Carboxyl-ester lipase is a digestive pancreatic enzyme encoded by the highly polymorphic CEL gene1. Mutations in CEL cause maturity-onset diabetes of the young (MODY) with pancreatic exocrine dysfunction2. Here we identified a hybrid allele (CEL-HYB), originating from a crossover between CEL and its neighboring pseudogene CELP. In a discovery cohort of familial chronic pancreatitis cases, the carrier frequency of CEL-HYB was 14.1% (10/71) compared with 1.0% (5/478) in controls (odds ratio [OR] = 15.5, 95% confidence interval [CI] = 5.1-46.9, P = 1.3 × 10−6). Three replication studies in non-alcoholic chronic pancreatitis cohorts identified CEL-HYB in a total of 3.7% (42/1,122) cases and 0.7% (30/4,152) controls (OR = 5.2, 95% CI = 3.2-8.5, P = 1.2 × 10−11; formal meta-analysis). The allele was also enriched in alcoholic chronic pancreatitis. Expression of CEL-HYB in cellular models revealed reduced lipolytic activity, impaired secretion, prominent intracellular accumulation and induced autophagy. The hybrid variant of CEL is the first chronic pancreatitis gene identified outside the protease/antiprotease system of pancreatic acinar cells.

SummaryBackgroundKCNJ11 mutations cause permanent neonatal diabetes through pancreatic ATP-sensitive potassium channel activation. 90% of patients successfully transfer from insulin to oral sulfonylureas with excellent initial glycaemic control; however, whether this control is maintained in the long term is unclear. Sulfonylurea failure is seen in about 44% of people with type 2 diabetes after 5 years of treatment. Therefore, we did a 10-year multicentre follow-up study of a large international cohort of patients with KCNJ11 permanent neonatal diabetes to address the key questions relating to long-term efficacy and safety of sulfonylureas in these patients.MethodsIn this multicentre, international cohort study, all patients diagnosed with KCNJ11 permanent neonatal diabetes at five laboratories in Exeter (UK), Rome (Italy), Bergen (Norway), Paris (France), and Krakow (Poland), who transferred from insulin to oral sulfonylureas before Nov 30, 2006, were eligible for inclusion. Clinicians collected clinical characteristics and annual data relating to glycaemic control, sulfonylurea dose, severe hypoglycaemia, side-effects, diabetes complications, and growth. The main outcomes of interest were sulfonylurea failure, defined as permanent reintroduction of daily insulin, and metabolic control, specifically HbA1c and sulfonylurea dose. Neurological features associated with KCNJ11 permanent neonatal diabetes were also assessed. This study is registered with ClinicalTrials.gov, number NCT02624817.Findings90 patients were identified as being eligible for inclusion and 81 were enrolled in the study and provided long-term (>5·5 years cut-off) outcome data. Median follow-up duration for the whole cohort was 10·2 years (IQR 9·3–10·8). At most recent follow-up (between Dec 1, 2012, and Oct 4, 2016), 75 (93%) of 81 participants remained on sulfonylurea therapy alone. Excellent glycaemic control was maintained for patients for whom we had paired data on HbA1c and sulfonylurea at all time points (ie, pre-transfer [for HbA1c], year 1, and most recent follow-up; n=64)—median HbA1c was 8·1% (IQR 7·2–9·2; 65·0 mmol/mol [55·2–77·1]) before transfer to sulfonylureas, 5·9% (5·4–6·5; 41·0 mmol/mol [35·5–47·5]; p<0·0001 vs pre-transfer) at 1 year, and 6·4% (5·9–7·3; 46·4 mmol/mol [41·0–56·3]; p<0·0001 vs year 1) at most recent follow-up (median 10·3 years [IQR 9·2–10·9]). In the same patients, median sulfonylurea dose at 1 year was 0·30 mg/kg per day (0·14–0·53) and at most recent follow-up visit was 0·23 mg/kg per day (0·12–0·41; p=0·03). No reports of severe hypoglycaemia were recorded in 809 patient-years of follow-up for the whole cohort (n=81). 11 (14%) patients reported mild, transient side-effects, but did not need to stop sulfonylurea therapy. Seven (9%) patients had microvascular complications; these patients had been taking insulin longer than those without complications (median age at transfer to sulfonylureas 20·5 years [IQR 10·5–24·0] vs 4·1 years [1·3–10·2]; p=0·0005). Initial improvement was noted following transfer to sulfo...

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.Most monogenic forms of diabetes are due to mutations in genes expressed in the pancreatic beta cell. Previously, Raeder et al.(1) reported a novel monogenic syndrome caused by mutations in the carboxyl ester lipase gene (CEL) (OMIM 609812; CEL-MODY 2 or MODY8), characterized by dominantly inherited childhood-onset pancreatic exocrine dysfunction and diabetes mellitus from adulthood. Notably, this gene is not transcribed in beta cells but is mainly expressed in pancreatic acinar tissue (2, 3) and lactating mammary glands (4, 5). The CEL enzyme (EC 3.1.1.13), also known as bile salt-stimulated/dependent lipase, is secreted into the intestine and activated by bile salts, playing a role in the hydrolysis and absorption of cholesterol-and lipid-soluble vitamins (6).The human CEL gene is ϳ10 kb in size and consists of 11 exons. In the last exon, there is a variable number of tandem repeats (VNTR) where the 33-bp nearly identical segments are repeated usually between 7 and 23 times (1, 7-9). The VNTR of the most common CEL allele has 16 repeats, thereby encoding a protein consisting of 745 amino acids with a predicted molecular mass of ϳ79 kDa. The rat Cel is secreted from the acinar cells and is thought to follow the classical pathway of secretory proteins (for review see Ref.3). In the endoplasmic reticulum (ER), the protein is co-translationally N-glycosylated at a conserved Asn residue (Asn-210), serving as a client protein for the molecular chaperone GRP94 and being associated with ER membranes (10, 11). The VNTR-encoded protein C terminus is heavily O-glycosylated in the mature protein, probably on Thr residues before or after PVPP motifs in the first 10 repeats (12). The O-glycosylation sites are present in a region enriched in the amino ...

Background: Human induced pluripotent stem cells (hiPSCs) can be harnessed for development of novel therapeutics for metabolic disorders. Results: Karyotypically normal hiPSCs were derived from patients with MODY1, MODY2, MODY3, MODY5, or MODY8. Conclusion: hiPSCs were successfully derived from a variety of MODY patients. Significance: MODY-hiPSCs can be used to explore the role of MODY genes in the development and function of pancreatic islet cells. Maturity onset diabetes of the young (MODY) is an autosomal dominant disease. Despite extensive research, the mechanism by which a mutant MODY gene results in monogenic diabetesis not yet clear due to the inaccessibility of patient samples. Induced pluripotency and directed differentiation toward the pancreatic lineage are now viable and attractive methods to uncover the molecular mechanisms underlying MODY. Here we report, for the first time, the derivation of human induced pluripotent stem cells (hiPSCs) from patients with five types of MODY: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY5 (HNF1B), and MODY8 (CEL) with a polycistronic lentiviral vector expressing a Cre-excisable human "stem cell cassette" containing the four reprogramming factors OCT4, KLF4, SOX2, and CMYC. These MODY-hiPSCs morphologically resemble human pluripotent stem cells (hPSCs), express pluripotency markers OCT4, SOX2, NANOG, SSEA-4, and TRA-1-60, give rise to derivatives of the three germ layers in a teratoma assay, and are karyotypically normal. Overall, our MODYhiPSCs serve as invaluable tools to dissect the role of MODY genes in the development of pancreas and islet cells and to evaluate their significance in regulating beta cell function. This knowledge will aid future attempts aimed at deriving functional mature beta cells from hPSCs. Maturity onset diabetes of the young (MODY)2 is an autosomal dominant monogenic diabetic disease typically affecting individuals before the age of 25 years. To date, 11 MODY genes have been identified, comprising both transcription factors and enzymes/hormones: MODY1 (HNF4A), MODY2 (GCK), MODY3 (HNF1A), MODY4 (PDX1), MODY5 (HNF1B), MODY6 (NEUROD1), MODY7 (KLF11), MODY8 (CEL), MODY9 (PAX4), MODY10 (INS), and MODY11 (BLK) (1). Various disease-causing genetic variants have been identified for each of the MODY genes, all of which affect pancreas and beta cell development, ultimately resulting in beta cell dysfunction and diabetes.The advent of induced pluripotency (2) has provided an opportunity to derive human induced pluripotent stem cells (hiPSCs) from MODY patients to model the disease in vitro. Several studies have reported that human somatic cells, commonly skin fibroblasts, can be reprogrammed into hiPSCs. A number of groups have reported the generation of hiPSCs from patients with mitochondrial diabetes, type 1 and type 2 diabetes mellitus (3, 4). However, given the complexity of type 1 and type 2 diabetes mellitus, the use of hiPSCs from these patients requires comparison with multiple controls that are appropriate for the genetic and environmenta...

A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.

Aims/hypothesis An accurate molecular diagnosis of diabetes subtype confers clinical benefits; however, many individuals with monogenic diabetes remain undiagnosed.Biomarkers could help to prioritise patients for genetic investigation. We recently demonstrated that highsensitivity C-reactive protein (hsCRP) levels are lower in UK patients with hepatocyte nuclear factor 1 alpha Diabetologia (2011) 54:2801-2810 DOI 10.1007/s00125-011-2261 (HNF1A)-MODY than in other diabetes subtypes. In this large multi-centre study we aimed to assess the clinical validity of hsCRP as a diagnostic biomarker, examine the genotype-phenotype relationship and compare different hsCRP assays. Methods High-sensitivity CRP levels were analysed in individuals with HNF1A-MODY (n=457), glucokinase (GCK)-MODY (n=404), hepatocyte nuclear factor 4 alpha (HNF4A)-MODY (n=54) and type 2 diabetes (n=582) from seven European centres. Three common assays for hsCRP analysis were evaluated. We excluded 121 participants (8.1%) with hsCRP values >10 mg/l. The discriminative power of hsCRP with respect to diabetes aetiology was assessed by receiver operating characteristic curvederived C-statistic. Results In all centres and irrespective of the assay method, meta-analysis confirmed significantly lower hsCRP levels in those with HNF1A-MODY than in those with other aetiologies (z score −21.8, p<5×10 −105 ). HNF1A-MODY cases with missense mutations had lower hsCRP levels than those with truncating mutations (0.03 vs 0.08 mg/l, p<5× 10 −5 ). High-sensitivity CRP values between assays were strongly correlated (r 2 ≥0.91, p≤1×10 −5 ). Across the seven centres, the C-statistic for distinguishing HNF1A-MODY from young adult-onset type 2 diabetes ranged from 0.79 to 0.97, indicating high discriminative accuracy.

SummaryPatients with an HNF1BS148L/+ mutation (MODY5) typically exhibit pancreatic hypoplasia. However, the molecular mechanisms are unknown due to inaccessibility of patient material and because mouse models do not fully recapitulate MODY5. Here, we differentiated MODY5 human-induced pluripotent stem cells (hiPSCs) into pancreatic progenitors, and show that the HNF1BS148L/+ mutation causes a compensatory increase in several pancreatic transcription factors, and surprisingly, a decrease in PAX6 pancreatic gene expression. The lack of suppression of PDX1, PTF1A, GATA4, and GATA6 indicates that MODY5-mediated pancreatic hypoplasia is mechanistically independent. Overexpression studies demonstrate that a compensatory increase in PDX1 gene expression is due to mutant HNF1BS148L/+ but not wild-type HNF1B or HNF1A. Furthermore, HNF1B does not appear to directly regulate PAX6 gene expression necessary for glucose tolerance. Our results demonstrate compensatory mechanisms in the pancreatic transcription factor network due to mutant HNF1BS148L/+ protein. Thus, patients typically develop MODY5 but not neonatal diabetes despite exhibiting pancreatic hypoplasia.

Carboxyl-ester lipase (CEL) maturity-onset diabetes of the young (MODY) is a monogenic form of diabetes and pancreatic exocrine dysfunction due to mutations in the CEL gene encoding CEL. The pathogenic mechanism for diabetes development is unknown. Since CEL is expressed mainly in pancreatic acinar cells, we asked whether we could find structural pancreatic changes in CEL-MODY subjects during the course of diabetes development. Furthermore, we hypothesized that the diseased pancreas releases proteins that are detectable in pancreatic fluid and potentially reflect activation or inactivation of disease-specific pathways. We therefore investigated nondiabetic and diabetic CEL-mutation carriers by pancreatic imaging studies and secretin-stimulated duodenal juice sampling. The secretin-stimulated duodenal juice was studied using cytokine assays, mass spectrometry (MS) proteomics, and multiplexed MS-based measurement of kinase activities. We identified multiple pancreatic cysts in all eight diabetic mutation carriers but not in any of the four nondiabetic mutation carriers or the six healthy controls. Furthermore, we identified upregulated mitogen-activated protein kinase (MAPK) target proteins and MAPK-driven cytokines and increased MAPK activity in the secretin-stimulated duodenal juice. These findings show that subjects with CEL-MODY develop multiple pancreatic cysts by the time they develop diabetes and that upregulated MAPK signaling in the pancreatic secretome may reflect the pathophysiological development of pancreatic cysts and diabetes.