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1.  Genetic Correction of Human Induced Pluripotent Stem Cells from Patients with Spinal Muscular Atrophy 
Science translational medicine  2012;4(165):165ra162.
Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Induced pluripotent stem cells (iPSCs) generated from skin fibroblasts from SMA patients and genetically corrected have been proposed to be useful for autologous cell therapy. We generated iPSCs from SMA patients (SMA-iPSCs) using nonviral, nonintegrating episomal vectors and used a targeted gene correction approach based on single-stranded oligonucleotides to convert the survival motor neuron 2 (SMN2) gene into an SMN1-like gene. Corrected iPSC lines contained no exogenous sequences. Motor neurons formed by differentiation of uncorrected SMA-iPSCs reproduced disease-specific features. These features were ameliorated in motor neurons derived from genetically corrected SMA-iPSCs. The different gene splicing profile in SMA-iPSC motor neurons was rescued after genetic correction. The transplantation of corrected motor neurons derived from SMA-iPSCs into an SMA mouse model extended the life span of the animals and improved the disease phenotype. These results suggest that generating genetically corrected SMA-iPSCs and differentiating them into motor neurons may provide a source of motor neurons for therapeutic transplantation for SMA.
doi:10.1126/scitranslmed.3004108
PMCID: PMC4722730  PMID: 23253609
2.  Usefulness of Multi-Parametric MRI for the Investigation of Posterior Cortical Atrophy 
PLoS ONE  2015;10(10):e0140639.
Background
Posterior Cortical Atrophy (PCA) is a neurodegenerative disease characterized by a progressive decline in selective cognitive functions anatomically referred to occipital, parietal and temporal brain regions, whose diagnosis is rather challenging for clinicians. The aim of this study was to assess, using quantitative Magnetic Resonance Imaging techniques, the pattern of regional grey matter loss and metabolism in individuals with PCA to improve pathophysiological comprehension and diagnostic confidence.
Methods
We enrolled 5 patients with PCA and 5 matched controls who all underwent magnetic resonance imaging (MRI) and spectroscopy (MRS). Patients also underwent neuropsychological and cerebrospinal fluid (CSF) assessments. MRI data were used for unbiased assessment of regional grey matter loss in PCA patients compared to controls. MRS data were obtained from a set of brain regions, including the occipital lobe and the centrum semiovale bilaterally, and the posterior and anterior cingulate.
Results
VBM analysis documented the presence of focal brain atrophy in the occipital lobes and in the posterior parietal and temporal lobes bilaterally but more pronounced on the right hemisphere. MRS revealed, in the occipital lobes and in the posterior cingulate cortex of PCA patients, reduced levels of N-Acetyl Aspartate (NAA, a marker of neurodegeneration) and increased levels of Myo-Inositol (Ins, a glial marker), with no hemispheric lateralization.
Conclusion
The bilateral but asymmetric pattern of regional grey matter loss is consistent with patients’ clinical and neuropsychological features and with previous literature. The MRS findings reveal different stages of neurodegeneration (neuronal loss; gliosis), which coexist and likely precede the occurrence of brain tissue loss, and might represent early biomarkers. In conclusion, this study indicates the potential usefulness of a multi-parametric MRI approach for an early diagnosis and staging of patients with PCA.
doi:10.1371/journal.pone.0140639
PMCID: PMC4610670  PMID: 26480294
3.  ISPD mutations account for a small proportion of Italian Limb Girdle Muscular Dystrophy cases 
BMC Neurology  2015;15:172.
Background
Limb Girdle Muscular Dystrophy (LGMD), caused by defective α-dystroglycan (α-DG) glycosylation, was recently associated with mutations in Isoprenoid synthase domain-containing (ISPD) and GDP-mannose pyrophosphorylase B (GMPPB) genes. The frequency of ISPD and GMPPB gene mutations in the LGMD population is unknown.
Methods
We investigated the contributions of ISPD and GMPPB genes in a cohort of 174 Italian patients with LGMD, including 140 independent probands. Forty-one patients (39 probands) from this cohort had not been genetically diagnosed. The contributions of ISPD and GMPPB were estimated by sequential α-DG immunohistochemistry (IHC) and mutation screening in patients with documented α-DG defect, or by direct DNA sequencing of both genes when muscle tissue was unavailable.
Results
We performed α-DG IHC in 27/39 undiagnosed probands: 24 subjects had normal α-DG expression, two had a partial deficiency, and one exhibited a complete absence of signal. Direct sequencing of ISPD and GMPPB revealed two heterozygous ISPD mutations in the individual who lacked α-DG IHC signal: c.836-5 T > G (which led to the deletion of exon 6 and the production of an out-of-frame transcript) and c.676 T > C (p.Tyr226His). This patient presented with sural hypertrophy and tip-toed walking at 5 years, developed moderate proximal weakness, and was fully ambulant at 42 years. The remaining 12/39 probands did not exhibit pathogenic sequence variation in either gene.
Conclusion
ISPD mutations are a rare cause of LGMD in the Italian population, accounting for less than 1 % of the entire cohort studied (FKRP mutations represent 10 %), while GMPPB mutations are notably absent in this patient sample. These data suggest that the genetic heterogeneity of LGMD with and without α-DG defects is greater than previously realized.
Electronic supplementary material
The online version of this article (doi:10.1186/s12883-015-0428-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12883-015-0428-8
PMCID: PMC4582941  PMID: 26404900
Isoprenoid synthase domain-containing gene; GDP-mannose pyrophosphorylase B; Limb Girdle Muscular Dystrophy; α-dystroglycan glycosylation
4.  Natural Selection at the Brush-Border: Adaptations to Carbohydrate Diets in Humans and Other Mammals 
Genome Biology and Evolution  2015;7(9):2569-2584.
Dietary shifts can drive molecular evolution in mammals and a major transition in human history, the agricultural revolution, favored carbohydrate consumption. We investigated the evolutionary history of nine genes encoding brush-border proteins involved in carbohydrate digestion/absorption. Results indicated widespread adaptive evolution in mammals, with several branches experiencing episodic selection, particularly strong in bats. Many positively selected sites map to functional protein regions (e.g., within glucosidase catalytic crevices), with parallel evolution at SI (sucrase-isomaltase) and MGAM (maltase-glucoamylase). In human populations, five genes were targeted by positive selection acting on noncoding variants within regulatory elements. Analysis of ancient DNA samples indicated that most derived alleles were already present in the Paleolithic. Positively selected variants at SLC2A5 (fructose transporter) were an exception and possibly spread following the domestication of specific fruit crops. We conclude that agriculture determined no major selective event at carbohydrate metabolism genes in humans, with implications for susceptibility to metabolic disorders.
doi:10.1093/gbe/evv166
PMCID: PMC4607523  PMID: 26319403
MGAM; SI; LCT; TREH; SLC2A2; natural selection
5.  Spinal muscular atrophy phenotype is ameliorated in human motor neurons by SMN increase via different novel RNA therapeutic approaches 
Scientific Reports  2015;5:11746.
Spinal muscular atrophy (SMA) is a primary genetic cause of infant mortality due to mutations in the Survival Motor Neuron (SMN) 1 gene. No cure is available. Antisense oligonucleotides (ASOs) aimed at increasing SMN levels from the paralogous SMN2 gene represent a possible therapeutic strategy. Here, we tested in SMA human induced pluripotent stem cells (iPSCs) and iPSC-differentiated motor neurons, three different RNA approaches based on morpholino antisense targeting of the ISSN-1, exon-specific U1 small nuclear RNA (ExSpeU1), and Transcription Activator-Like Effector-Transcription Factor (TALE-TF). All strategies act modulating SMN2 RNA: ASO affects exon 7 splicing, TALE-TF increase SMN2 RNA acting on the promoter, while ExSpeU1 improves pre-mRNA processing. These approaches induced up-regulation of full-length SMN mRNA and differentially affected the Delta-7 isoform: ASO reduced this isoform, while ExSpeU1 and TALE-TF increased it. All approaches upregulate the SMN protein and significantly improve the in vitro SMA motor neurons survival. Thus, these findings demonstrate that therapeutic tools that act on SMN2 RNA are able to rescue the SMA disease phenotype. Our data confirm the feasibility of SMA iPSCs as in vitro disease models and we propose novel RNA approaches as potential therapeutic strategies for treating SMA and other genetic neurological disorders.
doi:10.1038/srep11746
PMCID: PMC4485234  PMID: 26123042
6.  Post-Activation Brain Warming: A 1-H MRS Thermometry Study 
PLoS ONE  2015;10(5):e0127314.
Purpose
Temperature plays a fundamental role for the proper functioning of the brain. However, there are only fragmentary data on brain temperature (Tbr) and its regulation under different physiological conditions.
Methods
We studied Tbr in the visual cortex of 20 normal subjects serially with a wide temporal window under different states including rest, activation and recovery by a visual stimulation-Magnetic Resonance Spectroscopy Thermometry combined approach. We also studied Tbr in a control region, the centrum semiovale, under the same conditions.
Results
Visual cortex mean baseline Tbr was higher than mean body temperature (37.38 vs 36.60, P<0.001). During activation Tbr remained unchanged at first and then showed a small decrease (-0.20 C°) around the baseline value. After the end of activation Tbr increased consistently (+0.60 C°) and then returned to baseline values after some minutes. Centrum semiovale Tbr remained unchanged through rest, visual stimulation and recovery.
Conclusion
These findings have several implications, among them that neuronal firing itself is not a major source of heat release in the brain and that there is an aftermath of brain activation that lasts minutes before returning to baseline conditions.
doi:10.1371/journal.pone.0127314
PMCID: PMC4444346  PMID: 26011731
7.  The brain is hypothermic in patients with mitochondrial diseases 
We sought to study brain temperature in patients with mitochondrial diseases in different functional states compared with healthy participants. Brain temperature and mitochondrial function were monitored in the visual cortex and the centrum semiovale at rest and during and after visual stimulation in seven individuals with mitochondrial diseases (n=5 with mitochondrial DNA mutations and n=2 with nuclear DNA mutations) and in 14 age- and sex-matched healthy control participants using a combined approach of visual stimulation, proton magnetic resonance spectroscopy (MRS), and phosphorus MRS. Brain temperature in control participants exhibited small changes during visual stimulation and a consistent increase, together with an increase in high-energy phosphate content, after visual stimulation. Brain temperature was persistently lower in individuals with mitochondrial diseases than in healthy participants at rest, during activation, and during recovery, without significant changes from one state to another and with a decrease in the high-energy phosphate content. The lowest brain temperature was observed in the patient with the most deranged mitochondrial function. In patients with mitochondrial diseases, the brain is hypothermic because of malfunctioning oxidative phosphorylation. Neuronal activity is reduced at rest, during physiologic brain stimulation, and after stimulation.
doi:10.1038/jcbfm.2014.38
PMCID: PMC4013774  PMID: 24619278
activation; brain metabolism; brain temperature; mitochondrial diseases; recovery; visual stimulation
8.  Gene therapy rescues disease phenotype in a spinal muscular atrophy with respiratory distress type 1 (SMARD1) mouse model 
Science Advances  2015;1(2):e1500078.
Preclinical proof-of-principle data demonstrate the high efficacy of IGHMBP2 gene therapy in the SMARD1 mouse model as well as in an in vitro model of the human disease.
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an autosomal recessive motor neuron disease affecting children. It is caused by mutations in the IGHMBP2 gene (11q13) and presently has no cure. Recently, adeno-associated virus serotype 9 (AAV9)–mediated gene therapy has been shown to rescue the phenotype of animal models of another lower motor neuron disorder, spinal muscular atrophy 5q, and a clinical trial with this strategy is ongoing. We report rescue of the disease phenotype in a SMARD1 mouse model after therapeutic delivery via systemic injection of an AAV9 construct encoding the wild-type IGHMBP2 to replace the defective gene. AAV9-IGHMBP2 administration restored protein levels and rescued motor function, neuromuscular physiology, and life span (450% increase), ameliorating pathological features in the central nervous system, muscles, and heart. To test this strategy in a human model, we transferred wild-type IGHMBP2 into human SMARD1-induced pluripotent stem cell–derived motor neurons; these cells exhibited increased survival and axonal length in long-term culture. Our data support the translational potential of AAV-mediated gene therapies for SMARD1, opening the door for AAV9-mediated therapy in human clinical trials.
doi:10.1126/sciadv.1500078
PMCID: PMC4643829  PMID: 26601156
Spinal Muscular atrophy with Respiratory Distress Type 1; gene therapy
9.  OASes and STING: Adaptive Evolution in Concert 
Genome Biology and Evolution  2015;7(4):1016-1032.
OAS (2′–5′-oligoadenylate synthases) proteins and cyclic GMP–AMP synthase (cGAS, gene symbol: MB21D1) patrol the cytoplasm for the presence of foreign nucleic acids. Upon binding to double-stranded RNA or double-stranded DNA, OAS proteins and cGAS produce nucleotide second messengers to activate RNase L and STING (stimulator of interferon genes, gene symbol: TMEM173), respectively; this leads to the initiation of antiviral responses. We analyzed the evolutionary history of the MB21D1–TMEM173 and OAS–RNASEL axes in primates and bats and found evidence of widespread positive selection in both orders. In TMEM173, residue 230, a major determinant of response to natural ligands and to mimetic drugs (e.g., DMXAA), was positively selected in Primates and Chiroptera. In both orders, selection also targeted an α-helix/loop element in RNase L that modulates the enzyme preference for single-stranded RNA versus stem loops. Analysis of positively selected sites in OAS1, OAS2, and MB21D1 revealed parallel evolution, with the corresponding residues being selected in different genes. As this cannot result from gene conversion, these data suggest that selective pressure acting on OAS and MB21D1 genes is related to nucleic acid recognition and to the specific mechanism of enzyme activation, which requires a conformational change. Finally, a population genetics-phylogenetics analysis in humans, chimpanzees, and gorillas detected several positively selected sites in most genes. Data herein shed light into species-specific differences in infection susceptibility and in response to synthetic compounds, with relevance for the design of synthetic compounds as vaccine adjuvants.
doi:10.1093/gbe/evv046
PMCID: PMC4419793  PMID: 25752600
OAS; cGAS; STING; RNase L; positive selection
10.  Genetic adaptation of the human circadian clock to day-length latitudinal variations and relevance for affective disorders 
Genome Biology  2014;15(10):499.
Background
The temporal coordination of biological processes into daily cycles is a common feature of most living organisms. In humans, disruption of circadian rhythms is commonly observed in psychiatric diseases, including schizophrenia, bipolar disorder, depression and autism. Light therapy is the most effective treatment for seasonal affective disorder and circadian-related treatments sustain antidepressant response in bipolar disorder patients. Day/night cycles represent a major circadian synchronizing signal and vary widely with latitude.
Results
We apply a geographically explicit model to show that out-of-Africa migration, which led humans to occupy a wide latitudinal area, affected the evolutionary history of circadian regulatory genes. The SNPs we identify using this model display consistent signals of natural selection using tests based on population genetic differentiation and haplotype homozygosity. Signals of natural selection driven by annual photoperiod variation are detected for schizophrenia, bipolar disorder, and restless leg syndrome risk variants, in line with the circadian component of these conditions.
Conclusions
Our results suggest that human populations adapted to life at different latitudes by tuning their circadian clock systems. This process also involves risk variants for neuropsychiatric conditions, suggesting possible genetic modulators for chronotherapies and candidates for interaction analysis with photoperiod-related environmental variables, such as season of birth, country of residence, shift-work or lifestyle habits.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0499-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-014-0499-7
PMCID: PMC4237747  PMID: 25358694
11.  Albuminoid Genes: Evolving at the Interface of Dispensability and Selection 
Genome Biology and Evolution  2014;6(11):2983-2997.
The albuminoid gene family comprises vitamin D-binding protein (GC), alpha-fetoprotein (AFP), afamin (AFM), and albumin (ALB). Albumin is the most abundant human serum protein, and, as the other family members, acts as a transporter of endogenous and exogenous substances including thyroxine, fatty acids, and drugs. Instead, the major cargo of GC is 25-hydroxyvitamin D. We performed an evolutionary study of albuminoid genes and we show that ALB evolved adaptively in mammals. Most positively selected sites are located within albumin-binding sites for fatty acids and thyroxine, as well as at the contact surface with neonatal Fc receptor. Positive selection was also detected for residues forming the prostaglandin-binding pocket. Adaptation to hibernation/torpor might explain the signatures of episodic positive selection we detected for few mammalian lineages. Application of a population genetics–phylogenetics approach showed that purifying selection represented a major force acting on albuminoid genes in both humans and chimpanzees, with the strongest constraint observed for human GC. Population genetic analysis revealed that GC was also the target of locally exerted selective pressure, which drove the frequency increase of different haplotypes in distinct human populations. A search for known variants that modulate GC and 25-hydroxyvitamin D concentrations revealed linkage disequilibrium with positively selected variants, although European and Asian major GC haplotypes carry alleles with reported opposite effect on GC concentration. Data herein indicate that albumin, an extremely abundant housekeeping protein, was the target of pervasive and episodic selection in mammals, whereas GC represented a selection target during the recent evolution of human populations.
doi:10.1093/gbe/evu235
PMCID: PMC4255767  PMID: 25349266
albumin; vitamin D-binding protein; albuminoids; positive selection; vitamin D; hibernation
12.  Defective autophagy in spastizin mutated patients with hereditary spastic paraparesis type 15 
Brain  2013;136(10):3119-3139.
Hereditary spastic paraparesis type 15 is a recessive complicated form of the disease clinically characterized by slowly progressive spastic paraparesis and mental deterioration with onset between the first and second decade of life. Thinning of corpus callosum is the neuroradiological distinctive sign frequently associated with white matter abnormalities. The causative gene, ZFYVE26, encodes a large protein of 2539 amino acid residues, termed spastizin, containing three recognizable domains: a zinc finger, a leucine zipper and a FYVE domain. Spastizin protein has a diffuse cytoplasmic distribution and co-localizes partially with early endosomes, the endoplasmic reticulum, microtubules and vesicles involved in protein trafficking. In addition, spastizin localizes to the mid-body during the final step of mitosis and contributes to successful cytokinesis. Spastizin interacts with Beclin 1, a protein required for cytokinesis and autophagy, which is the major lysosome-mediated degradation process in the cell. In view of the Beclin 1–spastizin interaction, we investigated the possible role of spastizin in autophagy. We carried out this analysis by using lymphoblast and fibroblast cells derived from four different spastizin mutated patients (p.I508N, p.L243P, p.R1209fsX, p.S1312X) and from control subjects. Of note, the truncating p.R1209fsX and p.S1312X mutations lead to loss of spastizin protein. The results obtained indicate that spastizin interacts with the autophagy related Beclin 1–UVRAG–Rubicon multiprotein complex and is required for autophagosome maturation. In cells lacking spastizin or with mutated forms of the protein, spastizin interaction with Beclin 1 is lost although the formation of the Beclin 1–UVRAG–Rubicon complex can still be observed. However, in these cells we demonstrate an impairment of autophagosome maturation and an accumulation of immature autophagosomes. Autophagy defects with autophagosome accumulation can be observed also in neuronal cells upon spastizin silencing. These results indicate that autophagy is a central process in the pathogenesis of complicated forms of hereditary spastic paraparesis with thin corpus callosum.
doi:10.1093/brain/awt227
PMCID: PMC3784282  PMID: 24030950
spastizin; autophagy; Beclin 1; autophagosome maturation; SPG15
13.  A novel CCM1 mutation associated with multiple cerebral and vertebral cavernous malformations 
BMC Neurology  2014;14:158.
Background
Cerebral cavernous malformations are relatively rare vascular disorders that may affect any part of the central nervous system. This presentation has been associated with heterozygous mutations in CCM1/KRIT1, CCM2/malcavernin and CCM3/PDCD10. We aimed to investigate the genetic defect underlying multiple cerebral and vertebral cavernous malformations in a multigenerational Italian family.
Case presentation
The proband is a 49-year-old man who underwent cerebral MRI in his thirties for persistent haeadache and tingling in his left arm and leg and was diagnosed with multiple supratentorial cavernous angiomas. A right frontal angioma with radiological evidence of a recent bleeding was surgically removed when he was 39 years old and he was thereafter asymptomatic. Magnetic resonance imaging revealed multiple cerebral cavernous malformations in seven members of his familily. Four subjects were asymptomatic. Other family mambers displayed heterogeneous clinical features including seizures and recurrent brain haemorrhages. Sequence analysis in the proband disclosed a novel heterozygous nucleotide substitution (c.263-10A > G) in intron 5 of CCM1. This variant is predicted to create an abnormal acceptor splice site and segregated in affected relatives available for molecular screening. The analysis of CCM1 transcript in proband’s lymphocytes confirmed the partial retention of intron 3 resulting in a premature termination codon.
Conclusions
Our findings demonstrate that c.263-10A > G mutation is associated with cerebral cavernous malformations. A better knowledge of the disease-associated phenotype may lead to an early diagnosis and to an appropriate clinical surveillance in affected patients.
doi:10.1186/s12883-014-0158-3
PMCID: PMC4236643  PMID: 25086949
Cerebral Cavernous Malformations; CCM1 protein; Krev interaction trapped 1 protein; Hemangioma; Cavernous; Central Nervous System
14.  iPSC-Derived Neural Stem Cells Act via Kinase Inhibition to Exert Neuroprotective Effects in Spinal Muscular Atrophy with Respiratory Distress Type 1 
Stem Cell Reports  2014;3(2):297-311.
Summary
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a motor neuron disease caused by mutations in the IGHMBP2 gene, without a cure. Here, we demonstrate that neural stem cells (NSCs) from human-induced pluripotent stem cells (iPSCs) have therapeutic potential in the context of SMARD1. We show that upon transplantation NSCs can appropriately engraft and differentiate in the spinal cord of SMARD1 animals, ameliorating their phenotype, by protecting their endogenous motor neurons. To evaluate the effect of NSCs in the context of human disease, we generated human SMARD1-iPSCs motor neurons that had a significantly reduced survival and axon length. Notably, the coculture with NSCs ameliorate these disease features, an effect attributable to the production of neurotrophic factors and their dual inhibition of GSK-3 and HGK kinases. Our data support the role of iPSC as SMARD1 disease model and their translational potential for therapies in motor neuron disorders.
Graphical Abstract
Highlights
•Transplantation of human NSCs ameliorates the SMARD1 phenotype in a disease model•NSCs improve the disease features of SMARD1 motor neurons inhibiting GSK-3 and HGK
Corti and colleagues show that neural stem cells (NSCs) from human-induced pluripotent stem cells (hiPSCs) have therapeutic potential for Spinal Muscular Atrophy with Respiratory Distress Type 1 SMARD1. Transplanted NSCs engraft in the spinal cord of SMARD1 animals, ameliorating their phenotype. SMARD1 hiPSC-derived motor neurons recapitulate disease features that are improved by NSC coculture through GSK-3 and HGK kinase-modulated pathways.
doi:10.1016/j.stemcr.2014.06.004
PMCID: PMC4176534  PMID: 25254343
15.  Ancient and Recent Selective Pressures Shaped Genetic Diversity at AIM2-Like Nucleic Acid Sensors 
Genome Biology and Evolution  2014;6(4):830-845.
AIM2-like receptors (ALRs) are a family of nucleic acid sensors essential for innate immune responses against viruses and bacteria. We performed an evolutionary analysis of ALR genes (MNDA, PYHIN1, IFI16, and AIM2) by analyzing inter- and intraspecies diversity. Maximum-likelihood analyses indicated that IFI16 and AIM2 evolved adaptively in primates, with branch-specific selection at the catarrhini lineage for IFI16. Application of a population genetics–phylogenetics approach also allowed identification of positive selection events in the human lineage. Positive selection in primates targeted sites located at the DNA-binding interface in both IFI16 and AIM2. In IFI16, several sites positively selected in primates and in the human lineage were located in the PYD domain, which is involved in protein–protein interaction and is bound by a human cytomegalovirus immune evasion protein. Finally, positive selection was found to target nuclear localization signals in IFI16 and the spacer region separating the two HIN domains. Population genetic analysis in humans revealed that an IFI16 genic region has been a target of long-standing balancing selection, possibly acting on two nonsynonymous polymorphisms located in the spacer region. Data herein indicate that ALRs have been repeatedly targeted by natural selection. The balancing selection region in IFI16 carries a variant with opposite risk effect for distinct autoimmune diseases, suggesting antagonistic pleiotropy. We propose that the underlying scenario is the result of an ancestral and still ongoing host–pathogen arms race and that the maintenance of susceptibility alleles for autoimmune diseases at IFI16 represents an evolutionary trade-off.
doi:10.1093/gbe/evu066
PMCID: PMC4007548  PMID: 24682156
AIM2-like receptors; positive selection; long-standing balancing selection; IFI16
16.  An Evolutionary Analysis of Antigen Processing and Presentation across Different Timescales Reveals Pervasive Selection 
PLoS Genetics  2014;10(3):e1004189.
The antigenic repertoire presented by MHC molecules is generated by the antigen processing and presentation (APP) pathway. We analyzed the evolutionary history of 45 genes involved in APP at the inter- and intra-species level. Results showed that 11 genes evolved adaptively in mammals. Several positively selected sites involve positions of fundamental importance to the protein function (e.g. the TAP1 peptide-binding domains, the sugar binding interface of langerin, and the CD1D trafficking signal region). In CYBB, all selected sites cluster in two loops protruding into the endosomal lumen; analysis of missense mutations responsible for chronic granulomatous disease (CGD) showed the action of different selective forces on the very same gene region, as most CGD substitutions involve aminoacid positions that are conserved in all mammals. As for ERAP2, different computational methods indicated that positive selection has driven the recurrent appearance of protein-destabilizing variants during mammalian evolution. Application of a population-genetics phylogenetics approach showed that purifying selection represented a major force acting on some APP components (e.g. immunoproteasome subunits and chaperones) and allowed identification of positive selection events in the human lineage.
We also investigated the evolutionary history of APP genes in human populations by developing a new approach that uses several different tests to identify the selection target, and that integrates low-coverage whole-genome sequencing data with Sanger sequencing. This analysis revealed that 9 APP genes underwent local adaptation in human populations. Most positive selection targets are located within noncoding regions with regulatory function in myeloid cells or act as expression quantitative trait loci. Conversely, balancing selection targeted nonsynonymous variants in TAP1 and CD207 (langerin). Finally, we suggest that selected variants in PSMB10 and CD207 contribute to human phenotypes. Thus, we used evolutionary information to generate experimentally-testable hypotheses and to provide a list of sites to prioritize in follow-up analyses.
Author Summary
Antigen-presenting cells digest intracellular and extracellular proteins and display the resulting antigenic repertoire on cell surface molecules for recognition by T cells. This process initiates cell-mediated immune responses and is essential to detect infections. The antigenic repertoire is generated by the antigen processing and presentation pathway. Because several pathogens evade immune recognition by hampering this process, genes involved in antigen processing and presentation may represent common natural selection targets. Thus, we analyzed the evolutionary history of these genes during mammalian evolution and in the more recent history of human populations. Evolutionary analyses in mammals indicated that positive selection targeted a very high proportion of genes (24%), and revealed that many selected sites affect positions of fundamental importance to the protein function. In humans, we found different signatures of natural selection acting both on regions that are expected to regulate gene expression levels or timing and on coding variants; two human selected polymorphisms may modulate the susceptibility to Crohn's disease and to HIV-1 infection. Therefore, we provide a comprehensive evolutionary analysis of antigen processing and we show that evolutionary studies can provide useful information concerning the location and nature of functional variants, ultimately helping to clarify phenotypic differences between and within species.
doi:10.1371/journal.pgen.1004189
PMCID: PMC3967941  PMID: 24675550
17.  Molecular, genetic and stem cell-mediated therapeutic strategies for spinal muscular atrophy (SMA) 
Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease. It is the first genetic cause of infant mortality. It is caused by mutations in the survival motor neuron 1 (SMN1) gene, leading to the reduction of SMN protein. The most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment other than supportive care, although the past decade has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials. In this review, we would like to outline the most interesting therapeutic strategies that are currently developing, which are represented by molecular, gene and stem cell-mediated approaches for the treatment of SMA.
doi:10.1111/jcmm.12224
PMCID: PMC3930406  PMID: 24400925
spinal muscular atrophy; molecular therapy; antisense oligonucleotides; morpholino; gene therapy; stem-cell therapy; induced pluripotent stem cells
18.  Next-generation sequencing reveals DGUOK mutations in adult patients with mitochondrial DNA multiple deletions 
Brain  2012;135(11):3404-3415.
The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle.
doi:10.1093/brain/aws258
PMCID: PMC3501975  PMID: 23043144
DGUOK; mitochondrial DNA instability; autosomal recessive progressive external ophthalmoplegia; multiple mitochondrial DNA deletions
19.  Optic Neuritis as Isolated Manifestation of Leptomeningeal Carcinomatosis: A Case Report and Systematic Review of Ocular Manifestations of Neoplastic Meningitis 
Introduction. Leptomeningeal carcinomatosis occurs in about 5% of cancer patients. Ocular involvement is a common clinical manifestation and often the presenting clinical feature. Materials and Methods. We report the case of a 52-year old lady with optic neuritis as isolated manifestation of neoplastic meningitis and a review of ocular involvement in neoplastic meningitis. Ocular symptoms were the presenting clinical feature in 34 patients (83%) out of 41 included in our review, the unique manifestation of meningeal carcinomatosis in 3 patients (7%). Visual loss was the presenting clinical manifestation in 17 patients (50%) and was the most common ocular symptom (70%). Other ocular signs were diplopia, ptosis, papilledema, anisocoria, exophthalmos, orbital pain, scotomas, hemianopsia, and nystagmus. Associated clinical symptoms were headache, altered consciousness, meningism, limb weakness, ataxia, dizziness, seizures, and other cranial nerves involvement. All patients except five underwent CSF examination which was normal in 1 patient, pleocytosis was found in 11 patients, increased protein levels were observed in 16 patients, and decreased glucose levels were found in 8 patients. Cytology was positive in 29 patients (76%). Conclusion. Meningeal carcinomatosis should be considered in patients with ocular symptoms even in the absence of other suggestive clinical symptoms.
doi:10.1155/2013/892523
PMCID: PMC3816070  PMID: 24223306
20.  Growing Evidence about the Relationship between Vessel Dissection and Scuba Diving 
Case Reports in Neurology  2013;5(3):155-161.
Carotid and vertebral artery dissection are relatively frequent and risky conditions. In the last decade, different patients with extracranial (and in 1 case also intracranial) dissections associated with the practice of scuba diving were reported. The connection between the two conditions has not been fully explained so far. In the present article, we report the case of a patient presenting with Claude Bernard-Horner syndrome and homolateral XII cranial nerve palsy, manifesting a few days after diving in the cold water of a lake. The patient ended up having internal carotid artery dissection associated with the formation of a pseudoaneurysm. Here, we offer a summary of all cases reported in the literature about scuba diving and arterial dissection, and provide a critical discussion about which scuba diving-related factors can trigger the dissection of cervical vessels.
doi:10.1159/000354979
PMCID: PMC3806682  PMID: 24163671
Internal carotid dissection; Scuba diving; Claude Bernard-Horner syndrome; XII cranial nerve
21.  Minimally invasive transplantation of iPSC-derived ALDHhiSSCloVLA4+ neural stem cells effectively improves the phenotype of an amyotrophic lateral sclerosis model 
Human Molecular Genetics  2013;23(2):342-354.
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently, there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS, and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study, we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity, low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS, treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span, in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms, including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases.
doi:10.1093/hmg/ddt425
PMCID: PMC3869354  PMID: 24006477
22.  Selective DNA Methylation of BDNF Promoter in Bipolar Disorder: Differences Among Patients with BDI and BDII 
Neuropsychopharmacology  2012;37(7):1647-1655.
The etiology of bipolar disorder (BD) is still poorly understood, involving genetic and epigenetic mechanisms as well as environmental contributions. This study aimed to investigate the degree of DNA methylation at the promoter region of the brain-derived neurotrophic factor (BDNF) gene, as one of the candidate genes associated with major psychoses, in peripheral blood mononuclear cells isolated from 94 patients with BD (BD I=49, BD II=45) and 52 healthy controls. A significant BDNF gene expression downregulation was observed in BD II 0.53±0.11% P<0.05), but not in BD I (1.13±0.19%) patients compared with controls (CONT: 1±0.2%). Consistently, an hypermethylation of the BDNF promoter region was specifically found in BD II patients (CONT: 24.0±2.1% BDI: 20.4±1.7% BDII: 33.3±3.5%, P<0.05). Of note, higher levels of DNA methylation were observed in BD subjects on pharmacological treatment with mood stabilizers plus antidepressants (34.6±4.2%, predominantly BD II) compared with those exclusively on mood-stabilizing agents (21.7±1.8% P<0.01, predominantly BD I). Moreover, among the different pharmacological therapies, lithium (20.1±3.8%, P<0.05) and valproate (23.6±2.9%, P<0.05) were associated with a significant reduction of DNA methylation compared with other drugs (35.6±4.6%). Present findings suggest selective changes in DNA methylation of BDNF promoter in subjects with BD type II and highlight the importance of epigenetic factors in mediating the onset and/or susceptibility to BD, providing new insight into the mechanisms of gene expression. Moreover, they shed light on possible mechanisms of action of mood-stabilizing compounds vs antidepressants in the treatment of BD, pointing out that BDNF regulation might be a key target for their effects.
doi:10.1038/npp.2012.10
PMCID: PMC3358733  PMID: 22353757
brain-derived neurotrophic factor (BDNF); peripheral blood mononuclear cells (PBMCs); DNA methylation; gene expression; bipolar disorder (BD); mood stabilizers and antidepressants
23.  Mitochondrial Fusion Proteins and Human Diseases 
Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1) and 2 (MFN2), located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1), in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimer's and Parkinson's diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion.
doi:10.1155/2013/293893
PMCID: PMC3678461  PMID: 23781337
24.  Considerations on a mutation in the NOTCH3 gene sparing a cysteine residue: a rare polymorphism rather than a CADASIL variant 
Functional Neurology  2013;27(4):247-252.
Summary
Some missense mutations and small deletions in the NOTCH3 gene, not involving cysteine residues, have been described in patients considered to be affected by paucisymptomatic CADASIL. However, the significance of such molecular variants is still unclear. We describe a 49-year-old woman with a CADASIL-like phenotype, carrying a novel cysteine-sparing mutation in exon 29 of the NOTCH3 gene, and discuss the possible pathogenetic role of this molecular variant. Even though atypical clinical and MRI findings make a diagnosis of CADASIL unlikely in this patient, our report nevertheless underlines the intriguing genotype-phenotype relationship in NOTCH3 mutations and the importance of functional investigation to ascertain the role of new NOTCH3 mutations in CADASIL pathogenesis.
PMCID: PMC3861349  PMID: 23597439
CADASIL; cysteine residue; NOTCH3 mutations; white matter lesions
25.  The novel mitochondrial tRNAAsn gene mutation m.5709T>C produces ophthalmoparesis and respiratory impairment 
Although mutations in mitochondrial tRNAs constitute the most common mtDNA defect, the presence of pathological variants in mitochondrial tRNAAsn is extremely rare. We were able to identify a novel mtDNA tRNAAsn gene pathogenic mutation associated with a myopathic phenotype and a previously unreported respiratory impairment. Our proband is an adult woman with ophthalmoparesis and respiratory impairment. Her muscle biopsy presented several cytochrome c oxidase-negative (COX−) fibres and signs of mitochondrial proliferation (ragged red fibres). Sequence analysis of the muscle-derived mtDNA revealed an m.5709T>C substitution, affecting mitochondrial tRNAAsn gene. Restriction-fragment length polymorphism analysis of the mutation in isolated muscle fibres showed that a threshold of at least 91.9% mutated mtDNA results in the COX deficiency phenotype. The new phenotype further increases the clinical spectrum of mitochondrial diseases caused by mutations in the tRNAAsn gene.
doi:10.1038/ejhg.2011.238
PMCID: PMC3283170  PMID: 22189266
progressive external ophthalmoplegia; tRNA(Asn); mitochondrial myopathy

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