Maria Spadaro

Complete loss of P/Q calcium channel activity caused by a CACNA1A missense mutation carried by patients with episodic ataxia type 2

Familial hemiplegic migraine, episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 are allelic disorders of the CACNA1A gene (coding for the alpha(1A) subunit of P/Q calcium channels), usually associated with different types of mutations (missense, protein truncating, and expansion, respectively). However, the finding of expansion and missense mutations in patients with EA2 has blurred this genotype-phenotype correlation. We report the first functional analysis of a new missense mutation, associated with an EA2 phenotype-that is, T-->C transition of nt 4747 in exon 28, predicted to change a highly conserved phenylalanine residue to a serine at codon 1491, located in the putative transmembrane segment S6 of domain III. Patch-clamp recording in HEK 293 cells, coexpressing the mutagenized human alpha(1A-2) subunit, together with human beta(4) and alpha(2)delta subunits, showed that channel activity was completely abolished, although the mutated protein is expressed in the cell. These results indicate that a complete loss of P/Q channel function is the mechanism underlying EA2, whether due to truncating or to missense mutations.

A G301R Na+/K+-ATPase mutation causes familial hemiplegic migraine type 2 with cerebellar signs

Abstract.Familial hemiplegic migraine (FHM) is an autosomal dominant subtype of migraine with hemiparesis during the aura. In over 50% of cases the causative gene is CACNA1A (FHM1), which in some cases produces a phenotype with cerebellar signs, including ataxia and nystagmus. Recently, mutations in ATP1A2 on chromosome 1q23 encoding a Na+/K+-ATPase subunit were identified in four families (FHM2). We now describe an FHM2 pedigree with a fifth ATP1A2 mutation coding for a G301R substitution. The phenotype was particularly severe and included hemiplegic migraine, seizure, prolonged coma, elevated temperature, sensory deficit, and transient or permanent cerebellar signs, such as ataxia, nystagmus, and dysarthria. A mild crossed cerebellar diaschisis during an attack further supported the clinical evidence of a cerebellar deficit. This is the first report suggesting cerebellar involvement in FHM2. A possible role for CACNA1A in producing the phenotype in this family was excluded by linkage studies to the FHM1 locus. The study of this family suggests that the absence of cerebellar signs may not be a reliable indicator to clinically differentiate FHM2 from FHM1.

Riluzole in cerebellar ataxia A randomized, double-blind, placebo-controlled pilot trial

Background: The pleiotropic effects of riluzole may antagonize common mechanisms underlying chronic cerebellar ataxia, a debilitating and untreatable consequence of various diseases. Methods: In a randomized, double-blind, placebo-controlled pilot trial, 40 patients presenting with cerebellar ataxias of different etiologies were randomly assigned to riluzole (100 mg/day) or placebo for 8 weeks. The following outcome measures were compared: proportion of patients with a decrease of at least 5 points in the International Cooperative Ataxia Rating Scale (ICARS) total score after 4 and 8 weeks compared with the baseline score; mean changes from the baseline to posttreatment ICARS (total score and subscores at 8 weeks); and occurrence of adverse events. Results: Riluzole and placebo groups did not differ in baseline characteristics. The number of patients with a 5-point ICARS drop was significantly higher in the riluzole group than in the placebo group after 4 weeks (9/19 vs 1/19; odds ratio [OR] = 16.2; 95% confidence interval [CI ] 1.8–147.1) and 8 weeks (13/19 vs 1/19; OR = 39.0; 95% CI 4.2–364.2). The mean change in the riluzole group ICARS after treatment revealed a decrease (p < 0.001) in the total score (−7.05 [4.96] vs 0.16 [2.65]) and major subscores (−2.11 [2.75] vs 0.68 [1.94] for static function, −4.11 [2.96] vs 0.37 [2.0] for kinetic function, and −0.74 [0.81] vs 0.05 [0.40] for dysarthria). Sporadic, mild adverse events occurred. Conclusions: These findings indicate the potential effectiveness of riluzole as symptomatic therapy in diverse forms of cerebellar ataxia. Classification of evidence: This study provides Class I evidence that riluzole reduces, by at least 5 points, the ICARS score in patients with a wide range of disorders that cause cerebellar ataxia (risk difference 63.2%, 95% CI 33.5%–79.9%).

Monthly corticosteroids decrease neutralizing antibodies to IFNβ1b: A randomized trial in multiple sclerosis

Abstract Neutralizing antibodies (NAB) to interferon beta (IFNβ) occur in some multiple sclerosis (MS) patients, particularly during the first year of treatment. The presence of NAB may be associated with an attenuation of the therapeutic effect. The aim of this study was to compare the frequency of NAB occurrence in patients treated with IFNβ-1 b with that in patients treated with IFNβ-1 b combined with monthly pulses of intravenous methylprednisolone (MP).One hundred and sixty-one patients with relapsing-remitting MS were randomized in two treatment arms: 8 MIU of IFNβ-1 b subcutaneously injected every other day either alone or in combination with 1000 mg of monthly intravenous MP.NAB were evaluated at baseline and at months 3, 6, 9, 12 and 15 by the MxA assay in a specialized laboratory. Positivity was defined as a titer of ≥ 20 neutralizing units according to two different definitions: I) one or more non-consecutive positive samples, II) at least two consecutive positive samples.NAB (definition I) were observed in 26.8 % of patients in the IFNβ-1 b alone arm and in 12.1 % of patients in the combination therapy arm (p=0.05 by the chi-square test), which corresponds to a relative reduction of 54.9 %, whereas according to definition II, these figures dropped to 22.5 % for the IFNβ–1 b alone arm, and 10.6 % for the combination therapy arm (relative reduction 52.9 %, p=0.10, NS). A higher probability of remaining in the NAB-free status was observed in patients treated with the combination therapy (p=0.031 for definition I and p=0.049 for definition II, by the Wilcoxon-Gehan test).Methylprednisilone combined with IFNβ-1 b reduces the incidence of neutralizing bodies to interferon-β during the first year of treatment in MS patients.

Riluzole in patients with hereditary cerebellar ataxia: a randomised, double-blind, placebo-controlled trial

BACKGROUND Our previous study in patients with cerebellar ataxias of different causes showed significant benefit of riluzole after 8 weeks. We aimed to confirm these results in patients with spinocerebellar ataxia or Friedreichs ataxia in a 1-year trial. METHODS Patients with spinocerebellar ataxia or Friedreichs ataxia (2:1 ratio) from three Italian neurogenetic units were enrolled in this multicentre, double-blind, placebo-controlled trial, and randomly assigned to riluzole (50 mg orally, twice daily) or placebo for 12 months. The randomisation list was computer-generated and a centralised randomisation system was implemented. Participants and assessing neurologists were masked to treatment allocation. The primary endpoint was the proportion of patients with improved Scale for the Assessment and Rating of Ataxia (SARA) score (a drop of at least one point) at 12 months. An intention-to-treat analysis was done. This trial is registered at ClinicalTrials.gov, number NCT01104649. FINDINGS Between May 22, 2010, and Feb 25, 2013, 60 patients were enrolled. Two patients in the riluzole group and three in the placebo group withdrew their consent before receiving treatment, so the intention-to-treat analysis was done on 55 patients (19 with spinocerebellar ataxia and nine with Friedreichs ataxia in the riluzole group, and 19 with spinocerebellar ataxia and eight with Friedreichs ataxia in the placebo group). The proportion with decreased SARA score was 14 (50%) of 28 patients in the riluzole group versus three (11%) of 27 in the placebo group (OR 8·00, 95% CI 1·95-32·83; p=0·002). No severe adverse events were recorded. In the riluzole group, two patients had an increase in liver enzymes (less than two times above normal limits). In two participants in the riluzole group and two participants in the placebo group, sporadic mild adverse events were reported. INTERPRETATION Our findings lend support to the idea that riluzole could be a treatment for cerebellar ataxia. Longer studies and disease-specific trials are needed to confirm whether these findings can be applied in clinical practice. FUNDING Agenzia Italiana del Farmaco.

HLA-LINKED SPINOCEREBELLAR ATAXIA - A CLINICAL AND GENETIC-STUDY OF LARGE ITALIAN KINDREDS

Five families with late onset autosomal dominant spinocerebellar ataxia, were studied. Linkage between the disease and HLA loci on the short arm of chromosome 6 was shown in the two largest pedigrees. Clinical study of 26 patients and neuropathological study in one are reported. The disease was characterized by cerebellar and pyramidal involvement variably associated with cranial nerve and peripheral nervous system disorders. A remarkable concordance of the main clinical features was observed in patients with similar disease duration. Comparison with previous reports of HLA‐linked spinocerebellar ataxia kindreds showed differences in clinical phenotypes. Although these might be due to genetic variation, the hypothesis is suggested that the phenotype might appear more homogeneous if disease duration is taken into account.

AZT-induced mitochondrial myopathy

Histochemical, electron microscopy and biochemical studies were performed on muscle biopsy specimens from 11 AIDS patients treated with zidovudine. A peculiar association of structural abnormalities and mitochondrial dysfunction was found. Focal cytochrome c oxidase (COX) deficiency was evident in muscle sections from 9 patients, 8 of whom had received long-term treatment while one had been treated for 1 month only. Electron microscopy showed changes in number, size and structure of mitochondria. Biochemical studies proved partial COX and succinate cytochrome c reductase (SCR) deficiency in 4 patients; one patient had only reduced SCR activity. Our data confirm that AZT therapy can cause toxic myopathy with mitochondrial dysfunction.SommarioSono state esaminate le biipsie muscolari di 11 pazienti affetti da AIDS in trattamento con Zidovudina. Si è riscontrato una caratteristica associazione di anormalità strutturali delle fibre e di disfunzione mitocondriale.Un deficit focale di citocromo c ossidasi (COX) era presente nelle sezioni muscolari di nove pazienti, otto dei quali avevano subito un trattamento di lunga durata, mentre uno era stato trattato solo per un mese.La microscopia elettronica mostrava modificazioni della struttura, del numero e volume dei mitocrondri. Le indagini biochimiche hanno evidenziato un deficit parziale di COX e di succinato citocromo c reduttasi (SCR) di quattro pazienti, mentre in un paziente riduzione di attività della sola SCR. I nostri dati confermano che la terapia con AZT può causare una miopatia tossica con disfunzione dei mitocondri.

Impaired VEP after photostress response in multiple sclerosis patients previously affected by optic neuritis.

The aim of our work was to evaluate if an optic nerve involvement (multiple sclerosis patients previously affected by optic neuritis) may induce any change in visual evoked potential (VEP) after photostress response. VEP in basal conditions and after photostress were assessed in 10 patients with defined multiple sclerosis without a history of optic neuritis (MSWO); in 14 patients with defined multiple sclerosis previously affected by optic neuritis but with complete recovery of the visual acuity (MSON) and in 14 age-matched controls. In order to complete the investigation of the retinal function, Transient Pattern electroretinogram (PERG) and steady-state focal-ERG (counterphased gratings presented at 8 Hz in the macular region) were performed in MSON patients only. In MSWO eyes VEP parameters in basal condition and after photostress did not undergo significant changes compared to controls (ANOVA; P > 0.05). In MSON eyes we observed basal VEP with delayed P100 peak latency and reduced N75-P100 amplitude when compared with the control ones (P < 0.01). In MSON eyes the parameters of VEP after photostress underwent large changes and longer recovery time (RT) than in control and MSWO eyes (P < 0.01). In addition; in MSON eyes we found increased transient PERG P50 latency (P < 0.01) and reduced P50-N95 amplitude (P < 0.01); Focal-ERG (that displays a major component at 16 Hz; 2nd harmonic:2P) with reduced 2P amplitudes and delayed 2P phases (P < 0.01). Our results indicate that patients previously affected by optic neuritis present an abnormal VEP after photostress response and this may be ascribed predominantly to an involvement of the inner retinal layers as indicated by the concomitant impairment of PERG and focal-ERG responses.

A one-year study on the pharmacodynamic profile of interferon-β1a in MS

Interferon (IFN)-beta1a induction of neopterin and beta2-microglobulin (beta2-MG) were evaluated over 1 year in patients with MS. Neopterin and beta2-MG levels peaked 24 to 48 hours after weekly injections of IFNbeta1a over the entire study period. Predose levels of neopterin decreased significantly, consistent with a long-term decrease in IFNgamma expression and macrophage activation during IFNbeta-1a treatment. Predose levels of beta2-MG increased, the significance of which is as yet unclear.

Genetic fitness in Huntington's Disease and Spinocerebellar Ataxia 1: a population genetics model for CAG repeat expansions

An analysis of genetic fitness was performed in Huntingtons Disease (HD) and Spinocerebellar Ataxia 1 (SCA1) families. Two partially overlapping samples were used: clinically defined HD and SCA1 patients from families ascertained in definite geographical areas, and molecularly typed carriers of HD and SCA1 mutations (CAG trinucleotide expansions). In both cases, a control group of normal relatives was used. HD and SCA1 patients born before 1915–20 had more children than normal controls. Carriers of HD and SCA1 mutations, all in the low/medium expansion range (37–49 and 47–54 CAG repeats respectively), had a higher number of children than controls up to more recent times (1935–1950). The reproduction of heterozygotes for large expansions could be analysed only in subjects born after 1950 and provided indirect evidence of a lower than normal number of children. The above results fit a model based on a differential fitness according to the degree of expansion. Such a model predicts that 1) up to relatively recently the frequency of alleles in the low/medium range has been maintained or even increased by the increased fitness of their carriers, as well as by new mutations, and 2) the frequency of large expansions, part of which are lost at each generation, is maintained through further expansions of alleles in the low/medium expansion range. The implications of such a model on linkage disequilibrium and the possible spread of these diseases in future generations are discussed.