{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,7]],"date-time":"2026-04-07T08:31:08Z","timestamp":1775550668279,"version":"3.50.1"},"reference-count":50,"publisher":"American Diabetes Association","issue":"12","license":[{"start":{"date-parts":[[2020,11,20]],"date-time":"2020-11-20T00:00:00Z","timestamp":1605830400000},"content-version":"vor","delay-in-days":71,"URL":"https:\/\/www.diabetesjournals.org\/content\/license"}],"funder":[{"DOI":"10.13039\/100010663","name":"H2020 European Research Council","doi-asserted-by":"publisher","award":["ERC-2015-StG-680156-ZPR"],"award-info":[{"award-number":["ERC-2015-StG-680156-ZPR"]}],"id":[{"id":"10.13039\/100010663","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e a Tecnologia","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008530","name":"European Regional Development Fund","doi-asserted-by":"publisher","award":["SFRH\/BD\/147762\/2019"],"award-info":[{"award-number":["SFRH\/BD\/147762\/2019"]}],"id":[{"id":"10.13039\/501100008530","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008530","name":"European Regional Development Fund","doi-asserted-by":"publisher","award":["SFRH\/BD\/135957\/2018"],"award-info":[{"award-number":["SFRH\/BD\/135957\/2018"]}],"id":[{"id":"10.13039\/501100008530","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["diabetesjournals.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2020,12,1]]},"abstract":"<jats:p>Many single nucleotide polymorphisms (SNPs) associated with type 2 diabetes overlap with putative endocrine pancreatic enhancers, suggesting that these SNPs modulate enhancer activity and, consequently, gene expression. We performed in vivo mosaic transgenesis assays in zebrafish to quantitatively test the enhancer activity of type 2 diabetes\u2013associated loci. Six out of 10 tested sequences are endocrine pancreatic enhancers. The risk variant of two sequences decreased enhancer activity, while in another two incremented it. One of the latter (rs13266634) locates in an SLC30A8 exon, encoding a tryptophan-to-arginine substitution that decreases SLC30A8 function, which is the canonical explanation for type 2 diabetes risk association. However, other type 2 diabetes\u2013associated SNPs that truncate SLC30A8 confer protection from this disease, contradicting this explanation. Here, we clarify this incongruence, showing that rs13266634 boosts the activity of an overlapping enhancer and suggesting an SLC30A8 gain of function as the cause for the increased risk for the disease. We further dissected the functionality of this enhancer, finding a single nucleotide mutation sufficient to impair its activity. Overall, this work assesses in vivo the importance of disease-associated SNPs in the activity of endocrine pancreatic enhancers, including a poorly explored case where a coding SNP modulates the activity of an enhancer.<\/jats:p>","DOI":"10.2337\/db19-1049","type":"journal-article","created":{"date-parts":[[2020,9,11]],"date-time":"2020-09-11T00:25:58Z","timestamp":1599783958000},"page":"2794-2805","update-policy":"https:\/\/doi.org\/10.2337\/ada-journal-policies","source":"Crossref","is-referenced-by-count":13,"title":["In Vivo Reporter Assays Uncover Changes in Enhancer Activity Caused by Type 2 Diabetes\u2013Associated Single Nucleotide Polymorphisms"],"prefix":"10.2337","volume":"69","author":[{"given":"Ana","family":"Eufr\u00e1sio","sequence":"first","affiliation":[{"name":"i3S\u2013Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, and IBMC\u2013Instituto de Biologia Celular e Molecular, Porto, Portugal"}]},{"given":"Chiara","family":"Perrod","sequence":"additional","affiliation":[{"name":"i3S\u2013Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, and IBMC\u2013Instituto de Biologia Celular e Molecular, Porto, Portugal"}]},{"given":"F\u00e1bio J.","family":"Ferreira","sequence":"additional","affiliation":[{"name":"i3S\u2013Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, and IBMC\u2013Instituto de Biologia Celular e Molecular, Porto, Portugal"}]},{"given":"Marta","family":"Duque","sequence":"additional","affiliation":[{"name":"i3S\u2013Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, and IBMC\u2013Instituto de Biologia Celular e Molecular, Porto, Portugal"}]},{"given":"Mafalda","family":"Galhardo","sequence":"additional","affiliation":[{"name":"i3S\u2013Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, and IBMC\u2013Instituto de Biologia Celular e Molecular, Porto, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8591-7138","authenticated-orcid":false,"given":"Jos\u00e9","family":"Bessa","sequence":"additional","affiliation":[{"name":"i3S\u2013Instituto de Investiga\u00e7\u00e3o e Inova\u00e7\u00e3o em Sa\u00fade, Universidade do Porto, and IBMC\u2013Instituto de Biologia Celular e Molecular, Porto, Portugal"}]}],"member":"1167","published-online":{"date-parts":[[2020,9,10]]},"reference":[{"key":"2022031210025870600_B1","doi-asserted-by":"crossref","first-page":"2239","DOI":"10.1016\/S0140-6736(17)30058-2","article-title":"Type 2 diabetes","volume":"389","author":"Chatterjee","year":"2017","journal-title":"Lancet"},{"key":"2022031210025870600_B2","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1038\/s41586-019-1231-2","article-title":"Exome sequencing of 20,791 cases of type 2 diabetes and 24,440 controls","volume":"570","author":"Flannick","year":"2019","journal-title":"Nature"},{"key":"2022031210025870600_B3","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1126\/science.1142358","article-title":"Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels","volume":"316","author":"Saxena","year":"2007","journal-title":"Science"},{"key":"2022031210025870600_B4","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1038\/nature05616","article-title":"A genome-wide association study identifies novel risk loci for type 2 diabetes","volume":"445","author":"Sladek","year":"2007","journal-title":"Nature"},{"key":"2022031210025870600_B5","doi-asserted-by":"crossref","first-page":"423","DOI":"10.1038\/embor.2012.52","article-title":"Enhancers: emerging roles in cell fate specification","volume":"13","author":"Ong","year":"2012","journal-title":"EMBO Rep"},{"key":"2022031210025870600_B6","doi-asserted-by":"crossref","first-page":"136","DOI":"10.1038\/ng.2870","article-title":"Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants","volume":"46","author":"Pasquali","year":"2014","journal-title":"Nat Genet"},{"key":"2022031210025870600_B7","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1016\/j.cmet.2010.09.012","article-title":"Global epigenomic analysis of primary human pancreatic islets provides insights into type 2 diabetes susceptibility loci","volume":"12","author":"Stitzel","year":"2010","journal-title":"Cell Metab"},{"key":"2022031210025870600_B8","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1101\/gr.102038.109","article-title":"Genome-wide analysis of histone modifications in human pancreatic islets","volume":"20","author":"Bhandare","year":"2010","journal-title":"Genome Res"},{"key":"2022031210025870600_B9","doi-asserted-by":"crossref","first-page":"17921","DOI":"10.1073\/pnas.1317023110","article-title":"Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants","volume":"110","author":"Parker","year":"2013","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2022031210025870600_B10","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/ng.530","article-title":"A map of open chromatin in human pancreatic islets","volume":"42","author":"Gaulton","year":"2010","journal-title":"Nat Genet"},{"key":"2022031210025870600_B11","doi-asserted-by":"crossref","first-page":"1213","DOI":"10.1038\/nmeth.2688","article-title":"Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position","volume":"10","author":"Buenrostro","year":"2013","journal-title":"Nat Methods"},{"key":"2022031210025870600_B12","doi-asserted-by":"crossref","first-page":"279","DOI":"10.1038\/nature09692","article-title":"A unique chromatin signature uncovers early developmental enhancers in humans","volume":"470","author":"Rada-Iglesias","year":"2011","journal-title":"Nature"},{"key":"2022031210025870600_B13","doi-asserted-by":"crossref","first-page":"D88","DOI":"10.1093\/nar\/gkl822","article-title":"VISTA Enhancer Browser--a database of tissue-specific human enhancers","volume":"35","author":"Visel","year":"2007","journal-title":"Nucleic Acids Res"},{"key":"2022031210025870600_B14","doi-asserted-by":"crossref","first-page":"2409","DOI":"10.1002\/dvdy.22051","article-title":"Zebrafish enhancer detection (ZED) vector: a new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish","volume":"238","author":"Bessa","year":"2009","journal-title":"Dev Dyn"},{"key":"2022031210025870600_B15","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/bs.ctdb.2016.10.005","article-title":"Zebrafish pancreas development and regeneration: fishing for diabetes therapies","volume":"124","author":"Prince","year":"2017","journal-title":"Curr Top Dev Biol"},{"key":"2022031210025870600_B16","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/j.ygcen.2005.12.005","article-title":"The fish endocrine pancreas: review, new data, and future research directions in ontogeny and phylogeny","volume":"148","author":"Youson","year":"2006","journal-title":"Gen Comp Endocrinol"},{"key":"2022031210025870600_B17","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1006\/gcen.1999.7376","article-title":"Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fish","volume":"116","author":"Youson","year":"1999","journal-title":"Gen Comp Endocrinol"},{"key":"2022031210025870600_B18","doi-asserted-by":"crossref","first-page":"14241","DOI":"10.1038\/srep14241","article-title":"Diabetic pdx1-mutant zebrafish show conserved responses to nutrient overload and anti-glycemic treatment","volume":"5","author":"Kimmel","year":"2015","journal-title":"Sci Rep"},{"key":"2022031210025870600_B19","doi-asserted-by":"crossref","first-page":"2491","DOI":"10.1242\/dev.002691","article-title":"The transcription factors Nkx6.1 and Nkx6.2 possess equivalent activities in promoting beta-cell fate specification in Pdx1+ pancreatic progenitor cells","volume":"134","author":"Nelson","year":"2007","journal-title":"Development"},{"key":"2022031210025870600_B20","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1016\/j.ydbio.2010.01.025","article-title":"Nkx6.1 and nkx6.2 regulate alpha- and beta-cell formation in zebrafish by acting on pancreatic endocrine progenitor cells","volume":"340","author":"Binot","year":"2010","journal-title":"Dev Biol"},{"key":"2022031210025870600_B21","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1038\/s41588-018-0084-1","article-title":"Refining the accuracy of validated target identification through coding variant fine-mapping in type 2 diabetes","volume":"50","author":"Mahajan","year":"2018","journal-title":"Nat Genet"},{"key":"2022031210025870600_B22","doi-asserted-by":"crossref","first-page":"2070","DOI":"10.2337\/db09-0551","article-title":"Insulin storage and glucose homeostasis in mice null for the granule zinc transporter ZnT8 and studies of the type 2 diabetes-associated variants","volume":"58","author":"Nicolson","year":"2009","journal-title":"Diabetes"},{"key":"2022031210025870600_B23","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1038\/tpj.2010.22","article-title":"A low-risk ZnT-8 allele (W325) for post-transplantation diabetes mellitus is protective against cyclosporin A-induced impairment of insulin secretion","volume":"11","author":"Kim","year":"2011","journal-title":"Pharmacogenomics J"},{"key":"2022031210025870600_B24","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1007\/s00125-014-3405-7","article-title":"SLC30A8 mutations in type 2 diabetes","volume":"58","author":"Rutter","year":"2015","journal-title":"Diabetologia"},{"key":"2022031210025870600_B25","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/ng.2915","article-title":"Loss-of-function mutations in SLC30A8 protect against type 2 diabetes","volume":"46","author":"Flannick","year":"2014","journal-title":"Nat Genet"},{"key":"2022031210025870600_B26","doi-asserted-by":"crossref","first-page":"1596","DOI":"10.1038\/s41588-019-0513-9","article-title":"Loss of ZnT8 function protects against diabetes by enhanced insulin secretion","volume":"51","author":"Dwivedi","year":"2019","journal-title":"Nat Genet"},{"key":"2022031210025870600_B27","doi-asserted-by":"crossref","first-page":"26950","DOI":"10.1074\/jbc.M116.764605","article-title":"Lipid-tuned zinc transport activity of human ZnT8 protein correlates with risk for type-2 diabetes","volume":"291","author":"Merriman","year":"2016","journal-title":"J Biol Chem"},{"key":"2022031210025870600_B28","doi-asserted-by":"crossref","first-page":"R85","DOI":"10.1093\/hmg\/ddv264","article-title":"Recent advances in understanding the genetic architecture of type 2 diabetes","volume":"24","author":"Mohlke","year":"2015","journal-title":"Hum Mol Genet"},{"key":"2022031210025870600_B29","doi-asserted-by":"crossref","first-page":"981","DOI":"10.1038\/ng.2383","article-title":"Large-scale association analysis provides insights into the genetic architecture and pathophysiology of type 2 diabetes","volume":"44","author":"Morris","year":"2012","journal-title":"Nat Genet"},{"key":"2022031210025870600_B30","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1038\/ng.2385","article-title":"Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways","volume":"44","author":"Scott","year":"2012","journal-title":"Nat Genet"},{"key":"2022031210025870600_B31","doi-asserted-by":"crossref","unstructured":"Thurner\u2008M, van de Bunt\u2008M, Torres\u2008JM, et al. Integration of human pancreatic islet genomic data refines regulatory mechanisms at type 2 diabetes susceptibility loci. eLife\u20082018;7:e31977","DOI":"10.7554\/eLife.31977"},{"key":"2022031210025870600_B32","doi-asserted-by":"crossref","unstructured":"Mularoni\u2008L, Ramos-Rodr\u00edguez\u2008M, Pasquali\u2008L. The pancreatic islet regulome browser. Front Genet\u20082017;8:13","DOI":"10.3389\/fgene.2017.00013"},{"key":"2022031210025870600_B33","doi-asserted-by":"crossref","first-page":"11403","DOI":"10.1073\/pnas.97.21.11403","article-title":"Identification of a functional transposase of the Tol2 element, an Ac-like element from the Japanese medaka fish, and its transposition in the zebrafish germ lineage","volume":"97","author":"Kawakami","year":"2000","journal-title":"Proc Natl Acad Sci U S A"},{"key":"2022031210025870600_B34","doi-asserted-by":"crossref","first-page":"1025","DOI":"10.1002\/dvdy.21100","article-title":"Conditional targeted cell ablation in zebrafish: a new tool for regeneration studies","volume":"236","author":"Curado","year":"2007","journal-title":"Dev Dyn"},{"key":"2022031210025870600_B35","unstructured":"PME-MCS - Tol2kit\n          . Accessed 27 August 2019. Available from http:\/\/tol2kit.genetics.utah.edu\/index.php\/PME-MCS"},{"key":"2022031210025870600_B36","doi-asserted-by":"crossref","first-page":"615","DOI":"10.1038\/ncb3160","article-title":"TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors","volume":"17","author":"Cebola","year":"2015","journal-title":"Nat Cell Biol"},{"key":"2022031210025870600_B37","doi-asserted-by":"crossref","first-page":"D91","DOI":"10.1093\/nar\/gkh012","article-title":"JASPAR: an open-access database for eukaryotic transcription factor binding profiles","volume":"32","author":"Sandelin","year":"2004","journal-title":"Nucleic Acids Res"},{"key":"2022031210025870600_B38","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/B978-0-12-391938-0.00004-5","article-title":"4C technology: protocols and data analysis","volume":"513","author":"van de Werken","year":"2012","journal-title":"Methods Enzymol"},{"key":"2022031210025870600_B39","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1016\/j.ymeth.2012.04.009","article-title":"Determining long-range chromatin interactions for selected genomic sites using 4C-seq technology: from fixation to computation","volume":"58","author":"Splinter","year":"2012","journal-title":"Methods"},{"key":"2022031210025870600_B40","doi-asserted-by":"crossref","first-page":"222","DOI":"10.1126\/science.1207194","article-title":"The dynamic architecture of Hox gene clusters","volume":"334","author":"Noordermeer","year":"2011","journal-title":"Science"},{"key":"2022031210025870600_B41","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast gapped-read alignment with Bowtie 2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat Methods"},{"key":"2022031210025870600_B42","doi-asserted-by":"crossref","first-page":"1137","DOI":"10.1038\/s41588-019-0457-0","article-title":"Human pancreatic islet three-dimensional chromatin architecture provides insights into the genetics of type 2 diabetes","volume":"51","author":"Miguel-Escalada","year":"2019","journal-title":"Nat Genet"},{"key":"2022031210025870600_B43","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1016\/0092-8674(93)80052-G","article-title":"A 5\u2032 element of the chicken beta-globin domain serves as an insulator in human erythroid cells and protects against position effect in Drosophila","volume":"74","author":"Chung","year":"1993","journal-title":"Cell"},{"key":"2022031210025870600_B44","doi-asserted-by":"crossref","first-page":"3088","DOI":"10.1002\/dvdy.21343","article-title":"The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs","volume":"236","author":"Kwan","year":"2007","journal-title":"Dev Dyn"},{"key":"2022031210025870600_B45","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1042\/BJ20101488","article-title":"The pancreatic islet \u03b2-cell-enriched transcription factor Pdx-1 regulates Slc30a8 gene transcription through an intronic enhancer","volume":"433","author":"Pound","year":"2011","journal-title":"Biochem J"},{"key":"2022031210025870600_B46","doi-asserted-by":"crossref","first-page":"105","DOI":"10.14573\/altex.2010.2.105","article-title":"Pancreatic beta cell lines and their applications in diabetes mellitus research","volume":"27","author":"Skelin","year":"2010","journal-title":"ALTEX"},{"key":"2022031210025870600_B47","doi-asserted-by":"crossref","first-page":"1155","DOI":"10.1038\/sj.ki.5002156","article-title":"R990G polymorphism of calcium-sensing receptor does produce a gain-of-function and predispose to primary hypercalciuria","volume":"71","author":"Vezzoli","year":"2007","journal-title":"Kidney Int"},{"key":"2022031210025870600_B48","doi-asserted-by":"crossref","first-page":"5","DOI":"10.1016\/0092-8674(94)90227-5","article-title":"Transcriptional activation: a complex puzzle with few easy pieces","volume":"77","author":"Tjian","year":"1994","journal-title":"Cell"},{"key":"2022031210025870600_B49","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1038\/nrg2957","article-title":"Enhancer function: new insights into the regulation of tissue-specific gene expression","volume":"12","author":"Ong","year":"2011","journal-title":"Nat Rev Genet"},{"key":"2022031210025870600_B50","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1101\/gr.133546.111","article-title":"Coding exons function as tissue-specific enhancers of nearby genes","volume":"22","author":"Birnbaum","year":"2012","journal-title":"Genome Res"}],"container-title":["Diabetes"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/diabetes.diabetesjournals.org\/syndication\/doi\/10.2337\/db19-1049","content-type":"unspecified","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/journals.org\/diabetes\/diabetes\/article-pdf\/69\/12\/2794\/435266\/db191049.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/diabetesjournals.org\/diabetes\/article-pdf\/69\/12\/2794\/435266\/db191049.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,11,2]],"date-time":"2022-11-02T16:40:42Z","timestamp":1667407242000},"score":1,"resource":{"primary":{"URL":"https:\/\/diabetesjournals.org\/diabetes\/article\/69\/12\/2794\/16559\/In-Vivo-Reporter-Assays-Uncover-Changes-in"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,10]]},"references-count":50,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2020,9,10]]},"published-print":{"date-parts":[[2020,12,1]]}},"URL":"https:\/\/doi.org\/10.2337\/db19-1049","relation":{},"ISSN":["0012-1797","1939-327X"],"issn-type":[{"value":"0012-1797","type":"print"},{"value":"1939-327X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,9,10]]}}}