As in the prenatal stages, the postnatal period of neurodevelopment is also sensitive to environmental inputs. Often this sensitivity is reflected in children facing adverse conditions, such as maternal separation. This exposure to early life stress (ELS) is implicated in the manifestation of various behavioral abnormalities. Most FASD research has focused exclusively on the effect of prenatal ethanol exposure in isolation. Here, we review the research into the effect of prenatal ethanol exposure and ELS, with a focus on the continuum of epigenomic and transcriptomic alterations. Interestingly, a select few experiments have assessed the cumulative effect of prenatal alcohol and postnatal maternal separation stress. Regulatory regions of different sets of genes are affected by both treatments independently, and a unique set of genes are affected by the combination of treatments. Notably, epigenetic and gene expression changes converge at the clustered protocadherin locus and oxidative stress pathway. Functional studies using epigenetic editing may elucidate individual contributions of regulatory regions for hub genes and further profiling efforts may lead to the development of non-invasive methods to identify children at risk. Taken together, the results favor the potential to improve neurodevelopmental outcomes by epigenetic management of children born with FASD using favorable postnatal conditions with or without therapeutic interventions.Neurodevelopment in humans is a long, elaborate, and highly coordinated process involving three trimesters of prenatal development followed by decades of postnatal development and maturation. Throughout this period, the brain is highly sensitive and responsive to the external environment, which may provide a range of inputs leading to positive or negative outcomes. Fetal alcohol spectrum disorders (FASD) result from prenatal alcohol exposure (PAE). Although the molecular mechanisms of FASD are not fully characterized, they involve alterations to the regulation of gene expression  Also included in the scope of the epigenome are ncRNAs that act at the post-transcriptional level to interfere with the translation of mRNA into proteins, such as microRNAs (miRNAs).TET1 deficiency impairs neurogenesis and leads to the downregulation of neural progenitor proliferation genes , is critical for appropriate synapse formation and transmission through the regulation of genes such as the neurotransmitter receptor Htr5b  and the genome. It enables prenatal and postnatal events to shape neurodevelopment and related behavioral endophenotypes. At each stage of neurodevelopment, epigenetic players are at work. Neurogenesis relies on intergenic de novo DNA methylation by DNMT3A in neural stem cells to maintain active chromatin states for essential genes  are deregulated in the adult mouse brain , indicating that DNA methylation changes occur after PAE  at both the gene , together with adhesion and polarity genes DiGeorge syndrome critical region gene 2 (Dgcr2), and Par-6 family cell polarity regulator alpha , POU class 4 homeobox 3 (Pou4f3), and SRY-box transcription factor 7 (Sox7), are each hypomethylated following ethanol treatment  mRNA following PAE was accompanied by increased promoter methylation  alongside decreased promoter methylation  polycomb group ring finger 6 (Pcgf6), and ring finger protein 1 (Ring1), as well as transcription factor II D complex members general transcription factor II A, 2 (Gtf2a2), and TATA-box binding protein associated factors 8, 10, and 11 , nuclear transcription factor-Y gamma (Nfyc), peroxisome proliferator activator receptor delta (Ppard), and nuclear receptor subfamily 1, group H, member 4 and group D, member 2 , unc-51-like kinase 4 (Ulk4), disabled 2 interacting protein (Dab2ip), and rho/rac guanine nucleotide exchange factor (GEF) 2 (Arhgef2). Conversely, the combination of PAE and ELS led to increased promoter methylation of genes important for immune response, including 21 members of the cytokine-cytokine receptor interaction KEGG pathway  at the Pcdhb2 promoter and a corresponding decrease in gene expression (fold change = −1.23, p < 0.05; Laufer et al., Protocadherins are a group of cell-cell adhesion molecules that are highly expressed in the brain and are involved in establishing single-cell neuronal identity. Many of these protocadherins are encoded in a large genomic locus that consists of three multi-gene clusters (Chen and Maniatis, The clustered protocadherins have also been implicated in ELS, where a comparison of the hippocampal tissue from individuals that have experienced abuse as children and rats that received low levels of maternal care revealed DNA promoter hypermethylation and gene body DNA hypomethylation at the locus, corresponding with a repressive effect (Suderman et al., In an FASD model that includes PAE and ELS in adult mouse hippocampus both hyper- and hypo-methylation in the protocadherin region as well as decreased gene expression resulted from PAE (Alberry and Singh, Oxidative stress is a well-characterized part of FASD etiology. At the cellular level, ethanol acts directly on mitochondria to produce superoxide, hydroxide, and nitric oxide radicals (Wu and Cederbaum, via epigenetic manipulation.FASD is a common neurodevelopmental disorder that represents a societal burden with no effective treatments. Yet, it is entirely preventable. It requires the avoidance of alcohol before and during pregnancy. This is a challenging proposition given that the use of alcohol in reproductive-age women is increasing and many pregnancies are unintended, which is expected to lead to a corresponding increase in FASD (Alberry and Singh, Hoxa1 and its miRNA, miR-10a (Wang et al., Dnmt1 and Mecp2 in the hypothalamus (Bekdash et al., Set7/9, G9a, and Setdb1, as well as the detected changes in histone PTMs, H3K4me2, 3 and H3K9me2 (Bekdash et al., POMC promoter methylation and corresponding decreased gene expression in the hypothalamus (Bekdash et al., To this end, research regarding the prevention of FASD has centered on increasing the availability of methyl donors during development through supplementation of known methyl donors such as folate and choline. In an embryonic cell culture model, co-administration of folic acid prevents the morphological delays and deficits, together with the downregulation of Further consideration should also be paid to the types of experiments used in FASD research. To date, most studies have used bulk tissue or whole embryo inputs for molecular experiments. These have provided many valuable insights to this point, however, given the heterogeneity of the effects of PAE and preferential apoptosis of specific cell types, more focused studies are needed. Specifically, single-cell epigenetic and transcriptomic techniques are now viable for most FASD models. Single-cell or single-cell-type resolution may address many outstanding questions about the outcomes of ethanol exposure at the molecular level. Furthermore, single-cell studies and improved bioinformatic cellular deconvolution methods will aid future translational studies examining the DNA methylation signature of FASD in saliva (Turinsky et al., Neurodevelopment in mammals is a long-lasting, highly orchestrated, and coordinated continuum. It is highly sensitive and responsive to environmental exposures that may determine the outcome and resulting brain function, including neurodevelopmental disorders such as FASD. FASD is highly heterogeneous and a direct result of maternal alcohol consumption during pregnancy. Further, a stressful postnatal environment may worsen the outcomes. This review emphasizes the role of dynamic epigenetic changes during the neurodevelopmental continuum, from prenatal to postnatal stages in the development of FASD, including ncRNAs, DNA methylation, and histone PTMs. We argue that FASD is an epigenetic disorder. Additionally, the early postnatal environment may determine the severity of the effect of PAE. It may be possible to moderate this effect by postnatal epigenetic modifications, which may or may not be driven by pharmaceutical interventions. This perspective offers new hope for the development of effective treatment for FASD. Finally, the strategy of epigenetic intervention discussed here may apply to a variety of neurodevelopmental disorders involving epigenetic marks caused by environmental exposures.All authors contributed to the design and organization of this review. All authors performed a literature review, as well as interpretation and synthesis of findings. BA created the first draft, with all authors substantially contributing to revisions. All authors have approved the final revisions prior to submission. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The maternal diet during pregnancy is a key determinant of offspring health. Early studies have linked poor maternal nutrition during gestation with a propensity for the development of chronic conditions in offspring. These conditions include cardiovascular disease, type 2 diabetes and even compromised mental health. While multiple factors may contribute to these outcomes, disturbed epigenetic programming during early development is one potential biological mechanism. The epigenome is programmed primarily in utero, and during this time, the developing fetus is highly susceptible to environmental factors such as nutritional insults. During neurodevelopment, epigenetic programming coordinates the formation of primitive central nervous system structures, neurogenesis, and neuroplasticity. Dysregulated epigenetic programming has been implicated in the aetiology of several neurodevelopmental disorders such as Tatton-Brown-Rahman syndrome. Accordingly, there is great interest in determining how maternal nutrient availability in pregnancy might affect the epigenetic status of offspring, and how such influences may present phenotypically. In recent years, a number of epigenetic enzymes that are active during embryonic development have been found to require vitamin C as a cofactor. These enzymes include the ten-eleven translocation methylcytosine dioxygenases (TETs) and the Jumonji C domain-containing histone lysine demethylases that catalyse the oxidative removal of methyl groups on cytosines and histone lysine residues, respectively. These enzymes are integral to epigenetic regulation and have fundamental roles in cellular differentiation, the maintenance of pluripotency and development. The dependence of these enzymes on vitamin C for optimal catalytic activity illustrates a potentially critical contribution of the nutrient during mammalian development. These insights also highlight a potential risk associated with vitamin C insufficiency during pregnancy. The link between vitamin C insufficiency and development is particularly apparent in the context of neurodevelopment and high vitamin C concentrations in the brain are indicative of important functional requirements in this organ. Accordingly, this review considers the evidence for the potential impact of maternal vitamin C status on neurodevelopmental epigenetics. Most animals synthesise vitamin C  in the liver, and provide a steady supply to the body through the circulation. However, humans and a few other species including other primates and guinea pigs have lost this capability due to loss-of-function mutations in the gene encoding gulonolactone oxidase, the terminal enzyme in the vitamin C biosynthetic pathway ,2. ConseThe importance of vitamin C during pregnancy to support normal fetal growth and development has been recognised for decades. As early as 1938, it was reported that the fetus “acts as a parasite on the mother’s vitamin C pool” following the observation that concentrations of vitamin C in the umbilical cord plasma of newborn infants were 2–4 fold higher than that of their mothers at the time of delivery , which cVitamin C has many established functions in the nervous system. It acts as a co-factor for a plethora of different enzymes which are involved in diverse processes including epigenetic regulation, the biosynthesis of catecholamine neurotransmitters and hormones, collagen production and angiogenesis. Another function of the micronutrient is scavenging reactive oxygen species (ROS). Given the diverse roles of vitamin C in human biochemistry, the scope of this review is the role of vitamin C as an epigenetic regulator during neurodevelopment. This is a relatively narrow field, but one with broad implications for our understanding of health and disease.Vitamin C is a diacid-ascorbic acid. At a neutral pH, the nutrient is present almost exclusively (>99.9%) in the ascorbate monoanion form. Ascorbate is an electron donor that is readily oxidised firstly to the ascorbyl radical and then to dehydroascorbic acid . DehydroTissue vitamin C homeostasis is regulated by the sodium-dependent vitamin C transporters (SVCT) 1 and 2 that actively co-transport ascorbate with sodium across the cell membrane ,20. DietVitamin C displays a diverse distribution pattern across the body, with homeostatic concentrations ranging from 200 μM in the heart to ~10 mM in the brain and adrenal glands in humans  19,24,2,224,25. The developing fetus accesses vitamin C via SVCT2 transporters in the placenta and therefore relies on a sufficient maternal supply for the entire gestational period ,21,32. TGuinea pig studies indicate that plasma vitamin C concentrations in newborn pups are also significantly higher than in the mother, when the mother has an optimal dietary intake of vitamin C . Guinea In the late 1980’s Barker et al. demonstrated a significant association between decreased birth size and cardiovascular disease in adulthood that gave rise to the Developmental Origins of Health and Disease (DOHaD) phenomenon ,45. BarkA modern definition of epigenetics is “the study of changes in gene function that are mitotically and/or meiotically inherited without changes in DNA sequence, but with chemical modifications of DNA, or of the structural and regulatory proteins that are bound to it” ,55. WhilDuring DNA methylation, a methyl group is added at C5 of cytosine by the DNA methyltransferase (DNMT) enzymes, generating 5-methylcystosine (5 mC). CpG islands are by far the most heavily methylated regions of the mammalian genome . Most CpThe epigenome is established primarily in utero. DNA methylation patterns during gametogenesis and embryogenesis are remarkably dynamic . MethylaFollowing fertilization, global DNA demethylation occurs in both the maternal and paternal pronuclei, giving rise to the totipotent zygote ,69. DNA During morphogenesis, the totipotent zygote transforms into two distinct cell lines, the pluripotent inner cell mass and the trophectoderm, that form the primitive mammalian embryo. The inner cell mass eventually forms the embryo proper and the trophectoderm forms the placenta. Following implantation, the inner cell mass differentiates into the diverse assortment of cells such as myocytes, neurons, and epithelia that will form the tissues of the fetus. This remarkable process occurs in a controlled and stepwise fashion whereby individual cells are ‘programmed’ to begin forming the organs they are destined to become . It is aNutriepigenomics refers to the study of the effects of nutrients on health and development, as mediated through modulation of the epigenome. A famous example of nutriepigenomics is the immature honeybee that transforms into a queen or a worker bee, two very different phenotypes with identical genotypes, depending on whether the bee is fed royal jelly or pollen . By the Dnmt1 ,175Tet1 ntiation . Hypermeout mice . Tet3 increases rapidly during ESC and NPC differentiation [Tet3-knockout ESCs, self-renewal appears normal and cells can differentiate into NPCs but these rapidly undergo apoptosis, leading to an insufficient progenitor pool [NeuroD, Notch, and BMP2 that stimulate neural differentiation [DCX (Doublecortin) also become upregulated [The expression of ntiation . In Tet3tor pool . Notablyntiation ,180. Theegulated .Once a sufficient pool of neuroectoderm progenitors is established, the population of cells structurally transforms into the neural plate, the borders of which fold up and eventually converge to form the neural tube. Neurogenesis then proceeds in the walls of the neural tube to grow the brain and spinal cord ,173. NeuTet2 in regulating the balance between NPC self-renewal and neuronal differentiation has been recognised. The knockout of Tet2 leads to increased NPC proliferation and a significant reduction in the number of newly-born neurons indicating an impaired differentiation capacity [DCX-expressing neurons suggesting defective migration [Tet1 leads to decreased NPC proliferation with hypermethylation and downregulation of genes involved in progenitor maintenance [Tet3-knockout NPCs, the pluripotency genes Oct4 and Nanog become de-repressed [Tet-knockout studies described above. As mentioned previously, suboptimal vitamin C status in prenatal (beginning in the first trimester until birth) [Investigation of the distinct functions of the individual TET proteins during neurogenesis has been carried out in the adult mouse brain and derived NPCs. A specific role for capacity ,184. Thiigration . In contntenance . In Tet3epressed  and feweepressed  demonstrl birth)  or earlyl birth)  substantl birth) . These fTet1 does not completely abolish neurogenesis or cause neuroanatomical defects [Tet1/Tet2 or Tet1/Tet3 respectively causes exencephaly and holoprosencephaly in some embryos [Tet3 has been reported to cause neonatal lethality in mice [The knockout of  defects ,186. How embryos ,187. Thi in mice . Arc and Bndf, with the TETs specifically implicated in the regulation of spatial memory and fear memory extinction [The latter half of pregnancy is marked by synaptogenesis and myelination both in humans and precocial species such as guinea pigs . This cotinction ,195,196.tinction , highligSLC23A2 gene that encodes the SVCT2 membrane transporter protein, vitamin C fails to accumulate within Schwann cells and this results in a thinner layer of myelination on axon fibres and impaired synaptic transmission [The formation of the myelin sheath, mediated by Schwann cells in the periphery and oligodendrocytes in the CNS, is a critical step in the neuronal maturation pathway and is required for synaptic transmission. In the 1980s, Eldridge et al. revealed that adding vitamin C to co-cultures of Schwann cells and dorsal root ganglia facilitated the formation of myelin sheaths . Experimsmission . This fismission , supportTet3 mutations in a cohort of families with the common phenotypic features of intellectual disability, growth and craniofacial abnormalities, and autistic traits [DNMT3A [Further evidence validating the importance of the TETs in neurodevelopment and brain function has come from the recent discovery of c traits . While tc traits . The cli [DNMT3A ,199,203.Given the importance of maternal nutrition on epigenetic programming and health outcomes in later life, it is imperative that we develop the right paradigm to accurately study the effects of diet on early development. Numerous epidemiological studies have indicated that dietary variations during development can impact on health outcomes ,205,206.While rats and mice are convenient laboratory animals, they have the ability to endogenously synthesise vitamin C, meaning that mutant knockout animal models need to be established for dietary vitamin C studies ,210. TheThe pregnant guinea pig is in itself a well-established model that closely resembles human pregnancy and prenatal development, as previously reviewed . The strThe evidence is now conclusive—in utero experiences, particularly fetal nutrient availability as mediated by the maternal diet, can translate to phenotypic variation and future disease susceptibility that extends well into adulthood and even subsequent generations. The underlying biological mechanisms are indeed complex but huge advancements have been made with the advent of epigenetics. The TETs are a family of epigenetic enzymes that are highly expressed during mammalian development. As epigenetic erasers that promote the hydroxylation and demethylation of DNA, the enzymes function to modulate gene expression and coordinate complex processes such as cellular differentiation. A dozen years on from their initial discovery, there is a rapidly expanding body of literature that highlights the important roles of the TET enzymes in various aspects of neural physiology. The TET enzymes regulate NPC biology, neurogenesis, higher brain functions and behaviours, and have recently been implicated in the aetiology of neurological disorders. The dependency of the TETs on vitamin C for optimal catalytic activity becomes particularly relevant when considering the impact of nutrition on epigenetics in development. Future work examining the relationship between maternal vitamin C status and TET-mediated processes during development in utero will not only augment what is known about these prominent epigenetic enzymes but will greatly enhance our understanding of metazoan brain development and how early-life nutrition can program long-term cognitive outcomes. These questions are of great importance given the prevalence of hypovitaminosis C in pregnant women worldwide.


Restoring sensory circuit function after spinal cord injury (SCI) is essential for recovery of movement, yet current interventions predominantly target motor pathways. Integrated cortical sensorimotor networks, disrupted by SCI, are critical for perceiving, shaping, and executing movement. Corticocortical connections between primary sensory (S1) and motor (M1) cortices are critical loci of functional plasticity in response to learning and injury. Following SCI, in the motor cortex, corticocortical circuits undergo dynamic remodeling; however, it remains unknown how rehabilitation shapes the plasticity of S1-M1 networks or how these changes may impact recovery of movement. Sensory circuits provide essential components for accurate movement, including texture discrimination, spatial awareness, object perception, and tactile feedback . SensoryProprioceptive feedback transmitted through the dorsal column-medial lemniscal system is essential for movement control in healthy and injury conditions . Within Representations of somatosensory responses in S1 are highly plastic in response to nervous system damage, sensory experience, and learning. Cortical reorganization is a complex phenomenon that has been associated with both improved functional recovery and aberrant phantom sensations . Thus, tAfter incomplete lesions of the dorsal column, reactivation of S1 occurs with almost normal somatotopy . In contIn rodents using extracellular electrophysiology recordings and functional magnetic resonance imaging (fMRI) it has been shown that electrical stimulation of the forelimb after thoracic SCI elicits responses in deafferented hindlimb S1  and an eNeither the underlying neural architecture that supports S1 reorganization after SCI nor the extent of the reorganization has been clearly established. The reorganization of S1 after SCI appears to be less extensive than in M1. Forepaw S1 regions do not exhibit large-scale remodeling after SCI, but rather show a more limited expansion . Whetherfasciculus gracilis into the deafferented cuneate nucleus  in the rat has been shown to increase projections from  nucleus . In prim nucleus . Aberran nucleus .In vivo intracellular recordings in cats have shown that LTP can be induced in M1 layer 2/3 (but not in deeper layers neurons) by high frequency stimulation in S1. This plasticity is a likely mechanism underlying motor learning  cortex, layer 2/3 neurons are critical loci of functional plasticity in response to learning and injury . Forelimin vivo imaging tools and sensitive sensory and motor behavioral tasks (The circuit mechanisms underlying functional reorganization of sensory and motor cortex after SCI are not well-characterized . Studiesal tasks  will facYML and EH co-wrote the manuscript. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mollisonia symmetrica from the mid-Cambrian Burgess Shale. The fossilized neurological anatomy of M. symmetrica includes optic nerves connected to a pair of lateral eyes, a putative condensed cephalic synganglion, and a metameric ventral nerve cord. Each trunk tergite is associated with a condensed ganglion bearing lateral segmental nerves, and linked by longitudinal connectives. The nervous system is preserved as reflective carbonaceous films underneath the phosphatized digestive tract. Our results suggest that M. symmetrica illustrates the ancestral organization of stem-group Chelicerata before the evolution of the derived neuroanatomical characters observed in Cambrian megacheirans and extant representatives. Our findings reveal a conflict between the phylogenetic signals provided by neuroanatomical and appendicular data, which we interpret as evidence of mosaic evolution in the chelicerate stem-lineage.Recent years have witnessed a steady increase in reports of fossilized nervous tissues among Cambrian total-group euarthropods, which allow reconstructing the early evolutionary history of these animals. Here, we describe the central nervous system of the stem-group chelicerate  Here, the authors report a preserved central nervous system in the soft-bodied stem-group chelicerate Mollisonia symmetrica from the mid-Cambrian Burgess Shale. The neuroanatomy described here is proposed to represent the ancestral state for the stem group Chelicerata. The study of extinct soft-bodied organisms from marine deposits has become fundamental for understanding the sudden appearance of animal macrofossils during the Cambrian Explosion, and the increasing availability of material from different deposits reveals a broader spectrum of exceptional preservation than traditionally considered possible. Burgess Shale-type (BST) sites—defined by the preservation of non-biomineralizing organisms as carbonaceous compressions in marine shale6 demonstrate that in addition to external features such as eyes and limbs7, various aspects of the internal anatomy may also become fossilized and reveal substantial morphological details. For example, guts commonly occur as either carbonaceous compressions8 or three-dimensional mineralized replicates12, as are seldom muscle fibers14. An increasing number of studies published within the last decade have also reported the preservation of nervous systems in Cambrian macrofossils24, challenging the notion that brains and nerves are too delicate to be captured in the fossil record25.Exceptionally preserved fossils represent a valuable source of morphological information that allows us to reconstruct the origin and early evolutionary history of disparate animal groups that inhabit the modern biosphere27, most instances of exceptionally preserved nervous systems in Cambrian BST deposits are restricted to members of total-group Euarthropoda, including gilled lobopodians20, radiodonts17, fuxianhuiids28, megacheirans24, and bivalved forms30. These records encompass a broad phylogenetic sampling, which allows reconstructing the complex evolutionary history of the central nervous system (CNS) in this phylum, clarifying the segmental origin of euarthropod head structures, and illuminating the ancestral neuroanatomical organization of extant representatives31. Yet, further discoveries of palaeoneuroanatomical data in other euarthropod groups are still critically needed for comprehensively understanding the early macroevolution of the CNS in this megadiverse phylum, and testing competing phylogenetic hypotheses.With a few exceptionsMollisonia symmetrica from the mid-Cambrian (Wuliuan) Burgess Shale in British Columbia  with occurrences in various Cambrian-Ordovician BST deposits of North America, South China, Wales, Morocco, and possibly Australia (reviewed in refs. 33). Although mollisoniids are characterized by a distinctive exoskeletal morphology, including a head shield with deep anterolateral ocular notches, a parallel-sided thorax consisting of 7–14 freely articulating tergites, and a pygidial shield with marginal spines, until recently little had been known or even hypothesized concerning their relationships with other euarthropods32. The discovery of appendicular data in Mollisonia plenovenatrix from the Burgess Shale, suggesting a phylogenetic position within stem-group chelicerates, has prompted a substantial revaluation of the significance of mollisoniids for euarthropod evolution34. We explore the significance of our palaeoneuroanatomical data on mollisoniids within the context of the chelicerate stem-lineage, and address its broader implications for the evolution of the euarthropod CNS organization by the mid-Cambrian.Here, we describe the CNS organization in the mollisoniid euarthropod ia Figs. , 2. The 35. This specimen was designated as the holotype of the  species Houghtonites gracilis by P. Raymond35, who also included within this new genus specimen USNM 57661  based on the similarity with other small-sized specimens that have been previously attributed to this species32.MCZ 1811 is a completely articulated individual collected in 1925 by J. D. Houghton at the Raymond Quarry in Burgess Pass, and originally described by Percy Raymond in an upside-down orientation33, the body outline is elongate, subrectangular, and maintains a consistent width throughout . The exoskeleton consists of a head shield that represents approximately one-fifth of the body length (sag.), followed by a trunk composed of seven freely articulating tergites with lateral pleurae with subtriangular tips, and a pygidium  of similar dimensions to the head shield, and which bears two pairs of short marginal spines projecting from the posterior margin. The head shield is pentagonal in outline, and features a pair of anterolateral concave notches that accommodate well-developed, bulging lateral eyes. Between them, a pair of short, seemingly uniramous structures project anteriorly; their precise nature, especially whether they represent cephalic appendages, is uncertain owing to preservation  of 13 mm, and a maximum width (trans.) of 3.5 mm  of 7.5 mm and a maximum height (trans.) of 1.7 mm , and 11 pairs of trunk appendages  consisting of oval-shaped flattened exopods34. The prominent lateral eyes of M. plenovenatrix are also expressed in M. symmetrica, albeit with a less oblong shape, and our studied specimens clearly show how the optic nerves converge towards the median region of the head . Additional evidence for this proposal comes from the presence of three pairs of dorsal ridges and marginal spines in the pygidium, which are regarded as serial homologs of the pleural ridges and tips, respectively, of the thoracic tergites33. These observations indicate that the mollisoniid trunk follows the ancestral one-to-one correspondence between segmental ganglia and trunk tergites , rather than showing indications of substantial mismatch between the CNS and the dorsal exoskeleton as observed in some Cambrian euarthropods19.MCZ 1811 and USNM 305093 offer complementary dorsal and lateral views (respectively) that allow reconstructing the CNS of ety Fig. . A closead Figs. . Althoug093 Fig.  confirmsM. plenovenatrix has been interpreted as having seven pairs of appendages closely packed within the head shield34, the preserved CNS in USNM 305093 lacks evidence of substantial differentiation  expressed as a highly condensed synganglion-like structure, similar to that expressed in the prosoma of extant euchelicerates, but too compact to be discernible in such a small specimen through BST preservation. For context, Cambrian euarthropod specimens exhibiting conspicuous cephalic nerves are at least three times larger , and preserve significantly bigger brains  when compared to USNM 305093 . There is no clear evidence for encephalization in our studied specimens of Mollisonia, but future discoveries of neuroanatomical structures in larger specimens will most likely inform the CNS of the head region in greater detail. Despite these limitations, the fact that the organization of the CNS closely parallels that of the body  suggests that our data reliably capture the general neuroanatomy of these Cambrian euarthropods , a more recent analysis with smaller taxon sampling resolved Mollisonia as a stem-group chelicerate and the antennae-bearing artiopods as stem-group Mandibulata34. In order to explore the broader implications of M. symmetrica’s palaeoneuroanatomical data in the context of early chelicerate nervous system evolution, we performed Bayesian inference and maximum parsimony phylogenetic analyses  that incorporate all known Cambrian taxa with neurological preservation  share the presence of paired sets of eyes (or doublets), each containing a single neuropil that lies outside of the protocerebrum16, Mollisonia features a single set of lateral eyes   Chengjiangocaris kunmingensis19.ata Fig. . Mollisoata Fig. , and cou39) Fig.  suggestsoda Fig. , as for Habelia48, sanctacaridids50, and megacheirans52. Whereas there is growing support for the chelicerate affinities of Habelia and sanctacaridids based on their appendicular organization, and that both groups are close relatives of mollisoniids, the position of megacheirans has remained controversial54. Megacheirans are non-biomineralized euarthropods typified by the presence of a well-developed pair of raptorial “great appendages”, which have been interpreted as precursors of the chelicerate chelicerae . They also share with euchelicerates the presence of a reduced labrum58, as recently documented in Leanchoilia illecebrosa55, and several neuroanatomical features. Within Megacheira, neuroanatomical data is known from the leanchoiliids Alalcomenaeus and Leanchoilia, with the CNS organization in these taxa corroborated by multiple specimens from the lower and middle Cambrian of South China24 and North America22 that are preserved in dorsal and lateral views. The CNS of Alalcomenaeus supports a deutocerebral segmental origin for megacheiran great appendages, and also shows derived neurological characters shared with extant euchelicerates including the lateral eye doublets with a single neuropil outside the protocerebrum, a condensed synganglion-like structure that innervates the cephalic appendages, an extended esophageal foramen, and the presence of multiple connectives that extend into the posterior end of the body , the presence of paired axial protrusions on the tergites  and a paddle-shaped tailspine with marginal spines . Regardless of the position of megacheirans, both evolutionary scenarios carry the implication that the CNS of Pycnogonida41 appears to have undergone substantial secondary simplification relative to Cambrian stem-group representatives as informed by Mollisonia and Alalcomenaeus, whilst still retaining a distinctly ganglionated VNC organization that closely resembles that of M. symmetrica , and regarded as pervasive in the history of the biosphere64. Although the Cambrian fossil record of stem-group chelicerates remains too incomplete to properly test the rates of evolutionary change between the external and internal anatomy, future discoveries will continue providing additional data to test this hypothesis. In the meantime, our palaeoneurological data from Mollisonia symmetrica provides a valuable glimpse into the overall ground pattern for the CNS of crown-group Euarthropoda based on mid-Cambrian representatives.The description of the CNS of ges Fig. . The lacSpecimens were photographed with a Nikon D850 DSLR fitted with an AF-S Micro Nikkor 60 mm lens (MCZ), and an Olympus DSX-110 inverted digital microscope (USNM). Backscattered scanning electron microscopy and energy dispersive spectroscopy-based elemental mapping were performed with a QEMSCAN 650 F  at the Department of Earth Sciences at the University of Cambridge, and a custom Tescan Vega GMU  at Harvard University. Figures were produced in Photoshop CS and Inkscape.19, which is specifically designed to incorporate palaeoneuroanatomical data from Cambrian euarthropods. Character coding and discussion of results are available in the Supplementary Note.The character matrix used for the phylogenetic analyses includes 54 taxa and 106 characters (Supplementary Data 66 for 5 million generations (four chains), with every 1000th sample stored (resulting in 5000 samples), and 25% burn-in (resulting in 4000 retained samples). Convergence was diagnosed with the software Tracer67, with effective sample size values over 200. The parsimony analyses were run in TNT68 under New Technology Search, using Driven Search with Sectorial Search, Ratchet, Drift, and Tree fusing options activated with standard settings70. The analysis was set to find the minimum tree length 100 times and to collapse trees after each search. All characters were treated as unordered. For comparative purposes, analyses were performed under equal and implied weights (k = 3).The Bayesian analysis was run in MrBayes 3.2 using the Monte Carlo Markov-chain model for discrete morphological charactersFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


The interactions between the pulmonary ECM and BCs are, therefore, a vital component for understanding the mechanisms regulating BC stemness during health and disease. If we are able to replicate these interactions in airway models, we could significantly improve our ability to maintain basal cell stemness ex vivo for use in in vitro models and with prospects for cellular therapies. Furthermore, successful, and sustained airway regeneration in an aged or diseased lung by small molecules, novel compounds or via cellular therapy will rely upon both manipulation of the airway stem cells and their immediate niche within the lung. This review will focus on the current understanding of how the pulmonary ECM regulates the basal stem cell function, how this relationship changes in chronic disease, and how replicating native conditions poses challenges for ex vivo cell culture.The extracellular matrix (ECM) is not simply a quiescent scaffold. This three-dimensional network of extracellular macromolecules provides structural, mechanical, and biochemical support for the cells of the lung. Throughout life, the ECM forms a critical component of the pulmonary stem cell niche. Basal cells (BCs), the primary stem cells of the airways capable of differentiating to all luminal cell types, reside in close proximity to the basolateral ECM. Studying BC-ECM interactions is important for the development of therapies for chronic lung diseases in which ECM alterations are accompanied by an apparent loss of the lung’s regenerative capacity. The complexity and importance of the native ECM in the regulation of BCs is highlighted as we have yet to create an  If we are to look forward to regenerative strategies for the human lung it is critical that we understand the mechanisms by which the immediate cellular environment of the basal airway stem cell regulates its long-term regenerative capacity for restoring functional mucociliary clearance.The extracellular matrix (ECM) is a three-dimensional (3-D) network of extracellular macromolecules that provides structural, mechanical, and biochemical support for the cells and tissues that make up complex organisms. It is becoming increasingly apparent that the ECM plays a vital role in development and maintenance of healthy adult tissues. This review will focus on the ECM of the tracheo-bronchial airways, discussing our current knowledge of how the pulmonary ECM facilitates lung development, influences the stem cell niche, changes with chronic disease, and poses challenges for cell culture in an in vivo with varying capacity for differentiation  the appearance of the distal alveolar air sac (acinus), 2) the development of the air-blood interface and differentiation of the pulmonary epithelium, and 3) the commencement of surfactant production . Type 1 During the terminal saccular stage, air spaces continue to widen, and the air-blood interface becomes more closely related through the rearrangement of capillaries . A signiIn humans, alveolarization begins at approximately 20 weeks post-conception and is not completed until at least 7 years of age; in mice however, alveolarization takes place entirely postnatally . The sacTwo major structural types compose the adult pulmonary ECM. First, basement membranes, thin glycoprotein sheets, line the basal surface of the epithelia and endothelia, and also envelop muscle, peripheral nerve cells, and fat . Second,in vivo and in ex vivo models.Decoding the regenerative capacity of the lungs is a vital step toward identifying potential therapies for chronic lung diseases. This has proven to be difficult because the lung is relatively quiescent under normal conditions with infrequent divisions of progenitor cells to maintain the respiratory epithelium . As suchet al. has identified 5 BC sub-types in the native human proximal epithelium  that show upregulation of cytokeratin 8 (KRT8), and that these BLPs eventually differentiate to secretory or ciliated cells , or a combination of Matrigel and COLI or COLVI , decline   or in 3- decline . In ALI  decline . Often b decline . In addipithelia . While tpithelia . The prein vitro expansion. The addition of a combination of three small molecules to the media, Rho-associated protein kinase (ROCK) inhibitor, dorsomorphin homolog 1 (DMH-1) [inhibitor of bone morphogenic proteins (BMP) signaling], and A-83-01 , allows for extended airway BC expansion in the undifferentiated state; this is thought to be the result of dual SMAD inhibition  was the addition of Rho kinase inhibitor (Y-27632) and co-culture on a layer of fibroblast feeder cells, specifically NIH-3T3-J2 cells . Cells rxpansion .ex vivo, researchers are now exploring the decellularization of lung and airway tissues to more closely recapitulate the ECM and mechanical properties seen in vivo  when expanded on several artificial culture matrices, including Matrigel and COLI, and whole decellularized lung (in vivo (2010). Such comprehensive studies have yet to be carried out for human airways and will be essential if a functional lung derived from a repopulated ECM scaffold is to become reality.Although decellularization and recellularization techniques must be significantly refined before they are capable of generating a functional lung, researchers are demonstrating that there are advantages to using a native ECM scaffold for BC culture and epithelium generation . Recentlzed lung . They colatforms . Demonstlatforms . In the latforms . This stzed lung . The lunet al. reported that on hydrogels with increased elastic moduli, similar to native fibrotic ECM, ECM producing myofibroblasts showed heightened expression of myofibroblast transgenes and fibroblasts demonstrated a 60% increase in levels of collagen 1a1 and alpha smooth muscle actin compared to soft matrices  and ciliated cells shown directly through scanning electron microscopy and alpha-tubulin expression  and 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized bacterial cellulose (OBC) nanofibrils and engineered for 3D printing of lung tissue scaffolds . Anotherpression . The respression . Unfortupression   derigous BCs  (Firth egous BCs  or 4) acdiseases . All of Asthma is a chronic inflammatory disease of the airways marked by hyperresponsiveness of the airways to triggers or irritants such as allergens, viruses, and exercise resulting in exaggerated narrowing of the airways . This caIn vitro experiments replicate this phenotype with TGF-β-stimulated fibroblasts grown on polydimethylsiloxane (PDMS) substrates having increased expression of collagen 1 and alpha-smooth muscle actin indicative of their differentiation to myofibroblasts. As myofibroblast differentiation increased, substrate stiffness also increased from 1 to 50 kPa  in particular] , and uprin vitro models of asthma are supported by changes observed in the lungs of asthmatic patients. Recently, Vieira Braga et al. completed a comprehensive scRNAseq profile of biopsies from asthmatic airways. The data revealed a significant increase in goblet cell numbers compared healthy control samples supporting prior observations in asthmatic lung tissues . While still in early development, this seems to be the most promising avenue for restoring asthmatic airways and potentially eliminating completely the associated symptoms.Inhaled corticosteroids and beta-2-agonists are commonly used as asthma treatments to reduce inflammation and relax the airway smooth muscle; these treatment options are effective at preventing exacerbations, but they are not able to treat airway remodeling . More rein vitro assays, however, the abnormal mesenchymal phenotype only persists for two weeks in ALI models, after which mesenchymal markers progressively declined, suggesting that there are other in vivo microenvironmental factors necessary to maintain EMT or that EMT is specific to in vitro culture systems which mimic airway epithelial repair , and differentiation to produce functional gas exchange units. Advancements in our awareness of the often subtle, differences in ECM composition in different regions of the lung and how these differences produce diverse tissue types will aid in our ability to contain seeded cells to their designated area and direct them to differentiate appropriately.While at the surface level the ECM may appear to be simple scaffold, in reality it plays a critical role in lung physiology through mechanical and biochemical factors. Further study of the pulmonary ECM and its relationship with cells will progress airway modeling, therapy development for chronic diseases, and overall understanding of lung physiology.


Equitable access to essential medicines and diagnostic tools is crucial for improving global public health and promoting sustainable health development efforts. Many individuals suffering from health challenges around the world still do not have access to existing life-saving interventions due to lack of availability and high costs. The devastating effects of these inequities and discussions about their solutions have been at the forefront during the COVID-19 pandemic.Lancet Commission on Essential Medicines Policies, despite the existence of many industry-led AtM programs and the independent AtM Index that ranks 20 largest R&D-based pharmaceutical companies, rigorous evaluation remains a key challenge [Health experts have long been working to identify effective methods of increasing access to medicines (AtM) that also incentivize research and development (R&D) by drug manufacturers. In the last two decades, the number of industry-led AtM programs implemented has substantially increased , with thhallenge . In resphallenge . Figure hallenge . The Indhallenge . The lathallenge . SimilarIn addition to the AtM Index, another major effort to improve reporting on AtM programs is Access Accelerated and its Access Observatory, which attempts not only to collect data, which is made publicly available, but also to devise a standardized measurement framework . Their eA variety of factors continue to serve as barriers to conducting widespread, robust program evaluations, including the lack of specific data about drugs, quality medicine pricing data, and household usage of drugs. In addition, companies often lack incentives to collect and report high-quality impact data.Despite these challenges, there are a small number of pharmaceutical companies that have conducted robust evaluations in the past. While none of the 120 AtM initiatives evaluated by Rockers et al. in 2017 met the high-quality threshold under the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system, three studies were classified as moderate quality: (1) Merck MSD’s Mectizan Donation Program, (2) the Pfizer International Trachoma Initiative, and (3) Novartis’s Access program in Kenya. The Novartis Access study was the first randomized assessment of the impact of a pharmaceutical industry-led AtM program, and initial results indicate that even offering medicines at US$1 per treatment per month did not significantly improve overall access nor reduce prices of the medicine after one year . The stuProgram design varies widely between AtM initiatives, with the 120 AtM initiatives assessed in 2017 employing the following strategies: medicine donation (48%), price reduction (44%), licensing agreements (22%), and supply chain strengthening 11%). It has been found that bulk donations are more likely to be unsustainable and even cause harm in the long run by reducing competition [%. It hasThe urgent need to overcome COVID-19 has prioritized AtM among global leaders. In May 2020, the World Health Assembly adopted a resolution that calls for increased collaboration through “existing mechanisms for voluntary pooling and licensing of patents” to mitigate the COVID-19 pandemic and for “equitable access to and fair distribution of all essential health technologies and products to combat the novel coronavirus.” The resolution was co-sponsored by more than 130 countries and adopted by consensus [


Stroke is one of the leading causes of disability and death worldwide and places a significant burden on healthcare systems. There are significant racial/ethnic differences in the incidence, subtype, and prognosis of stroke, between people of European and African ancestry, of which only about 50% can be explained by traditional stroke risk facts. However, only a small number of genetic studies include individuals of African descent, leaving many gaps in our understanding of stroke genetics among this population. This review article highlights the need for and significance of including African-ancestry individuals in stroke genetic studies and points to the efforts that have been made towards this direction. Additionally, we discuss the caveats, opportunities, and next steps in African stroke genetics—a field still in its infancy but with great potential for expanding our understanding of stroke biology and for developing new therapeutic strategies.  Stroke is a complex and heterogeneous disease. Risk of stroke involves the interplay between genetic, epigenetic, and environmental factors. Stroke is the second leading cause of death and disability worldwide with over 13 million new strokes and 5.5 million deaths recorded in 2016 ,5,6. AnnThe incidence of stroke is twice as high in African Americans compared to Americans of European ancestry. This disparity in incidence is more pronounced for ages 45 to 54 years (Black/white Incidence rate ratio (IRR) = 4.02) and declines with increasing age  ,10 possiThe reasons for the above racial disparities in stroke occurrence and outcomes are multifaceted and remain largely unknown. It is hypothesized that differences in the rates of stroke risk factors, such as uncontrolled hypertension, might be a possible culprit. However, data from the biracial Reasons for Geographic and Racial Differences in Stroke (REGARDS) study found that only 50% of the increased stroke incidence reported in blacks could be explained by racial peculiarities in the prevalence of traditional risk factors such as the Framingham risk factors or by socioeconomic status, income and education . AdditioGenetic studies of stroke in populations of African ancestry are crucial not only for unveiling the reasons for the pronounced ethnic disparities in stroke incidence, subtype distribution and outcome but also for elucidating the genetic and biological mechanisms of stroke globally and even eventually moving towards developing precision medicine for stroke. Unfortunately, most genetic stroke studies involve primarily populations of European ancestry, although a few also include African Americans and only a couple focus on continental Africans. It is important to recognize the current state of stroke genetics in African-ancestry populations, the recent and upcoming advances and the work that still needs to be done. In this article we will review the findings of recent Genome-Wide Association Studies (GWAS), Whole-Exome Sequencing (WES) studies and candidate gene studies that have included individuals of African Ancestry , focusing on their advantages and shortcomings. We will also examine the existing knowledge gaps, challenges, opportunities and then discuss some next steps in the efforts to uncover the genetic underpinnings of the distinctive epidemiology and genetic epidemiology of stroke in these populations. A systematic literature review was conducted in accordance with the PRISMA guidelines using the PubMed and Google Scholar databases. Combinations of keywords used to generate the list of articles include: “African/African American/ Black stroke genetics”, “Stroke GWAS Africans /African Americans/Blacks”, “Ischemic stroke genetics Africans/African Americans/Blacks”, “Candidate gene studies stroke Africans/African Americans/Blacks”, “Exome Sequencing stroke Africans/African Americans/Blacks” and “Hemorrhagic stroke genetics Africans/African Americans/Blacks”. Please note that, in order to search more broadly, search phrases were not included in quotation marks and each keyword referring to individuals of African ancestry  was included in a separate search. Only studies published between January 2000 and May 2021 were selected. After eliminating duplicates, the studies were then characterized as GWAS, WES studies, candidate-gene, case-control, population-based and meta-analyses. Articles that were not written in English, did not pertain to studies of individuals of African ancestry or to stroke genetic studies and study protocols were excluded from the review . Non-speAPOL1) but are now shown to also increase susceptibility to non-communicable diseases [People of African ancestry, as a result of their longer history, have greater genetic diversity, higher levels of admixture and an intricate population substructure pattern compared to other ancestral groups. Because of this, individuals of African ancestry have the lowest levels of linkage disequilibrium and the smallest haplotype blocks compared to individuals of European ancestry. Due to the need to fight off certain pathogens and diseases, populations in Africa and therefore individuals of African Ancestry have developed genetic adaptations that have resulted in the presence of distinct allele frequencies, not seen in or among other ancestral groups/populations. Some of these once beneficial alleles can increase the risk of disease in certain environments. Examples include the APOE ε4 and APOL1 gene variants as well as sickle cell disease (SCD) all of which once provided a survival advantage  and against Trypanosoma brucei infections in the case of diseases ,17.The higher burden of stroke in people of African Ancestry might be partially attributable to differences in ancestry and genetic risk. Data from the SLESS study found that single nucleotide polymorphism (SNP) heritability of stroke is higher among African-ancestry populations compared with individuals of European ancestry, suggesting a stronger influence of genetics in stroke risk among this population . The preSCD has been known to significantly increase the risk for overt ischemic stroke, silent cerebral infarcts, and hemorrhagic stroke . More spThe role of the Sickle Cell Trait (SCT) (heterozygous state of the sickle cell mutation) in stroke risk is not as straightforward. An older, longitudinal single-cohort population study had suggested a link between SCT and ischemic stroke . HoweverAPOL1 risk allele, which has two variants [APOL1 high-risk variants significantly increased the risk for ischemic and small vessel disease-related stroke [Low-density lipoprotein receptor related proteins-1 and 6 variants which have been long linked to ischemic stroke. In a case-control study of the Ischemic Stroke Genetics Study (ISGS) cohort, the LRP1 rs11172113 variant was associated with stroke among African Americans without a replication of that association in the non-Hispanic White cohort [Interleukin-6 (IL-6) and CDKN2A/CDKN2B which have been implicated as sources of the racial disparities in stroke incidence. A candidate gene analysis of indigenous West African participants in the Stroke Investigative Research and Education Network (SIREN) Study revealed two genetic polymorphisms; rs1800796 in IL6, and rs2383207 in CDKN2A/CDKN2B, both of which were associated with increased stroke incidence in male participants [APOL1, CDKN2A/CDKN2B, and HDAC9 genes were significantly associated with small vessel disease ischemic stroke risk [NOS3 gene was linked with large artery ischemic stroke among African Americans in a small sample of 377 patients with ischemic stroke [NOS3 gene associated with ischemic stroke in the black women but not in white women [One way to uncover the ethnic differences in stroke prevalence is through targeted analyses of specific genes and alleles. One candidate allele which is found more frequently in African populations and is associated with several different phenotypes is the variants . In an ad stroke . Other ee cohort . Other cicipants . Anotheroke risk . Additioc stroke . Finallyte women . A revieALOX5AP) gene were associated with ischemic stroke in White Americans but not in African Americans [APOE locus which has been associated with lobar and deep Intracerebral Hemorrhage (ICH) in European populations [In contrast, other alleles that have been established to influence stroke risk in non-Hispanic White individuals do not seem to be genetic contributors to stroke risk in Blacks. For example, polymorphisms in the Arachidonate 5-lipoxygenase activating protein , the aldehyde dehydrogenase 1 family, member A2 (ALDH1A2) and hepatic lipase (LIPC) genes. This region has been linked with cerebral energy metabolism, brain ischemia, lipid levels and hypertension. 18 additional SNPs showed suggestive association and multiple loci had the same direction of effect in stroke risk as in European ancestry populations, pointing to common pathogenetic mechanisms of stroke across both ancestry groups [HNF1A gene that was significantly associated with stroke and 29 new suggestive variants, some of which were also replicated in European cohorts [GWAS, as opposed to candidate gene studies, have been successful in identifying novel variant-trait associations and can provide new insights into ethnic variation of complex traits such as stroke. Unfortunately, the results of GWAS and WES studies to date have been primarily driven by individuals of European ancestry. The first large-scale total stroke GWAS of African American individuals came from the Consortium of Minority Population genome-wide Association Studies of Stroke (COMPASS) collaboration. The analysis, which included 14,746 African Americans, found a novel SNP associated with total stroke in the 15q21.3 locus which is located near the y groups . A recenVWDE gene encoding von Willebrand factor D and EGF-domain–containing protein which was associated with early-onset stroke in both white and black individuals [Another GWAS of Africans and African Caribbeans in the SLESS study also showed that SNPs associated with ischemic stroke in Europeans had a similar impact in African populations . Additioividuals . An overIncluding African-ancestry individuals in stroke genetic studies presents several challenges. Firstly, the lack of reference panels that contain common variation of African-descent populations makes it difficult to characterize and as well as consider the genetic differences between populations. Secondly, the absence of an array which includes SNPs found in African populations makes SNP imputation a challenging task . ThirdlyAlthough including African ancestry populations in stroke genetics can seem a challenging task, the benefits and opportunities of such studies are immense and can have important global implications since all humans descended from Africa. Replication of European-descent population risk variants in African descent populations increases the generalizability and scope of the findings by indicating a true causal relationship between the candidate variant and the trait . AdditioThe important role of GWAS in discovering the biological pathways that lead to diseases, including stroke, has been highlighted. Although the number of GWAS performed increased more than 6-fold from 2009 to 2016, the number of African ancestry participants increased only by 2.5% . FurtherEfforts to include African populations specifically in stroke studies include SIREN which includes the largest population of Continental Africans with stroke and the COMPASS consortium which was founded to identify susceptibility loci for stroke in minority populations. Additional examples of studies that seek to elucidate the ethnic discrepancies in stroke incidence are The Genetics of Early Onset Stroke (GEOS) Study which includes a large number of African American stroke patients and the SLESS which includes a UK African ancestry population. The above cohorts would provide adequate statistical power for identifying the ancestry-specific genetic component of each of the stroke subtypes . As we look ahead, it will become important to perform gene and pathway-based analyses of diverse cohorts and to combine genomics with functional-omics such as proteomics, metabolomics, transcriptomics and epigenomics. This information could help us understand the complex pathophysiology of stroke and to uncover the complex gene-gene and gene-environment interactions responsible for the ethnic disparities in stroke risk . Based oThis systematic review explores the few efforts that have been made to better understand the distinct genetic and genomic underpinnings of stroke in individuals of African ancestry, an ethnic group that is disproportionately affected by stroke. These studies have uncovered points of divergence and convergence of stroke pathogenesis between different ethnicities and populations. By discussing this understudied, yet highly significant topic, we hope to emphasize the need for further, more focused research that will improve our understanding of stroke etiology and stroke management in African ancestry populations.


Triple negative breast cancer lacks targeted therapies and has poor prognosis; chemotherapy is currently the main treatment modality. Growing evidence has shown that breast cancer stem cells are associated with tumor initiation and metastasis and may play a critical role in chemoresistance. Multiple targets against breast cancer stem cells are now under investigation. Recent advances in the role of breast cancer stem cells in triple negative breast cancer and the identification of cancer stem cell biomarkers have paved the way for the development of new targeted therapies. The discovery of potential molecular signaling pathways targeting breast cancer stem cells to overcome chemoresistance and prevent metastasis will improve the overall survival of patients with triple negative breast cancer.Triple negative breast cancer (TNBC) remains an aggressive disease due to the lack of targeted therapies and low rate of response to chemotherapy that is currently the main treatment modality for TNBC. Breast cancer stem cells (BCSCs) are a small subpopulation of breast tumors and recognized as drivers of tumorigenesis. TNBC tumors are characterized as being enriched for BCSCs. Studies have demonstrated the role of BCSCs as the source of metastatic disease and chemoresistance in TNBC. Multiple targets against BCSCs are now under investigation, with the considerations of either selectively targeting BCSCs or co-targeting BCSCs and non-BCSCs (majority of tumor cells). This review article provides a comprehensive overview of recent advances in the role of BCSCs in TNBC and the identification of cancer stem cell biomarkers, paving the way for the development of new targeted therapies. The review also highlights the resultant discovery of cancer stem cell targets in TNBC and the ongoing clinical trials treating chemoresistant breast cancer. We aim to provide insights into better understanding the mutational landscape of BCSCs and exploring potential molecular signaling pathways targeting BCSCs to overcome chemoresistance and prevent metastasis in TNBC, ultimately to improve the overall survival of patients with this devastating disease. Stem cells are characterized by their self-renewal capacity and potential to differentiate into many more specialized mature cell types. A subpopulation of stem-like cells within tumors, known as cancer stem cells (CSCs), have been identified to exhibit characteristics of both stem cells and cancer cells. CSCs in human breast tumors were initially identified in 2003 by Al-Hajj et al. . Since tBreast cancer consists of four major molecular subtypes: luminal A (Hormone receptor (HR)+/HER2), luminal B (HR+/HER2+), HER2-enriched (HR−/HER2+) and triple-negative or basal-like (HR−/HER2−) ,12,13. TIt has been suggested that CSCs play an important role in the tumorigenesis and tumor biology of TNBC. Like other organs and tissues, CSCs originate from normal stem cells in breast tissue with increasing capacity for self-renewal and proliferation. CSCs, if present in TNBC, can enhance a tumor’s capacity for metastasis and risk of invasion . StudiesAlthough uncertainties about BCSCs exist, it has been suggested that CSCs should be considered as possible therapeutic targets. Emerging evidence demonstrates that CSCs usually escape current anti-cancer therapies, which may partly contribute to metastasis dissemination, tumor recurrence and poor prognosis ,19. ElimBCSCs are a small cell subpopulation among all tumor cells, with unique stem cell characteristics such as self-renewal, high proliferation and differentiation potential to multiple lineages within the breast. BCSCs interact with their tumor microenvironment (TME) as well as on the inducing factors and elements. Markers that have been identified in BCSCs include ALDH1+, CD24− and CD44+. Cells which simultaneously express these markers have the highest tumor initiating and proliferative capacity. Several theories have been proposed about the origin of BCSCs. One of them suggests mutations in dormant normal stem cells results in their transformation to cancer stem cells, a process similar to that of non-stem cells ,23.Several markers have been used for the isolation and identification of BCSCs. In 2003, Al-Hajj M et al. first described and isolated the BCSC phenotypes CD24− and CD44+ . CD24 isSix molecular subtypes of TNBC have been proposed: immunomodulatory, mesenchymal, mesenchymal stem-like, luminal androgen receptor and two basal-like subtypes . The CD4Cancer cells with the CD44+/CD24−/ALDH1+ phenotype in breast cancer can therefore be distinguished from other cancer cells by their stem-like features, ability to maintain survival and the role in cancer invasion and metastasis, particularly in TNBC.TNBC tumors are known as being enriched for BCSCs. Transcription factors, signaling pathways and tumor suppressor genes have played a pivotal role to maintain the state of stemness of BCSCs. The Wnt, Notch, and Shh pathways have been identified as playing an important role in BCSC and TNBC biology ,30,31. LThe canonical Notch signaling pathway is initiated by activating Notch receptors upon binding to Serrate- and Delta-like ligands present on the cell membranes of adjacent cells. Notch receptors have intracellular domains that are releases into the nucleus after proteolytic cleavages. These Notch intracellular domains can recruit MAML1 and histone acetyltransferase p300 to form active transcriptional complexes, which is the final regulator of the Notch target genes . It was Cyclin D1/2 [PDGFR, MYC [BCL2 [VEGF [MMP9 [SOX2 [The canonical Shh pathway is activated by releasing its ligands to bind and inhibit transmembrane receptor Patched-1 (PTCH1). PTCH1 can subsequent initiate an intracellular signal cascade to activate of the downstream transcription factors of glioma-associated oncogene (GLI) . Common lin D1/2 , PDGFR, GFR, MYC , BCL2 [4YC [BCL2 , VEGF [4L2 [VEGF , MMP9 [4GF [MMP9  and SOX2P9 [SOX2 . The nonP9 [SOX2 . Of noteThe self-renewal mechanism of CSCs, involving the signal transduction pathways noted above and including Wnt, Notch, and Shh , is illuUsing CSCs as a prognostic marker in patients with breast cancer has profound clinical significance and has been discussed in one of our prior reviews . The proThe epithelial–mesenchymal transition (EMT) has been recently recognized to have the ability to convert epithelial cells to mesenchymal cells and subsequently acquire motile and migratory propensity . This prIt has been shown that TNBC exhibits more traits possessed by CSC than other breast cancer subtypes and is more likely to develop metastases ,58. DrivAll the above mechanisms have been shown to be possibly involved in the high propensity for invasiveness and metastasis of BCSCs in TNBC. Targeting these mechanisms can potentially prevent metastasis and therefore improve survival in TNBC.TNBC, the most aggressive and deadliest type of breast cancer, lacks a targeted therapy and has therapy-resistance to the majority of chemotherapy regimens. Since none of the current chemotherapies specifically targets CSCs, CSCs serve as a tumor reservoir for self-renewal and proliferation. This causes inevitable tumor invasion and metastasis, which is more common in TNBC than non-TNBC, ultimately resulting in poor prognosis. Studies have shown CSCs are associated with chemoresistance, particularly in TNBC due to the higher propensity of developing stemness compared to those found in non-TNBC ,76.Doxorubicin (Dox), for instance, is widely used in the treatment of TNBC. However, resistance by tumor cells limits its effectiveness. CSCs have been found to be associated with Dox resistance. Signal transducer and activator of transcription 3 (Stat3) and its downstream pathway can convert non-CSCs to CSCs . More reSince the CD44+/CD24−/ALDH1+ phenotypes have been recognized as BCSC markers in many studies, a hypothetical approach to targeting CSCs against cell surface membrane antigens such as CD44 would be justified ,82. TargImmune evasion has also been suggested to result in chemoresistance in TNBC, as well as tumorigenesis in other tumor types. Tumor-infiltrating lymphocytes (TILs) have been shown to be present in some TNBC and non-TNBC breast cancers and have been associated with favorable prognosis ,86. ProgThe involvement of any of the above mechanisms may result in chemoresistance in TNBC. It is essential to take these mechanisms into account when developing novel targeted therapies to overcome chemoresistance.Recent studies have demonstrated the role of BCSCs as the source of metastatic disease and drug resistance in TNBC. Understanding the mechanisms and pathways that stimulate cancer cell proliferation can lead to the development of effective targeted treatment strategies. More importantly, in TNBC, as treatment failure is likely to be associated with BCSCs that possess unique survival mechanisms and pathways that bypass or evade the conventional chemotherapy, developing cancer stem cell-targeted therapies should focus on the specific signaling pathways unique to BCSCs. The three signaling pathways reviewed above, namely the Wnt, Notch, and Shh pathways are potential treatment targets . More anNon-CSC specific molecular signaling pathways have been proposed to be able to potentially eradicate CSCs and other cancer cells in TNBC. Due to the proven association between the EGFR pathway and TNBC metastasis , EGFR inThere are a few other mechanisms that have been proposed to tackle therapy-resistant TNBC. These mainly include the blockade or reversal of the EMT of cancer cells and the disruption of TME or the metabolism ,108,109.When being treated with conventional chemotherapy, therapy resistant BCSCs, non-BCSC tumor cells, stromal cells and immune cells can result in the minimal residual disease ,114. UnlAlthough BCSCs can evade the immune system, resulting in insufficient adaptive and inmate immune responses and subsequent chemoresistance, they are strongly antigenic so that naïve CD8+ effector T-cells, once activated, can still eliminate BCSCs. There is more and more evidence suggesting that empowering the CD8+ effector T-cells through the engagement of their programmed death receptor-1 (PD-1) with the PD1 ligand (PD-L1) would synergize with BCSC-directed therapy and in a combination with other target therapies ,118. A sLastly, gene therapy using advanced gene editing technology such as CRISPR targeting CSCs in TNBC could be a potential treatment option. The CRISPR/Cas9 system has been emerged as a leading technology to more quickly target specific genetic loci and more accurately modify genes of interest . Castro TNBC remains an aggressive disease due to the lack of targeted treatment and low rate of response to chemotherapy. BCSCs are a small subpopulation of breast tumors that play a vital role in metastatic disease and drug resistance. TNBC tumors are characterized as being enriched for BCSCs.Recent advances in BCSC biology in TNBC and the identification of cancer stem cell biomarkers have paved the way for the development of cancer stem cell-targeted therapies.Multiple targets against BCSCs are currently under investigation with the goals of either selectively targeting BCSCs or co-targeting BCSCs and non-BCSCs (majority of tumor cells).We hope this review provides insights into mutational landscape of BCSCs and the discovery of potential molecular signaling pathways targeting BCSCs to overcome chemoresistance and prevent metastasis, ultimately to improve the prognosis of TNBC.With the growing evidence of the role of BCSCs in TNBC, it is important to better define their biological characteristics, molecular pathways that sustain stemness, and mechanisms of drug resistance to discover effective cancer stem cell targeted therapies.The identification and quantification of existence of BCSCs using stem cell markers and deployment of CSC-targeted therapies may revolutionize the TNBC treatment paradigm. BCSCs may become potential prognostic and predictive biomarkers for metastasis and chemoresistance, and eradicating BCSCs in the TNBC may improve the overall survival of these patients. Although there have been more promising results recently, the shortcomings of the current research on BCSCs include a limited number of biomarkers and therapeutic targets. More clinical trials are also needed to demonstrate effectiveness and safety profiles of the new investigational drugs.


The syndrome of cerebellar ataxia with neuropathy and bilateral vestibular areflexia (CANVAS) has emerged progressively during the last 30 years. It was first outlined by the neurootology/neurophysiology community in the vestibular areflexic patients, through the description of patients slowly developing late-onset cerebellar ataxia and bilateral vestibulopathy. The characteristic deficit of visuo-vestibulo-ocular reflex (VVOR) due to the impaired slow stabilizing eye movements was put forward and a specific disease subtending this syndrome was suggested. The association to a peripheral sensory axonal neuropathy was described later on, with neuropathological studies demonstrating that both sensory neuropathy and vestibular areflexia were diffuse ganglionopathy. Clinical and electrophysiological criteria of CANVAS were then proposed in 2016. Besides the classical triad, frequent chronic cough, signs of dysautonomia and neurogenic pains were frequently observed. From the beginning of published cohorts, sporadic as well as familial cases were reported, the last suggestive of an autosomal recessive mode of transmission. The genetic disorder was discovered in 2019, under the form of abnormal biallelic expansion in the replication factor C subunit 1 (RFC1) in a population of late-onset ataxia. This pathological expansion was found in 100% of the familial form and 92% of sporadic ones when the triad was complete. But using the genetic criteria, the phenotype of CANVAS seems to expand, for exemple including patients with isolated neuronopathy. We propose here to review the clinical, electrophysiological, anatomical, genetic aspect of CANVAS in light of the recent discovery of the genetic aetiology, and discuss differential diagnosis, neuropathology and physiopathology.The online version of this article (10.1007/s12311-020-01192-w) contains supplementary material, which is available to authorized users. The association of vestibular dysfunction to chronic cerebellar ataxia has for long time been reported in patients with hereditary cerebellar ataxia, such as spinocerebellar ataxia type 3 (SCA3) , SCA1 22, FriedrIn the 1990s, neurootologists began to identify patients with a pattern of slowly progressive vestibular dysfunction and cerebellar ataxia that did not correspond to known etiologies. Their interest was first neurophysiological in showing absence of compensatory cervico-ocular reflex  and of ccerebellar ataxia with bilateral vestibulopathy, but still questioned the pure coincidence of the association. The authors did not include the peripheral neuropathy within the syndrome.The question of a distinct syndrome was raised in 2004, with the clinical and electrophysiological description of a series of 4 patients, with a quite specific pattern of slowly progressive adult-onset cerebellar ataxia with bilateral vestibulopathy . In thesand peripheral neuropathy was confirmed in 2007, by two studies from the same team. First, in a retrospective cohort of 255 patients with bilateral vestibulopathy, Zingler et al. [The non-coincidental association of cerebellar ataxia, bilateral vestibulopathy r et al.  demonstrr et al.  found 45Vestibular deficit in polyneuropathy is quite frequent and has notably been demonstrated for example in chronic inflammatory demyelinating polyneuropathy  and latecerebellar ataxia with neuropathy and bilateral vestibular areflexia syndrome (CANVAS) [In 2011, the specific entity associating late-onset slowly progressive cerebellar ataxia, bilateral vestibular areflexia and axonal sensory neuropathy was fully described in a cohort of 18 patients and renamed (CANVAS) . The sam(CANVAS) . Vestibu(CANVAS) . This su(CANVAS) . The inv(CANVAS) . The autEven though, proposed diagnostic criteria in 2016 of definite CANVAS were abnormal VVOR on videooculography, videonystagmography or rotational chair testing, and cerebellar atrophy on MRI displaying anterior and dorsal vermis atrophy and lateral hemispheric atrophy predominantly affecting crus I, and neurophysiologic evidence of a neuronopathy (ganglionopathy), and exclusion of genetic ataxias able to be gene tested, particularly SCA3 and Friedreich ataxia .Spasmodic cough was further shown to be an integral part of the clinical picture in CANVAS, antedating the appearance of imbalance in several decades  as well n = 23) and 92% of sporadic CANVAS. Furthermore, out of 150 cases diagnosed with familial or sporadic late-onset ataxia, 22% were tested positive for the biallelic repeat expansion. These patients all presented with neuronopathy, associated to cerebellar ataxia in 80%, with vestibular areflexia in 53%, with cough in 37% and dysautonomia in 23%. The biallelic expansion was found in 62% in case of association of neuronopathy and cerebellar ataxia. The presence of 100% of neuronopathy and quite low frequency of vestibular areflexia can be due to a biais in cases recrutement and/or lack of systematic eye movement recording [In 2019, Cortese et al. identified a recessive repeat expansion in intron 2 of RFC1 as the main cause of CANVAS . They inNevertheless, regarding the very high sensitivity of this RFC1 repeat expansion in full familial or sporadic CANVAS, the new definition of CANVAS has probably to be based on genetic.This review aims at describing and reviewing the clinical, electrophysiological, anatomical, genetic aspect of CANVAS in light of the recent discovery of the genetic aetiology, and discusses differential diagnosis and neuropathology.Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a slowly progressive late-onset ataxic disorder. According to the series of genetically confirmed cases, age of onset (cough excluded) is around 52 , 23 and The differences between clinically and genetically defined CANVAS syndromes are presented for 2 cohorts in Table The ataxic syndrome is explained by a combination of cerebellar postural disorder, proprioceptive deficit due to sensory axonal neuropathy and/or vestibular deficit. However, the timing of onset of the different components may vary, and the diagnosis may be challenging at the beginning of the affection. It was reported a delay of around 10 years to reach the characteristic triad . This meThe recent series of genetically confirmed cases found on clinical and investigations data the complete triad in 69% of cases, isolated neuronopathy in 15%, association of cerebellar ataxia and neuronopathy in 16%, association of vestibular areflexia and neuronopathy in 6% . There wThe presenting neurological symptoms are mainly postural imbalance (frequently increased in darkness), dizziness and/or falls, but patients may also primarily complain of sensory symptoms or oscillopsia. Oscillopsia can be permanent and mainly due to downbeat nystagmus associated to cerebellar ataxia. But in CANVAS, oscillopsia is mainly reported during head movements, due to impaired vestibulo-ocular reflex and/or visuo-vestibular ocular reflex. With the disease course, patients may develop dysarthria, dysphagia and symptoms of autonomic dysfuntion. The chronic cough can precede the onset of neurological symptoms by three decades , 20, 23.Gait ataxia is not specific and can be explained by all 3 of the cardinal features of CANVAS: cerebellar ataxia, vestibular ataxia, and a sensory ataxia. The positive Romberg test may reflect either vestibular or sensory deficit.Clinical evidence of cerebellar dysfunction is given by the combination of cerebellar-type dysarthria, appendicular ataxia, truncal ataxia and cerebellar-type ocular motor deficits. The main frequently reported ocular motor cerebellar signs are jerky smooth pursuit, gaze-evoked nystagmus, downbeat nystagmus and saccadic dysmetria , 23–25. Evidence of bilateral vestibular impairment is given by absent or severely reduced vestibulo-ocular reflex. This one can be clinically demonstrated by abnormal dynamic visual acuity , abnormaCompound deficit of vestibular and cerebellar ocular motor stabilizing functions leads to a quite specific deficit of visuo-vestibulo-ocular reflex (VVOR), which is the original feature first described in this syndrome , 8, 31. Evidence of sensory neuropathy is given by non-length-dependent multimodality sensory deficit including impairment of vibration sensation, joint position sense abnormalities, impairment of pinprick perception, limb ataxia (heel-knee and finger-nose with eyes closed) and less frequently deficit of pain/temperature . NeuropaMany patients report a long-standing chronic non-productive cough with normal ENT and pulmonary examination . This coClinical manifestations of dysautonomia, mainly orthostatic hypotension seem to be frequent , and morDisease progression is slow and 55% may need a walk stick after a mean of 10 years, and 25% a wheelchair after a mean of 15 years of disease duration .Different investigations are necessary, such as electroneuromyography to confirm or detect signs of neuronopathy, brain MRI to look for cerebellar atrophy and ocular motor vestibular testing to diagnose vestibular areflexia, visuo-vestibulo-ocular reflex deficit and search for cerebellar-induced ocular motor dysfunction.Initially, there was no mention of neuropathy in the first description of cerebellar ataxia and bilateral vestibulopathy (CABV) , but it .Electrophysiologically, a ganglionopathy results in a sensory axonal neuropathy without length-dependent pattern, a reduction or even abolition of the H reflexes but relative sparing of motor conduction and F-waves latencies. We propose a literature review of the published cohorts with electrophysiological studies in Table Diagnostic criteria of sensory neuronopathy have been proposed by Camdessanche et al.  and are The usefulness of cerebral MRI is closely correlated with cerebellar syndrome since it represents one of the most important investigations in the aetiological assessment of this type of disorder. Besides eliminating many differential diagnoses, a relatively reproducible pattern of cerebellar atrophy has been described , 17. ThiIf the clinical cerebellar syndrome appears to occur later than the peripheral symptomatology  , the MRIFinally, MRI spinal cord abnormalities (related to posterior cord injury) have been described later by Cortese et al. , reportiObjective testing in CANVAS patients can help to confirm bilateral vestibulopathy and quantify the severity and range of the impairment. Moreover, oculomotor testing can allow for early detection of cerebellar impairment in paucisymptomatic patients. Finally, objective measures can help to showcase impairement of the VVOR.In 2017, a consensus statement regarding diagnostic criteria for bilateral vestibulopathy (BVP) has been published by the Bárány Society in order to help clinicians and improve comparability with regard to scientific research . These cVideo head impulse test (or search-coil technique) allows for quantification of the VOR gain during HIT  by meansDuring rotatory chair testing, videonystagmography allows for quantification of the VOR in response to a stimulation of both horizontal semi-circular canals at low to middle frequencies. It is thus less sensitive for detection of a unilateral deficit. In BVH, however, VOR gain during rotatory chair testing is greatly reduced as none of the sides can compensate for the deficit of the other side. An impaired VOR gain of the horizontal canals is defined by horizontal angular VOR gain < 0.1 upon sinusoidal stimulation on a rotatory chair . In addition to VOR gain measurement, rotatory chair testing can also be used for quantification of the visio-vestibulo-ocular reflex (VVOR), cervico-ocular reflex (COR) and visually suppressed vestibulo-ocular reflex (VORS).Caloric testing has been first described in 1907 but is still used nowadays . The resAs stated above, diagnostic criteria for BVH do not take into account otolith function. Nevertheless, it can be evaluated with cervical and ocular vestibular evoked myogenic potential (cVEMPs and oVEMPs respectively testing the saccule and the utricule). In CANVAS, similarly to BVH, studies suggest a great variability in responses for VEMP testing –47. SubtVideonystagmography can help to identify ocular motor signs of cerebellar impairment. Saccadic testing in CANVAS patients often shows dysmetric saccades with a normal velocity , 19, 48.Cervico-ocular reflex (COR) is an ocular stabilization reflex that is elicited by rotation of the neck. It usually contributes little to gaze stability in normal subjects  but is gAs explained earlier, patients suffering from CANVAS have an impairment of all networks of slow stabilizing eye movements  resulting in a deficient visuo-vestibular ocular reflex (VVOR). Evaluation of VVOR can be done by using videonystagmography under rotational chair testing or a technique of portable high-speed videooculography equipment  Fig. 3b3b. ThresThe topicality of this syndrome is marked by the discovery of the genetic responsible abnormality of biallelic intronic AAGGG repeat expansion in the replication factor C subunit 1 (RFC1) .11 replaced by a variable number of (AAGGG) units (detected on both sides of the STR). The pathological repeat (AAGGG) has then been confirmed with repeat-primed PCR (RP-PCR) using specific primers. Standard flanking PCR failed to amplify this region in CANVAS patients suggesting the presence of a large expansion (the elongation time depends on the polymerase used and limits the amplification’s maximum size). This was confirmed by amplification with long range PCR (using a polymerase with a longer elongation time) and Sanger sequencing. The expansion sizes were estimated by southern blot with a mean of 1000 repeats (range 400 to 2000) and no correlation was found between age at onset and the size of the repeat. These results have been confirmed 3 months later by another independent study with a similar methodology using bioinfortmatics tools [Assuming the transmission was autosomal recessive, the study’s methodology consisted in combined use of classical genetic investigations and whole-genome sequencing (WGS). Indeed, the first step consisted in linkage analysis based on comparison between siblings affected or not haplotypes in order to reduce the genomic search zone to a 1.7 Mb region. Whereas whole-exome sequencing (WES) was inconclusive, whole-genome sequencing (WGS) was performed in this region and identified, by visual inspection of the aligned read pairs, a reduced read depth encompassing a short tandem repeat (STR) with as reference (AAAAG)cs tools .11 and 3 longer expansions sequences (AAAAG)n, (AAAGG)n and the pathological (AAGGG)n. Two other conformations were described later by Akcimen et al. [n and (AGAGG)n. Furthermore, it has been suggested that the conformation (AAAGG)n/(AAGGG)n could be associated with CANVAS and thus may correspond to a symptomatic compound heterozygote [First, four different repeat conformations have been observed : the wiln et al. , probablrozygote .The interest of this brief methodological description lies in the fact that the genetic tests routinely available today follow these steps and help to understand that a positive case is defined by an absence of possible amplification in standard PCR (short range) of the intron 2 of RFC1  with a positive target RP-PCR (AAGGG).Pentanucleotide expansion (AAGGG) occurs in the 3′ end of an Alu element: AluSx3. Alu elements are repeating elements about 300 bp, with a 3′ end part containing a long A-rich region considered highly polymorphic. Indeed, over the evolution, retrotransposons active elements degrade by poly(A) tail shortening or inactivation by guanine/cytosine interruption . The maiIn order to estimate the pathological allelic carrier frequency, Cortese et al. screened more than 300 controls and observed a heterozygous rate about 0.7% and thus an estimated homozygous prevalence of 1/20,000 . MoreoveProgressive ataxia due partial or complete combination of cerebellar ataxia, sensory neuropathy and vestibular deficit, as well as association to dysautonomia and chronic cough, is not specific to CANVAS syndrome. Differential etiologies or syndromes are declined below. Note however that, when present, the vestibular deficit is usually profound in CANVAS, resulting when associated to cerebellar ataxic ocular motor disorders, to the quite specific VVOR deficit. Regarding what was previously suggested as testing in diagnosing CANVAS syndrome , 19, theThe association of cerebellar ataxia, vestibular deficit and sensory neuropathy may be observed in known genetic disorders such as Friedreich ataxia, SCAs, mainly SCA3 and SCA1 and other phenotypes of mitochondrial diseases.In adult-onset Friedreich ataxia, cerebellar impairment and sensory neuronopathy can be accompanied by vestibular deficit, but much less profound than that of CANVAS , 60. IndVestibular areflexia in SCA3 has been reported later on , 2, and Within mitochondrial diseases, association of sensory neuropathy and cerebellar ataxia are prevalent notably in the phenotype of mutations in the nuclear POLG1 gene . While vBecause of dysautomic symptoms, the cerebellar form of multiple system atrophy (MSA-c) can be suspected, but the absence of vestibular deficit , 65, theThe association of neuropathy and vestibular deficit has been observed in numerous etiologies such as Charcot Marie Tooth –69, CIDPThere is a profuse literature on chronic cough resulting from neurogenic causes that could be secondary to local sensory nerve damage caused by inflammatory, infective and allergic factors . The assThe association of peripheral neuropathy and cerebellar ataxia can be observed in chronic alcoholic intoxication, and vestibular deficit has been observed in Wernicke’s encephalopathy , 77, butNeuropathology studies over these last 10 years help to better understand CANVAS and to confirm that axonal sensory neuropathy, vestibular areflexia and dysautonomia were related to a ganglionopathy.Nerve biopsy demonstrates severe axonal neuropathy , predomiAccording to Hannan et al. , the maiRFC1 codes for a subunit of replication factor C (RFC), which has a crucial role in DNA replication and repair . InteresIn summary, although still very uncertain and unproven, the main pathophysiological hypothesis suggests a mutation in a polymorphic zone, favouring during evolution the expansion of repeats modifying the expression of a gene coding for a protein involved in DNA repair, leading to damage to the most vulnerable tissues, notably the cerebellum and peripheral nerves, in particular the small fibres (Aδ and C). This differential fibre nerve damage may also explain the chronic cough. Physiologically, the cough corresponds to a reflex arc triggered by stimulation of polymodal receptors located mainly in the larynx and in the tracheobronchial tree. These nociceptors correspond to the myelinated Aδ (rather excitatory) and non-myelinated C (which may be excitatory or inhibitory) fibres which transmit information to the still poorly known centres of the brain stem, including the solitary nucleus. The efferent pathways are coming largely from the ambiguous nucleus and finally stimulate the muscles that cause the cough . In thatCANVAS is a nice story of a new disease which discovery involved the combined competences of specialists in neurootology, movement disorders/ataxia, peripheral nerve/neuromuscular diseases, neuropathology and finally neurogenetic. The last genetic discovery of biallelic RFC1 expansion explains most of the familial and sporadic cases. When the triad is complete, the sensitivity of the RFC1 expansion is very high, even in the sporadic cases. On the other hand, the core manifestation of the disease is the neuronopathy, and either cerebellar ataxia and/or vestibular areflexia may be lacking. Systematic clinical and electrophysiological search of eye movement disorders still lacks in the more recent cohort. And finally some of full phenotype of CANVAS may be explained by other diseases such as the novel variant of ELF2 gene or not explained yet. Therefore, one remaining question is whether the term CANVAS should be reserved to the clinically defined syndrome and/or the genetically (RFC1 expansion) defined disease.ESM 1(MP4 7161 kb)