Common single-base insertions in the VNTR of the carboxyl ester lipase (CEL) gene are benign and also likely to arise somatically in the exocrine pancreas.

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.

Deficiency of the metabolic enzyme SCHAD in pancreatic ß-cells promotes amino acid-sensitive hypoglycemia.

Congenital hyperinsulinism of infancy (CHI) can be caused by a deficiency of the ubiquitously expressed enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD). To test the hypothesis that SCHAD-CHI arises from a specific defect in pancreatic ß-cells, we created genetically engineered ß-cell-specific (ß-SKO) or hepatocyte-specific (L-SKO) SCHAD knockout mice. While L-SKO mice were normoglycemic, plasma glucose in ß-SKO animals was significantly reduced in the random-fed state, after overnight fasting, and following refeeding. The hypoglycemic phenotype was exacerbated when the mice were fed a diet enriched in leucine, glutamine, and alanine. Intraperitoneal injection of these three amino acids led to a rapid elevation in insulin levels in ß-SKO mice compared to controls. Consistently, treating isolated ß-SKO islets with the amino acid mixture potently enhanced insulin secretion compared to controls in a low-glucose environment. RNA sequencing of ß-SKO islets revealed reduced transcription of ß-cell identity genes and upregulation of genes involved in oxidative phosphorylation, protein metabolism, and Ca2+ handling. The ß-SKO mouse offers a useful model to interrogate the intra-islet heterogeneity of amino acid sensing given the very variable expression levels of SCHAD within different hormonal cells, with high levels in ß- and δ-cells and virtually absent α-cell expression. We conclude that the lack of SCHAD protein in ß-cells results in a hypoglycemic phenotype characterized by increased sensitivity to amino acid-stimulated insulin secretion and loss of ß-cell identity.

Characterisation of HNF1A variants in paediatric diabetes in Norway using functional and clinical investigations to unmask phenotype and monogenic diabetes.

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.

Unsupervised Clustering of Missense Variants in HNF1A Using Multidimensional Functional Data Aids Clinical Interpretation.

Exome sequencing in diabetes presents a diagnostic challenge because depending on frequency, functional impact, and genomic and environmental contexts, HNF1A variants can cause maturity-onset diabetes of the young (MODY), increase type 2 diabetes risk, or be benign. A correct diagnosis matters as it informs on treatment, progression, and family risk. We describe a multi-dimensional functional dataset of 73 HNF1A missense variants identified in exomes of 12,940 individuals. Our aim was to develop an analytical framework for stratifying variants along the HNF1A phenotypic continuum to facilitate diagnostic interpretation. HNF1A variant function was determined by four different molecular assays. Structure of the multi-dimensional dataset was explored using principal component analysis, k-means, and hierarchical clustering. Weights for tissue-specific isoform expression and functional domain were integrated. Functionally annotated variant subgroups were used to re-evaluate genetic diagnoses in national MODY diagnostic registries. HNF1A variants demonstrated a range of behaviors across the assays. The structure of the multi-parametric data was shaped primarily by transactivation. Using unsupervised learning methods, we obtained high-resolution functional clusters of the variants that separated known causal MODY variants from benign and type 2 diabetes risk variants and led to reclassification of 4% and 9% of HNF1A variants identified in the UK and Norway MODY diagnostic registries, respectively. Our proof-of-principle analyses facilitated informative stratification of HNF1A variants along the continuum, allowing improved evaluation of clinical significance, management, and precision medicine in diabetes clinics. Transcriptional activity appears a superior readout supporting pursuit of transactivation-centric experimental designs for high-throughput functional screens.

Congenital hyperinsulinism. / Medfødt hyperinsulinisme.

This clinical review will give doctors who work with children and neonates an introduction to the diagnosis and treatment of congenital hyperinsulinism, the most common cause of persistent neonatal hypoglycaemia. The condition is a rare monogenic disorder characterised by elevated insulin secretion and is a result of mutations in genes that regulate insulin secretion from pancreatic beta cells. The anabolic effect of insulin induces systemic glucose uptake and inhibits gluconeogenesis, glycogenolysis, ketogenesis and lipolysis. Low levels of glucose and ketone bodies in the blood are harmful to the central nervous system and can lead to brain damage or death. Early diagnosis and treatment of congenital hyperinsulinism are therefore crucial for a good prognosis.

The position of single-base deletions in the VNTR sequence of the carboxyl ester lipase (CEL) gene determines proteotoxicity.

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.

The genetic risk factor CEL-HYB1 causes proteotoxicity and chronic pancreatitis in mice.

BACKGROUND & AIMS: The CEL gene encodes the digestive enzyme carboxyl ester lipase. CEL-HYB1, a hybrid allele of CEL and its adjacent pseudogene CELP, is a genetic variant suggested to increase the risk of chronic pancreatitis (CP). Our aim was to develop a mouse model for CEL-HYB1 that enables studies of pancreatic disease mechanisms. METHODS: We established a knock-in mouse strain where the variable number of tandem repeat (VNTR) region of the endogenous mouse Cel gene was substituted with the mutated VNTR of the human CEL-HYB1 allele. Heterozygous and homozygous Cel-HYB1 mice and littermate wildtype controls were characterized with respect to pancreatic pathology and function. RESULTS: We successfully constructed a mouse model with pancreatic expression of a humanized CEL-HYB1 protein. The Cel-HYB1 mice spontaneously developed features of CP including inflammation, acinar atrophy and fatty replacement, and the phenotype became more pronounced as the animals aged. Moreover, Cel-HYB1 mice were normoglycemic at age 6 months, whereas at 12 months they exhibited impaired glucose tolerance. Immunostaining of pancreatic tissue indicated the formation of CEL protein aggregates, and electron microscopy showed dilated endoplasmic reticulum. Upregulation of the stress marker BiP/GRP78 was seen in pancreatic parenchyma obtained both from Cel-HYB1 animals and from a human CEL-HYB1 carrier. CONCLUSIONS: We have developed a new mouse model for CP that confirms the pathogenicity of the human CEL-HYB1 variant. Our findings place CEL-HYB1 in the group of genes that increase CP risk through protein misfolding-dependent pathways.

KRAS mutation analysis by droplet digital PCR of duodenal juice from patients with MODY8 and other pancreatic diseases.

BACKGROUND: Maturity-onset diabetes of the young type 8 (MODY8 or CEL-MODY) is an inherited pancreatic disease characterized by chronic inflammation of the pancreas and diabetes. It is not known whether MODY8 patients have increased risk for developing pancreatic cancer. We investigated KRAS mutation load in duodenal juice from MODY8 patients, comparing with other groups of pancreatic disease. METHODS: Droplet digital PCR (ddPCR) was used to detect KRAS codon 12/13/61 mutations in duodenal juice sampled from 11 MODY8 patients, nine healthy subjects and 100 patients clinically investigated due to suspected pancreatic disease. RESULTS: KRAS mutations were detected in 4/11 patients with MODY8 (36%), 1/9 healthy subjects (11%), 15/44 patients with chronic pancreatitis (CP, 34%), 3/5 patients with pancreatic ductal adenocarcinoma (PDAC, 60%), 3/20 patients with acute pancreatitis (15%), 0/13 patients with other pancreatic disorders and 2/18 patients with nonpancreatic gastrointestinal disease (11%). Of the 28 positive juice samples, 25 (89%) had low-abundance mutations in codons 12/13, with a variant allele frequency (VAF) less than 1%. KRAS-positive patients with MODY8 or CP had significantly lower VAFs than patients with PDAC (Mann-Whitney U test; p = 0.041). Although the overall mutation detection rate was higher for subjects ≥50 years old (26%) than for younger subjects (15%), the difference was not statistically significant. CONCLUSIONS: KRAS mutations were detectable in duodenal juice from MODY8 patients, but with low abundance and at the same frequency as in CP patients. The discriminative value of the analysis with regard to other pancreatic disease was limited.

Functional evaluation of 16 SCHAD missense variants: Only amino acid substitutions causing congenital hyperinsulinism of infancy lead to loss-of-function phenotypes in vitro.

Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), encoded by the HADH gene, is a ubiquitously expressed mitochondrial enzyme involved in fatty acid oxidation. This protein also plays a role in insulin secretion as recessive HADH mutations cause congenital hyperinsulinism of infancy (CHI) via loss of an inhibitory interaction with glutamate dehydrogenase (GDH). Here, we present a functional evaluation of 16 SCHAD missense variants identified either in CHI patients or by high-throughput sequencing projects in various populations. To avoid interactions with endogenously produced SCHAD protein, we assessed protein stability, subcellular localization, and GDH interaction in a SCHAD knockout HEK293 cell line constructed by CRISPR-Cas9 methodology. We also established methods for efficient SCHAD expression and purification in E. coli, and tested enzymatic activity of the variants. Our analyses showed that rare variants of unknown significance identified in populations generally had similar properties as normal SCHAD. However, the CHI-associated variants p.Gly34Arg, p.Ile184Phe, p.Pro258Leu, and p.Gly303Ser were unstable with low protein levels detectable when expressed in HEK293 cells. Moreover, CHI variants p.Lys136Glu, p.His170Arg, and p.Met188Val presented normal protein levels but displayed clearly impaired enzymatic activity in vitro, and their interaction with GDH appeared reduced. Our results suggest that pathogenic missense variants of SCHAD either make the protein target of a post-translational quality control system or can impair the function of SCHAD without influencing its steady-state protein level. We did not find any evidence that rare SCHAD missense variants observed only in the general population and not in CHI patients are functionally affected.

Single nucleotide polymorphisms in CEL-HYB1 increase risk for chronic pancreatitis through proteotoxic misfolding.

Genetic variants contribute to the risk of chronic pancreatitis (CP) in adults and children. The risk variant CEL-HYB1, a recombinant hybrid allele of CEL and its neighboring pseudogene (CELP), encodes a pathogenic variant of the pancreatic digestive enzyme carboxyl ester lipase (CEL). We previously identified combinations of two non-synonymous SNPs, c.1463T>C (p. Ile488Thr) and c.1643C>T (p. Thr548Ile), in the break point region of CEL-HYB1. Herein, we tested whether these missense variants alter CP risk and their impact on functional properties of the CEL-HYB1 protein. Examination of CEL-HYB1 haplotypes in European patients and controls revealed that the combinationThr488-Ile548 was present only in cases (p ≤ .001). The lipase activity of purified recombinant CEL-HYB1 variants showed normal or near normal activity. CEL-HYB variants expressed in HEK293T cells all had decreased secretion compared with CEL, formed intracellular protein aggregates, and triggered endoplasmic reticulum stress. Thus, we propose that the presence of missense variants in CEL-HYB increases the pathogenicity of CEL-HYB1 through misfolding and gain-of-function proteotoxicity. Interestingly, Thr488-Ile548 and Thr488-Thr548 were equally pathogenic in the functional assays even though only the Thr488-Ile548 haplotype was significantly enriched in cases. The explanation for the mismatch between genetic and functional data requires further investigation.

Characterization of CEL-DUP2: Complete duplication of the carboxyl ester lipase gene is unlikely to influence risk of chronic pancreatitis.

BACKGROUND/OBJECTIVES: Carboxyl ester lipase is a pancreatic enzyme encoded by CEL, an extremely polymorphic human gene. Pathogenic variants of CEL either increases the risk for chronic pancreatitis (CP) or cause MODY8, a syndrome of pancreatic exocrine and endocrine dysfunction. Here, we aimed to characterize a novel duplication allele of CEL (CEL-DUP2) and to investigate whether it associates with CP or pancreatic cancer. METHODS: The structure of CEL-DUP2 was determined by a combination of Sanger sequencing, DNA fragment analysis, multiplex ligation-dependent probe amplification and whole-genome sequencing. We developed assays for screening of CEL-DUP2 and analyzed cohorts of idiopathic CP, alcoholic CP and pancreatic cancer. CEL protein expression was analyzed by immunohistochemistry. RESULTS: CEL-DUP2 consists of an extra copy of the complete CEL gene. The allele has probably arisen from non-allelic, homologous recombination involving the adjacent pseudogene of CEL. We found no association between CEL-DUP2 carrier frequency and CP in cohorts from France (cases/controls: 2.5%/2.4%; P = 1.0), China (10.3%/8.1%; P = 0.08) or Germany (1.6%/2.3%; P = 0.62). Similarly, no association with disease was observed in alcohol-induced pancreatitis (Germany: 3.2%/2.3%; P = 0.51) or pancreatic cancer (Norway; 2.5%/3.2%; P = 0.77). Notably, the carrier frequency of CEL-DUP2 was more than three-fold higher in Chinese compared with Europeans. CEL protein expression was similar in tissues from CEL-DUP2 carriers and controls. CONCLUSIONS: Our results support the contention that the number of CEL alleles does not influence the risk of pancreatic exocrine disease. Rather, the pathogenic CEL variants identified so far involve exon 11 sequence changes that substantially alter the protein's tail region.

The mucinous domain of pancreatic carboxyl-ester lipase (CEL) contains core 1/core 2 O-glycans that can be modified by ABO blood group determinants.

Carboxyl-ester lipase (CEL) is a pancreatic fat-digesting enzyme associated with human disease. Rare mutations in the CEL gene cause a syndrome of pancreatic exocrine and endocrine dysfunction denoted MODY8, whereas a recombined CEL allele increases the risk for chronic pancreatitis. Moreover, CEL has been linked to pancreatic ductal adenocarcinoma (PDAC) through a postulated oncofetal CEL variant termed feto-acinar pancreatic protein (FAPP). The monoclonal antibody mAb16D10 was previously reported to detect a glycotope in the highly O-glycosylated, mucin-like C terminus of CEL/FAPP. We here assessed the expression of human CEL in malignant pancreatic lesions and cell lines. CEL was not detectably expressed in neoplastic cells, implying that FAPP is unlikely to be a glycoisoform of CEL in pancreatic cancer. Testing of the mAb16D10 antibody in glycan microarrays then demonstrated that it recognized structures containing terminal GalNAc-α1,3(Fuc-α1,2)Gal (blood group A antigen) and also repeated protein sequences containing GalNAc residues linked to Ser/Thr (Tn antigen), findings that were supported by immunostainings of human pancreatic tissue. To examine whether the CEL glycoprotein might be modified by blood group antigens, we used high-sensitivity MALDI-TOF MS to characterize the released O-glycan pool of CEL immunoprecipitated from human pancreatic juice. We found that the O-glycome of CEL consisted mainly of core 1/core 2 structures with a composition depending on the subject's FUT2 and ABO gene polymorphisms. Thus, among digestive enzymes secreted by the pancreas, CEL is a glycoprotein with some unique characteristics, supporting the view that it could serve additional biological functions to its cholesteryl esterase activity in the duodenum.

Mutation analysis by deep sequencing of pancreatic juice from patients with pancreatic ductal adenocarcinoma.

BACKGROUND: Reliable methods are needed to identify patients with early-stage cancer or high-grade precancerous lesions in the pancreas. Analysis of pancreatic juice to detect somatic mutations could represent one such approach. Here we investigated the concordance between mutations found in the primary tumor and pancreatic juice from the same patient. METHODS: Amplicon-based targeted deep sequencing was performed on samples from 21 patients with pancreatic ductal adenocarcinoma (PDAC) who had undergone Whipple's operation. Mutation profiles were determined in formalin-fixed sections of the primary tumor and in pancreatic juice sampled from the main pancreatic duct during surgery. RESULTS: Using a cut-off of 3% for variant allele frequency, KRAS mutations were detected in 20/21 primary tumors (95%) and in 15/21 (71%) juice samples. When also considering low-frequency variants, KRAS mutations were found in 20/21 juice samples. Most juice samples exhibited multiple KRAS variants not seen in the primary tumor, and only in 11 cases (52%) did the most abundant variant of the juice correspond to the KRAS mutation detected in the tumor. TP53 mutations were found in 16 tumors (76%) and six juice samples (29%). Among the positive juice samples, only one exhibited more than a single TP53 mutation. Detection of both KRAS and TP53 mutations was fully concordant in the primary tumor and juice sample in 7/21 cases (33%). CONCLUSIONS: Pancreatic juice from PDAC patients is rich in KRAS mutations often not seen in the primary tumor and possibly reflecting precancerous lesions in other regions of the pancreas. The inclusion of TP53 mutation detection and additional markers must therefore be considered for fully exploiting the clinical potential of pancreatic juice samples in early cancer detection.

The hybrid allele 1 of carboxyl-ester lipase (CEL-HYB1) in Polish pediatric patients with chronic pancreatitis.

OBJECTIVES: It has previously been reported in a European case-control study with patients from Germany and France that CEL-HYB1, a hybrid allele of the carboxyl ester lipase (CEL) gene and its pseudogene CELP, increases susceptibility to chronic pancreatitis (CP). Here, we aimed to replicate this finding in Polish pediatric patients with CP. METHOD: The distribution of the CEL-HYB1 allele in a CP pediatric cohort (n = 147, median age at CP onset 7.6 years) with no history of alcohol/smoking abuse was compared with ethnically matched healthy controls (n = 500, median age 46 years). Screening was performed using long-range PCR followed by agarose gel-electrophoresis. RESULTS: We observed no significant difference in the carrier frequency of the CEL-HYB1 allele between CP patients (7/147, 4.8%) and controls (12/500, 2.4%; P = 0.16). CONCLUSIONS: This study found no statistically significant association between CEL-HYB1 and chronic pancreatitis in a cohort of Polish pediatric CP patients.

The role of the carboxyl ester lipase (CEL) gene in pancreatic disease.

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.

Targeted next-generation sequencing reveals MODY in up to 6.5% of antibody-negative diabetes cases listed in the Norwegian Childhood Diabetes Registry.

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.

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.

Branched Fatty Acid Esters of Hydroxy Fatty Acids Are Preferred Substrates of the MODY8 Protein Carboxyl Ester Lipase.

A recently discovered class of endogenous mammalian lipids, branched fatty acid esters of hydroxy fatty acids (FAHFAs), possesses anti-diabetic and anti-inflammatory activities. Here, we identified and validated carboxyl ester lipase (CEL), a pancreatic enzyme hydrolyzing cholesteryl esters and other dietary lipids, as a FAHFA hydrolase. Variants of CEL have been linked to maturity-onset diabetes of the young, type 8 (MODY8), and to chronic pancreatitis. We tested the FAHFA hydrolysis activity of the CEL MODY8 variant and found a modest increase in activity as compared with that of the normal enzyme. Together, the data suggest that CEL might break down dietary FAHFAs.

SHORT syndrome with partial lipodystrophy due to impaired phosphatidylinositol 3 kinase signaling.

The phosphatidylinositol 3 kinase (PI3K) pathway regulates fundamental cellular processes such as metabolism, proliferation, and survival. A central component in this pathway is the p85α regulatory subunit, encoded by PIK3R1. Using whole-exome sequencing, we identified a heterozygous PIK3R1 mutation (c.1945C>T [p.Arg649Trp]) in two unrelated families affected by partial lipodystrophy, low body mass index, short stature, progeroid face, and Rieger anomaly (SHORT syndrome). This mutation led to impaired interaction between p85α and IRS-1 and reduced AKT-mediated insulin signaling in fibroblasts from affected subjects and in reconstituted Pik3r1-knockout preadipocytes. Normal PI3K activity is critical for adipose differentiation and insulin signaling; the mutated PIK3R1 therefore provides a unique link among lipodystrophy, growth, and insulin signaling.