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.
Aims/hypothesis
MODY can be wrongly diagnosed as type 1 diabetes in children. We aimed to find the prevalence of MODY in a nationwide population-based registry of childhood diabetes.
Methods
Using ...next-generation sequencing, we screened the
HNF1A
,
HNF4A
,
HNF1B
,
GCK
and
INS
genes in all 469 children (12.1%) negative for both GAD and IA-2 autoantibodies and 469 antibody-positive matched controls selected from the Norwegian Childhood Diabetes Registry (3882 children). Variants were classified using clinical diagnostic criteria for pathogenicity ranging from class 1 (neutral) to class 5 (pathogenic).
Results
We identified 58 rare exonic and splice variants in cases and controls. Among antibody-negative patients, 6.5% had genetic variants of classes 3–5 (vs 2.4% in controls;
p
= 0.002). For the stricter classification (classes 4 and 5), the corresponding number was 4.1% (vs 0.2% in controls;
p
= 1.6 × 10
−5
).
HNF1A
showed the strongest enrichment of class 3–5 variants, with 3.9% among antibody-negative patients (vs 0.4% in controls;
p
= 0.0002). Antibody-negative carriers of variants in class 3 had a similar phenotype to those carrying variants in classes 4 and 5.
Conclusions/interpretation
This is the first study screening for MODY in all antibody-negative children in a nationwide population-based registry. Our results suggest that the prevalence of MODY in antibody-negative childhood diabetes may reach 6.5%. One-third of these MODY cases had not been recognised by clinicians. Since a precise diagnosis is important for treatment and genetic counselling, molecular screening of all antibody-negative children should be considered in routine diagnostics.
The role of the carboxyl ester lipase (CEL) gene in pancreatic disease Johansson, Bente B.; Fjeld, Karianne; El Jellas, Khadija ...
Pancreatology : official journal of the International Association of Pancreatology (IAP) ... et al.,
January 2018, 2018-Jan, 2018-01-00, 20180101, Volume:
18, Issue:
1
Journal Article
Peer reviewed
The enzyme carboxyl ester lipase (CEL), also known as bile salt-dependent or -stimulated lipase (BSDL, BSSL), hydrolyzes dietary fat, cholesteryl esters and fat-soluble vitamins in the duodenum. CEL ...is mainly expressed in pancreatic acinar cells and lactating mammary glands. The human CEL gene resides on chromosome 9q34.3 and contains a variable number of tandem repeats (VNTR) region that encodes a mucin-like protein tail. Although the number of normal repeats does not appear to significantly influence the risk for pancreatic disease, single-base pair deletions in the first VNTR repeat cause a syndrome of endocrine and exocrine dysfunction denoted MODY8. Hallmarks are low fecal elastase levels and pancreatic lipomatosis manifesting before the age of twenty, followed by development of diabetes and pancreatic cysts later in life. The mutant protein forms intracellular and extracellular aggregates, suggesting that MODY8 is a protein misfolding disease. Recently, a recombined allele between CEL and its pseudogene CELP was discovered. This allele (CEL-HYB) encodes a chimeric protein with impaired secretion increasing five-fold the risk for chronic pancreatitis. The CEL gene has proven to be exceptionally polymorphic due to copy number variants of the CEL-CELP locus and alterations involving the VNTR. Genome-wide association studies or deep sequencing cannot easily pick up this wealth of genetic variation. CEL is therefore an attractive candidate gene for further exploration of links to pancreatic disease.
MODY8 (maturity onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes. This disease is associated with frameshift mutations in the carboxyl ester lipase (CEL) gene ...expressed by pancreatic acinar tissue. Patients with the mutation develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes in MODY8 patients. In this study, we investigated the potential crosstalk between exocrine acinar cells and endocrine beta cells in vitro. Co-culture of beta cells (EndoC-bH1 beta cells) with donor cells (HEK293 embryonic kidney cells or 266-6 acinar cells) overexpressing mutant or wild type CEL gene in an in vitro transwell system demonstrated transfer of mutant protein from donor cells to beta cells. While both the wild type and mutant CEL proteins were observed to be taken up by the beta cells there was a significantly higher amount of the mutant (11.4±2.3% cells) compared to the wild type protein (1.8±0.7% cells) (n=3, p<0.01 mutant vs. wild type). The mutant protein formed intracellular aggregates represented as punctuate structures within the cytoplasm of beta cells. Culturing beta cells in mutant donor cell-conditioned media for 12 days activated the unfolded protein response, and increased endoplasmic reticulum stress by promoting the gene expression of BiP, spliced XBP1, and caused beta cell secretory dysfunction (insulin stimulation index 16.7 mM/3.3 mM: 1.3±0.1 (mutant) vs. 2.1±0.3 fold (wild type), n=8, p=0.05). Our data indicate a unique crosstalk between pancreatic acinar and beta cells, which adversely affects the latter by increasing ER stress and impairing insulin secretion. This study provides one possible explanation of how a mutant gene expressed specifically in acinar cells causes endocrine cell dysfunction and diabetes in MODY8 patients.
Disclosure
S. Kahraman: None. D. Diegisser: None. B.B. Johansson: None. A. Molven: None. R. Kulkarni: None.
Funding
National Institutes of Health (R01DK67536)
Variable number of tandem repeat (VNTR) sequences in the genome can have functional consequences that contribute to human disease. This is the case for the CEL gene, which is specifically expressed ...in pancreatic acinar cells and encodes the digestive enzyme carboxyl ester lipase. Rare single-base deletions (DELs) within the first (DEL1) or fourth (DEL4) VNTR segment of CEL cause maturity-onset diabetes of the young, type 8 (MODY8), an inherited disorder characterized by exocrine pancreatic dysfunction and diabetes. Studies on the DEL1 variant have suggested that MODY8 is initiated by CEL protein misfolding and aggregation. However, it is unclear how the position of single-base deletions within the CEL VNTR affects pathogenic properties of the protein. Here, we investigated four naturally occurring CEL variants, arising from single-base deletions in different VNTR segments (DEL1, DEL4, DEL9, and DEL13). When the four variants were expressed in human embryonic kidney 293 cells, only DEL1 and DEL4 led to significantly reduced secretion, increased intracellular aggregation, and increased endoplasmic reticulum stress compared with normal CEL protein. The level of O-glycosylation was affected in all DEL variants. Moreover, all variants had enzymatic activity comparable with that of normal CEL. We conclude that the longest aberrant protein tails, resulting from single-base deletions in the proximal VNTR segments, have highest pathogenic potential, explaining why DEL1 and DEL4 but not DEL9 and DEL13 have been observed in patients with MODY8. These findings further support the view that CEL mutations cause pancreatic disease through protein misfolding and proteotoxicity, leading to endoplasmic reticulum stress and activation of the unfolded protein response.
Aims/hypothesis
Correctly diagnosing MODY is important, as individuals with this diagnosis can discontinue insulin injections; however, many people are misdiagnosed. We aimed to develop a robust ...approach for determining the pathogenicity of variants of uncertain significance in hepatocyte nuclear factor-1 alpha (HNF1A)
-
MODY and to obtain an accurate estimate of the prevalence of HNF1A-MODY in paediatric cases of diabetes.
Methods
We extended our previous screening of the Norwegian Childhood Diabetes Registry by 830 additional samples and comprehensively genotyped
HNF1A
variants in autoantibody-negative participants using next-generation sequencing. Carriers of pathogenic variants were treated by local healthcare providers, and participants with novel likely pathogenic variants and variants of uncertain significance were enrolled in an investigator-initiated, non-randomised, open-label pilot study (ClinicalTrials.gov registration no. NCT04239586). To identify variants associated with HNF1A-MODY, we functionally characterised their pathogenicity and assessed the carriers’ phenotype and treatment response to sulfonylurea.
Results
In total, 615 autoantibody-negative participants among 4712 cases of paediatric diabetes underwent genetic sequencing
,
revealing 19 with
HNF1A
variants. We identified nine carriers with novel variants classified as variants of uncertain significance or likely to be pathogenic, while the remaining ten participants carried five pathogenic variants previously reported. Of the nine carriers with novel variants, six responded favourably to sulfonylurea. Functional investigations revealed their variants to be dysfunctional and demonstrated a correlation with the resulting phenotype, providing evidence for reclassifying these variants as pathogenic.
Conclusions/interpretation
Based on this robust classification, we estimate that the prevalence of HNF1A-MODY is 0.3% in paediatric diabetes. Clinical phenotyping is challenging and functional investigations provide a strong complementary line of evidence. We demonstrate here that combining clinical phenotyping with functional protein studies provides a powerful tool to obtain a precise diagnosis of HNF1A-MODY.
Graphical Abstract
Maturity onset diabetes of the young, type 8 (MODY8) is associated with mutations in the CEL gene, which encodes the digestive enzyme carboxyl ester lipase. Several diabetes cases and families have ...in recent years been attributed to mutations in CEL without any functional or clinical evidence provided.
To facilitate correct MODY8 diagnostics, we screened 2 cohorts of diabetes patients and delineated the phenotype.
Young, lean Swedish and Finnish patients with a diagnosis of type 2 diabetes (352 cases, 406 controls) were screened for mutations in the CEL gene. We also screened 58 Czech MODY cases who had tested negative for common MODY genes. For CEL mutation-positive subjects, family history was recorded, and clinical investigations and pancreatic imaging performed.
Two cases (1 Swedish and 1 Czech) with germline mutation in CEL were identified. Clinical and radiological investigations of these 2 probands and their families revealed dominantly inherited insulin-dependent diabetes, pancreatic exocrine dysfunction, and atrophic pancreas with lipomatosis and cysts. Notably, hereditary pancreatitis was the predominant phenotype in 1 pedigree. Both families carried single-base pair deletions in the proximal part of the CEL variable number of tandem repeat (VNTR) region in exon 11. The mutations are predicted to lead to aberrant protein tails that make the CEL protein susceptible to aggregation.
The diagnosis of MODY8 requires a pancreatic exocrine phenotype and a deletion in the CEL VNTR in addition to dominantly inherited diabetes. CEL screening may be warranted also in families with hereditary pancreatitis of unknown genetic etiology.
The CEL gene encodes carboxyl ester lipase, a pancreatic digestive enzyme. CEL is extremely polymorphic due to a variable number tandem repeat (VNTR) located in the last exon. Single-base deletions ...within this VNTR cause the inherited disorder MODY8, whereas little is known about VNTR single-base insertions in pancreatic disease. We therefore mapped CEL insertion variants (CEL-INS) in 200 Norwegian patients with pancreatic neoplastic disorders. Twenty-eight samples (14.0%) carried CEL-INS alleles. Most common were insertions in repeat 9 (9.5%), which always associated with a VNTR length of 13 repeats. The combined INS allele frequency (0.078) was similar to that observed in a control material of 416 subjects (0.075). We performed functional testing in HEK293T cells of a set of CEL-INS variants, in which the insertion site varied from the first to the 12th VNTR repeat. Lipase activity showed little difference among the variants. However, CEL-INS variants with insertions occurring in the most proximal repeats led to protein aggregation and endoplasmic reticulum stress, which upregulated the unfolded protein response. Moreover, by using a CEL-INS-specific antibody, we observed patchy signals in pancreatic tissue from humans without any CEL-INS variant in the germline. Similar pancreatic staining was seen in knock-in mice expressing the most common human CEL VNTR with 16 repeats. CEL-INS proteins may therefore be constantly produced from somatic events in the normal pancreatic parenchyma. This observation along with the high population frequency of CEL-INS alleles strongly suggests that these variants are benign, with a possible exception for insertions in VNTR repeats 1-4.
Prostate cancer (PC) is a common cancer among men, and preventive strategies are warranted. Benzoxazinoids (BXs) in rye have shown potential against PC in vitro but human studies are lacking. The aim ...was to establish a quantitative method for analysis of BXs and investigate their plasma levels after a whole grain/bran rye vs refined wheat intervention, as well as exploring their association with PSA, in men with PC. A quantitative method for analysis of 22 BXs, including novel metabolites identified by mass spectrometry and NMR, was established, and applied to plasma samples from a randomized crossover study where patients with indolent PC (n = 17) consumed 485 g whole grain rye/rye bran or fiber supplemented refined wheat daily for 6 wk. Most BXs were significantly higher in plasma after rye (0.3-19.4 nmol/L in plasma) vs. refined wheat (0.05-2.9 nmol/L) intake. HBOA-glc, 2-HHPAA, HBOA-glcA, 2-HPAA-glcA were inversely correlated to PSA in plasma (p < 0.04). To conclude, BXs in plasma, including metabolites not previously analyzed, were quantified. BX metabolites were significantly higher after rye vs refined wheat consumption. Four BX-related metabolites were inversely associated with PSA, which merits further investigation.
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.
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