on neuronal differentiation in primary hippocampal neurons and retinoic acid-treated P19 cells by modulating the expression levels of wild-type senataxin and three different dominant mutant forms of the protein. Wild-type senataxin overexpression was required and sufficient to trigger neuritogenesis and protect cells from apoptosis during differentiation. These actions were reversed by silencing of senataxin. In contrast, overexpression of the dominant mutant forms did not affect the regular differentiation process in primary hippocampal neurons. Analysis of the cellular pathways leading to neuritogenesis and cytoprotection revealed a role of senataxin in modulating the expression levels and signalling activity of fibroblast growth factor 8. Silencing of senataxin reduced, while overexpression enhanced, fibroblast growth factor 8 expression levels and the phosphorylation of related target kinases and effector proteins. The effects of senataxin overexpression were prevented when fibroblast growth factor 8 signalling was inhibited, while exogenous fibroblast growth factor 8 reversed the effects of senataxin silencing. Overall, these results reveal a key role of senataxin in neuronal differentiation through the fibroblast growth factor 8 signalling and provide initial molecular bases to explain the neurodegeneration associated with loss-of-function mutations in senataxin found in recessive ataxia. The lack of effect on neuritogenesis observed with the overexpression of the dominant mutant forms of senataxin apparently excludes a dominant negative effect of these mutants while favouring haploinsufficiency as the pathogenic mechanism implicated in the amyotrophic lateral sclerosis 4-related degenerative condition. Alternatively, a different protein function, other than the one involved in neuritogenesis, may be implicated in these dominant degenerative processes.

Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin.

Ataxia with oculomotor apraxia (AOA) type 2 (AOA2 MIM 606002) is a recessive subtype of AOA characterized by cerebellar atrophy, oculomotor apraxia, early loss of reflexes, and peripheral neuropathy. Various mutations either in homozygous or compound heterozygous condition were so far identified in the associated gene SETX (MIM 608465). SETX encodes a large protein called senataxin with a DNA-RNA helicase domain and a putative N-terminus protein interaction domain. Here, we report the identification of two novel homozygous mutations in SETX gene, c.340_342delCTT (p.L114Del) and c.1669C > T (p.R557X), in two AOA2 families. The characterization of the mutant lymphoblastoid cell lines for sensitivity to oxidative DNA-damaging agents indicates that the p.L114Del deletion confers an increased sensitivity to H2O2, camptothecin, and mitomycin C, previously found to induce death in lymphoblasts harbouring other SETX mutations; the cells carrying the nonsense mutation display instead values within the normal range. Further analysis of a neuronal cell model SKNBE, transfected with the mutant senataxin proteins, reveals increased sensitivity also to staurosporine and excitotoxicity associated with the p.L114Del mutant only. We also demonstrate that the sensitizing effect of p.L114Del on apoptosis can be reversed by senataxin silencing. The ability of a single amino acid deletion to sensitize cells to death by different agents, compared to the lack of effect of a whole protein deletion, seems to exclude a protective role played by the native protein while suggesting that a specific mutation confers to the protein the ability to enhance the toxic effect of various cell damaging agents.

Cryptogenic epileptic syndromes related to SCN1A: twelve novel mutations identified.

BACKGROUND: Sodium channel alpha 1 subunit gene, SCN1A, is the gene encoding the neuronal voltage-gated sodium channel alpha 1 subunit (Na(v)1.1) and is mutated in different forms of epilepsy. Mutations in this gene were observed in more than 70% of patients with severe myoclonic epilepsy of infancy (SMEI) and were also found in different types of infantile epileptic encephalopathy. OBJECTIVE: To search for disease-causing mutations in SCN1A in patients with cryptogenic epileptic syndromes (ie, syndromes with an unknown cause). DESIGN: Clinical characterization and molecular genetic analysis of a cohort of patients. SETTING: University hospitals, rehabilitation centers, and molecular biology laboratories. PATIENTS: Sixty unrelated patients with cryptogenic epileptic syndromes. MAIN OUTCOME MEASURES: Samples of DNA were analyzed for mutations and for large heterozygous deletions encompassing the SCN1A gene. A search for microdeletions in the SCN1A gene was also performed in the subset of patients with SMEI/SMEI-borderland who had negative results at the point mutation screening. RESULTS: No large deletions at the SCN1A locus were found in any of the patients analyzed. In contrast, 13 different point mutations were identified in 12 patients: 10 with SMEI, 1 with generalized epilepsy with febrile seizures plus, and 1 with cryptogenic focal epilepsy. An additional search for SCN1A intragenic microdeletions in the remaining patients with SMEI/SMEI-borderland and no point mutations was also negative. CONCLUSIONS: These results confirm the role of the SCN1A gene in different types of epilepsy, including cryptogenic epileptic syndromes. However, large deletions encompassing SCN1A were not common disease-causing rearrangements in this group of epilepsies.