Diagrammatic representation of the amino acid sequence of human preproinsulin (signal peptide–green, B-chain–red, C-peptide–orange, A-chain–dark blue) indicating sites of mutations identified in patients with diabetes as well as hyperinsulinemia and hyperproinsulinemia. Mutations shown in black disrupt proinsulin folding and/or disulfide bond formation leading to permanent neonatal diabetes mellitus (PNDM); mutations in light blue do not impair folding but are associated with reduced insulin receptor binding potency (hyperinsulinaemia); mutations in light green are associated with hyperproinsulinaemia and either impair proteolytic processing to insulin or, in the case of H34D, aggregation and sorting into dense-core granules of the regulated secretory pathway. Mutations in pink and purple were found in patients with a diagnosis of maturity-onset diabetes of the young (MODY) and type 1b diabetes, respectively. The R55C mutation has been found in patients with a diagnosis of type 1b diabetes as well as MODY. The mutations shown in gray (A23S, A23T, L68M and G84R) are rare variants without functional effects on proinsulin/insulin biosynthesis. The mutations shown in yellow are recessive mutations that affect insulin biosynthesis (mutation of the translation initiation Met) or cause the synthesis of a nonsense protein (Q62X)

Summary of human insulin gene mutations and disease phenotype. The numbers in brackets indicate the number of probands with that specific mutation. PNDM, permanent neonatal diabetes mellitus; TNDM, transient neonatal diabetes mellitus; MODY, maturity-onset diabetes of the young; T1BDM, type 1b diabetes mellitus

A schematic of the human insulin gene showing the locations of mutations in the homozygous or compound heterozygous state in probands with PNDM and TNDM. Mutations involving coding regions are shown above the gene while those involving non-coding regions are shown below the gene. The protein coding regions of the gene are shown in gray and the regions encoding the 5′- and 3′-untranslated regions of insulin mRNA in yellow. The red and black text indicates mutations identified in probands with TNDM and PNDM, respectively. Exon 2 encodes amino acids (a.a.) 1–62 and exon 3 encodes amino acids 63–110 of the preproinsulin molecule. Adapted from Garin et al. [32]

Multiple sequence alignment of preproinsulin from fish to human. The ClustalW2 alignment was obtained using the online interface at EBI (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The sequence of the signal peptide is shown in green letters, the B-chain in red, the C-peptide in orange and A-chain in blue. The basic amino acids that flank the C-peptide and are the sites of proteolytic processing are shown in black letters. The conserved Cys residues are highlighted in yellow. Asterisks (*) indicate identical amino acids in all the sequences shown in this alignment, colons (:) indicate conserved residues and periods (.) indicate semi-conserved amino acids. Note that these do not represent all the known preproinsulin sequences but rather are a selection of representative sequences

Subcellular localization of wild-type (WT) and mutant human proinsulin expressed in rat INS-1 insulinoma cells [30]. The location of human C-peptide immunoreactive protein is shown in green, ER is shown in red and the Golgi marker in cyan. Nuclei were stained with DAPI (blue). The WT and R89H hyperproinsulinemia mutant proinsulins are localized to secretory granules. By contrast, the C96Y PNDM-associated mutant proinsulin is localized to the expanded ER and is not transported from the ER to the Golgi and packaged into secretory granules