Pathogenic viral infection represents a major challenge to human health. Due to the vast mucosal surface of respiratory tract exposed to the environment, host defense against influenza viruses has perpetually been a considerable challenge. Inflammasomes serve as vital components of the host innate immune system and play a crucial role in responding to viral infections. To cope with influenza viral infection, the host employs inflammasomes and symbiotic microbiota to confer effective protection at the mucosal surface in the lungs. This review article aims to summarize the current findings on the function of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) in host response to influenza viral infection involving various mechanisms including the gut–lung crosstalk. Influenza viruses are responsible for both seasonal epidemics and occasional pandemics, causing considerable health and economic burdens around the world ,2,3. InfInfluenza viruses are single-stranded negative-sense RNA (ssRNA) viruses  and beloBased on variations in HA and NA antigenicity, type A influenza viruses are further classified into different subtypes, such as H1N1 and H7N9. In total, 18 HAs and 11 NAs have been identified ,36,37, wInnate immune cells employ pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) generated during IAV infection, thereby initiating immune responses ,45. The During IAV infection, the surface glycoprotein haemagglutinins (HAs) of virions bind to host receptors first, and endocytosis then mediates virus entry ,30. FollDuring infection, the endocytic vesicles carrying IAV particles enter endosomes, where the genomic ssRNAs are released following the acidolysis of IAV particles. These ssRNAs are then detected by TLR7 within the endosomes, stimulating IRF7-dependent IFN-I production, as well as NF-κB-dependent pro-inflammatory cytokine synthesis ,46. IAV-While hosts attempt to eliminate the IAV, the virus also works diligently to suppress the antiviral immune responses. The envelope proteins HA and NA undergo constant mutation, allowing the IAV to escape neutralizing antibodies. Furthermore, in macrophages, the influenza virus nonstructural protein 1 (NS1) hinders caspase-1 activation through the N-terminal domain of NS1, and consequently inhibits the maturation of IL-1β and IL-18 . NS1 alsInflammasomes, as cytosolic protein complexes, play a critical role in the innate immune system, and provide a rapid response to pathogenic infections and cellular damage . The canNLRP3 plays a crucial role in both innate and adaptive immunity. In its inactive state, the NLRP3 protein is bound to the ubiquitin ligase-associated protein SGT-1 and heat shock protein 90 (HSP90), maintaining self-inhibition . Upon acNlrp3−/−, Caspase1−/−, and Asc−/− mice demonstrated survival rates of 20% [Nlrp3−/− mice had a significant disadvantage in clearing the virus compared to WT mice [Nlrp3−/− and WT mice [Nlrp3-R258W mutation, increases mouse survival rates and promotes virus clearance via IL-1β-mediated neutrophil recruitment [The roles of the NLRP3 inflammasome in the survival rates of experimental mice during influenza virus infection have been inconsistent. In PR8 (H1N1)-infected mice, wild-type (WT) mice showed a 70% survival rate, while s of 20%  (or 40%)s of 20% , 40%, ans of 20% ,16. Niges of 20% . Howevers of 20% . Researc WT mice , while Truitment . In addiruitment . This heruitment . Taking Nlrp3−/− mice is lower than that in WT mice, while the levels of MIP2, CXCL1, TNFα, and IL-6 are either lower than, or the same as, in WT mice on day 3 after infection [Nlrp3−/− and Caspase1−/− mice, while Ichinohe et al. [Nlrp3−/− than in WT mice. The different results obtained by different research groups could be due to the inconsistency in virus uptake by intranasal infection [The IL-1β level in the bronchoalveolar lavage fluid (BALF) of nfection ,16,49. Anfection  and Thomnfection .Nlrp3−/− mice displayed localized necrotic bronchiolar epithelial cells and bronchiole blockage by fibrin, neutrophils, macrophages, and necrotic cells, while bronchioles in WT mice remained largely unaffected [Nlrp3−/− and Caspase1−/− mice, whereas only minor collagen deposition occurred in the alveoli of WT mice [NLRP3 plays a vital role in repairing lung damage caused by influenza virus infection. On day 3 after infection with the influenza A virus, affected . Consist WT mice .Il18−/− mice exhibited significantly lower IFNγ levels, higher viral loads, significantly reduced NK cell-mediated cytotoxicity, and increased neutrophil infiltration compared to WT mice, but the humoral and cellular immunity remained unaffected [As downstream cytokines of inflammasomes, IL-1β and IL-18, have also been investigated for their roles in combating influenza virus infection. During infection, affected . This suaffected .Il1r1−/− mice exhibited increased mortality, reduced neutrophil infiltration, weakened IgM responses, impaired activation and migration of CD4+ T cells into the lungs, as well as higher viral loads [Il1b−/− mice showed higher viral antigen expression in the lungs [Compared to WT mice, al loads . Meanwhihe lungs . Coincidhe lungs . Moreovehe lungs , which pPyroptosis is a pro-inflammatory form of programmed cell death characterized by the formation of membrane pores, which leads to the release of pro-inflammatory cytokines and damage-associated molecular patterns (DAMPs) ,94,95. TRecent studies have shed light on the role of the new key players of NLRP3 inflammasomes in influenza virus infection. Specifically, DEAD-box helicase 3 X (DDX3X), a host protein, plays a critical role in orchestrating the antiviral innate immune response during IAV infection. DDX3X activates the NLRP3 inflammasome in response to the wild-type (WT) influenza A virus (IAV) that carries NS1, and thereby controls viral spread, clears the infected cells, and promotes lung tissue repair . HoweverDuring influenza virus infection, the activation of NLRP3 inflammasome is considered an essential component of the host’s antiviral defense mechanism. After viral infection, the formation of inflammasomes facilitates the autocatalytic processing of pro-caspase-1, leading to the cleavage and release of pro-inflammatory cytokines IL-1β and IL-18. The production of these cytokines helps to induce an antiviral immune environment, reducing viral replication and spread . HoweverThere are several possible ways to transit NLRP3 from detrimental to beneficial during influenza virus infection. (1) Regulating NLRP3 activation: The administration of specific inhibitors to prevent the overactivation of the NLRP3 inflammasome could help to minimize IAV pathogenesis . It is iIl1b and Il18, contributing to pulmonary protective immune responses during influenza virus infection [The symbiotic microbiota, which consists of trillions of microorganisms, plays a crucial role in regulating various host physiological functions, such as digestion, nutrient metabolism, and immune system development ,137,138.nfection . The comnfection . Moreovenfection ,147,148.nfection .Bacteroides fragilis triggers the expression of IFN-β via TLR4-TRIF signaling in colonic cDCs, thereby protecting against vesicular stomatitis virus (VSV) and IAV infections [Clostridium orbiscindens, combats IAV infection by amplifying IFN-I signaling [Bordetella pertussis expressing the influenza virus M2e elicited high titers of specific antibodies in mice [Bifidobacterium longum BB536 [Bifidobacterium pseudolongum NjM1 [Lactobacillus strains [Bifidobacterium longum BB536 and Bifidobacterium pseudolongum NjM1 significantly alleviate influenza virus infection by inhibiting virus replication [Lactobacillus strains, four strains have also been implicated in inhibiting virus replication, including Lactobacillus gasseri TMC0356 [Lactobacillus brevis KB290 [Lactobacillus acidophilus L-92 [Lactobacillus plantarum DK119 [Several mechanisms have been proposed to elucidate how the gut microbiota safeguards against influenza virus infection. Some researchers extensively studied mechanisms involving microbial components, such as lipooligosaccharide (LOS), or the production of microbial metabolites, including short-chain fatty acids (SCFAs), such as butyrate, acetate, and propionate, as well as desaminotyrosine (DAT) ,154,155.fections , whereasfections . Furtherignaling . Moreoveignaling ,159,160.ignaling . Furtherignaling ,163,164. in mice . Probiotum BB536 , Bifidobgum NjM1 , and var strains . Both Bilication ,165. Amo TMC0356 , Lactobais KB290 , Lactobalus L-92 , and Lacum DK119 ,172,173.um DK119 . This suNlrp3 deficiency, enhances the defense of WT mice against influenza A virus infection following the microbiota exchange between Nlrp3−/− and WT mice [The intricate interplay between the NLRP3 inflammasome and gut microbiota is vital in maintaining intestinal homeostasis , and dis WT mice . Meanwhi WT mice ,178 or N WT mice . Further WT mice . In retu WT mice ,180.Future research should explore the complex regulatory mechanisms and interactions of NLRP3 inflammasome with gut microbiota, cellular processes, and co-infections to develop targeted therapeutic strategies and improve patient outcomes. Given the critical role of the NLRP3 inflammasome and gut microbiota in influenza virus infection, targeting their interaction offers a promising therapeutic approach. For instance, modulation of the NLRP3 inflammasome with small molecules, such as nigericin and MCC950, may hold therapeutic potential in treating influenza virus infection. Additionally, strategies aimed at restoring gut microbiota balance, including dietary fiber, probiotics, prebiotics, and fecal microbiota transplantation (FMT), may help to improve morbidity and mortality caused by influenza virus infections. In particular, identifying specific bacterial strains, as well as their functional genes and metabolites that could regulate NLRP3 inflammasome activation using advanced sequencing methods, such as metagenomics, metatranscriptomics, and metabolomics, may provide a targeted approach to prevent or treat influenza virus infection.Particular attention should also be paid to the interplay between NLRP3 inflammasome activation and other inflammatory pathways, such as the RIG-I/TLR3 signaling pathway, to understand how these pathways cooperatively regulate antiviral immune responses. Additionally, researchers should investigate the connections between inflammasome activation and cellular processes (such as autophagy and pyroptosis) to provide a complete picture of intracellular signaling networks during influenza virus infection. Targeting key components of the pyroptotic pathway, such as NLRP3, caspase-1, or GSDMD, may provide novel treatment options for severe influenza viral infections.In addition, researchers should expand the current knowledge on the role of NLRP3 inflammasome in co-infections and secondary bacterial infections during influenza virus infection. These co-infections can result in severe clinical outcomes and often lead to increased morbidity and mortality. Understanding the interplay between NLRP3 inflammasome activation and the host response to co-infections will contribute to the development of more effective treatment strategies.The NLRP3 inflammasome is a critical component of the host immune response to influenza virus infection, and plays a pivotal role in orchestrating the intricate interplay between the immune system and the gut microbiota. This dynamic relationship holds immense potential for the development of innovative therapeutic strategies aimed at combating influenza viruses. However, to harness this potential effectively, further research is needed in order to gain a deeper understanding of the delicate balance required to control excessive inflammation while simultaneously preserving host immunity. By unraveling this delicate equilibrium, we could strive towards achieving optimal therapeutic outcomes in the battle against influenza viruses.


Concurrent cocaine and alcohol use is among the most frequent drug combination, and among the most dangerous in terms of deleterious outcomes. Cocaine increases extracellular monoamines by blocking dopamine (DA), norepinephrine (NE) and serotonin (5-HT) transporters . Likewise, ethanol also increases extracellular monoamines, however evidence suggests that ethanol does so independently of DAT, NET and SERT. Organic cation transporter 3 (OCT3) is an emergent key player in the regulation of monoamine signaling. Using a battery of in vitro, in vivo electrochemical, and behavioral approaches, as well as wild-type and constitutive OCT3 knockout mice, we show that ethanol’s actions to inhibit monoamine uptake are dependent on OCT3. These findings provide a novel mechanistic basis whereby ethanol enhances the neurochemical and behavioral effects of cocaine and encourage further research into OCT3 as a target for therapeutic intervention in the treatment of ethanol and ethanol/cocaine use disorders. Alcohol use disorder (AUD) is the most common substance use disorder (SUD) in the US . ProblemThe popularity of co-abuse of alcohol and cocaine could result from an increased “high” compared with taking either drug alone, as well as from a prolongation of cocaine-induced euphoria –18, possCocaine and ethanol each increase extracellular levels of DA, NE, and 5-HT, neurotransmitters linked to drug reward. Cocaine does this by blocking the high-affinity transporters for these neurotransmitters: the DA transporter (DAT), NE transporter (NET), and 5-HT transporter (SERT), respectively . While a3H]MPP+), into human epithelial colorectal adenocarcinoma (Caco-2) cells, which are rich in OCT3 ) in OCT3 .Literature evidence supports the premise that ethanol inhibits DA, NE, and 5-HT clearance from extracellular fluid but does so in a manner independent of high-affinity, “uptake-1” transporters, DAT, NET, and SERT , 34, 36.Naïve adult male and female OCT3+/+ and OCT3-/- mice bred on a C57BL/6 background were used for in vivo chronoamperometry, conditioned place preference (CPP) and locomotor sensitization studies. Male OCT3+/+, OCT3-/-, SERT+/+ and SERT-/- mice were used for loss of righting reflex (LORR) studies. All mice were from our in-house colonies. For details, see Supplementary Information  or hippocampal  homogenates, compared with high-affinity ligands for DAT, NET and SERT  using established methods MPP+ into hOCT3 HEK293 cells were analyzed using non-linear regression, and means were compared by ANOVA. Uptake of APP+ and ASP+ in cells, chronoamperometry and behavioral data were analyzed by ANOVA and post-hoc analyses described in the figure legends. Assumptions underlying the statistical tests were met. Results are expressed as mean and standard error (SEM) or 95% confidence interval (CI). Tests were performed with GraphPad Prism , with statistical significance defined as p < 0.05. See SI for details.Competition binding and uptake of [3H]WIN35,428, [3H]nisoxetine, and [3H]citalopram binding to DAT, NET, and SERT by GBR12909, reboxetine, and citalopram, respectively, in striatal (for DAT) or hippocampal (for NET and SERT) homogenates were in the low nanomolar range, consistent with previous reports [3H]WIN35,428, [3H]nisoxetine and [3H]citalopram, evidencing that ethanol does not interact with binding sites of well-established ligands at DAT, NET and SERT  = 10.66, p < 0.0001; ethanol concentration F = 17.3, p < 0.001; cell type F = 10.18, p = 0.013)  for 40 s to establish the initial uptake rate before cells were exposed to ethanol or well-established inhibitors of these transporters. As in previous studies, we used ASP+ to measure OCT3-mediated transport fluxes WIN35,428 binding to DAT in striatal homogenates, or against [3H]nisoxetine and [3H]citalopram binding to NET and SERT, respectively, in hippocampal homogenates. In contrast, high affinity ligands for these transporters  displaced [3H]WIN35,428, [3H]nisoxetine and [3H]citalopram to DAT, NET and SERT, respectively, with Ki values in the nM range. However, the pharmacology of monoamine transporters is complex and modulators may bind to ortho- and allosteric sites MPP+ into Caco-2 cells, which are rich in OCT3 [3H]MPP+ into hOCT3 HEK293 cells with a Ki of 4.2 ± 0.2 mM. Considering that brain concentrations of ethanol reported for rodents and human subjects are in the low mM range [3H]MPP+ as a substrate, it remains possible that the antagonistic activity of ethanol at OCT3 involves other effects, such as disruptions of membrane composition and intracellular signaling cascades affecting OCT3 more than members of the SLC6 family. However, the temporal resolution of the fluorescent tracer-based uptake experiments allowed us to uncover an instantaneous effect of ethanol on OCT3-mediated transport processes. This observation strongly supports the interpretation that ethanol directly and instantaneously perturbs the function of OCT3.Rapidly growing evidence supports an important role for OCT3 in monoamine neurotransmission , 45–54.  in OCT3 , we hypomM range , 76, thiTo interrogate the translational relevance of these in vitro findings we turned to constitutive OCT3-/- mice. Consistent with earlier reports , ethanolAlthough preclinical investigations using self-administration and CPP across species –28, 92, To examine the generality of our findings, we assessed the sedative/hypnotic effects of ethanol in wild-type, OCT3-/- and SERT-/- mice. We, and others, have shown OCT3 expression to be greater in SERT-/- mice than in wild-type mice , 55, andCollectively, our data provide compelling evidence for OCT3 as a novel and previously unsuspected player in the actions of ethanol to inhibit monoamine uptake. Here we identified OCT3 as an important mediator of the acute effects of ethanol to inhibit DA uptake and produce rewarding effects. These novel findings raise several avenues for exciting future studies, including investigations of (and SI for details) (1) the effect of chronic cocaine and ethanol use in combination or alone, (2) potential actions of cocaethylene, a psychoactive substance formed in the liver when cocaine and alcohol coexist in blood , at OCT3In sum, these data raise the provocative idea that it may be possible to pharmacologically prevent ethanol’s actions at OCT3, and/or to enhance OCT3 transporter function, as a therapeutic approach to treating alcohol dependence, and alcohol and cocaine co-abuse , and opeSupplemental Material


Congenital abnormalities in animals are a major concern for breeders due to the increased economic loss they entail. The aim of this article was to describe a congenital bilateral cleft lip and jaw and an abnormal opening of the penile urethra in a crossbred calf. Clinical examination, computed tomography, and whole genome sequencing were performed to describe and identify a possible cause of the abnormalities. The whole genome investigation indicates the involvement of multiple genes in the two birth defects in this case.Bos taurus crossbred Piedmontese × Wagyu calf. Clinical examination, computed tomography, and whole genome sequencing were performed to describe and identify a possible cause of the abnormalities. Clinical examination revealed a bilateral cheilognathoschisis of approximately 4 cm in length and 3 cm in width in the widest part, with computer tomography analyses confirming the bilateral absence of the processus nasalis of the incisive bone and the lateral deviation of the processus palatinus towards the left side. Genomic data analyses identified 13 mutations with a high impact on the products of the following overlapped genes: ACVR1, ADGRA2, BHMT2, BMPR1B, CCDC8, CDH1, EGF, F13A1, GSTP1, IRF6, MMP14, MYBPHL, and PHC2 with ADGRA2, EGF, F13A1, GSTP1, and IRF6 having mutations in a homozygous state. The whole genome investigation indicates the involvement of multiple genes in the birth defects observed in this case.Congenital abnormalities in animals, including abnormalities of the cleft lip and jaw and hypospadias have been reported in all domesticated species. They are a major concern for breeders due to the increased economic loss they entail. In this article, we described a congenital bilateral cheilognathoschisis (cleft lip and jaw) with campylognathia in association with penile hypospadias and preputial hypoplasia with failure of preputial fusion in a  Congenital oral cavity anomalies are disturbances or failures of the complex processes of craniofacial development. The stomodeum, a primitive oral cavity is formed from five processes: frontonasal, medial nasal, lateral nasal, maxillary, and mandibular, derived from the neural crest mesenchyme and ectomesenchyme . The oraBMP (bone morphogenetic proteins), FGFs (fibroblast growth factors), SHH (Sonic Hedgehog), and WNT (Wingless-int) genes, are involved in the growth and fusion of facial processes [BMP4, BMPR1A, SOX11, WNT9B [FGF8 [Failures in the fusion of the lateral and medial nasal processes produce cheiloschisis (lip cleft) and a failure of the medial fusion of the palatal shelves determines palatoschisis . Many molecular signals such as rocesses , and mut1, WNT9B , or FGF8Facial clefts, such as congenital lip and jaw (CLJ), can be classified depending on their location into unilateral right or left side, medial or bilateral CLJ ,9,10, wiNon-syndromic forms of orofacial defects are classified as cheilopalatoschisis , only cheiloschisis (cleft lip/CL), or only palatoschisis  ,14. TheyPossible causes for the non-syndromic forms are multifactorial diseases as a result of the interaction between genetic and environmental factors. They do not follow a mono-genic pattern of inheritance, but they reappear in future generations . SyndromIRF6 (Interferon regulatory factor 6), FGFR2 (fibroblast growth factor receptor 2), SUMO1 , BMP4 (bone morphogenetic protein 4), FGFR1 (fibroblast growth factor receptor 1), MSX1 (MSH homeobox 1), MAFB (MAF bZIP transcription factor B), ABCA4, VAX1 , WNT signaling, FOXE1 (forkhead box E1), ADAMTS20, DLX5, and DLX6 [DLX genes are essential genes for craniofacial, axial, and appendicular skeletal development; inactivation of DLX5 and DLX6 in mice results in severe abnormalities in these regions [Modern genomics techniques have made it possible to identify many candidate genes for CLJ abnormalities in both humans and animals, such as and DLX6 ,20,21,22 regions .In cattle, the frequency of congenital defects is estimated to be 0.25% to 3.6% and can be caused by genetic factors, environmental factors, or genetic–environmental interaction during embryonic development ,24,25.Numerous environmental factors have been reported to be associated with facial abnormalities during embryonic development, such as chemicals, toxic substances, infections, hormonal factors etc. ,4,9.The other congenital abnormality seen in the calf was hypospadias (OMIA 001187-9913), a developmental abnormality in which the male urethra opens on the ventral side of the penis or on the perineum and can be accompanied by a failed development of the median perineal raphe, and of scrotum, penile aplasia or hypoplasia, hypoplasia of the corpus cavernosum, and incomplete formation of the prepuce ,27,28. ABased on the literature data overview of hypospadias in large and small ruminants, it usually appears in single cases alone with urogenital anomalies  ,30,36, oIn the etiology of hypospadias, there are incriminated genetic, endocrinological, environmental factors and even epigenetic alterations of the genetic material ,28,38. DBMP2, BMP4, BMP7, FGF, FGF10, FGF8, HOXA13, HOXA4, HOXB6, HOXD13, TGF, and WNT5A genes [ESR1 and ESR2 genes through estrogens play a role in normal penile and clitoral development, and normal urethral development depends on the balance between androgens and estrogens [The morphogenesis of the genital tubercle involves different genes such as 5A genes ,42,43. Mstrogens ,45,46. Sstrogens  and the strogens .AR gene [SRD5A2 gene [Additionally, epigenetic alterations can be involved in hypospadias development such as the methylation of the AR gene , the SRD5A2 gene , or CpG 5A2 gene , determi5A2 gene .Bos taurus hybrid Piedmontese × Wagyu crossbred cattle using different paraclinical examinations.The purpose of this study was to investigate a male Piedmontese × Wagyu calf with congenital bilateral cheilognathoschisis and hypospadias using computer tomography analysis and whole genome sequencing in order to identify the possible cause of those congenital defects. To our knowledge, based on literature data, we present a unique case of congenital defects in a The case study was performed in compliance with national (Romanian Law 43/2014) and European guidelines (EU Directive 63/2010) for using animals for scientific purposes, and in accordance with the internal SOP  of USAMVBT-PG-001-R021  and Ethical Statement no. 73/2020.Bos taurus hybrid Piedmontese×Wagyu crossbred calf with a body weight of 32 kg was born alive after a normal pregnancy period of a heifer in September 2020, in the Didactic Farm of the University of Life Sciences ‘’King Mihai I’’ from Timişoara, Romania. Its mother was a primiparous Piedmontese crossbred  and the father was from the Wagyu breed. It was the first case with such congenital abnormalities in our Research Farm and, to our knowledge, the first such documented case in the world. The calf was euthanized a few days later due to severe neonatal diarrhea and anemia, as clinically it was impossible to feed properly with colostrum and it could not be kept in a quadrupedal position. Euthanasia was performed with Ketamidor 100 mg/mL from Richter Pharma, Austria, and Xylazin Bio 20 mg/mL from Bioveta, Cech Republic and followed by T61  1 mL/10 kg, intravenous, from Intervet International BV Wim de Korverstraat, Holland.The male The acquisition of CT scan images was performed postmortem with the body positioned such that the ventral aspect of the head was in contact with the CT table . CT scans were performed with a Siemens Somatom Definition AS 64 scanner, using conventional settings , and a slice thickness of 0.6 mm.® DNA Assay Kit on a Qubit® 2.0 Fluorometer .Genomic DNA was isolated from whole blood collected from the affected calf using a DNeasy Blood & Tissue Kit  for DNA extraction. Whole-genome sequencing was performed by Novogene, Cambridge, United Kindom. The sample was 41.13 µL in volume, 10.209 ng/µL concentration with a total amount of DNA of 0.41989 µg. The DNA concentration was measured using a QubitNext, the library preparation was performed. Briefly, the genomic DNA was randomly fragmented into 350 bp fragments, and then DNA fragments were end polished, A-tailed, and ligated with the adapters for Illumina sequencing, and further PCR enriched with P5 and P7 oligo primers. The PCR products as final construct of the libraries were purified (AMPure XP system), followed by a quality control test that included the size distribution using an Agilent 2100 Bioanalyzer  and the measurement of molarity using real-time PCR.Raw sequenced reads in FASTQ format were analyzed using the Bcbio-nextgen pipeline v. 1.2.9. The quality control of the raw data was performed with FastQC v. 0.11.p . Read deThe variants classified as having a high or moderate impact and the corresponding genes were retained for the downstream analysis, which was performed using various packages for the R programming language v. 4.1.3 . A list Homo Sapiens, using the select function from the AnnotationDbi package v. 1.56.2 [Bos taurus individual from this study.Finally, gene annotations were obtained via the AnnotationHub package v. 3.2.2  and mult. 1.56.2 . Gene anAt birth, the affected calf presented a cleft lip and jaw (OMIA 001714-9913) and hypospadias (OMIA 001187-9913). The bilateral cleft was characterized by a length of approximately 4 cm on each side and a width of 3 cm on the widest part. On the andrological examination, the calf showed penile hypospadias, the penile urethra ventrally opened at approximately 4 cm caudal of the normal opening of the urethra and the prepuce was incompletely developed and had failed to fuse normally a–d.The anus was open physiologically. The examination of the scrotum revealed that the testicles were not fully descended and at the necropsy, the testicles with a length of 2.2 cm were identified in the inguinal canal. The necropsy revealed that there was no other internal congenital abnormality in addition to the previously mentioned external defects.CT of the head  revealedBased on cleft lip-jaw scoring system in cattle existing in literature where different orofacial structures, such as lips, the processus nasalis of the os incisivum, the dental plate with adjacent segments of the hard palate, the facial bones—os incisivum, os maxillare, os nasale, os palatinum, and the mandibles, we scored the cleft lips and accompanying malformations in the face in this case as fallow. The score was 4  for the site ; a score of 5  for the depth of the cleft lip , a score of 6  for changes at the dental plate and hard palate , a score of 1 , for curvature to the right , and a score of 5  for changes in the processus nasalis of the os incisivum .Statistics regarding the proportion of reads mapped to the reference genome were computed. There was a total of 164.9 million mapped reads, a proportion of 98% of the total of reads. The percentage of PCR duplicate reads was 29%. The percentage of 1X coverage was 93.9%, for 5X coverage was 45%, for 10X coverage was 14.1%, with the mean genome coverage of 6.3X, as shown in Variant calling identified a total number of 5,598,215 variants, of which 4,951,834 were SNPs and 648,459 were indels. Predicted consequences of all variants (most severe per variant), computed by Ensembl Variant Effect Predictor (VEP), are presented in Most of the variants, such as non-coding transcript variants, intergenic variants, synonymous variants, are considered as unlikely to change protein behavior or with low or no impact on it. Conversely, as can be seen in An initial quality control checks on raw sequenced reads using FastQC revealed one “failure” result for the “Per sequence GC content” test, indicating a possible contamination with DNA from a different species. Subsequently, this scenario was checked with the Kraken2 tool, which classified 21.97% of the original raw reads as one of the taxons present in the reference database, consisting of all humans, bacterial, archaeal, viral, fungal, and protozoan sequences from the NCBI RefSeq database as of May 2022. Of the remaining, unclassified reads, which amounted to 164.9 million reads, 161.6 million reads (98%) mapped to the bovine reference genome ARS-UCD1.2.ADGRA2, BMPR1B, CCDC8, F13A1, GSTP1 and IRF6 genes are presented in IGV (integrative genome viewer) images for The raw sequencing data have been uploaded to the NCBI SRA database under the accession number PRJNA896881.Bos taurus reference genome. The list of 559 unique bovine genes that were found associated with hypospadias and CLJ in cattle and other mammals, according to literature and the CleftGeneDB database, are presented in an Excel sheet . The chrACVR1, ADGRA2, BHMT2, BMPR1B, CCDC8, CDH1, EGF, F13A1, GSTP1, IRF6, MMP14, MYBPHL, and PHC2.The list of SNPs reported by Ensembl VEP as having a high impact on any of the 559 target genes are presented in an Excel sheet  and the GO_high_moderate = Significant Gene Ontology terms for genes overlapped by variations with a high or moderate effect; KEGG_high_moderate = Significant KEGG terms for genes overlapped by variations with a high or moderate effect; MeSH_high = Significant MeSH terms for genes overlapped by variations with a high effect; MeSH_high_moderate = Significant MeSH terms for genes overlapped by variations with a high or moderate effect; Reactome_high_moderate = Significant Reactome terms for genes overlapped by variations with a high or moderate effect.Gene annotations based on GO, KEGG, MeSH, and Reactome, which were found significant after over-representation analyses based on genes with moderate and high impact SNPs , are included in The biological terms and pathways in the gene annotation databases consist of 5 GO terms, 2 KEGG pathways, 12 MeSH terms, and 5 Reactome pathways. A total of 490 genes were assigned to the Biological Process (BP) GO term category (3 terms), and 188 genes to the Molecular Function (MF) GO term category (2 terms). The analyses based on GO were highly enriched in genes associated with sensory perception of smell , olfactory receptor activity (GO:0004984), cargo receptor activity (GO:0038024), olfactory transduction, and glycosaminoglycan biosynthesis—chondroitin sulfate/dermatan sulfate (KEGG).TF (transferrin), EGF , MMP14 , CDH1 (Cadherin 1), and F13A1 (Coagulation factor XIII A chain) genes, related to CLJ and hypospadias.MeSH analyses captured several significant terms such as Gamma Catenin (MeSH:D051185), Glyoxal (MeSH:D006037), Sertoli Cell Tumor (MeSH:D012707), Desmogleins (MeSH:D051182), Factor VIII (MeSH:D005169), Hair Cells and Auditory (MeSH:D018072), Cytokinesis (MeSH:D048749), Glycopeptides (MeSH:D006020); Desmocollins (MeSH:D051187), Lung Diseases (MeSH:D008171). Several of these MeSH terms are associated with ADAM family ; Degradation of the extracellular matrix (R-HSA-1474228); Defective B3GALTL causes Peters-plus syndrome (PpS) (R-HSA-5083635); Diseases of metabolism (R-HSA-5668914); and O-glycosylation of TSR domain-containing proteins (R-HSA-517321). Notably, the latter four terms included numerous genes from the M family .ABCA4, ADAMTS20, BMP4, BRD1, CREBBP, CSK, DLX5, DLX6, DNM1L, FGFR1, FGFR2, FOXE1, IRF6, LOR, MAFB, MSX1, PTPN18, SND1, SUMO1, TGS1, VAX1, VIM, and WNT [AR, ATF3, ATF3, BMP4, BMP7, BNC2, CTGF, CYP1A1, CYR61, DGKK, EGESR1, ESR2, FGF8, FGFR2, GSTM1, GSTT1, HOXA4, HOXB6, HSD17B3, HSD3B2, MAMLD1, MID1, SF1, SRD5A2, WT1 [CYP11a1, CYP17a1, HES3, HSD3b1, INSL1, SF1, STAR, STAT3 [DGKK, STS, ZEB1 [EFNB2 [NR5A1 [Cleft lip and jaw and hypospadias are two congenital malformations which appear in populations due to various gene mutations, environmental factors, or both. The molecular mechanisms by which the two malformations appear are very complex and different. Among the genes known to be involved in cleft lip and jaw, we mention  and WNT ,70. For 5A2, WT1 ,59,71,72R, STAT3 , DGKK, STS, ZEB1 , EFNB2 [1 [EFNB2 , or NR5A2 [NR5A1 ), genes During CT analysis of this case, high scores were observed for orofacial defects according to the cleft lip and jaw scoring system developed by Reinartz et al.  in cattlFollowing the analysis of the genomic sequences, the genes with high, high/moderate, and moderate impact on the two congenital diseases were identified, as well as variations of these genes with high or moderate impact. We identified a large number of genes, 379 genes with 1103 genetic variations that can have a major impact on the phenotype. The genes overlapped by variations with high impact or moderate impact on all genes were in the number of 4177. and 221 were with impact on genes of interest, with 16,026 variants with high or moderate impact on all genes, as well as 4049 genes overlapped by variations with moderate impact, having 14,924 variations with moderate impact on the genes on which they were overlapped.EGF, IRF6, BMPR1B, MMP14, ADGRA2, F13A1, CCDC8, ACVR1, BHMT2, CDH1, GSTP1, PHC2, and MYBPHL where the EGF, IRF6, F13A1, ADGRA2, and GSTP1 genes were found with homozygous mutation, and the CCDC8 and BMPR1B genes were not reported in the literature  with imEGF , a prototypical ligand, and epidermal growth factor receptor (EGFR) are part of the EGF family, being important for cell motility, adhesion, proliferation by enhancing cell cycle progression through the G1 phase, differentiation, and survival being ubiquitously expressed in somatic tissues and in the gonads [EGFR has an important role in normal craniofacial development, EGFR-/- with decreased matrix metalloproteinases (MMPs) in mice embryos induced facial anomalies  [MMPs, part of a protease family that comprehends adamalysins (ADAMs), disintegrin, and metalloproteinase with thrombospondin motifs (ADAMTSs), with an important role in extracellular matrix remodeling, morphogenesis, have a possible implication in the etiology of CL/P [EGF, EGFR, and HOXA13 in the foreskin tissue and hypospadias cases indicates a possible cause of hypospadias [For the EGF gene (ENSBTAG00000048812), the identified mutation located in the position 6:g.15450370G>A (p.Arg105Ter) revealed a homozygous single nucleotide variant, and the consequence was a stop gained (premature stop codon). The protein encoded by EGF gene has 232 amino acids and the affected amino acid is located at position 105. e gonads . EGFR ha palate) . MMPs, p of CL/P . A correospadias .BMPR1B gene (ENSBTAG00000002081) (ACVR1 (activin A receptor type 1) in the palate epithelium and an increase of this signaling pathway causes a submucous palate [BMPR1A, BMPR1B, and BMPR2 class receptors with many other receptors in each class, and loss of function can be associated with muscular-skeletal and, cardiovascular diseases, cancer [EGF, play the role of paracrine mediators for epithelial–mesenchymal interactions during the embryonic development of various organs, including the male reproductive system, EGF stimulating androgen binding activity in the male genital tract, very important hormones for the fetal development of the reproductive system during the hormone-dependent period [BMP4 and BMP7—two factors that play a role in normal urethra development, along with HOXA4 and HOXB6, were identified in Chinese patients with hypospadias, but without a clear association [Another gene identified as having a potential high risk was the 0002081) , a membes palate . BMPs, c, cancer , or CP [t period ,42,82. Mociation .IRF6 gene (ENSBTAG00000002849), the homozygous state mutation identified was 16:g73512380-73512381insT, a frameshift variant consisting of an insertion of a nucleotide at position 162. The IRF6 gene (Interferon Regulatory Factor 6) plays an important role in normal early development. Its function is active in the cells from which the tissues of the face, head, skin, and genitals are formed. In the normal development of the epidermis, IRF6 regulates the balance between keratinocyte proliferation and differentiation, as well as in craniofacial morphogenesis, oral epithelium and breast epithelium [For the ithelium .IRF6 locus may increase the risk of syndromic and nonsyndromic forms of orofacial clefts; in humans, for example, mutations in the IRF6 gene are associated with Van der Wounde Syndrome (VWS) (OMIM # 119300) and Polipteal Pterygium syndrome (OMIM # 119500), rare orofacial cleft syndromes associated with lip pits, and cutaneous and limb defects [IRF6 gene are responsible for the disease and Zang et al. (2020) identified a frameshift mutation in IRF6 as a possible cause  [IRF6 gene variations are believed to affect the function of the IRF6 protein in its role as a transcription factor, which may interfere with the normal development of the face. Keratinocyte deficiency in IRF6 affects intracellular adhesion and cell colony morphology, an aspect observed clinically postoperatively in patients with cleft-lip/cleft palate by delaying wound healing [IRF6 SNP may play a major role in the pathogenesis and risk of developing non-syndromic cleft lip +/− palate in a South Indian population [IRF6 in mice leads to skin, craniofacial, and limb defects. A minimum level of IRF6 in the periderm and basal epithelial cells is necessary for orofacial development and a partial genetic rescue of the IRF6 knockout embryo with the KRT14 promoter can be performed [Different mutations in the  defects ,87. In a healing . An IRF6pulation . Loss oferformed .ADGRA2 gene (adhesion G protein-coupled receptor A2)(ENSBTAG00000008814) was found as having high-risk mutation at position 27:33105944C>T (p.Gln1341Thr). The protein consists of 1343 aa, and in our case, the aa at position 1341 was affected by the gene mutation. We found no data in the literature regarding its implication in CLJ in cattle and only little information in other species [F13A1 (coagulation factor XIII A chain) gene, the data from the literature does not evidence a linkage with CL/P [GSTP1 (glutathione S-transferase P1) was involved in the detoxification of electrophilic compounds by glutathione conjugation, I105V polymorphism in mothers and/or children with/without maternal smoking may increase the risk of CL/P [The  species . Regardiith CL/P , even ifith CL/P . The GST of CL/P .CCDC8 gene (ENSBTAG00000018181), the first being 18:g.53693827_53693828insCAGACAA, an insertion of seven nucleotides classified as a frameshift variant, identified at protein level p.Leu374ProfsTer5  and the second 18:g53693828-53693829 ins CAGAGGGCAGAGGCCCC, also classified as frameshift variant. In general, frameshift variants have a high impact on the protein, causing in most cases the production of an altered protein 3D structure. The biochemical and cellular functions of this gene are not clearly known, but the protein encoded by the gene acts as a cofactor required for p-53-mediated apoptosis after DNA damage and may also play a role in growth through interactions with the cytoskeletal adaptor protein obscurin-like 1. Deletion of CCDC8 caused perinatal lethality, intrauterine growth restriction, and placental defects [CCDC8 is associated with 3M syndrome dwarfism, a rare inherited disorder characterized by low body weight and short length at birth, short stature as adults, widespread skeletal abnormalities, and unusual facial features [CCDC8 proteins are rather conservative across species, suggesting their important functions.Two heterozygous insertions were identified for the  defects . In humafeatures ,95. The CDH1 gene (ENSBTAG00000015991), the mutation located in position 18:36101086-36101087insT had as a consequence a frameshift, splice region, and intron variant. The CDH1 gene, which encodes epithelial cadherin, is also very important for the cell–cell adhesions, mobility, and proliferation of epithelial cells, being highly expressed in the frontonasal prominence and in the lateral and medial nasal prominences of human embryos, during critical stages of lip and palate development. Pathogenic variants of CDH1 gene have been implicated in syndromic and non-syndromic cleft lip-palate, as well as in hereditary diffuse gastric cancer [In the c cancer ,97.ACVR1, ADGRA2, BHMT2, BMPR1B, CDH1, EGF, F13A1, GSTP1, IRF6, MMP14, MYBPHL, PHC2, except CCDC8 can be involved in CLJ. As for the hypospadias from the 13 genes, only CCDC8, EGF, IRF6, CDH1, and GSTP1 were reported in literature and in the Gene-Disease Associations dataset [EGF, IRF6, or CDH1 in various tissues is to stimulate the growth of epidermal and epithelial tissues or to sustain cell–cell adhesions, so mutation in them can induce congenital abnormalities in the skin in different anatomical regions, as in this case.Analyzing cleft lip and jaw independently by comparing the association of 13 major risk genes in this case with cleft lip and jaw reported in the literature and in the CleftGeneDB database, we can say that  dataset  as beingADGRA2, EGF, F13A1, IRF6, and GSTP1 genes mutation might encode mutant proteins, they do not provide a prediction of the phenotype, but may contribute to more accurate assessments of the orofacial clefts or hypospadias. It is difficult to predict the impact on the protein, because there are many characteristics of proteins that can be modified, such as folding, protein interactions, dynamics, post-translational modifications, or solubility [SRD5A2, AR, DGKK, NR5A1, and WNT5A, involved in the steroidogenesis of androgens and in the functionality of hormones, were not identified as having a major risk in this case, but we do not exclude the involvement of endocrine disruptors in the hormone-dependent period of the development of the genital segments external as a possible cause in hypospadias. New research data indicate that DNA methylation sites in the penile foreskin are involved in hypospadias etiology and a potential role of environmental factors that can act through epigenetic alterations and even epigenetic inheritance is suggested [Genomic data suggest the involvement of several mutated genes in the critical period of embryonic development of the affected regions. While these non-synonymous homozygous lubility . Knowinguggested ,100.Our study expands the gene spectrum toward new genes involved in cleft lip and jaw or in hypospadias, but it is possible that there are other molecular processes coordinated by other genes involved in the presented case that have not been identified. Despite these limitations, the present results provide a basis for further investigation of key genes in cleft lip and jaw or in hypospadias in animals.Bos taurus crossbred Piedmontese × Wagyu individual using different paraclinical examinations. The genomic analyses based on whole genome sequencing identified 13 mutations with a predicted high impact on the following overlapped genes: ACVR1, ADGRA2, BHMT2, BMPR1B, CCDC8, CDH1, EGF, F13A1, GSTP1, IRF6, MMP14, MYBPHL, and PHC2, of which ADGRA2, EGF, F13A1, GSTP1, and IRF6 showed homozygous mutations, and BMPR1B and CCDC8 had a low or no report in other clinical cases in bovine species regarding CLJ and hypospadias. In veterinary medicine, the genomic data for the two malformations are very rare, although there are numerous clinical reports in the literature about the existence of these congenital pathologies. Therefore, although we cannot specify exactly which of the high-risk genes identified in the study are responsible for the changes identified or if there were environmental factors involved in the appearance of these malformations, we believe that the present study expands the gene spectrum towards new genes involved in cheilognathoschisis or in hypospadias and that the obtained results increase the genetic contribution towards the knowledge of congenital defects in cattle. However, more studies are needed to better understand the specific effects of these genes variants on cheilognathoschisis or on hypospadias in bovine.In summary, in our study, we analyzed a unique case of congenital defects in a


SMARCA4 (BRG1)-deficient undifferentiated carcinoma is a rare and highly aggressive malignancy. It has been reported to occur in a multiple range of organs. However, to the best of our knowledge, SMARCA4 (BRG1)-deficient undifferentiated carcinoma of gallbladder has not yet been reported. Here, we describe a case of SMARCA4 (BRG1)-deficient undifferentiated carcinoma of gallbladder. Through comprehensive genetic analysis, we hypothesized that in addition to SMARCA4 (BRG1) deficiency, other genetic changes might also be involved in the tumorigenesis of undifferentiated gallbladder cancer in this patient, particularly somatic mutations in the CTNNB1, KRAS, PIK3CA, TP53, CREBBP, and FANCI genes. To the best of our knowledge, this is the first report of SMARCA4 (BRG1)-deficient undifferentiated carcinoma of gallbladder with genetic analysis. Gallbladder carcinoma (GBC) is a rare malignancy. Nearly 98% of GBCs are adenocarcinomas. Other rare histological types include mucinous, squamous, adenosquamous, small-cell, and undifferentiated carcinomas . UndiffeSMARCA2 (BRM), SMARCA4 (BRG1), SMARCC1, SMARCC2, SMARCB1 (INI1), ARID1A, and PBRM1. SWI/SNF complex exerts a crucial role in the differentiation, cell adhesion, motility of cancer cells and induces abnormal activation of the hedgehog signaling pathway (The switch/sucrose non-fermenting (SWI/SNF) complex is a family of ATP-dependent chromatin remodeling proteins that play a crucial role in regulating gene transcription. SWI/SNF complex consists of 12–15 subunits encoded by 29 genes, including SMARCA4 (BRG1) is one of the catalytic subunits of SWI/SNF complex. Inactivation of SMARCA4 (BRG1) has recently been suggested to be involved in the pathogenesis of some undifferentiated carcinomas. Previous studies have reported that SMARCA4 (BRG1)-deficient undifferentiated carcinomas could occur in the lung, ovary, gastrointestinal tract, uterus, and other organs (SMARCA4 (BRG1)-deficient undifferentiated carcinoma of gallbladder has not yet been reported.r organs –9. HowevSMARCA4 (BRG1)-deficient undifferentiated carcinoma of gallbladder and analyze comprehensively its genetic alterations. We also discussed the potential targeted therapy.Here, we report the first case of SMARCA4 (BRG1)-deficient undifferentiated carcinoma of gallbladder (stage II-III).On June 30, 2022, a 65-year-old female patient was admitted to the hospital due to right upper abdominal pain for more than one month and aggravation for half a month. A whole abdominal enhanced computer tomography (CT) scan showed a space-occupying lesion in the gallbladder, with enlargement of multiple peripheral and retroperitoneal lymph nodes, suspected a malignant lesion  status and medium tumor mutational burden . The genomic profile of the patient is demonstrated in On July 14, 2022, DNA and RNA were extracted from the resected formalin fixed and paraffin embedded tumor tissues of the patient, and were subjected to next-generation sequencing (NGS) using a 1123-gene panel and a 102-gene panel, respectively . The sequencing results using DNA samples revealed that the patient harbored somatic mutations in the TP53 (64%-73%), CDKN2A (11.0%-25.0%), ERBB2 (9.3%), and PIK3CA (10%-20%) in GBC  is an uncommon but highly fatal malignancy, and undifferentiated GBC is extremely rare. Some research had revealed the genomics profile of GBC. The common somatic mutation genes were d PIK3CA %-20% in , CDKN2A .0%-25.0%, CDKN2A .0%-25.0%ts study .SMRCA4 somatic mutations in NSCLC is 8% (407/4813); the major co-mutated genes were TP53 (56%), KEAP1 (41%), STK11 (39%), and KRAS (36%) in SMARCA4-mutant NSCLC. Patients with SMARCA4 co-mutations with STK11 or KEAP1 had a worse prognosis than those with single mutations, and patients with triple mutations had the worst prognosis  genes (KRAS (p.G12A) and TP53 (p.P152L) genes were also detected, but no aberrations in erbB-2.In addition, SMARCA4 (BRG1)-deficient in other undifferentiated cancers has been reported sporadically, such as colon , gastroi2) genes . In the KRAS, the mutation p.G12A in the KRAS has the potential capability to activate MEK/ERK- and PI3K/AKT-signaling pathways in cancer cells and is an oncogenic mutation  and PIK3CA genes, as well as six type III variations of CREBBP, ERCC5, FANCI, FANCM, FAP, and PTPRT genes.In addition, we also detected two type II variations of CTNNB1 encodes beta-catenin protein, which acts as an intracellular signal converter in the Wnt signaling pathway. Aberrant CTNNB1 activates proto oncogenes and cyclins, driving the occurrence, progress, survival, and recurrence of cancer. CTNNB1 is recurrently mutated in multiple cancer types . It plays an important role in the cell growth, proliferation, migration and survival through the PI3K/AKT/mTOR signaling pathway. Mutated PIK3CA gene leads to the continuously activated PI3K/Akt signaling pathway in various cancers  pathway  pathway, which is a DNA damage response (DDR) pathway  is a type II integral serine protease expressed specifically by activated fibroblasts. pression . MutatioPTPRT gene encodes a receptor-type tyrosine-protein phosphatase T enzyme. It is a tumor suppressor gene involved in signal transduction and cellular adhesion. PTPRT inhibits cell proliferation through the STAT3 pathway  deletion by NGS. The possible reason is that NGS can accurately detect small insertion/deletion mutations, point mutations and exonic copy-number changes. However, NGS cannot accurately detect large deletions. We speculate that the patient harbors a large SMARCA4 (BRG1) deletion. In addition, the alterations may occur in the regulatory regions of the SMARCA4 gene, such as the promoter region, CpG islands, enhancers, and so on, resulting in a loss of gene expression. Epigenetic modifications involving chemical alterations to chromatin or DNA molecules may also contribute to gene expression deficiency. These modifications include DNA methylation, histone modifications, and non-coding RNAs. For instance, DNA methylation refers to adding methyl groups to DNA molecules, often leading to gene silencing and suppressed expression.SMARCA4 (BRG1) deficiency, other genetic alterations might also participate in the occurrence and progress of the undifferentiated carcinoma of the gallbladder in this patient, particularly the mutations in the CTNNB1, KRAS, PIK3CA, TP53, CREBBP, and FANCI genes -deficient tumors. Based on the antagonism of SWI/SNF and polycomb repressive complex 2 (PRC2), SWI/SNF deletion leads to the loss of inhibition of EZH2 methyltransferase, which in turn results in PRC2-mediated tumorigenesis (SMARCB1 (INI1)-deficient rhabdoid tumors and epithelioid sarcomas have shown that EZH2 inhibitors have lasting anti-proliferative effects - and SMARCA2 (BRM)-deficient small-cell carcinomas of the ovary of hypercalcaemic type (SCCOHT) (SMARCA4 (BRG1)-deficient undifferentiated tumors. In addition to EZH2 inhibitors, other possible targeted therapies include histone deacetylase inhibitors and DNA methyltransferase inhibitors. Furthermore, another potential therapy is immune checkpoint inhibitor (ICI) therapy. The patient gave up the therapy after discharge from the hospital because of personal reasons.Conventional treatment is usually ineffective for igenesis . Targeteigenesis . Clinica effects . In a ph effects . Therefo(SCCOHT) , which bSMARCA4 (BRG1)-deficient undifferentiated carcinoma of gallbladder with genetic analysis. This case might contribute to the understanding of SWI/SNF-deficient carcinoma of gallbladder.To the best of our knowledge, this is the first case of The sample data of this patient for this study is included in the article/The work was approved by the Ethics Committee of Shengjing Hospital of China Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the participant/patient(s) for the publication of this case report.XM: conceptualization. JM: postoperative care. TY: Collection data. NM: writing the draft. BN: Editing the manuscript. All authors contributed to the article and approved the submitted version.


Patients were grouped based on phenotype and the presence of SCG variants. Screening of SCN3A, SCN7A-SCN11A, and SCN1B-SCN4B revealed 125 different  pathogenic variants in 194 patients . A potential pathogenic variant was present in 18.1% (n = 142/784) of painful neuropathy patients vs. 15.2% (n = 52/341) of painless neuropathy patients (17.3% (n = 41/237) for painful-DPN patients, 14.9% (n = 46/309) for painless-DPN patients, 18.5% (n = 101/547) for painful-SFN patients, and 18.8% (n = 6/32) for painless-SFN patients). Of the variants detected, 70% were in SCN7A, SCN9A, SCN10A and SCN11A. The frequency of SCN9A and SCN11A variants was the highest in painful-SFN patients, SCN7A variants in painful-DPN patients, and SCN10A variants in painless-DPN patients. Our findings suggest that rare SCG genetic variants may contribute to the development of painful neuropathy. Genetic profiling and SCG variant identification should aid in a better understanding of the genetic variability in patients with painful and painless neuropathy, and may lead to better risk stratification and the development of more targeted and personalized pain treatments.Neuropathic pain is a frequent feature of diabetic peripheral neuropathy (DPN) and small fiber neuropathy (SFN). Resolving the genetic architecture of these painful neuropathies will lead to better disease management strategies, counselling and intervention. Our aims were to profile ten sodium channel genes (SCG) expressed in a nociceptive pathway in painful and painless DPN and painful and painless SFN patients, and to provide a perspective for clinicians who assess patients with painful peripheral neuropathy. Between June 2014 and September 2016, 1125 patients with painful-DPN ( Neuropathic pain (NeuP) arises due to damage or a disease of the somatosensory nervous system involving peripheral fibers  and central neurons . NeuP isIn small fiber neuropathy (SFN), thinly myelinated Aδ and the unmyelinated C fibers are affected, resulting in sensory symptoms and autonomic dysfunction . Pure SFDiabetic peripheral neuropathy (DPN) is the most commonly reported complication of diabetes, affecting 50–90% of patients . ClinicaPathogenic genetic variants of voltage-gated sodium channel genes have been reported in patients with a painful neuropathy ,11,12,13SCN3A), Nav 1.6 (SCN8A), Nav 1.7 (SCN9A), Nav 1.8 (SCN10A), Nav 1.9 (SCN11A), Nax (SCN7A) and beta-subunits  were sequenced in patients with painful-DPN, painless-DPN, and painful and painless SFN. The aim of the current study was to profile these genes according to the different phenotypes and to provide a perspective for clinicians who assess patients with peripheral neuropathy.Single-molecule Molecular Inversion Probes-Next Generation Sequencing (smMIPs-NGS) has recently been introduced for the re-sequencing of gene panels in large cohorts of patients. Using this relatively inexpensive and reliable technique , ten genSCN3A, SCN7A-SCN11A, and SCN1B-SCN4B.A total of 1325 patients were recruited for this study. Two-hundred patients were excluded based on inclusion/exclusion criteria, incomplete clinical or diagnostic data, and/or low quality smMIPs-NGS data . EventuaAmong the 1125 patients, males (55.2%) were represented more than females. The mean age of the patient at recruitment for this study was 58.9 years (SD ± 14.0 years). The median age of onset of complaints was 53.0 years (SD ± 15.4 years) and the mean duration of neuropathy was 7.0 years (SD ± 8.1 years). One in five patients (20.2%) reported a family history of neuropathy.p = 0.005). The patient characteristics per subgroup are given in All patients diagnosed with DPN had an abnormal NCS. In 19.8% of patients the diagnosis of SFN was based on the clinical picture in combination with both an abnormal IENFD and QST, whereas in 26.7% of patients only the IENFD was abnormal, and in 53.5% of patients only the QST was abnormal. Of the DPN patients, 21.2% had type I diabetes, which was more prevalent in painless-DPN compared to painful-DPN .SCN3A, SCN7A-SCN11A, and SCN1B-SCN4B. Targeted regions enrichment, capture, and library sequencing were performed for all patients with painful and painless DPN and painful and painless idiopathic SFN. To calculate the performance and capture efficiency of the SCG-smMIPs-NGS, only the data of patients with complete clinical and diagnostic evaluation and sufficient smMIPs-NGS data were included  of ten sodium channel genes, n = 317/320 smMIPs) and, on average, 97.6% of the targeted regions were covered >30×. The average coverage of SCN3A = 99.6%, SCN7A = 100%, SCN8A = 97.6%, SCN9A = 98.7%, SCN10A = 99.9%, SCN11A = 99.9%, SCN1B = 93.6%, SCN2B = 100%, SCN3B = 100%, and SCN4B = 86.5%. No sequence reads were obtained for three smMIPs . We were not able to capture and sequence the first exon of SCN1B, SCN4B and SCN11A genes by smMIPs-NGS. Furthermore, exon 2 of SCN3B, exons 13, 16, 21 and 26 of SCN8A, exon 13 of SCN10A and exon 14 of SCN11A showed high variation in sequencing reads. To provide full coverage for SCN3A, SCN8A-SCN11A, SCN1B, and SCN4B, 2321 bp were subsequently tested by Sanger sequencing.The performance and capture efficiency for the ten genes’ smMIPs-NGS was 99.1%  pathogenic variants detected in our cohort of patients with painful-DPN have been reported in patients with epilepsy with or without neurodevelopment delay, autism spectrum disorder, atrial fibrillation, and sudden unexplained/infant death syndrome ,21,22,23n = 46/309, Among our cohort of 309 painless-DPN patients, 42 different potentially pathogenic heterozygous SCG variants were detected in 46 patients  pathogenic, illation , and thrillation ,28. Onlyn = 101/547, For the 547 painful-SFN patients, 71 different potentially pathogenic heterozygous variants were found in 101 patients  pathogenic variants detected in our patients with painful-SFN have been reported in patients with other neurological and cardiac disorders such as epilepsy with and without neurodevelopmental delay, autism spectrum disorder, Brugada syndrome, atrial fibrillation, and sudden infant death syndrome ,24,25,32n = 6/32, Ten different potentially pathogenic heterozygous variants were detected in six patients with painless-SFN  pathogenic SCG variants detected in our cohort of patients with painless-SFN have been reported in patients with epilepsy with or without neurodevelopmental delay, autism spectrum disorder, and several cardiac disorders ,25,33,34SCN9A c.2215A > G, c.3799C > G and c.4612T > C, SCN10A c.2972C > T, and SCN11A c.3473T > C were more often seen in patients with painful neuropathy, while SCN10A c.41G > T and c.4568G > A were more often seen in patients with painless neuropathy.In our cohort of 1125 patients with painful and painless DPN and painful and painless SFN, 74 potentially pathogenic variants in 92 patients were specific for a painful neuropathy, and 28 potentially pathogenic variants in 26 patients were specific for a painless neuropathy. Twenty-three potentially pathogenic variants were shared, either between painful-DPN and SFN, or painful-DPN/SFN and painless-DPN. However, n = 194/1125). The overall SCG mutation frequency for painful-DPN patients was 17.3% (n = 41/237), for patients with painless-DPN it was 14.9% (n = 46/309), for painful-SFN it was 18.5% (n = 101/547), and for painless-SFN it was 18.8% (n = 6/32).In total, 125 different  pathogenic SCG variants were detected in 194 patients  between patients with painful DPN, painless-DPN and painful-SFN  pathogenic variants were found in nful-SFN A. Data fSCN9A variants identified in our cohort of patients with painful SFN were clustered in transmembrane domains I (DI) and II (DII), and in the intracellular linker between DI and DII , the intracellular linker between DIII-DIV and the C-terminus of the protein in the patients with painful-SFN (SCN10A variants could be identified.For nful-SFN . In the nful-SFN . For patn = 28/237) for patients with painful-DPN, 7.4% (n = 23/309) for patients with painless-DPN, 11.7% (n = 64/547) for patients with painful-SFN, and 9.4% (n = 3/32) for patients with painless-SFN. For patients with painful-DPN and painful-SFN, approximately 80% of the  pathogenic variants were identified in SCN7A, SCN9A, SCN10A, and SCN11A, while for patients with painless-DPN, most variants were identified in SCN10A , no significant differences were seen for any of the patient characteristics for any of the subgroups.To provide a rationale for genetic profiling in patients with painful or painless DPN or painful or painless SFN, clinical features of the four different subgroups with and without SCG variants were compared. Except family history in patients with painful-DPN with and without SCG variants .When the comparison was corrected for variants that were not specific for a painful or painless phenotype, and the main patient characteristics were analyzed for all patients with or without SCG variants regardless of their phenotype, the patients with SCG variants more often reported a family history of neuropathy ; hot flashes . The data was further analyzed according to the presence or absence of SCG variants. The duration of neuropathy was shorter for patients with painful-DPN with SCG variants than for patients with painful-DPN without SCG variants (3.19 [SD ± 2.34] vs. 7.07 [SD ± 6.00), p < 0.001), while dry eyes were reported more often in patients with painful-SFN and painless-DPN with SCG variants than patients with painful-SFN and painless-DPN without SCG variants . All features analyzed after correction for non-specific painful or painless SCG variants are shown in Patients with and without SCG variants were grouped into those with painful or painless neuropathy. In this analysis, the occurrence of coldness and hot flashes was significantly higher in patients with painful neuropathy and SCG variants versus patients with painful neuropathy without SCG variants  neuron hyperexcitability. The pathogenic role of mutations in SCN9A, SCN10A, and SCN11A genes has triggered substantial interest in other sodium channel genes such as SCN3A, SCN7A, SCN8A, and SCN1B-SCN4B. To date, this is the first study that has investigated the presence of genetic variants in ten SCG  in a large cohort of painful and painless diabetic peripheral neuropathy (DPN) and painful and painless idiopathic small fiber neuropathy (SFN).There is a strong body of evidence implicating sodium channel genes in human painful disorders . Gain-ofn = 309) and painless-SFN patients (n = 32) were respectively 0.13 (SD ± 0.49) and 0.40 (SD ± 0.93). Of greater concern is the low number of painless-SFN patients enrolled in this study. During the inclusion period, only 32 patients with painless-SFN were seen in one of the participating centers. These patients most likely did not visit our clinics, because they do not request a diagnosis of idiopathic SFN and/or treatment of SFN-related symptoms. Due to this low number of painless-SFN patients, a comparison of clinical data of this patient group with the other 3 subgroups is unreliable and has not been performed. Data from painless-SFN patients were presented in this study only to exclude SCG variants not specific for a painful or painless neuropathy phenotype from the mutation frequencies and variant location analysis.Since no validation definition of painless or non-painful neuropathy has been established in the literature to date, in this prospective study we defined a ‘non-painful neuropathy’ as a neuropathy with a mean pain intensity <4 without analgesic drugs on PI-NRS. This cut-off of PI-NRS <4 was chosen because patients with a PI-NRS of 0 to 3 generally do not request treatment with analgesic drugs in clinical practice. Others may disagree with this definition and classify painless/non-painful neuropathy patients differently. If patients were incorrectly allocated as non-painful based on our definition of non-painful neuropathy, there is a potential risk that the interpretation of our clinical data and mutation frequencies per subgroup are biased. However, we assume that the risk is low, since the mean PI-NRS of the painless-DPN patients . Of the patients with painful neuropathy, 18.1% (n = 142/784) were heterozygous for ≥1 SCG variant, versus 15.2% (n = 52/341) of those with painless neuropathy. Ten patients harbored  pathogenic sodium channel variants in more than one sodium channel gene . Considering the presence of SCG variants, the duration of neuropathy was significantly shorter and dry eyes and hot flashes were significantly more often reported by patients with SCG variants.In our cohort of 1125 patients, we identified 125 different  pathogenic SCN9A variants were found more frequently than sodium channel variants in other genes, followed by SCN10A and SCN11A variants. These mutation frequencies for SFN patients were similar to those reported previously [SCN10A than in other genes , followed by SCN9A , SCN7A , and SCN11A . Less frequent variants (<6 variants) were identified in SCN3A, SCN8A, and SCN1B-SCN4B. The mutation frequencies for SCN3A, SCN8A, and SCN1B-SCN4B did not differ significantly between painful and painless DPN and painful idiopathic SFN.For painful-SFN,  pathogenic eviously ,4,11,18.SCN9A, SCN10A and SCN11A genes were 3.1% , 2.6%  and 2.6%  respectively, in the patients with painful neuropathy, while they were 0.9% , 3.2% painful-SFN, 3.2%, n = 10/309; painless-SFN, 3.1%, n = 1/32) and 0.9% , respectively, in the patients with painless neuropathy. Our data contrasts with the study by Wadhawan et al. (2017), who in a cohort of 457 patients showed no significant difference in the missense variant allele frequencies for SCN9A, SCN10A, and SCN11A between patients with painful and non-painful neuropathy [SCN9A variants were identified in patients with both painful-DPN and painless-DPN, whereas Blesneac et al. (2018) had found that 10 out of 111 patients with painful-DPN harbored rare Nav 1.7 variants  compared to none of their patients with painless-DPN (n = 78) [After correcting our data for variants that were not specific for a painful or painless phenotype, we show that the frequencies of  pathogenic variants in uropathy . Several(n = 78) . These dShared SCG variants were identified between painful-DPN and painful-SFN, painless-DPN and painless-SFN, as well as painful-DPN/SFN and painless-DPN/SFN. These findings suggest that a single genetic variant may be associated with different phenotypes depending on the presence or absence of other genetic variants in the SCG or other genomic regions and environmental factors. As several of these variants have been reported as gain-of-function mutations ,17,18, wKCNS1, SCN9A, ADRB2, H2TRA, CACNG2, and IL16 play a role in increasing the risk of chronic pain or an increase in pain sensitivity [KCNQ2, KCNQ3, KNQ3, COMT, OPRM1, TRPV1, MC1R, GCH1, and CACNA2D3 genes that have been associated with pain protection or pain resilience [A number of studies have shown that rare variants in genes belonging to sodium, calcium, potassium, and transient receptor potential channel gene families are implicated in determining the pain risk threshold and sensitivity ; while ositivity ,39,40. Tsilience ,42. Moresilience ,44.SCN9A and SCN10A variants in our painful-SFN patient cohort to specific protein domains was similar to that found in previously published data [SCN9A and SCN10A variants was also seen in patients with painless-DPN; however, it was located at different regions of SCN9A and SCN10A in our painful-SFN cohort, in DII and DIII of Nav1.7, and DI and DII of Nav1.8. In our cohort with painful-DPN, no hotspot regions were identified for SCN9A and SCN10A variants. After exclusion of the non-specific painful and painless SCG variants, the localization of SCN9A variants detected in the patients with painful-SFN and painless-DPN did not change. For SCN10A, the majority of variants identified in the patients with painful-SFN were localized to DIV, instead of DIII, DIV, the linker between DIII-DIV and C-terminus of the protein, whilst in patients with painless-DPN, the variants were located across the whole gene. Functional studies have shown that SCG variants are associated with both gain-of-function and loss-of-function leading to the impaired inactivation and hyperexcitability or hypoexcitability of neurons [The localization of identified hed data . The clu neurons ,45,46,47SCN9A, SCN10A and SCN11A variants [In our cohort, males were represented more in DPN than SFN, and the patients with DPN were older compared to patients with SFN. There was no difference in the duration of neuropathy between patients with DPN or SFN, although the duration of neuropathy was shorter in patients with painless compared to painful neuropathy. Of note, coldness and hot flashes were experienced more often by patients with painful neuropathy with SCG variants compared to those with painful neuropathy without SCG variants. Also, the unpleasantness of symptoms was higher in patients with painless neuropathy and SCG variants compared to patients with painless neuropathy without SCG variants. Furthermore, a complaint of dry eyes was reported more often in painful-SFN and painless-DPN patients with SCG variants rather than those without SCG variants. The duration of neuropathy was shorter in patients with painful-DPN and SCG variants than in patients with painful-DPN without SCG variants. This finding is in line with the results reported by Blesneac et al., who found that participants carrying rare Nav1.7 variants had been diagnosed for a significantly shorter duration . Howevern = 1325) were recruited in four different European centers from June 2014 until September 2016. Patients with DPN were recruited for this study at the University of Manchester (United Kingdom) and the Deutsche Diabetes Forschungsgesellschaft EV (Germany), and patients with SFN were recruited at the Fondazione IRCCS Instituto Neurologico “Carlo Besta”  and the Maastricht University Medical Center+ (The Netherlands). The local medical Ethical Committees of each center approved this study. Informed consent was given by patients to participate in this study.In this prospective study, patients (Patients who met the following inclusion criteria were eligible to participate in this study: (1) A diagnosis of sensory neuropathy, based on established clinical and neurological examination, nerve conduction studies (NCS), quantitative sensory testing (QST) and/or skin biopsy findings  with diaDPN patients were included in the study if the diagnosis of DPN was established based on clinically confirmed diabetes, clinical signs of sensory neuropathy, and abnormal NCS, and the absence of the previous mentioned known causes of neuropathy. If NCS was normal (no large fiber involvement), decreased IENFD was required to confirm DPN. Patients without confirmed DPN were excluded from the study.Idiopathic SFN patients were included in the study if the diagnosis SFN was established based on clinical signs of sensory neuropathy, decreased IENFD on skin biopsy and/or abnormal QST, with no evidence of large nerve fiber damage on neurological examination and NCS, and the absence of the previous mentioned known causes of neuropathy. If IENFD was normal, abnormal QST was required to confirm SFN. Patients without confirmed SFN were excluded from the study.Patients with pain, categorized as possible NeuP by Treede et al., 2008 , or <1 yAs at the start of this study no validated definition of ‘non-painful neuropathy’ was established in the literature, we defined DPN or SFN patients with NeuP by Treede et al., 2008   at the feet to determine cold, warm, heat-pain and cold-pain thresholds , skin biStudy participants were then allocated into four groups based on inclusion criteria 1 and 2: painful-DPN, painless-DPN, painful-SFN and painless-SFN, where idiopathic SFN is addressed as ‘SFN’, NeuP (possible NeuP excluded) for >1 year with PI-NRS ≥ 4 as ‘painful’, and no pain/NeuP (possible NeuP excluded) for >1 year with PI-NRS < 4 as ‘painless’ .Subsequently, DNA was isolated from blood (2 × 6 mL EDTA blood) for genetic analyses, and 10 qualities of neuropathic pain were evaluated on the 11-point Neuropathic Pain Scale  , and 13 Except for IENFD, which was optional for DPN patients, all tests were performed for all subgroups.® Blood Core Kit  or NucleoSpin® 8 Blood Isolation kit  according to the manufacturer’s instructions. Coding exons and exon-flanking intron sequences of SCN3A, SCN7A-SCN11A, and SCN1B-SCN4B were sequenced by smMIP-NGS. Three-hundred-twenty-four smMIPs were designed using a modified version of MIPgen (http://shendurelab.github.io/MIPGEN/ (accessed on 1 August 2014)). The gap-fill length between the extension—and ligation arm (region of interest) of the smMIPs was fixed at 220–230 nt. Probes were synthesized by Integrated DNA Technologies .Genomic DNA was extracted from whole blood using a QIAamp DNA Blood Maxi Kit/Puregenen = 9; coverage <30×, high false positive variant calling) were analyzed by Sanger sequencing [Targeted capture with smMIPs was performed according to standard protocols . In briequencing .SCN3A, SCN7A-SCN11A, and SCN1B-SCN4B were compared with the reference sequence GRCh37. Variants detected were annotated according to the guidelines of the Human Genome Variation Society (http://www.hgvs.org/varnomen/ (accessed on 13 April 2015)), and classified according to current classification guidelines [Patients’ coding and immediate flanking regions of idelines  using AlThe primary analysis was performed to compare patients with painful SFN, painless SFN, painful-DPN, and painless-DPN with and without SCG variants for gender, age, age of onset of neuropathy, duration of neuropathy, familial history of neuropathy, 10 qualities of neuropathic pain evaluated by NPS, and 13 SFN-related symptoms assessed by SFN-SIQ, and to measure the frequencies of the identified SCG variants in the tested patients.t-test was chosen. Equal variances between two groups were tested with the Levene’s test. For these analyses, a significance level of 0.05 was used.For categorical variables, the ÷2 test was used or the Fisher’s exact test when necessary. For continuous variables, the independent student’s Post hoc analyses were performed to investigate whether differences between patients with specific SCG variants and without variants were present. In total, six post hoc analyses per variable were executed. The analyses were performed in the same way as the primary analyses; however, the significance level was adjusted for multiple testing with the Bonferroni correction.In this large cohort of 1125 patients with painful-DPN and painless-DPN and painful-SFN and painless-SFN, 18.1% of patients with painful neuropathy and 15.2% of patients with painless neuropathy carried a potential pathogenic SCG genetic variant. The presence of SCG variants in patients with painful neuropathy was associated with symptoms of coldness and hot flashes. Furthermore, the occurrence of unpleasantness was higher in patients with painless neuropathy with SCG variants.The phenotypic expression and management of NeuP is undoubtedly complex. SCG variants appear to play a key role in the development of NeuP. As we move forward with applying the genetic testing of SCG, an exclusive wealth of data concerning DNA variations, deeper characterization of rare and common SCG variants, improved classification of disease-associated variants, and understanding of the functional consequences of many of these variants will be reached. Consequently, the utility of genetic screening of SCG for routine clinical use will rise, and possibilities for more effective disease treatment, such as the development of selective sodium channel blockers and optimal therapeutic modulation and pain relief, can be achieved. In addition, identifying the underlying genetic mechanism in families with NeuP has great clinical relevance, will improve genetic counseling, and may result in better clinical management in the respective families. Therefore, the genetic screening of SCG should be considered for patients with painful and painless diabetic peripheral neuropathy and painful and painless idiopathic small fiber neuropathy.


Despite the notable progress made in recent years, the understanding of the genetic control of gonadal sex differentiation and asymmetrical ovariogenesis in chicken during embryonic development remains incomplete. This study aimed to identify potential key genes and speculate about the mechanisms associated with ovary and testis development via an analysis of the results of PacBio and Illumina transcriptome sequencing of embryonic chicken gonads at the initiation of sexual differentiation . PacBio sequencing detected 328 and 233 significantly up-regulated transcript isoforms in females and males at E4.5, respectively. Illumina sequencing detected 95, 296 and 445 DEGs at E4.5, E5.5, and E6.5, respectively. Moreover, both sexes showed asymmetrical expression in gonads, and more DEGs were detected on the left side. There were 12 DEGs involved in cell proliferation shared between males and females in the left gonads. GO analysis suggested that coagulation pathways may be involved in the degradation of the right gonad in females and that blood oxygen transport pathways may be involved in preventing the degradation of the right gonad in males. These results provide a comprehensive expression profile of chicken embryo gonads at the initiation of sexual differentiation, which can serve as a theoretical basis for further understanding the mechanism of bird sex determination and its evolutionary process. SRY gene (sex-determining region Y) located on the Y chromosome to determine maleness, chickens employ a different strategy, possessing a ZW sex chromosome system, where females carry two different sex chromosomes (ZW) and males carry two of the same (ZZ) [Chickens and most other birds are sex-determined genetically via sex chromosomes. Unlike mammals, which employ a XX/XY sex chromosome system and rely on the presence of the ame (ZZ) . There aame (ZZ) ,3. The fame (ZZ) ,5. Even HINTW [FOXL2 [CYP19A1 [WNT4/RSPO1 [DMRT1 [SOX9 [AMH [HEMGN [AMH and SOX9. Ioannidis et al. [DMRT1-mutant chicken using CRISPR/Cas9, resulting in male chicken (ZZ) developing ovaries instand of testes, which proved that chicken sex fate is dependent on DMRT1 dosage and involves the regulation of estrogen signaling. In female chicken embryos, in the absence of a substantial DMRT1 expression, FOXL2 begins to be expressed at E5.5 and subsequently activates CYP19A1, further promoting the estrogen synthesis pathway [Wnt4, which stimulates the proliferation of follicular granulosa cells and also participate in chicken follicle selection [CAPN5, GPR56, and FGFR3, which potentially play significant roles in ovary development. However, the precise functional interactions among these genes are still unknown, and the existence of additional potential key candidates involved in sex determination remains uncertain.During embryonic development, gonads undergo morphological differentiation to form testes or ovaries. In chicken embryos, genital ridges begin to be generated in the anterior region of mesonephros on embryonic day 3 (E3-Hamburger and Hamilton stage 18) ,7, and sW [FOXL2 ,10, CYP1[CYP19A1  and WNT4T4/RSPO1 ,13, whic1 [DMRT1 , SOX9 [1T1 [SOX9 , or AMH OX9 [AMH , which aOX9 [AMH ,17, subs pathway ,12. Anotelection  conductePITX2 exhibits asymmetric expression in the female left cortex at an early stage (stage 18), which stimulates the proliferation of left cortex cells by regulating the expression levels of RALDH2, Ad4BP/SF-1, ERα, and cyclin D1 (stage 27) [Nanog, PouV and Cvh, which are related to the proliferation of primordial germ cells, (PGCs) [Besides differential expression of sex-related genes, there are also distinctions in gonadal morphology between the two sexes. In contrast to mammals, which develop a pair of either testes or ovaries, most female birds only develop ovaries on the left side, with the right side undergoing degeneration, while males develop bilateral testes . After stage 27) . Additio, (PGCs) . On the In this study, we collected chicken embryo gonads during the critical periods of sex differentiation at E4.5, E5.5, and E6.5. Total RNA was extracted and subjected to sequencing using the next-generation sequencing technology Illumina and the third-generation sequencing technology PacBio. Subsequently, we conducted an analysis of gene expression profiles to identify differentially expressed genes associated with sex bias and asymmetrical gonadal development.The total RNA of both sexes of chicken gonads at E4.5 was sequenced using the PacBio sequencing platform. We obtained 475,794 and 502,803 reads of inserts (RoI) for females and males, respectively. After clustering and merging, 13,178 and 14,813 collapsed isoforms for females and males were gained, respectively. Moreover, 99% of them were mapped to the genome. In total, 5593 genes for females and 6004 genes for males were annotated, and the total gene number was 6943 after merging. Additionally, 2334 and 2622 novel genes were unannotated for females and males, respectively .EIF4G2, HNRNPK and SPTLC1 showed male bias, and each of them had three different differentially expressed (DE) transcript isoforms. In females, HINTW, HNRNPKL, and HNRNPH2 also exhibited this type of expressive characteristics. Especially HINTW had 11 different DE transcript isoforms. Interestingly, 64 genes had up-regulated expression with different transcript isoforms in both sexes, including HMGB3, IST1, USP10, HMMR, etc. , 4 on mitochondria, and 337 on autosomes  database. 481 GO terms were significantly enriched , which wTo investigate the gene expression profiles in key sex-differentiated stages and analyze the sex determination mechanism, we respectively collected and extracted RNA from 210 paired gonads at developmental stages E4.5 , E5.5 , and E6.5 . The morphological characteristics of chicken embryonic gonads in these three stages are depicted in HINTW gene exhibited the highest expression levels in females, while PRLR (prolactin receptor) demonstrated the highest fold change in males. Secondly, at the later developed stage of E5.5, although sexual differentiation had not yet occurred, 296 genes showed significant differential expression between the two sexes, with 134 being up-regulated in females and 162 in males  compared to E5.5. In general, as the chicken embryos advanced in development, the total number of DEGs increased. At these three stages, the proportion of autosomal DEGs progressively rose, transitioning from 20% at E4.5 to 50% at E5.5, and peaking at 71% at E6.5  in left gonads included MSC, PIP5K1B, DAZL, NKAIN4 and CLDN1. Notably, PIP5K1B and DAZL are associated with the regulation of several cellular processes or germ cell growth. NKAIN4 and CLDN1 are related to the maintenance of the basic energy and protein required for cell growth. In the right gonad, one of the highly expressed DEGs was SLC25A37, which is involved in the negative regulation of angiogenesis and blood vessel endothelial cell migration. This indicated that these DEGs that collectively promote cell development and concurrently exert a negative regulatory influence on angiogenesis, thereby might contribute to the asymmetric development of female gonads. With regard to male left gonads at E5.5, a total of 151 DEGs were identified, with PIP5K1B exhibiting the highest expression levels among the DEGs at this developmental stage. At E6.5, there was a slight reduction in the number of DEGs, with a total of 173 DEGs identified. Meanwhile, the DEG count of the male left and right gonads dramatically increased to 388. Especially, HBA1, HBAD, HBBR and HBZ exhibited extremely high expression levels (FPKM > 4000). Furthermore, we observed that 46 DEGs in the left gonads exhibited overlapping expression across all three developmental stages in females, whereas only 2 exhibited overlap on the right side. In males, 19 DEGs in the left gonad displayed overlapping expression across these stages, while the right side had only 1 such DEG  in the left gonad included  protein , muscle such DEG . SurprisP2RY8 (purinergic receptor P2Y8), was found to be involved in blood coagulation and hemostasis pathways, suggesting that the apoptosis of right gonadal cells may have commenced at E4.5 and due to potential disruption of blood circulation. In males, a total of 1655 pathways were significantly enriched at the three stages. Among these pathways, 694 demonstrated significant enrichment at E4.5, while 450 pathways displayed significant enrichment at E5.5. At the two gonadal undifferentiated stages, DEGs were primarily involved in cellular communication and signaling pathways, tissue/cell morphogenesis, molecular regulations, etc. These pathways included cell-cell signaling (GO:0007267), cell communication (GO:0007154), blood vessel morphogenesis (GO:0048514), node of Ranvier (GO:0033268) and so on. Until the gonadal differentiation stage (E6.5), several pathways related to blood oxygen transport/binding, cell proliferation or differentiation, and signal transmission began to show significant enrichment. This included pathways such as hemoglobin complex (GO:0005833), oxygen transport (GO:0015671), cell morphogenesis (GO:0000902). A noteworthy discovery among the overlapping DEGs across all three stages in the left gonad of males was the highest expression of PIP5K1B, a gene previously linked to spermatogenesis in male mice. Previous research showed that PIP5K1B-knockout male mice exhibited sterile, confirming its role in spermatogenesis [GO enrichment analysis was conducted to elucidate the pathways involved in the asymmetric development of chicken gonads . In femag GO:00077, cell cZFAND5 and DMRT1 were male up-regulated DEGs; BTG1 and SNRPB2 were female up-regulated DEGs; PIP5K1B, SCAPER and C2orf88 were left gonadal up-regulated DEGs; CPNE8, C7, NPHS2 and P2RY8 were right gonadal up-regulated genes. The qRT-PCR results were consistent with the sequencing results  expressed two different transcript isoforms in both sexes. UBE2B encodes a ubiquitin-binding enzyme highly conserved in eukaryotes, and it is essential for DNA damage repair after replication. UBE2B has been validated to be implicated in spermatogenesis in male mice [UBE2B potentially has a repair effect on sex chromosome DNA replication and contribute to chicken gonadal development. Previous studies primarily focused more on the effect of sex chromosome genes in initiating sex differentiation [Notably, the full-length sequencing results of E4.5 gonads displayed that a significant portion of the up-regulated transcripts in females originated from the W chromosome, especially, certain genes expressed multiple transcript isoforms. For instance, lication , as welland SOX2 . In addiale mice , suggestntiation ,32,33. HDMRT1 is the key sex-determining gene dominating male sex differentiation [PRLR was the only overlapped DEG in the three stages in males. The PRLR gene, which is conserved in various species including mice, zebrafishes, dogs and humans, encodes the receptor of prolactin, an anterior pituitary peptide hormone essential for reproduction. The PRLR-knockout female mice displayed lactation failure or multiple reproductive abnormalities [PRLR is located on the Z chromosome and has been proven to be associated with broodiness and egg production, making it a potential molecular marker for chicken breeding [PRLR in male chicken embryos is related to sex determination. Moreover, our results suggest that female pathways exhibit more significant differential activity, with a higher number of DEGs, especially marked by a high expression of the W-linked gene HINTW. HINTW is expressed in multiple tissues of female chicken embryos [et al. [HINTW gene via retrovirus in chicken embryos did not reverse the sex of male embryos and had no influence on the normal development of female embryos. They also proposed that the protein encoded by HINTZ, a Z chromosome-homologous gene, could form a dimer with HITNW in vitro, thereby reducing the role of HINTZ in promoting testicular development [Although numerous studies have demonstrated that the Z-linked gene ntiation , interesmalities . In chicbreeding . However embryos  and has  [et al.  reportedelopment ,38. In aPITX2, the distribution of retinoic acid (RA) becomes uneven, resulting in a lack of meiotic division and subsequent apoptosis in PGCs of the left medullary and right gonadal regions following hatching. In our sequencing data, we found the asymmetrical expression of genes consistent with the asymmetrical development of gonads. However, both female and male left gonads differentially expressed plentiful genes, including PITX2. The PITX2 gene is conserved in multiple species and is involved in the asymmetric expression of the left-right axis. PITX2-knockout mice developed organ-asymmetric abnormalities [PITX2 is highly expressed in the female left gonadal cortex and up-regulates RALDH2 , ERα and cyclin D1 to stimulate cell proliferation [PITX2 in the right gonad can induce cortical differentiation and prevent degeneration [PITX2 may be the key gene that induces the asymmetric development of chicken ovaries. Estermann et al. [CYP17A1 serves as a steroidogenic marker and belongs to the theca cell lineage genes which possess the capability to synthesize substrates for androgens. In our research, we observed a significant up-regulation of CYP17A1 in male left gonads at E6.5, suggesting a potentially higher abundance of male left gonadal germ cells compared to those in the right gonads. Their study also mentioned DAZL as a marker for germ cells primarily localized within the cortical area. In females, the left gonadal cortex undergoes development while the right gonadal cortex regresses. Our sequencing results consistently demonstrated an up-regulated expression of the DAZL gene in the left gonads of females at E4.5, E5.5, and E6.5, aligning with previous research findings.Before the onset of sex differentiation, the gonadal morphology in both sexes is similar, and slight differences between male and female can be observed after E6–6.5. Primitive gonads contain two distinct layers, the cortex and medulla . Witschimalities . PITX2 iferation . Previouneration . Therefon et al.  also repPITX2, GDF8, LOC422926, etc. This observation suggested that both male and female gonads developed left-right asymmetry in the early stages, potentially sharing similar underlying mechanisms. However, several genes exhibited significantly higher expression and up-regulated in male right gonads at E6.5, which were related to blood oxygen transport, such as HBA1, HBAD, HBZ and HBBR. These genes were highly expressed in both male and female gonads at E4.5 and E5.5, with no significant differences between the left and right gonads, and their expression levels decreased as incubation progressed. Nevertheless, these genes were up-regulated in the male right gonad at E6.5, accompanied by a notable increase in fold change, suggesting that male chicken embryos may employ these blood oxygen transport pathways in the right gonads to prevent degeneration as they continue to develop. We have generated a hypothetical pathway diagram depicting the putative mechanism of asymmetric development in chicken embryos based on the DEGs analysis and enriched pathways in our study  exhibited ovarian morphology, where the right side underwent degeneration, while the left side developed normally. Although this study did not explore the phenomenon of gonadal asymmetric development after sex reversal, the results suggested that male chickens without one allele of DMRT1 did not develop symmetrical ovaries. This indicated that DMRT1, and potentially other male development genes, may also be involved in testicular symmetrical development.While most studies have primarily focused on elucidating the mechanisms behind the asymmetric development of the female gonads ,43, compur study . Besides  fertilized White Leghorns eggs  were incubated at 60–65% humidity and 37.8 °C in an OVA-Easy 380 incubator  until E4.5, E5.5, and E6.5. Under a microscope, the right and left gonads of the embryos at each stage were collected with sterilized microsurgical tweezers and scissors, and placed into 1.5 mL RNase-free centrifugal tubes individually. We collected 104, 55, and 51 paired gonads at E4.5, E5.5 and E6.5, respectively. Paired gonads were stored in a refrigerator at −80 °C. Additionally, a piece of extragonadal tissue from each embryo tissue was obtained in the meantime, for the subsequent sexing experiments.CHD1 [ATCB (β-actin). The sequences of the primers were as follows:The DNA extraction of extragonadal tissues was individually performed using an animal tissue DNA extraction kit  and sexing was performed using a PCR Taq master mix  with a primer of the sex-related gene CHD1  and the CHD1-F: 5′-ATCTACCACTTTT CTCACGG-3′;CHD1-R: 5′-TTCAGAGTGATAACGCATGG-3′;β-actin-F: 5′-TGG ATG ATG ATA TTG CTG C-3′;β-actin-R: 5′-ATC TTCTCC ATA TCA TCC C-3′.Using CHD1 as the sexing primers, the presence of two bands (400–500 bp and 300–400 bp) on the gel electrophoresis image indicated female embryos, while the presence of a single band (400–500 bp) indicated male embryos. After sexing, homolateral gonads of the same stage and sex were pooled , and total RNA was isolated using Trizol Reagent . RNA purity (OD260/280) was measured using a NanoDrop spectrophotometer .In PacBio sequencing, the total RNA of E4.5 gonads was reverse-transcribed to cDNA using SMARTer PCR cDNA Synthesis Kit . After PCR amplification, BluePippin was used to select long-length fragments. Library construction was conducted using SMRTbell™ Template Prep Kit 1.0 , and then was sequenced using Sequel™ Sequencing Kit 2.0. During Illumina sequencing, mRNA was separately purified from the total RNA of E4.5, E5.5, and E6.5 gonads using poly-T oligo-attached magnetic beads. cDNA was synthesized using random oligonucleotides and Super Script II for the first strand, and DNA Polymerase I and RNase H for the second strand. After the adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated. Fragments were purified using the AMPure XP system  for selecting 400–500 bp of cDNA, then PCR-amplified using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction and quantified using the Bioanalyzer 2100 system . The sequencing library was sequenced on Illumina NovaSeq 6000 platform. All transcriptome sequencing was performed by Shanghai Personal Biotechnology Cp. Ltd. .Gallus gallus reference assembly GRCg6a. Raw data (fastq file) from Illumina sequencing were generated using the software of the sequencing platform, the Cutadapt (v1.15) software [The reference chicken genome was the software , to filtsoftware , and trasoftware ; then, wsoftware , annotatp-value < 0.05 and |log2 foldchange| > 1. We used the R language Pheatmap (1.0.12) software package [The differentially expressed genes were analyzed using the DESeq2 R package . The cri package  to perfop-value was calculated via a hypergeometric test to confirm the main biological functions of the DEGs. GO terms with a p-value of <0.05 were considered significantly enriched.All DEGs were mapped to the terms of the GO database, and we calculated the numbers of differentially enriched genes in each term. GO enrichment analysis was performed using topGO for DEGs from Illumina and eggNOG-mapper for DEGs from PacBio . The p-vAdditional 300 paired chicken embryos gonads at E4.5, E5.5 and E6.5 were collected separately for verifying the DEGs of males/females and left/right gonads via qPCR. After sex identification, about 30 homolateral gonads were pooled for each biological replicate  and RNA was extracted using Fast Pure Cell/Tissue Total RNA Isolation Kit (Vazyme) in accordance with the manufacturer’s instructions.β-actin using the 2−∆∆Ct method [qRT-PCR analysis was performed using the CFX Connect Real-Time PCR detection system  and ChamQ Universal SYBR qPCR Master Mix . The primer sequences are listed in t method  and grapThis study investigated sex-biased gene expression and dynamic transcriptome changes during chicken sex differentiation. PacBio and Illumina sequencing platforms were used to sequence gonadal transcriptomes at different developmental stages. The analysis revealed the significant differential expression of genes between males and females, with sex chromosomes playing a crucial role. As gonads differentiate, autosomal genes also contribute to sex-specific expression. Enrichment analysis highlighted the genes involved in DNA, RNA, and protein synthesis, as well as pathways related to sex determination and gonad development. Left-right asymmetric gene expression was also explored. Overall, the study provides insights into the molecular mechanisms underlying avian sex determination.