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Permanent neonatal diabetes mellitus (PNDM) is a rare disorder usually presenting within 6 months of birth. Although several genes have been linked to this disorder, in almost half the cases documented in Italy, the genetic cause remains unknown. Because the Akita mouse bearing a mutation in the Ins2 gene exhibits PNDM associated with pancreatic beta cell apoptosis, we sequenced the human insulin gene in PNDM subjects with unidentified mutations. We discovered 7 heterozygous mutations in 10 unrelated probands. In 8 of these patients, insulin secretion was detectable at diabetes onset, but rapidly declined over time. When these mutant proinsulins were expressed in HEK293 cells, we observed defects in insulin protein folding and secretion. In these experiments, expression of the mutant proinsulins was also associated with increased Grp78 protein expression and XBP1 mRNA splicing, 2 markers of endoplasmic reticulum stress, and with increased apoptosis. Similarly transfected INS-1E insulinoma cells had diminished viability compared with those expressing WT proinsulin. In conclusion, we find that mutations in the insulin gene that promote proinsulin misfolding may cause PNDM.

OBJECTIVE:--Heterozygous, gain-of-function mutations of the insulin gene can cause permanent diabetes with onset ranging from the neonatal period through adulthood. The aim of our study was to screen for the insulin gene in patients who had been clinically classified as type 1 diabetic but who tested negative for type 1 diabetes autoantibodies. RESEARCH DESIGN AND METHODS--We reviewed the clinical records of 326 patients with the diagnosis of type 1 diabetes and identified seven probands who had diabetes in isolation and were negative for five type 1 diabetes autoantibodies. We sequenced the INS gene in these seven patients. RESULTS:--In two patients whose diabetes onset had been at 2 years 10 months of age and at 6 years 8 months of age, respectively, we identified the mutation GB⁸S and a novel mutation in the preproinsulin signal peptide (ASignal²³S). CONCLUSIONS:--Insulin gene mutations are rare in absolute terms in patients classified as type 1 diabetic (0.6%) but can be identified after a thorough screening of type 1 diabetes autoantibodies.

Maturity-Onset Diabetes of the Young in Children With Incidental Hyperglycemia: A multicenter Italian study of 172 families Renata Lorini , MD 1 , Catherine Klersy , MD 2 , Giuseppe d'Annunzio , MD 1 , Ornella Massa , PHD 3 , Nicola Minuto , MD 1 , Dario Iafusco , MD 4 , Christine Bellannè-Chantelot 5 , Anna Paola Frongia , MD 6 , Sonia Toni , MD 7 , Franco Meschi , MD 8 , Franco Cerutti , MD 9 , Fabrizio Barbetti , MD 3 , 10 , 11 and the Italian Society of Pediatric Endocrinology and Diabetology (ISPED) Study Group * 1 Department of Pediatrics, IRCCS Gaslini Children's Hospital, University of Genoa, Genoa, Italy; 2 Biometry and Clinical Epidemiology Service, IRCCS Fondazione Policlinico San Matteo, Pavia, Italy; 3 IRCCS Bambino Gesù Pediatric Hospital, Rome, Italy; 4 Department of Pediatrics, Second University of Naples, Naples, Italy; 5 Department of Genetics, AP-HP Hopital Pitié-Salpétrière, Univesité Pierre et Marie Curie, Paris, France; 6 Pediatric Division, Brotzu Hospital, Cagliari, Italy; 7 Meyer Pediatric Institute, Florence, Italy; 8 Department of Pediatrics, Scientific Institute, San Raffaele Hospital, Milan, Italy; 9 Department of Pediatrics, University of Turin, Turin, Italy; 10 Department of Laboratory Medicine, Tor Vergata University Hospital, University of Tor Vergata, Rome, Italy; 11 San Raffaele Biomedical Park Foundation, Rome, Italy. Corresponding authors: Renata Lorini, renatalorini{at}ospedale-gaslini.ge.it , and Fabrizio Barbetti, mody.2{at}libero.it . C.K. and G. d'A. contributed equally to this study. Abstract OBJECTIVE To investigate the prevalence of maturity-onset diabetes of the young (MODY) in Italian children with incidental hyperglycemia. RESEARCH DESIGN AND METHODS Among 748 subjects age 1–18 years with incidental hyperglycemia, minimal diagnostic criteria for MODY were met by 172 families. Mutational analyses of the glucokinase ( GCK ) and hepatocyte nuclear factor 1α ( HNF1 Α) genes were performed. RESULTS We identified 85 GCK gene mutations in 109 probands and 10 HNF1 Α mutations in 12 probands. In GCK patients, the median neonatal weight and age at the first evaluation were lower than those found in patients with HNF1A mutations. Median fasting plasma glucose and impaired fasting glucose/impaired glucose tolerance frequency after oral glucose tolerance testing were higher in GCK patients, who also showed a lower frequency of diabetes than HNF1A patients. CONCLUSIONS GCK mutations are the prevailing cause of MODY (63.4%) when the index case is recruited in Italian children with incidental hyperglycemia. Footnotes ↵ *A complete list of the members of the Italian Society of Pediatric Endocrinology and Diabetology (ISPED) Study Group can be found in the acknowledgments . The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received November 10, 2008. Accepted June 13, 2009. © 2009 by the American Diabetes Association.

OBJECTIVE: To investigate the prevalence of maturity-onset diabetes of the young (MODY) in Italian children with incidental hyperglycemia. RESEARCH DESIGN AND METHODS: Among 748 subjects age 1-18 years with incidental hyperglycemia, minimal diagnostic criteria for MODY were met by 172 families. Mutational analyses of the glucokinase (GCK) and hepatocyte nuclear factor 1α (HNF1A) genes were performed. RESULTS: We identified 85 GCK gene mutations in 109 probands and 10 HNF1A mutations in 12 probands. In GCK patients, the median neonatal weight and age at the first evaluation were lower than those found in patients with HNF1A mutations. Median fasting plasma glucose and impaired fasting glucose/impaired glucose tolerance frequency after oral glucose tolerance testing were higher in GCK patients, who also showed a lower frequency of diabetes than HNF1A patients. CONCLUSIONS: GCK mutations are the prevailing cause of MODY (63.4%) when the index case is recruited in Italian children with incidental hyperglycemia.

Biallelic insulin receptor ( INSR ) gene mutations cause congenital syndromes of severe insulin resistance (SIR) known as Donohue syndrome (DS) and Rabson–Mendenhall syndrome (RMS). At presentation, DS and RMS are difficult to differentiate since they share many clinical features; however, while patients with DS usually die within 1 year of birth, individuals classified as RMS can reach adult age. INSR mutations can be also found in pubertal females with hyperinsulinism, hyperandrogenism, and acanthosis nigricans (type A SIR). We studied the INSR gene in five subjects with congenital SIR and in a patient with type A SIR. Nine biallelic INSR gene mutations (eight novels, including an in-frame deletion of INSR signal peptide) were identified in patients with congenital SIR; a heterozygous, spontaneous INSR mutation was detected in the patient with type A SIR. Two probands, presenting severe hirsutism at birth, died at the age of 3 months and were classified as DS, while other 2, currently 2 and 3 years old, were diagnosed with RMS (patients 3 and 4). The fifth patient with congenital SIR died when 14 months old. Nephrocalcinosis, hyperaldosteronism, hyperreninemia, and hypokalemia, in the absence of hypertension, were discovered in patients 3 and 5 when 24 and 4 months old, respectively. Patient 3, now 3 years/3 months old, still shows hyperreninemic hyperaldosteronism requiring potassium supplementation. We conclude that renal abnormalities resembling antenatal Bartter’s syndrome type II, recently reported also by others, is a common observation in patients with congenital SIR.

Diabetes mellitus is a heterogeneous disorder that can occur at any age. 1 Neonatal diabetes mellitus, defined as insulin-requiring hyperglycemia within the first month of life, is a rare disorder that is usually associated with intrauterine growth retardation. 2 Like diabetes in general, neonatal diabetes is heterogeneous and can be either transient or permanent. Transient neonatal diabetes is associated with abnormalities of chromosome 6, 2 , 3 whereas mutations in insulin promoter factor 1 result in pancreatic agenesis and permanent neonatal diabetes. 4 We describe two patients in whom complete deficiency of the glycolytic enzyme glucokinase, a key regulator of glucose metabolism in pancreatic beta . . .

Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP‐dependent potassium (KATP) channel of the pancreatic β‐cell, were found in patients with PNDM. Closure of the KATP channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non‐autoimmune, insulin‐requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK‐PDMI, KCNJ11‐PDMI) to avoid confusion between patients with early‐onset, autoimmune type 1 diabetes. Hum Mutat 25:22–27, 2005. © 2004 Wiley‐Liss, Inc.

The Second Activating Glucokinase Mutation (A456V) Implications for Glucose Homeostasis and Diabetes Therapy Henrik B.T. Christesen 1 , Bendt B. Jacobsen 1 , Stella Odili 2 , Carol Buettger 2 , Antonio Cuesta-Munoz 2 , Torben Hansen 3 , Klaus Brusgaard 4 , Ornella Massa 5 , Mark A. Magnuson 6 , Chiyo Shiota 6 , Franz M. Matschinsky 2 and Fabrizio Barbetti 5 7 1 Department of Pediatrics, Odense University Hospital, Odense, Denmark 2 Department of Biochemistry and Biophysics and Diabetes Research Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 3 Steno Diabetes Center, Gentofte, Denmark 4 Department of Clinical Biochemistry and Genetics, Odense University Hospital, Odense, Denmark 5 IBCIT Biomedical Scientific Park S Raffaele, Rome, Italy 6 Department of Molecular Biophysics and Physiology, Vanderbilt University School of Medicine, Nashville, Tennessee 7 Bambino Gesù Pediatric Hospital, Rome, Italy Abstract In this study, a second case of hyperinsulinemic hypoglycemia due to activation of glucokinase is reported. The 14-year-old proband had a history of neonatal hypoglycemia, treated with diazoxide. He was admitted with coma and convulsions due to nonketotic hypoglycemia. His BMI was 34 kg/m 2 , and his fasting blood glucose ranged from 2.1 to 2.7 mmol/l, associated with inappropriately high serum levels of insulin, C-peptide, and proinsulin. An oral glucose tolerance test (OGTT) showed exaggerated responses of these peptides followed by profound hypoglycemia. Treatment with diazoxide and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, C-peptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening for mutations in candidate genes revealed a heterozygous glucokinase mutation in exon 10, substituting valine for alanine at codon 456 (A456V) in the proband and his mother. The purified recombinant glutathionyl S-transferase fusion protein of the A456V glucokinase revealed a decreased glucose S 0.5 (the concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant’s Hill coefficient was decreased, and its maximal specific activity k cat was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol/l. The theoretical and practical implications are manifold and significant. Footnotes Address correspondence and reprint requests to Henrik B.T. Christesen, Department of Pediatrics, Odense University Hospital, 5000 Odense C, Denmark. E-mail: thybo{at}dadlnet.dk . Received for publication 20 August 2001 and accepted in revised form 17 December 2001. CT, computed tomography; DGGE, denaturing gradient gel electrophoresis; EEG, electroencephalogram; GDH, glutamate dehydrogenase; GK, glucokinase; GSIR, glucose-stimulated insulin release; GST, glutathionyl S-transferase; MODY, maturity-onset diabetes of the young; OGTT, oral glucose tolerance test; PHHI, persistent hyperinsulinemic hypoglycemia in infancy; PND, permanent neonatal diabetes; S 0.5 , the concentration of glucose needed to achieve the half-maximal rate of phosphorylation; SSCP, single-strand conformation polymorphism. DIABETES

In this study, a second case of hyperinsulinemic hypoglycemia due to activation of glucokinase is reported. The 14-year-old proband had a history of neonatal hypoglycemia, treated with diazoxide. He was admitted with coma and convulsions due to nonketotic hypoglycemia. His BMI was 34 kg/m(2), and his fasting blood glucose ranged from 2.1 to 2.7 mmol/l, associated with inappropriately high serum levels of insulin, C-peptide, and proinsulin. An oral glucose tolerance test (OGTT) showed exaggerated responses of these peptides followed by profound hypoglycemia. Treatment with diazoxide and chlorothiazide was effective. His mother never had clinical hypoglycemic symptoms, even though her fasting blood glucose ranged from 2.9 to 3.5 mmol/l. Increases in serum insulin, C-peptide, and proinsulin in response to an OGTT suggested a lower threshold for glucose-stimulated insulin release (GSIR). Screening for mutations in candidate genes revealed a heterozygous glucokinase mutation in exon 10, substituting valine for alanine at codon 456 (A456V) in the proband and his mother. The purified recombinant glutathionyl S-transferase fusion protein of the A456V glucokinase revealed a decreased glucose S(0.5) (the concentration of glucose needed to achieve the half-maximal rate of phosphorylation) from 8.04 (wild-type) to 2.53 mmol/l. The mutant's Hill coefficient was decreased, and its maximal specific activity k(cat) was increased. Mathematical modeling predicted a markedly lowered GSIR threshold of 1.5 mmol/l. The theoretical and practical implications are manifold and significant.

Transglutaminase 2 (TG2) is a multifunctional protein with Ca 2+ -dependent transamidating and G protein activity. Previously, we reported that tgm2 −/− mice have an impaired insulin secretion and that naturally occurring TG2 mutations associated with familial, early-onset type 2 diabetes, show a defective transamidating activity. Aim of this study was to get a better insight into the role of TG2 in insulin secretion by identifying substrates of TG2 transamidating activity in the pancreatic beta cell line INS-1E. To this end, we labeled INS-1E that are capable of secreting insulin upon glucose stimulation in the physiologic range, with an artificial acyl acceptor (biotinamido-pentylamine) or donor (biotinylated peptide), in basal condition and after stimulus with glucose for 2, 5, and 8 min. Biotinylated proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. In addition, subcellular localization of TG2 in human endocrine pancreas was studied by electron microscopy. Among several TG2’s transamidating substrates in INS-1E, mass spectrometry identified cytoplasmic actin (a result confirmed in human pancreatic islet), tropomyosin, and molecules that participate in insulin granule structure (e.g., GAPDH), glucose metabolism, or [Ca 2+ ] sensing (e.g., calreticulin). Physical interaction between TG2 and cytoplasmic actin during glucose-stimulated first-phase insulin secretion was confirmed by co-immunoprecipitation. Electron microscopy revealed that TG2 is localized close to insulin and glucagon granules in human pancreatic islet. We propose that TG2’s role in insulin secretion may involve cytoplasmic actin remodeling and may have a regulative action on other proteins during granule movement. A similar role of TG2 in glucagon secretion is also suggested.