Allele-specific Transcript Quantification Research Articles

Variants of the IFI16 gene affecting the levels of expression of mRNA are associated with susceptibility to Behçet disease.

Behçet disease (BD) is a multifactorial disease in which infectious agents have been proposed as triggers in genetically predisposed individuals. The aim of our study was to investigate the role of innate immunity receptors, specifically the nucleic acid sensors, in susceptibility to BD. Seventy-four tag single nucleotide polymorphisms (tSNP) selected in 9 candidate genes (DDX58, IFIH1, TLR3, TLR7, TLR8, AIM2, IFI16, ZBP1, and TLR9) were genotyped in 371 patients and 854 controls. Assays of mRNA expression and allele-specific transcript quantification (ASTQ) were performed in 110 and 50 controls, respectively. Patients and controls were genotyped and 2 tSNP (rs6940 in IFI16 and rs855873 in AIM2) were associated with BD. To confirm this association, these tSNP were genotyped in 850 additional controls, and the total cohort was randomly divided into 2 cohorts. The association of these 2 tSNP with the disease remained in both cohorts. One haplotype (rs6940T-rs855873G) was identified as a risk factor (OR 1.41, 95% CI 1.06-1.86, p = 0.015), and another (rs6940A-rs855873A) as a protective factor (OR 0.65, 95% CI 0.47-0.90, p = 0.009). Samples with the risk haplotype had lower IFI16 expression levels than samples with the protective (0.99 ± 0.29 vs 1.23 ± 0.50, p = 0.022). Consistently, in the ASTQ assays performed with the nonsynonymous rs6940 SNP, the risk allele had lower IFI16 expression levels than the protective (p = 0.027). Our findings suggest association of IFI16, a cytosolic sensor of dsDNA and mediator of the AIM2 inflammasome-dependent pathway, in susceptibility to BD. Differences genetically determined in the levels of this molecule could be the cause of this association.

Thrombomodulin Has a Significant Impact on Transplant Outcomes after HLA-Fully-Matched Unrelated Bone Marrow Transplantation for Standard Risk Hematologic Malignancies

Background:Endothelial injury is involved in graft-versus-host-disease (GVHD), thrombotic microangiopathy, or sinusoidal obstruction syndrome. Thrombomodulin (THBD) plays a role of anti-coagulation or anti-inflammatory responses, and there are increasing evidences suggesting recombinant human soluble thrombomodulin (rTHBD) is effective on clinical outcomes of patients with coagulopathy after hematopoietic stem cell transplantation. THBD gene has several single nucleotide polymorphisms (SNP) which are associated with the risk of venous thrombotic events or coronary heart disease. In this study, we analyzed the impact of THBD polymorphism A2729C on transplant outcomes in recipients undergoing unrelated HLA-fully-matched T-cell-replete bone marrow transplantation through the Japan Donor Marrow Program and examined allele-specific gene expression of A2729C. Moreover, we investigated the effect of treatment with rTHBD prior to total body irradiation (TBI) on endothelial cells in vitro models.Methods and Results: The SNP A2729C was analyzed using the TaqMan system (Applied Biosystems), and the results were analyzed using the Allelic Discrimination software program (Applied Biosystems). The THBD polymorphisms were retrospectively analyzed in a cohort of 399 pairs of patients with hematologic malignancies and their unrelated donors. The genotype frequencies of A/A, A/C and C/C were 10%, 35% and 55% in recipients and 51%, 39% and 10% in donors (p=0.41). In standard risk disease group, the recipient C/C genotype was associated with a better overall survival (OS) than the recipient A/A and A/C genotype (p<0.10), while the recipient C/C genotype was not significantly associated with a better transplant-related mortality (TRM) than the recipient A/A and A/C genotype (p=0.11). On the multivariate analysis, the beneficial effects of the recipient C/C genotype remained significant for OS (hazard ratio [HR], 0.13; 95% confidence interval [CI], 0.02 to 0.99; p<0.05) and TRM (HR, 4.2x10-5; 95% CI, 1.3x10-5 to 1.4x10-4; p<0.0001). In standard risk disease group, moreover, the donor C/C genotype was associated with a better TRM than the donor A/A and A/C genotype (p<0.10), but was not associated with OS (p=0.80). On the multivariate analysis for TRM, there was the beneficial effect of the donor C/C genotype (HR, 3.6x10-5; 95% CI, 0.30 to 1.6; p<0.0001). The donor/recipient THBD genotype showed no significant effects on the relapse, aGVHD and cGVHD. To further investigate the effect of rs3176123 on transcription of mRNA, we performed Allele-specific transcript quantification on human primary monocytes from 6 healthy donors with heterozygous genotypes of rs3176123. In these cells the mean ratio (C allele vs A allele) was 0.53, which is significantly higher than that of DNA amplicons (ratio 0.50 ; p<0.01). In vitro, human umbilical vein endothelial cells (HUVECs) were pretreated with 1 μg/ml rTHBD for 1 hour, and irradiated with X-rays with single doses of 10 Gy. Total RNA was extracted 3 hours after radiation, and mRNA levels were determined by RT-PCR. Pretreatment with rTHBD significantly inhibited irradiation-induced tissue factor (p<0.03), IL-6 (p<0.02), E-selectin (p<0.03) and ICAM-1 (p<0.05) up-regulation in HUVECs.Conclusions: These results suggest an association of the recipient THBD genotype with overall survival and TRM after unrelated BMT in the standard risk disease group, and may substantiate that the higher THBD activity by donor with the THBD C allele likely accounts for a reduced risk for TRM in their recipients.These could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic BMT. DisclosuresNo relevant conflicts of interest to declare.

The rs1024611 Regulatory Region Polymorphism Is Associated with CCL2 Allelic Expression Imbalance

CC chemokine ligand 2 (CCL2) is the most potent monocyte chemoattractant and inter-individual differences in its expression level have been associated with genetic variants mapping to the cis-regulatory regions of the gene. An A to G polymorphism in the CCL2 enhancer region at position –2578 (rs1024611; A>G), was found in most studies to be associated with higher serum CCL2 levels and increased susceptibility to a variety of diseases such as HIV-1 associated neurological disorders, tuberculosis, and atherosclerosis. However, the precise mechanism by which rs1024611influences CCL2 expression is not known. To address this knowledge gap, we tested the hypothesis that rs1024611G polymorphism is associated with allelic expression imbalance (AEI) of CCL2. We used haplotype analysis and identified a transcribed SNP in the 3′UTR (rs13900; C>T) can serve as a proxy for the rs1024611 and demonstrated that the rs1024611G allele displayed a perfect linkage disequilibrium with rs13900T allele. Allele-specific transcript quantification in lipopolysaccharide treated PBMCs obtained from heterozygous donors showed that rs13900T allele were expressed at higher levels when compared to rs13900C allele in all the donors examined suggesting that CCL2 is subjected to AEI and that that the allele containing rs1024611G is preferentially transcribed. We also found that AEI of CCL2 is a stable trait and could be detected in newly synthesized RNA. In contrast to these in vivo findings, in vitro assays with haplotype-specific reporter constructs indicated that the haplotype bearing rs1024611G had a lower or similar transcriptional activity when compared to the haplotype containing rs1024611A. This discordance between the in vivo and in vitro expression studies suggests that the CCL2 regulatory region polymorphisms may be functioning in a complex and context-dependent manner. In summary, our studies provide strong functional evidence and a rational explanation for the phenotypic effects of the CCL2 rs1024611G allele.

A SNP in intron 1 of TSHR controls its thymic expression and susceptibility to Graves’ disease suggesting central tolerance failure in pathogenesis

Graves’ disease (GD) is the paradigm of an anti-receptor autoimmune disease with agonistic auto-antibodies against the thyrotropin receptor (TSHR) being the underlying pathogenic mechanism. TSHR belongs to the category of tissue-restricted antigens (TRAs), which are expressed by medullary thymic epithelial cells (mTECs) and thereby induce central T cell tolerance. In order to understand the association between TSHR gene polymorphisms and GD we tested the hypothesis that TSHR gene variants affect susceptibility to GD by influencing levels of TSHR transcription in the thymus. The results indicate that thymic glands from normal children homozygous for the rs179247 predisposing allele of TSHR had significantly fewer TSHR mRNA transcripts than carriers of the protective allele. In addition, in heterozygous, the TSHR predisposing allele was expressed at a lower level than the protective one as demonstrated by Allele Specific Transcript Quantification. The effect of TSHR SNP rs179247 was thymus-specific and not observed in thyroid glands. These results constitute first evidence for the involvement of central tolerance in the loss of tolerance to TSHR in GD and underscore the concept that variable expression levels of major target autoantigens in the thymus influence the predisposition to autoimmunity presumably by changing the threshold of tolerance.

Association of an SNP with intrathymic transcription of TSHR and Graves' disease: a role for defective thymic tolerance

Graves' disease (GD) is the paradigm of an anti-receptor autoimmune disease, with agonistic auto-antibodies against the thyrotropin receptor (TSHR-thyroid-stimulating hormone receptor) being the underlying pathogenic effector mechanism. The TSHR belongs to the category of tissue-restricted antigens, which are promiscuously expressed in the thymus and thereby induce central T cell tolerance. In order to understand the association between TSHR gene polymorphisms and GD, we tested the hypothesis that TSHR gene variants affect susceptibility to GD by influencing levels of TSHR transcription in the thymus. We show that thymic glands from non-autoimmune donors homozygous for the rs179247 SNP predisposing allele of TSHR had significantly fewer TSHR mRNA transcripts than carriers of the protective allele. In addition, in heterozygous individuals, the TSHR predisposing allele was expressed at a lower level than the protective one as demonstrated by allele-specific transcript quantification. This unbalanced allelic expression was detectable in both thymic epithelial cells and thymocytes. Since the level of self-antigen expression is known to influence the threshold of central tolerance, these results are compatible with the notion that defective central tolerance contributes to the pathogenesis of GD, a scenario already implicated in type 1 diabetes mellitus, myasthenia gravis and autoimmune myocarditis.

608 COMBINING DNA-SEQ AND RNA-SEQ FOR DISCOVERY OF NOVEL MUTATIONS IN HUMAN PROSTATE CANCER

rs1512268 on chromosome 8p21 is associated with PC susceptibility, which is located at 14kb downstream of a prostate tumor suppressor gene NKX3.1. In the present study, we aimed to identify functional or causative variants in this region conferring PC susceptibility. METHODS: To identify candidates of functional or causative variants, we performed re-sequencing and fine mapping of this region. The variants in the 3 UTR of NKX3.1 were further screened by RNA stability assay. The variants in the intron, the 5 UTR or the upstream of NKX3.1 were screened by electrophoretic mobility shift assay. The differences in the binding affinity to nuclear proteins between the alleles were confirmed by competition assays, and the nuclear protein that binds to this region was determined by supershift assays. The association between the alleles and transcriptional activities were assessed by reporter assays. NKX3.1 expression in relation to the alleles were examined in vivo by quantative real time PCR and allele specific transcript quantification assays using cDNA from normal prostate tissues. RESULTS: We identified 12 candidates of causative SNPs that were absolutely linked with each otherby re-sequencing and fine mapping of this region. Screening of these variants by RNA stability assay, electrophoretic mobility shift assay, and reporter assay indicated that rs11781886 in the 5 UTR of NKX3.1 displayed different binding affinity to nuclear proteins between the alleles, and that the transcriptional activity of the NKX3.1 promoter was significantly lower in the susceptible allele of this SNP. Sp1 was determined to be the transcription factor that binds to the susceptible G allele, but not to the nonsusceptible A allele. Allele-specific transcript quantification and quantitative PCR analyses showed that the expression of NKX3.1 transcript in the prostate was significantly lower in the subjects with the haplotype carrying the susceptible variant at rs11781886. CONCLUSIONS: These results suggest that the functional variant rs11781886 in the 5 UTR of NKX3.1 can affect its transcription by altering the binding affinity of a transcriptional factor Sp1, and results in PC susceptibility by lowering expression of NKX3.1 transcript in the prostate.

607 A FUNCTIONAL VARIANT IN NKX3.1 ASSOCIATED WITH PROSTATE CANCER SUSCEPTIBILITY DOWN-REGULATES NKX3.1 EXPRESSION

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011607 A FUNCTIONAL VARIANT IN NKX3.1 ASSOCIATED WITH PROSTATE CANCER SUSCEPTIBILITY DOWN-REGULATES NKX3.1 EXPRESSION Shusuke Akamatsu, Ryo Takata, Michiaki Kubo, Naoyuki Kamatani, Tomoaki Fujioka, Osamu Ogawa, Yusuke Nakamura, and Hidewaki Nakagawa Shusuke AkamatsuShusuke Akamatsu Kyoto, Japan More articles by this author , Ryo TakataRyo Takata Morioka, Japan More articles by this author , Michiaki KuboMichiaki Kubo Yokohama, Japan More articles by this author , Naoyuki KamataniNaoyuki Kamatani Yokohama, Japan More articles by this author , Tomoaki FujiokaTomoaki Fujioka Morioka, Japan More articles by this author , Osamu OgawaOsamu Ogawa Kyoto, Japan More articles by this author , Yusuke NakamuraYusuke Nakamura Tokyo, Japan More articles by this author , and Hidewaki NakagawaHidewaki Nakagawa Yokohama, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1460AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Genome-wide association studies (GWAS) identified multiple susceptible loci for prostate cancer (PC), and recent GWAS implicated that a common variant rs1512268 on chromosome 8p21 is associated with PC susceptibility, which is located at 14kb downstream of a prostate tumor suppressor gene NKX3.1. In the present study, we aimed to identify functional or causative variants in this region conferring PC susceptibility. METHODS To identify candidates of functional or causative variants, we performed re-sequencing and fine mapping of this region. The variants in the 3′UTR of NKX3.1 were further screened by RNA stability assay. The variants in the intron, the 5′UTR or the upstream of NKX3.1 were screened by electrophoretic mobility shift assay. The differences in the binding affinity to nuclear proteins between the alleles were confirmed by competition assays, and the nuclear protein that binds to this region was determined by supershift assays. The association between the alleles and transcriptional activities were assessed by reporter assays. NKX3.1 expression in relation to the alleles were examined in vivo by quantative real time PCR and allele specific transcript quantification assays using cDNA from normal prostate tissues. RESULTS We identified 12 candidates of causative SNPs that were absolutely linked with each otherby re-sequencing and fine mapping of this region. Screening of these variants by RNA stability assay, electrophoretic mobility shift assay, and reporter assay indicated that rs11781886 in the 5′UTR of NKX3.1 displayed different binding affinity to nuclear proteins between the alleles, and that the transcriptional activity of the NKX3.1 promoter was significantly lower in the susceptible allele of this SNP. Sp1 was determined to be the transcription factor that binds to the susceptible G allele, but not to the non-susceptible A allele. Allele-specific transcript quantification and quantitative PCR analyses showed that the expression of NKX3.1 transcript in the prostate was significantly lower in the subjects with the haplotype carrying the susceptible variant at rs11781886. CONCLUSIONS These results suggest that the functional variant rs11781886 in the 5′UTR of NKX3.1 can affect its transcription by altering the binding affinity of a transcriptional factor Sp1, and results in PC susceptibility by lowering expression of NKX3.1 transcript in the prostate. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e244-e245 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shusuke Akamatsu Kyoto, Japan More articles by this author Ryo Takata Morioka, Japan More articles by this author Michiaki Kubo Yokohama, Japan More articles by this author Naoyuki Kamatani Yokohama, Japan More articles by this author Tomoaki Fujioka Morioka, Japan More articles by this author Osamu Ogawa Kyoto, Japan More articles by this author Yusuke Nakamura Tokyo, Japan More articles by this author Hidewaki Nakagawa Yokohama, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Toll‐like receptor 7 rs179008/Gln11Leu gene variants in chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection affects an estimated 3% of the world's population. The natural outcome of infection and the natural course of disease are highly variable. Sensing of viral single-stranded RNA (ssRNA) by Toll-like receptor 7 (TLR7) is likely involved in early pathogen detection and host response to viral infections. This study analyzed epidemiological and clinical data from 136 patients with HCV infection with regard to rs179008/Gln11Leu, a non-synonymous polymorphism within exon 3 of the X-linked TLR7 gene, the variant allele of which is suggested to code for a functionally impaired protein. Allele-specific transcript quantification (ASTQ) analyses in heterozygous females revealed individual skewed mosaicism in peripheral blood mononuclear cells (PBMCs). Thus, analyses were restricted to homo- and hemizygous individuals. Among the clinical and histological parameters studied, the variant allele T was found to be solely associated with the presence of portal lymphoid aggregates. Whereas hepatic viral load and expression of genes known to be induced in chronic HCV infection were not found to differ in patients with wild-type or variant TLR7 rs179008 genotype, significant lower gene expression of interleukin-29 (IL-29)/lambda(1) interferon (IFN-λ(1)) and both of its receptor subunits was found for T homo- and hemizygous patients. Irrespective of the minor differences in disease phenotype including hepatic viral load, natural, and alpha interferon (IFN-α)-mediated outcome of infection, and disease activity and progression, the significant differences in hepatic IL-29/IFN-λ(1) and IFN-λ receptor gene expression between TLR7 rs179008 T and A allele patients might have implications for responsiveness to future IFN-λ-based approaches.

Functional relevance of the IRF-1 promoter polymorphism rs2549009 on transcriptional activity in a native genomic environment

Interferon regulatory factor-1 (IRF-1), a transcription regulator involved both in inducing and in mediating the effects of interferon, is encoded by a highly polymorphic gene in different ethnic populations. Some of these genetic variations have been described to be associated to disease traits in hepatitis C virus and in human immunodeficiency virus infection, including one single-nucleotide polymorphism rs2549009 within the promoter region. This study aimed at investigating the functional relevance of rs2549009 on IRF-1 transcriptional activity in peripheral blood mononuclear cells in its natural genomic environment. Haplotype-specific chromatin immunoprecipitation using antibodies directed against both the transcriptionally inactive and active RNA polymerase II (RNAPII) and allele-specific transcript quantification techniques were applied to ex vivo-derived samples from healthy heterozygous donors. Inactive serine 5 phosphorylated RNAPII was found to be preferentially bound to the rs2549009 A allele in all donors investigated. Active serine 2 phosphorylated (ser2-P) RNAPII, in contrast, was found to be precipitable, depending on the donor, preferentially either with the A or the G promoter variants or without any preference. The ratio of rs2549009 A/G promoter variants engaged by ser2-P RNAPII was closely related to the relative frequency of the respective IRF-1 transcripts, and relative allelic expression was found to be associated to total IRF-1 gene expression. These results provide evidence for a bidirectional IRF-1 gene expression imbalance that appears not to be solely controlled by rs2549009 in cis and may rely on a yet unidentified variant or haplotype or on environmental control in trans.

The Dilemma of Goodness: Acknowledging Beneficence Tension in the Search for Consensus

Patient care in family medicine and palliative medicine involves important communication with stakeholders. Beneficence is assumed to be crucial to achieving consensus in these encounters. However, stakeholders may have different perspectives of beneficence. The term beneficence tension is introduced to conceptualize these differing perspectives. Acknowledging beneficence tension is suggested as a way to enhance effective communication with patients and families.

A functional variant in NKX3.1 associated with prostate cancer susceptibility down-regulates NKX3.1 expression

Genome-wide association studies (GWAS) identified multiple susceptible loci for prostate cancer (PC), and recent GWAS implicated that a common variant rs1512268 on chromosome 8p21 is associated with PC susceptibility, which is located at 14 kb downstream of a prostate tumor suppressor gene NKX3.1. To clarify a susceptibility gene and functional variants in this locus, we performed re-sequencing and fine mapping of this region and identified 12 candidates of functional single nucleotide polymorphisms that were absolutely linked with each other. Screening of these variants by RNA stability assay, electrophoretic mobility shift assay (EMSA) and reporter assay indicated that rs11781886 in the 5'-UTR of NKX3.1 displayed different binding affinity to nuclear proteins between the alleles, and that the transcriptional activity of the NKX3.1 promoter was significantly lower in the susceptible allele of this variant. Sp1 was determined to be the transcription factor that binds to the susceptible G allele, but not to the non-susceptible A allele. Allele-specific transcript quantification (ASTQ) and quantitative PCR analyses showed that the expression of NKX3.1 in the prostate was significantly lower in the subjects with the haplotype carrying the susceptible allele. These results suggest that the functional variant rs11781886 in the 5'-UTR of NKX3.1 can affect its transcription by altering the binding affinity of a transcriptional factor Sp1, and might result in PC susceptibility by lowering expression of NKX3.1 in the prostate.

Loss of imprinting of IGF2 characterises high IGF2 mRNA-expressing type of fibroblast-like synoviocytes in rheumatoid arthritis

ObjectiveIncreased expression of insulin-like growth factor 2 (IGF2) by fibroblast-like synoviocytes (FLS) was associated with low inflammatory synovium of patients with rheumatoid arthritis (RA). The aim of this study was...

Functional impact of endotoxin receptor CD14 polymorphisms on transcriptional activity

The polymorphism rs2569190 within the CD14 endotoxin (lipopolysaccharide, LPS) receptor gene is associated with various disease conditions that are assumed to rely on endotoxin sensitivity. In vitro experiments suggest that the T allele sensitizes the host for exogenous or endogenous LPS via an enhanced CD14 expression. To prove the impact of this single nucleotide polymorphism in its natural genomic context in vivo, two parameters of gene transcription were analyzed in peripheral blood mononuclear cells (PBMC) from single healthy individuals: (a) recruitment of RNA polymerase II by haplotype-specific chromatin immunoprecipitation and (b) the relative amount of transcripts by allele-specific transcript quantification (ASTQ). RNA polymerase II was found to be twice as much bound to the most prevalent haplotype, C-T-C-G, the only one carrying a T at the position rs2569190 of interest. ASTQ employing two independent read-out assays revealed, however, similar transcript numbers originating from C-T-C-G and non-C-T-C-G haplotypes. Total CD14 mRNA levels from freshly isolated PBMC, moreover, were neither related to donors’ geno- nor haplogenotypes. Our data argue for a functional impact of the rs2569190 polymorphism in terms of a stronger transcription initiation on T allele gene variants even if preferential allele-specific binding does not result in an increase in transcript numbers. Endotoxin sensitivity associated with this genetic variation appears not to rely solely on a cis-acting regulatory impact of rs2569190 on CD14 gene transcription in PBMC.

A Polymorphism in MAPKAPK3 Affects Response to Interferon Therapy for Chronic Hepatitis C

This study aimed to identify host single nucleotide polymorphisms (SNPs) that are associated with the efficacy of interferon (IFN) therapy in patients with chronic hepatitis C. We examined whether 116 tagging-SNPs from 13 genes that are involved in type I IFN signaling associate with the outcome of IFN therapy in Japanese case-control groups; the study included 468 sustained responders and 587 nonresponders. We identified 2 SNPs (rs3792323 [A/T] and rs616589 [G/A]), located in intron 2 of mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) that were associated with the outcome of IFN therapy in patients infected with hepatitis C virus (HCV) genotype 1b (P = 4.6 x 10(-5) and 4.8 x 10(-5), respectively). The 2 SNPs were in strong linkage disequilibrium and multivariate logistic regression analysis showed that rs3792323 is an independent factor associated with the IFN efficacy (genotype 1b; P = .0011). MAPKAPK3 is a kinase involved in the mitogen and stress responses, but the biological significance of MAPKAPK3 in IFN responses is poorly understood. By using an allele-specific transcript quantification assay in liver biopsy, we showed that allele-specific expression of MAPKAPK3 messenger RNA, corresponding to the risk allele for nonresponse, was significantly higher than that of the other allele. Luciferase reporter assay data indicated that overexpression of MAPKAPK3 inhibits IFN-alfa-induced gene transcription via IFN-stimulated response element and IFN-gamma-activated site. The SNP rs3792323 in MAPKAPK3 associates with the outcome of IFN therapy in patients with HCV genotype 1b. Our functional analyses indicate that MAPKAPK3 inhibits IFN-alfa-induced antiviral activity.

Functional SNPs in CD244 increase the risk of rheumatoid arthritis in a Japanese population

Rheumatoid arthritis is a chronic autoimmune inflammatory disease with a complex genetic etiology. Members of the signaling lymphocyte activation molecule (SLAM) family carry out pivotal functions in innate immunity and in conventional lymphocytes. We identified a linkage disequilibrium block associated with rheumatoid arthritis in the chromosome 1q region containing multiple SLAM family genes. In this block, the association peaked at two functional SNPs (rs3766379 and rs6682654) in CD244 in two independent rheumatoid arthritis cohorts from Japan (P = 3.23 x 10(-8) and P = 7.45 x 10(-8)). We also identified a Japanese cohort with systemic lupus erythematosus that had a similar genotype distribution as the rheumatoid arthritis cohorts. We demonstrated that the rheumatoid arthritis-susceptible alleles of rs3766379 and rs6682654 and their haplotype increased their expression in luciferase and allele-specific transcript quantification assays. CD244 is a genetic risk factor for rheumatoid arthritis and may have a role in the autoimmune process shared by rheumatoid arthritis and systemic lupus erythematosus.

Nonsense Mutations in hERG Cause a Decrease in Mutant mRNA Transcripts by Nonsense-Mediated mRNA Decay in Human Long-QT Syndrome

Long-QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of the LQT2 mutations result in premature termination codons. Degradation of premature termination codon-containing mRNA transcripts by nonsense-mediated mRNA decay is increasingly recognized as a mechanism for reducing mRNA levels in a variety of human diseases. However, the role of nonsense-mediated mRNA decay in LQT2 mutations has not been explored. We examined the expression of hERG mRNA in lymphocytes from patients carrying the R1014X mutation using a technique of allele-specific transcript quantification. The R1014X mutation led to a reduced level of mutant mRNA compared with that of the wild-type allele. The decrease in mutant mRNA also was observed in the LQT2 nonsense mutations W1001X and R1014X using hERG minigenes expressed in HEK293 cells or neonatal rat ventricular myocytes. Treatment with the protein synthesis inhibitor cycloheximide or RNA interference-mediated knockdown of the Upf1 protein resulted in the restoration of mutant mRNA to levels comparable to that of the wild-type minigene, suggesting that hERG nonsense mutations are subject to nonsense-mediated mRNA decay. These results indicate that LQT2 nonsense mutations cause a decrease in mutant mRNA levels by nonsense-mediated mRNA decay rather than production of truncated proteins. Our findings suggest that the degradation of hERG mutant mRNA by nonsense-mediated mRNA decay is an important mechanism in LQT2 patients with nonsense or frameshift mutations.

Expression of human PTPN22 alleles

Considering the female predominance in most of the autoimmune disorders that associate with the PTPN22 Trp620 variant and the complexity by which this variant influences immunologic tolerance, the objective of this study was to ascertain if the allele-specific expression of the disease-associated Arg620Trp polymorphism is affected by cis-acting or sex-specific trans-acting factor/s (e.g. sex-hormones). The use of the allele-specific transcript quantification of the Arg620Trp encoding 1858T polymorphism revealed no difference in the expression of the 1858C- and T-alleles in non-stimulated peripheral blood mononuclear cells (PBMCs) from non-pregnant female subjects, male subjects or pregnant female subjects in first or third trimester (P=0.70), respectively. While the transcription of PTPN22 in anti-CD3/anti-CD28 stimulated PBMCs increased fourfold (P<0.0001) and 13-fold (P<0.0001) after 48 and 72 h of activation, respectively, the expression of PTPN22 1858C- and T-alleles increased to the same extent (P=0.64). The present result essentially excludes such phenomena as a partial explanation for the female predominance in most of the autoimmune disorders that associate with the PTPN22 Trp620 variant.

Allele-specific transcript quantification detects haplotypic variation in the levels of the SDF-1 transcripts

It has been suggested that SDF1-G801A, a single nucleotide polymorphism (SNP) in the 3' untranslated region (UTR) of the SDF1 gene, is associated with susceptibility to diseases such as AIDS and type-I diabetes. However, experimental studies examining the effect of SDF1-G801A on SDF-1 expression have not supported its functional importance. In this study, to examine whether other polymorphisms have a cis-acting effect on SDF1 expression, we carried out haplotype analyses of the SDF1 gene and the allele-specific transcript quantification utilizing Epstein-Barr virus-transformed lymphoblastoid cell lines with heterozygous genotype for SDF1-G801A. Haplotype-based analyses on the proportion of the allele-specific transcripts revealed the presence of haplotypes associated with a decreased amount of the transcripts. In addition, we observed haplotypic variation in response to dibutyl cyclic AMP and tetradecanoyl phorbol acetate that enhances the levels of SDF-1 transcripts probably through activation of transcription factors. Showing evidence that polymorphisms other than the SDF1-G801A have a cis-acting effect on expression of SDF-1 transcripts, the results of this study contribute to the interpretation of previous disease-association studies and to the selection of SNP markers for future studies. As shown in this study, allele-specific transcript quantification coupled with haplotype analyses can be an effective tool for detecting cis-acting polymorphisms in expressional regulation.

Role of IL-10 as a susceptibility factor for rheumatoid arthritis and cardiovascular disease

IL-10 is both an immunoinhibitory (monocytes and T cells) and an immunostimulatory (B cells) cytokine that may confer susceptibility and modulate disease expression in rheumatoid arthritis (RA). The capacity to produce IL-10 (as measured by production of IL-10 in a lipopolysaccharide-stimulated whole blood culture) differs about fourfold between individuals. By comparing variance between monozygotic and dizygotic twins it was estimated that 60% of these differences are caused by genetic factors. To determine relevance for susceptibility to diseases, we determined production levels of IL-10 in lipopolysaccharide-induced whole blood cultures from 76 new incident, non-treated RA patients and 63 healthy controls. RA patients had a 50% lower IL-10 production than controls (P < 0.001). In a cohort of women (≥ 85 years) with high risk of cardiovascular death [1], we observed that low innate IL-10 production yielded a threefold increase in risk of dying, which, interestingly, correlates with the presence of the A allele of the IL-10 -2849 promoter polymorphism (P = 0.021). To elucidate this genetic basis of IL-10 secretion and the functionality of IL-10 promoter polymorphisms, we used the technique of allele-specific transcript quantification to characterise the ratio between two alleles of the IL-10 gene in 15 healthy heterozygous individuals. We identified two groups whereby five healthy donors exhibited a 1:1 ratio whereas seven exhibited a ratio >1 (P < 0.0017) [2]. Donors heterozygous for haplotype IL-10.2 were only prevalent in the group with higher allelic expression ratios. The G allele of the IL-10 promoter single nucleotide polymorphism -2849 tags this haplotype, providing functional evidence that differential allelic transcription partly explains the constant associations found with IL-10 production levels. In conclusion, this study provides evidence that IL-10 haplotypes dictating production of IL-10 are involved not only in conferring susceptibility to RA, but also in risk for cardiovascular death in old age.

Limited impact of multiple 5′ single-nucleotide polymorphisms on the transcriptional control of the human β2-adrenoceptor gene

The human beta(2)-adrenoceptor (beta(2)-AR) is subject to agonist-induced down-regulation. The degree of down-regulation is associated with certain single-nucleotide polymorphisms (SNPs) through yet unknown mechanisms. The 5'-leader sequence of the beta(2)-AR gene contains several SNPs that are in strong linkage disequilibrium. The -367 T/C polymorphism, in particular, has been shown to affect transcriptional activity in reporter gene assays. In the present study we analysed the impact of this -367 SNP on the transcription rate of the beta(2)-AR gene in the context of the complete natural locus. Taking advantage of the additional full disequilibrium with the +79 SNP in the beta(2)-AR coding sequence, allele-specific transcript quantification was performed in PBMCs of six -367 heterozygous mild asthmatic patients. Our data are in line with the reported impact of the -367 SNP and give no indication of additional haplotype-related effects on beta(2)-AR transcription. We further show that the -367 SNP affects the binding of a yet unidentified transcription factor complex, whose binding activity is not modulated by pharmacological compounds that induce or down-regulate beta(2)-AR expression, suggesting a role in constitutive steady state expression rather than in inducible expression. As the beta(2)-AR allele with a higher transcription rate associates with stronger agonist-induced down-regulation, it is not likely that the -367 SNP is causally related to the degree of down-regulation.