Genome-wide differential genetic profiling characterizes colorectal cancers with genetic instability and specific routes to HLA class I loss and immune escape

Aim

We compared the expression of genes related to inflammatory and cytotoxic functions between MSI and MSS (HLA-class I-negative and HLA-class I-positive) colorectal cancers (CRCs), seeking evidence of differences in inflammatory mediators and cytotoxic T-cell responses. Twenty-two CRCs were divided into three study groups as a function of HLA class I expression and MSI phenotype: 8 MSI tumours, 6 MSS/HLA? tumours and 6 MSS/HLA+ tumours (controls).

Findings

A first comparison between eight MSI and six MSS/HLA-positive (control) cancers, based on microarray analysis on an Affymetrix^? HG-U133-Plus-PM plate, identified 1974 differentially expressed genes (P?P?=?5.5·10^?3), leucocyte activation (43 genes, P?=?1.8·10^?5), T-cell activation (24 genes, P?=?6.3·10^?4), inflammatory response (40 genes, 2.3·10^?2) and cytokine production (10 genes, P?=?1.9·10^?2). Real-time PCR and immunohistochemical evaluation were used to validate the data, finding that increased mRNA levels of pro-inflammatory cytokines and cytotoxic mediators were associated with greater infiltration by CD8+T lymphocytes in the MSI group (P?AP2, B2m) were downregulated in MSS/HLA-class I-negative CRCs (n?=?6) in comparison to controls.

Conclusions

In conclusion, microarray and immunohistochemical data may be useful to comprehensively assess tumour–host interactions and differentiate MSI from MSS cancers. The two types of tumour, MSI/HLA-class I-negative and MSS/HLA-class I-negative, showed marked differences in the composition and intensity of infiltrating leucocytes, suggesting that their immune escape strategies involve distinct pathways.     

HLA class I and H ferritin gene polymorphisms in normal subjects and patients with haemochromatosis

Summary The gene for idiopathic haemochromatosis is located on the short arm of chromosome 6 within 1 cM of the HLA-A locus. In this region there are many HLA class I genes, and there may also be a gene for the H subunit of ferritin. Both HLA class I and H ferritin genes are therefore candidates for the abnormal gene in idiopathic haemochromatosis. In 15 unrelated patients the frequency of HLA-A3 was 80% compared with 24% for 600 unrelated individuals from South Wales. The most common haplotype involved is probably HLA-A3, B7. DNA was prepared from leucocytes from 12 of these patients and from 85 normal subjects. After digestion with Taq1, electrophoresis, and Southern blotting, class I sequences were detected by hybridisation to an HLA class I probe (pHLA-A). Of the 34 restriction fragments detected, 22 were polymorphic. Particular fragments correlated with the presence of HLA-A antigens A1, 2, 3, 10, 11, w19, and 28, but there was little correlation with B antigens. Restriction fragment patterns specific for haemochromatosis were not found with TaqI or during less extensive studies with other restriction enzymes. No differences in restriction fragment patterns were found between four patients and four normal subjects apparently homozygous for HLA-A3 and B7. Examination of Southern blotting patterns for genomic DNA from patients and normal subjects with a panel of 12 restriction enzymes and a probe for the H ferritin gene (pDBR-2) revealed no polymorphisms associated with either idiopathic haemochromatosis or particular HLA phenotypes. These studies provide no support for either HLA class I genes or the H ferritin gene as candidates for the haemochromatosis gene.     

Altered structure of HLA class I heavy chains associated with mouse beta-2 microglobulin

The serological reactivities of HLA-A3, -B7, and -CW3 heavy chains associated with either mouse, bovine, or human beta-2 microglobulin ( ₂m) and expressed on the surface of transfected mouse fibroblasts were analyzed. All reactivities associated with one cluster (defined by monoclonal antibody W6/32) of antigenic determinants expressed by these HLA class I molecules were lost, or profoundly reduced, after each heavy chain associated with mouse ₂-m. Expression by the transfected fibroblasts of the HLA-A3, -B7, and -CW3 heavy chains in association with human ₂m restores these reactivities. Since most of the amino acid differences between mouse and human ₂m probably correspond to externally oriented hydrophilic residues, these results suggest that critical interactions in the three-dimensional structure of HLA class I molecules occur between the light chain and the first two external domains of the class I heavy chains, to which some of the altered reactivities have been mapped.     

The transport of class I major histocompatibility complex antigens is determined by sequences in the α1 and α2 protein domains

The transport of human-mouse hybrid class I histocompatibility antigens has been studied in a mutant human cell line, 174 × CEM.T2 (T2). T2, a somatic cell hybrid of human B- and T-lymphoblastoid cell lines (B-LCL and T-LCL, respectively), synthesizes HLA-A2 and HLA-B5 glycoproteins, but expresses only low levels of A2 and undetectable levels of B5 at the cell surface. We have previously shown that the products of human class I genes introduced into T2 by transfection behave like the endogenous HLA-B5 glycoproteins, while the products of mouse class I alleles similarly introduced are transported normally to the cell surface. We have now determined that the surface expression of class I glycoproteins in T2 depends on the origin of the ₁ and ₂ domains. Human (HLA-B7) and mouse (H-2D ^p ) hybrid class I genes, encoding the leader, ₁, and ₂ sequences of one species fused to the ₃, transmembrane, and cytoplasmic domains of the other, were transfected into T2. Normal surface expression of the hybrid class I molecule was observed in T2 only when the leader, ₁, and ₂-encoding exons were derived from the mouse gene. The reciprocal construct, encoding human leader, ₁, and ₂ domains fused to the mouse ₃, transmembrane, and cytoplasmic regions, resulted in biosynthesis of a hybrid glycoprotein which was not transported to the cell surface. The products of both constructs were expressed normally in control cells. The effects of glycosylation on class I antigen transport were also studied using mutant class I constructs with altered glycosylation sites. Two mutant B7 genes encoding either an extra glycosylation site at position 176 or no glycosylation sites were transfected into T2. These mutant products were expressed at the cell surface in control cells, but were synthesized and not surface-expressed in T2. These data demonstrate that the HLA/H-2 transport dichotomy in T2 is a function of the origin of the ₁ and/or ₂ domains of the class I glycoprotein, and is not a reflection of glycosylation differences between the human and mouse molecules. Offprint requests to: P. Cresswell.     

Immunochemical ANALYSIS OF GLYCOSYLATED AND nonglycosylated dla class I antigens

Dog peripheral blood lymphocytes, when cultured with ³⁵S-methionine in the presence of tunicamycin, synthesize DLA molecules consisting of ₂-microglobulin and a heavy chain approximately 3000 daltons lower in apparent mol. wt. than observed in control cases. This difference in mol. wt. is consistent with the fact that a single N-linked carbohydrate side chain is present on the heavy chain of DLA class I antigens. There is no evidence of polymorphism in the DLA light chain ( ₂m). Both glycosylated and nonglycosylated forms of the heavy chain, however, show microheterogeneity, which can be related to tissue-type. Analysis by two-dimensional electrophoresis shows that the biochemical heterogeneity in the DLA heavy chain is less than expected from DLA serology, and less than found in HLA class I antigens. The data are consistent with the fact that the products of only a single DLA class I locus are detected.Abbreviations used in this paper ₂m beta-2-microglobulin - 2D two dimensional - DLA dog MHC - HLA human MHC - Ia I-region associated - MHC major histocompatibility complex - PBL peripheral blood lymphocytes - PHA-M phytohaemagglutinin-muco - pI isoelectric point - RLA rabbit MHC - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Correlation of HLA-A02* genotype and HLA class I antigen down-regulation with the prognosis of epithelial ovarian cancer

Background

In recent years, evidence is accumulating that cancer cells develop strategies to escape immune recognition. HLA class I HC down-regulation is one of the most investigated. In addition, different HLA haplotypes are known to correlate to both risk of acquiring diseases and also prognosis in survival of disease or cancer. We have previously shown that patients with serous adenocarcinoma of the ovary in advanced surgical stage disease have a particularly poor prognosis if they carry the HLA-A02* genotype. We aimed to study the relationship between HLA-A02* genotype in these patients and the subsequent HLA class I HC protein product defects in the tumour tissue.

Materials and methods

One hundred and sixty-two paraffin-embedded tumour lesions obtained from Swedish women with epithelial ovarian cancer were stained with HLA class I heavy chain (HC) and β₂-microglobulin (β₂-m)-specific monoclonal antibodies (mAb). Healthy ovary and tonsil tissue served as a control. The HLA genotype of these patients was determined by PCR/sequence-specific primer method. The probability of survival was calculated using the Kaplan–Meier method, and the hazard ratio (HR) was estimated using proportional hazard regression.

Results

Immunohistochemical staining of ovarian cancer lesions with mAb showed a significantly higher frequency of HLA class I HC and β₂-m down-regulation in patients with worse prognosis (WP) than in those with better prognosis. In univariate analysis, both HLA class I HC down-regulation in ovarian cancer lesions and WP were associated with poor survival. In multivariate Cox-analysis, the WP group (all with an HLA-A02* genotype) had a significant higher HR to HLA class I HC down-regulation.

Conclusions

HLA-A02* is a valuable prognostic biomarker in epithelial ovarian cancer. HLA class I HC loss and/or down-regulation was significantly more frequent in tumour tissues from HLA-A02* positive patients with serous adenocarcinoma surgical stage III–IV. In multivariate analysis, we show that the prognostic impact is reasonably correlated to the HLA genetic rather than to the expression of its protein products.     

Analysis of human class I antigens by two-dimensional gel electrophoresis

Class I antigens were isolated by immunoprecipitation from cell extracts prepared from mitogenically stimulated and internally radiolabeled peripheral blood lymphocytes (PLBs). The precipitating antibodies used are monomorphic and recognize a determinant on the heavy chain of HLA-A, B, C antigens regardless of their allelic specificities when complexed with ₂m, or determinants on ₂m itself. Comparison of class I molecules isolated from 25 different homozygous typing cels (HTC) and analyzed by two-dimensional (2-D) gel electrophoresis allowed the identification of those HLA-A,13 locus specificities most common in the European Caucasoid population. Class I antigens isolated from HTC that are HLA identical are biochemically indistinguishable also. Evidence was obtained for the expression of additional class I antigens besides the HLA-A, B, C locus products: for some haplotypes, up to six class I genes may be active in mitogenically activated PBLs. No differences in molecular weight and isoelectric point of the class I heavy chains were observed between the antigens recognized by W6/32, the anti-heavy chain reagent, and anti- ₂m reagents. The nature of the mitogenic stimulus, i. e., pokeweed mitogen or phytohemagglutinin, was irrelevant with respect to the class I antigens isolated by this method. Using the HTCs as reference, a panel of HLA-B27 positive heterozygous cells was analyzed. Two types of HLA-B27 antigens, distinct by CML typing were represented. These two forms differed also in their biochemical properties. In addition, we obtained evidence for the existence of an A2 variant. This finding was likewise confirmed by CML typing.     

Hierarchy in the assembly of HLA-B27 and HLA-Cw3 molecules in transgenic mice

Cell surface expression of human class I molecules in transgenic mice is dependent upon the available pool of ₂-microglobulin (₂m) and the affinity between mouse ₂m and human class I molecules. HLA-B27 and HLA-Cw3 transgenes can be expressed in mouse strains of the H-2 haplotypes b,f,k, and s which encode two endogenous class I genes mapping to H-2K and H-2D. The human class I genes cannot be expressed on H-2 ^dand H-2 ^qhaplotypes which encode three endogenous class I molecules (K,D,L). This suggests that there may be only enough mouse ₂m molecules to support three class I molecules. When both the HLA-B27 and HLA-Cw3 genes are introduced into H-2 ^bmice, only HLA-Cw3 reaches the cell surface. This suggests that HLA-Cw3 has a higher affinity than HLA-B27 for mouse ₂m. The possible implications of our findings regarding the assembly, transport, and expression of class I MHC molecules in vivo are discussed.     

Relationships linking amplification level to gene over-expression in gliomas

Background

Gene amplification is thought to promote over-expression of genes favouring tumour development. Because amplified regions are usually megabase-long, amplification often concerns numerous syntenic or non-syntenic genes, among which only a subset is over-expressed. The rationale for these differences remains poorly understood.

Methodology/Principal Finding

To address this question, we used quantitative RT-PCR to determine the expression level of a series of co-amplified genes in five xenografted and one fresh human gliomas. These gliomas were chosen because we have previously characterised in detail the genetic content of their amplicons. In all the cases, the amplified sequences lie on extra-chromosomal DNA molecules, as commonly observed in gliomas. We show here that genes transcribed in non-amplified gliomas are over-expressed when amplified, roughly in proportion to their copy number, while non-expressed genes remain inactive. When specific antibodies were available, we also compared protein expression in amplified and non-amplified tumours. We found that protein accumulation barely correlates with the level of mRNA expression in some of these tumours.

Conclusions/Significance

Here we show that the tissue-specific pattern of gene expression is maintained upon amplification in gliomas. Our study relies on a single type of tumour and a limited number of cases. However, it strongly suggests that, even when amplified, genes that are normally silent in a given cell type play no role in tumour progression. The loose relationships between mRNA level and protein accumulation and/or activity indicate that translational or post-translational events play a key role in fine-tuning the final outcome of amplification in gliomas.     

Characterization of the endothelial cell cytoskeleton following HLA class I ligation

Background

Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood.

Methodology and Principal Findings

The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin.

Conclusions/Significance

Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes.     

T-Cell Tolerance for Variability in an HLA Class I-Presented Influenza A Virus Epitope

To escape immune recognition, viruses acquire amino acid substitutions in class I human leukocyte antigen (HLA)-presented cytotoxic T-lymphocyte (CTL) epitopes. Such viral escape mutations may (i) prevent peptide processing, (ii) diminish class I HLA binding, or (iii) alter T-cell recognition. Because residues 418 to 426 of the hypervariable influenza A virus nucleoprotein (NP_418-426) epitope are consistently bound by class I HLA and presented to CTL, we assessed the impact that intraepitope sequence variability has upon T-cell recognition. CTL elicited by intranasal influenza virus infection were tested for their cross-recognition of 20 natural NP_418-426 epitope variants. Six of the variant epitopes, of both H1N1 and H3N2 origin, were cross-recognized by CTL while the remaining NP_418-426 epitope variants escaped targeting. A pattern emerged whereby variability at position 5 (P5) within the epitope reduced T-cell recognition, changes at P4 or P6 enabled CTL escape, and a mutation at P8 enhanced T-cell recognition. These data demonstrate that substitutions at P4 and/or P6 facilitate influenza virus escape from T-cell recognition and provide a model for the number, nature, and location of viral mutations that influence T-cell cross-recognition.Cytotoxic T-lymphocytes (CTL) kill virus-infected cells and release antiviral cytokines upon recognition of short viral peptides displayed on the cell surface by the class I HLA molecule (36). Virus-derived peptides are processed in the cytoplasm by proteasome degradation of viral proteins (25), shuttled into the lumen of the endoplasmic reticulum (ER) by the transporter-associated protein, and loaded into the basket-like groove of the class I molecule. Class I HLA molecules await peptide loading in the ER and demonstrate specificity for viral peptides with particular anchor residues representing a good fit for the class I HLA binding groove. Once stable class I HLA-peptide complexes are formed, the class I molecule and its peptide cargo are transported via the Golgi apparatus to the cell surface, where the complex is anchored to the plasma membrane (21, 36-38). CTL then survey class I HLA-presented peptides on the cell surface. Viral peptides must therefore be processed, specifically bound by class I HLA, and presented at the plasma membrane for CTL to distinguish infected cells from uninfected tissue.A high mutation rate is one of many mechanisms utilized by viruses to escape detection by the immune system. Mutations within the genome allow viruses to accumulate and select for amino acid substitutions that (i) inhibit proteasome processing and viral peptide generation (2, 23), (ii) alter anchor residues within viral peptides to diminish class I HLA binding specificity (3, 14, 24, 32), or (iii) reduce immune recognition of the class I HLA-peptide complex by varying amino acids that come in contact with the T-cell receptor (6, 10, 27, 30, 35). While viral mutations might be advantageous for escaping immune detection, such flexibility can cost the virus in terms of replicative fitness. In order to maintain reproductive fitness and structural integrity, viruses must temper their use of genetic flexibility as a means of immune escape.Influenza viruses have the well-documented ability to escape detection by various immune epitopes (3, 10, 27). A priori, investigators often assume that variable regions of the virus represent poor immune targets because such regions will not be consistently processed, presented, or recognized (15, 20). However, we along with others continue to find that a hypervariable stretch of the influenza virus nucleoprotein consisting of residues 418 to 426 (NP_418-426) is presented to CTL by different HLA-B alleles (B*0702 and B*3501) in spite of extensive viral variability within this epitope (8, 10, 27, 34). Moreover, NP_418-426 is a dominant immune epitope (8, 10, 27, 34). The consistent processing and presentation of NP_418-426 by class I HLA can be explained by the finding that different influenza virus isolates cannot mutate the proline located at position 2 (P2) within the epitope because elimination of this proline reduces viral fitness (4, 5). Little to no variability is found at the methionine P9 anchor as well. These facts lead to the unique observation that strain-to-strain variability does not abrogate class I HLA presentation of the influenza virus NP_418-426 epitope and that CTL respond to this consistently presented viral epitope in an immunodominant fashion.In this study we took advantage of the anchor residue conservation that prompts the NP_418-426 epitope to be consistently presented to CTL by investigating the functional impact that influenza virus intraepitope variability has on CTL recognition. The amino acid alignment of human influenza A (H1N1 and H3N2) virus nucleoprotein molecules identifies 20 unique NP_418-426 peptide sequences which demonstrate amino acid diversity between the anchors. We infected HLA-transgenic mice intranasally with influenza virus and tested CTL from these animals for their ability to recognize each of the 20 NP_418-426 variants. These 20 NP_418-426 sequences represent a natural “recombinant library” of viral epitopes that the immune system has and will face. The resulting data demonstrate a gradient of viral substitutions whereby CTL recognition diminishes depending upon the number of viral substitutions and their location within the epitope. Understanding how intraepitope variability impacts CTL recognition is discussed in terms of eliciting immune responses to variants of influenza.     

Molecular evidence for five distinct MHC class II α genes in the rabbit

Human HLA class 11 gene probes were used to identify five distinct genes encoding the class 11 heavy chain (a chain) in the rabbit. The rabbit genes were defined by both mapping data and hybridization studies of genomic clones derived from the inbred B/J rabbit strain. Analysis of the clones by hybridization at graded stringencies indicated that one group of clones corresponded to HLA-DR, one group to HLA-DQ, and two groups to HLA-DP. Clones within a fifth group, designated DN, hybridized weakly to HLA-DR and may carry a fourth species of class II genes in the rabbit. Clones within the group showing high homology to HLA-DR were found to also contain sequences hybridizing with a probe for HLA-DR . No HLA-DP, -DQ, or -DR sequences were detected in any of the other class II clones. Distinct banding patterns observed in Southern blot analyses using either human or rabbit class II probes revealed restriction fragment length polymorphism for the different rabbit haplotypes studied. TheDN, DQ, andDR genes appear to be present as single copies whereas there are two distinctDP-like genes in the rabbit.Abbreviations used in this paper RLA major histocompatibility complex of the rabbit - RFLP restriction fragment length polymorphism - RL-5 rabbit T-cell line - SSC 0.15 M sodium chloride, 0.015 M sodium citrate     

Frequent alterations of SLIT2–ROBO1–CDC42 signalling pathway in breast cancer: clinicopathological correlation

The aim of the study was to understand the role of SLIT2–ROBO1/2–CDC42 signalling pathways in development of breast cancer (BC). Primary BC samples (n=150), comprising of almost equal proportion of four subtypes were tested for molecular alterations of SLIT2, ROBO1, ROBO2 and CDC42, the key regulator genes of this pathway. Deletion and methylation frequencies of the candidate genes were seen in the following order: deletion, SLIT2 (38.6%) >ROBO1 (30%) >ROBO2 (7.3%); methylation, SLIT2 (63.3%) >ROBO1 (26.6%) >ROBO2 (9.3%). Majority (80%, 120/150) of the tumours showed alterations (deletion/methylation) in at least one of the candidate genes. Overall, alterations of the candidate genes were as follows: SLIT2, 75.3% (101/150); ROBO1, 45.3% (68/150); ROBO2, 15.3% (23/150). Significantly, higher alteration of SLIT2 locus was observed in triple negative breast cancer (TNBC) over HER2 subtype (P=0.0014). Similar trend is also seen in overall alterations of SLIT2 and/or ROBO1, in TNBC than HER2 subtype (P=0.0012); of SLIT2 and/or ROBO2 in TNBC than luminal A (P=0.014) and HER2 subtype (P=0.048). Immunohistochemical analysis of SLIT2, ROBO1/2 showed reduced expression, concordant with their molecular alterations. Also, high expression of total CDC42 (49/52; 94.2%) and reduced expression of phospho Serine-71 CDC42 (41/52; 78.8%) was observed. Coalterations of SLIT2 and/or ROBO1, SLIT2 and/or ROBO2 had significant association with reduced expression of phospho Serine-71 CDC42 (P=0.0012–0.0038). Alterations of SLIT2 and/or ROBO1, reduced expression of phospho Serine-71 CDC42 predicted poor survival of BC patients. Results indicate the importance of SLIT2–ROBO1–CDC42 signalling pathway in predicting tumour progression.     

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

Background

Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available.

Methodology/Principal Findings

We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers.

Conclusions/Significance

Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers.     

Application of isoelectric focusing for studies of major histocompatibility complex class I antigen expression on human carcinomas and sarcomas

Summary By one-dimension isoelectric focusing we analysed the major histocompatibility complex class I antigen expression on human tumours. Blood lymphocytes of the patients, processed in parallel, served as a basis for comparison. The prerequisite for the analysis is the preparation of metabolically active tumour cell suspensions devoid of significant leucocyte contamination. The method was found to be suitable for study of the expression of HLA alleles on ex vivo tumour cells and allowed the detection of changes imposed by in vitro treatment with interferon and tumour necrosis factor .     

Lack of association between integrin α<Subscript>v</Subscript>β<Subscript>3</Subscript> gene polymorphisms and hemorrhagic fever with renal syndrome in Han Chinese from Hubei,China

Hantaviruses belong to the family Bunyaviridae and cause hemorrhagic fever with renal syndrome (HFRS) in humans. β₃ integrins, including α_Vβ₃ and α_IIbβ₃ integrins, act as receptors on endothelial cells and play key roles in cellular entry during the pathogenesis of hantaviruses. Previous study demonstrated that the polymorphisms of integrin α_IIbβ₃ are associated with susceptibility to hantavirus infection and the disease severity of HFRS in Shaanxi Province of China, rather than in Finland. However, the polymorphisms of integrin α_vβ₃ in patients with HFRS was incompletely understood. Here, we aimed to investigate the associations between polymorphisms in human integrin α_vβ₃ and HFRS in Han Chinese individuals. Ninety patients with HFRS and 101 healthy controls were enrolled in this study. Analysis of five single nucleotide polymorphism (SNP) sites (rs3768777 and rs3738919 on ITGAV; rs13306487, rs5921, and rs5918 on ITGB3) was performed by TaqMan SNP genotyping assays and bi-directional PCR allele-specific amplification method. No significant differences were observed between the HFRS group and controls regarding the genotype and allele frequency distributions of any of the five SNP sites, and no associations were found between ITGAV polymorphisms/genotypes and disease severity. In conclusion, our results implied that these five SNPs in the integrin α_vβ₃ gene were not associated with HFRS susceptibility or severity in Han Chinese individuals in Hubei Province.

Open image in new window