Human islets expressing HNF1A variant have defective β cell transcriptional regulatory networks
Rachana Haliyur, Xin Tong, May Sanyoura, Shristi Shrestha, Jill Lindner, Diane C. Saunders, Radhika Aramandla, Greg Poffenberger, Sambra D. Redick, Rita Bottino, Nripesh Prasad, Shawn E. Levy, Raymond D. Blind, David M. Harlan, Louis H. Philipson, Roland W. Stein, Marcela Brissova, Alvin C. Powers
Rachana Haliyur, Xin Tong, May Sanyoura, Shristi Shrestha, Jill Lindner, Diane C. Saunders, Radhika Aramandla, Greg Poffenberger, Sambra D. Redick, Rita Bottino, Nripesh Prasad, Shawn E. Levy, Raymond D. Blind, David M. Harlan, Louis H. Philipson, Roland W. Stein, Marcela Brissova, Alvin C. Powers
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Concise Communication Endocrinology

Human islets expressing HNF1A variant have defective β cell transcriptional regulatory networks

Abstract

Using an integrated approach to characterize the pancreatic tissue and isolated islets from a 33-year-old with 17 years of type 1 diabetes (T1D), we found that donor islets contained β cells without insulitis and lacked glucose-stimulated insulin secretion despite a normal insulin response to cAMP-evoked stimulation. With these unexpected findings for T1D, we sequenced the donor DNA and found a pathogenic heterozygous variant in the gene encoding hepatocyte nuclear factor-1α (HNF1A). In one of the first studies of human pancreatic islets with a disease-causing HNF1A variant associated with the most common form of monogenic diabetes, we found that HNF1A dysfunction leads to insulin-insufficient diabetes reminiscent of T1D by impacting the regulatory processes critical for glucose-stimulated insulin secretion and suggest a rationale for a therapeutic alternative to current treatment.

Authors

Rachana Haliyur, Xin Tong, May Sanyoura, Shristi Shrestha, Jill Lindner, Diane C. Saunders, Radhika Aramandla, Greg Poffenberger, Sambra D. Redick, Rita Bottino, Nripesh Prasad, Shawn E. Levy, Raymond D. Blind, David M. Harlan, Louis H. Philipson, Roland W. Stein, Marcela Brissova, Alvin C. Powers

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Figure 2

Expression and functional characterization of HNF1AT260M variant.

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Expression and functional characterization of HNF1AT260M variant. (A) An...
(A) Analysis of donor’s native pancreatic tissue for HNF1A compared with controls (n = 4 donors; ages 10–55 years) revealed HNF1A protein in donor β cells. Scale bar: 50 μm. (B) Electrophoretic mobility shift assay (EMSA) shows that the HNF1AT260M variant has impaired DNA binding, with loss of the HNF1A-specific DNA binding complex (arrow) in Myc-tagged HNF1AT260M–transfected HeLa cells compared with Myc-tagged HNF1AWT. Specificity of this complex (arrow) was shown by exclusive elimination of these species by adding either Myc antibody (Myc-Ab) or unlabeled oligonucleotide (WT Oligo) containing the HNF1A consensus recognition motif, but not a mutated form of this oligonucleotide (Comp Oligo). Moreover, HNF1A antibody (HNF1A-Ab) only supershifted (s.s.) this complex. All samples in B include oligonucleotide labeled with 32P as described in the supplemental material. Asterisk indicates nonspecific complexes. NT, nontransfected HeLa cells. One representative experiment of 3 is shown. (C) Molecular modeling of the HNF1AT260 variant in PyMOL predicts that the hydroxyl group (red) on threonine 260 (Thr-260) stabilizes arginine 263 (Arg-263) by hydrogen bonding to nitrogen (blue). Arg-263 H-bonds to the DNA backbone of the fifth adenosine of the HNF1A consensus recognition motif (5′-CTTGGTTAATAATTCACCAGA-3′) in control conditions (18). A missense mutation from threonine to methionine at position 260 is predicted to result in the loss of this interaction by destabilizing Arg-263 and subsequently DNA binding. Results of control samples are expressed as mean ± SEM. See complete unedited blots in the supplemental material.