Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups , representing five new subgroups . The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology. Phytoplasmas are a large group of phloem-restricted, cell wall-less bacteria that infect nearly a thousand plant species and cause serious economic loss worldwide. In nature, phytoplasmas are transmitted by phloem sap feeding insect vectors, mainly leafhoppers, in a persistent-propagative manner ,2,3. FirCandidatus status is used for reserving the putative lower rank taxa (Genus and Species). Phytoplasmas are currently assigned in the provisional genus ‘Candidatus Phytoplasma’ [Candidatus Phytoplasma’ species have been formally named under this provisional genus based on the 16S rRNA gene sequence [rp), protein translocase subunit SecY, and translation elongation factor Tu-EF [rp, and secY genes revealed azalea little leaf phytoplasmas belonged to a distinct lineage within the aster yellows phytoplasma group [Despite numerous efforts, pure phytoplasma culture has not been established in vitro thus far. Like many other unculturable bacteria, higher rank taxa of phytoplasmas  were named in the absence of type genus and species, and the oplasma’ . So far,sequence ,6,7,8,9.sequence ,11,12,13or Tu-EF . For exama group .Dalbulus maidis expanded after acquiring an aster yellows phytoplasma strain [Hyalesthes obsoletus from field bindweed to stinging nettle was observed in northern Italy [In plants, phytoplasma infection induces various symptoms including virescence , phyllody (leafy flowers), cauliflower-like inflorescence, and witches’-broom, thereby altering plant morphology, growth patterns, and architecture ,16,17,18a strain . In addirn Italy ,22. Due rn Italy .Recently, Trivellone et al.  used quaLeofa dispar, Pravistylus exquadratus, Neoaliturus opacipennis, Macrosteles sordidipennis, Mayawa capitata, Mayawa affinifacialis, and Acharis ussuriensis , and ‘Candidatus Phytoplasma brasiliense’, respectively. The similarity of 16S rRNA sequence was 98.7%, 99.1% and 97.7%, respectively. Strains PLH078-1, PLH078-12, PLH082-1, PLH082-2, PLH139-1, PLH143-1, and PLH143-5 were closely related to the reference strain of ‘Candidatus Phytoplasma cynodontis’, sharing 98.2–98.6% sequence similarity in 16S rRNA gene.PCR products amplified from ten leafhopper samples were sequenced and the obtained sequences covered a nearly full-length 16S rRNA gene and a partial 16S-23S RNA intergenic region. The sequences were deposited to the GenBank  16S rRNA gene fragment was conducted by assifier , and sixHhaI alone was able to distinguish strain PLH102-1 from 23 previously established 16SrI subgroups. Virtual RFLP patterns from MseI, AluI, and HpaII digestions separated strain PLH102-1 from the remaining ten 16SrI subgroups. Similarity coefficients derived from virtual RFLP analysis of 16S rRNA genes of strain PLH102-1 and other 16SrI subgroups were less than or equal to 0.97, the threshold for a new subgroup delineation [Strain PLH102-1 belonged to 16SrI group and exhibited a collective RFLP profile different from those of all 33 previously established subgroups in the group 16SrI. As shown in ineation . Therefoineation ,48,49,50MseI digestion alone, and 16SrXIV-E and 16SrXIV-F can be separated by BfaI digestion  group and represented a new subgroup 16SrXV-C. 16SrXV-C and previously reported 16SrXV-A and 16SrXV-B subgroups can be differentiated by on alone c.Strain PLH098-1 exhibited a collective virtual RFLP profile identical to that of subgroup J of the X-disease (16SrIII) group b. TherefCandidatus Phytoplasma’ species were examined by phylogenetic analysis of 16S rRNA gene sequences using Mega 6 [Candidatus Phytoplasma asteris’, ‘Candidatus Phytoplasma lycopersici’, and ‘Candidatus Phytoplasma tritici’ (rrnA and rrnB), which are classified in 16SrI group. Strain PLH098-1 and ‘Candidatus Phytoplasma pruni’ (rrnA and rrnB) reference strains grouped together and fell into 16SrIII group , and then ‘Candidatus Phytoplasma oryzae’ reference strain (16SrXI group), and ‘Candidatus Phytoplasma cirsii’ reference strain (16SrXI group). In the process of updating the phytoplasma classification, it was difficult to determine the classification of some phytoplasma strains that are related to the groups 16SrXI and 16SrXIV. This is because the similarity coefficients between two groups of strains are not always less than 0.85  were detected in two different leafhopper species, Acharis ussuriensis, in Zhouzhi county, Shaanxi, China  which induces white leaf disease in bermudagrass (Cynodon dactylon) was found in Yangling, Shaanxi, China . Amo16SrXV-C c and shaMayawa affinifacialis [Candidatus Phytoplasma cynodontis’ reference strain (16SrXIV-A) and represented a new subgroup 16SrXIV-D . They all belonged to a new subgroup 16SrXIV-F  were detected in leafhoppers collected from Arthurs Seat Hill, KwaZulu-Natal Province, and Wemmershoek Dam, North of Franschhoek in Western Cape province . Althoug6SrXIV-F . These pCandidatus Phytoplasma cynodontis’-related phytoplasma strains have been found in South Africa.Phytoplasma disease is rarely reported in South Africa. Prior to this study, only two reports had been published. One reported a mixed infection of 16SrII and 16SrXII groups of phytoplasmas in grapevine  (sequencCandidatus Phytoplasma cynodontis’-related BGWL strains (16SrXIV group) have been reported in several African countries, such as Sudan [Candidatus Phytoplasma cynodontis’-related phytoplasmas (16SrXIV-F) in leafhopper samples collected at two geographical locations in South Africa  group and represented three distinct genetic subgroup lineages . Bermudagrass is a fast-growing grass that is spread by seeds and its vegetative propagules, stolons, and rhizomes, and quickly colonize new areas where it grows into dense mats ,64. As sas Sudan , Kenya, as Sudan . Identifh Africa a furtherSaccharum officinarum, [Saccharum, is one of the important sources of raw materials for sugar and ethanol production [Candidatus Phytoplasma cynodontis’-related strains were found in South Africa, Australia, and China. Considering that BGWL disease has already occurred in Australia and China, it is likely to occur in South Africa. The bermudagrass and sugarcanes in the vicinity of the collection sites where the leafhoppers tested positive for BGWL phytoplasma strains should be investigated as a priority in future studies.On the other hand, bermudagrass is also considered as one of the most “troublesome” agricultural and environmental invasive weeds in the world. This is because bermudagrass is drought tolerant, flood resistant and can regenerate quickly after fire, and compete for space and nutrients with many crops, especially, sugarcane . Sugarcoduction . Bermudaoduction , and sugoduction . In addioduction ,59,77,79Although phytoplasma-harboring leafhoppers examined in the present study are not new species, none of the them were unveiled as phytoplasma “hosts”. It is worth mentioning that phytoplasma detection in the present study was based on the DNA extraction from the dissected abdomen of a single insect specimen, instead of pooled insect specimens commonly adopted to screen insect vectors. Nine of the ten phytoplasma-positive leafhoppers could be detected by direct PCR  accessed on 29 November 2019.A total of 76 leafhopper specimens collected in natural habitats were used in this study . Among t2, 2.5 units of LATaq DNA polymerase . PCRs were operated for 38 cycles and the following conditions were used: denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min and extension at 72 °C for 3 min . Diluted PCR product (1:20) from the first amplification was used as template in the semi-nested PCR. Amplicons were analyzed by electrophoresis through an agarose gel, incorporated with SYBR Safe DNA Gel Stain  and visualized with a UV transilluminator. PCR amplified products were cleaned by gel extraction using the QIAquick Gel Extraction Kit  and sequenced with an automated DNA sequencer . Raw sequence assembly and analysis was conducted by Seqman, an application of DNAStar LaserGene software package . All sequences were deposited into NCBI nucleotide database  followeiPhyClassifier [AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI (MboI), MseI, RsaI, SspI, and TaqI) were simulated. A phylogenetic tree was constructed using the Minimum Evolution (ME) method implemented in the software package MEGA-6 [Candidatus Phytoplasma’ species except for a new Candidatus species because the sequence has not been released yet. Acholeplasma palmae served as an out-group during the phylogenetic tree construction.In order to further identify and classify the phytoplasma strains, computer simulated virtual RFLP analysis and similarity coefficient calculations were performed for the obtained 16S rRNA gene sequences by using the online tool, assifier . Digestie MEGA-6 . The ME


BCR-ABL1 translocation leading to a diagnosis of chronic myeloid leukemia (CML), or somatic mutations in JAK2, CALR, or MPL resulting in Philadelphia-chromosome-negative MPNs with constitutive activation of the JAK-STAT signaling pathway. In the Philadelphia-chromosome-negative MPNs, modern sequencing panels have identified a vast molecular landscape including additional mutations in genes involved in splicing, signal transduction, DNA methylation, and chromatin modification such as ASXL1, SF3B1, SRSF2, and U2AF1. These additional mutations often influence prognosis in MPNs and therefore are increasingly important for risk stratification. This review focuses on the molecular alterations within the WHO classification of MPNs and laboratory testing used for diagnosis.Chronic myeloproliferative neoplasms (MPNs) are hematopoietic stem cell neoplasms with driver events including the  BCR-ABL1-positive (CML); chronic neutrophilic leukemia (CNL); polycythemia vera (PV); essential thrombocythemia (ET); primary myelofibrosis (PMF); chronic eosinophilic leukemia (CEL); and MPN–unclassifiable (MPN-U) [BCR-ABL1 as well. These three entities have overlapping morphologic features and thus are together referred to as BCR-ABL1-negative myeloproliferative neoplasms or Philadelphia chromosome-negative myeloproliferative neoplasms (Ph- MPNs) [JAK2, which encodes Janus kinase 2, in Ph- MPNs, our understanding of the molecular and genetic basis for these diseases has substantially improved [MPL and CALR mutations has further advanced our understanding and diagnostic criteria [Myeloproliferative neoplasms (MPNs) are characterized by clonal proliferation of hematopoietic precursors in the bone marrow. The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues classifies these myeloproliferative neoplasms as chronic myeloid leukemia,  (MPN-U) ,3,4. Simh- MPNs) . With thimproved ,7. Subsecriteria ,9,10,11.criteria . This excriteria . In thisABL gene (located on chromosome 9) and the breakpoint cluster region BCR gene (located on chromosome 22) [BCR-ABL1 oncogene constitutively encodes an oncoprotein with increased tyrosine kinase activity [BCR-ABL1 mRNA transcripts is initiated by the BCR-ABL oncogene [BCR-ABL1 transcripts encode fusion proteins of different sizes with tyrosine kinase activity and can result in different phenotypes. The most commonly reported major breakpoint region p210 BCR-ABL1 proteins are encoded by e13a2 and e14a2 mRNA transcripts, but the resulting p210 proteins have slightly different sizes and induce slightly different phenotypes—patients with e14a2 transcripts have significantly higher platelet counts compared to patients with the e13a2 transcript [BCR-ABL1 proteins are encoded by the e19a2 transcript, and these patients present with more prominent neutrophilic maturation and/or increased platelet counts. While p190 BCR-ABL1 proteins (caused by the minor breakpoint and encoded by e1a2 transcript) most frequently result in Ph+ Acute Lymphoblastic Leukemia/Lymphoma, they can rarely be seen as the main protein in CML. These patients present with monocytosis without basophilia, which can be misclassified as Chronic Myelomonocytic Leukemia, and have a higher risk of progressing to the lymphocytic blast phase [BCR gene [CML is an MPN that is characterized by the clonal proliferation of myeloid elements (predominantly granulocytes) resulting from a reciprocal translocation of the some 22) ,13. Thisactivity . Dependioncogene . Multiploncogene ,17. Thesanscript ,19,20. pBCR gene .Along with inducing different phenotypic features, different transcripts may also have varying sensitivities to tyrosine kinase inhibitors (TKIs) . Some smBCR-ABL1 transcript level to achieve Major Molecular Remission [ABL1 kinase domain (KD) mutations, which include M244V, G205E, Q252H, Y253F/H, E255K/V, D276G, F311L, T315, F317L, M351T, E355G, F359V, L384M, L387F, H396R/P, E459K, and F486S, but many other missense mutations have been identified [BCR-ABL1 kinase domain. T315I is considered a gatekeeper mutation as it is resistant to all TKIs other than ponatinib [Since the advent of TKIs, the management and treatment of patients with CML have significantly improved with dramatically reduced rates of stem cell transplantation . TKI theemission . Currentemission ,31. Howeentified ,32,33. TBCR-ABL1 transcripts ranging from ≤ 0.01% (MR4) to ≤ 0.001% (MR5), is not achieved or if the response is suboptimal, it is imperative to assess for BCR-ABL1 KD mutations [If complete or deep molecular response (CMR), which is defined as utations ,35. Simiutations ,35.ASXL1, which was already present in these patients at the time of CML diagnosis. They also noticed that the ASXL1 mutational load in these patients correlated with BCR-ABL1 transcripts. Other genes identified were RUNX1, IDH1, and DNMT3A [KD mutations account for about 30–90% of cases of TKI resistance . In orded DNMT3A . Howeverd DNMT3A .BCR-ABL1 transcripts periodically, starting with 3-month intervals [BCR-ABL1 transcript to >1%, or suggestion of loss of hematologic response [BCR-ABL1 gene fusion [BCR-ABL1 transcript levels, and loss of MMR, or if there is disease progression to accelerated or blast phase [As outlined by ELN and the National Comprehensive Cancer Network (NCCN) guidelines, the current gold standard to monitor treatment is the use of quantitative reverse-transcriptase PCR (RQ-PCR) to quantify the ntervals ,38,39,40response . This ape fusion . Additioe fusion ,42. Juste fusion . Some ste fusion ,44. Thuse fusion . Kinase st phase .Ph- MPNs consist of three different entities, Essential Thrombocythemia (ET), Polycythemia Vera (PV), and Primary Myelofibrosis (PMF), which are all characterized by overproduction of differentiated cells of various lineages, which can typically be identified in the peripheral blood ,45. All MPL) and G-CSFR by Colony-Stimulating Factor 3 Receptor (CSF3R). Driver mutations that affect this pathway are predominantly JAK2 V617F and JAK2 exon 12 mutations, MPL mutations, CALR exon 9 mutations, and mutations in CSF3R [CBL (casitas B-lineage lymphoma proto-oncogene) [MPL, THPO, and JAK2 [The genetic hallmark of Ph- MPN is the constitutive activation of the JAK-STAT pathway ,47. Hemain CSF3R ,51,52,53in CSF3R . Such nencogene) ,55,56. Mand JAK2 .JAK2 V617F mutation is diagnostic and is present in greater than 95% of cases; however, approximately 3–4% of cases are associated with various JAK2 exon 12 mutations, which are considered functionally similar, resulting in an increased RBC production [JAK2-negative harbor either driver mutations that activate the JAK-STAT pathway or non-driver mutations involved in DNA methylation [JAK2-negative cases have shown mutations in genes involved in histone modification as well [PV presents with erythrocytosis in the peripheral blood and is suggested clinically by hemoglobin levels greater than 16 g/dL in women and greater than 16.5 g/dL in men, along with hyperplasia of all the marrow lineages resulting in panmyelosis, but may be most morphologically apparent by increased megakaryocytes and erythroid precursors . The preoduction ,58. Rarehylation ,58. A sm as well .9 /L and morphologically atypical megakaryocytes, with no or minimal increase in marrow cellularity [JAK2 or GSN (gelsolin gene) [ET presents with platelet counts greater than 450 × 10lularity . LeukocyJAK2 V617F  mutations, while 15–30% patients present with CALR and 1–4% have the MPL mutations [MPL gene have been identified [Genetically, 50–60% of ET patients harbor the JAK2 V617F mutations, 20–24% have CALR mutations, and 8–10% have MPL mutations. However, about 12% of cases have none of these and are regarded as “triple-negative”. With the increased use of NGS, multiple other cooperating mutations in genes associated with myeloid malignancies have been identified, including ASXL1, EZH2, TET2, IDH1, IDH2, SRSF2, and SF3B1 [SRSF2, ASXL1, and U2AF1-Q157 mutations have been shown to predict poor survival independent of other risk factors [CALR type-1-like mutations, the presence of high-risk mutations such as ASXL1, SRSF2, EZH2, IDH1, or IDH2, and the presence of two or more high-molecular-risk mutations. MIPSSv2 uses mutations, karyotypes, and clinical variables to assess prognosis. Additionally, this model uses U2AF1 Q157 as a high-molecular-risk mutation. Karyotype, very high-risk mutations, and the absence of CALR type 1-like mutation and other clinical variables have been included in this prognostic model [PMF is the proliferation of bizarre megakaryocytes and myeloid hyperplasia within the bone marrow, with evolving bone marrow fibrosis, until the overt stage where patients present with leukoerythroblastosis and extramedullary hematopoiesis, predominantly in the liver and spleen, resulting in hepatosplenomegaly . Moleculic model .JAK2, located on the short arm of chromosome 9, encodes the non-receptor tyrosine kinase Janus kinase 2 (JAK2) which is associated with cytokine receptors [JAK2 is an important mediator of erythropoiesis and is the most frequent gene implicated in BCR-ABL1-negative myeloproliferative neoplasms [eceptors ,63. Uponeoplasms .JAK2 mutation encountered in PV is the V617F somatic mutation in exon 14, which results in the substitution of valine to phenylalanine at position 617 in the pseudokinase domain [JAK2 V617F mutation arises in a multipotent hematopoietic stem cell and thus is present in all myeloid and lymphoid (B and NK) cells, with a later rare presentation in T cells [The most common e domain ,47,48,63e domain . The JAK T cells .JAK2 V617F-negative cases of PV, JAK2 exon 12 mutations have been identified [JAK2 exon 12 mutations are younger and present with erythrocytosis, but have similar symptoms and rates of survival when compared to patients with JAK2 V617F mutations [In entified ,46,48. TJAK2 mutations are not specific for PV and can be identified in up to 60% cases of ET and PMF [JAK2 V617F expression levels correlated with an ET-like phenotype, while high expression levels were shown to induce a PV-like phenotype in mouse models [JAK2 V617F can also be detected in the general population at very low levels or in refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) or in clonal hematopoiesis of indeterminate potential (CHIP) [ and PMF ,46,63. M and PMF . However and PMF . This wae models ,65. JAK2l (CHIP) ,67,68.9/L and longer duration of disease are both implicated in predicting the risk of post-PV MF, while leukocytosis (>15 × 109/L) and advanced age are poor prognostic factors implicated in progression to AML [JAK2 V617F allelic burden influences the risk of thrombotic events, not just in PV but in other BCR-ABL1-negative myeloproliferative neoplasms (Ph- MPNs) as well. A prospective study by Passamonti et al. looked at JAK2-mutated cases of PV and compared allelic burden with multiple factors such as hemoglobin concentration, WBC count, bone marrow cellularity, size of spleen, and platelet counts. They analyzed the allelic burden in 320 cases of PV with the JAK2 V617F mutation and found that higher allelic burden was directly related to hemoglobin concentration, marrow cellularity, splenomegaly, and WBC counts but inversely related to platelet counts. The study concluded that the JAK2 V617F mutant allelic burden greater than 50% was significantly related to the risk of developing post-PV MF but not to the risk of developing AML or thrombosis [JAK2 V617F allelic burden in 126 patients with Ph- MPN and the mutation was analyzed with the intention of assessing its role in the risk stratification of future vascular complications. They quantified the allelic burden according to percentages: levels >51% were considered homozygous for the mutation, and the highest levels were found in patients with PV. They concluded that as allelic burden increased the risk of venous thromboembolism increased as well. At allelic burden greater than 25%, they observed a 7.4-fold increase in the risk of thrombosis as compared to patients with <25% JAK2 V617F allelic burden [Thrombotic complications, which include venous thromboembolism or arterial thrombosis, occur more commonly in about 12–39% of PV patients but can also be seen in a small percentage of ET patients . In factn to AML . Multiplrombosis . In contc burden .CALR is the second most frequently mutated gene encountered in Ph- MPNs, although these mutations have not been identified in PV patients [CALR mutations were recently discovered in 2013 by two separate studies that utilized whole-exome sequencing [CALR in 70–80% ET and PMF patients who were negative for the two known driver mutations, JAK2 and MPL. In an early study by Klampfl et al., amongst 1107 patients with MPN, CALR mutations were identified in 25% ET and 35% PMF patients, but not in PV patients or other myeloid neoplasms [CALR, JAK2, and MPL mutations are considered to be mutually exclusive; however, recently, few cases with concurrent JAK2 V617F, MPL, and/or CALR mutations have been reported [patients . CALR muquencing ,9. Both reported ,74,75,76CALR, which encodes a highly conserved calcium-binding chaperone protein calreticulin, is located on chromosome 19 [CALR have shown that it retains localization to the endoplasmic reticulum showing that there are other KDEL-independent mechanisms as well [CALR has multiple functions—maintaining calcium homeostasis and assisting in protein folding, cell adhesion, immune response, and phagocytosis [CALR mutations in ET and PMF are classified as type 1-like or type 2-like, depending on their corresponding similarities in structure, extent of amino acid deletions, and effect on the KDEL motif at the C-terminus [osome 19 . Calreti as well ,63,77. Cocytosis . The remterminus ,49,77.CALR-driven ET cases, mutated calreticulin had a lower calcium-binding affinity and showed a loss of the ER retention KDEL motif [JAK2-mutated ET, CALR allelic burden was higher. Clinically, CALR-mutated ET patients have much higher platelet counts (>1000 × 109/L) and presented at a younger age; however, their risk of thrombosis was lower than patients with JAK2 mutated ET [CALR-mutated PMF have been shown to have a more indolent clinical course [CALR mutations have a better overall survival when compared to JAK2- or MPL-mutated cases [CALR mutations were younger, with a higher platelet count, and presented with lower DIPSS-plus score; additionally, they were also less likely to require blood transfusions for anemia or present with leukocytosis [Clinically, different mutations have distinguishing presentations as well. In one study by Rumi et al., driver mutations in 1135 patients with either PV or ET were analyzed. For EL motif . The alltated ET . Similarl course ,78,80. Focytosis .MPL mutations were the second driver mutations to be identified in JAK2-negative MPNs [MPL) is located on chromosome 1p34 and consists of 12 exons that encode the 70kDa thrombopoietin receptor protein (TpoR) [MPL have been associated with both thrombocytosis and thrombocytopenia [MPL-activating mutation in exon 10, MPL W515L, was discovered in 2006 in a JAK2-negative ET/PMF [MPL W515L mutation results from G to T substitution at position 1544, leading to a leucine residue at codon 515 [MPL W515L retroviral mouse models were bone-marrow-transplanted, Pikman et al. observed that these mouse models presented with thrombocytosis, which was followed by myelofibrosis, a similar evolution of disease observed in ET [MPL W515 mutations are not only found in MPNs, they have also been described in RARS-T [ive MPNs ,61,81. Mn (TpoR) . This ren (TpoR) ,82. Mutaytopenia . The firodon 515 ,63,77,82odon 515 . This reodon 515 . When MPed in ET ,63,83. Sn RARS-T ,85.MPL variants outside of exon 10 have also been identified in sporadic MPN cases. Such acquired mutations result in amino acid substitutions in either the extracellular  or intracellular  domains of the MPL/TpoR but have shown a lower gain-of-function activity when compared to MPL W515L mutants [JAK2. Another rare somatic mutation that has been reported in less than 1% of ET and PMF cases is MPL S505N, which is part of the TpoR transmembrane domain, and the mutation leads to constitutive dimerization of MPL/TpoR, leading to activation of the downstream signaling pathways [MPL and THPO genes have been identified in hereditary thrombocytosis as well as inherited thrombocytopenia [MPL or THPO germline mutations are a result of gain-of-function mutations, which lead to defective MPL/TpoR, either because of loss of signaling or impaired stability, which constitutively activates the MPL signaling pathways. Similarly, thrombocytopenia results from loss-of-function germline mutations and leads to no production of TPO or impaired binding of MPL to TPO, impairing the MPL signaling pathways and decreasing platelet production [MPL genes in cases of ET. mutants ,57,86,87pathways ,57,84. Tpathways ,57,88. Gytopenia ,57. Throoduction ,90,91,92The current WHO classification of Ph- MPNs does not require the identification of a driver mutation in order to make a diagnosis; however, they are typically a major criterion because morphology and clinical presentation alone can overlap with reactive myelopoiesis . The preJAK2 V617F mutations, the most commonly used method is real-time allele-specific PCR (rt-PCR) [JAK2 V617F mutation when detected by rt-PCR [JAK2 V617F mutations without any known clinical significance [Currently, there are no Food and Drug Administration (FDA)-approved detection methods for driver mutations . For JAK(rt-PCR) ,94. The (rt-PCR) ,96,97. Q(rt-PCR) ,94. Drop(rt-PCR) ,99,100. y rt-PCR . Howeverificance .JAK2 exon 12 mutations are rare, and many of the mutations detected are of uncertain clinical significance [ificance ,104. Henificance ,104,105.CALR exon 9 mutations are more commonly detected by fragment analysis, which has relatively high sensitivity and also evaluates mutant allelic burden [CALR exon 9 mutations is the use of a standardized PCR-HRM analysis method or fragment analysis combined with Taqman Real-Time quantitative PCR (RT-qPCR) [CALR mutations, such a sensitive method may not be necessary for driver mutation detection in routine cases [c burden ,106,107.c burden . Howeverc burden . AnotherRT-qPCR) . Even thne cases ,94. Nevene cases .MPL mutations are detected in exon 10, and the methods of detection for these mutations depend on targeted identification of the individual mutations or sequencing of exon 10 entirely [MPL mutations, namely W515L, W515K, W515A, andS505N, has been developed [The most frequent entirely . Identifentirely ,112,113.eveloped . The speeveloped ,114,115.With the advent of genome-wide NGS, our understanding of the different genetic alterations involved in human cancers has exponentially increased. NGS has several advantages over traditional sequencing methods of detection as well. First, NGS is a high-throughput method of analysis that can detect multiple different mutations in the same run. Secondly, this mode of detection does not require high-input DNA/RNA samples and also has much higher sensitivity (<1%). NGS can also detect many other genomic deviations such as single-nucleotide variants (SNVs), insertions, deletions, and copy number variations (CNV) with high accuracy. The gene fragments detected through different available NGS platforms are then compared to the human reference genome through bioinformatics analyses ,116. ThuTET2, DNMT3A, and IDH1/2) and histone/chromatin modifiers (ASXL1 and EZH2), splicing factors , signal transduction factors (SH2B3/LNK and CBL), and transcription factors (TP53 and RUNX1) [The vast majority of Ph- MPNs can be attributed to the three known driver mutations. However, the heterogeneity of PH- MPNs cannot be explained by mutations in just these three genes. Ph- MPNs have often been shown to contain at least one additional non-driver mutation when samples were analyzed with NGS. Additional mutations have been detected in about 50% of ET and PV patients and 80% of PMF patients ,118 Thesd RUNX1) ,117,118.d RUNX1) . These gSRSF2, SF3B1, and U2AF1) were most frequently mutated in PMF [ASXL1, EZH2, TET2, DNMT3A, and IDH1/2) were more frequently mutated in PMF patients than in ET or PV patients. Out of these, ASXL1 and TET2 were associated with poor survival, which was in accordance with other studies [Delic et al. studied 100 cases of MPNs and concluded that splicing genes  and the demethylating (TET2) at cytosine residues in the cytosine-guanine (CpG) dinucleotide sequences [JAK2 positive MPNs and is identified in about 10–15% MPNs [TET2 mutation have progressed to AML; however, other studies have not shown a clear increase in the risk of thrombosis or overall poor survival in MPN patients [DNMT3A mutations only occur in about 5–10% of MPNs and mostly precede JAK2 or MPL mutations [JAK2 V617F [JAK2 mutated hematopoietic stem cells, thus playing a key role in disease initiation; however, if they occur as secondary mutations, they aid in disease progression [TET2, patients with DNMT3A mutation first are more likely to develop ET-like phenotype, while patients with JAK2 first are more likely to develop PV-like phenotype [IDH1/2 genes are also included in this group. Mutations in these genes commonly occur at R132 (IDH1) or R140/R172 (IDH2) and result in the inappropriate conversion of a-ketoglutarate to 2-hydroglutarate, leading to dysregulation of downstream targets affecting leukemogenesis [IDH1/2 mutations can predict progression to myelofibrosis and AML transformation [ASXL1 and EZH2. ASXL1 or additional sex combs-like 1, interacts with Polycomb group complexes (PgC), which are involved in histone modifications to continue gene expression [ASXL1 in normal hematopoiesis, but along with TET2, this gene is the second most common to be implicated in MPNs [ASXL1 are frameshift or nonsense mutation (exon 12) in the N-terminal, which results in a C-terminal truncated protein that affects the subsequent binding of BAP1, which binds to chromatin and brings about additional epigenetic modifications [EZH2 encodes for a histone methyltransferase, which also interacts with the PgC and is normally involved in the suppression of H3K27 trimethylation [EZH2 are commonly identified in 5–10% PMF patients, but such mutations have also been identified in other myeloid malignancies [ASXL1 and EZH2 mutations can occur as early events in hematopoietic stem cells and can exist simultaneously in the same clone. Patients with such double mutations presented with decreased hemoglobin levels and increased WBC counts. Both mutations are also associated with an increased risk of transformation to MF and AML, thus indicating a poor clinical outcome, especially when present along with JAK2 V617F [equences ,93,123. patients ,124,125.utations . R882H iK2 V617F ,127. Botgression . Similarhenotype ,128. IDHogenesis ,130,131.ormation ,132. Witpression ,133,134. in MPNs . Most muications ,134. Suchylation . Loss ofgnancies ,136,137.K2 V617F ,139,140.SF3B1, SRSF2, and U2AF1. Most documented mutations in this group are heterozygous missense mutations, which lead to altered function and can affect RNA splicing [SRSF2 mutations have been reported in up to 20% of PMF [EZH2, which subsequently leads to myelofibrosis and overall poor survival [The second group of mutations involves spliceosome/splicing genes, which are detected in 3–5% MPNs, with a higher frequency in PMF ,141. Gensplicing . SF3B1 msplicing ,143. Thisplicing . Somaticsplicing . SF3B1 m% of PMF . This ge% of PMF . Mutatiosurvival ,147. U2Asurvival ,149. Mutsurvival .ASXL1, EZH2, IDH1/2, SRSF2, and U2AF1. The mutational frequencies of these cooperating genes were highest in ASXL1 (31%), U2AF1 (16%), SRSF2 (12%), SF3B1 (7%), EZH2 (6%), and IDH (4%) [CALR-mutated patients have favorable prognostic features such as decreased anemia and leukocytosis, while CALR-/ASXL1+ have decreased survival. They also identified that the frequency of U2AF1 mutations was lower in CALR-mutated PMF cases but higher in CALR-mutated ET cases, which may account for the opposing trends in anemia [ASXL1, SRSF2, EZH2, IDH1/2, and U2AF1 as high-risk mutations and their inclusion in the MIPSSv2 prognostic model for risk stratification in PMF [Tefferi et al. compared the clinical and molecular features of 254 patients with PMF. These patients were screened for IDH (4%) . Based on anemia ,150,151.n in PMF ,76,152.JAK2 negative PV cases is LNK, which encodes for the lymphocyte adapter protein [LNK  plays an important role as a negative regulator of the JAK-STAT and the TPO/MPL pathways and thus can affect both erythropoiesis and megakaryopoiesis [LNK somatic mutations are present in JAK2 negative MPN cases, with an increased frequency in ET and PMF [LNK mutations have also been implicated in idiopathic erythrocytosis and familial MPNs [LNK mutations and familial cases of JAK-negative PV, 94 MPN families were studied by Rumi et al. [JAK2 mutation-negative, but LNK (E208Q)-positive. When the remaining 93 families were scanned for this mutation using Sanger sequencing, one other patient was also found to harbor this mutation. Germline testing then revealed that both patients carried the LNKE208Q mutation, but the family members were negative for this germline mutation. Thus, it is recommended that patients with JAK2-V617F and exon 12-negative PV be tested with LNK gene sequencing to find any underlying germline causes for erythrocytosis [Another important gene that has been implicated in  protein . LNK  [CSF3R gene, located in the membrane region of exon 14, and frameshift or nonsense mutation in exon 17, which results in the truncation of the protein’s cytoplasmic tail [CSF3R mutation as one of the diagnostic requirements to diagnose CNL. Other causes of reactive neutrophilia or myeloid neoplasms must be ruled out before making this diagnosis.Chronic neutrophilic leukemia (CNL) is defined by the WHO as absolute neutrophilia without left-shift or dysplasia identified . Up to 8(G-CSFR) . Thus, t(G-CSFR) ,52,93. Tmic tail . The WHOBCR-ABL1 fusion and driver mutations in JAK2, CALR, and MPL must be ruled out. Mutations in CSF3R and rearrangements in PDGFRA, PDGFRB, FGFR1, and PCM1-JAK2 fusion have to be assessed as well. Cooperating mutations are common in CNL as well. Approximately 50–60% cases have ASXL1 mutations, while 30–40% case harbor SETBP1 [CSF3R along with other known mutations such as ASXL1 and SETBP1.In order to make the diagnosis, r SETBP1 ,161. BotBCR-ABL1, PCM1-JAK2, ETV6-JAK2, or BCR-JAK2 fusions are absent, as are PDGFRA, PDGFRB, and FGFR1 rearrangements [KIT, ASXL1, U2AF1, EZH2, SETBP1, ETV6, and CSF3R [Chronic eosinophilic leukemia, not otherwise specified , is a rare clonal proliferation of eosinophils and their precursors, which accumulate in blood and marrow and cause tissue damage secondary to the cellular infiltration and subsequent protein, cytokine, and enzyme release. CEL, NOS does not have a specifically identified driver mutation and therefore is a diagnosis of exclusion, where nd CSF3R ,163,164.Due to the rapidly growing field of molecular technology and bioinformatics, our knowledge about disease diagnosis, prognosis, and therapeutic options has increased substantially. Our proposed algorithm for molecular workup of MPNs and commonly used methods are presented in BCR-ABL1 translocation. JAK2 V617F is the most common mutation identified in all three Ph- MPNs and should be identified (or ruled out) using DNA-based quantitative assays. In PV, JAK2 V617F negative samples can then undergo high-resolution melt analysis (HRM) along with Sanger Sequencing to detect JAK2 exon 12 mutations. In rare PV cases that are negative for both above mutations, the next step would be to sequence the JAK2 gene and use allele-specific quantitative PCR (AS-qPCR) or digital PCR (ddPCR) to detect any LNK gene mutations. In JAK2 V617F negative ET and PMF cases, the next step is to identify MPL exon 10 and CALR exon 9 mutations. HRM with Sanger sequencing can be used for both, while fragment analysis capillary electrophoresis can also be used for CALR mutation detection. If both of these are negative as well, the next steps would include MPL gene sequencing and NGS with a broad panel of non-driver genes. Alternatively, NGS panels that includes the driver genes along with the non-driver genes can be used as well.This figure shows a proposed diagnostic algorithm for the presence of known driver and non-driver mutations along with the method of detection. When there is a clinical suspicion of MPN with morphologic evidence, the first step is to rule out


Xenopus laevis based on the T3-induced Xenopus metamorphosis assay we established previously, as well as the Xenopus Eleutheroembryonic Thyroid Assay (XETA). Stage 48 tadpoles were treated with a series of concentrations of T3 in 6-well plates for 24 h and the expression of six TH-response genes was analyzed for choosing a proper T3 concentration. Next, bisphenol A (BPA) and tetrabromobisphenol A (TBBPA), two known TH signaling disruptors, were tested for determining the most sensitive TH-response gene, followed by the detection of several suspected TH signaling disruptors. We determined 1 nM as the induction concentration of T3 and thibz expression as the sensitive endpoint for detecting TH signaling disruptors given its highest response to T3, BPA, and TBBPA. And we identified betamipron as a TH signaling agonist, and 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) as a TH signaling antagonist. Overall, we developed a multiwell-based assay for rapidly screening TH signaling disruptors using thibz expression as a sensitive endpoint in X. laevis.There is a need for rapidly screening thyroid hormone (TH) signaling disruptors in vivo considering the essential role of TH signaling in vertebrates. We aimed to establish a rapid in vivo screening assay using  Xenopus [Thyroid hormones THs, T3 and T4) produced by the thyroid gland are critical for growth and development in vertebrates . THs exe and T4 pXenopus ,9,10,11,Xenopus ,13.Several in vitro binding assays  and in vXenopus Eleutheroembryonic Thyroid Assay (XETA) [X. laevis bearing a TH/bZIP-GFP construct are exposed to chemicals in 6-well plates for 72 h with or without 5 nM T3, and fluorescent intensity is detected to assess the thyroid disrupting activity of the test chemicals. However, the TH/bZIP-GFP transgenic line is unavailable for many laboratories, which restricts the application of the XETA. Previously, we developed a T3-induced Xenopus metamorphosis assay (TIXMA) for the detection of TH signaling disruptors [TH-dependent amphibian metamorphosis offers an opportunity to investigate thyroid disruption including TH signaling disruption in vivo ,17. Specy (XETA) , which hsruptors ,22. In tXenopus tadpoles, referencing the XETA and the TIXMA. Considering their high sensitivity to exogenous TH due to the high expression of the thyroid hormone receptor (thr) gene [The present study aimed to develop a simple and rapid screening assay for detecting TH signaling disruptors using wild-type hr) gene ,24 and lhr) gene ,26, tadphr) gene , was rephr) gene  and requhr) gene  and TCS hr) gene  are two hr) gene  and BP-3hr) gene  are two hr) gene  as a brokruppel-like factor 9 (klf9), thyroid hormone induced bZip protein (thibz), thyroid hormone receptor beta (thrb), stromelysin-3 (st3), type 3 deiodinase (dio3), and matrix metallopeptidase 13 (mmp13), in a concentration-dependent manner. The changes of klf9, thibz, thrb, st3, dio3, and mmp13 in the highest group of T3 (5 nM) were about 5-, 280-, 21-, 6-, 6-, and 4-fold of the control, respectively. Moreover, among the six TH-response genes, thibz was the most sensitive, with a 200-fold change following 1 nM T3 treatment. Therefore, 1 nM was selected as the induction concentration of T3 in the following tests.The effects of a series of concentrations of T3  on the expression of six TH-response genes were investigated. As shown in thibz expression. Then, 1000 nM BPA and TBBPA significantly upregulated the expression of st3. However, there were no obvious effects on other genes, , thrb, dio3klf9, and mmp13. thibz expression was significantly induced by as low as 100 nM BPA and TBBPA, indicating that thibz was the most sensitive gene among six TH-response genes. Furthermore, T3-induced gene expression of thibz, thrb, and dio3 were significantly inhibited by 100 nM BPA, and 1000 nM BPA significantly downregulated all six T3-induced TH-response genes expression. Similar to BPA, 100 nM TBBPA antagonized the T3-induced gene expression of klf9, thibz, and thrb, and 1000 nM TBBPA significantly antagonized all T3-induced genes we tested. In the co-exposure groups, only thibz and thrb were significantly antagonized by both 100 nM BPA and TBBPA, and T3-induced expression of the other four TH-response genes was antagonized by the higher concentrations of BPA and TBBPA. Taken together, thibz expression was chosen as a sensitive endpoint for screening TH signaling disruptors.The expression of six TH-response genes was measured in tadpoles with BPA or TBBPA exposure in the absence or presence of 1 nM T3. As shown in thibz expression as an endpoint. As shown in thibz expression compared with the control. In the absence of T3, 10–1000 nM betamipron significantly promoted the expression of thibz in a concentration-dependent manner, and in the presence of T3, 100–1000 nM betamipron increased the expression of thibz compared with the T3 group. BDE-47 inhibited the T3-induced thibz expression but did not affect thibz expression in the absence of T3. TCC, TCS, BP, and BP-3 had a similar mode of action on thibz expression both in the absence and presence of T3. These four chemicals at high concentrations significantly resulted in down-regulation of T3-induced expression of thibz expression and high concentrations of these four chemicals alone increased thibz expression.We analyzed the TH signaling disruption activity of betamipron, BDE-47, TCC, TCS, BP, and BP-3 using thibz expression analysis using Xenopus tadpoles at stage 48. The concentration-dependent upregulation of all tested TH-response gene expressions by T3 treatment has demonstrated the sensitivity of stage 48 tadpoles to T3 within 24 h. Given that 1 nM T3 dramatically induced upregulation of TH-response gene expression but did not reach the expression climax, we determined 1 nM as the induction concentration of T3, avoiding the possibility that the higher concentrations of T3 would cover up the antagonistic actions of tested chemicals. When 1 nM T3 upregulated the expression of all the test TH-response genes, 1000 and/or 100 nM both BPA and TBBPA antagonized T3 actions, showing that this screening assay is effective in detecting TH signaling antagonists. As mentioned above, the XETA employs transgenic TH/bZIP-GFP tadpoles to indicate TH signaling disruption by measuring GFP fluorescence. Considering the finding that thibz expression was the most sensitive to T3 and highly responsive to BPA and TBBPA, we chose thibz expression as a sensitive endpoint to detect TH signaling disruptors. To our current understanding, thibz is believed to be a gene that specifically responds to TH signaling [thibz expression. Therefore, the screening assay we developed is more sensitive or comparable with the XETA assay for detecting antagonistic actions of chemicals on TH signaling.We developed a multiwell-based screening assay to detect TH signaling disruptors based on ignaling . Moreoveignaling ,26. Therignaling  reportedthibz expression even at 10 nM in our screening assay, providing the first in vivo evidence that betamipron is a TH signaling agonist. In contrast to betamipron, the screening assay revealed that BDE-47 inhibited T3-induced thibz expression as BDE-47 exposure alone had no effect, indicating TH signaling antagonism. Previous studies reported that BDE-47 inhibited TH-dependent development in X. laevis [thrb and klf9 in X. laevis [Using this screening assay, we examined several chemicals for TH signaling disrupting activity. In the Tox21 project, betamipron was for the first time reported as a TR agonist in the in vitro reporter gene assay , with th. laevis ,36, and . laevis . Togethethibz expression in either the absence or presence of T3, i.e., they stimulated thibz gene expression in the absence of T3 but antagonized T3-induced expression in the presence of T3. The effects of these chemicals are similar to those of BPA and TBBPA, implying that they, like BPA and TBBPA, exerted TH signaling antagonistic action in the presence of T3, but could have agonistic action in the absence of T3. Previously, several studies reported that TCS altered TH-response gene expression and TH-dependent growth in amphibians, despite seemly inconsistent effects [Rana rugose and suppressed the T3-induced EGFP activity in transgenic X. laevis tadpoles [In the screening assay, TCC, TCS, BP, and BP-3 exerted similar effects on  effects ,38,39. Stadpoles , but behtadpoles . Given t3,3′,5-triiodo-L-thyronine  was obtained from Geel Belgium  and a stock solution of T3 was prepared by dissolving into ultrapure water. Dimethyl sulfoxide  and 3-aminobenzoic acid ethyl ester  were purchased from Sigma-Aldrich . Bisphenol A , tetrabromobisphenol A , betamipron , 2,2′,4,4′-tetrabromodiphenyl ether , triclocarban , triclosan , benzophenone  and benzophenone-3  were dissolved into DMSO to prepare 10 mM stock solutions. The stock solutions for chemicals were stored at −20 °C for future exposure experiments. An RNA Extraction kit (AU1201) was obtained from BioTeke Corporation . PCR primers were synthesized by BGI Tech Solutions . DNase/RNase-free water (RT121), Quantscript RT Kit (KR116), and a Real Master Mix (SYBR Green) Kit (FP205) were obtained from TIANGEN Biotech . Human chorionic gonadotropin (HCG) was purchased from the Ningbo Second Hormone Factory  and dissolved into 0.6% sodium chloride.X. laevis were described in our previous study with some adjustments [X. laevis were raised at 20 °C with a light/dark cycle of 12 h:12 h in charcoal-filtered tap water. Breeding was induced by injection HCG into a pair of adult frogs . After spawning, fertilized eggs were incubated in glass aquariums containing charcoal-filtered tap water with a 12 h light/12 h dark cycle at 22 ± 1 °C. Developmental stages of Xenopus tadpoles were identified according to the Nieuwkoop and Faber table [Housing and breeding conditions for ustments . In brieer table . This st, thibz, thrb, st3, dio3klf9, and mmp13 [To reduce the test size, we employed stage 48 tadpoles in 6-well plates in place of stage 52 tadpoles in glass tanks in the TIXMA. Our previous study has shown that stage 48 tadpoles are highly sensitive to T3 . To detend mmp13 . Then, sX. laevis.Finally, stage 48 tadpoles were exposed to betamipron, BDE-47, TCC, TCS, BP, and BP-3  in the absence or presence of T3 to screen their TH signaling disrupting activity. Exposures and sampling were conducted as described above. Each experiment was independently repeated three times using offspring from different pairs of , and RNA quality was examined by A260/A280 ratios and agar-gel electrophoresis. The first-strand cDNA was synthesized from 1000 ng total RNA using the Fast RT Kit according to the manufacturer’s instructions . Following this, the first-strand cDNA was stored at −20 °C for gene expression analysis. Expression of TH-response genes was analyzed using SYBR Green I with the Real-time Polymerase Chain Reaction system , with Ribosomal protein L8 (rpl8) as a reference gene [rpl8 was not affected by treatments  following the manufacturer’s instructions. RNA concentration was measured using a NanoDrop 2000 nce gene ,44,45 aneatments . In 10 µ methods .p value < 0.05. The statistical analysis of the experimental data was performed using SPSS software version 16.0 . The data were checked for normal distribution (Kolmogorov–Smirnov test) and homogeneity of variance (Levene test). Data for relative expression of genes are presented as mean ± standard error of the mean (SEM). Statistical differences in relative expression of genes were analyzed by two-way analysis of variance (ANOVA) followed by Tukey’s HSD test. Statistical significance was defined as We developed a multiwell-based assay for rapidly screening TH signaling disruptors using thibz expression as a sensitive endpoint in X. laevis which effectively detected the agonistic effect of T3 and the antagonistic effect of BPA and TBBPA on TH signaling within 24 h. Using this screening assay, we identified betamipron as a TH signaling agonist and BDE-47 as a TH signaling antagonist, while TCC, TCS, BP, and BP-3 appeared to exert complex TH signaling disrupting actions in a TH-dependent manner. Overall, all results indicate that this assay is suitable for screening the TH signaling disrupting activity of chemicals.


Until recently, astrocytes were thought to be a part of a simple “brain glue” providing only a supporting role for neurons. However, the discoveries of the last two decades have proven astrocytes to be dynamic partners participating in brain metabolism and actively influencing communication between neurons. The means of astrocyte-neuron communication are diverse, although regulated exocytosis has received the most attention but also caused the most debate. Similar to most of eukaryotic cells, astrocytes have a complex range of vesicular organelles which can undergo exocytosis as well as intricate molecular mechanisms that regulate this process. In this review, we focus on the components needed for regulated exocytosis to occur and summarise the knowledge about experimental evidence showing its presence in astrocytes. Astrocytes, together with oligodendrocytes and microglia, form three main categories of glial cells in the central nervous system. They are found throughout the brain and occupy around half of the brain’s volume. Astrocytes have a bushy morphology with few main processes, starting from a relatively small cell body and extending many fine processes that invade extracellular space. It has been estimated that an astrocyte can contact over 100,000 synapses in rats and almost two million in the human cortex .2+ waves in cultured astrocytes. This raised the possibility that astrocytes may contribute to modulating neuronal activity [2+ levels in astrocytes can cause a release of glutamate and a subsequent Ca2+ increase in neurons [3) production and Ca2+ release from the endoplasmic reticulum (ER) [2+ levels lead to a release of gliotransmitters, including glutamate, ATP, d-serine and γ-aminobutyric acid (GABA) [d-aspartate receptor (NMDAR), synchronising neuronal spiking [For a long time, neuroscientists considered glia as merely supportive cells in the brain that control ion and water homeostasis, produce and remove neurotransmitters, induce synaptogenesis, provide trophic factors for neurons and maintain the blood–brain barrier. Their view as “passive” cells was reinforced by observations that astrocytes expressed potassium and sodium channels and could exhibit evoked inward currents, but they did not “fire” or propagate action potentials . Therefoactivity . This obactivity ,6,7,8,9. neurons ,11 led t neurons . The clalum (ER) . Increasd (GABA) . The rel spiking , as well spiking ,16,17,18 spiking ,20).2+) leads to fusion, during which the vesicle and the plasma membrane combine with each other. After fusion, the secretory vesicle can either be recycled by the closing of the fusion pore, a model termed “kiss-and-run”, or it can collapse and fully integrate with the plasma membrane [Exocytosis is a process in which the cargo of a secretory vesicle is released across the cell membrane. This universal process, which is common to all eukaryotic cells, can be generally divided into two types: unregulated and regulated exocytosis. Unregulated (constitutive) exocytosis typically involves the formation of membranous secretory vesicles within the cell, in which the cargo is packaged and then continually released through the cell membrane. In regulated exocytosis, the secretory vesicle with its cargo is stored until a signal triggers the process of secretion ,22,23. Tmembrane ,24. Rega2+ sensors which can trigger membrane fusion.Similar to all eukaryotic cells, astrocytes contain different types of vesicular organelle which cargo can be released to the environment. Generally, secretory vesicles form a complex network originating from the ER or the Golgi apparatus, as well as from endosomes. Astrocytes are not different, and they contain several secretory organelles, including synaptic-like microvesicles (SLMV) ,26,27,282+ concentration released from the ER can trigger a release of gliotransmitters. It should be noted, however, that ER is usually not present in perisynaptic processes [2+ causes vesicle docking [2+-regulated mobility of vesicular glutamate transporter 1 (VGluT1)-positive vesicles have been found also in other studies [Astroglial SLMVs are small (30–100 nm in dimeter) electron-lucent vesicles, similar to neuronal synaptic vesicles. They were first identified in the brain tissue in the molecular layer of the dentate gyrus using electron microscopy . They arrocesses . Additio docking . Similar studies .+ pump (V-ATPase), a large complex composed of two domains, VO  and V1 (responsible for ATP hydrolysis) [SLMVs contain mainly small signalling molecules: glutamate and D-serine . Fillingrolysis) . Expressrolysis) ,42, immurolysis)  and funcrolysis) ,45,46. Trolysis) ,48,49. Mrolysis) ,46,50,51rolysis) ,39,51 anrolysis) , by meanrolysis) ,53, generolysis) , or by crolysis) . It has rolysis) ,57.d-serine vesicular transporter has been identified in astrocytes in situ; however, it was shown to be present in immunopurified astrocytic vesicles in one study [d-serine/chloride co-transporter, which uses the H+ gradient created by V-ATPase concentrated d-serine in the vesicles [d-serine is loaded together with glutamate to the same SLMVs. This process of vesicular synergy is present in neurons where glutamate is co-released with dopamine, serotonin or acetylcholine [d-serine are located in astrocytic VAMP2- and VAMP3-positive vesicles, which raises the possibility that these may be the same vesicles [d-serine application modulates the uptake of glutamate to immunopurified SLMVs and vice versa [d-serine had no effect on glutamate uptake into immunopurified synaptic vesicles [d-serine and glutamate are stored in distinct vesicles within the same astrocyte [d-serine is mainly neuronal, as serine racemase (SR)—the enzyme which converts l-serine into d-serine—is expressed almost entirely in the neurons [d-serine transport from neurons to astrocytes and secondary astroglial release of d-serine [d-serine may be important in pathological conditions [So far, no ne study . The idevesicles . It is slcholine . VGluTs vesicles ,49,59,60ce versa . This suvesicles . Contrarstrocyte . Notably neurons ,62. Thisd-serine . Additionditions .3) receptors (IP3Rs) in DCVs, suggesting that they can serve as IP3-sensitive intracellular Ca2+ reservoirs. As they are relatively small, they could be directed to many distinct processes of the astrocyte, including perisynaptic processes [2+ for regulated exocytosis. Similar to SMLVs, DCVs have also been found to be highly mobile, and an increase in cytosolic Ca2+ levels seems to decrease this mobility [Dense-core vesicles (DCVs) have been well studied in a variety of tissues, where they are responsible for the storage and secretion of biogenic amines, peptides and neurotrophins, including catecholamines released from adrenal chromaffin cells or insulin released from DCVs in pancreatic β cells . In astrrocesses , and promobility ,78.Of all molecules present in DCVs, ATP has attracted the most attention, as it is a potent transmitter influencing glial and neuronal signalling, as well as behaviour ,80. Its 2+-dependent manner [2+-regulated manner, although small vesicles are exocytosed more efficiently than lysosomes [Secretory lysosomes have diameters ranging from 300 to 500 nm ,88 and ht manner ,35,89. Mysosomes . Secretoysosomes ,37,90,91ysosomes ,93, variysosomes ,94—as weysosomes . In thisysosomes ,34; howeysosomes , sialin,ysosomes  and VAMPysosomes ,91. Downysosomes , which ttrans” configuration (SNARE proteins are on opposite membranes) with amino- to carboxy-terminal zippering of proteins, which brings the vesicle closer to the plasma membrane. Assembly of the full trans-SNARE complex, together with the action of the SM protein, opens the fusion pore. After fusion pore opening, the membrane of the vesicle and the plasma membrane completely merge, and trans-SNARE complexes are converted into cis-SNARE complexes, where the SNARE complexes are on a single membrane. Next, the cis-SNARE complex proteins are bound by N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment proteins  to catalyse SNARE complex dissociation into monomers. This allows for endocytosis of the v-SNARE and recycling of the individual t-SNAREs back to their respective plasma membrane compartments.Secretory vesicle docking, priming and fusion with a plasma membrane are generally mediated by SNARE proteins  and SM proteins (Sec1/Munc18-like proteins) that undergo a cycle of association and dissociation during the fusion reaction. The mechanism of regulated SNARE complex assembly is conserved in many different cell types, including neuronal, exocrine, haematopoetic and endocrine cells  and botulinum neurotoxin (BoNT), which cleave SNARE proteins. In particular, application of TeNT to cultured astrocytes attenuated the exocytotic release of glutamate ,105,106.d-serine  or glutad-serine ,107, whid-serine . This med-serine  and was d-serine . Experimd-serine ,112,113.d-serine  found thd-serine ,116). Thd-serine . Later, d-serine . This reOther v-SNARE proteins have also been identified in astrocytes. In particular, VAMP7 was shown in astrocytes in vitro as a v-SNARE associated with lysosomes ,59,91, aFor t-SNARE proteins, astrocytes were shown to express syntaxin1; however, the results of various studies have been mixed. Syntaxin1 has been demonstrated in cultured astrocytes ,66, albeAs mentioned above, the SNARE complex requires SM proteins for its formation, in particular Munc-18 is a prominent and well-studied example. It was shown to be significant for vesicle docking in chromaffin cells where Munc18-1 knockout reduces fraction of docked vesicles from 30% to 5% ,127. Morhttp://astrocyternaseq.org/ accessed on 1 January 2021) of sequenced astrocytic RNAs from different brain structures [Other components of the exocytosis machinery in astrocytes have not been intensively studied. Notable exceptions are the Rab proteins—small GTPases that are localised on synaptic vesicles and are important for their docking. Cultured astrocytes were shown to express Rab3 ,129,130,ructures , we wereructures ).2+ channels to docked and primed synaptic vesicles at the active zone. Most of the active zones in vertebrates are specialised disc-like structures at the plasma membrane with a 0.2–0.5 μm diameter. Active zones are surrounded by a perisynaptic zone containing transsynaptic cell adhesion molecules and receptors regulating neurotransmitter release, which is the site of synaptic vesicle endocytosis [2+ channels to the docked and primed vesicles, tethers the vesicles and Ca2+ channels to synaptic cell adhesion molecules, and mediates synaptic plasticity. Astrocytes do not have an active zone that could be observed under an electron microscope; however, the fact that there have been observations of small groups of SMLVs in astrocytes (see above) [Ultrafast neurotransmitter release, which occurs in neurons in response to an action potential, can only be achieved by bringing Caocytosis . The core above) ,38,39 sue above)  found the above) ,134. Sime above) . Resultse above) . The inte above) . Homer1 e above) ) and itse above) . Interese above) .2+; therefore, to trigger the final stage of the fusion reaction, a Ca2+ sensor is required at the site of exocytosis. Since the discovery of synaptotagmin1 (Syt1) [2+ sensor for regulated exocytosis. Syts are evolutionarily conserved proteins containing a short amino-terminal sequence directed toward the vesicle lumen, followed by a transmembrane region, a central linker sequence of variable length, and two carboxy-terminal C2 domains that bind Ca2+. [2+/phospholipid-binding domains. In addition, C2 domains constitute protein interaction domains and, in the case of Syt1, bind to syntaxin-1 and to SNARE complexes. There are 16 Syts expressed in the brain and eight of them bind Ca2+: Syt1, Syt2, Syt3, Syt5, Syt6, Syt7, Syt9 and Syt10 [2+-triggered exocytosis, Syt1, Syt2 and Syt9 are involved in chromaffin cells, neuropeptide secretion in neurons or mast cell degranulation. However, Syt7 has also been shown to be involved in triggering exocytosis in chromaffin cells [2+-triggered asynchronous release [2+ [2+-dependent glutamate release in cultured astrocytes [2+-dependent lysosome exocytosis in injured astrocytes [2+ elevation, arguing against the existence of Ca2+-dependent exocytosis in astrocytes  , it has nd Ca2+. ,140. C2 nd Syt10 ,141,142.in cells , pancreain cells ,145, or in cells . In neur release . For ast release  or even  release . However release ,149,150. release ,53 and Rease [2+ , Zhang atrocytes . It shouews, see ,115,152)Synaptotagmins, however, do not act alone in triggering fusion but require complexin as a cofactor . Complex2+ is not the only signal that can trigger vesicle secretion, as many cells show GTP-dependent DCV secretion. Non-hydrolysable GTP was shown to trigger secretion in a Ca2+-independent manner in chromaffin cells [2+-dependent exocytosis in terms of the signal sensor for triggering, the final fusion steps are still dependent on the SNARE proteins [http://astrocyternaseq.org/ accessed on 1 January 2021), we found that all these proteins are expressed at a significantly different level from the expression threshold set as fragments per kilobase per million (FPKM) >10 [2+-binding synaptotagmins in astrocytes and their slow exocytosis [It should also be noted that Cain cells , mast cein cells  and pancin cells . Even thproteins . The majproteins . Ral proproteins ,159. It proteins , which iproteins . Additioproteins , directlproteins , and witproteins . InteresPKM) >10 . Howeverocytosis ,161, GTPIn nearly all brain pathologies, such as traumatic brain injury, stroke, ischemia, infectious disease, neuroinflammatory and neurodegenerative disease, epilepsy, brain tumours, schizophrenia, migraine or depression, there is clearly a presence of “reactive astrogliosis” ,166. Thi2+-regulated exocytosis only after activation by microglia , tumour necrosis factor (TNF) and complement component 1, subcomponent q (C1q)), subsequently trigger astrocytic activation ,169. Actexample, ,175). Inexample,  showed t release ,177. Int release  has show release .2+-dependent manner. They act through activation of their respective receptors—CXCR4 and-, TNFR (both Gi/o–associated GPCRs)—or prostaglandin E (EP) receptors (Gi/Gs–associated GPCRs) [2+-dependence of the exocytosis of glutamate after activation of these receptors is based on an observation that the response is blocked by intracellular Ca2+ chelators or inhibitors of exocytosis [SDF-1α, TNF or PGE2 by themselves are also sufficient to induce glutamate release from astrocytes, albeit still in a Cad GPCRs) ,180,181.ocytosis .PCO2/pH and they respond to physiological decreases in pH with vigorous elevations in intracellular Ca2+ and regulate the exocytosis of ATP [PO2 by Ca2+ signalling, accompanied by increases in mitochondrial reactive oxygen species (ROS) production and ATP exocytosis [2+-sensing EF-domain, controls ATP-induced Ca2+ signalling and gliotransmission. This effect is mediated by hypoxia-inducible factor 1 (HIF1), which is a heterodimeric transcription factor. In normoxia, prolyl 4-hydroxylase (P4H) hydroxylates two prolyl residues located on HIF’s α subunit. This hydroxylation leads to von Hippel–Lindau (VHL)-targeted degradation of HIFa, which suppresses the transcription of hypoxia-responsive genes. However, in hypoxia, P4H is inactive, which leads to stabilisation, accumulation and activation of HIF and induction of hypoxia-responsive genes. One study [2+ signalling and vesicular transport/docking pathways, thus influencing receptor-operated calcium entry (ROCE) and store-operated calcium entry (SOCE), as well as calcium re-uptake by mitochondria. Moreover, in an in vitro model of cerebral ischemia, oxygen–glucose deprivation (OGD), there was a biphasic increase in Ca2+ signalling in astrocytes as well as neurons and a subsequent accumulation of pro-inflammatory factors, such as IL-1b and TNFα, leading to hyperexcitation of the neurons and their death after reoxygenation [2+-regulated exocytosis of ATP [In physiological conditions, astrocytes maintain constant levels of blood and brain s of ATP . Howeverocytosis ,184. In ocytosis  has showne study  showed tgenation . Interess of ATP .Reactive astrocytes have been also shown to release complement system proteins . The comFinally, the activation of astrocytes is often associated with their morphological and biochemical remodelling. The reactivity is manifested by an increased expression of intermediate filaments (most notably GFAP and vimentin). It has been suggested that such an upregulation of intermediate filaments allows faster and therefore more efficient delivery of major histocompatibility complex (MHC) Class II molecules to the cell surface. Exposure of astrocytes to INF-γ induced MHC Class II expression in late endosomes/lysosomes ,96.2+ mechanisms of triggering exocytosis. Moreover, much attention needs to be given to designing new experiments to bring them to the more physiology-relevant environment of neural tissue. New techniques of astrocyte culturing also give hope for creating new study models which will resemble astrocytes in the brain. Regardless of the discussion on Ca2+ sources for triggering exocytosis and gaps in our knowledge about the elements of the exocytotic molecular machinery, astrocytes have proven to be an important component of neuronal networks.Astrocytes are receiving increasing attention as their complex role in the central nervous system becomes more prominent and accepted. In this review, we presented only a fraction of the studies which not only show that astrocytes have various types of secretory vesicles but also that they express a complex molecular machinery associated with those vesicles which is sufficient for their regulated exocytosis. For other excellent in-depth reviews, please see ,134,161.


SACS and KIF1C) and with ataxia and neuropathy or AOA (PNKP). We also identified three families with autosomal dominant (AD) forms arising from de novo variants in KIF1A, CACNA1A, or ATP1A3, reinforcing the importance of differential diagnosis (AR vs. AD forms) in families with only one affected member. Moreover, 10 novel causal-variants were identified, and the detrimental effect of two splice-site variants confirmed through functional assays. Finally, by reviewing the molecular mechanisms, we speculated that regulation of cytoskeleton function might be impaired in spastic ataxia, whereas DNA repair is clearly associated with AOA. In conclusion, our study provided a genetic diagnosis for HCA families and proposed common molecular pathways underlying cerebellar neurodegeneration.Hereditary cerebellar ataxia (HCA) comprises a clinical and genetic heterogeneous group of neurodegenerative disorders characterized by incoordination of movement, speech, and unsteady gait. In this study, we performed whole-exome sequencing (WES) in 19 families with HCA and presumed autosomal recessive (AR) inheritance, to identify the causal genes. A phenotypic classification was performed, considering the main clinical syndromes: spastic ataxia, ataxia and neuropathy, ataxia and oculomotor apraxia (AOA), ataxia and dystonia, and ataxia with cognitive impairment. The most frequent causal genes were associated with spastic ataxia ( Hereditary cerebellar ataxias (HCA) comprise a heterogeneous group of neurological disorders. The cardinal cerebellar syndrome of HCA may be present in isolation or accompanied by a wide constellation of neurological and non-neurological symptoms and signs. All modes of inheritance have been recognized, with the autosomal recessive (AR) forms being the most complex, both from a clinical and genetic point of view . PrevaleDespite the diversity of HCA-genes and their ubiquitous expression throughout the brain, genetic defects in HCA predominantly affect the cerebellum, suggesting that cerebellar cells  are more vulnerable. Therefore, the identification of shared molecular pathways between mutated genes is crucial to understand the basis of cerebellar neurodegeneration ,7. Many This study focused on 19 Portuguese families with apparent AR-HCA, previously identified during a large population-based survey and studied by targeted testing for the most common genes , and nowA national, systematic, population-based survey was conducted in Portugal from 1994 until 2004, aiming at identifying Portuguese families with HCA and hereditary spastic paraplegia (HSP) . Its detPNKP [MAG [RFC1 [In the national survey, 250 families (344 patients) with presumed autosomal recessive ataxia were identified. Targeted genetic testing was conducted ab initium, according to the main clinical phenotype; 61 families (83 patients), however, remained without a genetic diagnosis . PathogePNKP  were latNKP [MAG  in one fAG [RFC1  was exclFrom the 61 undiagnosed families, we only had genomic DNA available for 27 families, with a final diagnosis being reached in the 19 here described. We performed whole-exome sequencing (WES) in at least one patient of each family (depending on the availability of genomic DNA). Exome sequencing libraries were prepared using a SureSelect Exome Capture Kit v7 (Agilent), and sequencing was performed on NextSeq550 with 100 bp paired-end reads, according to the manufacturer’s instructions. After sequencing, reads were then aligned to the human reference genome hg19/GRCh37 using Burrows-Wheeler Aligner (bwa) v0.7.1  and varihttps://www.ncbi.nlm.nih.gov/snp (accessed on 11 February 2022)), Genome Aggregation Database ), and the 1000 Genome Project ((http://www.1000genomes.org (accessed on 11 February 2022)). Non-synonymous and splice-site variants, and variants for AR forms were filtered for primary analysis. This analysis was performed using Exomiser v7.2.1 [http://www.ncbi.nlm.nih.gov/clinvar (accessed on 11 February 2022)) and Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php (accessed on 11 February 2022)) were also used for variant classification.Annotated variants were filtered to include rare variants with a minor allele frequency (MAF) <1% in population databases such as Nucleotide Polymorphism Database , purified products with Exo/SAP , and performed Sanger sequencing using Big Dye Terminator Cycle Sequencing v1.1  in an ABI 3130xl Genetic Analyzer . Sequencing analysis was carried out using the SeqScape v2.6 software .SPG11 and KIF1C minigenes constructs were obtained by cloning the exonic and intronic sequences flanking the variants of interest (c.3039-5T > G in SPG11 and c.1166-2A > G in KIF1C) into the pCMVdi vector, kindly provided by Dr Alexandra Moreira [SPG11 exons 16, 17, and 18 with intronic regions or KIF1C exons 13, 14, and 15 with intronic regions were PCR amplified from patients’ genomic DNA and cloned into the pCMVdi vector, using the Gibson assembly method. Sequences were modified by site-directed mutagenesis to generate the wild-type constructs, using a QuikChange II Kit , according to the manufacturer’s protocol.The  Moreira . BrieflyHEK293T cells were transfected with the minigene constructs using jetPRIME , according to the manufacturer’s protocol. RNA was extracted 48 h after transfection, using NZYol , as per manufacturer’s recommendations, followed by purification of the RNA aqueous phase, using an RNeasy mini kit . RNA quantification was performed on NanoDrop 2000 . cDNA was synthesized by reverse transcription-PCR of 2 µg total RNA with oligo (dT), using a SuperScript III first-strand synthesis system , according to the manufacturer’s protocol. The resulting cDNA was amplified by PCR and loaded on agarose gel for extraction with a Zymoclean Gel DNA Recovery Kit . The resulting products were sequenced by Sanger sequencing using Big Dye Terminator Cycle Sequencing v1.1  in an ABI 3130xl Genetic Analyzer .We used WES to identify variants and genes causing HCA in 19 Portuguese families with family history compatible with AR transmission (including 9 with known consanguinity), in a total of 30 individuals: 19 index cases, one affected and 10 non-affected relatives. At least, one relative of seven index cases was available. All relevant variants  were confirmed by Sanger sequencing, and segregation analysis was performed with all available family members. Phenotypic classification considered the main clinical symptom associated with ataxia: spasticity, neuropathy, oculomotor apraxia, dystonia, or cognitive impairment.SACS, KIF1C, ANO10, SPG11, SYNE1, and CACNA1A were related to spastic ataxia ; KIF1A, POLG, SETX, and PNKP to ataxia and neuropathy ; PNKP to AOA ; HEXB and ATP1A3 to ataxia and dystonia ; and FA2H to ataxia with cognitive impairment . The most frequent genes were those associated with spastic ataxia, ataxia, and neuropathy or AOA: SACS , KIF1C , and PNKP ; the remaining genes were identified in single families only.Overall, we identified 24 rare nucleotide variants in 13 genes : SACS, KSPG11 and c.1166-2A > G in KIF1C—both in families with spastic ataxia; their detrimental effect on splicing was confirmed by minigene splicing-assays, concluding that both are expected to result in frameshifts (Variants were classified as pathogenic when having been previously reported as disease-causing (14/24), or by causing an early stop codon  in genes where loss-of-function is a known disease mechanism . In addimeshifts .CACNA1A , at an amino-acid residue where a different missense change has been determined to be pathogenic, located in a well-established functional (transmembrane) domain , a gene with low rate of benign missense variation, not reported in population databases, located in a mutation hotspot within a transmembrane domain (CACNA1A (Arg1666) and ATP1A3  are conserved across species, while the mutated residues are predicted to change their interactions, probably affecting protein structure . Unfortunately, we did not have parental samples to test for the CACNA1A variant (family AR267), but this variant probably occurs de novo, as these are a recurrent cause of CACNA1A-related ataxia [KIF1A  in a patient with spastic ataxia, and confirmed the unaffected parents and sibling did not carry it (family AR49).We also identified two novel missense variants , both in) domain  and not e domain  and predtructure . Besidesd ataxia . In addiThus, we reported 10 novel disease-associated variants in nine families . All varBeyond the 20 patients tested, we had clinical information (but no DNA sample) from nine additional affected relatives of probands (detailed data SACS) and AR267 (CACNA1A), all patients had onset in the first decade of life. Its presenting symptoms comprised ataxia (AR53 (SACS), AR120 (SACS), AR103 (ANO10), AR109 (SYNE1), AR267(CACNA1A)), delayed motor milestones (AR77 (SACS), AR252 (SACS), AR111 (KIF1C)), cognitive regression (AR108 (SPG11)), and upper limb tremors (AR96(KIF1C)); we highlight, due to its rarity, the presence of seizures in AR77 (SACS) and the spastic ataxia phenotype in AR267 (CACNA1A). Regarding imaging, notably, family AR96 (KIF1C) showed basal ganglia hypointensities on T2/FLAIR sequences and AR111 (KIF1C) had brainstem atrophy.Spastic ataxia was the largest phenotypic group, comprising 10 families (12 patients). With the exception of AR120 (KIF1A) and AR92 (PNKP), second decade in AR4 (SETX), and third decade in AR126 (POLG). Ataxia was the presenting feature in all families, with neuropathy developing later. It is noteworthy that in AR4 (SETX) and AR92 (PNKP) none of the patients had oculomotor apraxia. All patients from AR4 presented diplopia and nystagmus, but none from AR92 displayed such features. The patient from AR49 (KIF1A) had an extensor plantar response in the absence of spasticity. AR126 had a POLG-related ataxia, with the typical optic atrophy, vertical gaze palsy, and epilepsy. One of the members from AR92 died at age 35, with 29 years of disease duration. Imaging was only available in two families , all displaying cerebellar atrophy.This group included four families (10 patients). Onset was in the first decade in AR49 ; when tested, total proteins/albumin level was decreased, and cholesterol increased. In AR117, death occurred between 37 and 55 years of age, with a mean disease duration of 37 years.Both families (4 patients) had onset in the first decade of life, and disease-causing variants in HEXB) and AR278 (ATP1A3), with one patient each, presented ataxia and dystonia. AR2 (HEXB) had adult-onset ataxia, very prominent oromandibular dystonia and muscle wasting, while AR278 (ATP1A3) had onset at age 11 years, with paroxysmal lower limbs dystonia induced by walking, and progressive ataxia four years later. Over the disease course, dystonia became permanent and generalized. Both AR2 and AR278 had cerebellar atrophy on MRI.AR2 (FA2H) was the only family with ataxia and cognitive impairment as the major phenotype, in a patient with delayed motor and cognitive milestones, who later developed ataxia and seizures. Cortical and cerebellar atrophy was present on MRI, and increased latency was identified in visual evoked potentials.AR16  recessive cerebellar ataxia, aiming at providing a conclusive genetic diagnosis for them. These families had been identified during our national population survey in Portugal , but remith AOA4 , MAG in uropathy , and DABth SCA37 .SACS, KIF1C, and PNKP. To note, Friedreich ataxia, AOA (AOA1 and 2) and L-2-hydroxyglutaric aciduria had been previously screened and found to be the most prevalent types of AR-HCA [KIF1C and PNKP were described only a few years later to be the causative genes for SPAX2 and AOA4 [PNKP-related ataxia as one of the most prevalent AR-HCA in Portugal [PNKP and SETX were universally associated with neuropathy, but not with AOA; the reason why AR4 had not been previously tested for SETX. This has been described in the meantime [SACS and two in KIF1C, broadening the genetic spectrum of ARSACS and SPAX2.In this report, pathogenic and likely-pathogenic variants in several genes were identified and clearly associated with HCA . Divisiof AR-HCA ; KIF1C aand AOA4 ,39. PresPortugal . Both PNmeantime . OculomoSPG11 c.3039-5T > G variant creates a new 3′ acceptor splice site, while KIF1C c.1166-2A > G abolished the canonical 3′ acceptor slice site. Both were predicted to modify the open reading-frame, resulting in a premature stop codon.We also provided functional data to validate the detrimental effect of two new splice-site variants  that wouKIF1A [CACNA1A and ATP1A3 were classified as likely pathogenic. KIF1A has been associated with AR and de novo AD diseases [KIF1A-related variants have shown a complex phenotype, including developmental delay, ataxia, spastic paraplegia, and neuropathy, with onset in the first year of life [KIF1A should be considered in ‘Friedreich ataxia-like’ patients.All families analyzed had been classified as AR-HCA, based either on the presence of affected relatives in the same generation with unaffected parents; absence of other affected relatives; or consanguinity. Nevertheless, de novo variants in heterozygosity were identified (or presumed) in three families with only one affected member , highligKIF1A , and twodiseases . Patient of life ,43,44. OKIF1A, CACNA1A and ATP1A3 are known to cause only autosomal dominant disorders [CACNA1A cause SCA6, and missense mutations affecting the calcium channel-encoding sequence cause progressive cerebellar ataxia, familial hemiplegic migraine (FHM1), and episodic ataxia type 2 (EA2) [CACNA1A-related disorders. Interestingly, CACNA1A variants were one of the most prevalent causes of AD-HCA, after SCA3 and dentatorubral-pallidoluysian atrophy (DRPLA), in our population-based survey [ATP1A3 produce a wide spectrum of AD neurological and psychiatric disorders, ranging from infantile to adult onset. Cerebellar ataxia is classically present in CAPOS syndrome , but atypical phenotypes with ataxia  have been increasingly recognized [Opposed to isorders ,46,47,48 2 (EA2) . A conge 2 (EA2) . A spast 2 (EA2) , probabld survey . Variantcognized . The procognized .HCA can be caused either by loss-of-function, gain-of function, or a dominant-negative effect, in a multitude of apparently unrelated genes. Generally, AR-HCA is associated with loss-of-function variants, whereas AD-HCA can be caused by a combination of gain and/or loss of function . Our stuSACS, KIF1C, SPG11, and SYNE1 interact with the cytoskeleton and may function to ensure proper vesicle/organelle trafficking and/or neuronal structure.Spasticity and pyramidal signs are hallmarks of several cerebellar ataxias ,54. As dSACS encodes the large protein sacsin, the function of which has not yet been clearly established. The presence of both ubiquitin-like (UBL) and DNaJ domain (implicated in chaperon-mediated folding process), and a potential role in the degradation of aberrant ataxin-1, suggest that sacsin may integrate the ubiquitin–proteasome system and Hsp70 chaperone function [SACS knockout mice, revealed that sacsin regulates the neurofilament cytoskeleton and mitochondria dynamics [SACS variants lead to the complete loss of sacsin, although some missense variants are associated with low levels of the protein [function . Moreovedynamics ,57. Most protein ,59. Sinc protein . ConcordKIF1C) is a ubiquitously expressed motor protein involved in both anterograde transport of vesicles and retrograde transport from Golgi to the ER [KIF1C variants, but it was suggested that variants in the motor domain may affect microtubule binding and impair ATP hydrolysis, while truncating variants lead to reduced protein levels and may impair cargo binding [Kinesin family member 1C , also known as nuclear envelope spectrin 1 (nesprin 1). This protein mediates the link from the nuclear envelope to the actin cytoskeleton (LINC complex), being important for nuclear migration [SYNE1 variants associated with ARCA1 are nonsense or frameshift, and span the giant nesprin-1 isoform, but exclude the C-terminal Klarsicht-ANC-Syne-homology (KASH) [igration ,67. Mosty (KASH) . Thus, Ay (KASH) . NeverthANO10, which encodes a putative calcium-activated chloride channel, named anoctamin-10 (ANO10), are associated with SCAR10/ARCA3 [ANO10 mRNA expression in patient’s skin fibroblasts [ANO10 variants remains unexplored.In addition, variants in 10/ARCA3 , with deroblasts , but thePOLG, SETX, and PNKP) with various molecular functions, causing recessive ataxia and neuropathy.Peripheral neuropathy is a common feature associated to HCAs; but, as far as we know, this combination has not been linked with specific disease pathways. In this study, we found three genes , which functions in the replication and repair of mtDNA [POLG variant identified in our study (p.Trp748Ser) is within the C-terminal domain of the polymerase, an important domain for its activity [of mtDNA . Functioof mtDNA ,74.The Pactivity . We can,SETX and PNKP are discussed in the following sub-section.XRCC1-AOA, and spinocerebellar ataxia with axonal neuropathy 1 are all caused by genes with a role in DNA repair [SETX and PNKP, causing AOA2 and AOA4, respectively. Interestingly, in one family with pathogenic variants in SETX and one in PNKP, oculomotor apraxia was not present. Both had been phenotypically classified as ‘ataxia with neuropathy’, due to severe neuropathy. This possible occurrence has been previously described and is reinforced in this study.AOA has been associated with the DNA damage repair pathway. Ataxia telangiectasia, AOA1, AOA2, AOA4, A repair . We idenSETX encodes a large nuclear protein termed senataxin, with an RNA/DNA helicase domain. Senataxin is involved in RNA metabolism, DNA maintenance, and damage response. Several types of biallelic variants in SETX were identified in AOA2 [SETX cause amyotrophic lateral sclerosis (ALS) with juvenile onset (ALS4) [SETX variants [ in AOA2 ,77,78, pt (ALS4) , suggestvariants ,80,81,82variants , but as PNKP, encoding the nuclear polynucleotide kinase 3′ phosphatase protein (PNKP), result in a range of AR disorders. The enzymatic activity of PNKP is involved in the repair of both DNA double strand-break (DSB) and single-strand break (SSB); impaired function can cause neurodevelopmental dysfunction ) [PKNP variants. Nevertheless, most variants seem to cause reduced stability and levels of PNKP, and reduced DNA phosphatase activity in MCSZ or reduced kinase activity in neurodegenerative diseases. These ultimately lead to reduced DNA repair [PNKP; most are in the kinase domain, particularly in neurodegenerative diseases [Variants in  (MCSZ))  and neur (MCSZ)) ,36. Few A repair ,86. Morediseases ,87, as sHEXB in one family with Sandhoff disease, and a new variant in FA2H causing ataxia with cognitive impairment.Additionally, other neurological signs may be present in AR-HCA, such as dystonia, and cognitive impairment. In our study, we identified a known variant in HEXB encoding the β subunit of the enzyme hexosaminidase (HEXB) [HEXB variants is wide, but most reduce GM2 hydrolysis, resulting in the accumulation of GM2 ganglioside [Sandhoff disease is caused by variants in e (HEXB) . HEXB isglioside ,90. Moreglioside ,92,93. Pglioside .FA2H encodes the fatty acid 2-hydroxylase (FA2H) protein, which is involved in the synthesis of 2-hydroxy fatty acid galactolipids, the most abundant lipids in the myelin sheath [FA2H are speculated to cause neurodegeneration through a loss-of-function mechanism, since there is evidence of decreased hydroxylation of myelin lipids [FA2H variant we identified is predicted to cause a frameshift, it is also possible it leads to loss-of-function.n sheath . Pathogen lipids . Moreoven lipids . Since tPNKP associated with either ataxia and neuropathy or AOA was also amongst the most frequent causal-genes. We highlight three families with de novo variants in KIF1A, CACNA1A, and ATP1A3, despite having been initially classified has AR. These results evidence the importance of performing a differential diagnosis (AR vs. AD forms) in the absence of affected relatives of the proband. All the remaining families were associated with AR-HCA related genes. In addition, we provided a review on potential common mechanisms underlying neurodegeneration in AR-HCA, namely cytoskeleton function in spastic ataxia, and DNA repair in AOA. Translation of genetic findings into a better understanding of HCA mechanisms may help the development of effective therapies [Our study provided the molecular diagnosis of 19 families with various types of HCA through WES, expanding the genetic and phenotypic spectrum of HCA. Most of the mutated genes caused a spastic ataxia phenotype, but herapies ,7.