Title: IDDM12 LOCUS ANALYSIS IN T1DM: IDENTIFICATION AND CHARACTERIZATION OF FUNCTIONAL POLYMORPHISMS Keywords: T1D, CTLA4, ICOS Disease: Type 1 diabetes Research type: Basic science Authors: Ruberti G, Belfiore F, Malquori L, Carsetti L and Cascino I. Institute of Cell Biology Via E. Ramarini, 00016 Monterotondo scalo (RM) Tel. 06-90091201, Fax 06-90091260, Abstract: Type1 Diabetes (T1D) is a polygenic disease caused by the immune-mediated destruction of pancreatic insulin-secreting-cells. Increasing evidence both in humans and in animal models has shown that defect(s) in immunoregulation underlie T1D, similarly to other immune-mediated disorders. T cell activation results from the integration of signals generated through the T cell antigen receptor-CD3 complex with those from additional positive and negative regulatory pathways mainly mediated by engagement of costimulatory receptors on T cells. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system. Genetic studies of human T1D indicate the presence of a disease locus in a restricted region on chromosome 2q33 (IDDM12) and in the orthologous region of murine chromosome 1. CD28, CTLA4 and ICOS genes, coding for lymphocyte co-stimulatory receptors, map in IDDM12 locus (1-3). Data obtained by our group in nuclear families and in a case-control study show an association with T1D of a series of markers in the IDDM12 region. These appear to belong to two separate association groups, one centered on the CTLA4 locus and the other on the ICOS locus. The specific aims of this project are to: 1. Identify and functionally characterize the disease variation in the ICOS region; 2. Study the functional role of the T1D-associated CTLA4 3'UTR (AT)n repeat and the CT60 polymorphism on mRNA stability and targeting. Aim 1. For the analysis of the region comprised between ICOS gene and d2s2189 microsatellite (150 kb) we have selected 12 SNPs, already described in the NCBI SNP database. The SNPs were selected for being located in the sequences that were at least 80% identical to the orthologous mouse region over a 100 bp. The underlying rationale is that essential regulatory elements are conserved among mammals, suggesting that cross-species sequence comparisons should identify them (4). We have set up the conditions for multiple PCRs and multiple microsequencing on 10 DNA control samples.Then, we have tested for association the 12 SNP in a case-control study. Three SNP who showed a trand of association with the disease, are now under TDT study on our T1D family panel with at least one affected child. Aim 2. CTLA-4 plays a critical role in downregulating T cell responses. T1D and other autoimmune diseases have shown genetic linkage to the CTLA4 locus. Here, we report that the 3'UTR of CTLA4 regulates Luciferase reporter gene expression, and CTLA4 mRNA decay and translation efficiency, providing the cells with tools to rapidly respond to changes in intracellular and extracellular stimuli. Intriguingly, we also observed that the length of the (AT)n repeat contributed to gene expression regulation. The 3'UTR T1D-associated (AT)n repeat medium (16AT) and long (24AT) alleles decreased the luciferase activity of 80% and 70% respectively in Jurkat and Hek 293T cells; while the short (6AT) allele decreased the activity of 70% and 60% in the hematopoietic and epithelial cell lines. The difference observed was statistically significant in both cell types (p<0.0001). A contribution of the medium and long (AT)n alleles on CTLA-4 protein expression was also observed. UV-crosslinking experiments suggest that the length of the (AU)n repeat influences the recruitment of trans-acting factors potentially involved in gene expression regulation. Finally, we also show that the "CT60" region of 736 bp, located 3' to the major poly(A) site of the 3'UTR of CTLA4, regulates mRNA and protein expression levels. However, the T1D-associated CT60 polymorphic SNP does not appear to contribute to this regulation in our experimental conditions. References: 1) Nistico L., Buzzetti R., Pritchard L.E., Van der Auwera B., Giovannini C., Bosi E., Larrad M.T., Rios M.S., Chow C.C., Cockram C.S., Jacobs K., Mijovic C., Bain S.C., Barnett A.H., Vandewalle C.L., Schuit F., Gorus F.K., Tosi R., Pozzilli P., Todd J.A. The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry. Hum Mol Genet. 5, 1075-80, 1996. 2) Ueda H., Howson J.M., Esposito L., Heward J., Snook H., Chamberlain G., Rainbow D.B., Hunter K.M., Smith A.N., Di Genova G., Herr M.H., Dahlman I., Payne F., Smyth D., Lowe C., Twells R.C., Howlett S., Healy B., Nutland S., Rance H.E., Everett V., Smink L.J., Lam A.C., Cordell H.J., Walker N.M., Bordin C., Hulme J., Motzo C., Cucca F., Hess J.F., Metzker M.L., Rogers J., Gregory S., Allahabadia A., Nithiyananthan R., Tuomilehto-Wolf E., Tuomilehto J., Bingley P., Gillespie K.M., Undlien D.E., Ronningen K.S., Guja C., Ionescu-Tirgoviste C., Savage D.A., Maxwell A.P., Carson D.J., Patterson C.C., Franklyn J.A., Clayton D.G., Peterson L.B., Wicker L.S., Todd J.A., Gough S.C.. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature. 423, 506-11, 2003. 3) Ihara K., Ahmed S., Nakao F., Kinukawa N., Kuromaru R., Matsuura N., Iwata I., Nagafuchi S., Kohno H., Miyako K., Hara T. Association studies of CTLA-4, CD28, and ICOS gene polymorphisms with type 1 diabetes in the Japanese population. Immunogenetics. 53, 447-54, 2001. 4) Dermitzakis E.T., Reymond A., Scamuffa N., Ucla C., Kirkness E., Rossier C., Antonarakis S.E. Evolutionary discrimination of mammalian conserved non-genic sequences (CNGs). Science. 302, 1033-5, 2003.
IDDM12 Locus analysis in T1DM: identification and characterization of functional polymorphisms
Ruberti G;Cascino I
2007
Abstract
Title: IDDM12 LOCUS ANALYSIS IN T1DM: IDENTIFICATION AND CHARACTERIZATION OF FUNCTIONAL POLYMORPHISMS Keywords: T1D, CTLA4, ICOS Disease: Type 1 diabetes Research type: Basic science Authors: Ruberti G, Belfiore F, Malquori L, Carsetti L and Cascino I. Institute of Cell Biology Via E. Ramarini, 00016 Monterotondo scalo (RM) Tel. 06-90091201, Fax 06-90091260, Abstract: Type1 Diabetes (T1D) is a polygenic disease caused by the immune-mediated destruction of pancreatic insulin-secreting-cells. Increasing evidence both in humans and in animal models has shown that defect(s) in immunoregulation underlie T1D, similarly to other immune-mediated disorders. T cell activation results from the integration of signals generated through the T cell antigen receptor-CD3 complex with those from additional positive and negative regulatory pathways mainly mediated by engagement of costimulatory receptors on T cells. Disruption of this balance leads to a defective immune response or alternative over-activation of the immune system. Genetic studies of human T1D indicate the presence of a disease locus in a restricted region on chromosome 2q33 (IDDM12) and in the orthologous region of murine chromosome 1. CD28, CTLA4 and ICOS genes, coding for lymphocyte co-stimulatory receptors, map in IDDM12 locus (1-3). Data obtained by our group in nuclear families and in a case-control study show an association with T1D of a series of markers in the IDDM12 region. These appear to belong to two separate association groups, one centered on the CTLA4 locus and the other on the ICOS locus. The specific aims of this project are to: 1. Identify and functionally characterize the disease variation in the ICOS region; 2. Study the functional role of the T1D-associated CTLA4 3'UTR (AT)n repeat and the CT60 polymorphism on mRNA stability and targeting. Aim 1. For the analysis of the region comprised between ICOS gene and d2s2189 microsatellite (150 kb) we have selected 12 SNPs, already described in the NCBI SNP database. The SNPs were selected for being located in the sequences that were at least 80% identical to the orthologous mouse region over a 100 bp. The underlying rationale is that essential regulatory elements are conserved among mammals, suggesting that cross-species sequence comparisons should identify them (4). We have set up the conditions for multiple PCRs and multiple microsequencing on 10 DNA control samples.Then, we have tested for association the 12 SNP in a case-control study. Three SNP who showed a trand of association with the disease, are now under TDT study on our T1D family panel with at least one affected child. Aim 2. CTLA-4 plays a critical role in downregulating T cell responses. T1D and other autoimmune diseases have shown genetic linkage to the CTLA4 locus. Here, we report that the 3'UTR of CTLA4 regulates Luciferase reporter gene expression, and CTLA4 mRNA decay and translation efficiency, providing the cells with tools to rapidly respond to changes in intracellular and extracellular stimuli. Intriguingly, we also observed that the length of the (AT)n repeat contributed to gene expression regulation. The 3'UTR T1D-associated (AT)n repeat medium (16AT) and long (24AT) alleles decreased the luciferase activity of 80% and 70% respectively in Jurkat and Hek 293T cells; while the short (6AT) allele decreased the activity of 70% and 60% in the hematopoietic and epithelial cell lines. The difference observed was statistically significant in both cell types (p<0.0001). A contribution of the medium and long (AT)n alleles on CTLA-4 protein expression was also observed. UV-crosslinking experiments suggest that the length of the (AU)n repeat influences the recruitment of trans-acting factors potentially involved in gene expression regulation. Finally, we also show that the "CT60" region of 736 bp, located 3' to the major poly(A) site of the 3'UTR of CTLA4, regulates mRNA and protein expression levels. However, the T1D-associated CT60 polymorphic SNP does not appear to contribute to this regulation in our experimental conditions. References: 1) Nistico L., Buzzetti R., Pritchard L.E., Van der Auwera B., Giovannini C., Bosi E., Larrad M.T., Rios M.S., Chow C.C., Cockram C.S., Jacobs K., Mijovic C., Bain S.C., Barnett A.H., Vandewalle C.L., Schuit F., Gorus F.K., Tosi R., Pozzilli P., Todd J.A. The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry. Hum Mol Genet. 5, 1075-80, 1996. 2) Ueda H., Howson J.M., Esposito L., Heward J., Snook H., Chamberlain G., Rainbow D.B., Hunter K.M., Smith A.N., Di Genova G., Herr M.H., Dahlman I., Payne F., Smyth D., Lowe C., Twells R.C., Howlett S., Healy B., Nutland S., Rance H.E., Everett V., Smink L.J., Lam A.C., Cordell H.J., Walker N.M., Bordin C., Hulme J., Motzo C., Cucca F., Hess J.F., Metzker M.L., Rogers J., Gregory S., Allahabadia A., Nithiyananthan R., Tuomilehto-Wolf E., Tuomilehto J., Bingley P., Gillespie K.M., Undlien D.E., Ronningen K.S., Guja C., Ionescu-Tirgoviste C., Savage D.A., Maxwell A.P., Carson D.J., Patterson C.C., Franklyn J.A., Clayton D.G., Peterson L.B., Wicker L.S., Todd J.A., Gough S.C.. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature. 423, 506-11, 2003. 3) Ihara K., Ahmed S., Nakao F., Kinukawa N., Kuromaru R., Matsuura N., Iwata I., Nagafuchi S., Kohno H., Miyako K., Hara T. Association studies of CTLA-4, CD28, and ICOS gene polymorphisms with type 1 diabetes in the Japanese population. Immunogenetics. 53, 447-54, 2001. 4) Dermitzakis E.T., Reymond A., Scamuffa N., Ucla C., Kirkness E., Rossier C., Antonarakis S.E. Evolutionary discrimination of mammalian conserved non-genic sequences (CNGs). Science. 302, 1033-5, 2003.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
