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Smith-Magenis syndrome (SMS) is a complex neurodevelopmental disorder with a birth incidence of 1:25,000. SMS is caused by haploinsufficiency of the retinoic acid-induced retinoic acid1 (RAI1) gene, determined by an interstitial deletion of ∼ 3.7 Mb (17p11.2, including the RAI1 gene) in 90 % of cases and a mutation on the RAI1 gene in only 10 % of cases. We generated and characterized a human pluripotent stem cell line (hIPSCs) derived from primary fibroblasts of a 17-year-old woman carrying a 17p11.2 deletion including the RAI1 gene.

Hypomyelinating leukodystrophies (HLD) are a group of heterogeneous genetic disorders characterized by a deficit in myelin deposition during brain development. Specifically, 4H-Leukodystrophy is a recessive disease due to biallelic mutations in the POLR3A gene, which encodes one of the subunits forming the catalytic core of RNA polymerase III (PolIII). The disease also presents non-neurological signs such as hypodontia and hypogonadotropic hypogonadism. Here, we report the generation of a human induced pluripotent stem cell (hiPSC) line from fibroblasts of the first identified carrier of the biallelic POLR3A variants c.1802 T > A and c.4072G > A.

Abstract Background This Phase 1 study evaluates the intra- and peritumoral administration by convection enhanced delivery (CED) of human recombinant Bone Morphogenetic Protein 4 (hrBMP4) – an inhibitory regulator of cancer stem cells (CSCs) – in recurrent glioblastoma. Methods In a 3 + 3 dose escalation design, over four to six days, fifteen recurrent glioblastoma patients received, by CED, one of five doses of hrBMP4 ranging from 0·5 to 18 mg. Patients were followed by periodic physical, neurological, blood testing, magnetic resonance imaging (MRI) and quality of life evaluations. The primary objective of this first-in-human study was to determine the safety, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of hrBMP4. Secondary objectives were to assess potential efficacy and systemic exposure to hrBMP4 upon intracerebral infusion. Results Intra- and peritumoral infusion of hrBMP4 was safe and well-tolerated. We observed no serious adverse events related to this drug. Neither MTD nor DLT were reached. Three patients had increased hrBMP4 serum levels at the end of infusion, which normalized within 4 weeks, without sign of toxicity. One patient showed partial response and two patients a complete (local) tumor response, which was maintained until the most recent follow-up, 57 and 30 months post-hrBMP4. Tumor growth was inhibited in areas permeated by hrBMP4. Conclusion Local delivery of hrBMP4 in and around recurring glioblastoma is safe and well-tolerated. Three patients responded to the treatment. A complete response and long-term survival occurred in two of them. This warrants further clinical studies on this novel treatment targeting glioblastoma CSCs. Trial registration : ClinicaTrials.gov identifier: NCT02869243.

Abstract Mitochondrial dysfunction has pleiotropic effects and is frequently caused by mitochondrial DNA mutations. However, factors such as significant variability in clinical manifestations make interpreting the pathogenicity of variants in the mitochondrial genome challenging. Here, we present APOGEE 2, a mitochondrially-centered ensemble method designed to improve the accuracy of pathogenicity predictions for interpreting missense mitochondrial variants. Built on the joint consensus recommendations by the American College of Medical Genetics and Genomics/Association for Molecular Pathology, APOGEE 2 features an improved machine learning method and a curated training set for enhanced performance metrics. It offers region-wise assessments of genome fragility and mechanistic analyses of specific amino acids that cause perceptible long-range effects on protein structure. With clinical and research use in mind, APOGEE 2 scores and pathogenicity probabilities are precompiled and available in MitImpact. APOGEE 2’s ability to address challenges in interpreting mitochondrial missense variants makes it an essential tool in the field of mitochondrial genetics.

Abstract Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.

Circadian rhythm impairment may play a role in Parkinson's disease (PD) pathophysiology. Recent literature associated circadian rhythm features to the risk of developing Parkinson and to its progression through stages. The association between the chronotype and the phenotype should be verified on a clinical and biological point of view. Herein we investigate the chronotype of a sample of 50 PD patients with the Morningness Eveningness Questionnaire and monitor their daily activity with a motion sensor embedded in a smartphone. Fibroblasts were collected from PD patients (n = 5) and from sex/age matched controls (n = 3) and tested for the circadian expression of clock genes (CLOCK, BMAL1, PER1, CRY1), and for cell morphology, proliferation, and death. Our results show an association between the chronotype and the PD phenotype. The most representative clinical chronotypes were “moderate morning” (56%), the “intermediate” (24%) and, in a minor part, the “definite morning” (16%). They differed for axial motor impairment, presence of motor fluctuations and quality of life (p

Familial Hypocalciuric Hypercalcemia (FHH1) is a rare autosomal dominant disease with low penetrance, caused by inactivating mutations of the calcium-sensing receptor (CaSR) gene, characterized by significant hypercalcemia, inappropriately normal serum PTH levels and a low urinary calcium level. Human induced pluripotent stem cells (hiPSCs) from a patient carrying a previously identified heterozygous mutation, a p.T972M amino acid substitution in cytoplasmic tail of CasR, were produced using a virus, xeno-free and non-integrative protocol.

KDM6A is the disease causative gene of type 2 Kabuki Syndrome, a rare multisystem disease; it is also a known cancer driver gene, with multiple somatic mutations found in a few cancer types. In this study, we looked at eleven missense variants in lung squamous cell carcinoma, one of the most common lung cancer subtypes, to see how they affect the KDM6A catalytic mechanisms. We found that they influence the interaction with histone H3 and the exposure of the trimethylated Lys27, which is critical for wild-type physiological function to varying degrees, by altering the conformational transition.

The human α7 neuronal nicotinic acetylcholine receptor gene (CHRNA7) is widely expressed in the central and peripheral nervous systems. This receptor is implicated in both brain development and adult neurogenesis thanks to its ability to mediate acetylcholine stimulus (Ach). Copy number variations (CNVs) of CHRNA7 gene have been identified in humans and are genetically linked to cognitive impairments associated with multiple disorders, including schizophrenia, bipolar disorder, epilepsy, Alzheimer’s disease, and others. Currently, α7 receptor analysis has been commonly performed in animal models due to the impossibility of direct investigation of the living human brain. But the use of model systems has shown that there are very large differences between humans and mice when researchers must study the CNVs and, in particular, the CNV of chromosome 15q13.3 where the CHRNA7 gene is present. In fact, human beings present genomic alterations as well as the presence of genes of recent origin that are not present in other model systems as well as they show a very heterogeneous symptomatology that is associated with both their genetic background and the environment where they live. To date, the induced pluripotent stem cells, obtained from patients carrying CNV in CHRNA7 gene, are a good in vitro model for studying the association of the α7 receptor to human diseases. In this review, we will outline the current state of hiPSCs technology applications in neurological diseases caused by CNVs in CHRNA7 gene. Furthermore, we will discuss some weaknesses that emerge from the overall analysis of the published articles.

Charcot-Marie-Tooth type 4B3 (CMT4B3) is a rare subtype of hereditary neuropathy associated with variants in the MTMR5/SBF1 gene. Herein, we report the generation and characterization of a hiPSC line from a 12-year-old Italian girl with early onset severe polyneuropathy with motor and axonal involvement, harboring biallelic variants in the MTMR5/SBF1 gene. Fibroblasts were reprogrammed using non-integrating episomal plasmids, and iPSCs successfully passed the stemness and pluripotency tests. Patient-specific hiPSCs were produced to obtain a disease model for the study of this rare condition.

Amyotrophic Lateral Sclerosis (ALS) is a fatal disease affecting both upper and lower motoneurons. The transactive response DNA binding protein (TARDBP) gene, encoding for TDP-43, is one of the most commonly mutated gene associated with familial cases of ALS (10%). We generated a human induced pluripotent stem cell (hiPSC) line from the fibroblasts of an asymptomatic subject carrying the TARDBP p.G376D mutation. This mutation is very rare and was described in a large Apulian family, in which all ALS affected members are carriers of the mutation. The subject here described is the first identified asymptomatic carrier of the mutation.

Mucopolysaccharidosis type II (Hunter Syndrome) is a rare X-linked inherited lysosomal storage disorder presenting a wide genetic heterogeneity. It is due to pathogenic variants in the IDS gene, causing the deficit of the lysosomal hydrolase iduronate 2-sulfatase, degrading the glycosaminoglycans (GAGs) heparan- and dermatan-sulfate. Based on the presence/absence of neurocognitive signs, commonly two forms are recognized, the severe and the attenuate ones. Here we describe a line of induced pluripotent stem cells, generated from dermal fibroblasts, carrying the mutation c.479C>T, and obtained from a patient showing an attenuated phenotype. The line will be useful to study the disease neuropathogenesis.

IntroductionIn central nervous system neurodegenerative disorders, stem cell-based therapies should be considered as a promising therapeutic approach. The safe use of human Neural Stem Cells (hNSCs) for the treatment of several neurological diseases is currently under evaluation of phase I/II clinical trials. Clinical application of hNSCs require the development of GMP standardized protocols capable of generating high quantities of reproducible and well characterized stem cells bearing stable functional and genetic properties.AimThe aim of this study was to evaluate possible instabilities or modifications of the microsatellite loci in different culture passages because high culture passages represent an in vitro replicative stress leading to senescence. Experimental method: The hNSCs were characterized at different culture time points, from passage 2 to passage 25, by genetic typing at ten microsatellite loci.ConclusionWe showed that genetic stability at microsatellite loci is maintained by the cells even at high passages adding a further demonstration of the safety of our hNSCs GMP culture method.

Mutations in genes encoding for histone methylation proteins are associated with several developmental disorders. Among them, KDM6A is the disease causative gene of type 2 Kabuki Syndrome, a rare multisystem disease. While nonsense mutations and short insertions/deletions are known to trigger pathogenic mechanisms, the functional effects of missense mutations are still uncharacterized. In this study, we demonstrate that a selected set of missense mutations significantly hamper the interaction between KDM6A and the histone H3, by modifying the dynamics of the linker domain, and then causing a loss of function effect.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative condition with phenotypic and genetic heterogeneity. It is characterized by the selective vulnerability and the progressive loss of the neural population. Here, an induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of an individual carrying the p.G376D mutation in the TDP-43 protein. Fibroblasts were reprogrammed using non-integrating episomal plasmids. There were no karyotype abnormalities, and iPSCs successfully differentiated into all three germ layers. This cell line may prove useful in the study of the pathogenic mechanisms that underpin ALS syndrome.

Among the known causative genes of familial ALS, SOD1 mutation is one of the most common. It encodes for the ubiquitous detoxifying copper/zinc binding SOD1 enzyme, whose mutations selectively cause motor neuron death, although the mechanisms are not as yet clear. What is known is that mutant-mediated toxicity is not caused by loss of its detoxifying activity but by a gain-of-function. In order to better understand the pathogenic mechanisms of SOD1 mutation, a human induced pluripotent stem cell (hiPSC) line was generated from the somatic cells of a female patient carrying a missense variation in SOD1 (L145F).

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of the upper and lower motor neurons (MNs). About 10% of patients have a family history (familial, fALS); however, most patients seem to develop the sporadic form of the disease (sALS). SOD1 (Cu/Zn superoxide dismutase-1) is the first studied gene among the ones related to ALS. Mutant SOD1 can adopt multiple misfolded conformation, lose the correct coordination of metal binding, decrease structural stability, and form aggregates. For all these reasons, it is complicated to characterize the conformational alterations of the ALS-associated mutant SOD1, and how they relate to toxicity. In this work, we performed a multilayered study on fibroblasts derived from two ALS patients, namely SOD1L145F and SOD1S135N, carrying the p.L145F and the p.S135N missense variants, respectively. The patients showed diverse symptoms and disease progression in accordance with our bioinformatic analysis, which predicted the different effects of the two mutations in terms of protein structure. Interestingly, both mutations had an effect on the fibroblast energy metabolisms. However, while the SOD1L145F fibroblasts still relied more on oxidative phosphorylation, the SOD1S135N fibroblasts showed a metabolic shift toward glycolysis. Our study suggests that SOD1 mutations might lead to alterations in the energy metabolism.

Abstract EphB2 and EphA2 control stemness and differentiation in the intestinal mucosa, but the way they cooperate with the complex mechanisms underlying tumor heterogeneity and how they affect the therapeutic outcome in colorectal cancer (CRC) patients, remain unclear. MicroRNA (miRNA) expression profiling along with pathway analysis provide comprehensive information on the dysregulation of multiple crucial pathways in CRC. Through a network-based approach founded on the characterization of progressive miRNAomes centered on EphA2/EphB2 signaling during tumor development in the AOM/DSS murine model, we found a miRNA-dependent orchestration of EphB2-specific stem-like properties in earlier phases of colorectal tumorigenesis and the EphA2-specific control of tumor progression in the latest CRC phases. Furthermore, two transcriptional signatures that are specifically dependent on the EphA2/EphB2 signaling pathways were identified, namely EphA2, miR-423-5p, CREB1, ADAMTS14, and EphB2, miR-31-5p, mir-31-3p, CRK, CXCL12, ARPC5, SRC. EphA2- and EphB2-related signatures were validated for their expression and clinical value in 1663 CRC patients. In multivariate analysis, both signatures were predictive of survival and tumor progression. The early dysregulation of miRs-31, as observed in the murine samples, was also confirmed on 49 human tissue samples including preneoplastic lesions and tumors. In light of these findings, miRs-31 emerged as novel potential drivers of CRC initiation. Our study evidenced a miRNA-dependent orchestration of EphB2 stem-related networks at the onset and EphA2-related cancer-progression networks in advanced stages of CRC evolution, suggesting new predictive biomarkers and potential therapeutic targets.

CHRNA7, encoding the neuronal alpha7 nicotinic acetylcholine receptor (a7nAChR), is highly expressed in the brain, particularly in the hippocampus. It is situated in the 15q13.3 chromosome region, frequently associated with a Copy Number Variation (CNV), which causes its duplication or deletion. The clinical significance of CHRNA7 duplications is unknown so far, but there are several research data suggesting that they may be pathogenic, with reduced penetrance. We have produced an iPS cell line from a single healthy donor's fibroblasts carrying a 15q13.3 CNV, including CHRNA7 in order to study the exact role of this CNV during the neurodevelopment.

Juvenile Onset Huntington's Disease (JOHD) is a rare variant of HD withage of onset ≤20 years, accounting for 3–10% of all HD patients. The rarity occurrence of JOHD cases, who severely progress towards mental and physical disability with atypical clinical manifestations compared to classical HD, are responsible of general lack of knowledge about this variant. We obtained a fully reprogrammed iPS cell line from fibroblasts of a JOHD patient carrying 65 CAG repeats and age at onset at age 15. At the biopsy time, the patient showed an advanced stage after 10 years of disease.

Smith-Magenis syndrome (SMS) is a complex genetic disorder characterized by developmental delay, behavioural problems and circadian rhythm dysregulation. About 90% of SMS cases are due to a 17p11.2 deletion containing retinoic acid induced1 (RAI1) gene, 10% are due to heterozygous mutations affecting RAI1 coding region. Little is known about RAI1 role.

Huntington's disease (HD) is an incurable, autosomal dominant, hereditary neurodegenerative disorder that typically manifests itself in midlife. This pathology is linked to the deregulation of multiple, as yet unknown, cellular processes starting before HD onset. A human iPS cell line was generated from skin fibroblasts of a subject at the presymptomatic life stage, carrying a polyglutamine expansion in HTT gene codifying Huntingtin protein. The iPSC line contained the expected CAG expansion, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had a normal karyotype.

Huntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT gene beyond 35 repeats. The juvenile form (JHD) may begin before the age of 20 years and is associated with expanded alleles as long as 60 or more CAG repeats. In this study, induced pluripotent stem cells were generated from skin fibroblasts of a 8-year-old child carrying a large size mutation of 84 CAG repeats in the HTT gene. HD appeared at age 3 with mixed psychiatric (i.e. autistic spectrum disorder) and motor (i.e. dystonia) manifestations.

Joubert Syndrome (JS) is a rare autosomal recessive or X-linked condition characterized by a peculiar cerebellar malformation, known as the molar tooth sign (MTS), associated with other neurological phenotypes and multiorgan involvement. JS is a ciliopathy, a spectrum of disorders whose causative genes encode proteins involved in the primary cilium apparatus. In order to elucidate ciliopathy-associated molecular mechanisms, human induced pluripotent stem cells (hiPSCs) were derived from a patient affected by JS carrying a homozygous missense mutation in the AHI1 gene (p.H896R) that encodes a protein named Jouberin.

COVID-19 is a viral infection, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and characterized by a complex inflammatory process and clinical immunophenotypes. Nowadays, several alterations of immune response within the respiratory tracts as well as at the level of the peripheral blood have been well documented. Nonetheless, their effects on COVID-19-related cell heterogeneity and disease progression are less defined. Here, we performed a single-cell RNA sequencing of about 400 transcripts relevant to immune cell function including surface markers, in mononuclear cells (PBMCs) from the peripheral blood of 50 subjects, infected with SARS-CoV-2 at the diagnosis and 27 healthy blood donors as control. We found that patients with COVID-19 exhibited an increase in COVID-specific surface markers in different subsets of immune cell composition. Interestingly, the expression of cell receptors, such as IFNGR1 and CXCR4, was reduced in response to the viral infection and associated with the inhibition of the related signaling pathways and immune functions. These results highlight novel immunoreceptors, selectively expressed in COVID-19 patients, which affect the immune functionality and are correlated with clinical outcomes.

Dentato-Rubral-pallidoluysian atrophy (DRPLA) is a rare autosomal, dominant, progressive neurodegenerative disease that causes involuntary movements, mental and emotional problems. DRPLA is caused by a mutation in the ATN1 gene that encodes for an abnormal polyglutamine stretch in the atrophin-1 protein. DRPLA is most common in the Japanese population, where it has an estimated incidence of 2 to 7 per million people. This condition has also been seen in families from North America and Europe. We obtained a reprogrammed iPSC line from a Caucasian patient with a juvenile onset of the disease, carrying 64 CAG repeat expansion in the ATN1 gene.

Joubert syndrome (JS) is an autosomal recessive neurodevelopmental disorder, characterized by congenital cerebellar and brainstem defects, belonging to the group of disorders known as ciliopathies, which are caused by mutations in genes encoding proteins of the primary cilium and basal body. Human induced pluripotent stem cells (hiPSCs) from a patient carrying a homozygous missense mutation (c.2168G > A) in AHI1, the first gene to be associated with JS, were produced using a virus-free protocol.

24,189 are all the possible non-synonymous amino acid changes potentially affecting the human mitochondrial DNA. Only a tiny subset was functionally evaluated with certainty so far, while the pathogenicity of the vast majority was only assessed in-silico by software predictors. Since these tools proved to be rather incongruent, we have designed and implemented APOGEE, a machine-learning algorithm that outperforms all existing prediction methods in estimating the harmfulness of mitochondrial non-synonymous genome variations. We provide a detailed description of the underlying algorithm, of the selected and manually curated training and test sets of variants, as well as of its classification ability.

In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinson's disease (PD), Schizophrenia (SCZ), and Bipolar Disorder (BD), when compared to healthy controls (about 200 post-mortem samples). Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the putamen of patients suffering from PD showed a significant 35% reduction of this transcript, compared to healthy subjects. Interestingly, in line with observations obtained in humans, we found 27% decrease in Rhes mRNA levels in the putamen of MPTP-treated primates. Based on the established inhibitory influence of Rhes on dopamine-related responses, we hypothesize that its striatal downregulation in PD patients and animal models of PD might represent an adaptive event of the dopaminergic system to functionally counteract the reduced nigrostriatal innervation.

In questo saggio, tramite l'analisi del cantiere della chiesa di Sant'Andrea, viene ripercorso lo sviluppo di Parma tra XII e XIII secolo, nell'ampliamento delle mura e nella creazione della "platea".