Mutations Disrupting the Kennedy Phosphatidylcholine Pathway in Humans with Congenital Lipodystrophy and Fatty Liver Disease
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Mutations disrupting the Kennedy phosphatidylcholine pathway in humans with congenital lipodystrophy and fatty liver disease Felicity Paynea,1, Koini Limb,1, Amandine Girousseb,1, Rebecca J. Brownc, Nora Koryd, Ann Robbinsb, Yali Xuea, Alison Sleighe, Elaine Cochranc, Claire Adamsb, Arundhati Dev Bormanf, David Russel-Jonesg, Phillip Gordenc, Robert K. Sempleb, Vladimir Saudekb, Stephen O’Rahillyb,2, Tobias C. Waltherd, Inês Barrosoa,b,2,3, and David B. Savageb,2,3 aMetabolic Disease Group, The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Cambridge CB10 1SA, United Kingdom; bUniversity of Cambridge, Metabolic Research Laboratories, Wellcome Trust–Medical Research Council Institute of Metabolic Science, Cambridge CB2 0QQ, United Kingdom; cDiabetes Endocrinology and Obesity Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892; dDepartment of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520; eWolfson Brain Imaging Centre, University of Cambridge, Cambridge CB2 0QQ, United Kingdom; fMoorfields Eye Hospital National Health Service Trust, London EC1V 2PD, United Kingdom; and gCentre for Endocrinology, Diabetes and Research, Royal Surrey County Hospital, Guildford GU2 7XX, United Kingdom Contributed by Stephen O’Rahilly, May 12, 2014 (sent for review March 14, 2014) Phosphatidylcholine (PC) is the major glycerophospholipid in eukary- which the dominant feature was very low HDL cholesterol levels, otic cells and is an essential component in all cellular membranes. The insulin-resistant diabetes, and short stature (Table 1) (4, 5). biochemistry of de novo PC synthesis by the Kennedy pathway is Proband 1 (P1) was the only affected child of unrelated un- well established, but less is known about the physiological functions affected parents and had four unaffected sisters (Fig. 1B). She of PC. We identified two unrelated patients with defects in the initially presented, aged 18 y, with type 2 diabetes. Her body 2 Kennedy pathway due to biallellic loss-of-function mutations in mass index (BMI) was 17.9 kg/m , and her total fat mass (6.5%) phosphate cytidylyltransferase 1 alpha (PCYT1A), the rate-limiting was very low in keeping with lipodystrophy (Fig. S1A). However, enzyme in this pathway. The mutations lead to a marked reduction in contrast to most patients with congenital lipodystrophy, her height was significantly less than predicted (height SD score, in PCYT1A expression and PC synthesis. The phenotypic consequen- − ces include some features, such as severe fatty liver and low HDL 2.8), and her lean mass was not increased. At presentation, she cholesterol levels, that are predicted by the results of previously was extremely insulin resistant (requiring very high-dose insulin reported liver-specific deletion of murine Pcyt1a. Both patients also therapy to control her diabetes) hypertriglyceridemic, and had had lipodystrophy, severe insulin resistance, and diabetes, providing severe hepatic steatosis (Table 1). Her HDL cholesterol level was particularly low and remained low even after normalization evidence for an additional and essential role for PCYT1A-generated of her triglyceride levels. We used stable isotopes to measure her PC in the normal function of white adipose tissue and insulin action. rate of hepatic de novo lipogenesis and found this to be signifi- cantly elevated (Table 1). ll living cells are surrounded by a lipid membrane. Eukaryotic Proband 2 (P2) was also the only affected member of a smaller Acells also contain several internal membrane-bound organ- family with one unaffected brother (Fig. 1B). She presented at elles, which enable them to compartmentalize related biological age 3 mo with pneumonia and was noted to have abnormal liver functions and thereby to enhance the efficiency of these processes. enzyme levels. A liver biopsy revealed hepatic steatosis. Her Phospholipids are the predominant component of these mem- height was below the third centile for age throughout childhood, branes. Their hydrophilic head groups interact with the cytosol, and near-adult height was 148 cm (at age 16.8 y, bone age, 15.5 y, whereas their hydrophobic side chains are either buried within the suggesting growth 99.6% complete). At age 9 y, progressive limb hydrophobic interior of a typical membrane bilayer or interact fat loss developed. Subsequent evaluation at the National Institutes B with the hydrophobic neutral lipid core of lipoproteins and lipid of Health revealed lipodystrophy (Fig. S1 ), hypertriglyceridemia, droplets (LDs). Phospholipids are generally defined by their or- ganic head group with phosphatidylcholine (PC) constituting over Significance 50% of all membrane phospholipids. PC was first isolated in the 19th century and the major enzymatic pathway involved in its The characterization of rare monogenic human disorders can synthesis was revealed by Kennedy and Weiss (1) in the 1950s. Cells and has yielded unique biological insights. Our phenotypic synthesize PC in three consecutive steps (Fig. 1A): choline kinase description and functional characterization of human loss-of- PCYT1A phosphorylates choline before choline phosphate cytidylyltransferase function mutations in is both clinically important for 1 α (encoded by the PCYT1A gene) generates the high-energy patients and their families afflicted with this rare but serious donor CDP-choline in the rate-limiting step of the pathway. In the metabolic disease and biologically helpful in advancing un- derstanding of the physiological consequences of impaired last step, DAG:CDP-choline cholinephosphotransferase (CPT) PCYT1A activity in humans. uses CDP-choline and diacylglycerol (DAG) to form PC (2, 3). Membrane phospholipids are a defining feature of advanced Author contributions: R.K.S., S.O., I.B., and D.B.S. designed research; F.P., K.L., A.G., R.J.B., life-forms so it is perhaps not surprising that the pathways in- N.K., A.R., Y.X., A.S., E.C., C.A., A.D.B., D.R.-J., P.G., R.K.S., V.S., I.B., and D.B.S. performed volved in their synthesis are ancient, and mutations affecting research; F.P., K.L., A.G., R.J.B., N.K., A.R., Y.X., S.O., T.C.W., I.B., and D.B.S. analyzed data; MEDICAL SCIENCES them are rarely tolerated in evolution. Here, we describe the and F.P., K.L., A.G., R.J.B., N.K., S.O., T.C.W., I.B., and D.B.S. wrote the paper. identification and characterization of pathogenic human loss-of- The authors declare no conflict of interest. function mutations affecting the eponymous Kennedy pathway. 1F.P., K.L., and A.G. contributed equally to this work. 2To whom correspondence may be addressed. E-mail: [email protected], ib1@ Case Histories sanger.ac.uk, or [email protected]. We studied two female patients with similar metabolic syn- 3I.B. and D.B.S. contributed equally to this work. dromes characterized by childhood presentation with severe This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. nonalcoholic fatty liver disease, lipodystrophy, dyslipidemia in 1073/pnas.1408523111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1408523111 PNAS | June 17, 2014 | vol. 111 | no. 24 | 8901–8906 Downloaded by guest on September 27, 2021 A N+ OH B Choline WT/p.Glu280del WT/p.Val142Met ATP Ht 1.85m Ht 1.63m CK BMI 22.8 BMI 29.1 ADP O- O N+ P OH Proband 1 O Fig. 1. Cosegregation of biallelic PCYT1A mutations Phosphocholine ND ND WT/p.Glu280del WT/p.Val142Met p.Val142Met/ p.Glu280del with fatty liver, low HDL cholesterol levels, lipodys- CTP Ht 1.68m Ht 1.63m PCYT1A BMI 29.4 BMI 18.8 Ht 1.46m trophy, insulin-resistant diabetes, and short stature. Pi BMI 17.9 (A) Schematic illustration of the Kennedy PC synthe- O sis pathway. CK, choline kinase; CPT, CDP-choline:1,2- N+ CDP WT/p.Glu280del WT/p.Ser333Leufs*164 Fatty liver diacylglycerol cholinephosphotransferase; PCYT1A, CDP-choline Ht 1.66m Ht 1.65m Low-HDL-cholesterol BMI 34.8 BMI 31.5 choline-phosphate cytidylyltransferase A, CTP:phos- DG CPT lipodystrophy phocholine-cytidylyltransferase. (B) Family pedigrees of CMP both probands demonstrating that only compound Proband 2 Insulin resistance/type 2 diabetes O- heterozygous carriers of PCYT1A mutations manifest O O N+ P O O WT/p.Glu280del p.Glu280del/ fatty liver (red), low HDL cholesterol (blue), lipodys- O O Ht 1.66m p.Ser333Leufs*164 O BMI 23.0 Ht 1.23m trophy (yellow), and insulin resistance/type 2 diabetes BMI 17.9 (T2DM) (green). PCYT1A mutation status, height (Ht.), Phosphatidylcholine and body mass index (BMI) are indicated below each individual’s symbol. ND, not determined; WT, wild type. C Nuclear localisation (C) The location of PCYT1A mutations E280del, V142M, signal Catalytic domain Domain M Domain P and 333fs in relation to known functional domains Wildtype of PCYT1A. Domain M, membrane binding domain; 1 73 236 298 315 367 Mutation 1 domain P, phosphorylated region. (D) Conservation p.Glu280del 1 73 236 297 314 366 around the V142(red*) and E280(red*) mutation sites. Mutation 2 Sequence alignment of representative metazoan se- p.Val142Met 1 73 236 298 315 367 quences in the region surrounding the mutated resi- RYVD->RYMD Mutation 3 dues. Hydrophobic (blue) and polar (green) residues p.Ser333Leufs*164 1 73 236 298 315 497 interacting with V142 are highlighted. Only residues frameshift mutated carboxyl terminal sequence different from the human sequence are shown. Se- mutation site quence IDs: human (Homo sapiens) P49585, zebrafish D * * (Danio rerio) F1QEN6, sea squirt (Ciona intestinalis) XP_002130773.1, sea urchin (Strongylocentrotus pur- puratus) H3I3V9, water flee (Daphnia pulex) E9G1P5, Drosophila (D. melanogaster) Q9W0D9, Caenorhabditis (C. elegans) P49583, Trichoplax (T. adherens) B3RI62. (E and F) Structure of the catalytic domain of PCYT1A E F highlighting the role of V142M in the core packing. The Monomer a two chains in the dimer are shown in yellow and gray; the residues and the secondary structure units are highlighted in color in the yellow monomer A: loop L3 α V136 with V142, red; -helix, green; and the interacting V110 β-sheet, blue.