Chiara Pane

A Combined Nucleic Acid and Protein Analysis in Friedreich Ataxia: Implications for Diagnosis, Pathogenesis and Clinical Trial Design

Background Friedreichs ataxia (FRDA) is the most common hereditary ataxia among caucasians. The molecular defect in FRDA is the trinucleotide GAA expansion in the first intron of the FXN gene, which encodes frataxin. No studies have yet reported frataxin protein and mRNA levels in a large cohort of FRDA patients, carriers and controls. Methodology/Principal Findings We enrolled 24 patients with classic FRDA phenotype (cFA), 6 late onset FRDA (LOFA), all homozygous for GAA expansion, 5 pFA cases who harbored the GAA expansion in compound heterozygosis with FXN point mutations (namely, p.I154F, c.482+3delA, p.R165P), 33 healthy expansion carriers, and 29 healthy controls. DNA was genotyped for GAA expansion, mRNA/FXN was quantified in real-time, and frataxin protein was measured using lateral-flow immunoassay in peripheral blood mononuclear cells (PBMCs). Mean residual levels of frataxin, compared to controls, were 35.8%, 65.6%, 33%, and 68.7% in cFA, LOFA, pFA and healthy carriers, respectively. Comparison of both cFA and pFA with controls resulted in 100% sensitivity and specificity, but there was overlap between LOFA, carriers and controls. Frataxin levels correlated inversely with GAA1 and GAA2 expansions, and directly with age at onset. Messenger RNA expression was reduced to 19.4% in cFA, 50.4% in LOFA, 52.7% in pFA, 53.0% in carriers, as compared to controls (p<0.0001). mRNA levels proved to be diagnostic when comparing cFA with controls resulting in 100% sensitivity and specificity. In cFA and LOFA patients mRNA levels correlated directly with protein levels and age at onset, and inversely with GAA1 and GAA2. Conclusion/Significance We report the first explorative study on combined frataxin and mRNA levels in PBMCs from a cohort of FRDA patients, carriers and healthy individuals. Lateral-flow immunoassay differentiated cFA and pFA patients from controls, whereas determination of mRNA in q-PCR was sensitive and specific only in cFA.

Long-term effect of epoetin alfa on clinical and biochemical markers in friedreich ataxia

Friedreich ataxia is an autosomal recessive disease with no available therapy. Clinical trials with erythropoietin in Friedreich ataxia patients have yielded conflicting results, and the long‐term effect of the drug remains unknown.

Dimethyl fumarate mediates Nrf2-dependent mitochondrial biogenesis in mice and humans

The induction of mitochondrial biogenesis could potentially alleviate mitochondrial and muscle disease. We show here that dimethyl fumarate (DMF) dose-dependently induces mitochondrial biogenesis and function dosed to cells in vitro, and also dosed in vivo to mice and humans. The induction of mitochondrial gene expression is more dependent on DMFs target Nrf2 than hydroxycarboxylic acid receptor 2 (HCAR2). Thus, DMF induces mitochondrial biogenesis primarily through its action on Nrf2, and is the first drug demonstrated to increase mitochondrial biogenesis with in vivo human dosing. This is the first demonstration that mitochondrial biogenesis is deficient in Multiple Sclerosis patients, which could have implications for MS pathophysiology and therapy. The observation that DMF stimulates mitochondrial biogenesis, gene expression and function suggests that it could be considered for mitochondrial disease therapy and/or therapy in muscle disease in which mitochondrial function is important.

The Multiple Faces of Spinocerebellar Ataxia type 2

Spinocerebellar ataxia type 2 (SCA2) is among the most common forms of autosomal dominant ataxias, accounting for 15% of the total families. Occurrence is higher in specific populations such as the Cuban and Southern Italian. The disease is caused by a CAG expansion in ATXN2 gene, leading to abnormal accumulation of the mutant protein, ataxin‐2, in intracellular inclusions. The clinical picture is mainly dominated by cerebellar ataxia, although a number of other neurological signs have been described, ranging from parkinsonism to motor neuron involvement, making the diagnosis frequently challenging for neurologists, particularly when information about the family history is not available. Although the functions of ataxin‐2 have not been completely elucidated, the protein is involved in mRNA processing and control of translation. Recently, it has also been shown that the size of the CAG repeat in normal alleles represents a risk factor for ALS, suggesting that ataxin‐2 plays a fundamental role in maintenance of neuronal homeostasis.

The EDSS integration with the Brief International Cognitive Assessment for Multiple Sclerosis and orientation tests

Objective: Despite cognitive tests have been validated in multiple sclerosis (MS), a neuropsychological evaluation is not implemented in the Expanded Disability Status Scale (EDSS) scoring. Methods: We used the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) and orientation tests (OTs) to measure the cerebral functional system (CFS) score and to evaluate its impact on the EDSS. We compared EDSS calculated as usual (Native-EDSS) and after the use of the BICAMS and OT (NPS-EDSS). Results: We tested 604 MS patients with BICAMS, OTs, and EDSS. In all, 384 patients (63.6%) had at least one altered test at the BICAMS. Older age, lower education, higher Native-EDSS, and male gender were independently associated with at least one impaired BICAMS test. Native-EDSS was different from NPS-EDSS (−0.112; p < 0.001) in 99 patients (16%). When considering patients with a Native-EDSS ⩽ 4.0, the proportion of miscalculated EDSS was 25%. Conclusion: The use of brief neuropsychological tests leads to a more accurate CFS assessment in two-thirds of MS patients, and a more accurate EDSS calculation in 25% of patients with a score ⩽4.0. This may help clinicians to better recognize cognitive impairment in everyday clinical practice, especially in the case of isolated cognitive worsening.

Lamivudine and fingolimod co-administration in two patients with multiple sclerosis and occult hepatitis B virus infection

Multiple sclerosis (MS) is a chronic autoimmune, neurodegenerative disease affecting up to 2.5 million people worldwide, with an estimated prevalence of 1 in 1000. Recently, Fingolimod, a sphingosine 1-phosphate receptor modulator, was approved for relapsing–remitting MS (RRMS) patients non-responders to first line disease modifying therapies (DMTs) or rapidly evolving severe MS. FTY prevents the egress of lymphocytes from lymph nodes. Circulating lymphocytes up to 200/mL are considered acceptable during treatment. Lower respiratory tract infections, and rare cases of severe infections with herpes viruses have been reported during fingolimod development [1]. Before FTY treatment, patients are usually screened for the presence of Varicella-zoster IgG antibodies, and for hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV). Natural history of HBV infection accounts for five different and non necessarily sequential phases: immune tolerant, immune reactive, inactive HBV carrier state, reactivation phase, and the occult hepatitis B virus infection (OBI) [2]. During OBI hepatitis B surface antigen (HBsAg), liver enzymes, and circulating HBV-DNA are usually normal or negative. Positivity for antibodies against core antigens, with or without antibodies against HBsAg are hallmarks of the OBI. The majority of OBI cases are secondary to HBV infection and show residual low or absent viremia level suppressed by strong immune response. Long-term maintenance of an active anti-viral T cell response, mainly due to central memory T cells (CD45RO), is necessary several years after clinical recovery from acute hepatitis B for keeping the persisting virus under control [3]. CD45RO are typically decreased after fingolimod treatment and this may pose a serious risk of HBV reactivation in OBI patients [4]. Nevertheless, no guidelines are available for the screening and management of OBI in MS patients when starting an immunosuppressive therapy with fingolimod or other DMTs.

Stability of erythropoietin repackaging in polypropylene syringes for clinical use

Introduction: Epoetin alfa (Eprex®) is a subcutaneous, injectable formulation of short half-life recombinant human erythropoietin (rHuEPO). To current knowledge there are no published studies regarding the stability of rHuEPO once repackaging occurs (r-EPO) for clinical trial purposes. Materials and methods: We assessed EPO concentration in Eprex® and r-EPO syringes at 0, 60, 90, and 120 days after repackaging in polypropylene syringes. R-EPO was administered to 56 patients taking part in a clinical trial in Friedreich Ataxia. Serum EPO levels were measured at baseline and 48 h after r-EPO administration. Results: No differences were found between r-EPO and Eprex® syringes, but both globally decreased in total EPO content during storage at 4 °C. Patients receiving r-EPO had similar levels in EPO content as expected from previous trials in Friedreich Ataxia and from pharmacokinetics studies in healthy volunteers. Discussion: We demonstrate that repackaging of EPO does not alter its concentration if compared to the original product (Eprex®). This is true both for repackaging procedures and for the stability in polypropylene tubes. The expiration date of r-EPO can be extended from 1 to 4 months after repackaging, in accordance with pharmacopeia rules.

Mobitz type I and II atrioventricular blocks during fingolimod therapy

We investigated patients who showed a second-degree atrioventricular block (S-AVB) after the first fingolimod administration. We observed six patients with S-AVB, three Mobitz type I, and three type II. Monitoring continued on the second day for all patients. Three patients showed persistence of the S-AVB, with resolution on the second or third day. One patient had a persistent S-AVB up to the fourth day when fingolimod was discontinued. We conclude that Mobitz type II S-AVB is possible during fingolimod therapy. Patients with S-AVB could be monitored until resolution of the S-AVBs, as these may persist several days after the first fingolimod administration.

Emotion Recognition and Psychological Comorbidity in Friedreich’s Ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive disease presenting with ataxia, corticospinal signs, peripheral neuropathy, and cardiac abnormalities. Little effort has been made to understand the psychological and emotional burden of the disease. The aim of our study was to measure patients’ ability to recognize emotions using visual and non-verbal auditory hints, and to correlate this ability with psychological, neuropsychological, and neurological variables. We included 20 patients with FRDA, and 20 age, sex, and education matched healthy controls (HC). We measured emotion recognition using the Geneva Emotion Recognition Test (GERT). Neuropsychological status was assessed measuring memory, executive functions, and prosopagnosia. Psychological tests were Patient Health Questionnaire-9 (PHQ-9), State Trait Anxiety Inventory–state/−trait (STAI-S/−T), and Structured Clinical Interview for DSM Disorders II. FRDA patients scored worse at the global assessment and showed impaired immediate visuospatial memory and executive functions. Patients presented lower STAI-S scores, and similar scores at the STAI-T, and PHQ-9 as compared to HC. Three patients were identified with personality disorders. Emotion recognition was impaired in FRDA with 29% reduction at the total GERT score (95% CI − 44.8%, − 12.6%; p < 0.001; Cohen’s d = 1.2). Variables associated with poor GERT scores were the 10/36 spatial recall test, the Ray Auditory Verbal Learning Test, the Montreal Cognitive Assessment, and the STAI-T (R2 = 0.906; p < 0.001). FRDA patients have impaired emotion recognition that may be secondary to neuropsychological impairment. Depression and anxiety were not higher in FRDA as compared to HC and should not be considered as part of the disease.

Dissociative identity disorder: Restoration of executive functions after switch from alter to host personality: Letters to the Editor

D ISSOCIATIVE IDENTITY DISORDER (DID) is a psychiatric illness presenting with two or more personalities in the same patient, causing an alteration in the sense of self, with amnesia for events of life and personal information. We report a diagnosed case of DID in a 31-year-old woman. The patient had had 13 years of education, was employed full-time, was the mother of a 10-year-old child, was not married, and was living with her parents in a working-class family with no known psychiatric disorders. She provided informed consent for publication. During a bus trip, she had experienced a near-accident without physical trauma. She was brought to a local hospital, where a spatial–temporal–bodily disorientation was found together with a widespread retrograde amnesia (from the age of 14 to 31 years) and anterograde amnesia. Three days later, she was able to fixate new memories, and was taken to our clinic for further evaluations. She showed a retrograde amnesia, and could not remember the nearaccident with frequent depersonalizations/-realizations. Her mother and sisters described her personality as different from usual. She was anhedonic, apathetic, behaved as a teenager, with non-fluent and aprosodic voluntary speech. Medical and psychiatric history, brain magnetic resonance imaging, electroencephalogram, blood exams, and drug and infectious disease screening were negative. Neuropsychological assessment showed normal global functioning, aboveaverage working memory, and impaired selective attention. Altered tests were the Attentional Matrices (27/60), Semantic Fluency (3.5), Frontal Assessment Battery (14/18), the Trail Making Test B (179 s), and the Stroop test (32.5 s). Three weeks later, without any specific intervention, she abruptly recovered her retrograde memory from her first memories until the near-accident. We re-examined her within 6 h and found that memories from the near-accident were lost, including her first hospitalization and our examination. Relatives confirmed she had switched to her usual personality. Neuropsychological tests were repeated and showed normal selective attention abilities, with normal or above normal scores, and above normal global executive functions. Previously altered tests were now normal: the Attentive Matrices (48/60), Semantic Fluency (11.75), Frontal Assessment Battery (17/18), the Trail Making Test B (140 s), and the Stroop test (26.5 s). With the limitations of a single case report, personality switch can affect executive functions, and this can be measured with available scales. A few cases linking executive function to dissociative disorders have been previously reported, but they did not explore the effect of personality switch on neuropsychological performance. Awareness of this may be helpful for clinicians, who should not consider these symptoms as secondary to organic diseases.