Christopher Faulk1, Kate R Mueller2, David Cheishvili3, Mathia Colwell1, Anne-Sophie Pepin3, Moshe Syzf3, Bernhard J Hering2, Christopher Burlak2. 1. Department of Animal Sciences, College of Food, Agricultural and Natural Resource Sciences, University of Minnesota, Saint Paul, MN, USA. 2. Department of Surgery, Schulze Diabetes Institute, School of Medicine, University of Minnesota, Minneapolis, MN, USA. 3. Department of Pharmacology & Therapeutics, McGill University, Montreal, QC, Canada.
Abstract
BACKGROUND: Xenotransplantation of porcine islets has emerged in recent decades as a potential treatment for type 1 diabetes (T1D). Current methods of detection, indicative of successful engraftment, occur downstream of actual islet death. Epigenetic biomarkers can be detected in circulating cell-free DNA (cfDNA) to provide an earlier indication of graft dysfunction. AIMS: The present study identified a biomarker of islet death using differential methylation of the insulin gene, INS, originating from β-cells in porcine islets. MATERIALS & METHODS: Pyrosequencing primers specific for porcine INS were designed to quantify hypomethylation along 12 cysteine-guanine dinucleotide (CpG) sites, including three sites in the cyclic adenosine monophosphate (cAMP) response element (CRE) binding protein 2 (CRE2) binding region of the 5' untranslated region (UTR) and nine sites within intron 2. RESULTS: PCR amplification of bisulfite-converted DNA combined with pyrosequencing data support the conclusion that hypomethylated porcine INS is specific to islet origin. CONCLUSION: Moreover, the results of this study indicate a highly specific epigenetic biomarker, capable of detecting a single islet, supporting the measurement of cfDNA as a biomarker for transplanted islet death. Defining the epigenetic characteristics of porcine-derived islets within cfDNA will be crucial to develop a better understanding of graft survival immunology for transplantation.
BACKGROUND: Xenotransplantation of porcine islets has emerged in recent decades as a potential treatment for type 1 diabetes (T1D). Current methods of detection, indicative of successful engraftment, occur downstream of actual islet death. Epigenetic biomarkers can be detected in circulating cell-free DNA (cfDNA) to provide an earlier indication of graft dysfunction. AIMS: The present study identified a biomarker of islet death using differential methylation of the insulin gene, INS, originating from β-cells in porcine islets. MATERIALS & METHODS: Pyrosequencing primers specific for porcine INS were designed to quantify hypomethylation along 12 cysteine-guanine dinucleotide (CpG) sites, including three sites in the cyclic adenosine monophosphate (cAMP) response element (CRE) binding protein 2 (CRE2) binding region of the 5' untranslated region (UTR) and nine sites within intron 2. RESULTS: PCR amplification of bisulfite-converted DNA combined with pyrosequencing data support the conclusion that hypomethylated porcine INS is specific to islet origin. CONCLUSION: Moreover, the results of this study indicate a highly specific epigenetic biomarker, capable of detecting a single islet, supporting the measurement of cfDNA as a biomarker for transplanted islet death. Defining the epigenetic characteristics of porcine-derived islets within cfDNA will be crucial to develop a better understanding of graft survival immunology for transplantation.
Authors: Eitan M Akirav; Jasmin Lebastchi; Eva M Galvan; Octavian Henegariu; Michael Akirav; Vitaly Ablamunits; Paul M Lizardi; Kevan C Herold Journal: Proc Natl Acad Sci U S A Date: 2011-11-09 Impact factor: 11.205
Authors: Christoph M Koch; Robert M Andrews; Paul Flicek; Shane C Dillon; Ulaş Karaöz; Gayle K Clelland; Sarah Wilcox; David M Beare; Joanna C Fowler; Phillippe Couttet; Keith D James; Gregory C Lefebvre; Alexander W Bruce; Oliver M Dovey; Peter D Ellis; Pawandeep Dhami; Cordelia F Langford; Zhiping Weng; Ewan Birney; Nigel P Carter; David Vetrie; Ian Dunham Journal: Genome Res Date: 2007-06 Impact factor: 9.043
Authors: Melanie L Graham; Eric F Rieke; Michele Dunning; Lucas A Mutch; Angela M Craig; Elizabeth K Zolondek; Bernhard J Hering; Henk-Jan Schuurman; Richard W Bianco Journal: J Med Primatol Date: 2009-02-10 Impact factor: 0.667
Authors: Kevan C Herold; Sahar Usmani-Brown; Tara Ghazi; Jasmin Lebastchi; Craig A Beam; Melena D Bellin; Michel Ledizet; Jay M Sosenko; Jeffrey P Krischer; Jerry P Palmer Journal: J Clin Invest Date: 2015-02-02 Impact factor: 14.808
Authors: Melanie L Graham; Lucas A Mutch; Eric F Rieke; Michele Dunning; Elizabeth K Zolondek; Aaron W Faig; Bernhard J Hering; Henk-Jan Schuurman Journal: Comp Med Date: 2010-12 Impact factor: 0.982
Authors: Lucas A Mutch; Samuel T Klinker; Jody J Janecek; Melanie N Niewinski; Rachael M Z Lee; Melanie L Graham Journal: J Invest Surg Date: 2018-12-13 Impact factor: 2.533
Authors: Melanie L Graham; Eric F Rieke; Lucas A Mutch; Elizabeth K Zolondek; Aaron W Faig; Theresa A Dufour; James W Munson; Jessica A Kittredge; Henk-Jan Schuurman Journal: J Med Primatol Date: 2011-12-12 Impact factor: 0.667