Intestinal failure and aberrant lipid metabolism in patients with DGAT1 deficiency.
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2018-07
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BACKGROUND & AIMS:Congenital diarrheal disorders arerare inherited intestinal disorders characterized by intractable,sometimes life-threatening, diarrhea and nutrient malab-sorption; some have been associated with mutations indiacylglycerol-acyltransferase 1(DGAT1), which catalyzesformation of triacylglycerol from diacylglycerol and acyl-CoA.We investigated the mechanisms by which DGAT1 deficiencycontributes to intestinal failure using patient-derived organo-ids.METHODS:We collected blood samples from 10 patients,from 6 unrelated pedigrees, who presented with early-onsetGastroenterology 2018;155:130–143BASIC ANDTRANSLATIONAL AT
Jorik M. van RijnRico Chandra ArdyZarife KulogluBettina HärterDésirée Y. van Haaften-VisserHubert P. J. van der DoefMarliek van HoeselAydan KansuAnke H. M. van VugtMarini ThianFreddy T. M. KokkeAna KroloMeryem Keçeli BasaranNeslihan Gurcan KayaAysel Ünlüsoy AksuBuket DalgıçFigen OzcayZeren BarisRenate KainEdwin C. A. StigterKlaske D. LichtenbeltMaarten P. G. MassinkKaren J. DuranJoke B. G. M VerheijDorien LugtenbergPeter G. J. NikkelsHenricus G. F. BrouwerHenkjan J. VerkadeRené ScheenstraBart SpeeEdward E. S. NieuwenhuisPaul J. CofferAndreas R. JaneckeGijs van HaaftenRoderick H. J. HouwenThomas MüllerSabine MiddendorpKaan Boztug
severe diarrhea and/or vomiting, hypoalbuminemia, and/or(fatal) protein-losing enteropathy with intestinal failure; weperformed next-generation sequencing analysis of DNA from 8patients. Organoids were generated from duodenal biopsiesfrom 3 patients and 3 healthy individuals (controls). Caco-2cells and patient-derived dermalfibroblasts were transfectedor transduced with vectors that express full-length or mutantforms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption ofDGAT1in control intestinal organo-ids. Cells and organoids were analyzed by immunoblot,immunofluorescence,flow cytometry, chromatography, quan-titative real-time polymerase chain reaction, and for the activityof caspases 3 and 7.RESULTS:In the 10 patients, we identified5 bi-allelic loss-of-function mutations inDGAT1. In patient-derivedfibroblasts and organoids, the mutations reducedexpression of DGAT1 protein and altered triacylglycerol meta-bolism, resulting in decreased lipid droplet formation afteroleic acid addition. Expression of full-length DGAT2 in patient-derivedfibroblasts restored formation of lipid droplets. Orga-noids derived from patients withDGAT1mutations were moresusceptible to lipid-induced cell death than control organoids.CONCLUSIONS:We identified a large cohort of patients withcongenital diarrheal disorders with mutations inDGAT1thatreduced expression of its product; dermalfibroblasts and in-testinal organoids derived from these patients had altered lipidmetabolism and were susceptible to lipid-induced cell death.Expression of full-length wildtype DGAT1 or DGAT2 restorednormal lipid metabolism in these cells. Thesefindings indicatethe importance of DGAT1 in fat metabolism and lipotoxicity inthe intestinal epithelium. A fat-free diet might serve as thefirstline of therapy for patients with reduced DGAT1 expression. Itis important to identify genetic variants associated withcongenital diarrheal disorders for proper diagnosis and selec-tion of treatment strategies.Keywords:CDD; Genomic; PLE; 3-D Culture Model.Congenital diarrheal disorders (CDDs) are a group ofrare inherited intestinal disorders that are charac-terized by intractable, sometimes life-threatening, diarrheaand nutrient malabsorption. CDDs can be classified based ontheir aberrations in absorption and transport of nutrientsand electrolytes, enterocyte differentiation and polarization,enteroendocrine cell differentiation, or dysregulation of theintestinal immune response.1Congenital protein-losing en-teropathy (PLE) is a type of CDD that is characterized byincreased protein loss from the gastrointestinal (GI) system.Patients with PLE often suffer from hypoproteinemia, fatmalabsorption, fat-soluble vitamin deficiencies, and malnu-trition. Recently, we have identified germline loss-of-function mutations inCD55as a major monogenic etiologyfor congenital PLE.2Previously, mutations in the gene encoding diacylglycerol-acyltransferase 1 (DGAT1) were found to underlie a syn-drome of diarrhea and congenital PLE.3–6DGAT1 and itsisozyme DGAT2 (encoding for diacylglycerol-acyltransferase2) are responsible for the conversion of diacylglycerol (DG)and fatty acyl-CoA to triacylglycerol (TG) in humans.7,8TGis the main energy substrate stored in human adipose tissue, isessential for milk production in the mammary gland, and ispartofthe verylow-density lipoprotein–mediated transport oflipids to peripheral tissue.9,10In the human small intestine,DGAT1istheonlyhighlyexpressedenzyme,whereasDGAT2ismainlyexpressed in the liver.3,11Inenterocytes, TGis stored inlipid droplets or packaged into chylomicrons before transportinto the lymphatic system.8,12,13The pathomechanism responsible for intestinal failureand PLE in DGAT1 deficiency has remained unclear.Through next-generation sequencing, we identified 10additional patients from 6 unrelated pedigrees with 5different, novel bi-allelic mutations inDGAT1leading tosevere, sometimes fatal course of PLE and fat intolerance.We took this unique opportunity to further shed light on thefundamental pathomechanisms of human DGAT1 deficiency.Materials and MethodsStudy ApprovalThe study was approved by the responsible local ethicscommittees (Ethics Commission of the Medical University ofWHAT YOU NEED TO KNOWBACKGROUND AND CONTEXTMutations inDGAT1have recently been identified inpatients with congenital diarrheal disorders (CDDs), butthe underlying molecular pathomechanisms haveremained largely elusive.NEW FINDINGSThe authors identified 10 patients with DGAT1 deficiencyrepresenting the largest cohort to date, linking gutepithelial lipid metabolism and lipotoxicity to CDD; andrescued aberrant lipid metabolism with isoenzyme DGAT2.LIMITATIONSAlthough the authors show exogenous DGAT1 or DGAT2expression or proteasome inhibitors may overcomedefects, future studies may need to address how thatknowledge can be translated to targeted therapies.IMPACTThe authors highlight the importance of identifying thegenetic defect in patients with CDD, and showcasefurther use of gut organoid technology to study rarediseases of the gastrointestinal tract.*Authors share co-first authorship;§Authors share co-senior authorship.Abbreviations used in this paper:B-LCL, B lymphoblastoid cell line; BSA,bovine serum albumin; CDD, congenital diarrheal disorder; cDNA, com-plementary DNA; DG, diacylglycerol; DGAT1, diacylglycerol-acyltransfer-ase 1; hSI-EM, human small intestine expansion medium; FFA, free fattyacid; GI, gastrointestinal; OA, oleic acid; PB, PiggyBac transposon; PBS,phosphate-buffered saline; PLE, protein-losing enteropathy; sgRNA, sin-gle-guide RNA; SSC, Side Scatter; TG, triacylglycerol; WT, wild-type.Most current article© 2018 by the AGA Institute. Published by Elsevier Inc. This is an openaccess article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).0016-5085https://doi.org/10.1053/j.gastro.2018.03.040July 2018DGAT1 Deficiency Impairs Lipid Metabolism 131BASIC ANDTRANSLATIONAL AT
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CDD; Genomic; PLE; 3-D Culture Model