Identification of immune feature genes and intercellular profiles in diabetic cardiomyopathy

BACKGROUND Diabetic cardiomyopathy(DCM)is a multifaceted cardiovascular disorder in which immune dysregulation plays a pivotal role.The immunological molecular mechanisms underlying DCM are poorly understood.AIM To examine the immunological molecular mechanisms of DCM and construct diagnostic and prognostic models of DCM based on immune feature genes(IFGs).METHODS Weighted gene co-expression network analysis along with machine learning methods were employed to pinpoint IFGs within bulk RNA sequencing(RNA-seq)datasets.Single-sample gene set enrichment analysis(ssGSEA)facilitated the analysis of immune cell infiltration.Diagnostic and prognostic models for these IFGs were developed and assessed in a validation cohort.Gene expression in the DCM cell model was confirmed through real time-quantitative polymerase chain reaction and western blotting techniques.Additionally,single-cell RNA-seq data provided deeper insights into cellular profiles and interactions.RESULTS The overlap between 69 differentially expressed genes in the DCM-associated module and 2483 immune genes yielded 7 differentially expressed immune-related genes.Four IFGs showed good diagnostic and prognostic values in the validation cohort:Proenkephalin(Penk)and retinol binding protein 7(Rbp7),which were highly expressed,and glucagon receptor and inhibin subunit alpha,which were expressed at low levels in DCM patients(all area under the curves>0.9).SsGSEA revealed that IFG-related immune cell infiltration primarily involved type 2 T helper cells.High expression of Penk(P<0.0001)and Rbp7(P=0.001)was detected in cardiomyocytes and interstitial cells and further confirmed in a DCM cell model in vitro.Intercellular events and communication analysis revealed abnormal cellular phenotype transformation and signaling communication in DCM,especially between mesenchymal cells and macrophages.CONCLUSION The present study identified Penk and Rbp7 as potential DCM biomarkers,and aberrant mesenchymal-immune cell phenotype communication may be an important aspect of DCM pathogenesis.

Prediction of Tumor Microenvironment Characteristics and Treatment Response in Lung Squamous Cell Carcinoma by Pseudogene OR7E47P-related Immune Genes

Objective Pseudogenes are initially regarded as nonfunctional genomic sequences,but some pseudogenes regulate tumor initiation and progression by interacting with other genes to modulate their transcriptional activities.Olfactory receptor family 7 subfamily E member 47 pseudogene(OR7E47P)is expressed broadly in lung tissues and has been identified as a positive regulator in the tumor microenvironment(TME)of lung adenocarcinoma(LUAD).This study aimed to elucidate the correlation between OR7E47P and tumor immunity in lung squamous cell carcinoma(LUSC).Methods Clinical and molecular information from The Cancer Genome Atlas(TCGA)LUSC cohort was used to identify OR7E47P-related immune genes(ORIGs)by weighted gene correlation network analysis(WGCNA).Based on the ORIGs,2 OR7E47P clusters were identified using non-negative matrix factorization(NMF)clustering,and the stability of the clustering was tested by an extreme gradient boosting classifier(XGBoost).LASSO-Cox and stepwise regressions were applied to further select prognostic ORIGs and to construct a predictive model(ORPScore)for immunotherapy.The Botling cohorts and 8 immunotherapy cohorts(the Samstein,Braun,Jung,Gide,IMvigor210,Lauss,Van Allen,and Cho cohorts)were included as independent validation cohorts.Results OR7E47P expression was positively correlated with immune cell infiltration and enrichment of immune-related pathways in LUSC.A total of 57 ORIGs were identified to classify the patients into 2 OR7E47P clusters(Cluster 1 and Cluster 2)with distinct immune,mutation,and stromal programs.Compared to Cluster 1,Cluster 2 had more infiltration by immune and stromal cells,lower mutation rates of driver genes,and higher expression of immune-related proteins.The clustering performed well in the internal and 5 external validation cohorts.Based on the 7 ORIGs(HOPX,STX2,WFS,DUSP22,SLFN13,GGCT,and CCSER2),the ORPScore was constructed to predict the prognosis and the treatment response.In addition,the ORPScore was a better prognostic factor and correlated positively with the immunotherapeutic response in cancer patients.The area under the curve values ranged from 0.584 to 0.805 in the 6 independent immunotherapy cohorts.Conclusion Our study suggests a significant correlation between OR7E47P and TME modulation in LUSC.ORIGs can be applied to molecularly stratify patients,and the ORPScore may serve as a biomarker for clinical decision-making regarding individualized prognostication and immunotherapy.

Differential Expression of Immune Genes between Body Side Skin and Groin Skin of Aohan Fine Wool Sheep

[Objective] To get major genes for wool traits regulation from immune genes. [Methods] Microarray technology was used to detect differentially expressed immune genes between body side skin （more wool growing） and groin skin （no wool growing） of Aohan fine wool sheep. [Results] 46 immune genes （fold change 〉2.0） were identified and classified, and then 6 of which were selected for QPCR confir- mation. The degree of consistency of the QPCR and microarray results was 66.67%, [Conclusion] Immune privilege may participate in wool growth regulation.

Differential Gene Expression Profiles in Acute Hepatic Failure Model in Mice Infected with MHV-3 Virus Intervened by Anti-hepatic Failure Compound

Differential gene expression profiles in Balb/cJ mouse model of acute hepatic failure infected with MHV-3 virus intervened by anti-hepatic failure compound （AHFC） and the changes of cytokines regulated by genes were investigated. The Balb/cj mice were divided into AHFC-intervened group and control group randomly. Acute hepatic failure model of Balb/cJ mice infected with MHV-3 virus was established. The survival rate in the two groups was observed. It was found that the survival rate in the AHFC-intervened group and control group was 90% and 50% respectively 48 h after intraperitoneal injection of MHV-3 （P〈0.05）. Before and after the experiment, the cytokines in peripheral blood of the survival mice were determined, and RNA was extracted from survival mouse liver tissue for the analysis of the differential gene expression by a 36 kb mouse oligonuleotide DNA array. In all the genes of microarray there were 332 genes expressed differently in the two groups, in which 234 genes were up-regulated and 78 genes down-regulated. Through clustering analysis, the differential expression of immune related genes, including TNF receptor superfamily, Kctd9, Bcl-2, Fgl2, IL-8, IL-6, IFN-7, TNF-α etc. might be related with the curative effectiveness of AHFC. It was suggested that AHFC can balance the immune state of mouse model of acute hepatic failure infected with MHV-3 virus mainly through regulating the expression of immune related genes, decrease the immune damage and inhibit liver cell apoptosis of mouse acute hepatic failure model obviously so as to increase the survival rate of mouse models of acute hepatic failure.

Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells

To construct the recombinant expression functionally in Chinese hamster ovary cells in order vector for CD80-IgG fusion gene and to express it to be used as an effective method to eliminate the immune escape of leukemic cells, the cDNA encoding the signal and extracellular domains of murine CD80 was generated by PCR amplification from plasmid pcDNMB7 containing the full length cDNA of murine CD80 and those of murine IgG1, in which the Fc fragment was obtained through RT-PCR amplification from murine spleen cells. These two cDNAs were then cloned in tandem into eukaryotic expression vector pcDNA3.0 and the resultant recombinant plasmid pcDNA/CD80-IgG was then transfected to Chinese hamster ovary cells with liposome transfection reagent. The cell clones constitutively expressing CD80-IgG fusion protein were obtained by G418 screening. Western blotting and dot ELISA assay were used to detect the expression of the fusion protein in the supernatants of these cells. Meanwhile, the fusion protein expressed was then purified with affinity chromatography, and its biological activity was demonstrated by flow cytometry, MTr colorimetry and ELISA assay. The experimental resuits showed that these two inserts were successfully cloned into plasmid pcDNA3.0, and the highly purified fusion protein was obtained. This fusion protein was proved to be able to upregulate the density of CD80 on leukemic cells, deliberately promote the proliferative reactions of mouse allogenic lymphocytes and increase the killing activity against WEHI-3 cells from 49.7 % up to 84.6 %. In addition, this fusion protein could also enhance the IL-2 secretion from allogenic lymphocytes activated by tumorspecific antigens. It is concluded that the recombinant vector constructed can be functionally expressed in the mammalian cells, thus providing a solid foundation for the further investigation on the mechanism to eliminate the immune escape of leukemic cells in vivo.

Transcriptome of intraperitoneal organs of starry flounder Platichthys stellatus challenged by Edwardsiella ictaluri JCM1680

Platichthys stellatus is an economically important marine bony fish species that is cultured in China on a large scale.However,very little is known about its immune-related genes.In this study,the transcriptome of the immune organs ofP.stellatus that were intraperitoneally challenged with the pathogen Edwardsiella ictaluri JCM1680 is analyzed.Total RNA from four tissues(spleen,kidney,liver,and intestine) was mixed equally and then sequenced on an Illumina HiSeq 2000 platform.Overall,28 465 813 quality reads were generated and assembled into 43 061 unigenes.Similarity searches against public protein sequence databases were used to annotate 28 291 unigenes(65.7%of the total),368 of which were associated with immunoregulation,including 188 related to immunity response.Additionally,the transcript levels of immunity response unigenes annotated as related to tumor necrosis factor(TNF),TNF receptor,chemokine,major histocompatibility complex,and interleukin-6 were investigated in the different tissues of normal and infected P.stellatus by real-time quantitative PCR.The results confirmed that the unigenes identified in the transcriptome database were indeed expressed and up-regulated in infected P.stellatus.To our knowledge,this is the first report of the sequencing and analysis of the transcriptome of P.stellatus.These findings provide insights into the transcriptomics and immunogenetics of bony fish.

Construction of a risk score prognosis model based on hepatocellular carcinoma microenvironment

BACKGROUND Hepatocellular carcinoma(HCC)is a common cancer with a poor prognosis.Previous studies revealed that the tumor microenvironment(TME)plays an important role in HCC progression,recurrence,and metastasis,leading to poor prognosis.However,the effects of genes involved in TME on the prognosis of HCC patients remain unclear.Here,we investigated the HCC microenvironment to identify prognostic genes for HCC.AIM To identify a robust gene signature associated with the HCC microenvironment to improve prognosis prediction of HCC.METHODS We computed the immune/stromal scores of HCC patients obtained from The Cancer Genome Atlas based on the ESTIMATE algorithm.Additionally,a risk score model was established based on Differentially Expressed Genes(DEGs)between high and lowimmune/stromal score patients.RESULTS The risk score model consisting of eight genes was constructed and validated in the HCC patients.The patients were divided into high-or low-risk groups.The genes(Disabled homolog 2,Musculin,C-X-C motif chemokine ligand 8,Galectin 3,B-cell-activating transcription factor,Killer cell lectin like receptor B1,Endoglin and adenomatosis polyposis coli tumor suppressor)involved in our risk score model were considered to be potential immunotherapy targets,and they may provide better performance in combination.Functional enrichment analysis showed that the immune response and T cell receptor signaling pathway represented the major function and pathway,respectively,related to the immune-related genes in the DEGs between high-and low-risk groups.The receiver operating characteristic(ROC)curve analysis confirmed the good potency of the risk score prognostic model.Moreover,we validated the risk score model using the International Cancer Genome Consortium and the Gene Expression Omnibus database.A nomogram was established to predict the overall survival of HCC patients.CONCLUSION The risk score model and the nomogram will benefit HCC patients through personalized immunotherapy.

Eukaryotic expression of outer membrane protein Gpd from Treponema pallidum and preliminary studies on its immune response in rabbits

The Gpd gene was amplified from the genomic DNA of Treponema the appropriate site of pcDNA3.1 （ ＋ ） vector. The expression of pcDNA3. I was tested with Western blotting and technology of immunoeytochemisty. New munized with the eukaryotic expression recombinant pcDNA3, 1 （ ＋ ）-Gpd pallidum and cloned into （ ＋ ）-Gpd in Hel,a cells Zealand rabbits were imA fusion protein of C, pd with 4.1 kDa has been effectively expressed in HeLa cells, which were detected bv Western blotting and the immunocytochemistry techniques. The New Zealand rabbits were able to elicit the specific antibody after immunization with the nucleic acid vaccine. The antibody titer could reach as high as 1 ： 1024 after 2 weeks of the third injection; and the splenocytes proliferated evidently due to the Gpd protein stimulation. Both the antibody titer and the splenocytes proliferation were higher substantially than those of controls （ P 〈 0.01 ）. All above data will contribute to an experimental basis of further study of the biological function of Gpd protein as well as DNA vaccine for syphilis.

Construction of regulatory network for alopecia areata progression and identification of immune monitoring genes based on multiple machine-learning algorithms

Objectives Alopecia areata(AA)is an autoimmune-related non-cicatricial alopecia,with complete alopecia(AT)or generalized alopecia(AU)as severe forms of AA.However,there are limitations in early identification of AA,and intervention of AA patients who may progress to severe AA will help to improve the incidence rate and prognosis of severe AA.Methods We obtained two AA-related datasets from the gene expression omnibus database,identified the differentially expressed genes(DEGs),and identified the module genes most related to severe AA through weighted gene co-expression network analysis.Functional enrichment analysis,construction of a protein–protein interaction network and competing endogenous RNA network,and immune cell infiltration analysis were performed to clarify the underlying biological mechanisms of severe AA.Subsequently,pivotal immune monitoring genes(IMGs)were screened through multiple machine-learning algorithms,and the diagnostic effectiveness of the pivotal IMGs was validated by receiver operating characteristic.Results A total of 150 severe AA-related DEGs were identified;the upregulated DEGs were mainly enriched in immune response,while the downregulated DEGs were mainly enriched in pathways related to hair cycle and skin development.Four IMGs(LGR5,SHISA2,HOXC13,and S100A3)with good diagnostic efficiency were obtained.As an important gene of hair follicle stem cells stemness,we verified in vivo that LGR5 downregulation may be an important link leading to severe AA.Conclusion Our findings provide a comprehensive understanding of the pathogenesis and underlying biological processes in patients with AA,and identification of four potential IMGs,which is helpful for the early diagnosis of severe AA.

THE EUKARYOTIC EXPRESSION OF HUMAN PERFORIN PROTEIN AND THE ESTABLISHMENT OF HYBRIDOMAS PRODUCING ANTI-HUMAN PERFORIN PROTEIN

Objective To prepare the monoclonal antibody against human perforin(HP). Methods Recombi- nant eukaryotic expression plasmid pCDM8-HP was extracted and purified, and the BALB/C mice were immunized with the plasmid. The hybridomas producing anti-HP McAbs were established by using hybridoma technique, then the specificity of the McAbs was identified by using immunocytochemical technique and Western blot. Results Three hy- bridoma cell lines secreting McAbs against human perforin were established, and the three McAbe showed positive only with LAK cells containing human perforin protein, and showed negative with inactive human peripheral blood lymphocytes (PBLs). The subclasses of the three McAbs were determined as IgG2bK. Western blot results showed that the three McAbs recognized a specific band of LAK cell lysateds with molecular weight of 70.0Kd. Conclusion The three hybridoma cell lines secreting McAbs against human perforin were established and the secreted McAbs were specific.

Hyperbaric oxygen treatment on keloid tumor immune gene expression

Background:Hyperbaric oxygen treatment(HBOT)has been demonstrated to influence the keloid recurrence rate after surgery and to relieve keloid symptoms and other pathological processes in keloids.To explore the mechanism of the effect of HBOT on keloids,tumor immune gene expression and immune cell infiltration were studied in this work.Methods:From February 2021 to April 2021,HBOT was carried out on keloid patients four times before surgery.Keloid tissue samples were collected and divided into an HBOT group(keloid with HBOT before surgery[HK]group,n=6)and a non-HBOT group(K group,n=6).Tumor gene expression was analyzed with an Oncomine Immune Response Research Assay kit.Data were mined with R package.The differentially expressed genes between the groups were compared.Hub genes between the groups were determined and verified with Quantitative Real-time PCR.Immune cell infiltration was analyzed based on CIBERSORT deconvolution algorithm analysis of gene expression and verified with immunohistochemistry(IHC).Results:Inflammatory cell infiltration was reduced in the HK group.There were 178 upregulated genes and 217 downregulated genes.Ten hub genes were identified,including Integrin Subunit Alpha M(ITGAM),interleukin(IL)-4,IL-6,IL-2,Protein Tyrosine Phosphatase Receptor Type C(PTPRC),CD86,transforming growth factor(TGF),CD80,CTLA4,and IL-10.CD80,ITGAM,IL-4,and PTPRC with significantly downregulated expression were identified.IL-10 and IL-2 were upregulated in the HK group but without a significant difference.Infiltration differences of CD8 lymphocyte T cells,CD4 lymphocyte T-activated memory cells,and dendritic resting cells were identified with gene CIBERSORT deconvolution algorithm analysis.Infiltration levels of CD4 lymphocyte T cell in the HK group were significantly higher than those of the K group in IHC verification.Conclusion:HBOT affected tumor gene expression and immune cell infiltration in keloids.CD4 lymphocyte T cell,especially activated memory CD4+T,might be the key regulatory immune cell,and its related gene expression needs further study.

Integrative Characterization of Immune-relevant Genes in Hepatocellular Carcinoma

Background and Aims:Tumor microenvironment plays an essential role in cancer development and progression.Cancer immunotherapy has become a promising approach for the treatment of hepatocellular carcinoma(HCC).We aimed to analyze the HCC immune microenvironment characteristics to identify immune-related genetic changes.Methods:Key immune-relevant genes(KIRGs)were obtained through integrating the differentially expressed genes of The Cancer Genome Atlas,immune genes from the Immunology Database and Analysis Portal,and immune differentially expressed genes determined by single-sample gene set enrichment analysis scores.Cox regression analysis was performed to mine therapeutic target genes.A regulatory network based on KIRGs,transcription factors,and immune-related long non-coding RNAs(IRLncRNAs)was also generated.The outcomes of risk score model were validated in a testing cohort and in clinical samples using tissue immunohistochemistry staining.Correlation analysis between risk score and immune checkpoint genes and immune cell infiltration were investigated.Results:In total,we identified 21 KIRGs,including programmed cell death-1(PD-1)and cytotoxic T-lymphocyte associated protein 4(CTLA4),and found IKBKE,IL2RG,EDNRA,and IGHA1 may be equally important to PD-1 or CTLA4.Meanwhile,KIRGs,various transcription factors,and IRLncRNAs were integrated to reveal that the NRF1-AC127024.5-IKBKE axis might be involved in tumor immunity regulation.Furthermore,the immune-related risk score model was established according to KIRGs and key IRLncRNAs,and verified more obvious discriminating power in the testing cohort.Correlation analysis indicated TNFSF4,LGALS9,KIAA1429,IDO2,and CD276 were closely related to the risk score,and CD4 T cells,macrophages,and neutrophils were the primary immune infiltration cell types.Conclusions:Our results highlight the importance of immune genes in the HCC microenvironment and further unravel the underlying molecular mechanisms in the development of HCC.

The genetics of Behcet’s disease in a Chinese population

Behcet’s disease is defined as a multisystemic inflammatory disease.Although the precise pathogenesis and etiology is still a mystery,accumulating evidence shows that genetic variants of immune-related genes have a profound influence on the development of Behcet’s disease.To explore the genetic factors for Behcet’s disease,our group investigated the association of Behcet’s disease with multiple immune response genes and has identified multiple Behcet’s disease-related immunoregulatory pathways in the Chinese Han population.A large number of gene polymorphisms were studied including STAT4,IL23R,CD40,CCR1/CCR3,STAT3,OPN,IL17,JAK2,MCP-1,CTLA4,PD-1,PD-L1,PD-L2,TGRBR3,CCR6,PTPN22,FCRL3,IRF5,SUMO4 and UBAC2.Significant associations were found between Behcet’s disease and STAT4,IL23R,CD40,CCR1/CCR3,STAT3,MCP-1,TGFBR3,FCRL3,SUMO4,UBAC2.These genetic predisposition studies support an important role for both lymphocyte differentiation as well as ubiquitination pathways.These findings are helpful in elucidating the pathogenesis of Behcet’s disease and hopefully will allow the development of novel treatment regimes.

Application of a recombinant replicase to localize the Trionyx sinensis hemorrhagic syndrome virus and evaluate its effects on antiviral genes of T.sinensis

Trionyx sinensis Hemorrhagic Syndrome Virus(TSHSV)is an arterivirus newly discovered in Chinese softshell turtles.Little is known about the effect of antibodies against the virus or the distribution of the virus in different organs of infected turtles.In this study,a partial protein of TSHSV-HP4 was produced using a prokaryotic expression system,and its polyclonal antibody was generated.The polyclonal antibody was confirmed by western blot and dot enzyme-linked immunosorbent assay(dot-ELISA).The distribution of TSHSV in different organs of T.sinensis was examined by immunohistochemistry(IHC)and the expression of immune-related genes was analyzed using quantitative real-time polymerase chain reaction(qRT-PCR).The results indicated that the recombinant TSHSV-HP4 protein was successfully expressed,and the generated polyclonal antibody showed specific binding to viral particles in the lung tissues of infected turtles.The IHC assay indicated that the virus was highly localized in various cells,including intestinal lymphocytes,enterocytes,kidney epithelial cells,spleen cells,lung macrophages,and cardiomyocytes.The qRT-PCR analysis revealed that TSHSV was detected in all organs tested,including the lungs,liver,kidneys,spleen,and heart.The numbers of viral mRNA copies in lung and heart tissues were significantly higher in the virus-antibody group than in the virus group.The interferonstimulated genes(ISGs),myxovirus resistance protein 2(MX2)and radical S-adenosyl methionine domain containing 2(RSAD2)were highly upregulated in all groups of infected turtles.Antibody-dependent enhancement(ADE)seemed to occur after stimulation by the polyclonal antibody,because significantly greater expression of the two genes was detected in the virus-antibody group than in the virus group.Overall,these results are important in understanding the cell localization of TSHSV and the immune response of infected turtles.

Understanding the lipopolysaccharide induced liver proteome changes and identification of immune genes in Lampetra morii

Lamprey is considered to be an excellent model for studying the origin of adaptive immunity.In the present study,three up-regulated and ten down-regulated proteins were identified in the lipopolysaccharide(LPS)induced liver proteome of Lampetra morii.Quantitative real-time PCR(qPCR)was used to characterize in the liver the abundance of transcripts encoding the differentially expressed proteins.The abundance of transcripts and the corresponding differentially expressed proteins revealed that expression of cystathionase and Zgc:112210-201 was not correlated with protein expression level.Polyl 4-hydroxylase,serum lectin,Zgc:112210-201,and cofilin 1 mRNA expression was significantly upregulated in liver tissue after treatment with LPS.Transcript abundance of differentially expressed proteins in the gill,kidney,heart,intestine,liver,and supraneural body was determined.Transcripts of Zgc:112210-201 were almost undetectable in the tissues while polyl 4-hydroxylase,protein disulfide isomerase family A(member 4),cystathionase,and serum lectin were low abundance unlike the protein which was easily detected.This study represents the first attempt at understanding the LPS induced liver proteome of L.morii and our findings contribute to the identification of novel immune genes in lamprey.

A Rapid and Simple Approach to Preparation of Monoclonal Antibody Based on DNA Immunization

Inoculation with purified specific protein is usually the first step for preparation of monoclonal antibody (mAb). But it is quite difficult to obtain pure proteins especially with natural structures.Here we attempt to replace the protein inoculation with DNA immunization in the preparation of mAb.The eukaryotic expression vectors pcDNA3-PreS2/S and pVAX-PreS2/S encoding the HBV M protein were constructed and prepared for DNA immunization.Female BALB/c mice developed a well antibody response to the target antigen after muscle injection with corresponding plasmids.The mice with effective antibodies induced were used for preparation of mAb.We found the mice immunized with three administrations of pcDNA3-PreS2/S and boosted by intrasplenic injection with the same plasmid could be exploited for preparation of mAb.And positive hybridoma cell 2D3 that can secrete specific mAb was cloned and analyzed.Our studies demonstrate that gene immunization may provide a convenient and efficient way to prepare mAbs.Cellular & Molecular Immunology. 2004;1(4):295-299.

Uncovering the immunological repertoire of the carpet shell clam Ruditapes decussatus through a transcriptomic-based approach

The grooved carpet-shell clam Ruditapes decussatus is native to the Northern Atlantic and Mediterranean Sea and has a high commercial value.It is one of the main native bivalve species cultured in Europe.The main objective of the present study was to gain further insights into the immunological repertoire of R.decussatus through a transcriptomic approach.Pooled mantle samples of eight R.decussatus individuals were sequenced using Illumina platform.A total of 67132 contigs with more than 800 bp were obtained.Manual annotation of these contigs revealed 146 immune-related genes.The gene families in which the highest number of immune-related genes was observed were:C1q domain-containing proteins(63),tumor necrosis factors(15)and toll-like receptors(TLRs,10).A total of 5359 putative single nucleotide polymorphisms(SNPs)were identified in the 146 immune-related genes.The density of SNPs ranged between 0.04 and 7.92 SNPs/100 bp.The highest and the lowest SNP density were observed in genes of the C1q domain-containing protein family.Due to the importance of TLRs in innate immunity,we focused our attention on these membrane receptors.Ten TLRs were identified based on protein domain organization.Phylogenetic analysis revealed that R.decussatus TLRs were diverse and only 3 showed orthology with TLRs of known immune functions in other bivalve species.Moreover,our analysis suggests that lineage restricted-expansions of TLRs occurred in all mollusc taxa analysed including in venerids.