Insulin Gene Mutations Resulting in Early-Onset Diabetes: Marked Differences in Clinical Presentation, Metabolic Status, and Pathogenic Effect Through Endoplasmic Reticulum Retention

OBJECTIVE

Heterozygous mutations in the human preproinsulin (INS) gene are a cause of nonsyndromic neonatal or early-infancy diabetes. Here, we sought to identify INS mutations associated with maturity-onset diabetes of the young (MODY) or nonautoimmune diabetes in mid-adult life, and to explore the molecular mechanisms involved.

RESEARCH DESIGN AND METHODS

The INS gene was sequenced in 16 French probands with unexplained MODY, 95 patients with nonautoimmune early-onset diabetes (diagnosed at <35 years) and 292 normoglycemic control subjects of French origin. Three identified insulin mutants were generated by site-directed mutagenesis of cDNA encoding a preproinsulin–green fluorescent protein (GFP) (C-peptide) chimera. Intracellular targeting was assessed in clonal β-cells by immunocytochemistry and proinsulin secretion, by radioimmunoassay. Spliced XBP1 and C/EBP homologous protein were quantitated by real-time PCR.

RESULTS

A novel coding mutation, L30M, potentially affecting insulin multimerization, was identified in five diabetic individuals (diabetes onset 17–36 years) in a single family. L30M preproinsulin-GFP fluorescence largely associated with the endoplasmic reticulum (ER) in MIN6 β-cells, and ER exit was inhibited by ∼50%. Two additional mutants, R55C (at the B/C junction) and R6H (in the signal peptide), were normally targeted to secretory granules, but nonetheless caused substantial ER stress.

CONCLUSIONS

We describe three INS mutations cosegregating with early-onset diabetes whose clinical presentation is compatible with MODY. These led to the production of (pre)proinsulin molecules with markedly different trafficking properties and effects on ER stress, demonstrating a range of molecular defects in the β-cell.Misfolding of insulin, and consequently defective trafficking to secretory granules, has been recognized for a number of years as the likely underlying cause of β-cell dysfunction and death in several rodent models of nonimmune diabetes. These include the Akita mouse (1,2), in which a heterozygous mutation in the Ins2 gene (CA7Y) disrupts interchain disulphide bond formation leading to the engorgement of the endoplasmic reticulum (ER) with misfolded proteins and ER stress. A similar mechanism appears to pertain to the diabetic munich mouse, in which intrachain disulphide bond formation is blocked by a C95S mutation (3).Mutations in the human preproinsulin (INS) gene were first identified more than 20 years ago (4–6) and although some of these led to hyperproinsulinemia (7), none was found to be associated with frank diabetes (4–7). More recently, Støy et al. (8) described a group of patients presenting with permanent neonatal diabetes or early infancy–onset diabetes (median age at diagnosis of 13 weeks) who were carriers of a missense INS mutation. Most of these mutations were novel, and three were inherited in an autosomal dominant manner. Subsequently, we and three other reports (8–11) described additional INS mutations linked to permanent neonatal diabetes or nonautoimmune early infancy–onset diabetes. The majority, although not all, of the mutations led to diabetes onset in the first 6 months of life (8–11). In vitro analyses by Colombo et al. revealed that six of the mutations identified led to ER retention in HEK293T cells and to mild ER stress and at least two led to apoptosis (12).Some of the INS mutations, including R6C (9) and A23S (13) in the signal peptide, R46Q in the B chain, and R55C at the B/C junction (11), were described with later ages at diagnosis (up to 20 years), and these are presumed also to cause insulin misfolding with consequent ER retention, an unfolded protein response (UPR), and ER stress (2). Of note, two individuals with the R55C mutation were diagnosed with diabetes at ages 10 and 13 years, both with severe clinical symptoms including hyperglycemia and ketoacidosis, but abundant circulating C-peptide levels were detected in each case subject (11). The impact of these mutations on protein folding, ER stress, and β-cell death has, until now, not been examined.In this study, we aimed to determine the prevalence and phenotype of INS mutations that may lead to diabetes at a later age, including in maturity-onset diabetes of the young (MODY), or in patients presenting with nonautoimmune diabetes in mid-adult life (14). We describe here three families with two novel and one previously described INS mutations. The L30M mutation is predicted by structural analysis in silico to be better tolerated within the insulin hexamer than a previously described mutation at this residue (L30P), which causes severe diabetes within the first 6 months of life (12). Thus, the L30M mutation was associated with relatively mild diabetes (age at diagnosis in the proband: 17 years). By confocal imaging of chimeric insulin-GFP constructs in which GFP is fused in-frame with C-peptide (plasmid hProCpepGFP) (15), we show that this mutation causes clear insulin retention in the ER and concomitant ER stress. By contrast, insulin-GFP bearing the R6H mutation in the signal peptide exited the ER normally and was properly targeted to secretory granules, but induced significant ER stress. The previously reported R55C mutation (11), described here in a MODY family, was also substantially retained in the ER in clonal β-cells. These findings reveal an unexpected divergence in the effects of INS mutations at the molecular and cellular level, with the clinical presentation and the severity of the disease.