Waldenström Macroglobulinemia (WM) is an indolent lymphoplasmacytic lymphoma, characterized by the production of excess immunoglobulin M monoclonal protein. WM belongs to the spectrum of IgM gammopathies, ranging from asymptomatic IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), through IgM-related disorders and asymptomatic WM to symptomatic WM. In recent years, its complex genomic and transcriptomic landscape has been extensively explored, hereby elucidating the biological mechanisms underlying disease onset, progression and therapy response. An increasing number of mutations, cytogenetic abnormalities, and molecular signatures have been described that have diagnostic, phenotype defining or prognostic implications. Moreover, cell-free nucleic acid biomarkers are increasingly being investigated, benefiting the patient in a minimally invasive way. This review aims to provide an extensive overview of molecular biomarkers in WM and IgM-MGUS, considering current shortcomings, as well as potential future applications in a precision medicine approach. Molecular biomarkers are broadly used for diagnosis, treatment selection and disease monitoring in many clinical settings ,2. In thWM belongs to the spectrum of IgM gammopathies, encompassing a wide and heterogeneous group of hematological conditions, ranging from asymptomatic IgM monoclonal gammopathies of undetermined significance (IgM-MGUS), through symptomatic IgM related disorders , to asymptomatic WM (aWM), and ultimately to symptomatic WM ,10,11,12Patients with IgM-MGUS can progress to WM at a rate of 1.5% to 2% per year . ApproxiMYD88L265P) in 90% of WM and 10% of IgM-MGUS cases .CXCR4 gene  [CXCR4 mutations, is a non-sense C > A or C > G transversion in a highly conserved region at nucleotide position 1013, responsible for the generation of a stop codon (S338X), resulting in the loss of 15 amino acids at the C-terminal region of the CXCR4 protein [S338XCXCR4 nonsense mutations affect the expression and activity of CXCR4 mainly through the PI3K-AKT-NF-κB and the MEK1/2 and ERK 1/2 pathways, involved in cell proliferation, migration, and survival [More than 40 non-sense (NS) or frameshift (FS) mutations have been observed in the XCR4MUT) ,87. The  protein ,89. Thessurvival ,91.MUTCXCR4 patients harbor L265PMYD88, suggesting the subclonal nature of MUTCXCR4 with respect to L265PMYD88 acquisition, and only rare cases of MUT/MYD88WTCXCR4 have been reported  (UVRAG gene), tumor suppression , chromosome instability , cell cycle regulation (SCAPER) and post-translational protein modifications (LNX1 and DCUN1D4). However, further analysis of 46 WM patients did not show a significantly different distribution of these mutations between WTCXCR4 vs. MUTCXCR4 patients [In a recent case study in a patients .L265PMYD88 and MUTCXCR4 were initially assessed on CD19+ (CD19-selected cells) BM samples using allele-specific quantitative polymerase chain reaction (AS-qPCR) and Sanger sequencing. Subsequently, many studies have used unselected BM samples and distinct assays and methods with different levels of sensitivity  (63% vs. 16%) [L265PMYD88 detection (98% and 69% respectively), confirming the subclonal nature of S338XCXCR4 but also indicating a direct dependence of MPS performance on the level of BM involvement [As of today, a gold standard molecular method for ratories ,49,60,97vs. 16%) . Additioolvement ,67,92.L265PMYD88/WTMYD88 ratio might also be proposed as a quantitative marker and useful diagnostic tool for MRD analysis. Recently, digital PCR (dPCR) has been described as more sensitive than AS-qPCR across different specimen types (including plasma-cfDNA), for L265PMYD88 screening and MRD analysis, suggesting that the implementation of dPCR assay in routine diagnostic laboratories might avoid the need for CD19+ selection [Consistently, a strong correlation between the mutational burden and the number of pathological cells has been demonstrated in unsorted material ,42. Therelection ,99.Small CNAs involving B-cell regulatory genes are highly prevalent in WM . Paiva eThe 6q21 deletion (del(6q)) is the most frequent cytogenetic aberration and is detected by fluorescence in situ hybridization (FISH) in up to 30–50% of WM cases ,103,104.CXCR4 mutations are mutually exclusive, conflicting data have been reported [CXCR4 mutations, as detected by chromosomal banding analysis (CBA), FISH, and targeted MPS. Moreover, a significantly more complex karyotype was shown in patients with del(6q) [So far, limited and discordant data linking molecular and cytogenetic information are available. Despite the initial observation that del(6q) and reported ,84,103.  del(6q) .Cytogenetic studies may be useful to detect del(6q) as well as other abnormalities that might aid in differential diagnosis and outcome prediction . HoweverARID1A (17%), CD79B, KMT2D (or MLL2), MYBBP1A and TP53 (<15% of cases each)  C [26,27,KMT2D (16%), TP53 (8%), NOTCH2 (7%), PRDM1 (4%), ARID1A (3%), CD79B (3%) and TRAF3 (1%), while no mutations were found in MYBBP1A and TNFAIP3 [KMT2D mutations was significantly higher in WM compared to IgM-MGUS patients. A subsequent study evaluating the 12 most frequently mutated genes confirmed an increased mutational load in different stages of monoclonal gammopathies: 21% in IgM-MGUS , 35% in aWM (by 8/12 genes) and 50% in symptomatic WM (by 12/12 genes) [Using MPS in a large series of WM and IgM-MGUS patients, Varettoni et al. (2017) demonstrated subclonal mutations in  TNFAIP3 . Of note2 genes) . A recen2 genes) . This fi2 genes) .MYD88 and CXCR4 mutations have both diagnostic and therapeutic implications in WM. So far, four distinct subsets of WM patients with peculiar clinical features, different outcomes and drug responses have been identified: L265PMYD88/MUTCXCR4, L265PMYD88/WTCXCR4, WTMYD88/WTCXCR4 and WTMYD88/MUTCXCR4.It has been widely demonstrated that L265PMYD88/S388XCXCR4 patients show a higher BM disease burden, higher serum IgM levels and are more likely to have symptomatic disease compared to L265PMYD88/FS-MUTCXCR4, L265PMYD88/WTCXCR4 or WTMYD88/WTCXCR4 patients that show respectively a lower and the lowest (WT/WT) BM disease involvement. Discordances between studies in WM have been reported, but the most supported observation is that L265PMYD88/MUTCXCR4 patients show lower, later and less deep responses to BTK inhibitors (mainly ibrutinib), eventually resulting in shorter PFS compared to L265PMYD88/WTCXCR4 patients [WTMYD88/WTCXCR4 cases, on the other hand, show resistance to targeted drugs  and are characterized by an increased risk of disease transformation to high-grade lymphoma or of developing a therapy-related myelodysplastic syndrome (t-MDS), both leading to a poor OS [WTMYD88 patients with DDR mutations represent a subgroup with ultra-high-risk disease [WTMYD88/MUTCXCR4 patients [patients ,113. MYD poor OS ,114,115. disease ,114. Laspatients .WTMYD88 patients, by showing overall and major response  rates comparable to L265PMYD88 cases [Recently, multicenter phase II and III trials comparing the efficacy and safety of novel BTK inhibitors to ibrutinib, hinted at a higher efficacy of acalabrutinib and zanubrutinib in a small subset of 5P cases ,118,119.ARID1A mutations are associated with greater tumor involvement [CD79B are mainly observed in L265PMYD88/MUTCXCR4 patients, with the exception of two studies in which the co-expression of CD79B and MYD88L265P/WTCXCR4 was associated with disease transformation and progression [SPI1 and TP53 mutations have been associated with aggressive disease course and shorter OS. Data concerning the coexistence of TP53 mutations with both L265PMYD88 and MUTCXCR4 and the activity of ibrutinib in this group of patients are conflicting [Within the uncommon mutations, olvement . Mutatiogression ,120. Desflicting ,111,121.flicting ,105,110.Approximately 80% of the human genome is transcribed into RNA, of which only 1.5% is protein-coding mRNA, with the rest being termed non-coding RNA (ncRNA). Current classifications differentiate between short ncRNAs  and large ncRNAs  . RNA molGene expression profiling studies showed that lymphoplasmacytic WM cells (CD138+/CD19+) have a homogeneous transcription profile with an mRNA signature that resembles CLL and normal B cells but clearly differs from MM and normal plasma cells (NPC). Both WM and CLL are indolent lymphomas that are likely to be derived from memory B-cells, which might explain the similar B-cell-like signature ,124. AmoCXCR4 and its ligand CXCL12 have been reported to increase cell adhesion to VCAM1, which might explain the homotypic cell clustering in WM patients [As the WM clone is comprised of B lineage cells ranging from B lymphocytes (BL) to plasma cells (PC), several studies have compared separate expression profiles of clonal CD19+ WM B-cells (WM-BL) and CD138+ WM plasma cells (WM-PC) with their respective normal counterparts . Using MPS, Hunter et al. presented the first transcriptional landscape of WM-BL compared to NBL. Upregulated genes included RAG1, RAG2, DNTT and IGLL1, involved in VDJ recombination, BCR signaling and somatic mutation. The class switch recombination gene AICDA was not observed, which is consistent with the lack of immunoglobulin class switching. Furthermore, upregulation of patients . Gutiérrpatients . Of intepatients . Lastly,patients . Increaspatients .WTMYD88 patients were relatively heterogeneous with downregulation of NF-κB signaling-associated genes and upregulation of PIK3 signaling. Of the 1155 genes that were differentially expressed between L265PMYD88 and WTMYD88 patients, only 603 were identified in the L265PMYD88/WTCXCR4 vs. L265PMYD88/WHIMCXCR4 signature. As WHIMCXCR4 mutations are found almost exclusively in L265PMYD88 patients, WHIMCXCR4 mutations appear to counteract tumor suppressor upregulation in response to mutant L265PMYD88 signaling, as evidenced by the normalization for TLR4 signaling associated gene expression and upregulation of IRAK3. In the L265PMYD88/CXCR4WT genotype, a marked increase of GPER1, WNT5A, IGF1 and IL17RB expression was shown, in which IL17RB and IGF1 activate NF-κB and AKT1/MAPK signaling, respectively [L265PMYD88/CXCRWHIM4 patients were the upregulation of CXCR7 and TSPAN33, as well as suppression of IL-15 [NF-κB negative regulator HIVEP2, as well as BCLAF1, FOXO3 and ARID1B [Regarding the impact of recurrent genetic alterations on transcriptional regulation, a study showed that expression profiles in ectively ,131. Theof IL-15 . Concernof IL-15 . This isd ARID1B .CXCL13, TP53, CXCR4, MYD88, CDC23 and AKAP1 were significantly associated with BM disease involvement [Regarding their potential use as biomarkers in WM, a study has shown that the level of mRNA expression was greatly dependent on the extent of BM involvement, with the expression profile of cases with low infiltration, clustering with normal plasmacytes . In the olvement  (Table 4MicroRNAs (miRNAs) are short non-coding RNAs of ~22 nucleotides that play essential roles in almost all biological pathways, negatively regulating gene expression by targeting mRNA, typically at the 3′-untranslated region. Since miRNAs can target up to several hundred mRNAs, aberrant expression can influence a multitude of cell signaling pathways, including cancer onset and progression . SeveralIRS-PI3K signaling pathway that plays a role in the growth and survival of WM cells: miR-29c (PIK3R1); miR-155 (SHIP1); miR-21 ; miR-145 (IRS1); and miR-126-3p  [MYC [c-MYC acts jointly with SP1 to downregulate miR-23b expression [Bouyssou et al. have shown that a 12-miRNA signature from extracellular vesicles was able to discriminate asymptomatic WM patients from healthy controls . Hunter  PIK3R2) . The comR2) [MYC ,142. Fulpression . Gain ofpression .PRDM1, a significant regulator of B-cell development [SMAD5, SOCS1 and CEBPβ) as well as three novel targets  [FOXO3 transcription factor and pro-apoptotic BCL2L11 in cells with augmented miR-155-5p expression, hereby blocking apoptosis. Furthermore, mitochondrial priming can be induced by antagonism of miR-155, lowering the apoptosis threshold [Roccaro et al. identified a WM-specific miRNA signature characterized by increased expression of miR-363-5p/-206/-494/-155/-184/-542-3p, and decreased expression of miR-9-3p . Decreaselopment . MiR-155elopment ,143. In elopment ,144. Inthreshold . Lastly,hreshold . Figure Hodge et al. investigated specific miRNA signatures of different WM cellular subgroups. The miRNA signature of WM-BL consisted of mostly downregulated miRNAs compared to CLL and non-malignant B-cells, including miR-151, miR-335 and miR-342, whereas miR-373 was clearly increased in WM-BL. Most WM-PC clustered with MM-PC, yet retained a distinct miRNA profile of their own, characterized by the increased expression of more than 40 candidate miRNAs. No differentially expressed miRNA was detected between WM-BL and WM-PC, and no clear signature for lymphoplasmacytic (CD19+/CD138+) WM cells could be identified, possibly due to the clone’s morphologic diversity, sharing features of both BL and PC. After combining WM-BL, WM-PC and lymphoplasmacytic cells to mimic the heterogeneity observed in WM tumors, six miRNAs were differentially expressed in WM compared to nonmalignant B lineage cells  .Increased expression levels of miR-192-5p, miR-21-5p and miR-320b have been associated with disease progression, while expression of let-7d decreased with disease stage . IncreasSHIP1, which acts as a negative regulator of the PI3K/AKT and mTOR pathway [Roccaro et al. illustrated ex vivo that everolimus targeted mTOR downstream signaling pathways in responders. Furthermore, everolimus induced toxicity, supported by cell-cycle arrest and caspase-dependent and -independent induction of apoptosis, even in the context of BM milieu, affecting migration, adhesion and angiogenesis. Through miR-155 loss-of-function studies, everolimus-dependent anti-WM activity was shown to be partially driven by targeting miR-155 . It has  pathway . Moreove pathway   in WM showed reduced cell proliferation in a dose-dependent manner. There was only a moderate effect on cell viability, which may be explained by increased anti-apoptotic BCL-2 expression, suggesting that targeting BCL-2 may be effective in inducing WM cell death. Indeed, combined treatment of JQ1 and venetoclax enhanced apoptosis. Notably, the efficacy was not compromised in the presence of the TME. Moreover, BET inhibitors were also shown to decrease stromal cell proliferation. This suggests that BET inhibition may influence the epigenetic regulation of both the tumor and the TME. When HDAC and BET inhibitors were combined, synergistic effects on cell death were shown, even with a very low dose of panobinostat (LBH589) [In recent years, there has been increased interest in targeting epigenetic modulators by small molecule inhibitors. Different HDAC inhibitors , and sirtinol) demonstrated dose-dependent killing and had at least additive antitumor effects when combined with bortezomib in a WM cell line . In a ph(LBH589) .Long non-coding RNAs (lncRNAs) are a large and heterogeneous class of non-coding transcripts, greater than 200 nucleotides in length. Based on their relative positions to protein-coding genes, lncRNAs are commonly classified as intergenic, intronic, sense overlapping or antisense overlapping . AlthougNF-κB signaling and via induction by p53. BIC acts as an oncomiR progenitor by being a host of miR-155 [OCT4 [p53 gene expression. MALAT1 and TUG1 have been reported to act as oncogenes by respectively regulating the bioavailability of TGF-β and affecting the expression of cell cycle regulatory genes by binding PRC2 [MCL-1 transcriptional translation and inhibiting apoptosis [To date, lncRNA expression in WM has not yet been investigated. In CLL and MM, however, dysregulation of multiple lncRNAs has been shown. In CLL, DLEU2 , BM74240 miR-155  and MIAT55 [OCT4 . Other d55 [OCT4 . In MM, 55 [OCT4 ,165, DLE55 [OCT4  and MEG355 [OCT4  have beeing PRC2 . Dysreguing PRC2 . Furtherpoptosis ,168. SinCircular RNAs (circRNAs) are a more recently discovered subclass of large ncRNA, with covalently closed ends and lengths between ~100 to thousands of nucleotides. CircRNAs originate from a host gene and are formed through a backsplicing event, ligating the 3′ end of an exon to the 5′ end of the same or an upstream exon. Being highly evolutionary conserved, circRNAs may function as direct or indirect regulators of host gene expression at the transcriptional level, as sponges or decoys for miRNAs or RNA binding proteins, regulators of protein translation or under certain circumstances even as templates for translation. Altered expression has been shown in different solid and hematological cancers and increasing evidence shows that circRNAs can be used as reliable (cell-free) biomarkers, as they are present in different human body fluids, are highly tissue-specific and are more resistant to exonucleases due to their closed structure ,169.RAF1 levels, activating MAPK signaling and promoting cell growth [Wnt/β-catenin pathway by binding miR-607 and thereby derepressing production of FZD3, stimulating proliferation [PML, a known regulator of gene expression and cell viability [MAPK4, slowing down progression. Upregulation of circ_000190 was associated with longer PFS and improved OS [To date, differential expression of circRNAs has not been studied in WM. In CLL, circRPL15 has recently been shown to sponge miR-146b-3p, thereby increasing l growth . Upregulferation . Moreoveferation ,171. Ciriability . In MM, iability . CircSMAiability . Circ_00roved OS . As the L265PMYD88 in IgM-MGUS patients (50–80%) suggests that this somatic mutation is most likely an early oncogenic driver , which usually occur in a later stage, indicates that L265PMYD88 by itself is insufficient to explain the malignant transformation from IgM-MGUS to WM [HIST1H1B, EZH2, CHECK1, LEF1, ADAM23, RASGRP3, ADRB2, PIK3AP1, CDHR3) might highlight new candidate markers in IgM-MGUS responsible for the risk of progression to WM [L265PMYD88. As levels of miR-320a negatively correlated with lymphoplasmacytic cells infiltration in the BM, the expression profiles in samples with higher BM involvement were more similar to their malignant counterparts, whereas patients with less involvement had samples that tended to cluster with the respective non-malignant cells [Patients with IgM-MGUS have a significantly lower number of mutations than patients with WM. The high prevalence of c driver . Meanwhitations <%, which US to WM ,178,179,US to WM . A more on to WM . Furtheron to WM . The asson to WM . The comnt cells .Liquid biopsy is the process of investigating tumor-derived cells, cell-free nucleic acids, metabolites, proteins or extracellular vesicles through biofluid sampling without the need for tissue biopsy. Biological sources for liquid biopsy include PB, urine, cerebrospinal fluid, saliva and pleural effusions, among other body fluids .In the past decade, there have been major advances in the identification of diagnostic, phenotype-defining and prognostic biomarkers in lymphoproliferative diseases, which might complement current classification and prognostic tools, as well as guide therapy choice in a precision medicine approach ,184. In Potential advantages of liquid biopsy include its minimally invasive nature, its ability to reflect spatial inter- and intra-tumor heterogeneity and the possibility of longitudinal profiling. To date, however, their analysis has only been implemented as a therapy decision-maker in solid tumors ,188.In WM, ctDNA may represent a reliable “echo” of the tumor-specific genomic and epigenomic aberrations of the BM compartment, and even of extramedullary sites, and might be useful in assessing disease status, guiding therapeutic decisions and monitoring minimal residual disease (MRD) .To this day, only a few studies have investigated the use of cfDNA in the characterization of the mutational landscape of patients with IgM monoclonal gammopathies.MYD88 and CXCR4 in paired gDNA (from BM CD19+ selected cells) and cfDNA samples of patients with IgM monoclonal gammopathies. Qualitative allele-specific PCR and direct sequencing showed an overall concordance rate, between gDNA and cfDNA, of 94% and 90% for the most common MYD88 and CXCR4 mutations, respectively. These results were consistent among patient subgroups according to disease status  [L265PMYD88 and S338XCXCR4 mutations by AS-qPCR, revealed only one discordance, related to an aWM patient with a slight BM infiltration [L265PMYD88 and S338XCXCR4 against CD19-selected and unselected BM and PB tissue fractions, strengthening the evidence that cfDNA can reliably be used to identify these two most common variants in WM patients [L265PMYD88 mutation in paired unselected BM and cfDNA samples from 60 WM patients. Their L265PMYD88 dPCR assay showed an overall mutation detection rate on baseline unselected mononuclear cells samples of 95.3% in BM and 71.2% in PB. Interestingly, dPCR detected a log10 higher median L265PMYD88 mutated/WT ratio in ctDNA compared to PB, while no statistically significant difference was observed between ctDNA and BM samples [L265PMYD88 mutation  . A subsepatients . Of notepatients ,190. Lik samples . These dmutation .MYD88 detection. Moreover, an algorithm was provided to suggest the most convenient PCR method based on available specimens and laboratory equipment [L265PMYD88 mutation rate detected in cfDNA by dPCR is equivalent to the rate detected in BM.Up to now, there is a lack of consensus regarding the optimal specimen and analytical method for mutational detection in WM, in terms of operating procedures, test sensitivity and result interpretation ,192. Resquipment . AlthougL265PMYD88 recruits WTMYD88 and triggers the activation of the NF-kB and the inflammatory pathway, both important for WM pathogenesis [Although most studies have focused on cfDNA, there has been an increased interest in different forms of circulating-free RNA biomarkers. Several mechanisms, such as encapsulation within extracellular vesicles (EVs) or ribonucleoprotein RNA-binding proteins complexes, protect circulating-free RNAs (cfRNA) from nuclease activity. Although the source and function of cfRNAs remain largely unknown, RNAs seem to be selectively packaged according to the viability and origin of the cells. Living cells seem to actively release RNA encased in large lipoprotein complexes, such as exosomes or microvesicles, while circulating RNA from dying cells is enclosed within apoptotic bodies or bound in protein complexes . Besidesogenesis .In WM, three studies have investigated circulating EV-derived miRNA expression and showed an upregulation of miR-192-5p, miR-93-5p, miR-15a-5p, miR-16-5p, miR-20a-5p, miR-378a-3p and miR-155 and a downregulation of miR-199a-5p, miR-145-5p, miR199a-3p, miR-221-3p, miR-335-5p, let-7d-5p, miR-320a-3p, miR-320b-3p, miR-151-5p and let-7a-5p, compared to healthy controls ,138,140.This review aimed to provide an overview of the molecular and cytogenetic landscape of WM and IgM-MGUS, as well as its clinical applications. In recent years, major progress has been made in genomic and transcriptomic profiling, hereby shedding light on the origin and evolution of the disease, unraveling involved pathways and dissecting the heterogeneity within the WM clone, as well as of the TME. Moreover, (cf)DNA and (cf)RNA biomarkers have been proposed that are able to define disease subgroups, are associated with disease progression and therapy response and allow minimally invasive detection of mutations.WTMYD88/WTCXCR4 subgroup. Therefore, we encourage all authors to report sufficient experimental design details in order to allow a reliable comparison among studies. Likewise, there is a current lack of consensus between diagnostic laboratories on how to perform profiling analyses in WM and IgM-MGUS patients, which is the main reason why molecular data are not yet included in the diagnostic criteria. Lastly, studies in patients with familial WM are very limited. Potential contributors to genetic predisposition have been identified and represent possible candidates for validation within different familial WM populations. Family history information should therefore be routinely collected.An important limitation, however, is the discordancy between studies concerning diagnostic criteria, detection methods with ranging sensitivities and types of specimens analyzed . Moreover, experimental details are often not reported, making data comparison challenging, especially in small patient cohorts such as the infrequent In terms of future research directions, since the temporal acquisition of genomic mutations remains unclear, longitudinal studies are needed to explore the dynamics of clonal architecture and to identify driver mutations that play a role in disease course or chemoresistance. Moreover, deciphering the mechanisms of progression in premalignant IgM gammopathies will help to monitor patients at risk of progression. The use of circulating nucleic acids as minimally invasive, disease-specific and dynamic biomarkers is highly promising, but further research is needed to develop signatures with high specificity and sensitivity that can be routinely implemented in clinical practice. In the same regard, an interesting future perspective is combining cell-free DNA and RNA, as well as other cellular molecules, in a multi-omics approach. Lastly, in contrast to different solid and hematological tumors, the role of (epi)miRNAs interaction, lncRNAs and circRNAs have been very sparsely investigated in WM and IgM-MGUS. Including these dimensions could significantly contribute to our understanding of these diseases and ultimately to the development of new biomarkers and therapies.


Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations. It is the most common cause of secondary glaucoma, resulting in widespread global blindness. The largest global meta-analysis of XFS in 123,457 multi-ethnic individuals from 24 countries identified seven loci with the strongest association signal in chr15q22–25 region near LOXL1. Expression analysis have so far correlated coding and a few non-coding variants in the region with LOXL1 expression levels, but functional effects of these variants is unclear. We hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes and clarify biology that underlie XFS.Transcriptomic Wide Association Studies (TWAS) using PrediXcan models trained in 48 GTEx tissues leveraging on results from the multi-ethnic and European ancestry GWAS were performed. To eliminate the possibility of false-positive results due to Linkage Disequilibrium (LD) contamination, we i) performed PrediXcan analysis in reduced models removing variants in LD with LOXL1 missense variants associated with XFS, and variants in LOXL1 models in both multiethnic and European ancestry individuals, ii) conducted conditional analysis of the significant signals in European ancestry individuals, and iii) filtered signals based on correlated gene expression, LD and shared eQTLs, iv) conducted expression validation analysis in human iris tissues. We observed twenty-eight genes in chr15q22–25 region that showed statistically significant associations, which were whittled down to ten genes after statistical validations. In experimental analysis, mRNA transcript levels for ARID3B, CD276, LOXL1, NEO1, SCAMP2, and UBL7 were significantly decreased in iris tissues from XFS patients compared to control samples. TWAS genes for XFS were significantly enriched for genes associated with inflammatory conditions. We also observed a higher incidence of XFS comorbidity with inflammatory and connective tissue diseases.Our results implicate a role for connective tissues and inflammation pathways in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.The online version contains supplementary material available at 10.1186/s12864-023-09179-7. Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations , 2. The lysyl oxidase-like 1 gene (LOXL1) [LOXL1, rs1048661 (encoding LOXL1 p.Leu141Arg) and rs3825942 (p.Gly153Asp), are likely to confer risk of developing XFS, with very high heterogeneity across populations because the alleles show an effect reversal [LOXL1 expression levels, however, (iv) no clear functional effects for the haplotypes that represent the two variants have been shown [LOXL1 and the neighboring STRA6 gene [Genetic mechanisms have substantial influence on XFS etiology as evidenced in family and twin studies , 14. The (LOXL1) , 21, whi (LOXL1) . The sig942 p.Gly3Asp, arereversal –26, (ii)942 p.Gly3Asp, arereversal , 21, 24,en shown , 28. TheRA6 gene , 30.) project tissues: Adipose - Subcutaneous, Adipose - Visceral (Omentum), Adrenal Gland, Artery - Aorta, Artery - Coronary, Artery - Tibial, Brain - Amygdala, Brain - Anterior cingulate cortex (BA24), Brain - Caudate , Brain - Cerebellar Hemisphere, Brain - Cerebellum, Brain - Cortex, Brain - Frontal Cortex (BA9), Brain - Hippocampus, Brain - Hypothalamus, Brain - Nucleus accumbens , Brain - Putamen , Brain - Spinal cord , Brain - Substantia nigra, Breast - Mammary Tissue, Cells - Cultured fibroblasts, Cells - EBV-transformed lymphocytes, Colon - Sigmoid, Colon - Transverse, Esophagus - Gastroesophageal Junction, Esophagus - Mucosa, Esophagus - Muscularis, Heart - Atrial Appendage, Heart - Left Ventricle, Liver, Lung, Minor Salivary Gland, Muscle - Skeletal, Nerve - Tibial, Ovary, Pancreas, Pituitary, Prostate, Skin - Not Sun Exposed (Suprapubic), Skin - Sun Exposed (Lower leg), Small Intestine - Terminal Ileum, Spleen, Stomach, Testis, Thyroid, Uterus, Vagina, Whole Blood [After considering all the reports on genetic architecture of XFS to date, we hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes in XFS. We used a gene-based TWAS method, PrediXcan , implemele Blood , 34. GTEle Blood , 34. We le Blood . The phele Blood  hamperinle Blood , 31. We P < 2.02 × 10− 7 after Bonferroni corrections) on chromosome 15: CYP1A2, CYP1A1, STOML1, LOXL1, ISLR2, RPP25, INSYN, ISLR, STRA6, CD276, NEO1, ARID3B, COX5A, PML, CPLX3, LMAN1L, UBL7, MPI, CLK3, CSK, SEMA7A, TBC1D21, and NPTN that mapped to region 15q22–25 region that spans ~ 3 Megabases  summary data, identifying 23 genes  were associated in the multi-tissue analysis: ADPGK (p = 7.32E-07), CYP11A1 (p = 1.36E-16), HEXA (p = 1.03E-06), PARP6 (p = 1.82E-06), SCAMP2 (p = 1.65E-10) .To determine the joint effects of gene expression variation predicted across all 48 tissues analyzed, we performed a multivariate regression multi-tissue analysis. Each of the 23 associations from the single-tissue PrediXcan analysis remained significant in multi-tissue analysis , 6 (PRRT1 p = 9.10E-07), 8 (PRSS55 p = 4.18E-13), 10  and 19 (CALM3 p = 2.60E-07), were significantly associated in the multi-tissues analysis   Fig. . These rLOXL1 and shown to play regulatory roles [LOXL1 and STOML1 signals on chr15q22–25 region observed associations in each tissue. We also determined the effect of shared variants between prediction models for the genes in the region.To dissect the potential source of LD contamination in the PrediXcan analysis, we looked into the effect of the two GWAS missense variants implicated in XFS that have mostly been linked to ry roles , follower2 = 0.982) with the rs1048661 missense variant, and iii) all the variants in our gene models that were in LD with the two variants (r2 > 0.1). The two missense variants had wide ranging effect on the genetically predicted expression of many chr15q22–25 region genes, with the largest effect on LOXL1. The strength of the association signals diminished in six of the nine tissues for which we had the gene’s predicted expression. Association signals in three of these tissues fell below genome-wide threshold in the global dataset (Suppl. Table SSTOML1 lost genome-wide significance: CD276 (2 tissues), COX5A, CYP1A1, LMAN1L, MPI, SCAMP2 and TBC1D21.We modified our prediction models by excluding: i) rs3825942 missense variant, ii) rs4886776 intronic variant, which is at near perfect LD   with any variants in LOXL1 or STOML1 tissue models. We also excluded variants that were shared between two or more genes in their original prediction models. Seven of the genes that were associated with XFS at genome-wide threshold in their original models showed diminished signals, including four below significance levels: UBL7, ISLR, LMAN1L and COX5A in reduced models , while some of the signals were masked in the original models . There was inconsistent result for UBL7, where there was no effect in its association signal in a tissue, enhanced effect in another tissue, and diminished signal in two other tissues, one of which went below the genome-wide threshold, albeit the reduced model had only a single variant in the prediction   Table . After cely Fig. . This inSTOML1 predicted gene expression, we observed significant association signals for 8 other genes (in addition to STOML1)  remained, while association with four other genes  disappeared. In addition, associations with five more genes  attained genome-wide significance after the conditional analysis.In 17 tissues with LOXL1, ARID3B, CPLX3, CYP1A1, CYP1A2, INSYN1, NEO1, PML, SCAMP2, and UBL7, all of which, except for INSYN1, have been shown to be highly expressed in eye tissues [. However, INSYN1 has enhanced expression in brain tissues [LOXL1 and its “proxy” STOML1 signal.Overall, conditional PrediXcan analysis of genetic signals in the chromosome 15 region in the European dataset in a limited number of tissues was mostly consistent with PrediXcan analysis using the reduced models above. The analysis confirms the associations for  tissues   and nominal significance (p < 0.05) were evaluated for enrichment of known pathways, using Enrichr [CYP1A1 and CYP1A2 [Genes at genome-wide significance  and  Enrichr  traits.  Enrichr . Some ofd CYP1A2 , are invhttps://diseases.jensenlab.org/).Our gene set is also enriched for genes associated with carcinoma and three inflammatory conditions: rheumatoid arthritis, Type 1 diabetes, vitiligo in Jensen Diseases, a database that integrates evidence on disease-gene associations from automatic text mining, manually curated literature, cancer mutation data, and GWAS (http://tanlab.ucdenver.edu/DSigDB), a gene set resource that relates drugs/compounds and their target genes. Our gene set is enriched for genes that are targets of cyclosporin A (p = 9.66E-11), and genes that are targets for compounds that are either 1) carcinogenic: Aflatoxin B1, potassium chromate, methyl methanesulfonate and copper sulfate, 2) neuroactive: valproic acid and methamphetamine, 3) neuroprotective: quercetin and epigallocatechin gallate, or 4) analgesic: acetaminophen  returned significant enrichment for the endosomal-vacuolar pathway (p = 8.14E-11), an enrichment that was replicated in gene sets that were predicted to be downregulated (p = 3.24E-8). Our results broadly recapitulated results above, even after excluding genes in HLA and chr17 inversion regions from the enrichment analysis of the gene set (p < 0.05)  using a ranked association gene list based on effect sizes confirmed some of the enrichment observations using Enrichr. GSEA besides replicating enrichment for acetaminophen, showed enrichment for: 1) six synthetic estrogens, 2) estrogen regulators (Clomifene), 3) antiarrhythmic (quinidine), and 4) an anti-fungal (ketaconazole) Table . The gen5) Table .ARID3B, CD276, INSYN1, LOXL1, NEO1, SCAMP2, STOML1 and UBL7 were measured in XFS (N = 12) and control (N = 19) eye tissues. We selected iris tissue because it is one of the tissues that are part of the anterior segment structures bathed by aqueous humor and upon which the flaky XFS materials are deposited [ARID3B, CD276, LOXL1, NEO1, SCAMP2 and UBL7 (p < 0.05) . INSYN1 and STOML1 were not significantly downregulated in diseased eyes relative to normal eyes in validation analysis. STOML1 is the closest gene to and potentially proxy for LOXL1 among those that show association in our PrediXcan results within the chr15q22–25 region. We included it as a negative control in the validation analysis, while LOXL1 was a positive control considering that it had already been shown to exhibit pattern of downregulation in gene expression in diseased relative to normal tissues [CD276 was selected for functional validation in eye tissue despite no significant association with XFS in single-tissue analysis in the European ancestry data because it was significantly associated with XFS in multi-tissue analysis in European data. In addition, it was one of the gene associations signals which were not affected by excluding variants that were in LD with LOXL1/STOML1 model SNPs in the multi-ethnic global dataset. Overall, our validation results replicate the associations found using the genetically determined gene expression.Expression levels of eposited . Other aeposited . All tra tissues . CD276 wTo gain further biological insights into the gene associations we observed in our PrediXcan analysis, we performed logistic regression analysis of both XFS ICD9/10 diagnosis, and Polygenic Risk Score generated from the multi-ethnic summary data across the BioVU individuals, Vanderbilt University’s electronic health records database linked to genetic information, as the target dataset . XFS diagnosis was associated with an increased risk of 96 phenotypes in BioVU, including 12 musculoskeletal phenotypes, 4 infectious diseases, and 1 cardiovascular phenotype. These results are consistent with higher comorbidity of diseases affecting inflammation, connective tissue, and the circulatory system in individuals with XFS  and steroid derivatives: protectin (Reactome), omega fatty acid and estrogen (WikiPathways) are also consistent with the potential role of inflammation in XFS. Protectin, a derivative of PUFA including Omega-3 that are major components of fish oil, has an anti-inflammatory, anti-amyloidogenic, and anti-apoptotic activities in human neural cells , 62, 63.IL-6) and IL-8 [TNF-α) and YKL-40 [The association with inflammation is consistent with studies in limited numbers of XFS patients that found elevated inflammatory markers relative to controls, including cytokines, and markers such as interleukin-6  signaling pathway, and that siRNA-mediated downregulation of RA signaling induces upregulation of LOXL1 and XFS-associated matrix genes in XFS-relevant cell types [STRA6 and impaired retinoid metabolism are involved in the pathophysiology of XFS syndrome. Retinoic acid, the active metabolite involved in the signaling pathway implicated by Berner et al. [ISLR2, STRA6 and LOXL1, has been shown to control critical checkpoints in inflammation and to promote an inflammatory environment [Second, only a third of the signals identified in the larger data were robustly confirmed in a European dataset at genome-wide significance. This raised the possibility that most of the initial signals identified an artefact of local LD leakage or shared eQTLs with the sentinel nd STRA6 . ISLR2 all types . These dr et al.  in XFS tironment –109.Our analysis of predicted gene expression and extensive functional analysis in eye tissue prioritized six genes in association with XFS. Our results further confirmed the role of connective tissues and highlighted the importance of inflammation in the etiology of XFS. Thus, molecular elements that underlie the interaction of connective tissue biosynthesis and inflammatory pathways may play a central role in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.We used an extension of PrediXcan  that useWe used the gene-based method, PrediXcan, that provides a framework for correlating imputed gene expression with phenotype . Gene exP < 0.0001. We used Bonferroni adjustment for multiple hypothesis testing. Genome-wide significance for a gene-level association in single-tissue and multi-tissue PrediXcan analysis were defined as p < 2.02e-7 and p < 3.02e-6, respectively.We used S-PrediXcan  to analyTo determine whether multiple association signals within the same locus are due to independent causal genes or statistical artefacts of correlation in measured expression and predicted gene expression for adjacent genes , we examLOXL1 missense variants in our PrediXcan analysis, we excluded them and all the variants in our gene models that were in LD with them (defined as pairwise r2 > 0.1) to generate “reduced models”. We predicted gene expression and performed association analysis using reduced models in both the global multiethnic and the European subset for the genes in chromosome 15 region. To assess whether the association signals in the chr15q22–25 region for each tissue are independent of the ‘classical’ LOXL1 signal, we excluded variants in the prediction models of genes in the region that were in LD (pairwise r2 > 0.1) with any variant in the LOXL1 model. In tissues without a LOXL1 model , we excluded variants for chr15q22–25 region genes that were in LD (r2 > 0.1) with variants in the STOML1 models. In addition, we excluded variants that were shared between prediction models for genes in the region. In each case, we performed association analysis using the reduced models and compared the results with the original models.To measure potential regulatory effects of the two classical LOXL1 and STOML1) identified in the primary analysis, we performed conditional analysis using ancestrally ‘homogenous’ individual-level genotype data that included our BioVU cohort and a subset of Aung, et al. consisting of three European ancestry cohorts. For each tissue with a significant association, the conditional analysis was performed on the gene that was the most statistically significant as identified from the initial PrediXcan analysis. We generated genetically determined expression for each individual in the dataset and then performed association analysis using Genetic Association Analysis Under Complex Survey Sampling (SUGEN: version 8.8) [To determine whether additional genes within the region were significantly associated with XFS, independently of the most highly associated genes (ion 8.8)  on the ip < 0.05) in single-tissue analysis were checked for enrichment of particular categories in several databases using the web-based enrichment tools, Enrichr [GPX6 – CUTA genes (238 genes) and ~ 2.5 Mb chromosome 17 region that encompassed CCDC43 – NPPEPS that include the 900 kb inversion common in population of European ancestries (72 genes).Genes that were predicted to be associated with XFS at genome-wide significance in both single-tissue and multi-tissue analysis, and at nominal significance  and 19 normal-appearing control eyes without any known ocular disease  were used. All XFS tissues donors were previously confirmed XFS patients based on routine ophthalmologic examination after pupillary dilation. The presence of characteristic XFS material deposits was assessed by macroscopic inspection of anterior segment structures and confirmed by electron microscopic analysis of small tissue sectors. Iris tissues were prepared under a dissecting microscope and frozen rapidly in liquid nitrogen.N = 31, 12 XFS and 19 control eyes) were extracted using the Precellys 24 homogenizer and lysing kit  together with the AllPrep DNA/RNA kit  according to the manufacturer’s instructions including an on-column DNaseI digestion step using the RNase-free DNase Set (Qiagen). First-strand cDNA synthesis and PCR reaction was performed as previously described [http://bioinfo.ut.ee/primer3/), are summarized in Suppl. Table Shttps://genome.ucsc.edu/) and amplification specificity was checked using melt curve, agarose gel and sequence analyses with the Prism 3100 DNA-sequencer . For normalization of gene expression levels, mRNA ratios relative to the house-keeping gene GAPDH were calculated.For quantitative real-time PCR, iris tissues . n = 52,251) on the polygenic risk score generated from the Aung et al’s. [To determine the comprehensive health consequences of high genetic risk to XFS, we performed a phenome-wide association analysis (PheWAS) . First, et al’s.  XFS globAdditional file 1.


YU-Pingfeng San (YPFS) can regulate inflammatory response to alleviate the symptoms of nasal congestion and runny rose in allergic rhinitis (AR). However, the mechanism of action remains unclear. In this study, 30 active ingredients of three effective herbs included in YPFS and 140 AR/YPFS-related genes were identified by database analysis. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the targets were mainly enriched in immune inflammatory-related biological processes and pathways. Finally, three hub gene targeting epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B1 (AKT1) related to YPFS and AR were identified by network pharmacology analysis. YPFS treatment decreased the expression of EGFR, MAPK1, and AKT1 in ovalbumin (OVA)-induced AR mice and impaired the production of inflammatory factors interleukin (IL)-4, IL-5, and IL-13, thus alleviating immunoglobulin E (IgE) production and the symptoms of scratching nose in AR. Through molecular docking analysis, we found that the active ingredients decursin, anomalin, and wogonin of YPFS could bind to EGFR, MAPK1, and AKT1 proteins. Moreover, decursin treatment impaired the expression of IL-4 and IL-5 in human PBMCs. These results suggested that YPFS could alleviate the AR inflammatory responses by targeting EGFR, MAPK1, and AKT1, showing the mechanism of action of YPFS in AR treatment. Allergic rhinitis (AR) is an allergic disease of the nasal mucosa caused by a variety of factors. Clinical manifestations are characterized by nasal itching, sneezing, hypersecretion, and nasal mucosal swelling . AR is oAstragali Radix , Atractylodis Macrocephalae Rhizoma , and Saposhnikoviae Radix , at a content ratio of 3:1:1 . Those chemical compounds of YPFS with identifiable properties of absorption, distribution, metabolism, and excretion (ADME) and druggable properties, were selected as active components for further target prediction, with a cutoff of oral bioavailability of 30% and drug-likeness (DL) of 0.18  and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of AR/YPFS-related genes was performed to identify genes in the biological processes (BP), cellular components (CC), molecular functions (MF) categories, and pathways using the ClusterProfiler R Package . The dat8 .9 a protein interaction functional enrichment analysis website, to construct the Protein–protein interaction (PPI) network with a cutoff of high confidence of 0.7  2014-0007). All mice were raised in a specific pathogen-free level environment in the Research Building of Yuhuangding Hospital, Yantai. All experimental animals were fed with standardized sterile feed and water, which was sterilized by high-pressure steam and cooled to room temperature. All experimental animals were dissected after fasting for 12 h. All animal experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Yantai Yuhuangding Animal Ethics Committee.Ovalbumin-induced AR mice were induced through two stages: The basic sensitization stage and the stimulation stage. In the basic sensitization stage, 200 μl of a mixture comprising 40 μg OVA  and 2 mg aluminum hydroxide  gel were injected intraperitoneally into the OVA-induced AR group seven times on days 1, 3, 5, 7, 9, 11, and 13. In the stimulation stage, mice received nasal drops comprising 20 μl of 5% OVA solution following atomization with 5% ovalbumin saline solution in a homemade simple atomizer for 30 min, from days 21 to 27 . The micYPFS comprises three kinds of herbs, HQ , BZ , and FF  at a content ratio of 3:1:1. The three herbs were mixed, soaked in water for 1 h, and then decocted two times using an automatic decocting machine for 1.5 h each time. The decoctions  were filtered through double gauze to concentrate the filtrate to the final concentration of 1 g/ml and stored at 4°C until use .YPFS was intragastrically administered to the AR mice in the YPFS group at a dose of 130 mg/20 g body weight for 14 days continuously . The samThe serum was separated from the upper layer of the blood after centrifugation for 20 min at 900 g. The concentrations of immunoglobulin E (IgE), interleukin (IL)-4, IL-5, IL-13, and interferon-gamma (IFN-γ) in mouse serum were detected using an Enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer’s instructions.–ΔΔCT method  using the Trizol reagent  and reverse transcribed into cDNA using a reverse transcription kit . The qPCR step was performed using an SYBR Green qPCR Mix kit (SparkJade) in a StepOnePlus fluorescence quantitative PCR instrument . The expression levels of the genes encoding IL-4, IL-5, epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), and protein kinase B1 (AKT1) were detected. The relative expression level was calculated using the 2T method . The pri10 Ingredients involved in regulating immune inflammation were selected for molecular docking with the three hub genes decursin (11). The eutectic ligands isolated from the receptor and the active pocket of each target were identified using AutodockTools-1.5.6  was used to isolate Human PBMCs according to the manufacturer’s instructions. The Human PBMCs were cultured in 10% fetal bovine serum and 90% Roswell Park Memorial Institute-1640 medium for 48 h. Then, decursin  was added to PBMCs at concentrations of 0, 5, and 10 μM, respectively, and incubated for 48 h.t-tests, and p-value < 0.05 was considered statistically significant. The results are shown as the mean and standard deviation.The experimental data were analyzed using GraphPad Prism 8  using unpaired The workflow chart for network pharmacology analysis and experimental verification is shown in A total of 49 AR-associated target genes were obtained from the DrugBank database, 1,311 AR-associated target genes were obtained from the Genecards database, and 214 AR-associated target genes were obtained from the OMIM database. After combining the related target genes from the three databases and the removal of duplicates, a total of 945 AR-associated target genes were obtained . About 1Functional enrichment analysis was performed on the 140 AR/YPFS-related genes using GO, which showed that these genes were mainly enriched in processes involved in immune regulation, such as regulation of inflammatory response, leukocyte migration and chemotaxis, regulation of leukocyte migration, lymphocyte differentiation, and T cell activation . KEGG paTo understand the interaction regulation of the 140 AR/YPFS-related genes, we uploaded them to the String database to obtain a PPI network diagram . Using CCompared with those in the control group, serum IgE, IL-4, IL-5, and IL-13 levels were significantly higher in the OVA-induced AR group . The IFNCompared with those in the control group, the frequency and times of nasal scratching in the OVA-induced AR group mice increased significantly , whereasEgfr, Mapk1, and Akt1 in the nasal mucosa of the OVA-induced AR group were significantly increased, and no significant expression differences were found between the YPFS treatment group and the control group  and 2 types (Th2) immune responses and T cell cytokine production. Through + T cell differentiation to Th2 cells, which produce the cytokines IL-4, IL-5, and IL-13, thus inducing a classical type 2 inflammatory response  to produce the type 2 cytokines IL-5 and IL-13 and also acting on dendritic cells (DCs). DCs present antigens to resident lymph nodes to activate CD4response . In our AR is an allergic disease of the nasal mucosa caused by the imbalance of differentiation of T cells. YPFS is recommended for the management of AR by the Chinese medicine clinical practice guidelines and can relieve the clinical symptoms of AR. However, how YPFS functions is still not clear. YPFS could impair the inflammatory response by reducing thymic stromal lymphopoietin (TSLP), tumor necrosis factor-alpha (TNFα), and IL-6 production . YPFS alKEGG pathway enrichment analysis showed that 140 AR/YPFS-related genes were enriched in pathways involved in regulating AR, such as the PI3K/AKT pathway, the hypoxia-inducible factor 1 alpha (HIF1α) signaling pathway, and the MAPK, RAS, and JAK/STAT signaling pathways. The PI3K/AKT signaling pathway is involved in a variety of AR inflammatory responses, including Th2-mediated inflammation , the perThree hub genes, EGFR, MAPK1, and AKT1, were identified through PPI analysis and Cytoscape analysis. Accumulating studies have shown that EGFR, MAPK1, and AKT1 participate in AR regulation. EGFR was significantly elevated in patients with AR , and thein vivo by reducing the number of eosinophils, increasing eosinophil apoptosis, reducing airway mucus production, and reducing airway hyperresponsiveness. Studies have shown that wogonin might play a role in the treatment of allergic diseases by regulating Th1/Th2 cytokine imbalance and histamine release in mast cells (The compounds decursin, anomalin, and wogonin, screened from YPFS, can potentially bind to EGFR, MAPK, and AKT and inhibit their expression. Decursin has been reported to be associated with inflammatory immunity . A novelst cells . These sIn vivo experiments showed that YPFS reduced the IgE level in serum, decreased the production of Th2-type cytokines, and relieved AR symptoms. Meanwhile, EGFR, MAPK1, and AKT1 were found to be downregulated by YPFS in OVA-induced AR mice, and be targeted by active ingredients decursin, anomalin, and wogonin from YPFS through molecular docking prediction. These results showed that YPFS could relieve the AR inflammatory response and symptoms mainly via active ingredients decursin, anomalin, and wogonin targeting and inhibiting EGFR, MAPK1, and AKT1, which may provide novel ingredients for AR treatment. However, these results should be further verified using more detailed molecular mechanism studies, contributing to the in-depth research and application of traditional Chinese medicines.In summary, we identified the active ingredients in YPFS and 140 AR/YPFS-related genes through network pharmacology analysis. The original contributions presented in this study are included in the article/The studies involving human participants were reviewed and approved by Yantai Yuhuangding Animal Ethics Committee. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Yantai Yuhuangding Animal Ethics Committee.YY and YL designed and directed the study. ZL and QS organized the public data and wrote the manuscript. ZL, XL, XS, QS, and ZS performed the experimental work and analyzed the data. FS, CL, and QS took charge of data visualization. XS, YZ, and YL revised the manuscript. All authors contributed to the article and approved the submitted version.


Recently, antiplatelet agents have been shown to have anticancer effects, especially for hepatocellular carcinoma (HCC) but have never been studied in liver transplantation (LT) recipients. We investigated 468 LT patients to ensure that antiplatelet drugs (aspirin or clopidogrel) could prevent HCC recurrence after LT. In matched patients, the 5-year incidence of HCC recurrence (15.8% versus 20.4%) and death from HCC (18.3% versus 15.4%) were not significantly different between the antiplatelet and non-antiplatelet groups. When adjusted for other risk factors of HCC recurrence, antiplatelet use was not associated with HCC recurrence. Unlike in non-recipients with liver disease, antiplatelet therapy did not affect HCC recurrence or death from HCC when used after LT.p = 0.876) and non-antiplatelet groups (p = 0.701). All-cause and non-HCC deaths were also similar between the two groups . In multivariable analyses of the entire cohort, antiplatelet use was not associated with HCC recurrence  or HCC-specific death . Therefore, unlike the usual setting with liver disease, antiplatelet therapy did not affect HCC recurrence or HCC-specific mortality when used after LT.Previous studies reported suppressive effects of antiplatelet agents on hepatocellular carcinoma (HCC); however, this has never been assessed in patients who underwent liver transplantation (LT). This retrospective observational study used data from LT recipients with pre-transplant HCC in a single tertiary hospital. The study population was divided into two groups according to the use of antiplatelet agents for >90 days within the study period (377 antiplatelet groups versus 91 non-antiplatelet groups). Matched groups containing 79 patients in each group were also compared regarding HCC-recurrence and HCC-related mortality, which were analyzed by treating non-HCC death as a competing risk. In Kaplan–Meier analyses of the matched cohort, the 5-year cumulative incidences of HCC recurrence and HCC-specific death were similar between the antiplatelet ( Liver transplantation (LT) is an optimal treatment option for unresectable hepatocellular carcinoma (HCC) that can simultaneously cover underlying liver diseases. However, 10–15% of overall recurrence after LT  have focRecent studies reported that higher platelet counts negatively affect the prognosis of patients with HCC ,4. PlateIn patients diagnosed with HCC, antiplatelet therapy has been reported to be effective in increasing survival . FurtherAntiplatelet drugs have been used after LT for thromboprophylaxis to reduce hepatic artery thrombosis ,14 althon = 4). Patients who used antiplatelet drugs for >30 days were excluded from the non-antiplatelet group (n = 35). From the entire cohort of 377 antiplatelet and 91 non-antiplatelet groups, we established a 1:1 matched cohort with propensity scores  age < 18 years, (2) patient death or HCC recurrence within 90 days, (3) hospital stay > 90 days , (4) combined transplantation with other solid organs, (5) malignant portal vein tumor thrombus, (6) combined with cholangiocellular carcinoma, and (7) incomplete data. We then divided the patients into two groups according to the use of antiplatelet drugs for >90 days before HCC recurrence within the study period. Patients who started antiplatelet therapy >90 days after LT were excluded from the antiplatelet group . Hospital stay was recorded as the duration from LT to the first discharge in days. Immunosuppressants were categorized according to the regimen used 3 months after LT: tacrolimus, tacrolimus+mycophenolate, and tacrolimus+mTOR inhibitor. As the ordinary target trough level of tacrolimus was 5~8 ng/mL during the first month, and 4~6 thereafter in our institution, we compared serial values of it throughout the study period between the antiplatelet users and non-antiplatelet users.To compare whether antiplatelet therapy was indicated by platelet volume and to evaluate the inflammation score associated with tumor recurrence, the platelet count, platelet-lymphocyte ratio, and neutrophil-lymphocyte ratio immediately before LT were recruited. Laboratory results indicating liver function, such as AST, ALT, total bilirubin, and INR, were recorded 1 month after LT.The primary outcomes were HCC recurrence and HCC-specific death. HCC recurrence was defined as the first notification of recurrent HCC in imaging studies that were screened at least every 3 months for the first year post-transplantation, at 3–6 month intervals until 2 years, and then annually. HCC-specific death was defined as death after HCC recurrence. The secondary outcomes were all-cause and non-HCC death. Non-HCC death was defined as death without HCC recurrence after LT.http://cran.r-project.org/) , with the threshold for significance set at p < 0.05.Data are presented as median (interquartile range [IQR]) for continuous variables according to their normality and number (proportion) for categorical variables. For group comparison in the entire cohort, the Mann–Whitney U test and χ2 test were used when appropriate. For matched analyses, propensity score matching was performed using the nearest neighbor method with a caliper of 0.2 . To geneIn the entire cohort, most baseline characteristics were similar between the antiplatelet and non-antiplatelet groups, except for pre-transplant MELD and hospital stay . Liver gn = 357), clopidogrel (n = 3), and dual antiplatelet (n = 17). Reasons for the use of dual antiplatelet were related with cardiovascular disease (n = 6), need for more potent thromboprophylaxis due to compromised anastomosis of hepatic vein/vena cava (n = 4), hepatic artery (n = 2), portal vein (n = 1), and unclear reason (n = 3). The median duration of antiplatelet drug use was 613  days. In the matched antiplatelet group, patients started antiplatelet agents at a median of 7  days after LT. The antiplatelet drugs were aspirin (n = 74) and dual antiplatelet agents (n = 5). The duration of antiplatelet drug use was 520  days.In the entire antiplatelet group, patients started antiplatelet agents at a median of 7  days after LT. The subclasses of antiplatelet drugs were aspirin (p = 0.895), bleeding , and bile duct complication  were not different between two groups. Those complications were similar between the two matched cohort, either We analyzed post-postoperative complications which occurred during the follow-up period . In the Of 158 matched patients, 27 (17.1%) experienced recurrence of HCC, and 32 (20.3%) died during a mean follow-up of 43.9 ± 19.6 months. Among the patients who experienced HCC recurrence, the median time from LT to HCC recurrence was 12.0 months , with cumulative incidences of 11.9%, 17.5%, and 18.5% at 1, 3, and 5 years in the antiplatelet group versus 8.2%, 14.0%, and 20.4% at 1, 3, and 5 years in the non-antiplatelet group, respectively. HCC-specific deaths were also not significantly different (p = 0.701), with cumulative incidences of 2.7%, 11.6%, and 18.3% at 1, 3, and 5 years in the antiplatelet group versus 1.4%, 10.1%, and 15.4% at 1, 3, and 5 years in the non-antiplatelet group, respectively. In Kaplan–Meier analyses , both map = 0.867), with cumulative incidences of 5.2%, 15.3%, and 23.5% at 1, 3, and 5 years, respectively, in the antiplatelet group versus 6.4%, 17.4%, and 24.3% at 1, 3, and 5 years, respectively, in the non-antiplatelet group (p = 0.413), with cumulative incidences of 2.6%, 4.2%, and 6.4% at 1, 3, and 5 years, respectively, in the antiplatelet group versus 5.1%, 8.2%, and 10.5% at 1, 3, and 5 years, respectively, in the non-antiplatelet group.All-cause death did not differ significantly between the matched groups  patients experienced HCC recurrence and 66 patients (14.1%) died during the mean follow-up period of 43.1 ± 20.9 months. Among patients whose HCC recurred, the median time from LT to recurrence was 13.1 (IQR 9.2–23.1) months. Of the 71 recurrences, 52 were extrahepatic . Other factors associated with HCC recurrence were age , diabetes , pre-transplant MELD , AFP by log scale , bridging therapy , viable tumor number , maximum tumor size , microvascular invasion , and platelet-lymphocyte ratio .As shown in p = 0.310). Other factors associated with HCC-specific death were donor age , AFP by log scale , bridging therapy , viable tumor number , satellite nodule , and immunosuppressants . In the analyses of secondary outcomes, antiplatelet use was not associated with all-cause death -2, nuclear factor-kB signaling, and inhibition of protein kinase 3 have been suggested as the mechanisms of the tumor suppression effect of aspirin ,18,19. IHowever, the suppressive effect of antiplatelet agents on HCC recurrence was not observed in our LT-only population. We hypothesized this discrepancy with previous studies with non-LT populations. First, many more confounding factors affect HCC outcomes in LT patients than in the non-LT population. Aside from pre-transplant treatment, underlying disease, tumor markers, explant pathology, and post-transplant management, such as immunosuppression, might vary across patients and could affect HCC outcomes more than the effect of antiplatelet . AnotherThe duration of antiplatelet exposure could be another important issue in this study compared to previous studies in patients with liver cirrhosis. In a recent Korean population-based study, aspirin use was defined as more than 3 years of aspirin prescription during 7.5 years of mean follow-up . In anotTo our knowledge, only one population-based study has evaluated the effect of aspirin in patients who received specific treatments for HCC . They deAmong other factors, mTOR inhibitor appeared as a significant protective factor for HCC-specific death, although it did not reduce HCC recurrence itself in the current study. mTOR inhibitor is known for its anticancer effect via decreasing the production of vascular endothelial growth factor and inhibiting vascular endothelial cell response to it . The larThe relatively small sample size, single-center, and retrospective design were the limitations of our study. However, the recruitment of numerous variables associated with LT and HCC prognosis was a strength of this study, which was not performed in a large number of population-based studies. The heterogeneous indication for antiplatelet use in our study population was another limitation to hinder an intention-to-treat approach. Lastly, the relatively short duration of antiplatelet use before primary endpoints in our LT subjects might have made our results differ from those of prior studies with non-LT subjects whose antiplatelet use had a longer duration. This study was the first to investigate the effect of antiplatelet therapy on the prognosis of HCC patients after LT. Unlike usual settings with cirrhosis or hepatitis, we found that antiplatelet therapy did not affect HCC recurrence or HCC-specific mortality when used after LT. Further studies with larger sample size and intentional purposes should be implemented to clarify the relationship between antiplatelet therapy and HCC in LT patients.


Lactobacillus helveticus VHProbi Y21 confers beneficial effects in the prevention and treatment of acne. Here, we report the whole-genome sequence of this bacterium, which contains a chromosome and two plasmids, with a GC content of 37.52%.The probiotic strain  The inoculum was incubated at 37°C for 72 h under anaerobic conditions to enrich the bacteria. A colony named VHProbi Y21 was picked up and identified as the species Lactobacillus helveticus using 16S rRNA gene sequencing, as described by Estifanos et al.  were generated using the PacBio platform. The Illumina raw reads were quality filtered with Sickle v1.33 (https://github.com/najoshi/sickle). The PacBio raw reads were processed using single-molecule real-time (SMRT) Analysis v2.3.0 (https://github.com/tseemann/barrnap) were used to predict the protein-coding genes, tRNA genes, and rRNA genes, respectively. Genome annotations were performed using the NCBI Prokaryotic Genomic Annotation Pipeline (PGAP) v6.2 (Spontaneous kimchi fermentation leads to the growth of lactic acid bacteria, which results in variations in the taste and sensory characteristics of kimchi products. In this study, we present the complete genome sequence of a bacterium that was isolated from kimchi and has the ability to produce propionic acid. A 1-mL sample of kimchi soup collected from homemade kimchi in Yanji city, China, was serially diluted in saline solution, and the 10s v2.3.0 . If therL. helveticus VHProbi Y21 comprises 2,171,060 bp in a circular chromosome and two circular plasmids, with a GC content of 37.52%. There were 1,952 protein-coding genes, 12 rRNA genes, 63 tRNA genes, 3 noncoding RNA genes, and 188 pseudogenes in the genome. The characteristics of the genome are shown in The complete genome sequence of L. helveticus VHProbi Y21 has been deposited in GenBank with accession numbers CP103857, CP103858, and CP103859, SRA accession numbers SRR21166832 and SRR21166831, BioProject accession number PRJNA871029, and BioSample accession number SAMN30399899.The whole-genome sequence of


Salmonella Typhimurium is associated with foodborne diseases worldwide, including in Peru, and its emerging antibiotic resistance (AMR) is now a global public health problem. Therefore, country-specific monitoring of the AMR emergence is vital to control this pathogen, and in these aspects, whole genome sequence (WGS)—based approaches are better than gene-based analyses. Here, we performed the antimicrobial susceptibility test for ten widely used antibiotics and WGS-based various analyses of 90 S. Typhimurium isolates  from 14 cities of Peru isolated from 2000 to 2017 to understand the lineage and antimicrobial resistance pattern of this pathogen in Peru. Our results suggest that the Peruvian isolates are of Typhimurium serovar and predominantly belong to sequence type ST19. Genomic diversity analyses indicate an open pan-genome, and at least ten lineages are circulating in Peru. A total of 48.8% and 31.0% of isolates are phenotypically and genotypically resistant to at least one antibiotic, while 12.0% are multi-drug resistant (MDR). Genotype–phenotype correlations for ten tested drugs show >80% accuracy, and >90% specificity. Sensitivity above 90% was only achieved for ciprofloxacin and ceftazidime. Two lineages exhibit the majority of the MDR isolates. A total of 63 different AMR genes are detected, of which 30 are found in 17 different plasmids. Transmissible plasmids such as lncI-gamma/k, IncI1-I(Alpha), Col(pHAD28), IncFIB, IncHI2, and lncI2 that carry AMR genes associated with third-generation antibiotics are also identified. Finally, three new non-synonymous single nucleotide variations (SNVs) for nalidixic acid and eight new SNVs for nitrofurantoin resistance are predicted using genome-wide association studies, comparative genomics, and functional annotation. Our analysis provides for the first time the WGS-based details of the circulating S. Typhimurium lineages and their antimicrobial resistance pattern in Peru. Salmonella Typhimurium, and other non-typhoidal Salmonella (NTS), are responsible for foodborne illnesses worldwide [Salmonella a high-priority pathogen due to increased resistance to first-line antibiotics, fluoroquinolones, and third-generation cephalosporins [S. enterica serovars, mainly Infantis, Enteritidis, and Typhimurium, were associated with gastroenteritis in Lima hospitals during 2008–2013 and 2015–2017 [Salmonella Infantis isolates resistant to first-line antibiotics, third-generation cephalosporins, and ciprofloxacin antibiotics are highly prevalent in Peru [Salmonella.orldwide . NTS canosporins . In Peruosporins ,4. S. en015–2017 . AdditioS. Typhimurium has a broad host range and emerging dominant MDR phenotypes. For example, the MDR DT104 group disseminated rapidly globally [Salmonella isolated in various countries largely contain genes for β-lactam, tetracycline, aminoglycoside, and quinolone resistance on plasmids [globally , and ST3globally ,10,11,12plasmids ,14. S. Typhimurium exhibits a broad and diverse host range, pathogenicity, and risk to human health [S. Typhimurium infections [The use of WGS in molecular epidemiology and AMR surveillance has several advantages as compared to conventional PCR, other molecular methods, or phenotypic approaches ,16,17. Sn health ,19,20; Wfections ,22,23,24fections ,25,26. Cfections .S. Typhimurium isolates from a Peruvian surveillance study to determine its Peru-specific lineage and antimicrobial resistance pattern. More specifically, we classified the S. Typhimurium serovars using serotyping, multi-locus sequence typing, and average nucleotide identity (ANI) analysis approaches. Further, the nucleotide diversity, phylogenetic, pan-genome, and population structure analyses were carried out to determine the genome diversity of the circulating Peruvian S. Typhimurium isolates. Finally, identification and characterisation of AMR genes, GWAS-based prediction of new non-synonymous single nucleotide variation (SNVs) for AMR, prediction of AMR effect of newly identified SNVs, and genotype–phenotype correlations were performed for antimicrobial resistome profiling of these isolates. The overview of the methods applied, and the objectives of this study are represented in In this study, we used whole genome sequences of S. Typhimurium isolated from humans (n = 78), animals (n = 3) and the environment (n = 9). The isolates were collected between 2000 and 2017 from 14 cities in Peru, as shown in the global map . We examined 90 pathogenic The disk diffusion method  was usedSalmonella Typhimurium genome (90 isolates) sequence raw reads generated under the 10K Salmonella Project (BioProject: PRJEB35182). After assembly and annotation, we submitted these genomes to the BioProject (PRJNA635403) as “Peruvian Salmonella spp. Genome sequencing and assembly”. The genome sequencing was performed within the 10K Salmonella Project as described by Perez-Sepulveda et al., 2021 [We used Peruvian l., 2021 . Addition = 90 out of a total n = 109) for core genome analysis, variant determination, and monitoring of infectious outbreaks in other previous studies [https://github.com/tseemann/abricate; accessed on 4 April 2022) were also used to crosscheck the MOB-suite results.The raw fastq sequences from the Illumina 150 bp paired-end were checked for quality using FastQC v0.11.8 . De novo studies ,36,37. P studies  and clas studies  and ABRihttps://github.com/tseemann/mlst; accessed on 5 April 2022) [https://ggplot2.tidyverse.org/reference/index.html; accessed on 5 April 2022). The respective tools’ assembled genomes and default parameters were used for these analyses.The serovar and sequence types were predicted using SeqSero  and MLSTil 2022) , respectil 2022)  to deterS. Typhimurium isolates from 29 countries distributed on six continents . We used S. Typhimurium  as the reference. Core genome-SNPs were predicted using Snp-sites [Core and accessory genes were identified using Roary v3.12  with defnp-sites  from thenp-sites  core gennp-sites , maximumS. Typhimurium populations was calculated using the pairwise similarity  and median pairwise similarities using the core genome SNPs and the MEGA-X tool [The population structure for two sample sets was determined separately using core gene SNPs: (i) Peruvian and (ii) Peruvian + 50 global samples. The Bayesian analysis of population structure (BAPS)  model wahttps://github.com/tseemann/abricate; accessed on 4 April 2022) using CARD [Salmonella spp. sequences, and were included to corroborate the results. The AMR genes were classified according to resistance mechanisms and drug class using the CARD database and manual curation [For each genome, the AMR-associated genes were identified in the chromosome and plasmids using ABRicate  analysis using disc diffusion antimicrobial susceptibility test results in combination with the WGS-informed AMR analysis. These assays were compared with the occurrence of known AMR genes and alleles associated with the resistance to the respective drug. False-positive, false-negative, sensitivity, specificity, and accuracy of the GPC were calculated as described previously . Finallyhttps://github.com/tseemann/snippy; accessed on 16 April 2022) was used for variant calling, and the SNPs and indels were filtered considering a minimum sequencing depth > 20x and minor allele frequency less than 0.02 were removed. A significant association was considered when p < 0.05, and the values were automatically corrected for multiple testing using the Bonferroni method [https://github.com/vicbp1/Genetic-Arquitecture-of-Zika; accessed on 16 April 2022). The population structure was predicted using principal component analysis (PCA) and PLINK v1.90b6.9 [In isolates where genotype–phenotype association did not match, we used the genome-wide association study (GWAS) approach to identify possible genes and alleles for specific AMR. A similar approach, as described by Bandoy and Weimer , was usei method . The res1.90b6.9 , consideThe GWAS represent a powerful approach to identifying new genetic variants in isolates that demonstrated phenotypic drug resistance but did not contain determinants associated with known antimicrobials. We examined the potential for additional gene variants that may explain the specific drug resistance in those isolates. When found, we adopted the strategy described by Ferla et al. ,60. Addin = 140) were >99.4% identical  and contained 11,168 orthologous genes with 3455 core genes and 7713 accessory genes  that represent the 30.9% and 69.1%, respectively (n = 90), the core and accessory genome constitutes 42.6% and 57.4%, respectively, where there are 7462 orthologous genes, 3181 core genes and 4281 accessory genes   C,D. We i = 2611)  between n = 47) includes the sequences cluster SC13 (n = 24), SC14 (n = 9), SC15 (n = 7), SC16 (n = 4), and SC9 (n = 3); while the second clade (n = 43) includes SC2 (n = 6), SC3 (n = 4), SC8 (n = 6), SC17 (n = 26), and SC18 (n = 1)  .While we further analysed the sequence clusters only for the 90 Peruvian samples, we identified four additional sub-populations without much phylogenetic difference, where SC2 consists of three sub-sequence clusters and SC3 and SC8 had two sub-sequence clusters each . Therefon = 18, 40.9%; or 20.0% considering total 90 isolates), TE , AM , N , and 11 samples  show MDR (n = 5), followed by SC9 (n = 2), SC15 (n = 2), SC17 (n = 1). In sample collection site-based analysis, Lima shows most (n = 10) of the MDR isolates and only one from Huánuco  contained resistance to at least one drug from the tested drugs using the disc diffusion assay. Out of these 44 isolates, the highest phenotypic resistance profile was for NA  different AMR genes as per drug class and resistance mechanisms. We found 31 were chromosomal, 32 were acquired in the 90 samples . Howevern = 17, 65.4% or 18.9% considering total 90 isolates), tetracycline , and ampicillin  (n = 5) and SC9 (n = 3)  . These M (n = 3) . The detn = 2), multi-drug resistance (n = 2) and additional drugs not testing (n = 2). Therefore, we considered a total of 50 isolates  for our further analysis  isolates had no correlation between the genotype and phenotype. In these 22 phenotypically drug-resistant isolates, ten isolates showed resistance exclusively to N, 3 to AM, 2 to CTX, and one each to NA, TE, AMC, AM + N, AM+ CTX+ C, AM + C+ TE, and AM+ SXT  . These in = 1/SC8) carrying blaCTX-M-15 gene and IncI1-I(Alpha)_P14 plasmid (n = 1/SC3), blaSHV-12 and blaSHV-134 genes  showed presence of Col(pHAD28) plasmid carrying fluoroquinolone-resistant either qnrB19 or qnrB5 gene. The fluoroquinolone-resistant qnrE2 gene was found in three isolates belonging to the SC9 group that contained the IncHI2_P1 plasmid _P12 (n = 1/SC15), and mcr-1 gene for colistin resistance was found in IncHI2_P1 (n = 1/SC9), lncI2_P3 (n = 2/SC9)  .n = 11, Huanuco n = 1). From these twelve MDR isolates, six isolates were from Lima, Hospital Emergencias Pediatrica, two were from Instituto Nacional de Salud del niño and another four MDR isolates were distributed in four different hospitals in Lima. AM, NA and TE resistance were the most prevalent in these MDR isolates. Three isolates showed phenotypic resistance to maximum five antibiotics  and were prevalent at Hospital Emergencias Pediatrica and Instituto Nacional de Salud del niño. Importantly, we also found that Salmonella acquired MDR genes after 2015  had 17 SNVs and the microcin resistance gene (yojI) showed two SNVs. However, we were unable to proceed with these variations as we did not perform the phenotypic tests for these drugs , and sdiA (K104Q for NA), were selected for structure-based functional annotation  in 11 AMR genes in our samples. Among these, four aminoglycoside resistance genes . It is also important to note that these 11 isolates belong to SC13, and the SC13 group had 24 isolates. Therefore, any SC13 isolate containing all eight variations has a 46.0% chance of showing the N-resistant phenotype. The SNVs were MdtH L15P, hypothetical protein G6V, QseC1 L19V, PpnN R116C, BioH A236T, WecA B284I, PurA A103G, and Tsr2 D161G. The details of these genes and SNVs are provided in We tested the effect of the newly identified three SNVs from two core AMR genes (for NA resistance) and eight new SNVs from eight genes (N resistance) from our GWAS analysis following the methods we described.tsr_2. Therefore, we succeed in checking the effect of twelve new SNVs in nine genes  between 2000 and 2017 from different cities in Peru to examine the diversity, WGS-AST correspondence, resistome profile, and emerging lineages. Our study concludes that a considerable nucleotide, gene, and phylogenetic group diversity circulates in Peru.Our study is the first using WGS analysis and antimicrobial susceptibility test (AST) of S. Typhimurium isolates in Peru belong to sequence type ST19. This was expected because ST19 is among the most predominant ST associated with gastroenteritis cases worldwide, and it is the most ancestral and diverse phylogenetically ST for the serovar Typhimurium [Peruvian samples belong predominant to ST19, with a diverse accessory content, constituted by plasmids and segregated by phylogenetic groups. We reported that 94.4% of himurium ,19,20.S. Typhimurium is mainly based on the diversity contribution of prophage genes, up 23.4%, followed by other elements such as plasmids and mobile islands, up 13.3% [The population contained an open pan-genome with many repertoire genes for global and Peruvian isolates comparable to previous reports ,63,64. Hup 13.3% ,64. In aInitially, a low accumulation nucleotide of 400–600 SNPs was reported for the serovar . AlthougThe phylogenetic topology showed two high-order clades and population structures identified at least 10 lineages supported by subclades with depth branches to multiple branches. This topology has already been discussed previously in studies; this includes an alpha clade basal with livestock samples with a diversity of terminal branches corresponding to clonal expansions. A distinct beta clade is characterised by multiple lineages from the vast host (including wild avian) that are deeply rooted ,20. LikeS. Typhimurium presents considerable resistance to first-line antibiotics, susceptibility to cephalosporins and ciprofloxacin, and low sensitivity value of phenotypic prediction due to the unknown resistance mechanism. This study explored resistance trends, transmission, profile genotypic and phenotypic, correlation, and discovery of new genetic bases for resistance phenotypes with phylogeny. The study reports a high number of strains (48.8%) with phenotypic resistance to at least one antibiotic compared to the variable prevalence  for NTS clinical and food samples from Peru, US, and England, respectively [Salmonella [ectively ,22,67. Wlmonella .S. Typhimurium strains show a significant number (24.3%-43.0%) of MDR strains to first-line antibiotics except for nitrofurantoin, and with the addition of sulphonamide, streptomycin, and chloramphenicol (in some cases) in American Latin, USA, and England [S. Infantis has been the most predominant serovar detected in clinical samples associated with high resistance to first-line antibiotics, third-generation cephalosporins [S. Typhimurium, compared to other serovars, still presents a reduced resistance to priority antibiotics. On the contrary, S. Typhimurium is a relevant pathogen in guinea pigs with distinct and moderate AMR profiles that include colistin and enrofloxacin [Salmonella isolates from chicken meat show high quinolone resistance (enrofloxacin and NA). Currently, the resistance spectra of the MDR strains of Salmonella serovars have been emerging in farm animals [Previous studies of NTS and  England ,24,66,68osporins ,8, and cosporins . Althougfloxacin , erythrofloxacin . In addi animals ,72,73,74qnrB5, qnrE2, qnrB19) or mutations  in the gyrA gene, and presence combined of both markers are associated with ciprofloxacin resistance [blaCTX-M-15, blaSHV-12, blaSHV-134). Additional WGS allowed the detection of genes associated with additional drug resistance as aminoglycoside, aminocoumarin, bacitracin, nitroimidazole, microcin, lincosamide, bleomycin, fosfomycin, colistin, and multiclass.WGS showed the content of AMR chromosomal genes with an impact on the resistance based on their expression. These AMR genes encoded most efflux pumps reported by Seribelli . Complemsistance . Interessistance , and thesistance . In onlySalmonella. Despite them, our study shows susceptibility to ciprofloxacin and third-generation cephalosporins, reported in other works [S. Typhimurium infections would be recommended.A worrying trend is an increase in resistance to treatment antibiotics  for er works ,67,68,73WGS strategy allows monitoring and complementing the prediction of phenotypic AMR profiles ,73. We fMismatch categories with a lower sensitivity, where an isolate is genotypically predicted to be susceptible but exhibits phenotypic resistance, highlights limitations based on sequence quality , partialSalmonella isolates from food animals in the USA predicted 212 resistance plasmids [S. Typhimurium [S. Infantis [S. Typhimurium isolates are prevalent that contains the virulence plasmid (pSV) [The diversity of plasmid families carrying AMR genes in isolates belong to SC8 and SC9 lineages and was detected in two hospital centres considered the focus of transmission of antimicrobial resistance. WGS also allows identifying AMR genes commonly present on plasmids, primary transmission resources, and related to the emergency lineages. Here, we reported at least 17 resistant plasmid families, including conjugative and mobilisable plasmids, with the potential threat of spreading AMR genes in NTS ,22. Theshimurium  and MDR id (pSV) ,81. HoweMCR-1-carrying lncI2 and IncHI2 belong to the SC9 lineage. These dominant mobilisable plasmids show colistin resistance [fosA3-carrying IncFIB plasmid. Previous studies have reported antibiotic’s last line resistance as the fosA3 gene in AMR plasmids [Salmonella, playing a role in the acquisition of ARGs, and has been reported recently encoding ESBLs [Interestingly, the genotypic profile is linked to family plasmid in some lineages. For example, two only strains contain replicon plasmid carrying ESBLs genes and unnamed replicon plasmid P4 carrying beta-lactamases genes in lineage SC8. Lineages S8 and SC2 harbour PMQR genes in Col(pHAD28) plasmid. Col(pHAD28) plasmids related to fluoroquinolone resistance were reported . MCR-1-csistance ,84. Likeplasmids  and IncFplasmids ,87. Intrng ESBLs ,88,89,90ng ESBLs . TherefoparC p.R360H, R365H gene, and sdiA p.K104Q that show destabilising effect protein with a possible impact on the protein function. Mutations in gyrA-parC genes decrease the binding affinity of quinolones with DNA-topoisomerase enzymes [sdiA gene acts as a positive regulator of the constitutive expression of the AcrAB–TolC pump system [acrR regulator or multi-drug pump AcrAB were associated with high-level fluoroquinolone resistance [GWAS analysis allows us to identify new non-synonymous mutations that can potentially improve resistance fitness; however other resistance confirmation strategies are necessary. We reported new SNVs in  enzymes ,93. Effl enzymes . sdiA gep system . Nucleotp system  act in hp system ,95. Genesistance .We found that considerable nitrofurantoin resistance could not be associated with known AMR markers such as those previously reported . This woS. Typhimurium. However, due to low sensitivity values from genotype–phenotype resistance correlation, it is still necessary to evaluate the use of WGS to predict AMR susceptibility. We recommend the third-generation cephalosporin antibiotic as a potential treatment against infection by S. Typhimurium. We can reinforce that WGS constitutes a complement but not an alternative to traditional methods to infer antimicrobial susceptibility, as a powerful tool that allows genome-based epidemiological study, monitoring AMR genes, virulence, plasmid typing, outbreaks, understanding of resistance mechanism, and transmission patterns. Pathogen genomic surveillance should be expanded globally and continuously monitored for better treatment of Salmonellosis and control strategies against the disseminating AMR. Future work should be based on better discordant prediction due to the absence of AMR genes on resistance phenotypes, adding new resistance mechanisms and improving the database’s reliability, replicating assay, large-scale samples, and supplementary technical methods.Our work based on the WGS analysis has allowed us to understand the dynamics and determinants of antimicrobial resistance distinguished by the population diversity for