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.

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.

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.

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.

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.

Fat Malabsorption and Ursodeoxycholic Acid Treatment in Children With Reduced Organic Solute Transporter-α (SLC51A) Expression.

Objectives: A bile acid homeostasis disorder was suspected in 2 siblings and their second cousin who presented in infancy with fat malabsorption, severe fat-soluble vitamin deficiency, rickets, and mild liver involvement. Our aims were to identify the genetic cause, describe the disease, and evaluate the response to ursodeoxycholic acid (UDCA) treatment. Methods: Whole exome sequencing, immunohistochemistry of duodenal biopsies and candidate variant testing in a cell-based model was performed. Fecal fat excretion, serum bile acids, 7α-hydroxy-4-cholesten-3-one (C4), and fibroblast growth factor 19 (FGF19) were quantified in both siblings on and off UDCA treatment. Results: A novel homozygous variant of SLC51A, which encodes the bile acid carrier organic solute transporter (OST)-α, was identified in all affected children. OSTα protein expression was readily detected by immunohistochemistry in duodenum of pediatric control subjects but not in the affected siblings. The siblings had low serum levels of bile acids and C4 and high serum levels of FGF19 consistent with repression of hepatic bile acid synthesis. On treatment with UDCA, fecal fat excretion was reduced and serum levels of C4, FGF19, and liver enzymes normalized. Conclusions: We report an apparent deficiency of OSTα associated with early onset fat malabsorption and mild liver involvement. The clinical presentation partially overlaps previous reports for 3 patients with OSTα or OSTß deficiency and extends the clinical spectrum associated with loss of SLC51A expression. Our data suggest that repression of hepatic bile acid synthesis contributes to fat malabsorption in OSTα-OSTß deficiency but can be partly reversed with UDCA treatment.

Novel hybridization- and tag-based error-corrected method for sensitive ctDNA mutation detection using ion semiconductor sequencing.

Circulating tumor DNA (ctDNA) analysis has emerged as a clinically useful tool for cancer diagnostics and treatment monitoring. However, ctDNA detection is complicated by low DNA concentrations and technical challenges. Here we describe our newly developed sensitive method for ctDNA detection on the Ion Torrent sequencing platform, which we call HYbridization- and Tag-based Error-Corrected sequencing (HYTEC-seq). This method combines hybridization-based capture with molecular tags, and the novel variant caller PlasmaMutationDetector2 to eliminate background errors. We describe the validation of HYTEC-seq using control samples with known mutations, demonstrating an analytical sensitivity down to 0.1% at > 99.99% specificity. Furthermore, to demonstrate the utility of this method in a clinical setting, we analyzed plasma samples from 44 patients with advanced pancreatic cancer, revealing mutations in 57% of the patients at allele frequencies as low as 0.23%.

C/EBPB-dependent adaptation to palmitic acid promotes tumor formation in hormone receptor negative breast cancer.

Epidemiological studies have established a positive association between obesity and the incidence of postmenopausal breast cancer. Moreover, it is known that obesity promotes stem cell-like properties of breast cancer cells. However, the cancer cell-autonomous mechanisms underlying this correlation are not well defined. Here we demonstrate that obesity-associated tumor formation is driven by cellular adaptation rather than expansion of pre-existing clones within the cancer cell population. While there is no correlation with specific mutations, cellular adaptation to obesity is governed by palmitic acid (PA) and leads to enhanced tumor formation capacity of breast cancer cells. This process is governed epigenetically through increased chromatin occupancy of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPB). Obesity-induced epigenetic activation of C/EBPB regulates cancer stem-like properties by modulating the expression of key downstream regulators including CLDN1 and LCN2. Collectively, our findings demonstrate that obesity drives cellular adaptation to PA drives tumor initiation in the obese setting through activation of a C/EBPB dependent transcriptional network.

Abnormal exocrine-endocrine cell cross-talk promotes ß-cell dysfunction and loss in MODY8.

MODY8 (maturity-onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes associated with mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar cells. MODY8 patients develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes. In the present study, we report the transfer of CEL proteins from acinar cells to ß-cells as a form of cross-talk between exocrine and endocrine cells. Human ß-cells show a relatively higher propensity for internalizing the mutant versus the wild-type CEL protein. After internalization, the mutant protein forms stable intracellular aggregates leading to ß-cell secretory dysfunction. Analysis of pancreas sections from a MODY8 patient reveals the presence of CEL protein in the few extant ß-cells. The present study provides compelling evidence for the mechanism by which a mutant gene expressed specifically in acinar cells promotes dysfunction and loss of ß-cells to cause diabetes.

Two New Mutations in the CEL Gene Causing Diabetes and Hereditary Pancreatitis: How to Correctly Identify MODY8 Cases.

CONTEXT: 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. OBJECTIVE: To facilitate correct MODY8 diagnostics, we screened 2 cohorts of diabetes patients and delineated the phenotype. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

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.

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.

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.

Protein misfolding in combination with other risk factors in CEL-HYB1-mediated chronic pancreatitis.

OBJECTIVES: The hybrid allele of the carboxyl ester lipase gene (CEL-HYB1) is a genetic risk factor for chronic pancreatitis (CP) although the mechanism promoting disease development is largely unknown. Here, we aimed to clinically describe subjects carrying the CEL-HYB1 allele and to elucidate why the protein product is pathogenic by analyzing pancreatic secretions and cellular models. METHODS: Norwegian cases (n = 154) diagnosed with recurrent acute pancreatitis or CP were subjected to genetic screening by a CEL-HYB1-specific PCR assay followed by Sanger sequencing. For investigation of CEL-HYB1 protein secretion, duodenal juice samples from cases and controls were analyzed by western blotting. HEK293cells were transfected with constructs expressing CEL-HYB1 or the normal CEL protein (CEL-WT) and analyzed by qPCR, cell fractionation and western blotting. RESULTS: Two CEL-HYB1-positive families were identified. In both pedigrees, CEL-HYB1 did not fully co-segregate with disease. One proband had recurrent acute pancreatitis and was an active smoker. Her mother was a CEL-HYB1 carrier who had suffered from several attacks of acute pancreatitis until she stopped smoking. The other proband was diagnosed with CP and pancreas divisum. Her CEL-HYB1-positive parent was symptom-free but exhibited pancreatic imaging changes. When analyzing the CEL protein in duodenal juice, CEL-WT was readily detectable but no band corresponding to the risk variant was seen. In CEL-HYB1-transfected cells, we observed impaired protein secretion, protein aggregation and endoplasmic reticulum stress. CONCLUSION: Our data suggest that CEL-HYB1, in combination with well-known pancreatitis risk factors, causes disease through the misfolding-dependent pathway of genetic CP risk.

Association of HERV-K and LINE-1 hypomethylation with reduced disease-free survival in melanoma patients.

Aim: To evaluate CpG methylation of long interspersed nuclear elements 1 (LINE-1) and human endogenous retrovirus K (HERV-K) retroelements as potential prognostic biomarkers in cutaneous melanoma. Materials & methods: Methylation of HERV-K and LINE-1 retroelements was assessed in resected melanoma tissues from 82 patients ranging in age from 14 to 88 years. In addition, nevi from eight patients were included for comparison with nonmalignant melanocytic lesions. Results: Methylation levels were lower in melanomas than in nevi. HERV-K and LINE-1 methylation were decreased in melanoma patients with clinical parameters associated with adverse prognosis, while they were independent of age and gender. Hypomethylation of HERV-K (but not LINE-1) was an independent predictor of reduced disease-free survival. Conclusion: HERV-K hypomethylation can be a potential independent biomarker of melanoma recurrence.

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.

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.

Golgi-Localized PAQR4 Mediates Antiapoptotic Ceramidase Activity in Breast Cancer.

The metabolic network of sphingolipids plays important roles in cancer biology. Prominent sphingolipids include ceramides and sphingosine-1-phosphate that regulate multiple aspects of growth, apoptosis, and cellular signaling. Although a significant number of enzymatic regulators of the sphingolipid pathway have been described in detail, many remained poorly characterized. Here we applied a patient-derived systemic approach to identify and molecularly define progestin and adipoQ receptor family member IV (PAQR4) as a Golgi-localized ceramidase. PAQR4 was approximately 5-fold upregulated in breast cancer compared with matched control tissue and its overexpression correlated with disease-specific survival rates in breast cancer. Induction of PAQR4 in breast tumors was found to be subtype-independent and correlated with increased ceramidase activity. These findings establish PAQR4 as Golgi-localized ceramidase required for cellular growth in breast cancer. SIGNIFICANCE: Induction of and cellular dependency on de novo sphingolipid synthesis via PAQR4 highlights a central vulnerability in breast cancer that may serve as a viable therapeutic target.

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.