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

Huntingtin polyQ Mutation Impairs the 17\u3b2-Estradiol/Neuroglobin Pathway Devoted to Neuron Survival

Among several mechanisms underlying the well-known trophic and protective effects of 17\u3b2-estradiol (E2) in the brain, we recently reported that E2 induces the up-regulation of two anti-apoptotic and neuroprotectant proteins: huntingtin (HTT) and neuroglobin (NGB). Here, we investigate the role of this up-regulation. The obtained results indicate that E2 promotes NGB-HTT association, induces the localization of the complex at the mitochondria, and protects SK-N-BE neuroblastoma cells and murine striatal cells, which express wild-type HTT (i.e., polyQ7), against H2O2-induced apoptosis. All E2 effects were completely abolished in HTT-knocked out SK-N-BE cells and in striatal neurons expressing the mutated form of HTT (mHTT; i.e., polyQ111) typical of Huntington\u2019s disease (HD). As a whole, these data provide a new function of wild-type HTT which drives E2-induced NGB in mitochondria modulating NGB anti-apoptotic activity. This new function is lost by HTT polyQ pathological expansion. These data evidence the existence of a novel E2/HTT/NGB neuroprotective axis that may play a relevant role in the development of HD therapeutics

Genes involved in regulation of stem cell properties: studies on their expression in a small cohort of neuroblast oma patients

Cancer stem cells have been isolated from many tumors. Several evidences prove that neuroblastoma contains its own stem cell-like cancer cells. We chose to analyze 20 neuroblastoma tumor samples in the expression of 13 genes involved in the regulation of stem cell properties to evaluate if their misregulation could have a clinical relevance. In several specimens we detected the expression of genes belonging to the OCT3/SOX2/NANOG/KLF4 core circuitry that acts at the highest level in regulating stem cell biology. This result is in agreement with studies showing the existence of malignant stem cells in neuroblastoma. We also observed differences in the expression of some stemness-related genes that may be useful for developing new prognostic analyses. In fact, preliminary data suggests that the presence/absence of UTF1 along with differences in BMI1 mRNA levels could distinguish low grade neuroblastomas from IV stage tumors

High grade glioblastoma is associated with aberrant expression of ZFP57, a protein involved in gene imprinting, and of CPT1A and CPT1C that regulate fatty acid metabolism

The diagnosis of glioblastoma is still based on tumor histology, but emerging molecular diagnosis is becoming an important part of glioblastoma classification

<i>R</i>. <i>graveolens</i> a.e. is able to induce ERK1/2 phosphorylation in glioma and in A1 proliferating neural cells.

<p>Western blotting detection of p-ERK1/2 and ERK1/2 proteins in C6 glioma cells (A), in U138 glioma cells (B), in A1 cells (C) and in U87MG (D) treated with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 5, 10, 30 and 60 minutes. Two specific bands are observed respectively at 44 and 42 kDa. Each blot is representative of three separate experiments. The graphs show the relative quantitation of p-ERK1/2 and ERK1/2 in the different cell lines. Data are expressed as ratios of p-ERK/ERK. Asterisks represent p<0.05 <i>vs</i> controls. (G-H) MTT assay in U87MG cells (E), A1 cells (F), C6 glioma cells (G) and U138 cells (H) treated for 48 hours with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (R48), 10μM PD98059 (PD) or in combination (PD+R48); *p<0.01 <i>vs</i> control conditions.°p<0.05 <i>vs</i> R48.</p

Rutin does not influence proliferating A1 cells viability.

<p>Trypan blue exclusion test on proliferating A1 cells treated with or without (control) increasing concentrations of rutin (3μg/ml, 30μg/ml and 300μg/ml) for 24, 48 and 72h.</p

<i>R</i>. <i>graveolens</i> a.e. induces cell death in proliferating but not in differentiated A1 cells.

<p>(A) Microphotographs of the mouse mesencephalic embryonic cell line A1 mes c-myc (A1). They are proliferating/undifferentiated in the presence of serum (left panel) but acquire a neuronal phenotype upon serum withdrawal and stimulation with cAMP (right panel). (B) MTT assay on proliferating A1 cells in control conditions (♦) or treated with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (■) for 24, 48, 72 and 96 hours. *p<0.01 <i>vs</i> controls. (C) MTT assay on differentiated A1 cells in control conditions (♦) or treated with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (■) for 48 and 72 hours *p<0.01 <i>vs</i> controls. (D) Trypan blue exclusion test on proliferating A1 cells treated (light grey) or not (dark grey) with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 24, 48 and 72h; *p<0.01 <i>vs</i> controls. (E) Trypan blue exclusion test on differentiated A1 cells treated (light gray) or not (dark gray) with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 48 and 72h; *p<0.01 <i>vs</i> controls.</p

<i>R</i>. <i>graveolens</i> extract induces cell death of U87MG human glioma cells.

<p>(A) MTT assay on proliferating U87MG human glioma cells treated with vehicle (♦) or with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. (■) for 24, 48, 72, 96h, *p<0,01 vs control conditions. (B) Trypan blue exclusion test on U87MG glioma cells treated (light gray) or not (dark gray) with 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 24, 48 and 72h; *<0,01 <i>vs</i> control conditions.</p

<i>R</i>. <i>graveolens</i> a.e. induces apoptosis in A1 cells.

<p>(A) Cell cycle was analyzed by means of Tali image-based cytometry on proliferating A1 cells in control conditions (dark grey) and 48h after 1mg/ml <i>R</i>. <i>graveolens</i> a.e. treatment (light grey) *p<0.01 <i>vs</i> controls (B) Number of apoptotic nuclei/100 cells treated (R48) or not (CTRL) with 1 mg/ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours. *p<0.01 <i>vs</i> controls. (C-E) Caspase 3 activity expressed as absorbance at 400 nm in A1 cells (C), U87MG cells (D) and C6 cells (E) treated with vehicle (CTRL), 1mg/ml <i>R</i>. <i>graveolens</i> a.e. for 24 (R24) or 48 (R48) hours, 10μM PD98059 in combination with ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours (PD+R48), 1μM wortmannin in combination with ml <i>R</i>. <i>graveolens</i> a.e. for 48 hours (W+R48) or the combination of the two inhibitors (PD+W+R48) for 48 hours. *p<0.01 <i>vs</i> control conditions.</p

Temozolomide and cisplatin affect viability of proliferating and differentiated A1.

<p>(A-B) MTT assay on proliferating A1 cells treated with increasing concentrations of temozolomide (A) and cisplatin (B) for 48 (♦) and 72 (■) hours; *p<0.01 <i>vs</i> controls; (C-D) MTT assay on differentiated A1 cells treated with increasing concentrations of temozolomide (C) and cisplatin (D) for 48 (♦) and 72 (■) hours; *p<0.01 <i>vs</i> controls.</p