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.     

Reference values for thromboelastometry (ROTEM®) in cynomolgus monkeys (Macaca fascicularis)

Introduction

The imbalance in clotting homeostasis, tending towards hypercoagulation, is recognized as the real barrier to the long-term survival of porcine xenografts in pig-to-primate xenotransplatation. The present study aimed to validate in primate blood the applicability of whole blood rotation thromboelastometry, performed by ROTEM®, which evaluates the characteristics of clot formation by dynamic monitoring.

Materials and Methods

ROTEM® (Pentapharm GmbH, Munich, Germany) was used to investigate native coagulation (NATEM®), the intrinsic (INTEM®) and extrinsic (EXTEM®) pathways, the function of fibrinogen (FIBTEM®), and the presence of fibrinolysis in 40 naïve cynomolgus monkeys. Using classic validation approaches, the normal thromboelastographic profile was defined and the influence of haematocrit (Hct,%), platelet count (x10⁹/L), fibrinogen (mg/dl), and factor VIII (FVIII,%) was evaluated.

Results

In all four (NATEM®, INTEM®, EXTEM®, FIBTEM®) assays considered, Clotting Time (CT, sec) and Clot Formation Time (CFT, sec) were shorter in primates than humans. Moreover, α-angle (°), Maximum Clot Firmness (MCF, mm), and MaxVel (mm/min) were also higher in primates than humans. No substantial difference was observed for Hct and platelet count between the two species. On the contrary, FVIII was higher in primates than in humans whereas, interestingly enough, fibrinogen levels were lower in monkeys than in humans.

Conclusion

ROTEM® depicts a hypercoagulable profile in primates as compared to humans. Taken together these data suggest that, with regard to coagulation, xenotransplantation in cynos may represent a much more difficult situation than xenotransplantation in humans.     

Psychiatric comorbidity and suicidal behavior in epilepsy: A community‐based case–control study

Purpose: To provide information about psychiatric comorbidity and suicidal behavior in people with epilepsy compared to those without epilepsy from a community sample in Brazil. Methods: An attempt was made to evaluate all 174 subjects with epilepsy (cases) identified in a previous survey. For every case identified, an individual without epilepsy (control) matched by sex and age was selected in the same neighborhood. A structured interview with validated psychiatric scales was performed. One hundred and fifty‐three cases and 154 controls were enrolled in the study. Results: People with epilepsy had anxiety more frequently [39.4% vs. 23.8%, odds ratio (OR) 2.1, 95% confidence interval (CI) 1.2–3.5; p = 0.006], depression (24.4% vs. 14.7%, OR 1.9, 95% CI 1.01–3.5; p = 0.04), and anger (55.6% vs. 39.7%, OR 1.9, 95% CI 1.2–3.1; p = 0.008). They also reported more suicidal thoughts [36.7% vs. 23.8%, OR 1.8, 95% CI 1.1–3.1; p = 0.02), plans (18.2% vs. 3.3%, OR 2.0, 95% CI 1.0–4.0; p = 0.04), and attempts (12.1% vs. 5.3%, OR 2.4, 95% CI 1.1–3.2, p = 0.04) during life than controls. Conclusions: These findings call attention to psychiatric comorbidity and suicidal behavior associated with epilepsy. Suicide risk assessment, mental evaluation, and treatment may improve quality of life in epilepsy and ultimately prevent suicide.     

miR-21 functionally interacts with the 3'UTR of chemokine CCL20 and down-regulates CCL20 expression in miR-21 transfected colorectal cancer cells

As deregulation of miRNAs and chemokine CCL20 was shown to play a role in colorectal cancer (CRC) pathogenesis, we analyzed the functional interactions of candidate miRNAs with CCL20 mRNA. After target prediction software programs indicated a role for miR-21 in CCL20 regulation, we applied the luciferase reporter assay system to demonstrate that miR-21 functionally interacts with the 3′UTR of CCL20 mRNA and down-regulates CCL20 in miR-21 mimic transfected CRC cell lines (Caco-2, SW480 and SW620). Thus, regulation of CCL20 expression by miR-21 might be a regulatory mechanism involved in progression of CRC.     

The effect of multiple doses of rifampin and ketoconazole on the single-dose pharmacokinetics of ridaforolimus

Purpose

Ridaforolimus is an inhibitor of the mammalian target of rapamycin protein, with potent activity in vitro and in vivo. Ridaforolimus is primarily cleared by metabolism via cytochrome P450 3A (CYP3A) and is a P-glycoprotein (P-gp) substrate. Since potential exists for ridaforolimus to be co-administered with agents that affect CYP3A and P-gp activity, this healthy volunteer study was conducted to assess the effect of rifampin or ketoconazole on ridaforolimus pharmacokinetics.

Methods

Part 1: single-dose ridaforolimus 40?mg followed by rifampin 600?mg daily for 21?days and single-dose ridaforolimus 40?mg on day 14. Part 2: single-dose ridaforolimus 5?mg followed by ketoconazole 400?mg daily for 14?days and single-dose ridaforolimus 2?mg on day 2.

Results

Part 1: the geometric mean ratios (GMRs) (90% confidence interval [CI]) for ridaforolimus area under the concentration–time curve to the last time point with a detectable blood concentration (AUC_0–∞) and maximum blood concentration (C _max) (rifampin?+?ridaforolimus/ridaforolimus) were 0.57 (0.41, 0.78) and 0.66 (0.49, 0.90), respectively. Both time to C _max (T _max) and apparent half-life (t _1/2) were similar. Part 2: the GMRs (90% CI) based on dose-normalized AUC_0–∞ and C _max (ketoconazole?+?ridaforolimus/ridaforolimus alone) were 8.51 (6.97, 10.39) and 5.35 (4.40, 6.52), respectively. Ridaforolimus apparent t _1/2 was ~1.5-fold increased for ketoconazole?+?ridaforolimus; however, T _max values were similar.

Conclusions

Rifampin and ketoconazole both have a clinically meaningful effect on the pharmacokinetics of ridaforolimus.     

The role of MTHFR and RFC1 polymorphisms on toxicity and outcome of adult patients with hematological malignancies treated with high-dose methotrexate followed by leucovorin rescue

Purpose

In the last years, the influence of different genes involved in metabolism of chemotherapeutic agents has been studied. Methotrexate (MTX) is a key compound of chemotherapeutic regimens used in the treatment of acute lymphoblastic leukemia (ALL), primary central nervous system lymphoma (PCNSL) and Burkitt??s lymphomas (BL). This study aims to evaluate the role of MTHFR C677T and A1298C polymorphisms and G80A reduced folate carrier gene (RFC1) in a cohort of adult patients with lymphoproliferative malignancies submitted to high-dose MTX followed by leucovorin rescue.

Methods

We performed the analysis of these polymorphisms on genomic DNA with RFLP?CPCR.

Results

Patients carrying MTHFR A1298C variant showed decreased hepatic and hematological toxicity (P?=?0.03). Overall survival (OS) and progression-free survival (PFS) between homozygous wild-type and variant patients for the RFC1 G₈₀A were significantly different (P?=?0.035 and P?=?0.02, respectively). A significant correlation between hematological toxicity and age (P?=?0.003) was observed. There was no significant influence of MTHFR C677T genotype on toxicity, OS and PFS.

Conclusions

Leucovorin rescue given after high-dose MTX probably accounts for the lack of influence of C677T polymorphism. To better define a role of RFC1 polymorphism on patients outcome, it would be worthwhile to perform a study on intracellular MTX level and RFC1 substrate binding affinities in different genotypes.