Erling Tjora

Derivation of Human Induced Pluripotent Stem Cells from Patients with Maturity Onset Diabetes of the Young

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 diabetes is 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 MODY-hiPSCs 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.

A recombined allele of the lipase gene CEL and its pseudogene CELP confers susceptibility to chronic pancreatitis.

Carboxyl ester lipase is a digestive pancreatic enzyme encoded by the CEL gene. Mutations in CEL cause maturity-onset diabetes of the young as well as pancreatic exocrine dysfunction. Here we describe a hybrid allele (CEL-HYB) originating from a crossover between CEL and its neighboring pseudogene, CELP. In a discovery series of familial chronic pancreatitis cases, we observed CEL-HYB in 14.1% (10/71) of cases compared to 1.0% (5/478) of controls (odds ratio (OR) = 15.5; 95% confidence interval (CI) = 5.1–46.9; P = 1.3 × 10−6 by two-tailed Fishers exact test). In three replication studies of nonalcoholic chronic pancreatitis, we 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 showed reduced lipolytic activity, impaired secretion, prominent intracellular accumulation and induced autophagy. These findings implicate a new pathway distinct from the protease-antiprotease system of pancreatic acinar cells in chronic pancreatitis.

Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile

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.

Diabetes and Pancreatic Exocrine Dysfunction Due to Mutations in the Carboxyl Ester Lipase Gene-Maturity Onset Diabetes of the Young (CEL-MODY) A PROTEIN MISFOLDING DISEASE

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.

Assessment of exocrine pancreatic function by secretin-stimulated magnetic resonance cholangiopancreaticography and diffusion-weighted imaging in healthy controls

To characterize and quantify exocrine pancreatic function by secretin‐stimulated magnetic resonance cholangiopancreaticography (s‐MRCP) and diffusion‐weighted imaging (DWI) in healthy subjects and compare these findings to morphological features, ie, pancreatic volume and secretin‐stimulated peak bicarbonate concentration measured in pancreatic juice.

Quantification of pancreatic function using a clinically feasible short endoscopic secretin test.

Objectives Clinical and morphological criteria are not precise enough to diagnose early chronic pancreatitis (CP). We investigated if short endoscopic pancreas function testing as a part of routine upper endoscopy could improve clinical diagnostics. Methods Patients with suspected CP underwent modified secretin-stimulated upper endoscopy (short endoscopic secretin test, or EST). Duodenal juice was collected during 15 minutes starting 30 minutes after stimulation. A modified scoring system for CP after Layer with bicarbonate and fecal elastase 1 (FE1) was used. We tested with receiver operating characteristic curves the diagnostic accuracy of bicarbonate and FE1 and with analysis of variance how precise the 2 parameters can discriminate the groups. Results Fifty-two patients aged 19 to 67 years and 25 healthy controls aged 19 to 64 years were included. Twenty-four patients fulfilled the modified Layer Score for CP or non-CP. The overall accuracy of the EST versus FE1 test was 85%/71%, with positive and negative predictive values of 100%/79% and 80%/69%, respectively. Conclusions Short EST is rapid and easy to perform and can be incorporated in daily routines. We demonstrate that EST is superior to FE1 in the assessment of pancreatic insufficiency and may prove to be useful in diagnosing early or mild CP.

Exocrine pancreatic function in hepatocyte nuclear factor 1β‐maturity‐onset diabetes of the young (HNF1B‐MODY) is only moderately reduced: compensatory hypersecretion from a hypoplastic pancreas

To examine the exocrine pancreatic function in carriers of the hepatocyte nuclear factor 1β gene (HNF1B) mutation by direct testing.

Early Developmental Perturbations in a Human Stem Cell Model of MODY5/HNF1B Pancreatic Hypoplasia

Summary Patients 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 Maturity-Onset Diabetes of the Young Is Associated With Development of Pancreatic Cysts and Upregulated MAPK Signaling in Secretin-Stimulated Duodenal Fluid

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.

Severe pancreatic dysfunction but compensated nutritional status in monogenic pancreatic disease caused by carboxyl-ester lipase mutations.

Objectives The impact of pancreatic dysfunction in several diseases of the pancreas, including chronic pancreatitis and cystic fibrosis, is obscured by concomitant extra-pancreatic disease. Carboxyl-ester lipase–maturity-onset diabetes in the young (CEL-MODY) is a monogenic, highly penetrant and progressive pancreatic disease with no known primary extrapancreatic manifestations. It is characterized by low fecal elastase, steatorrhea, and development of diabetes mellitus. We sought to determine the nature of the exocrine dysfunction in CEL-MODY and relate the findings to clinical parameters of malnutrition. Methods We examined CEL-MODY patients and control subjects by rapid, endoscopic secretin test and dynamic magnetic resonance imaging of the pancreas. The findings were related to the subjects’ clinical status. Results The CEL-MODY patients displayed severely reduced acinar function and moderately reduced ductal function of the pancreas compared with control subjects. Surprisingly, CEL-MODY patients did not have clinical or biochemical signs of malnutrition, except for subnormal levels of vitamin E. Vitamin E levels seemed to be directly related to pancreatic acinar function. Conclusions Pancreatic exocrine dysfunction in CEL-MODY is associated with severely reduced acinar and moderately reduced ductal dysfunction. Despite severely reduced exocrine pancreatic function, CEL-MODY patients revealed only minor signs of malnutrition.