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The objective of this study was to test if a novel platform could be used for isotype-specific autoantibody testing in humans. Further, we evaluated if testing with this novel platform enables earlier detection of insulin autoantibodies in individuals that have first-degree relatives with type-1 diabetes than currently used approaches. Longitudinal serum samples from participants were collected before and after they converted to become positive for insulin autoantibodies by the current standardly used assays. Using a novel plasmonic gold chip platform, we tested these samples for IgM isotype-specific autoantibodies. Serial serum samples from individuals without diabetes were also tested as a comparison control cohort. Our results demonstrate proof-of-concept that a plasmonic gold chip can specifically detect the IgM insulin autoantibody. Five out of the six individuals that converted to being positive for insulin autoantibodies by standard testing had significant IgM autoantibodies on the plasmonic chip platform. The plasmonic chip platform detected IgM autoantibodies earlier than standard testing by up to 4 years. Our results indicate that the plasmonic gold platform can specifically detect the IgM isotype autoantibodies and suggest that combining isotype-specific testing with currently used approaches enables earlier detection of insulin autoantibodies in individuals that have first-degree relatives with type 1 diabetes., Competing Interests: The authors have declared that no competing interests exist.

Patients with pancreatic agenesis are born without a pancreas, causing permanent neonatal diabetes and pancreatic enzyme insufficiency. These patients require insulin and enzyme replacement therapy to survive, grow, and maintain normal blood glucose levels. Pancreatic agenesis is an uncommon condition but high-throughput sequencing methods provide a rare opportunity to identify critical genes that are necessary for human pancreas development. Here we present the clinical history, evaluation, and the genetic and molecular analysis from two patients with pancreatic agenesis. Both patients were born with intrauterine growth restriction, minor heart defects and neonatal diabetes. In both cases, pancreatic agenesis was confirmed by imaging studies. The patients are clinically stable with pancreatic enzymes and insulin therapy. In order identify the etiology for their disease, we performed whole exome sequencing on both patients. For each proband we identified a de novo heterozygous mutation in the GATA6 gene. GATA6 is a homeobox containing transcription factor involved in both early development of the pancreas and heart. In vitro functional analysis of one of the variants revealed that the mutation creates a premature stop codon in the coding sequence resulting in the production of a truncated protein with loss of activity. These results show how genetic mutations in GATA6 may lead to functional inactivity and pancreatic agenesis in humans.

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.