Targeted Next-Generation Resequencing Panel in inherited ataxias

Hereditary cerebellar ataxias are a group of neurodegenerative diseases with clinical and genetic heterogeneity and variable age at onset. Next-Generation Sequencing technology has revolutionized the paradigm of clinical diagnostics, helping to end the long search for a genetic cause. A Targeted Next-Generation Resequencing Panel, containing 273 genes known or supposed to be related to hereditary ataxias, was performed in 39 index ataxic patients, recruited from the Ataxias Clinic of Neuroscience, Reproductive and Odontostomatological Science Department of the Federico II University. We identified eighteen probands out of 39 (46%) carrying pathogenic variants in ten different genes: SPG7 (five probands), PNPLA6 (three probands), SYNE1 (two probands), SETX (two probands), RNF216, ZFYVE26, ANO10, PMM2, ATP13A2 and TGM6 (one proband each). Variants of unknown significance were found in nine probands (23%). In the remaining twelve out of 39 cases (31%) no candidate variants or variants with unlikely pathogenicity were identified. Our findings suggest that after exclusion of repeat expansion ataxias, ataxia Targeted Next-Generation Resequencing Panel, could be a good first tier diagnostic line, when a specific single gene is not immediately suspected to be causative

Complex phenotype in an Italian family with a novel mutation in SPG3A.

Mutations in the SPG3A gene represent a significant cause of autosomal dominant hereditary spastic paraplegia with early onset and pure phenotype. We describe an Italian family manifesting a complex phenotype, characterized by cerebellar involvement in the proband and amyotrophic lateral sclerosis-like syndrome in her father, in association with a new mutation in SPG3A. Our findings further widen the notion of clinical heterogeneity in SPG3A mutations

Epoetin alfa increases frataxin production in Friedreich's ataxia without affecting hematocrit.

Objective of the study was to test the efficacy, safety, and tolerability of two single doses of Epoetin alfa in patients with Friedreich's ataxia. Ten patients were treated subcutaneously with 600 IU/kg for the first dose, and 3 months later with 1200 IU/kg. Epoetin alfa had no acute effect on frataxin, whereas a delayed and sustained increase in frataxin was evident at 3 months after the first dose (+35%; P < 0.05), and up to 6 months after the second dose (+54%; P < 0.001). The treatment was well tolerated and did not affect hematocrit, cardiac function, and neurological scale. Single high dose of Epoetin alfa can produce a considerably larger and sustained effect when compared with low doses and repeated administration schemes previously adopted. In addition, no hemoglobin increase was observed, and none of our patients required phlebotomy, indicating lack of erythropoietic effect of single high dose of erythropoietin. © 2010 Movement Disorder Society

Pulmonary disease caused by Mycobacterium marseillense, Italy

Mycobacteriummarseillense was recently described as a new species belonging to the Mycobacterium avium complex (MAC).We describe a case of pulmonary disease caused by M. marseillense in an immunocompetent patient. All strains isolated from the patient were preliminarily identified as M. intracellulare; however, a retrospective molecular analysis corrected the identification to M. marseillense

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

BACKGROUND: Friedreich's 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

Next Generation Molecular Diagnosis of Hereditary Spastic Paraplegias: An Italian Cross-Sectional Study

Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that they facilitate the diagnostic approach to HSP, but the power of NGS as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity-there are over 80 potential disease-associated genes-and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 4 years, 239 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we obtained a positive diagnostic yield of 29% (70/239), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 cases remained unsolved. This study is among the largest screenings of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy

Metabolic Ataxias in Adults.

Metabolic ataxias are rare. They usually start in the childhood and often have autosomal recessive inheritance. They may also present in adulthood. The diagnosis is important since some patients may be successfully managed with diet and treatments

Friedreich ataxia: 150 years of bench and bedside studies

Friedreich ataxia is the most frequent hereditary ataxia among Caucasians. Almost invariably, the disease is caused by homozygous GAA triplet repeat expansions in the first intron of the frataxin gene, FXN, whereas point mutations or deletions in conjunction with an expanded GAA tract account for the remaining cases. The expanded intronic alleles interfere with FXN transcription, decreasing the production of normally functioning frataxin protein to 5-20% of normal. Deficient frataxin levels result in excessive mitochondrial iron accumulation, reduced iron-sulfur clusters vital for mitochondrial energy production, and increased intracellular oxidative damage. To date, no cure has emerged and treatments remain largely supportive, despite extensive ongoing research and several rationale strategies have been attempted