Honggang Ye

Insulin gene mutations as a cause of permanent neonatal diabetes

We report 10 heterozygous mutations in the human insulin gene in 16 probands with neonatal diabetes. A combination of linkage and a candidate gene approach in a family with four diabetic members led to the identification of the initial INS gene mutation. The mutations are inherited in an autosomal dominant manner in this and two other small families whereas the mutations in the other 13 patients are de novo. Diabetes presented in probands at a median age of 9 weeks, usually with diabetic ketoacidosis or marked hyperglycemia, was not associated with β cell autoantibodies, and was treated from diagnosis with insulin. The mutations are in critical regions of the preproinsulin molecule, and we predict that they prevent normal folding and progression of proinsulin in the insulin secretory pathway. The abnormally folded proinsulin molecule may induce the unfolded protein response and undergo degradation in the endoplasmic reticulum, leading to severe endoplasmic reticulum stress and potentially β cell death by apoptosis. This process has been described in both the Akita and Munich mouse models that have dominant-acting missense mutations in the Ins2 gene, leading to loss of β cell function and mass. One of the human mutations we report here is identical to that in the Akita mouse. The identification of insulin mutations as a cause of neonatal diabetes will facilitate the diagnosis and possibly, in time, treatment of this disorder.

Alterations in TCF7L2 expression define its role as a key regulator of glucose metabolism.

Genome-wide association studies (GWAS) have consistently implicated noncoding variation within the TCF7L2 locus with type 2 diabetes (T2D) risk. While this locus represents the strongest genetic determinant for T2D risk in humans, it remains unclear how these noncoding variants affect disease etiology. To test the hypothesis that the T2D-associated interval harbors cis-regulatory elements controlling TCF7L2 expression, we conducted in vivo transgenic reporter assays to characterize the TCF7L2 regulatory landscape. We found that the 92-kb genomic interval associated with T2D harbors long-range enhancers regulating various aspects of the spatial-temporal expression patterns of TCF7L2, including expression in tissues involved in the control of glucose homeostasis. By selectively deleting this interval, we establish a critical role for these enhancers in robust TCF7L2 expression. To further determine whether variation in Tcf7l2 expression may lead to diabetes, we developed a Tcf7l2 copy-number allelic series in mice. We show that a null Tcf7l2 allele leads, in a dose-dependent manner, to lower glycemic profiles. Tcf7l2 null mice also display enhanced glucose tolerance coupled to significantly lowered insulin levels, suggesting that these mice are protected against T2D. Confirming these observations, transgenic mice harboring multiple Tcf7l2 copies and overexpressing this gene display reciprocal phenotypes, including glucose intolerance. These results directly demonstrate that Tcf7l2 plays a role in regulating glucose tolerance, suggesting that overexpression of this gene is associated with increased risk of T2D. These data highlight the role of enhancer elements as mediators of T2D risk in humans, strengthening the evidence that variation in cis-regulatory elements may be a paradigm for genetic predispositions to common disease.

Diagnosis and treatment of neonatal diabetes: an United States experience

Background/objective:  Mutations in KCNJ11, ABCC8, or INS are the cause of permanent neonatal diabetes mellitus in about 50% of patients diagnosed with diabetes before 6 months of age and in a small fraction of those diagnosed between 6 and 12 months. The aim of this study was to identify the genetic cause of diabetes in 77 consecutive patients referred to the University of Chicago with diabetes diagnosed before 1 yr of age.

Clinical and molecular genetics of neonatal diabetes due to mutations in the insulin gene

Over the last decade our insight into the causes of neonatal diabetes has greatly expanded. Neonatal diabetes was once considered a variant of type 1 diabetes that presented early in life. Recent advances in our understanding of this disorder have established that neonatal diabetes is not an autoimmune disease, but rather is a monogenic form of diabetes resulting from mutations in a number of different genes encoding proteins that play a key role in the normal function of the pancreatic beta-cell. Moreover, a correct genetic diagnosis can affect treatment and clinical outcome. This is especially true for patients with mutations in the genes KCNJ11 or ABCC8 that encode the two protein subunits (Kir6.2 and SUR1, respectively) of the ATP-sensitive potassium channel. These patients can be treated with oral sulfonylurea drugs with better glycemic control and quality of life. Recently, mutations in the insulin gene (INS) itself have been identified as another cause of neonatal diabetes. In this article, we review the role of INS mutations in the pathophysiology of neonatal diabetes.

Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted.

Mutations in the preproinsulin protein that affect processing of preproinsulin to proinsulin or lead to misfolding of proinsulin are associated with diabetes. We examined the subcellular localization and secretion of 13 neonatal diabetes-associated human proinsulin proteins (A24D, G32R, G32S, L35P, C43G, G47V, F48C, G84R, R89C, G90C, C96Y, S101C and Y108C) in rat INS-1 insulinoma cells. These mutant proinsulin proteins accumulate in the endoplasmic reticulum (ER) and are poorly secreted except for G84R and in contrast to wild-type and hyperproinsulinemia-associated mutant proteins (H34D and R89H) which were sorted to secretory granules and efficiently secreted. We also examined the effect of C96Y mutant proinsulin on the synthesis and secretion of wild-type insulin and observed a dominant-negative effect of the mutant proinsulin on the synthesis and secretion of wild-type insulin due to induction of the unfolded protein response and resulting attenuation of overall translation.

Neonatal Diabetes, Gallbladder Agenesis, Duodenal Atresia, and Intestinal Malrotation Caused by a Novel Homozygous Mutation in RFX6

Recently, bi‐allelic mutations in the transcription factor RFX6 were described as the cause of a rare condition characterized by neonatal diabetes with pancreatic and biliary hypoplasia and duodenal/jejunal atresia. A male infant developed severe hyperglycemia (446 mg/dL) within 24 h of birth. Acute abdominal concerns by day five necessitated exploratory surgery that revealed duodenal atresia, gallbladder agenesis, annular pancreas and intestinal malrotation. He also exhibited chronic diarrhea and feeding intolerance, cholestatic jaundice, and subsequent liver failure. He died of sepsis at four months old while awaiting liver transplantation. The phenotype of neonatal diabetes with intestinal atresia and biliary agenesis clearly pointed to RFX6 as the causative gene; indeed, whole exome sequencing revealed a novel homozygous RFX6 mutation c.779A>C; p.Lys260Thr (K260T). This missense mutation also changes the consensus 5′ splice donor site before intron 7 and is thus predicted to cause disruption in splicing. Both parents, who were not known to be related, were heterozygous carriers. Targeted genetic testing based on consideration of phenotypic features may reveal a cause among the many genes now associated with heterogeneous forms of monogenic neonatal diabetes. Our study demonstrates the feasibility of using modern sequencing technology to identify one such rare cause. Continued research is needed to determine the possible cost‐effectiveness of this approach, especially when clear phenotypic clues are absent. Further study of patients with RFX6 mutations should clarify its role in pancreatic, intestinal and enteroendocrine cellular development and explain features such as the diarrhea exhibited in our case.

Calpain-10 is a component of the obesity-related quantitative trait locus Adip1.

We previously mapped Adip1, an obesity quantitative trait locus (QTL), to the central portion of murine chromosome 1 containing the calpain-10 (Capn10) gene. Human studies have associated calpain-10 (CAPN10) variants with type 2 diabetes and various metabolic traits. We performed a quantitative hybrid complementation test (QHCT) to determine whether differences attributed to Adip1 are the result of variant Capn10 alleles in LG/J and SM/J mice. We crossed LG/J and SM/J to wild-type (C57BL/6J) and Capn10 knockout (Capn10(-/-)) mice to form four F(1) hybrid groups: LG/J by wild-type, LG/J by Capn10(-/-), SM/J by wild-type, and SM/J by Capn10(-/-). We performed a two-way ANOVA with the experimental strain, tester strain, and their interaction as the factors. Significant interaction indicates a quantitative failure to complement. We found failure to complement for fat, organ, and body weights, and leptin, female free fatty acid, and triglyceride levels. Capn10(-/-) resulted in heavier weights and higher serum levels in LG/J crosses but not in SM/J crosses. For glucose tolerance and insulin response tests, the Capn10(-/-) allele resulted in lower glucose levels in crosses with SM/J but had no effect in the LG/J crosses. Differences between the LG/J and SM/J Capn10 alleles are the likely source of some of the QTL effects mapped to Adip1 in the LG/J-by-SM/J cross. Capn10 plays an important role in regulating obesity and diabetes in mice.

Case Report: Preservation of Reduced Numbers of Insulin-Positive Cells in Sulfonylurea-Unresponsive KCNJ11-related Diabetes

Context The most common genetic cause of permanent neonatal diabetes mellitus is activating mutations in KCNJ11, which can usually be treated using oral sulfonylureas (SUs) instead of insulin injections, although some mutations are SU unresponsive. In this work, we provide a report of the pancreatic islet endocrine cell composition and area in a patient with an SU-unresponsive KCNJ11 mutation (p.G334D), in comparison with age-matched controls. Case Description Pancreatic autopsy tissue sections from a 2-year-old female child diagnosed with KCNJ11-related diabetes at 4 days of age and 13 age-matched controls were stained with insulin, glucagon, somatostatin, pancreatic polypeptide, and Ki67 antibodies to determine islet endocrine cell composition and area. β-cell ultrastructure was assessed by electron microscopic (EM) analysis. The patients pancreas (sampling from head to tail) revealed insulin-positive cells in all regions. The pancreatic β-cell (insulin) area was significantly reduced compared with controls: 0.50% ± 0.04% versus 1.67% ± 0.20%, respectively (P < 0.00001). There were no significant differences in α-cell (glucagon) or δ-cell (somatostatin) area. EM analysis revealed secretory granules with a dense core typical of mature β-cells as well as granules with a lighter core characteristic of immature granules. Conclusions Our results suggest that mechanisms exist that allow preservation of β-cells in the absence of insulin secretion. It remains to be determined to what extent this reduction in β-cells may be reversible.

Continued lessons from the INS gene: an intronic mutation causing diabetes through a novel mechanism

Background Diabetes in neonates usually has a monogenic aetiology; however, the cause remains unknown in 20–30%. Heterozygous INS mutations represent one of the most common gene causes of neonatal diabetes mellitus. Methods Clinical and functional characterisation of a novel homozygous intronic mutation (c.187+241G>A) in the insulin gene in a child identified through the Monogenic Diabetes Registry (http://monogenicdiabetes.uchicago.edu). Results The proband had insulin-requiring diabetes from birth. Ultrasonography revealed a structurally normal pancreas and C-peptide was undetectable despite readily detectable amylin, suggesting the presence of dysfunctional β cells. Whole-exome sequencing revealed the novel mutation. In silico analysis predicted a mutant mRNA product resulting from preferential recognition of a newly created splice site. Wild-type and mutant human insulin gene constructs were derived and transiently expressed in INS-1 cells. We confirmed the predicted transcript and found an additional transcript created via an ectopic splice acceptor site. Conclusions Dominant INS mutations cause diabetes via a mutated translational product causing endoplasmic reticulum stress. We describe a novel mechanism of diabetes, without β cell death, due to creation of two unstable mutant transcripts predicted to undergo nonsense and non-stop-mediated decay, respectively. Our discovery may have broader implications for those with insulin deficiency later in life.

Microcephaly, epilepsy, and neonatal diabetes due to compound heterozygous mutations in IER3IP1: insights into the natural history of a rare disorder

Neonatal diabetes mellitus is known to have over 20 different monogenic causes. A syndrome of permanent neonatal diabetes along with primary microcephaly with simplified gyral pattern associated with severe infantile epileptic encephalopathy was recently described in two independent reports in which disease‐causing homozygous mutations were identified in the immediate early response‐3 interacting protein‐1 (IER3IP1) gene. We report here an affected male born to a non‐consanguineous couple who was noted to have insulin‐requiring permanent neonatal diabetes, microcephaly, and generalized seizures. He was also found to have cortical blindness, severe developmental delay and numerous dysmorphic features. He experienced a slow improvement but not abrogation of seizure frequency and severity on numerous anti‐epileptic agents. His clinical course was further complicated by recurrent respiratory tract infections and he died at 8 years of age.