Mitchell-Riley syndrome iPSC exhibit reduced pancreatic endoderm differentiation due to an RFX6 mutation.

Mitchell-riley syndrome (MRS) is caused by recessive mutations in the Regulatory Factor X, 6 (RFX6) gene and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why MRS patients specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSC and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6 HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridization for RFX6 in the dorsal pancreatic bud of a Carnegie Stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in MRS patients specifically impairs formation of endocrine cells of the pancreas head and tail.

[1]  D. Schaffer,et al.  Gene Editing to Generate Versatile Human Pluripotent Stem Cell Reporter Lines for Analysis of Differentiation and Lineage Tracing , 2019, Stem cells.

[2]  Y. Hasegawa,et al.  A heterozygous protein‐truncating RFX6 variant in a family with childhood‐onset, pregnancy‐associated and adult‐onset diabetes , 2019, Diabetic medicine : a journal of the British Diabetic Association.

[3]  Gopika G. Nair,et al.  Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived β cells , 2019, Nature Cell Biology.

[4]  Jinkui Yang,et al.  Identification of Rfx6 target genes involved in pancreas development and insulin translation by ChIP-seq. , 2019, Biochemical and biophysical research communications.

[5]  T. Meitinger,et al.  Genome-wide analysis of PDX1 target genes in human pancreatic progenitors , 2018, Molecular metabolism.

[6]  Dave T. Gerrard,et al.  Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos , 2017, Stem cell reports.

[7]  Heiko Lickert,et al.  Cellular and molecular mechanisms coordinating pancreas development , 2017, Development.

[8]  L. Stanton,et al.  Long-Term Culture of Self-renewing Pancreatic Progenitors Derived from Human Pluripotent Stem Cells , 2017, Stem cell reports.

[9]  Laura J. Scott,et al.  Genetic regulatory signatures underlying islet gene expression and type 2 diabetes , 2017, Proceedings of the National Academy of Sciences.

[10]  Danwei Huangfu,et al.  Genome Editing of Lineage Determinants in Human Pluripotent Stem Cells Reveals Mechanisms of Pancreatic Development and Diabetes. , 2016, Cell stem cell.

[11]  Dave T. Gerrard,et al.  Human pancreas development , 2015, Development.

[12]  N. Hanley,et al.  TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors , 2015, Nature Cell Biology.

[13]  L. Stanton,et al.  PDX1 Binds and Represses Hepatic Genes to Ensure Robust Pancreatic Commitment in Differentiating Human Embryonic Stem Cells , 2015, Stem cell reports.

[14]  Aliz R. Rao,et al.  Rich annotation of DNA sequencing variants by leveraging the Ensembl Variant Effect Predictor with plugins , 2015, Briefings Bioinform..

[15]  Gopika G. Nair,et al.  Controlled induction of human pancreatic progenitors produces functional beta‐like cells in vitro , 2015, The EMBO journal.

[16]  P. Rorsman,et al.  RFX6 regulates insulin secretion by modulating Ca2+ homeostasis in human β cells. , 2014, Cell reports.

[17]  G. Rutter,et al.  Rfx6 Maintains the Functional Identity of Adult Pancreatic β Cells , 2014, Cell reports.

[18]  James D. Johnson,et al.  Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells , 2014, Nature Biotechnology.

[19]  V. Schwitzgebel.  Many faces of monogenic diabetes , 2014, Journal of diabetes investigation.

[20]  T. Hearn,et al.  Development of the Human Pancreas From Foregut to Endocrine Commitment , 2013, Diabetes.

[21]  Y. Kawaguchi.  Sox9 and programming of liver and pancreatic progenitors. , 2013, The Journal of clinical investigation.

[22]  S. Ellard,et al.  Clinical characterization of a newly described neonatal diabetes syndrome caused by RFX6 mutations , 2011, American journal of medical genetics. Part A.

[23]  A. Gonzalez-Perez,et al.  Improving the assessment of the outcome of nonsynonymous SNVs with a consensus deleteriousness score, Condel. , 2011, American journal of human genetics.

[24]  M. DePristo,et al.  The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. , 2010, Genome research.

[25]  Philipp Bucher,et al.  The Transcription Factor Rfx3 Regulates β-Cell Differentiation, Function, and Glucokinase Expression , 2010, Diabetes.

[26]  P. Bork,et al.  A method and server for predicting damaging missense mutations , 2010, Nature Methods.

[27]  C. Orvain,et al.  Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development , 2010, Development.

[28]  K. Dewar,et al.  Rfx6 Directs Islet Formation and Insulin Production in Mice and Humans , 2009, Nature.

[29]  S. Henikoff,et al.  Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm , 2009, Nature Protocols.

[30]  Nansheng Chen,et al.  Identification and characterization of novel human tissue-specific RFX transcription factors , 2008, BMC Evolutionary Biology.

[31]  Carine Benadiba,et al.  Novel Function of the Ciliogenic Transcription Factor RFX3 in Development of the Endocrine Pancreas , 2007, Diabetes.

[32]  S. L. Wong,et al.  Towards a proteome-scale map of the human protein–protein interaction network , 2005, Nature.

[33]  C. Polychronakos,et al.  Neonatal diabetes, with hypoplastic pancreas, intestinal atresia and gall bladder hypoplasia: search for the aetiology of a new autosomal recessive syndrome , 2004, Diabetologia.

[34]  R. Beddington,et al.  Hex homeobox gene-dependent tissue positioning is required for organogenesis of the ventral pancreas , 2004, Development.

[35]  Samuel L. Pfaff,et al.  Independent requirement for ISL1 in formation of pancreatic mesenchyme and islet cells , 1997, Nature.

[36]  William L. Clarke,et al.  Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence , 1997, Nature Genetics.

[37]  H. Edlund,et al.  The morphogenesis of the pancreatic mesenchyme is uncoupled from that of the pancreatic epithelium in IPF1/PDX1-deficient mice. , 1996, Development.

[38]  B. Hogan,et al.  PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. , 1996, Development.

[39]  H. Edlund,et al.  Insulin-promoter-factor 1 is required for pancreas development in mice , 1994, Nature.