Copy number variants and VNTR length polymorphisms of the carboxyl-ester lipase (CEL) gene as risk factors in pancreatic cancer.

BACKGROUND/OBJECTIVES: We have recently described copy number variants (CNVs) of the human carboxyl-ester lipase (CEL) gene, including a recombined deletion allele (CEL-HYB) that is a genetic risk factor for chronic pancreatitis. Associations with pancreatic disease have also been reported for the variable number of tandem repeat (VNTR) region located in CEL exon 11. Here, we examined if CEL CNVs and VNTR length polymorphisms affect the risk for developing pancreatic cancer. METHODS: CEL CNVs and VNTR were genotyped in a German family with non-alcoholic chronic pancreatitis and pancreatic cancer, in 265 German and 197 Norwegian patients diagnosed with pancreatic adenocarcinoma, and in 882 controls. CNV screening was performed using PCR assays followed by agarose gel electrophoresis whereas VNTR lengths were determined by DNA fragment analysis. RESULTS: The investigated family was CEL-HYB-positive. However, an association of CEL-HYB or a duplication CEL allele with pancreatic cancer was not seen in our two patient cohorts. The frequency of the 23-repeat VNTR allele was borderline significant in Norwegian cases compared to controls (1.2% vs. 0.3%; P = 0.05). For all other VNTR lengths, no statistically significant difference in frequency was observed. Moreover, no association with pancreatic cancer was detected when CEL VNTR lengths were pooled into groups of short, normal or long alleles. CONCLUSIONS: We could not demonstrate an association between CEL CNVs and pancreatic cancer. An association is also unlikely for CEL VNTR lengths, although analyses in larger materials are necessary to completely exclude an effect of rare VNTR alleles.

In Vitro Functional Analysis Can Aid Precision Diagnostics of HNF1B-MODY.

Precision medicine relies on accurate and consistent classification of sequence variants. A correct diagnosis of hepatocyte nuclear factor (HNF) 1B maturity-onset diabetes of the young, caused by pathogenic variants in the HNF1B gene, is important for optimal disease management and prognosis, and it has implications for genetic counseling and follow-up of at-risk family members. We hypothesized that the functional characterization could provide valuable information to assist the interpretation of pathogenicity of HNF1B variants. Using different in vitro functional assays, variants identified among 313 individuals, suspected to have monogenic diabetes with or without kidney disease, were characterized. The data from the functional assays were subsequently conjugated with obtained clinical, biochemical, and in silico data. Two variants (p.A167P, p.H336Pfs∗22) showed severe loss of function due to impaired transactivation, reduced DNA binding (p.A167P), and mRNA instability (p.A167P). Although both these variant carriers were diagnosed with diabetes, the p.H336Pfs∗22 carrier also had congenital absence of a kidney, which is a characteristic trait for HNF1B maturity-onset diabetes of the young. Functional analysis of the p.A167P variant revealed damaging effects on HNF-1B protein function, which may warrant imaging of the kidneys and/or pancreas. In addition, the current study has generated important data, including evidence supporting the benign functional impact of five variants (p.D82N, p.T88A, p.N394D, p.V458G, and p.T544A), and piloting new approaches that will prove critical for the growth of HNF1B-diabetes diagnosis.

Glycogenin-2 is dispensable for liver glycogen synthesis and glucagon-stimulated glucose release.

CONTEXT: The synthesis of glycogen is initiated by glycogenin. In humans, glycogenin-1 is expressed ubiquitously, whereas glycogenin-2 (GN2) is highly expressed in liver. It has therefore been suggested that GN2 is a liver isoform of glycogenin. In a search for possible copy number variations associated with monogenic diabetes, we identified a 102-kb deletion of the X chromosome involving the entire GYG2 gene (encoding GN2) in 2 families. OBJECTIVE: The purpose of this study was to test whether male GYG2 deletion carriers had abnormal glucose metabolism and/or glycogen synthesis. DESIGN, SETTING, AND PATIENTS: Two families with diabetes and a GYG2 deletion were investigated with medical history and examination, glucagon stimulation tests, and liver biopsies. RESULTS: We identified a GYG2 deletion in 3 members of family 1, 8 members of family 2, and 1 blood donor. The deletion showed no clear cosegregation with diabetes. Deletion carriers reported no symptoms related to fasting. Results of cardiac examination and abdominal ultrasound imaging were normal. A glucagon stimulation test in 4 male deletion carriers showed a mean rise in plasma glucose of 3.6 mmol/L (95% confidence interval, 2.9-4.2) compared with 2.8 mmol/L (95% confidence interval, 2.2-3.4) in control subjects. Liver biopsy specimens did not show clear morphologic changes by light microscopy and showed the presence of both α- and ß-glycogen by electron microscopy. We detected GYG1 but not GYG2 mRNA expression in the liver biopsy specimens. CONCLUSIONS: This is the first evaluation of humans without GN2 expression. Our data indicate that GN2 is not required for liver glycogen synthesis and glucagon-stimulated glucose release.

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 Fisher's 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 the insulin gene can cause MODY and autoantibody-negative type 1 diabetes.

OBJECTIVE: Mutations in the insulin (INS) gene can cause neonatal diabetes. We hypothesized that mutations in INS could also cause maturity-onset diabetes of the young (MODY) and autoantibody-negative type 1 diabetes. RESEARCH DESIGN AND METHODS: We screened INS in 62 probands with MODY, 30 probands with suspected MODY, and 223 subjects from the Norwegian Childhood Diabetes Registry selected on the basis of autoantibody negativity or family history of diabetes. RESULTS: Among the MODY patients, we identified the INS mutation c.137G>A (R46Q) in a proband, his diabetic father, and a paternal aunt. They were diagnosed with diabetes at 20, 18, and 17 years of age, respectively, and are treated with small doses of insulin or diet only. In type 1 diabetic patients, we found the INS mutation c.163C>T (R55C) in a girl who at 10 years of age presented with ketoacidosis and insulin-dependent, GAD, and insulinoma-associated antigen-2 (IA-2) antibody-negative diabetes. Her mother had a de novo R55C mutation and was diagnosed with ketoacidosis and insulin-dependent diabetes at 13 years of age. Both had residual beta-cell function. The R46Q substitution changes an invariant arginine residue in position B22, which forms a hydrogen bond with the glutamate at A17, stabilizing the insulin molecule. The R55C substitution involves the first of the two arginine residues localized at the site of proteolytic processing between the B-chain and the C-peptide. CONCLUSIONS: Our findings extend the phenotype of INS mutation carriers and suggest that INS screening is warranted not only in neonatal diabetes, but also in MODY and in selected cases of type 1 diabetes.

Toll-like receptor 3 associates with c-Src tyrosine kinase on endosomes to initiate antiviral signaling.

Double-stranded RNA (dsRNA) is produced during the replication cycle of most viruses and triggers antiviral immune responses through Toll-like receptor 3 (TLR3). However, the molecular mechanisms and subcellular compartments associated with dsRNA-TLR3-mediated signaling are largely unknown. Here we show that c-Src tyrosine kinase is activated by dsRNA in human monocyte-derived dendritic cells, and is recruited to TLR3 in a dsRNA-dependent manner. DsRNA-induced activation of interferon-regulatory factor 3 and signal transducer and activator of transcription 1 was abolished in Src kinase-deficient cells, and restored by adding back c-Src, suggesting a central role of c-Src in antiviral immunity. We also provide evidence that TLR3 is localized in the endoplasmic reticulum of unstimulated cells, moves to dsRNA-containing endosomes in response to dsRNA, and colocalizes with c-Src on endosomes containing dsRNA in the lumen. These results provide novel insight into the molecular mechanisms of TLR3-mediated signaling, which may contribute to the understanding of innate immune responses during viral infections.