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Background: The mechanisms of genotype-phenotype interaction in Familiar Hemiplegic migraine type 2 (FHM2) are still far from clear. Different ATP1A2 mutations have been described, with a spectrum of phenotypes ranging from mild to severe. No genotype-phenotype correlations have been attempted. Case presentation: We describe an Italian family with FHM and a missense ATP1A2 variant (L425H) not previously described. The clinical picture was mild in all the affected members. Conclusions: Co-segregation of the variant with the aura phenotype was complete in this family, suggesting a 100% penetrance. In silico protein prediction softwares indicate that this variant may change the 3D structure of ATPA1A2 at the cytoplasmic loop between the two central transmembrane helices. Milder FHM phenotypes are rarely reported in literature, likely because case reports are biased towards the most severe phenotypes, with milder forms possibly misdiagnosed as sporadic migraine with aura forms (MAs), even with complex auras. Further studies taking into account intra-familiar variability and functional consequences on the channel protein may help clarify genotype-phenotype correlations. [ABSTRACT FROM AUTHOR]

Background: Sporadic Hemiplegic Migraine is a rare form of migraine headache. Mutations in three different genes, two ion-channel genes and one encoding an ATP exchanger, CACNA1A, ATP1A2 and SCN1A are all responsible for the FHM phenotype, thus indicating a genetic heterogeneity for this disorder. Here, we described a de novo exonic duplication of ATP1A2 in an Italian patient with Hemiplegic Migraine. Case presentation: We describe the case of a young woman (33 year old) who suffered from the age of 8 years of episodic weakness of the limbs, associated to other subjective and objective features. From aged 25, she developed neurological symptoms, like dizziness, blurred vision and an MRI scan revealed aspecific peritrigonal white matter hyperintensities. Aged 32 she suffered of right hemisomatic sudden-onset paresthesias, hypoesthesia and hyposthenia and the patient was genetically investigated for sporadic hemiplegic migraine. Conclusions: Here we report, for the first time, an exonic duplication in the ATP1A2 associated with hemiplegic migraine. The variation identified involves exon 21 of the ATP1A2 and is expected to alter the function of the alpha(2) subunit of the Na(+)/K(+) pump; the de novo nature of the duplication further supports its pathogenic role. To date, no other CNVs have been described in the ATP1A2 but only point mutations are reported. The novel mutation may result impaired M9 transmembrane domain, in a loss-of-function of the alpha(2) Na(+)/K(+)-ATPase with glutamate accumulation, alteration of synaptic function and neurotransmission. [ABSTRACT FROM AUTHOR]

Background: Amplicon-based targeted resequencing is a commonly adopted solution for next-generation sequencing applications focused on specific genomic regions. The reliability of such approaches rests on the high specificity and deep coverage, although sequencing artifacts attributable to PCR-like amplification can be encountered. Between these artifacts, allele drop-out, which is the preferential amplification of one allele, causes an artificial increase in homozygosity when heterozygous mutations fall on a primer pairing region. Here, a procedure to manage such artifacts, based on a pipeline composed of two steps of alignment and variant calling, is proposed. This methodology has been compared to the Illumina Custom Amplicon workflow, available on Illumina MiSeq, on the analysis of data obtained with four newly designed TruSeq Custom Amplicon gene panels. Results: Four gene panels, specific for Parkinson disease, for Intracerebral Hemorrhage Diseases (COL4A1 and COL4A2 genes) and for Familial Hemiplegic Migraine (CACNA1A and ATP1A2 genes) were designed. A total of 119 samples were re-sequenced with Illumina MiSeq sequencer and panel characterization in terms of coverage, number of variants found and allele drop-out potential impact has been carried out. Results show that 14 % of identified variants is potentially affected by allele drop-out artifacts and that both the Custom Amplicon workflow and the procedure proposed here could correctly identify them. Furthermore, a more complex configuration in presence of twomutations was simulated in silico. In this configuration, our proposed methodology outperforms Custom Amplicon workflow, being able to correctly identify two mutations in all the studied configurations. Conclusions: Allele drop-out plays a crucial role in amplicon-based targeted re-sequencing and specific procedures in data analysis of amplicon data should be adopted. Although a consensus has been established in the elimination of primer sequences from aligned data (e.g., via primer sequence trimming or soft clipping), more complex configurations need to be managed in order to increase the retrieved information from available data. Our method shows how to manage one of these complex configurations, when two mutations occur. [ABSTRACT FROM AUTHOR]

Channelopathies are a heterogeneous group of neurological disorders resulting from dysfunction of ion channels located in cell membranes and organelles. The clinical scenario is broad and symptoms such as generalized epilepsy (with or without fever), migraine (with or without aura), episodic ataxia and periodic muscle paralysis are some of the best known consequences of gain- or loss-of-function mutations in ion channels. We review the main clinical effects of ion channel mutations associated with a significant impact on migraine headache. Given the increasing and evolving use of genetic analysis in migraine research--greater emphasis is now placed on genetic markers of dysfunctional biological systems--we also show how novel information in rare monogenic forms of migraine might help to clarify the disease mechanisms in the general population of migraineurs. Next-generation sequencing (NGS) and more accurate and precise phenotyping strategies are expected to further increase understanding of migraine pathophysiology and genetics. [ABSTRACT FROM AUTHOR]

The Brown-Vialetto-Van Laere syndrome (BVVLS) is a rare neurological disorder characterized by progressive pontobulbar palsy, sensorineural deafness and mixed spinal and upper motor neuropathy. Mutations in the C20orf54 gene have been linked to the disease and recently we reported the first Italian case of a BVVLS family with an intriguing C20orf54 genotype. However, the pathomechanisms underlying BVVLS are still unknown. Here we present the particular disease course with partial response to immunosuppressive therapy of our BVVLS patient for whom we hypothesize that dysimmune factors may have played a role in disease physiopathology. [ABSTRACT FROM AUTHOR]

Objectives Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease mainly involving cortical and spinal motor neurones. Molecular studies have recently identified different mutations in the ubiquilin-2 (UBQLN2) gene as causative of a familial form of X-linked ALS, 90% penetrant in women. The aim of our study was to analyse the UBQLN2 gene in a large cohort of patients with familial (FALS) and sporadic (SALS) amyotrophic lateral sclerosis, with or without frontotemporal dementia (FTD), and in patients with FTD. Methods We analysed the UBQLN2 gene in 819 SALS cases, 226 FALS cases, 53 ALS-FTD patients, and 63 patients with a clinical record of FTD. Molecular analysis of the entire coding sequence was carried out in all FALS and ALS-FTD patients, while SALS and FTD patients were analysed specifically for the genomic region coding for the PXX repeat tract. Healthy controls were 845 anonymous blood donors and were screened for the PXX repeat region only. Results We found five different variants in the UBQLN2 gene in five unrelated ALS patients. Three variants, including two novel ones, involved a proline residue in the PXX repeat region and were found in three FALS cases. The other two were novel variants, identified in one FALS and one SALS patient. None of these variants was present in controls, while one control carried a new heterozygous variant. Conclusions Our data support the role of the UBQLN2 gene in the pathogenesis of FALS, being conversely a rare genetic cause in SALS even when complicated by FTD. [ABSTRACT FROM AUTHOR]

Several methods have been developed to detect common prothrombotic mutations, including factor V Leiden (G1691), prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677C. In this study, we compared the accuracy of three different molecular techniques, i.e.: (1) restriction enzyme digestion (RFLP), (2) real time with hybridization probes and final melting curve (Fluorescence Resonance Energy Transfer, FRET), and (3) real time with hydrolysis probes (TaqMan). Sequencing was used as the reference standard. Our data showed that RFLPs analysis for the detection of prothrombotic mutations, albeit easy-to-perform, had a limited reliability for assessing correct genotypes. FRET analysis displayed higher resolution than RFLPs. Additionally, FRET analysis was faster and less tedious than sequencing. [ABSTRACT FROM AUTHOR]

The article reports on the identification of novel TUBA4A gene variants in patients with sporadic amyotrophic lateral scierosis (SALS) in Italy. Topics discussed include the cause and clinical characteristics of ALS, the result of functional studies on TUBA4A mutations, and details of the findings of the mutational sceening of the Italian patients.

• SARS-CoV-2 in frontal cortex may be detected only by extremely sensitive method. • Genes associated to hypoxia are down-regulated in COVID-19 patients. • Up-regulation of hemoglobin genes may act as response system to mTOR activity. • Oxidative stress, hydrogen peroxide processed are involved pathways. SARS-Cov-2 infection is frequently associated with Nervous System manifestations. However, it is not clear how SARS-CoV-2 can cause neurological dysfunctions and which molecular processes are affected in the brain. In this work, we examined the frontal cortex tissue of patients who died of COVID-19 for the presence of SARS-CoV-2, comparing qRT-PCR with ddPCR. We also investigated the transcriptomic profile of frontal cortex from COVID-19 patients and matched controls by RNA-seq analysis to characterize the transcriptional signature. Our data showed that SARS-CoV-2 could be detected by ddPCR in 8 (88%) of 9 examined samples while by qRT-PCR in one case only (11%). Transcriptomic analysis revealed that 11 genes (10 mRNAs and 1 lncRNA) were differential expressed when frontal cortex of COVID-19 patients were compared to controls. These genes fall into categories including hypoxia, hemoglobin-stabilizing protein, hydrogen peroxide processes. This work demonstrated that the quantity of viral RNA in frontal cortex is minimal and it can be detected only with a very sensitive method (ddPCR). Thus, it is likely that SARS-CoV-2 does not actively infect and replicate in the brain; its topography within encephalic structures remains uncertain. Moreover, COVID-19 may have a role on brain gene expression, since we observed an important downregulation of genes associated to hypoxia inducting factor system (HIF) that may inhibit the capacity of defense system during infection and oxigen deprivation, showing that hypoxia, well known multi organ condition associated to COVID-19, also marked the brain. [ABSTRACT FROM AUTHOR]

Abstract: GM1 gangliosidosis and Morquio B syndrome, both arising from beta-galactosidase (GLB1) deficiency, are very rare lysosomal storage diseases with an incidence of about 1:100,000–1:200,000 live births worldwide. Here we report the beta-galactosidase gene (GLB1) mutation analysis of 21 unrelated GM1 gangliosidosis patients, and of 4 Morquio B patients, of whom two are brothers. Clinical features of the patients were collected and compared with those in literature. In silico analyses were performed by standard alignments tools and by an improved version of GLB1 three-dimensional models. The analysed cohort includes remarkable cases. One patient with GM1 gangliosidosis had a triple X syndrome. One patient with juvenile GM1 gangliosidosis was homozygous for a mutation previously identified in Morquio type B. A patient with infantile GM1 gangliosidosis carried a complex GLB1 allele harbouring two genetic variants leading to p.R68W and p.R109W amino acid changes, in trans with the known p.R148C mutation. Molecular analysis showed 27 mutations, 9 of which are new: 5 missense, 3 microdeletions and a nonsense mutation. We also identified four new genetic variants with a predicted polymorphic nature that was further investigated by in silico analyses. Three-dimensional structural analysis of GLB1 homology models including the new missense mutations and the p.R68W and p.R109W amino acid changes showed that all the amino acid replacements affected the resulting protein structures in different ways, from changes in polarity to folding alterations. Genetic and clinical associations led us to undertake a critical review of the classifications of late-onset GM1 gangliosidosis and Morquio B disease. [Copyright &y& Elsevier]

Abstract: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative multifactorial disease characterized, like other diseases such as Alzheimer''s disease (AD), Parkinson''s disease (PD) or frontotemporal dementia (FTD), by the degeneration of specific neuronal cell populations. Motor neuron loss is distinctive of ALS. However, the causes of onset and progression of motor neuron death are still largely unknown. In about 2% of all cases, mutations in the gene encoding for the Cu/Zn superoxide dismutase (SOD1) are implicated in the disease. Several alterations in the expression or activation of cell cycle proteins have been described in the neurodegenerative diseases and related to cell death. In this work we show that mutant SOD1 can alter cell cycle in a cellular model of ALS. Our findings suggest that modifications in the cell cycle progression could be due to an increased interaction between mutant G93A SOD1 and Bcl-2 through the cyclins regulator p27. As previously described in post mitotic neurons, cell cycle alterations could fatally lead to cell death. [Copyright &y& Elsevier]

Abstract: The mutated Cu,Zn-superoxide dismutase gene (SOD1) (E.C. No. 1.15.1.1) is generally recognized as a pathological cause of 20% of the familial form of Amyotrophic Lateral Sclerosis (ALS). However, several pieces of evidence also show that wild-type SOD1, under conditions of cellular stress, is implicated in a significant fraction of sporadic ALS cases, which represent 90% of ALS patients. Herein, we describe an abnormally high level of SOD1 transcript in spinal cord, brain stem and lymphocytes of sporadic ALS patients. Protein expression studies show a similar or lower amount of SOD1 in affected brain areas and lymphocytes, respectively. No differences are found in brain regions (cerebellum and non-motor cerebral cortex) not involved in the ALS neurodegenerative processes. In this report, cell and disease specificity are shown since no mRNA SOD1 increase is observed in sporadic ALS fibroblasts or in lymphocytes of patients affected by Alzheimer''s disease. These findings provide new insight and understanding of the pathologic causes of sporadic forms of ALS and allow a possible explanation for the molecular involvement of wild-type SOD1. [Copyright &y& Elsevier]

Abstract: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease associated with a positive familial history in 5–10% of ALS cases. Mutations in the superoxide dismutase-1 (SOD1) gene have been found in 12%–23% of patients diagnosed with familial ALS. Here we report a novel mutation in exon 4 of SOD1 gene in a 55-year-old ALS patient belonging to a large Italian family with ALS first clinically described in 1968. In the family the clinical presentation was characterized by relatively early age of onset, spinal onset with proximal distribution weakness, bulbar involvement and a rapid disease course. Molecular analysis showed a heterozygous mutation at codon 106 resulting in a substitution of phenylalanine for leucine in the SOD1 protein (L106F). In analogy with the previously reported L106V mutation, we propose that the L106F causes a relevant destabilization of the protein chain around the mutation site, able to affect the SOD1 monomer and dimer structures suggesting a pathogenic role for this novel mutation. [Copyright &y& Elsevier]

Abstract: Mutations in the prion-like domain (PrLD) of hnRNPA1 and A2/B1 genes were recently identified in 2 families with inclusion body myopathy associated with Paget disease of bone, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis, and in ALS patients. These mutations were shown to increase the propensity of hnRNPA1 and A2/B1 proteins, which are TDP-43–binding partners, to self-aggregate. hnRNPA3 protein contains a similar PrLD and was recently described in the p62-positive/TDP-43–negative inclusions in affected tissues of C9orf72-mutated ALS/FTD patients. We screened hnRNPA1, A2/B1, and A3 genes in a cohort of 113 familial ALS (FALS) individuals without mutations in other known ALS-causative genes. We extended our analysis to 108 FALS with mutations in other ALS-associated genes and to 622 sporadic cases by screening specifically the PrLDs of hnRNPA1, A2/B1, and A3. We failed to find variants in each cohort. Our results suggest that mutations in hnRNPA1, A2/B1, and A3 genes are a rare finding in ALS. [Copyright &y& Elsevier]

Abstract: A hexanucleotide repeat expansion (RE) in C9ORF72 gene was recently reported as the main cause of amyotrophic lateral sclerosis (ALS) and cases with frontotemporal dementia. We screened C9ORF72 in a large cohort of 259 familial ALS, 1275 sporadic ALS, and 862 control individuals of Italian descent. We found RE in 23.9% familial ALS, 5.1% sporadic ALS, and 0.2% controls. Two cases carried the RE together with mutations in other ALS-associated genes. The phenotype of RE carriers was characterized by bulbar-onset, shorter survival, and association with cognitive and behavioral impairment. Extrapyramidal and cerebellar signs were also observed in few patients. Genotype data revealed that 95% of RE carriers shared a restricted 10-single nucleotide polymorphism haplotype within the previously reported 20-single nucleotide polymorphism risk haplotype, detectable in only 27% of nonexpanded ALS cases and in 28% of controls, suggesting a common founder with cohorts of North European ancestry. Although C9ORF72 RE segregates with disease, the identification of RE both in controls and in patients carrying additional pathogenic mutations suggests that penetrance and phenotypic expression of C9ORF72 RE may depend on additional genetic risk factors. [Copyright &y& Elsevier]

Abstract: Mutations in valosin-containing protein (VCP) gene, already known to be associated with the multisystemic disorder, inclusion body myopathy with Paget''s disease and frontotemporal dementia (IBMPFD), have been recently found also in familial cases of amyotrophic lateral sclerosis (ALS). To further define the frequency of VCP mutations in ALS Italian population, we screened a cohort of 166 familial ALS and 14 ALS-frontotemporal dementia (FTD) individuals. We identified a previously reported synonymous mutation (c.2093A>C; p.Q568Q), 2 intronic variants (c.1749-14C>T; c.2085-3C>T), and 1 nucleotide change (c.2814G>T) in the 3′ untranslated region (UTR). Bioinformatical analyses predicted no changes in splicing process or microRNA binding sites. Our results do not confirm a main contribution of VCP gene to familial ALS in the Italian population. [Copyright &y& Elsevier]

Abstract: Motor neurons in amyotrophic lateral sclerosis (ALS) are characterized by the presence of inclusion bodies composed of intermediate filament (IF) proteins. Peripherin protein is as components of these inclusions and rare mutations in peripherin gene (PRPH) were identified in sporadic ALS cases. The aim of this study was to further define the spectrum of PRPH mutations in a cohort of 122 Italian ALS patients. We screened the coding sequence, the exon/intron boundaries, and the 5′–3′ un-translated regions (UTRs) in 122 ALS patients. Eighteen sequence variations were detected. Seven variants were not identified in a panel of at least 245 matched controls, including 2 missense variations, namely p.R133P and p.D141Y, each identified in one heterozygous patient. p.R133P was newly identified whereas p.D141Y was previously described in one homozygous sporadic ALS patient. These 2 variants were predicted to have a deleterious effect on protein structure or function. This work contributes to determine the role of PRPH gene variants in ALS. Further studies are necessary to define the mechanisms through which the mutant peripherin could cause ALS phenotype. [Copyright &y& Elsevier]