Yuki Hitomi1,2, Yoshihiro Aiba3, Kazuko Ueno4, Nao Nishida4,5, Yosuke Kawai4, Minae Kawashima6, Makoto Tsuiji7, Chisato Iwabuchi8, Sanami Takada8, Noriko Miyake8, Masao Nagasaki9, Katsushi Tokunaga4, Minoru Nakamura3,10,11. 1. Department of Human Genetics, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-Ku, Tokyo, 162-8655, Japan. yhitomi@ri.ncgm.go.jp. 2. Department of Microbiology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan. yhitomi@ri.ncgm.go.jp. 3. Clinical Research Center, National Hospital Organization (NHO) Nagasaki Medical Center, Omura, Japan. 4. Genome Medical Science Project, Research Institute, National Center for Global Health and Medicine, Tokyo, Japan. 5. The Research Center for Hepatitis and Immunology, Research Institute, National Center for Global Health and Medicine, Ichikawa, Japan. 6. Japan Science and Technology Agency (JST), Tokyo, Japan. 7. Department of Microbiology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan. 8. Department of Human Genetics, Research Institute, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-Ku, Tokyo, 162-8655, Japan. 9. Human Biosciences Unit for the Top Global Course Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Kyoto, Japan. 10. Department of Hepatology, Nagasaki University Graduate School of Biomedical Sciences, Omura, Japan. 11. Headquarters of PBC Research in NHO Study Group for Liver Disease in Japan (NHOSLJ), Clinical Research Center, NHO Nagasaki Medical Center, Omura, Japan.
Abstract
BACKGROUND: Ligation of CD28 with ligands such as CD80 or CD86 provides a critical second signal alongside antigen presentation by class II major histocompatibility complex expressed on antigen-presenting cells through the T cell antigen receptor for naïve T cell activation. A number of studies suggested that CD28 plays an important role in the pathogenesis of various human diseases. Recent genome-wide association studies (GWASs) identified CD28 as a susceptibility locus for lymphocyte and eosinophil counts, multiple sclerosis, ulcerative colitis, celiac disease, rheumatoid arthritis, asthma, and primary biliary cholangitis. However, the primary functional variant and molecular mechanisms of disease susceptibility in this locus remain to be elucidated. This study aimed to identify the primary functional variant from thousands of genetic variants in the CD28 locus and elucidate its functional effect on the CD28 molecule. RESULTS: Among the genetic variants exhibiting stronger linkage disequilibrium (LD) with all GWAS-lead variants in the CD28 locus, rs2013278, located in the Rbfox binding motif related to splicing regulation, was identified as a primary functional variant related to multiple immunological traits. Relative endogenous expression levels of CD28 splicing isoforms (CD28i and CD28Δex2) compared with full-length CD28 in allele knock-in cell lines generated using CRISPR/Cas9 were directly regulated by rs2013278 (P < 0.05). Although full-length CD28 protein expressed on Jurkat T cells showed higher binding affinity for CD80/CD86, both CD28i and CD28Δex2 encoded loss-of-function isoforms. CONCLUSION: The present study demonstrated for the first time that CD28 has a shared disease-related primary functional variant (i.e., rs2013278) that regulates the CD28 alternative splicing that generates loss-of-function isoforms. They reduce disease risk by inducing anergy of effector T cells that over-react to autoantigens and allergens.
BACKGROUND: Ligation of CD28 with ligands such as CD80 or CD86 provides a critical second signal alongside antigen presentation by class II major histocompatibility complex expressed on antigen-presenting cells through the T cell antigen receptor for naïve T cell activation. A number of studies suggested that CD28 plays an important role in the pathogenesis of various human diseases. Recent genome-wide association studies (GWASs) identified CD28 as a susceptibility locus for lymphocyte and eosinophil counts, multiple sclerosis, ulcerative colitis, celiac disease, rheumatoid arthritis, asthma, and primary biliary cholangitis. However, the primary functional variant and molecular mechanisms of disease susceptibility in this locus remain to be elucidated. This study aimed to identify the primary functional variant from thousands of genetic variants in the CD28 locus and elucidate its functional effect on the CD28 molecule. RESULTS: Among the genetic variants exhibiting stronger linkage disequilibrium (LD) with all GWAS-lead variants in the CD28 locus, rs2013278, located in the Rbfox binding motif related to splicing regulation, was identified as a primary functional variant related to multiple immunological traits. Relative endogenous expression levels of CD28 splicing isoforms (CD28i and CD28Δex2) compared with full-length CD28 in allele knock-in cell lines generated using CRISPR/Cas9 were directly regulated by rs2013278 (P < 0.05). Although full-length CD28 protein expressed on Jurkat T cells showed higher binding affinity for CD80/CD86, both CD28i and CD28Δex2 encoded loss-of-function isoforms. CONCLUSION: The present study demonstrated for the first time that CD28 has a shared disease-related primary functional variant (i.e., rs2013278) that regulates the CD28 alternative splicing that generates loss-of-function isoforms. They reduce disease risk by inducing anergy of effector T cells that over-react to autoantigens and allergens.
Keywords:
Alternative splicing; CD28; CRISPR/Cas9; Genome-wide association study (GWAS); Immunological-trait; Linkage disequilibrium; Primary functional variant
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