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

Abstract

OBJECTIVEHeterozygous 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 METHODSThe 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.RESULTSA 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.CONCLUSIONSWe 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.

Inefficient Translocation of Preproinsulin Contributes to Pancreatic β Cell Failure and Late-onset Diabetes

“…Using GFP-tagged preproinsulin constructs, a recent study reported no defect of R6C/R6H in the targeting of preproinsulin to insulin granules but suggested that ER stress might contribute to ␤ cell death and diabetes in patients carrying these mutations (14). Importantly, fusion with GFP (238 amino acids) triples the length of preproinsulin (110 amino acids) that may alter (increase) translocation efficiency (32)(33)(34).…”

“…Recently, two preproinsulin SP mutants, preproinsulin R6C and R6H (here simply called R6C or R6H) have been linked to autosomal dominant late-onset diabetes in humans (14 -16). The molecular mechanism by which these mutants bring about diabetes has been speculated to involve ER stress (14), but the actual defect has remained unknown.…”

“…Nevertheless, to date, no human disease has been shown to be caused by a loss of the n region positive charge. Indeed, a recent study reported that neither R6C nor R6H results in defective insulin granule targeting of a preproinsulin-GFP chimera, thus leaving the diabetes linked to these mutations largely unexplained (14).…”

Diabetes caused by insulin gene (INS) deletion: clinical characteristics of homozygous and heterozygous individuals

“…1) in which diabetes at adult age appeared to segregate as a dominant trait, while neonatal diabetes occurred in a recessive way. Screening for INS mutation in these two cases resulted in a lack of the PCR product of the coding exon 2 and the noncoding exon 1, while exon 3 and the neighboring tyrosine hydroxylase gene (TH) were well amplified (11). Thus, sequencing with several primer sets within the region between INS and TH was performed to characterize size and exact location of the deletion.…”

“…Recent studies indicated that autosomal dominant insulin gene mutations can cause permanent neonatal diabetes (PND) but also autoantibody-negative diabetes that is classified then as type 1B or maturity-onset diabetes of the young (MODY)-type diabetes (8)(9)(10). The molecular mechanism behind these cases with a MODY-like presentation was suggested to be defective trafficking of proinsulin and increased endoplasmic reticulum (ER) stress (11). Recessive mutations of the preproinsulin (INS) gene have also been identified as a novel cause of neonatal diabetes and were the commonest cause of isolated PND in the offspring of consanguineous parents (12).…”