Scophthalmus maximus), a representative species of Pleuronectiformes, is one of the most commercially important fish species cultured in Europe and North China. However, the mechanisms by which the Kiss/Kissr system regulates the reproductive axis of turbot according to seasonal changes, especially photoperiod, have not been clearly characterized. In the current study, the cDNA sequences of kiss2/kissr2, along with kiss1/kissr3 which was thought to be lost in flatfish species, were cloned and functionally characterized. The kiss1, kiss2, and kissr3 transcripts were highly detected in the brain and gonad, while kissr2 mRNA was only abundantly expressed in the brain. Moreover, kiss/kissr mRNAs were further examined in various brain areas of both sexes. The kiss1, kissr2, kissr3 mRNAs were highly expressed in the mesencephalon, while a substantial degree of kiss2 transcripts were observed in the hypothalamus. During annual reproductive cycle, both kiss and kissr transcript levels declined significantly from the immature to mature stages and increased at the degeneration stage in the brains of both sexes, especially in the mesencephalon and hypothalamus. The ovarian kiss1, kiss2, and kissr2 mRNA levels were highest at the vitellogenic stage (mature stage), while expression of kissr3 was highest at the immature stage. The testicular kiss and kissr transcripts were highest in the immature and degeneration stages, and lowest at the mature stage. In addition, intraperitoneal injection of Kiss1-10 and Kiss2-10 significantly stimulated mRNA levels of pituitary lhβ, fhsβ, and gthα. In summary, two Kiss/Kissr systems were firstly proven in a flatfish species of turbot, and it has a positive involvement in controlling the reproduction of the Kiss/Kissr system in turbot. The results will provide preliminary information regarding how the Kiss/Kissr system controls seasonal reproduction in turbot broodstock.Seasonal reproduction is generally controlled by the hypothalamus-pituitary-gonadal (HPG) axis in fish. Previous studies have demonstrated that the kisspeptin (Kiss)/kisspeptin receptor (Kissr) system, a positive regulator of the HPG axis, mediates the responses to environmental cues. Turbot ( In vertebrates, seasonal reproduction, as the most important biological rhythm, is classically controlled by the hypothalamus-pituitary-gonadal (HPG) axis. Hormones secreted from the HPG axis are regulated by seasonally changing patterns, such as photoperiod, which is an environmental signal that can trigger organism to sexually mature. The discovery of the kisspeptin/GPR54 signaling system has greatly improved our understanding of reproductive endocrinology –3. Therekissr1) was characterized in rats  syndrome could cause sterile in mice , and its receptor, G protein-coupled receptor 54 (GPR54), now renamed kisspeptin receptor (Kissr) . The fir in rats . Further in mice , 9. In m in mice , 11.kissr2 was co-expressed in GnRH neurons in Nile tilapia (Oreochromis niloticus), providing valuable insights into the kisspeptin system during normal sexual and reproductive development  and three kissr genes  have been identified in various teleosts, including zebrafish (Danio rerio) (Oryzias latipes) (Carassius auratus) (Dicentrarchus labrax) (Epinephelus coioides) (Scomber japonicas)  (Anguilla anguilla) (Odontesthes bonariensis) (Hippocampus erectus) (Amphiprion clarkia)  (Hippoglossus hippoglossus)  (Cynoglossus semilaevis)  –15, medalatipes) , 17, golauratus) , 19, Eur labrax) , 21, oraoioides) , chub maponicas) , 24, yellalandi) , 26, Eurnguilla) , 28, pejriensis) , 30, linerectus) , yellowtclarkia)  as well alensis) , Atlantiglossus) , Japanesivaceus) , and halilaevis) , 37.kiss/kissr mRNAs in golden mahseer (Tor putitora) and rohu (Labeo rohita) indicated that they have a role in gonadal development and annual reproductive cycle , European sea bass and grass puffer (Takifugu niphobles)  –50.Scophthalmus maximus) is classified into the Pleuronectiformes, and is one of the most commercially important fish species cultured in Europe and North China. During turbot aquaculture, photoperiod manipulation is used to induce egg and sperm production on a year-round basis . Three males and three females were collected for sampling every month throughout the reproductive season. Before sampling, turbots were anesthetized with a 0.05% MS222 (Sigma-Aldrich). The gonad from each turbot was divided into two parts, one part was fixed in Bouin’s fluid for histological analysis and the other was frozen in liquid nitrogen and then stored at -80°C. Besides gonads, brain and pituitary gland were also collected for qPCR analysis. In addition, various tissues, including brain, eye, skin, muscle, gill, liver, heart, spleen, kidney, stomach, intestine, liver, and gonad from immature male turbot were collected and stored at -80°C to examine the tissue distribution of the Kiss/Kissr system.The turbot individuals used in this study were obtained from a fish farm in Shandong Province, China. All of the broodstock were over 2 years old and had previously undergone gonadal maturation. The fish were cultured under a simulated natural photoperiod (12L:12D) and ambient temperature of 17 ± 0.8°C before the reproductive season. During the breeding season, from November 2018 to April 2019, the lighting regime progressed through three stages: an inhibitory photoperiod (8L:16D) for about 1.5 months, a natural photoperiod (12L:12D) for about 0.5 months, and a prolonged photoperiod (16L:8D) for about 4 months. The fish were fed with enhanced nutrition  and treated with HE (Haematoxylin and Eosin) stain. Stained sections were examined using a microscope Axio Scope A1 . The developmental stages of gonads were confirmed based on ovarian and testicular histology. All of the collected turbots were classified into II, III, IV, V, and VI stages according to the dominant gamete type and characteristics , 57.kiss1, kiss2, kissr2, and kissr3 were amplified with primers  . Quality and concentration of RNA were measured using 1.0% agarose gels electrophoresis and assessed by spectrophotometry . The first-strand cDNA was synthesized using a TransScript First-Strand cDNA synthesis kit (AT301-02)  following the manufacturer’s instructions. Subsequently, cDNA fragments of turbot http://www.ncbi.nlm.nih.gov/). The putative signal peptides were predicted by SignalP4.1. The putative transmembrane domain was predicted using the TMHMM server V2.0. The percentages of similarity and identity were calculated using LALIGN (http://www.ch.embnet.org/software/LALIGN-form.html). Multiple alignments of predicted amino acid sequences were conducted using BioEdit with the ClustalW alignment tool. A phylogenetic tree was constructed in the Mega7 software by the neighbor-joining method using bootstrapping with over 1,000 iterations.Homology searches of the deduced turbot Kiss1, Kiss2, Kissr2, and Kissr3 sequences were performed using the National Center for Biotechnology Information website (http://asia.ensembl.org/index.html) and Genomicus (https://www.genomicus.bio.ens.psl.eu/genomicus-83.01/cgi-bin/search.pl).The syntenic analysis of kiss1 and kiss2 genes among turbot, zebrafish, medaka and tilapia was conducted basing on the comparison the neighboring genes. Briefly, the protein sequences of neighbor genes of Kiss were predicted from the turbot chromosome sequences by FGENESH program. The identified neighboring protein sequences were annotated against NCBI by BLASTP. The conserved syntenic pattern of Kiss genes in other species were determined in Ensembl database  = 9.575 ± 0.47 g) were used, at a rearing temperature of 16 ± 0.5°C with dissolved oxygen > 7.0 mg/L and a 12L:12D photoperiod.According to the sequence analysis, turbot Kiss1-10 (YNLNSFGLRY-NH2) and Kiss2-10 (FNFNPFGLRF-NH2) peptides were synthetized by ChinaPeptides Co., Ltd.  with purities of 98% and 97%, respectively, as determined by HPLC. For lhβ and fshβ. Therefore, doses of 100ng/g BW and 1000 ng/g BW with intraperitoneal injection were chosen. The synthetized peptides were dissolved in phosphate-buffered saline (PBS) and injected intraperitoneally into turbot anesthetized with 0.05% MS222. PBS alone acted as the negative control. At 3 and 6 h post-injection, sample fish were collected (n = 6) and their pituitary glands quickly dissected, frozen in liquid nitrogen, and stored at -80°C for qPCR analysis.The doses of Kiss peptides referred the administration experiments in some fish species, including goldfish and cinnamon clownfish , 31, 42.β-actin as the reference gene. Specific primers used for each target gene were designed by the Primer Premier 5.0 software  with a SYBR Premix Ex Taq Kit  using the standard curve method with P < 0.05. All assays were carried out independently in triplicate.Statistical analysis was carried out using SPSS version 21.0. All results are presented as means ± SEM. Gene expression during gonadal stages were analyzed using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range tests. Differences were considered to be significant at kiss1 (GenBank accession no. MW057929) was 580 bp in size, with an open reading frame (ORF) of 312 bp encoding a preprohormone of 104 amino acids. The full-length cDNA of turbot kiss2 (GenBank accession no. MW057930) was 622 bp, with an ORF of 369 bp encoding a precusor of 123 amino acids. Amino acid sequence analysis demonstrated that an obvious Kiss1 domain-YNLNSFGLRY (Kiss1-10) and Kiss2 domain-FNFNPFGLRF (Kiss2-10) were involved in turbot kisspeptin system , rare minnow (Gobiocypris rarus), and Senegalese sole. However, turbot had Phenylalanine and Glycine at positions 6 and 7 from the N-terminus, which differed from the Leucine and Threonine in Japanese flounder.Notably, although the Kiss-10 regions are highly conserved across vertebrates, there were still 2 positions in Kiss1-10 and 3 positions in Kiss2-10 with different amino acids. For Kiss1-10, at the 3kiss1 gene was usually positioned in the genomic regions, including plekha6, cntn2, psma5, foxp4, mapkapk2, and pik3c2b, and kiss2 gene was usually positioned including gys2, spx, golt1b, ldhba, slc25a3a, and strap  was 2329 bp, with an ORF of 1092 bp encoding a peptide with 363 amino acids. The full-length cDNA of turbot kissr2 (GenBank accession no. MW057931) was 1578 bp, with an ORF of 1128 bp encoding a peptide with 375 amino acids. The deduced amino acids of Kissr3 and Kissr2 all contained a seven transmembrane domain  and kissr  genes in various tissues and different brain regions were detected by qPCR. Firstly, expression of tissues of brain, pituitary, gonad, eye, skin, muscle, gill, heart, spleen, kidney, stomach, intestine, and liver from adult male were shown in kiss1 mRNA was highly expressed in the brain and gonad, but had low expression in the skin, pituitary, and intestine. The kiss2 mRNA was particularly highly expressed in brain and gonad, and barely expressed in other tissues. The kissr3 mRNA was significantly expressed in brain, gonad, and eye. While the kissr2 mRNA was abundantly expressed in brain and eye, but barely in gonad.Expression patterns of turbot kiss/kissr mRNAs in central brain areas, including olfactory bulbs (Ob), telencephalon (Te) with preoptic area (POA), mesencephalon (Me) with thalamus, hypothalamus (Hy) with saccus vasculosus, cerebellum (Ce), medulla oblongata (Mo), and pituitary gland (Pi) were also analyzed  and hepatosomatic index  were calculated , mature stage (IV-V), and degeneration stage (VI), were analyzed    in turbot, and provided preliminary information on its roles throughout the seasonal reproductive period. To date, numerous studies have reported on the Kiss/Kissr system in vertebrates. In most mammals, there is only one kisspeptin gene, kiss1, except in the platypus which contains two forms, kiss1 and kiss2 , Nile tilapia (Oreochromis niloticus), and orange-spotted grouper (Epinephelus coioides)  , 60, 61.kiss and four kissr genes have been identified, indicating the Kiss/Kissr system has a high diversity in vertebrates (kiss genes (kiss1 and kiss2) and two kissr genes (kissr3 and kissr2) were isolated in turbot. However, previous studies in other Pleuronectiformes only found kiss2 and kissr2, including Japanese flounder  and ‘flatfish-like Psettodoidei’ (FLP) lineages by genome analyses, and it was confirmed a polyphyletic origin for these two lineages in recent research   , which skiss and kissr genes have been reported in many fish species , and Nile tilapia, among others  the expression of kissr2 mRNA was low in the advanced gonadal stages in males  at detection time. In the present study, Kiss1-10 and Kiss2-10 stimulated the expression of lhβ, fhsβ, and gthα in the pituitary of turbot, indicating that the Kiss/Kissr system could elevate GTH release in turbot, which is conserved among vertebrates  of the Kiss/Kissr system were present in this species, which was different from other Pleuronectiformes species. The expression levels of kiss and kissr in HPG during the reproductive stages and in the pituitary gland after administration of synthetic Kiss1-10 and Kiss2-10 suggest the positive involvement of the Kiss/Kissr system in controlling the seasonal reproductive cycle in turbot.In conclusion, the present study investigated the Kiss/Kissr system and the changes in the expression of The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Animal Research and Ethics Committees of Qingdao Agricultural University.CZ: Performed experiments, analyzed the data, wrote, and approved the manuscript; BW: Performed experiments, analyzed the data, edited and approved the manuscript; YL, CF, SX, WW, and QL: Performed experiments and reviewed the manuscript; JL: Designed research, edited and approved the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by National Natural Science Foundation of China , Advanced Talents Foundation of QAU (6631119055), Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (GML2019ZD0402), China Agriculture Research System (CARS-47).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.


African American (AA) women experience higher rates of maternal morbidity and mortality compared to US women of other racial/ ethnic groups. Cardiometabolic complications of pregnancy  are leading contributors to maternal morbidity and mortality. Marked changes in circulating lipids are known to accompany cardiometabolic complications of pregnancy. Serum concentrations of docosahexaenoic acid (DHA) have been shown to be inversely correlated with risk for preeclampsia. DHA is a biosynthetic precursor of a class of specialized pro-resolving mediators (SPMs), resolvins, that have anti-inflammatory properties and are also associated with hypertensive disorders of pregnancy. We employed targeted lipidomics to characterize the distribution of DHA-containing phospholipids and SPMs in maternal serum collected in early and late pregnancy  to identify key lipids that are dysregulated during pregnancy in AA women who develop cardiometabolic complications. We identified a lipid signature in early pregnancy serum samples of AA women that is predictive of cardiometabolic complications of pregnancy with 74% accuracy. These are Resolvin D1, Resolvin E1, 2-AG, PGE2-glyerol ester, and 36:6 PC. These findings suggest that there are blood-based markers detectable in early pregnancy that can potentially identify persons at risk and tailor clinical interventions. DHArtension ,8,9. DHArtension ; the Intrtension . Despitertension ,13. DHA rtension . Interesrtension , which irtension ,17,18.Gestational diabetes, gestational hypertension and preeclampsia are common cardiometabolic complications of pregnancy ,20,21 anb), interleukin-6 receptor (IL6R), interleukin-8 (IL8), macrophage inflammatory protein-1a (MIP1a), tissue inhibitor of metalloproteinases -1 (TIMP1), TNFa, and tumor necrosis factor receptor −1 (TNFR-I), whereas white women had significant increases in biomarkers associated with hematological factors and inflammation, including interleukin-1 receptor antagonist (IL1RA), interleukin-8 (IL8), TNFR-I, and vascular endothelial growth factor (VEGF). Combined, these findings suggest that the predominant pathways that result in preterm delivery and cardiometabolic complications of pregnancy vary significantly between African American and white women [Biomarker investigations that are associated with preterm delivery have yielded different biomarker patterns for African American compared with white women ,24,33,34te women . Intereste women ,37.In this study, we used targeted lipidomics to selectively target the subset of DHA-containing phospholipids and SPMs in the serum of pregnant African American women with and without cardiometabolic complications of pregnancy to determine if key DHA-derived lipids were altered over the course of pregnancy. Secondarily, we used a Receiver operating characteristic (ROC) curve to determine if the serum abundance of the identified lipids is predictive of cardiometabolic complications of pregnancy in early pregnancy.n = 7), gestational hypertension (n = 9), or preeclampsia (n = 11), for a total n = 27, while controls were those whose pregnancy resulted in a full-term birth (between 39–0/7 and 40–6/7 weeks of gestation) without cardiometabolic complications of pregnancy (n = 27).This nested case–control study was undertaken on a subset of pregnant women who were invited to participate in an on-going study cohort in the Emory University African American Vaginal, Oral, and Gut Microbiome in Pregnancy Cohort Study, which is described in detail elsewhere . BrieflyData collection occurred at the enrollment Visit 1 (between 8–14 weeks gestation) and Visit 2 (24–30 weeks gestation) via medical record abstraction completed post-delivery to capture the birth outcomes and complications that occurred over the course of the pregnancy. Measures collected as part of the larger cohort study that are relevant to this study include the following:Socio-demographic Survey based on maternal self-report and prenatal administrative record review was used to ascertain maternal age, years of education, marital status, health insurance status, and household income in relation to the federal poverty level. Relevant patient demographic information is de-identified and provided in Medical Chart Abstraction was completed by the research team using a standardized chart abstraction tool to ascertain for the following pre- and peri-natal conditions and birth outcomes: (1) Pre-pregnancy BMI, calculated from measured height at the first prenatal visit and patient report of prepregnancy weight and categorized according to accepted definitions ; (2) Gestational age at birth. All participants received early pregnancy dating by last menstrual period (LMP) and/or early ultrasound, given enrollment criteria. Gestational age at birth was determined from the delivery record using the best obstetrical estimate determined by comparing the date of delivery in relation to the estimated date of confinement established by the 8–14 week prenatal visit considering last menstrual period and/or ultrasound according to standard clinical criteria [Cardiometabolic complications of pregnancy. Women were considered to have gestational hypertension if they had new onset hypertension (defined as systolic blood pressure ≥ 140 mm Hg and/or diastolic blood pressure ≥ 90 mm Hg) at ≥20 weeks gestation in the absence of proteinuria or new signs of end-organ dysfunction (with blood pressure readings documented on at least two occasions at least four hours apart), and they were considered to have preeclampsia if they met the same blood pressure criteria along with proteinuria or new signs of end-organ dysfunction [criteria . Gestatifunction . Women wVenous blood draw was completed by a phlebotomist during the study visits. Serum aliquots from the venous blood were obtained and stored at −80 degrees until later analysis of serum lipid profiles.Chemical reagents and HPLC grade organic solvents were purchased from Sigma Aldrich . These include HPLC grade Methanol, Chloroform, Acetonitrile, Isopropanol, Butylated Hydroxytoluene, Ammonium Formate, and Formic Acid. Synthetic lipid standards 21:0–22:6 PC was purchased from Avanti Polar Lipids . Oxylipin standards 9,10-DIHOME, 20-HETE, 9-HETE, 8,9-DHET, 14,15-DHET, 5-HETE, 12-HETE, 11,12-DHET, 5,6-DHET, 13-KODE, 13-HpODE, 9-HpODE, 9-HODE, 9-HoTre, 13-HoTre, 14(15)-EET, 8(9)-EET, 11(12)-EET, 12(13)-EPOME, 13-HODE, Prostaglandin E2, Prostaglandin E2 Ethanolamide, Prostaglandin E2 Glycerol, ARA-Ethanolamide (AEA), 2-AG, Oxy-AEA, Oleamide, Docosanoyl Ethanolamide, Docosahexaneoyl Ethanolamide (DHEA), Linoleoyl Ethanolamide (LEA), dihomo-gamma Linolenoyl Ethanolamide, a-Linolenoyl Ethanolamide (ALEA), Resolvin D3, Resolvin D1, Resolvin D2, Resolvin E1, Thromboxane B2, Leukotriene B4, Prostaglandin F2a, 8-Isoprostane, Oleoyl Ethanolamide (OEA), Palmitoyl Ethanolamide (PEA), Stearoyl Ethanolamide (SEA) were purchased from Cayman Chemical . The targeted lipidomics workflow used for analysis is depicted in Patient serum samples were extracted according to a modified Bligh and Dyer lipid extraction protocol . BrieflyOxilipins and endocannabinoids were isolated from serum samples using an automated C18 solid phase extraction manifold . For this, 100 μL of patient samples was transferred to a deep well 96 well plate. To the sample, 300 μL of 20:80 methanol:water, 55 μL 1% BHT, and 80 μL of glacial acetic acid was added to adjust the pH to 3.0. Samples are centrifuged at 4000 rpm for 10 min at 4 °C to pellet any precipitated solids. The supernatant was transferred to a preconditioned C18 solid phase extraction (SPE) plate . The sample was then rinsed with 800 μL water, followed by 800 μL hexane. Oxylipins and endocannabinoids were then eluted with 400 μL methyl formate, dried under nitrogen gas, and subsequently reconstituted in 200 μL methanol for LC/MS analysis.To quantify DHA containing phospholipids, 20 μL of extracted lipids were injected onto a Thermo Acclaim C30 column  and lipid classes were separated by a 12 minute linear gradient using HPLC Grade water:acetonitrile  and isopropanol:acetonitrile , each with 0.1 mM Ammonium Formate and 0.1% Formic Acid added as solvent modifiers. To quantify oxylipins and endocannabinoids in patient serum samples, a total of 11 μL oxy/endo extract  was deposited onto an Accucore C18 column  and resolved on a 16 minute linear gradient using water as Solvent A and acetonitrile as Solvent B, both containing 10 mM ammonium acetate. For each experiment, the analytical column was heated to 50 °C in a temperature-controlled column chamber and 0.5 mL/min flow rate was used. All relevant gradient information used for chromatographic resolution is provided in m/z 327.4, corresponding to the molecular weight of DHA, was conducted to selectively target the subset of phospholipids that contain esterified DHA  with binary pump and autosampler coupled to a QTrap 5500  mass spectrometer was used to complete the lipidomics analyses. For identification of DHA containing phospholipids, the mass spectrometer was operated in the negative ion mode. Precursor ion scanning for fied DHA . The masTo detect oxylipins and endocannabinoids, the mass spectrometer was operated in the negative and positive ion modes, respectively. For each, a multiple reaction monitored (MRM) based method was used for detection of species. The table of identified species and their respective MRM transitions are reported in All instrumental parameters such as electrospray (ESI) voltage, ion spray gases (GS1 and GS2), declustering potential, and collision energy (CE) were optimized using synthetic standards and held consistent over the course of analysis. All instrumental parameters are listed in To quantify DHA containing phospholipids, an external calibration curve in the concentration range of 0.23 μM–4.23 μM was prepared and the equation of the linear trendline was used for quantification. Similarly, for oxylipin and endocannabinoid quantification, the area under the curve of each identified lipid is calibrated against an external calibration curve in the linear range of 0.1 nM–10 nM. The quantified lipids are then used to create a lipid profile and for statistical analysis.Identification of DHA containing lipids was done in using LipidView version 1.2 . Raw mass spectrometry data was uploaded into the database and peak areas were extrapolated and aligned. Parent and fragment ion data was searched against an extensive lipid database, containing over 25,000 lipid species in more than 50 lipid classes, to make putative lipid identifications. All ions with a signal-to-noise ratio above 5 were selected and, as an additional quality filter, only MS/MS confirmed lipid species are reported. All putatively identified lipids were manually confirmed by visual inspection of extracted data. Aligned peak areas were exported and processed using Excel, whereby a table of positively identified lipids, corresponding peak areas, and corrected concentrations was created. Statistical analysis of the resulting table was completed using open source statistics platform, MetaboAnalyst . This ann = 27 African American women with cardiometabolic complications of pregnancy to n = 27 AA women who delivered at term and did not have such complications; the latter is used as the control group for this study. Relevant patient metadata is provided in We began our analysis by first comparing patient metadata, such as age, body mass index (BMI), and gestational age of birth, of t-test, we identified statistically significant increases in the overall abundance of DHA in serum of both control and CMD patients, comparing Visit 1 to Visit 2  though also appears to be decreased in CMD serum compared to control patients.A volcano plot of lipid distribution in later pregnancy (Visit 2) demonstrates that the abundance of endocannabinoid 2-AG is significantly decreased in the serum of CMD patients compared to control patients . At VisiWe then sought to evaluate the use of differentially abundant lipids as biomarkers of cardiometabolic diseases of pregnancy by plotting the Receiver Operating Characteristic (ROC) and used the area under the curve (AUC) as the predictive measure . Figure p-value < 0.05) .A longitudinal analysis of the patient’s serum samples, comparing the lipidome of each patient at Visit 1 and Visit 2, is shown in  < 0.05) b. The ab of 0.07 b. A simiDocosahexaenoic acid (DHA) is an omega-3, long chain polyunsaturated fatty acid (LCPUFA) with 22 acyl carbons and 6 non-conjugated double bonds. It is basally synthesized from essential fatty acid α-linolenic acid along the Sprecher pathway . DHA is The link between DHA and pregnancy outcomes is well established. Generally, it is accepted that increasing plasma levels of DHA is beneficial to pregnancy and is correlated with positive birth outcomes ,12,13. HFor this study, AA women with gestational diabetes, gestational hypertension, or preeclampsia were grouped together for the cardiometabolic complications group. While each of these complications contributes to cardiovascular risk, the etiology varies. Gestational diabetes stems from dysregulation of insulin and altered fatty acid metabolism while hypertension and preeclampsia may arise from underlying kidney disease or changes in the renin–angiotensin or sympathetic nervous systems ,47,48. OThe participants of our study experience increases in DHA-containing phospholipids over the course of pregnancy, comparing Visit 1 to Visit 2 a. Howeven = 60 women in the study is unclear [To identify key lipids that are dysregulated in early pregnancy that are indicative of cardiometabolic diseases of pregnancy, we investigated the distribution of DHA phospholipids, oxylipins, and endocannabinoids in serum of pregnant AA women at Visit 1.  unclear ,53. In a unclear . In addi unclear . The rol unclear ,55.p-value 0.069). At Visit 2, concentrations of RvD1 and PGE2-Gly are no longer significantly altered in the plasma of CMD patients compared to controls. Lipid metabolism is variable in late pregnancy as the body prepares for delivery [The distribution of 2-AG is dysregulated in late pregnancy . Similardelivery ,61 and wTo evaluate the predictive ability of 2-AG, RvD1, and RvE1, we plotted ROC curves at Visit 1 and Visit 2 . Panel 8Combined, we present data demonstrating that pregnant AA women with cardiometabolic complications of pregnancy have increased levels of serum DHA phospholipids, driven by the abundance of 36:6 PC. Despite increased DHA phospholipids, CMD patients have decreased serum abundance of DHA derived pro-resolving lipid mediator, RvD1, as well as RvE1, 2-AG, and PGE2-Gly. This suggests that these patients have either decreased activity of phospholipase A2 isoforms or separate enzymes, e.g., epoxygenases, necessary for biosynthesis of SPMs. Alterations in PLA2 activity have been reported previously in pregnant women with preeclampsia and gestational diabetes, though the specific PLA2 isoform and mechanism requires further investigation ,63. We p


Glioblastoma multiforme (GBM) is an aggressive form of glioma characterized by poor survival rates. The main cause of the limited efficacy of the current treatments and tumor recurrence is associated with the infiltration of GBM cells into the surrounding brain tissue. Until recently, peripheral nerves were believed to play a passive role in tumorigenesis; however, over the last decade, pioneering studies have highlighted their involvement in cancer initiation and progression by releasing neurotransmitters (NTs). In this study, we hypothesized that dysregulated genes encoding for neurotransmitter receptors (NTRs) could have a different association with patient survival in GBM and low-grade glioma (LGG). We identified 10 prognostic NTR genes that are progressively downregulated across cancer grades and are negatively correlated with genes associated with immune response, inflammation, and brain cancer hallmarks in LGG but not in GBM. We believe our findings shed new light on the role of neurotransmitters and their interactions with inflammation and immune response in the malignant progression of human gliomas.DRD1, HTR1E, HTR3B, GABRA1, GABRA4, GABRB2, GABRG2, GRIN1, GRM7, and ADRA1B) predicted a positive prognosis in LGG and a negative prognosis in GBM. These genes were progressively downregulated across glioma grades and exhibited a strong negative correlation with genes associated with immune response, inflammasomes, and established cancer hallmarks genes in lower grade gliomas, suggesting a putative role in inhibiting cancer progression. This study might have implications for the development of novel therapeutics and preventive strategies that target regulatory networks associated with the link between the autonomic nervous system, cancer cells, and the tumor microenvironment. Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer. Neurotransmitters (NTs) have recently been linked with the uncontrolled proliferation of cancer cells, but the role of NTs in the progression of human gliomas is still largely unexplored. Here, we investigate the genes encoding for neurotransmitter receptors (NTRs) by analyzing public transcriptomic data from GBM and LGG (low-grade glioma) samples. Our results showed that 50 out of the 98 tested NTR genes were dysregulated in brain cancer tissue. Next, we identified and validated NTR-associated prognostic gene signatures for both LGG and GBM. A subset of 10 NTR genes ( The most prevalent intracranial primary tumors in adults are gliomas, which originate from glial support cells ,2. In thUntil recently, the role of the peripheral nerves in tumorigenesis has been understudied, as they were believed to play only a passive role . Over thThe complex interactions between cancer cells, NTs, and other matrisomal components are increasingly recognized . Variouswww.genenames.org, accessed on 21 January 2021) was used to identify the genes encoding for classical neurotransmitter receptors. Then, a differential gene expression (DEG) analysis between tumor and normal tissue was conducted for each of the selected TCGA malignancies. The web-based tool GEPIA2 was used . Specifically, the “Expression DIY” module was used. Each DE gene was inputted individually as “Gene” in GEPIA2, and all the TCGA cancer datasets available in the “Dataset selection” pane were added to the analysis. The |Log2FC| of 1 and the p-value cut-off of 0.01 were selected as the parameters. The option “Match TCGA normal and GTEx data” was also chosen. Therefore, TCGA cancer transcriptomics data were either matched with TCGA normal data or with data from the Genotype-Tissue Expression (GTEx) project, which is a public database containing tissue-specific gene expression and regulation data from 54 healthy tissue sites across more than 1000 individuals. The method for differential analysis is one-way ANOVA, using disease state  as the variable for calculating differential expression. Only the statistically significant genes were used for further analyses.The HUGO Gene Nomenclature Committee , where each TCGA tumor analyzed using GEPIA2 was accessed: (1) the TCGA pan-cancer (PANCAN) study was selected; (2) the “Genomic” data type was selected as the “first variable”; (3) the DE genes identified for each cancer type were inputted; and (4) the gene expression was selected. The normalized RNA-seq data were also downloaded as a spreadsheet file. Gene expression data accompanied by the survival data and clinicopathological annotation were downloaded from XENA and imported into Excel for further data curation. The selected data were then imported into R (version 4.0.2) and RStudio (version 1.3.1073) where: (1) an optimal cut-off score, defined as the gene expression level with the most significant split using a log-rank test, was selected to demonstrate the prognostic performance of each gene. The optimal cut-off was computed using the “maxstat.test” function (“maxstat” package [p ≤ 0.05. (4) The effect on the overall survival probabilities of each NTR gene was assessed using the Cox proportional-hazard model. The assumption of the model was evaluated using the scaled Schoenfeld residual test through the “cox.zph” function . (2) Pa package  was used package  in R/Biohttps://artyomovlab.wustl.edu/phantasus/, accessed on 25 January 2021), gene expression profiles were normalized, log2-transformed, collapsed, and exported into spreadsheet files for further analyses. The dataset was collected using the platform Illumina HumanHT-12 WG-DASL V4.0 R2 expression Beadchip, and the Affymetrix Human Genome U133A Array for the dataset GSE4412-GPL96.The NTR-associated LGG and GBM prognostic indexes were computed as  package ). These p = 0.01).The GO enrichment analysis of the 10 NTR genes that exhibited an opposite association with clinical outcomes in LGG and GBM was performed in Orange (Version 3.26). Significance was determined by a hypergeometric test  was used to perform the network analysis of the 10 NTR genes with opposite associations with clinical outcomes between LGG and GBM. Specifically, tissue-specific (whole brain) protein–protein interaction (PPI) analysis was selected.The NetworkAnalyst . A pairwise, two-sided, unpaired two-sample Wilcoxon test was performed among the “normal”, “LGG”, and “GBM” groups.t-tests between the two groups were computed.The proteins encoded by the 10-NTR genes were inputted into the University of Alabama at Birmingham cancer data analysis portal (UALCAN). The expression levels of these proteins were compared between the GBM and normal tissue. The proteomics datasets belong to the Clinical Proteomic Tumor Analysis Consortium (CPTAC). No LGG dataset is currently available in this database. Seven out of the ten inputted proteins were found. Box plots were then visualized where on the y axis the Z-values were shown. The Z-values were the standard deviations from the medians across the samples for each group. The spectral count ratio values from CPTAC were first log2-transformed, normalized within each sample profile, and then normalized across samples. Lastly, two-sided www.genenames.org, accessed on 21 January 2021), 98 genes  and LGG (N = 518) were matched with normal brain samples (N = 207).We found that 50 out of 98 genes were differentially expressed in brain cancer tissue. The results of the differential expression (DE) analysis are shown in p ≤ 0.05; (2) 0.05 < p ≤ 0.07; (3) non-significant (NS). The prognostic value of each of these genes in CHRNA7, GABRA3, GRIA3, and HTR5A; whereas the following genes had an HR > 1: ADRA1B, ADRA2A, ADRA2C, CHRM3, CHRM4, CHRNA5, CHRNB2, DRD1, GABRA1, GABRA2, GABRA4, GABRB2, GABRD, GABRG3, GRIN1, GRIN3A, GRM5, GRM7, HTR1E, and HTR3B. For LGG, the following genes had an HR < 1: ADRA1B, CHRM1, CHRNA2, DRD1, GABRA1, GABRA4, GABRA5, GABRB2, GABRG2, GRID2, GRIK4, GRIN1, GRIN2A, GRIN2B, GRM2, GRM3, GRM4, GRM7, HTR1E, HTR2A, HTR3B, and HTR5A; whereas the following genes had an HR > 1: CHRM3, CHRNB1, and GABRA2. Next, we sought to understand whether the differentially expressed NTR genes had an impact on the patients’ survival. Kaplan–Maier (KM) plots were generated for each DEG, for each malignancy, as shown in We next identified and validated NTR-associated gene signatures using independent datasets. First, we calculated the NTR-LGG prognostic index by multiplying the expression level by the hazard ratio of each gene and summing these values for all the prognostic NTR genes identified for LGG in HTR5A, HTR3B, HTR1E, GRM7, GRIN1, GABRG2, GABRB2, GABRA4, GABRA2, GABRA1, DRD1, CHRM3, and ADRA1B). Three of them  were associated with the same clinical outcomes in both cancers, whereas the remaining 10 genes  exhibited an opposite association with clinical outcomes. Specifically, these genes predicted positive prognosis in LGG and negative prognosis in GBM. We then explored how the expression levels of this 10-gene panel, which we defined as “10-NTR”, varied between normal tissues and across progressive WHO grades. For the former analysis, we used GEPIA2 to access the TCGA and GTEx datasets, whereas for the latter analysis we accessed both the TCGA and the CGGA (Chinese Glioma Genome Atlas) databases using GlioVis [N = 515 for TCGA and N = 625 for CGGA) to IV , as shown in  GlioVis  and sortp ≤ 0.01) involving the genes in the network in The top 10 enrichment scores in the gene ontology (GO) enrichment analysis of the 10-NTR genes are shown in HTR3B, GABRA4, GABRB2, GABRG2, GRM7, whereas DRD1, HTR1E, and GRIN1 had very low expression levels in this dataset.Furthermore, we performed gene expression analysis of the 10-NTR genes on a single-cell RNA-seq GBM dataset (GEO accession number GSE131928) from Neftel et al.  that wasCALR, CANX, CD4, CD74, CTSS, FCER1G, FCGRT, HLA-A, HLA-B, HLA-C, HLA-DMA, HLA-DOA, HLA-DPB1, HLA-DQA2, HLA-DRA, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-H, HSPA1B, HSPA5, HSP90AB1, KLRC2, NFYA, PSMB8, PSMC6, RFXAP, TAP1, TAPBC, KLRC4, IFI30, PROCR, ERAP1, PDIA2, and CXCL16. We tested the correlation between the 10-NTR genes and the IRGs, both in LGG and GBM. In Furthermore, 36 immune-related genes (IRGs) with prognostic values in LGG have been recently identified by Zhang et al. : CALR, CAIM2, CARD8, CASP1, CASP4, CASP5, CASP12, DDX3X, GSDMD, NLRC4, NLRP1, NLRP3, and PYCARD. A strong negative correlation was found between most of the genes composing these two panels in the case of LGG  dataset. Our results provide a landscape of the dysregulated genes encoding for NT receptors and their impact on brain cancer patients’ overall survival in low-grade glioma and glioblastoma multiforme.A differential expression of a subset of NTR genes was found when comparing cancer and normal brain tissues. Specifically, we found that the majority of the differentially expressed NTR genes were under-expressed in both LGG and GBM. This could be possibly explained by the downregulation of neurotransmitter receptors via receptor-ligand-induced desensitization , resultiDRD1, HTR1E, HTR3B, GABRA1, GABRA4, GABRB2, GABRG2, GRIN1, GRM7, and ADRA1B) were associated with favorable prognostics for LGG, but were unfavorable for GBM. This suggests that the direct or indirect involvement of these 10 NTR genes might exist in the progression from LGG to GBM. The decreasing expression levels of these genes across successive histology grades are presumably associated with a decreased density of nerve fiber infiltration in the tumor microenvironment moving from low to higher histology grades. Previous studies have shown that white matter fiber infiltration tends to be destroyed in higher-grade tumors [Of the NTR genes which had prognostic values for LGG and GBM, most of them belonged to the following classes: acetylcholine, glutamate, and GABA. Two 25-gene NTR-associated signatures allowed us to stratify brain cancer patients into low and high-risk groups within LGG and GBM, independently. For both cancer types, the prognostic genes strongly correlated with each other, suggesting that biological associations among neurotransmitters of different classes exist. A subset of 13 prognostic genes was found to be shared by LGG and GBM, 10 of which (e tumors ,49 compae tumors  found the tumors  and glioe tumors , suggeste tumors . Serotone tumors . Glutamae tumors . Dopamine tumors . Epinephe tumors ,58. TherPAK1 was positively correlated with all the 10-NTR genes. This gene was previously identified as an unfavorable prognostic marker in glioma and encodes for a protein kinase involved in cell adhesion, cell migration, proliferation, mitosis, cytoskeleton dynamics, apoptosis, and vesicle-mediated transport processes.Finally, we investigated the correlation between the 10-NTR genes and the brain cancer-specific hallmarks recently published by Nagy et al. . Most ofThis study is limited to the investigation of classical NT receptors, and did not include neuropeptides, which are released by nerves in both the central and peripheral nervous system and play an important role in mediating the communication between the nervous system and the tumor microenvironment . SpecifiTaken together, this study confirms that neurotransmitters and their receptors have an important role in tumor progression and survival—as observed in recent studies on the role of acetylcholine  and dopa


Giardia lamblia and Leishmania major, and histone-like proteins from the Marseilleviridae amoeba virus family. The presented data confirm the sharing of the overall DNA compaction system among evolutionally distant species and clarify the deviations from the species-specific nature of the nucleosome.In eukaryotes, genomic DNA is bound with histone proteins and packaged into chromatin. The nucleosome, a fundamental unit of chromatin, regulates the accessibility of DNA to enzymes involved in gene regulation. During the past few years, structural analyses of chromatin architectures have been limited to evolutionarily related organisms. The amino acid sequences of histone proteins are highly conserved from humans to yeasts, but are divergent in the deeply branching protozoan groups, including human parasites that are directly related to human health. Certain large DNA viruses, as well as archaeal organisms, contain distant homologs of eukaryotic histone proteins. The divergent sequences give rise to unique and distinct nucleosome architectures, although the fundamental principles of histone folding and DNA contact are highly conserved. In this article, we review the structures and biophysical properties of nucleosomes containing histones from the human parasites  In eukaryotes, highly negatively charged DNA binds to positively charged histone proteins, thereby packaging genomic DNA within the nucleus. The packing architecture, called chromatin, forms a fibrous “beads-on-a-string” structure, in which linker DNAs connect nucleosomes . In the Homo sapiens [Xenopus laevis [Gallus gallus [Drosophila melanogaster [Archaea, the nucleosome-like particle formed by the self-octamerization of one histone-like protein (HMfB) is similar to the eukaryotic nucleosome, although the archaeal chromatin adopts a unique polymeric architecture [To date, numerous nucleosome structures from various eukaryotic species, such as  sapiens , Xenopuss laevis ,9, Gallus gallus , Drosophnogaster , and yeanogaster ,13, havenogaster ,15,16. Titecture ,18,19.Giardia parasite, and a hybrid nucleosome structure containing a Leishmania parasite histone protein.In the phylogenic tree of eukaryotes, the evolutionary distance between yeasts and humans is shorter than that of the deeply branching protozoan group. Many previous studies of chromatin structures are limited to relatively evolutionarily close model organisms, and thus the chromatin organization of the protozoan group, including human parasites that are directly related to human health, remains unclear. Intriguingly, histones have also been identified among proteins secreted into host cells by multiple protozoan parasites ,21. HereArchaea, as the basic structures of the folded histones are shared in various species. In the nucleosome, the central ‘disc’ architecture is composed of four core histones, H2A, H2B, H3, and H4, sharing well-conserved motifs termed histone-fold domains comprising three α-helices, α1, α2, and α3, separated by two loops, L1 and L2 [2 tetramer in physiological ionic strength solutions by the interactions of the C-terminal halves of the α2 helices of H3 and H3′ (adjacent copy of H3) and the α3 helix of H3. A single H3–H4 tetramer and two H2A–H2B dimers are incorporated into the nucleosome core particle to form the histone octamer, around which the double-stranded DNA is wrapped  and the αC helix of H2B (Glu105 and Glu113 in human) form a negatively charged patch exposed to the solvent E. The acHistone proteins contain intrinsically disordered regions at the N-termini of H2A, H2B, H3, and H4, and the C-terminus of H2A A,E. ThesGiardia lamblia is a flagellated unicellular eukaryote that parasitizes the small intestines of humans and animals. Giardia causes a diarrheal disease termed giardiasis, which is one of the most common parasitic infections throughout the world [Giardia life cycle consists of two main forms: the non-infectious trophozoite that causes the primary symptoms of diarrhea by proliferating on the surfaces of the host intestinal cells, and the highly infective cyst form, which is environmentally resistant and can survive outside the host. This parasite is classified in the order diplomonadia within the Excavata supergroup. In the eukaryotic tree of life, Giardia is in a deeply branched position, with a simplification of most cellular processes, and lacking certain organelles, such as mitochondria and peroxisomes [Giardia has two nuclei with equivalent activities. Consistent with higher eukaryotes, genomic regulation occurs on the “beads-on-a-string” chromatin structure [Giardia genome contain 4963 open reading flames with few introns, short intergenic and 3′ and 5′ untranslated regions, and small promoter regions that are close to transcriptional start sites [Giardia has a simple transcriptional initiation complex [Giardia [Giardia possesses an RNA interference system, which is involved in antigenic switching, conferring a pathogenic ability to evade the host immune system [Giardia. In the encystation process, the transformation from trophozoite to cyst stage, histone modifications such as ubiquitylation, deacetylation, and methylation have been verified, indicating that the chromatin-based regulation of gene expression is essential for biological processes in Giardia [he world ,40. The oxisomes . Giardiatructure . The 12 rt sites ,44,45. G complex , with fe complex  and chro complex . A genom[Giardia . In addi Giardia ,52,53,54Giardia genome encodes two copies of the H2A, H2B, and H3 genes, and three copies of the H4 genes for canonical histones, and their corresponding mRNAs are polyadenylated [Giardia, the “Ser-Gln-Asp-Leu” motif, within the H2A.X variant in higher eukaryotes, is present in the H2A C-terminus. The four core histones share the typical eukaryotic features of histone-fold domains, containing three α helices separated by two loops [Giardia lamblia (G. lamblia) histone-fold domains of H2A, H2B, H3, and H4 compared to the human histone-fold domains are 48, 49, 60, and 78%, respectively. As in other eukaryotes, the Giardia core histones contain intrinsically disordered “tail” regions that undergo conserved post-translational modifications. Emery-Corbin and colleagues identified Giardia histone modifications, including methylation, acetylation, and phosphorylation, using mass spectrometry [Giardia histone tails are highly modified (more than 50 sites were identified) and that these histone modifications in Giardia are largely equivalent to those in many other eukaryotes, suggesting that similar epigenetic mechanisms exist in this parasite [Giardia.The enylated ,55. Histenylated . In highenylated ,36,57. Awo loops ,59. IndeG. lamblia nucleosome was determined at a 3.6 angstrom resolution, after reconstitution with 145 base pairs of modified Widom 601 (601L) palindromic DNA [G. lamblia nucleosome resembles that of the human nucleosome , which mimics LmaH3 released in infected cells, is incorporated into human chromatin in HeLa cells [Many proteomic studies have demonstrated the release of virulence factors by various protozoan parasites into the host cell environment, suggesting that these proteins could play an important role for parasite survival in the host ,89,90,91echanism ,94,95,96ost cell ,82,83,84La cells . In addiLa cells . This isLeishmania H3, the amino acid residues Trp35 and Gln57 replace the respective human H3 Tyr41 and Arg63 residues, which interact with DNA  with a 1.2 mega base pair DNA genome encoding approximately 1200 open reading frames [Marseilleviridae, including Melbournevirus and Marseillevirus, is an established family among the large amoebal NCLDVs, which have icosahedral shapes with a diameter of ~250 nm, and double-stranded circular DNA genomes that are more than 300 kilo base pairs in length [Marseilleviridae genomes [Marseilleviridae histone proteins, Hα, Hβ, Hγ, and Hδ, correspond to H2A, H2B, H3, and H4, respectively, based on the homology of their histone-fold domains. The N-termini of Hα and Hγ fuse with the C-termini of Hβ and Hδ, respectively, through a connector, and these Hβ–Hα and Hδ–Hγ fusions are described as histone doublets  in vitro. Velencia-Sanchez and Abini-Agbomson et al. determined the cryo-EM structure of the nucleosome containing histone doublets of  601 DNA . Liu et  601 DNA . These s 601 DNA . In Melbresidues . The virresidues . Additiocleosome . These sMarseillevirus nucleosome [Melbournevirus nucleosome [In viral nucleosome structures, approximately ~120 base pairs of DNA are wrapped around the viral histones, although the main principles of histone–DNA interactions are conserved. In eukaryotes, the H3 αN helix guided by the H2A C-terminal domain contributes toward stabilizing the last turn of the 147 base pair superhelical DNA organized in the nucleosome C. The Hγcleosome   and H2B (Glu105 and Glu113). In viral nucleosomes, six residues in Hα  and Hβ (Asp84 and Glu92) form the acidic patch, resulting in size and charge reductions, compared to eukaryotic canonical nucleosomes E. The acMelbournevirus histone doublets accumulated in the host amoeba cytoplasm after infection, indicating that viral histone doublets are recruited for viral DNA packaging and virion production in the viral factory [Marseillevirus DNA in virions [Marseillevirus virions, the viral genome was digested with micrococcal nuclease, which predominantly produced 121 base pair DNA fragments [Liu et al. detected the localization of the virus histone doublets in host cells. Transfected GFP-fused  factory . Further factory . Their r virions . They reragments . Based oragments ,109. Theragments . These fragments . Interesragments ,114,115.G. lamblia parasite and histone-like proteins from Marseilleviridae of the large DNA virus family, both with highly divergent histone sequences. As in the ancestral histone-like proteins of HMfB from Archaea, and even viruses, the overall structures are well-conserved in the nucleosome or nucleosome-like particles, in which the DNA is left-handedly wrapped around handshake motifs with predominantly conserved histone–DNA contact sites. It would be quite interesting to determine whether the high degree of histone sequence divergence influences the complex relationship between chromatin structure and gene regulatory mechanisms. In addition, expanding the knowledge of the chromatin structures of parasite species will help fill the knowledge gaps in the chromatin characteristics of related parasites and also reveal new targets for future drug development strategies. A novel function has been proposed for the secreted L. major histone H3, which may potentially modify the host chromatin structure by infiltrating the host nucleosome. The secreted histone incorporation may cause aberrations of host chromatin homeostasis and thus change the host cell status to one suitable for parasite life. Therefore, it is exciting to imagine that several chromatin proteins released by Leishmania, or other parasites, could also serve as virulence factors by disrupting the host’s epigenetic system, thus actively participating in the infection mechanism. Further studies to clarify the unknown functions of these chromatin proteins to better understand parasite biology are awaited.Over the past three decades, many eukaryotic nucleosome structures have been revealed at atomic resolutions, including novel structures of nucleosomes containing histones from the


Richter’s syndrome represents the progression of chronic lymphocytic leukemia (CLL) to more aggressive diseases, most frequently diffuse large B-cell lymphoma, while Hodgkin’s lymphoma (HL) and hairy cell leukemia (HCL) are rarely described. The first case involved a 67-year-old man with a diagnosis of a high-risk stage-II CLL treated with rituximab and ibrutinib, developed a HL nodular sclerosis variant after three months of therapy for CLL. After achieving a complete remission for HL and ibrutinib cessation because of drug-related cardiotoxicity, the patient relapsed after five months off-therapy and died due to disease progression after two cycles of brentuximab-vedotin. The second case involved an 83-year-old female with a diagnosis of stage-IV CLL treated with rituximab plus bendamustine who developed a HCL eight years later. Pentostatin was unsuccessfully employed as upfront HCL therapy, and the patient was then switched to rituximab while in remission for CLL. In conclusion, Richter’s transformation risk rate might be higher in patients treated with novel targeted therapies, and multiparametric flow cytometry and lymph node biopsy at relapse could help in early identifying small clones. The treatment of predominant neoplasia is mandatory, and disease-specific drugs are administered; however, clinical efficacy might be lower in these patients. B-cell chronic lymphocytic leukemia (CLL), a clonal malignant hematological disease of mature B lymphocytes, is the most frequent leukemia in adults and the elderly in Western Countries and is characterized by various biological features and clinical outcomes, mostly with indolent courses, with 5-year overall survivals ranging from 23% to 93% ,2,3,4,5.+CD20+CD5+CD23+CD200+ by flow cytometry analysis, and he had TP53 mutation and unmutated immunoglobulin heavy chain (IGHV) status, identifying a high-risk disease. CT scan imaging showed multiple bilateral lymphadenopathies in the lateral cervical  area, paratracheal and abdominal region, and splenomegaly , with a radiotracer uptake of 4.9 at Positron Emission Tomography (PET) scan. The patient was monitored until July 2020, when a PET/CT scan re-evaluation was performed showing an increase in lymph node and spleen size and in radiotracer uptake. Therefore, therapy with rituximab, an anti-CD20 monoclonal antibody, and ibrutinib, a Bruton’s tyrosine kinase (BTK) inhibitor, was commenced. After three months of treatment, no improvements were observed, and PET/CT scan re-evaluation displayed a further increase in lymph node size and radiotracer uptake . A lateral cervical lymph node biopsy was performed showing an altered tissue architecture characterized by an inflammatory infiltrate predominantly composed of CD3+ T lymphocytes, and rare PAX5+CD20-CD23- B cells, eosinophils, and plasma cells. Large mono-/bi-nucleated Hodgkin and Reed-Sternberg-like cells were identified, and they were positive for CD30, PAX5, IRF4/MUM1, and BCL6, with increased PD-L1 expression but negative for CD20, CD10, CD3, and CD23 (A 67-year-old man arrived at our observation for absolute lymphocytosis (4850 cells/µL) and lateral cervical lymphadenopathy without any other symptoms in December 2019, and a diagnosis of CLL stage II was made according to 2016 World Health Organization criteria and the RAI staging system ,25. A miand CD23 . A histoThe R-CHOP regimen  was initiated, and ibrutinib administration was continued. After six cycles of therapy, the patient achieved a complete remission (CR), and radiotherapy was performed as consolidation. Ibrutinib was stopped in July 2021, after one year of treatment because of cardiotoxicity requiring hospitalization for heart failure, as reported as BTK inhibitor-related adverse event . After f+CD20+CD23+SmIg κ+ by flow cytometry. The patient had grade I normocytic anemia  and mild thrombocytopenia  without lymphocytosis . Spleen enlargement  was detected by ultrasound with a radiotracer uptake of 4.7 by PET scan. Moreover, several lymph node enlargements were observed by CT scan: diaphragmatic (12 × 8 mm); hepatic hilum (23 × 12 mm); celiac; pericaval; lumbar aortic; mesenteric; and parasternal . The patient was diagnosis with CLL stage-IV disease, and rituximab and bendamustine were started for a total of six cycles achieving a CR.An 83-year-old female with a history of arterial hypertension, atrial fibrillation, and Hashimoto’s disease diagnosed in 1966 and treated with tapazole for 38 years arrived at our observation in 2014 with a diagnosis of CLL made in another Institution. Flow cytometry immunophenotyping on peripheral blood at diagnosis identified a CLL clone positive for CD5, CD20, CD23, and SmIg κ , and negative for CD38, CD200, CD10, and CD103. Fluorescence in situ hybridization (FISH) analysis did not detect any chromosomal abnormalities. BM aspirate showed the presence of 29% of small size lymphocytes with compact chromatin by light microscopy and CD52); however, no pathological lymph nodes were found, and the patient remained under observation. In September 2019, five years from diagnosis, pathological CD19+CD5+CD23+SmIg κ+ lymphocytes were observed by flow cytometry accounting for 18% of total circulating cells. Blood counts were normal, and no signs of lymphadenopathies were present. In February 2022, because of worsening blood counts and increased spleen size , a PET/CT scan re-evaluation was carried out that confirmed splenomegaly  with increased radiotracer uptake . No pathological lymph nodes were detected. Peripheral blood flow cytometry immunophenotyping displayed a total of 58% lymphocytes, composed as follows: 17% of total CD3+ T cells; 6% of CD4+ and 10% of CD8+ cells with an abnormal ratio of 0.6; 18% of CD7+; 17% of CD5+; and 70% of CD19+ without any CD5+CD19+ B cells. A more detailed immunophenotyping was carried out on B cells, showing negativity for CD10, CD5, CD23, CD43, and CD38, and positivity for CD20, CD22, FMC7, CD103, CD11c, CD25, CD49d, CD200, and SmIg κ , and Richter Scoring System values could identify high-risk CLL patients with HL transformation [TP53 mutation and unmutated IGHV status, identifying a high-risk disease. Therefore, our patient showed unfavorable prognostic markers for both CLL and HL.HL arising from CLL is documented in less than 0.4% of all cases, especially in older males presenting with systemic symptoms, lymphadenopathies, and liver and spleen enlargements . CLL proormation . Our HL-+CD5+ T cells and fewer CD20-CD23-PAX5+ B cells, eosinophils, and plasma cells.Two different post-CLL HL variants can be observed: type I is characterized by few Hodgkin and Reed-Sternberg (HRS) cells within a CLL background, suggesting the neoplastic transformation of a CLL clone into a HRS cell, especially when these latter express B-cell surface markers ,14,22. IDifferent pathogenetic hypotheses have been proposed for Richter’s transformation to HL, including a chronic Epstein–Barr virus infection that might induce a prolonged immunosuppression, thus favoring CLL progression to a high-grade lymphoma ,10,31. ITo date, there are no current guidelines for the treatment of HL arising from CLL, even though high-dose chemotherapies are usually employed in fitting patients. The most commonly used chemotherapy regimens are doxorubicin (Adriamycin), bleomycin, vinblastine, and dacarbazine association; cyclophosphamide, vinblastine, procarbazine, and prednisone chemotherapy; or other cytotoxic drugs in combination with rituximab, an anti-CD20 monoclonal antibody . HoweverBRAF gene [HCL is an infrequent indolent lymphoproliferative disorder with an 8-year overall survival of 85%, and its coexistence with CLL is even rarer and anecdotally reported ,22,23,37RAF gene  and freqRAF gene . Second RAF gene ,46,47,48RAF gene ,49.The clinical significance of these different manifestations remains unclear as patients show similar outcomes . In our In conclusion, Richter’s transformation is a rare event and might be determined by different pathogenetic mechanisms, such as TP53 pathway alterations in HL post-CLL or different unknown causes in other variants . However, the clinical guard should be always high, especially with patients treated with novel targeted therapies for which Richter’s transformation risk rate is still unclear. Multiparametric flow cytometry and immunohistochemistry on lymph node biopsy at relapse might help with the early identification of small clones; therefore, a complete wide antibody panel should always be performed and not be limited to minimal residual disease monitoring. The treatment of predominant neoplasia is mandatory, and disease-specific drugs are administered, such as ABVD regimen for HL-transformed CLL; however, the clinical efficacy of these drugs might be lower in patients with Richter’s syndrome. Therefore, larger studies or metanalysis are needed to outline clinical and therapeutic guidelines for the treatment of Richter’s disease.


CLEC5A is a member of the C-type lectin superfamily. It can activate macrophages and lead to a series of immune-inflammation reactions. Previous studies reveal the role of CLEC5A in infection and inflammation diseases.via GSCALite, cBioPortal, and TIMER 2.0 platforms or software. Single-cell sequencing analysis was performed for quantifying the tumor microenvironment of several types of cancers.We acquire and analyze data from The Cancer Genome Atlas (TCGA) database, Genotype-Tissue Expression (GTEx) database, and other comprehensive databases CLEC5A is differentially expressed in a few cancer types, of which overexpression accompanies low overall survival of patients. DNA methylation mainly negatively correlates with CLEC5A expression. Moreover, CLEC5A is positively related to immune infiltration, including macrophages, cancer-associated fibroblasts (CAFs), and regulatory T cells (Tregs). Immune checkpoint genes are significantly associated with CLEC5A expression in diverse cancers. In addition, CLEC5A expression correlates with mismatch repair (MMR) in several cancers. Tumor mutation burden (TMB), microsatellite instability (MSI), and neoantigens show a positive association with CLEC5A expression in several cancers. Furthermore, CLEC5A in cancer correlates with signal transduction, the immune system, EMT, and apoptosis process. The drug sensitivity analysis screens out potential therapeutic agents associated with CLEC5A expression, including FR-180204, Tivozanib, OSI-930, Linifanib, AC220, VNLG/124, Bexarotene, omacetaxine mepesuccinate, narciclasine, leptomycin B, PHA-793887, LRRK2-IN-1, and CR-1-31B.CLEC5A overexpresses in multiple cancers in contrast to normal tissues, and high CLEC5A expression predicts poor prognosis of patients and immune infiltration. CLEC5A is a potential prognostic biomarker of diverse cancers and a target for anti-tumor therapy. Cancer has the second mortality rate worldwide, and immunotherapy is one of the most promising cancer therapeutic strategies. Despite significant advances and successful application of immunotherapy across a wide range of human cancers, most patients had limited or no response to these therapies. Previous research indicated tumor mutation burden (TMB), the proportion of immune cell infiltration, and PD-L1/PD-L1 expression are widely recognized indicators for cancer immunotherapy response . HoweverHuman C‐type lectin domain family 5 member A (CLEC5A), also known as myeloid DAP12-associating lectin-1 (MDL-1), is a II-type transmembrane protein and only expresses on the myeloid lineages including macrophages, monocytes, neutrophiles, and dendritic cells (DCs) . As a paCLEC5A silencing or knockdown results in decreased cell growth, colony-formation capability, and tumor volume and weight, which is consistent with the findings in GBM  database and genotype-tissue expression . To make gene expression data more comparable between samples, fragments per kilobase million (FPKM) values were converted to transcript per kilobase million (TPM) values, normalized by Log2 conversion. The expression difference of CLEC5A in cancer and normal tissues was visualized.The gene expression data of CLEC5A expression in cancer cell lines were acquired from the Cancer Cell Line Encyclopedia (CCLE) platform (https://sites.broadinstitute.org/ccle/tools)  website (The gene expression profile data of e/tools) . The CLE website , 24.CLEC5A expression and patient survival in different cancers . A single-cell sequencing dataset of LUAD was downloaded from the NCBI BioProject (PRJNA591860), and a single-cell sequencing dataset of PAAD, CRA001160, was downloaded from the Genome Sequence Archive (GSA) database (https://ngdc.cncb.ac.cn/gsa/browse/CRA001160). The detailed information on these GEO datasets and R packages for data integration and quality control are shown in CLEC5A expression.Single-cell sequencing datasets of BLCA, BRCA, CHOL, COAD, GBM, HNSCC, KIRC, LIHC, OV, SKCM, and STAD were acquired from the Gene Expression Omnibus (GEO) database (http://bioinfo.life.hust.edu.cn/web/GSCALite/) was used to analyze differential methylation of CLEC5A in tumors and normal tissues across various TCGA cancer types   and mutation landscape of tal.org) , 28. Thuhttp://timer.cistrome.org/) was utilized to analyze the relationship between CLEC5A expression and tumor-infiltration immune cells in tumors across various cancer types , and cancer-associated fibroblast (CAF). A P-value less than 0.05 indicated a significant correlation. We evaluated the relationship between CLEC5A gene expression and immune checkpoint (ICP), TMB, microsatellite instability (MSI), and neoantigens using the Pearson correlation analysis. Pearson correlation analysis was also implemented to evaluate the correlation between CLEC5A gene expression and MMR genes  and DNA methyltransferases .TIMER2.0 (er types . Tumor phttps://string-db.org/). The OPEN TARGET platform  was used to identify CLEC5A related diseases network  and the ethics was approved. The primary antibodies were CLEC5A , CD68 , CD163 , CD8 . The secondary antibody was horseradish peroxidase-conjugated secondary antibody incubation , and the tyramide signal amplification was TSA . Multispectral images were analyzed, and positive cells were quantified at single-cell levels by Caseviewer  and Pannoramic viewer (PV 1.15.3) image analysis software.CLEC5A expression levels were compared across tumor cell lines between pre- and post-cytokine treated samples and between pre- and post-ICB treated samples. The differences between groups were statistically evaluated by Wald test using DESeq2 , and the comparison results were summarized in boxplots. Tumor models included were: mammary cancer: 4T1, E0771, EMT6, T11, KPB25L, p53-2225L, p53-2336R; colorectal carcinoma: CT26, MC38; gastric adenocarcinoma: YTN16; head and neck squamous cell carcinoma: MOC22; hepatocellular carcinoma: BNL-MEA; lung carcinoma: LLC; melanoma: B16, YUMM1.7, D3UV2, D4M.3A.3; sarcoma: 402230.via GSCALite platform to explore the correlation between drug sensitivity and CLEC5A expression. Spearman correlation analysis was used to evaluate the correlation. Positive correlation indicated that tumor cells with high CLEC5A expression are prone to drug resistance, while negative correlation suggested that tumor cells with high CLEC5A expression are sensitive to drugs.GSCALite integrates over 750 small molecule drugs from the Genomics of Drug Sensitivity in Cancer (GDSC)/Therapeutics Response Portal (CTRP). We conducted the drug sensitivity analysis P-value ≤ 0.05 indicated statistically significant.The statistical significance of differences between groups was analyzed by Student’s t test, and the comparison among groups was analyzed by one-way ANOVA. CLEC5A expression in tumors and paired normal tissues in different cancers with TCGA and GTEx gene expression data. The results showed that CLEC5A mRNA expression was significantly up-regulated in 23 tumors , BRCA (breast invasive carcinoma), CESC , CHOL (cholangiocarcinoma), COAD (colon adenocarcinoma), ESCA , GBM (glioblastoma), HNSC (head and neck squamous cell cancer), KIRC , KIRP , LAML (acute myeloid leukemia), LGG (lower grade glioma), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), OV (ovarian serous cystadenocarcinoma), PAAD (pancreatic adenocarcinoma), READ (rectum adenocarcinoma), SKCM (skin cutaneous melanoma), STAD (stomach adenocarcinoma), TGCT (testicular germ cell tumors), THCA (thyroid carcinoma), UCEC , and UCS (uterine carcinosarcoma)) and down-regulated in 4 tumors   were identified  between tumors and normal tissues in various cancers were evaluated with the GSCALite platform. The results indicated that CLEC5A methylation was significantly down-regulated in UCEC, LIHC, KIRC, KIRP , LUAD, HNSC, BRCA, BLCA, and PRAD (prostate adenocarcinoma)  in pan-cancer. The results exhibited that the CLEC5A mRNA expression was mainly negatively correlated with its DNA methylation in most tumors but only positively correlated with its DNA methylation in only some tumors, such as SKCM (cutaneous skin melanoma), TGCT (testicular germ cell tumors), LIHC and HNSC  and T cells biomarker genes  also mainly showed a negative correlation with their DNA methylation  frequency in 32 TCGA cancers types. The results showed that the alteration frequency of CLEC5A varies with tumor type. As shown in CLEC5A was found to have a high alteration frequency of nearly 8% in both SKCM and OV, but no related alteration was found in the other 10 cancer types . Moreover, in the four alteration types  of CLEC5A, copy-number alteration  frequency was the highest, followed by mutation and considerable alteration. Then, we explored the correlation between the putative CNA of CLEC5A and its gene expression in tumors. The putative CNA of CLEC5A in pan-cancer were shown in .To study the mutation landscape of CD68, MRC1, CD8A, CD8B, CD163, and CLEC5A) was more frequent in various cancers  module of the GSCALite platform provided heterozygous and homozygous CNV profiles in 33 cancer types. The pie chart showed that the heterozygous CNVs of all 6 genes  in various cancers. As the waterfall plots exhibited, the SNV frequency of CD163 was 68%, and that of CLEC5A was 12%, with missense mutation accounting for the majority  is a common gene mutation associated with gene expression or function. GSCALite was utilized to identify the SNV frequency of all 6 genes  play important roles in tumor infiltration and immunotherapy . To deteCLEC5A expression was positively correlated with the expression of MLH1, MSH2, MSH6, and PMS2 in KIRC, BRCA, LGG, LIHC, PADD, and PRAD, and negatively correlated with EPCAM in CESC, COAD, KIRC, LGG, LUAD, LUSC, PADD, and THCA  is essential to DNA replication and genetic recombination . MMR defCLEC5A expression was significantly correlated with the expression of DNMT signatures in more than 20 tumors. The result suggested CLEC5A may respond to oncogenesis by influencing DNA methylation.Numerous studies have found that abnormal DNA methylation catalyzed by DNMTs is involved in the occurrence and progression of various cancers , 38. We CLEC5A-related diseases network. The results showed that CLEC5A is highly associated with nervous system diseases, immune system diseases, infectious diseases, and musculoskeletal or musculoskeletal or connective tissue diseases  had a potential impact on pathway activity were also summarized in CLEC5A mediated activation of the EMT signaling pathway in 35% of cancers, while CLEC5A mediated inhibition of DNA damage response and hormone AR pathway in 18% and 25% of cancers, respectively.Then, GSCALite was utilized to investigate the impact of the gene set on tumor pathway activity. The pie chart showed that CLEC5A expression in tumor cells and their sensitivity to immunotherapy agents, CLEC5A expression levels across tumor cell lines were compared between pre- and post-cytokine-treated samples. As shown in CLEC5A expression in MOC2 cells, while IFNγ, IFNβ, TNFα, and TGFb1 treatment did not change substantially CLEC5A expression in 4T1, B16, CT26, E0771, EMT6, KPC, LLC, MC38, MOC1, MOC22, Panc02, and Renca cells. We then compared the CLEC5A expression level across different tumor cell lines between pre- and post-ICB treatment and responders and non-responders. CLEC5A expression was significantly increased in B16 cells and T11 cells that responded to anti-CTLA4 and anti-PD-1 treatment. In addition, CLEC5A expression was increased in MOC22 cells that responded to anti-PD-1 therapy, and CLEC5A expression was increased in EMT6 that responded to anti-PDL-1 treatment. In contrast, CLEC5A expression was significantly decreased in CT26 cells that responded to anti-PD-1 therapy , CD8 score (AUC > 0.5 in 18 immunotherapy cohorts), IFNG score (AUC > 0.5 in 17 immunotherapy cohorts), and Merck 18 score (AUC > 0.5 in 18 immunotherapy cohorts). Above all, CLEC5A expression can function as an effective biomarker for predicting immunotherapy response.Next, we evaluated the correlation between CLEC5A in pan-cancer, we confirmed that CLEC5A might play an essential role in tumorigenesis and tumor immunity, suggesting its enormous potential as a tumor therapeutic target. To identify potential therapeutic agents associated with CLEC5A expression, we conducted a Spearman correlation analysis between CLEC5A expression and drug sensitivity (IC50) via the GSCALite website. The results indicated that CLEC5A expression was negatively correlated with IC50 of small molecule drugs such as FR-180204, Tivozanib, OSII-930, Linifanib, AC220, VNLG/124, Bexarotene, Omacetaxine mepesuccinate, narciclasine, leptomycin B, PHA-793887, LRRK2-IN-1, and CR-1-31B, but positively associated with IC50 of JW-55 , and CLEC5A high expression appears in diverse diseases, especially tumors . Both genetic mutation and epigenetic modification can induce abnormal gene expression during tumorigenesis. In this study, CLEC5A gene alteration occurs in most cancer types, predominately amplification, followed by mutation. CLEC5A expression positively correlates with CNV in LGG but negatively correlates with CNV in PAAD and COAD. Besides, we found that the SNV frequency of CLEC5A reaches 12%, of which missense mutations were the most common, mainly occurring in SKCM, COAD, UCEC, LUAD and LUSC. Moreover, our results found that high CLEC5A expression indicated poor OS and DFS in patients with THCA, LGG, PAAD, GBM, CESC, LICH, KIRC, and OV but predicted better OS and RFS in patients with SKCM, UCS, and KIRP.Previous studies indicated that CLEC5A is overexpressed in some cancers and involves tumorigenesis and progression –20. ConsCLEC5A expression was correlated with five MMR genes expression in pan-cancer, such as KIRC, BRCA, CESC, COAD, LGG, LIHC, LUAD, LUSC, PADD, PRAD, and THCA. Besides, TMB, MSI, and neoantigens all reflect the mutation level of organisms contributing to cancer initiation. They are also considered predictors of immunotherapy. We found that in specific cancer types, the CLEC5A expression also significantly correlated with TMB, MSI, and neoantigens. Abnormal DNA methylation is a common epigenetic feature of cancer and is associated with the development and progression of the tumor , an essential part of TME, intimately correlates with tumor immunity accounting for tumor progression –48. Immual score . The preIt is no doubt that CLEC5A participates in tumorigenesis; the underlying mechanism remains unclear. We implemented the functional enrichment analysis to identify the biological roles of CLEC5A in pan-cancer. The results showed that in addition to activation of EMT and apoptosis and the inhibition of DNA damage response, CLEC5A high expression was mainly related to leukocyte trans-endothelial migration, inflammatory response, and two signaling pathways . It implicated that CLEC5A is related to tumor initiation and metastasis. Previous studies on dengue virus infection have revealed the role of CLEC5A in disease development and the efficacy of CLEC5A silencing or inhibition in disease treatment , 59. WheEven though the pan-cancer analysis covers comprehensive information, this study is still limited. Most results were based on data analysis, and more experimental verification is essential to provide solid evidence. Previous studies have shown that anti-CLEC5A mAb can be applied to treat viral infection with good clinical efficacy. However, whether it can be used as a tumor treatment drug remains to be studied further.In summary, we proved that CLEC5A expression is associated with immune infiltration and affects immunotherapy sensitivity patient’s prognosis in pan-cancer, suggesting that CLEC5A can serve as a biomarker for tumor immunity and prognosis of a potential promising anti-tumor therapeutic target.The original contributions presented in the study are included in the article/WW performed the data analysis and drafted the figures. S-YC wrote the manuscript. ZZL revised the manuscript. RC, QC, and W-JZ conceived and designed the study. Z-PW, ZL, JZ, PL, JY, and LZ supervised the study. All authors approved the submitted version of the manuscript.This study was supported by the National Natural Science Foundation of China  the Hunan Provincial Natural Science Foundation of China , and the Hunan Provincial Health Committee Foundation of China (No. 202204044869).This work was supported in part by the High Performance Computing Center of Central South University.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.


The time of survival in patients with amyotrophic lateral sclerosis (ALS) varies greatly, and the genetic factors that contribute to the survival of ALS are not well studied. There is a lack of a comprehensive study to elucidate the role of genetic factors in the survival of ALS.The published studies were systematically searched and obtained from PubMed, EMBASE, and the Cochrane Library without any language restrictions from inception to Oct 27, 2021. A network meta-analysis for ALS causative/risk genes and a systematic review and pairwise meta-analysis for other genetic modifiers were conducted. The PROSPERO registration number: CRD42022311646.ATXN2 (HR: 3.6), C9orf72 (HR: 1.6), and FUS (HR:1.8), were associated with short survival of ALS, but such association was not identified in SOD1, TARDBP, TBK1, NEK1, UBQLN2, and CCNF. In addition, UNC13A rs12608932 CC genotype and ZNF521B rs2275294 C allele also caused a shorter survival of ALS; however, APOE ε4 allele and KIFAP3 rs1541160 did not be found to have any effect on the survival of ALS.A total of 29,764 potentially relevant references were identified, and 71 papers were eligible for analysis based on pre-decided criteria, including 35 articles in network meta-analysis for 9 ALS causative/risk genes, 17 articles in pairwise meta-analysis for four genetic modifiers, and 19 articles described in the systematic review. Variants in three genes, including Our study summarized and contrasted evidence for prognostic genetic factors in ALS and would help to understand ALS pathogenesis and guide clinical trials and drug development.The online version contains supplementary material available at 10.1186/s12916-022-02411-3. Amyotrophic lateral sclerosis (ALS) is one of the most devastating neurodegenerative diseases, characterized by degeneration of the upper and lower motor neurons. It eventually results in progressive muscle atrophy and death in 3–5 years after disease onset . About 5UNC13A rs12608932 and CAMTA1 rs2412208 [C9orf72, SOD1, FUS, TARDBP) might modify the disease course as well [As we all know, aging, environmental and genetic factors play an essential role in the development of ALS. However, as a rare disease, we still don’t have an excellent strategy for preventing it from developing due to the limited knowledge of its etiology. Hence, more attention has been paid to the associated factors that affect the survival time. In our recent study, twenty-five non-genetic factors associated with ALS survival were identified, such as age at onset, onset site, the time between onset and diagnosis, et al. . HoweverDifferent genetic variants are considered as different interventions in this study, so we employed a NMA following the International Society for Pharmacoepidemiology and Outcomes Research (ISPOR) guidelines  and repoThe published studies were systematically searched and obtained from PubMed, EMBASE, and the Cochrane Library without any language restrictions, by using the term: “(gene* OR geno* OR variant* OR mutation OR haplotyp* OR polymorphism* OR SNP OR Allel*) AND  AND  OR (motor neuron disease) OR (Lou Gehrig’s disease) OR (Gehrig Disease))”. Reference lists of full review articles were also reviewed to search for additional articles. All randomized clinical trials (RCTs), quasi-RCTs, and observational studies were eligible, but no RCTs were identified. The articles were updated to October 27, 2021.According to previous survival research in ALS, survival was defined as the time between the onset of symptoms and noninvasive ventilation (NIV) for more than 23 hours per day, or tracheostomy or death , 17, 18.First author’s name, year of publication or online, patients’ nation, number of patients, type of disease, diagnosis criteria, age at onset and gender distribution of patients, median survival time, and HR with their 95% CI were extracted. When HR was unavailable directly from the articles, the Kaplan-Meier curve was evaluated by Engauge Digitizer version 12.1, and HRs and 95%CI were estimated using Richard Steven’s excel workbook . Data frSOD1 mutation) against the remaining groups.Only observational studies were included for analysis in this study, and their quality was appraised with the Newcastle-Ottawa Scale (NOS) . As for SMN2 deletion, SMN1 and SMN2 copy numbers, CX3CR1 V249I, CX3CR1 T280M, haplotype in CX3CR1, ABCC8 rs4148646, KCNJ11 rs5219, LXRs rs2279238, LXRs rs7120118, LXRs rs35463555, LXRs rs2695121, PRGN rs9897526, PRGN rs34424835, haplotype in PRGN, HTR2B rs10199752, STMN2 CA repeat, BDNF C270T, C7 gene rs3792646, PON1 rs854560, PON1 rs662, NIPA1 polyalanine repeat expansions, SLC11A2 rs407135, CAMTA1 rs2412208, GSTP1 rs1695, CNTF, HLA-DRA or HLA-DRB5 rs9268856, rs4623951, EPHA4 rs6436254, and IDE rs139550538). All computations were conducted on R (V4.1.1) package “gemtc,” “rjags,” and STATA/MP 16.0.HR was used for each dichotomous outcome, and traditional pairwise meta-analyses were performed for studies, which directly compared different groups. And from known ALS causative/risk genes variants, outcome data were pooled using NMA models through the R 4.1.1 software. A network relationship diagram was drawn, among which parameters such as each node and line thickness respectively represent a certain gene’s variants and the researches sizes were considered from the included studies. The model fit was assessed using three criteria based on the deviance and node-based residuals. We evaluated the inconsistency, which means the difference between the pooled direct and indirect evidence of a particular comparison, using inconsistency factors based on a modified back-calculation approach . In addi.We identified 29,764 potentially relevant references from PubMed, EMBASE, and Cochrane Library and three additional records from reference lists Fig. . Of thesC9orf72 was the most frequently investigated regimen with 40 comparisons . In addition, patients with other two genes variants, ATXN2 and FUS, presented a short survival time compared to those without known ALS causative genes variants . However, SOD1, TARDBP, TBK1, NEK1, UBQLN2, and CCNF did not affect the survival of ALS. The detailed features for each gene are shown in Table C9orf72 repeats expansion, patients presented with SOD1 or TARDBP variants or without genetic variants seem to possess a better prognosis. However, no difference between C9orf72 repeats expansion and FUS variants and ATXN2 polyQ were identified. The detailed results are illustrated in Fig. A total of 35 articles including 37 trials with nine known ALS causative/risk genes were involved in this network meta-analysis, which is shown in Fig. ATXN2 polyQ repeats (≥31) had the largest probability of being the worst genetic variants . TBK1(70.0%), FUS (69.5%), CCNF (67.7%), and C9orf72 (63.4%) had a similar probability of being the worst. The result of pairwise comparison among these genes is shown in Fig. The rankogram plots are shown in Fig. repeats ≥1 had theC9orf72 expansion in patients with ALS compared to those without ALS-related mutation  and ZNF512B rs2275294 CC+CT (dominant model) could accelerate the death of ALS patients  did not show any modificatory effect on ALS survival  in ATXN2 have been known to be related to spinocerebellar ataxia type 2 (SCA2) [ATXN2 ployQ may render C9orf72 repeat expansion carriers more susceptible to ALS [ATXN2 ployQ expansion or with other genetic variants. Therefore, several gene therapies targeted for ATXN2 used in cell or ALS animal models to prevent or delay its progression were reported [This current study provided robust evidence that genetic factors affected the survival of ALS. For NMA, among the nine ALS causative/risk genes involved, we found 2 (SCA2) , but the2 (SCA2) , not onl2 (SCA2) . Meanwhie to ALS . It was e to ALS . In thise to ALS . In ALS,e to ALS . Motor nreported .C9orf72 GGGGCC (G4C2) hexanucleotide repeat expansion was identified as the most common mutation in Europe ALS and the second most common mutation in Asia ALS [C9orf72 was the most studied gene in the ALS research field in the last ten years. Although healthy individuals can have from 2 to 25 G4C2 repeats, ALS or FTD patients harbor hundreds to even thousands of these repeats [C9orf72 expansion was also associated with shorter survival . In addition, cases with C9orf72 expansion may have a more rapid rate of cognitive decline and a higher risk of developing FTD [C9orf72 has been carried out in preclinical studies [Thanks to advances in the next-generation technology, Asia ALS . Hence,  repeats , 113. Fu repeats . Here, aping FTD , which w studies , 116, anSOD1, FUS, and TARDBP, the other three common ALS causative genes, usually present as missense variants. When all the reported mutation sites were analyzed together, we found only FUS would shorten the survival time of ALS. However, we could not ignore that different variants on these genes have different effects on ALS survival. Based on previous genetic studies, we summarized the characteristics of several common variants in these genes in Additional file SOD1 with more than 150 ALS-associated SOD1 variants described. SOD1 was believed to cause ALS through toxic gain of function caused by aggregation of misfolded SOD1 [SOD1A4V and SOD1H46R associated, with the shorter and significantly longer durations, respectively, were identified not only in Caucasians but also in Asians. Our recent cohort study has yielded similar results [SOD1. There, it does not mean that SOD1 variants do not affect the survival of ALS, for individuals, specific variants should be identified. Further, we found a similar but slight difference from a meta-analysis of published FUS-ALS cases [FUSR521, FUSP525L, frameshift/truncation, and the remaining variants in FUS [FUS reported that would prolong the survival of ALS  may be associated with the prognosis of ALS, however, they cannot be performed by meta-analysis due to the limited studies.In addition, four genetic factors were studied by pairwise meta-analysis. Consistent with most previous studies , 60, 124tibility  and may n UNC13A , 128. HeALS risk . The ZNFignaling . SeveralAPOE polymorphism can change the course of ALS [APOE ε4 allele does not modify the clinical course of ALS as well under meta-analysis, so does KIFAP3 rs1541160. As for the remaining genetic factors, although SMN genes have been reported to be associated with ALS survival [SMN1 and SMN2 copy number variation in ALS [SMN1 and SMN2 are implicated in motor neuron death in spinal muscular atrophy, but SMN expression levels in the physiological range may not modify the progression of ALS [It is controversial whether e of ALS , 57. Howsurvival , 131, Thn in ALS . .The gen of ALS . Addition of ALS , suggestZNF521B rs2275294 were reported in only three articles, the results should be explained with caution. Therefore, more high-quality prospective studies are warranted. Finally, the rules of the review methodology of its restriction to articles published in English, and the low specificity of the search strategy, increase the risk of missing relevant studies.Although we find some potential genetic factors that affected the survival of ALS, these findings should be interpreted with caution, given some limitations. First and foremost, different variants in the same gene might have different effects on survival, it may result in information missing when a gene was analyzed as a whole factor. What is more, as we discussed above, whether some of the variants in these genes are ALS-associated mutations remains in doubt. Therefore, the results were not absolutely correct. Besides, some studies did not report the HR directly, when we get HR from the survival curve, there may be some slight gap with the accurate results. Some differences in follow-up time, the included population, and the definition of outcomes may lead to publication bias and the non-genetic factors related to ALS survival may also play a role . MoreoveThe present meta-analysis summarized and contrasted evidence for genetic prognostic factors in patients with ALS and will help to understand ALS genetics. Genetic prognostic factors deserve attention and careful consideration as the field moves forward to combat and prevent this devastating disease.Additional file 1: Table S1. Characteristics of the articles included in network meta-analysis for variants in ALS causative genes. Table S2. Characteristics of the articles included in pairwise meta-analysis for other modification loci. Table S3. Characteristics of the articles included in systematic review for other modification loci. Table S4. The quality of articles included in network meta-analysis and pairwise meta-analysis by NOS. Table S5. The feathers for different variants in SOD1, FUS, TARDBP. Figure S1. Forest plot for inconsistency test in network meta-analysis. Figure S2-S12. Heterogeneity analysis n network meta-analysis. Figure S13. publication bias for studies reporting C9orf72 expansion in patients with ALS compared to those without ALS-related mutation.