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Peroxisome Proliferator-Activated Receptor- C190S Mutation Causes Partial Lipodystrophy

Departments of Medicine and Anatomy and Cell Biology (A.L., W.W., A.M., H.J.W.), College of Physicians and Surgeons, Columbia University, New York, New York 10032; Medizinische Klinik mit Schwerpunkt Gastroenterologie, Hepatologie, and Endokrinologie (A.L., J.B., H.H.-J.S.), Charité Universitätsmedizin Berlin, Campus Mitte, 10117 Berlin, Germany; Diabetesnetz Kraichgau Nord (A.S.), 69168 Wiesloch, Germany; Département d’Ingénierie et d’Etude des Protéines (S.Z.-J.), Commissariat à l’Energie Atomique Saclay, 91191 Gif-sur-Yvette Cedex, France; Abteilung für Neurologie (S.S.), Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany; and Transplantationshepatologie (H.H.-J.S.), Universitätsklinikum Münster, 48149 Münster, Germany

Address all correspondence and requests for reprints to: Howard J. Worman, M.D., Departments of Medicine and Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, P&S Building 10-508, 630 West 168th Street, New York, New York 10032. E-mail: hjw14{at}columbia.edu.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Context: Mutations in PPARG are associated with insulin resistance and familial partial lipodystrophy, a disease characterized by altered distribution of sc fat and symptoms of the metabolic syndrome. The encoded protein, peroxisome proliferator-activated receptor (PPAR)-, plays a pivotal role in regulating lipid and glucose metabolism, the differentiation of adipocytes, and other cellular regulatory processes.

Objectives: The objective of the study was to detect a novel PPARG mutation in a kindred with partial lipodystrophy and analyze the functional characteristics of the mutant protein.

Patients and Methods: In three subjects with partial lipodystrophy, one unaffected family member, and 124 unaffected subjects, PPARG was screened for mutations by direct sequencing. Body composition, laboratory abnormalities, and hepatic steatosis were assessed in each affected subject. Transcriptional activity was determined, and EMSA was performed to investigate DNA binding capacity of the mutant protein.

Results: We identified a PPARG mutation, C190S, causing partial lipodystrophy with metabolic alterations in three affected family members. The mutation was absent in the unaffected family member and unaffected controls. The mutation is located within zinc-finger 2 of the DNA binding domain. C190S PPAR has a significantly lower ability to activate a reporter gene than wild-type PPAR in absence and presence of rosiglitazone. A dominant-negative effect was not observed. Compared with wild-type PPAR, C190S PPAR shows a reduced capacity to bind DNA.

Conclusion: Mutation of a zinc-binding amino acid of PPAR leads to an altered protein-DNA binding pattern, resulting in a partial loss of function, which in turn is associated with partial lipodystrophy.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
AUTOSOMAL DOMINANT FAMILIAL partial lipodystrophy is a rare disease, which in most cases is caused by mutations in LMNA (MIM 151330), encoding the nuclear intermediate filament proteins lamin A and C. This form of lipodystrophy is referred to as Dunnigan-type lipodystrophy (MIM 151660) (1, 2, 3). It is characterized by loss of fat from the extremities and accumulation of sc fat around neck and face. Patients also frequently suffer from diabetes mellitus, hyperlipoproteinemia, and hepatic steatosis (4, 5, 6). Subjects with partial lipodystrophy are often diagnosed because of insulin-resistant diabetes mellitus or after suffering acute pancreatitis due to hypertriglyceridemia. In addition to LMNA mutations, several mutations in the peroxisome proliferator-activated receptor (PPAR)-, encoded by PPARG (MIM 601487), are also associated with a form of partial lipodystrophy (MIM 604367), which is phenotypically very similar to the Dunnigan-type partial lipodystrophy (7, 8, 9, 10, 11, 12).

PPAR belongs to the superfamily of nuclear hormone receptors and is closely related to two other human PPAR isotypes, PPAR and PPAR (13, 14). PPAR is expressed ubiquitously except for the liver. PPAR is located mainly in the liver and to a lesser extent in heart and muscle, whereas PPAR is widely expressed with predominance in liver and adipose tissue. PPAR and PPAR are mainly involved in lipid metabolism and energy homeostasis (15, 16). PPAR is known to play a crucial role in lipid and glucose metabolism, differentiation of adipocytes, cellular energy homeostasis, inflammation, cell differentiation, and carcinogenesis (17, 18). It regulates transcription of numerous PPAR-responsive genes by forming a heterodimer with retinoid X receptor (RXR)- and subsequently binding to PPAR-responsive elements (PPREs) (19). The first natural PPRE was identified in the promoter of the acyl coenzyme A (acyl-CoA) oxidase gene (20). Like other nuclear hormone receptors, PPAR contains a ligand binding domain (LBD), a DNA binding domain (DBD), and an A/B domain. The DBD possesses nine conserved cysteines, eight of them coordinating two zinc ions in a tetrahedral configuration (21). Mutations in zinc-finger 1, which do not alter the zinc-finger’s structure, do not change the protein’s ability to bind DNA and activate PPREs. In contrast, a mutation leading to a disrupted structure of zinc-finger 1 results in loss of the DNA binding capacity and PPRE activation (22). The PPARG gene encodes four different PPAR isoforms, PPAR1–4, generated by use of alternate promoters and differential mRNA splicing (23, 24, 25). Whereas PPAR1 and PPAR3 are widely expressed in most differentiated cells (23, 26), PPAR2 is expressed in adipose tissue only, emphasizing its functional importance for the metabolism of adipocytes (26). PPAR4 is also expressed in adipose tissue (25).

Seven different disease-causing mutations have been identified in PPARG, causing insulin resistance, hypertriglyceridemia, and lipodystrophy. These mutations are located in either the promoter region of PPAR4 or the LBD, affecting all four isoforms (7, 8, 9, 10, 11, 12). We therefore examined PPARG of a patient with partial lipodystrophy and identified a mutation, C190S, located in a highly conserved region of the DBD. The same mutation was present in two additional family members who also presented with partial lipodystrophy, and it was not detected in a clinically unaffected fourth family member. Although the wild-type (WT) and mutant proteins are similarly expressed, this mutation leads to a loss of PPAR transcriptional activity. The mutant form has negligible DNA binding capacity, which is likely responsible for the reduced transcriptional activity.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients and clinical evaluations

The index patient had been referred to the Charité Hospital by her general practitioner for evaluation of lipodystrophy. Consent was obtained from all subjects. The study was approved by the Ethic Committee of the Charité Hospital (Berlin, Germany).

In all subjects, body weight and height were obtained to calculate body mass index. Body composition was evaluated by measuring triceps skinfold thickness with a Lange caliper (Cambridge Scientific Industries, Cambridge, MD) and performing dual-energy x-ray absorptiometry (DEXA; Lunar DXA; GE Healthcare, Fairfield, CT). Laboratory results were obtained from the clinical laboratory facility at the Charité Hospital, and analyses were done according to standard procedures. Hepatic steatosis was assessed sonographically in all subjects and liver magnetic resonance imaging scans were obtained from two of the three subjects.

Mutational analysis

DNA was isolated from EDTA containing blood samples. LMNA was directly sequenced as previously described (27). For amplification of PPARG, we designed primers for each exon, and after amplification, the PCR products were purified and sequenced by cycle sequencing with fluorescent dye terminators on an ABI 310 automatic sequencer (Applied Biosystems, Darmstadt, Germany). The mutation within PPARG was confirmed by restriction fragment length polymorphism analysis using the restriction enzyme Pci1.

Sequence analysis

Homology search for the DBD of PPAR was performed using PSI-BLAST (28). A three-dimensional model of the dimeric DBDs of PPAR/RXR in interaction with their cognate DNA sequences was created using PyMOL (29).

Plasmid constructions

For the reporter gene assay, constructs expressing WT PPAR and the C190S mutation were cloned into the plasmid pSVK3 (Pharmacia Biotech, Inc., Piscataway, NJ). A human PPAR cDNA clone (ATCC MGC-5014), encoding PPAR isoform 1, was obtained from American Type Culture Collection (Manassas, VA) and used as a template. WT human PPAR cDNA was generated by PCR, digested with XbaI and XhoI and ligated into the pSVK3 plasmid. The C190S mutation was introduced at the respective site of PPAR isoform 1 by using the Transformer site-directed mutagenesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA).

For immunoblot analysis, cDNA encoding WT and C190S PPAR were cloned into the vector pSVF, which is identical with pSVK3 except for a FLAG tag coding sequence that has been inserted in the multiple cloning site. PCR using WT and C190S PPAR cDNA in pSVK3 as templates was performed, and PPAR cDNA was ligated into the vector after digestion with KpnI and XhoI.

Reporter gene assays

293T cells were cultured at 37 C in DMEM containing 10% fetal bovine serum. For transient transfection, cells were grown to 70–80% confluency on 6-well plates and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).

For transcription assays, cells were cotransfected with 2 µg reporter plasmid acyl-CoAx3-TK-LUC (kindly provided by Dr. M. A. Lazar, University of Pennsylvania, Philadelphia, PA), 1 µg pSV-ß-galactosidase plasmid (Promega, Madison, WI), and 1 µg of plasmids expressing WT PPAR, C190S PPAR, or empty vector as control. Approximately 24 h after transfection, cells were treated with DMEM containing vehicle [dimethylsulfoxide (DMSO)] or 10 µM rosiglitazone (Cayman Chemical, Ann Arbor, MI) for 24 h. Relative luciferase activities in extracts were measured using the luciferase assay system (Promega) and a luminometer (Wallac Victor 1420 multilabel counter; PerkinElmer Life Sciences, Boston, MA). To correct for differences in the transfection efficiency between experiments, luciferase activities were normalized to ß-galactosidase activities. Activity measured in cells transfected with only empty vector was subtracted from the results, and transcriptional activity was expressed relative to the maximum obtained with the WT form treated with vehicle. To determine whether C190S PPAR interfered with activity of WT PPAR, 293T cells were transfected with 2.4 µg empty vector or 1.2 µg WT PPAR cDNA and 1.2 µg of empty vector, WT, or C190S PPAR cDNA. This experiment was performed in absence and presence of rosiglitazone. Rosiglitazone dose response was assessed by cotransfecting 293T cells with either empty vector or plasmid encoding WT or mutant PPAR. Increasing concentrations of rosiglitazone were added and reporter gene activity was measured. Results were compared using Student’s t test with a statistically significant difference defined as a value of P < 0.05. Results are given as mean ± SD of three independent experiments.

Immunoblot analysis

Cos7 cells were cultured at 37 C in DMEM containing 10% fetal bovine serum. Cells were transiently transfected with empty vector or pSVF containing WT or mutant PPAR cDNA in frame with a FLAG epitope coding sequence. Transfection was performed using Lipofectamine 2000 (Invitrogen). After 24 h, protein was extracted with a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2.5 µM sodium phosphate, 1 mM sodium orthovanadate, and antiprotease mix (Sigma, St. Louis, MO). Proteins were separated by SDS-PAGE and blotted on a nitrocellulose membrane. Primary antibody was monoclonal anti-FLAG antibody M5 (Sigma), and antimouse-IgG (Amersham, Pittsburgh, PA) was used as secondary antibody. Blots were processed with enhanced chemiluminescence (Amersham).

EMSA

WT or C190S PPAR cDNA cloned into pSVK3 and pCMX containing human RXR cDNA (a kind gift from Dr. Alan Tall, Columbia University, New York, NY) were used for in vitro synthesis using the TNT-coupled reticulocyte lysate system (Promega). Detection of the proteins was performed using the Transcend nonradioactive translation detection system (Promega). EMSA was performed as described (30), using the following PPREs: acyl-CoA (31) (identical with the sequence of the plasmid that encodes the reporter gene), phosphoenolpyruvate carboxykinase 2 (32), adipocyte protein 2 (33), adiponectin (34), lipoprotein lipase (35), liver X receptor- (36), and the synthetic PPRE FLJ10079 (37).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

The index subject (subject II.2) was a 26-yr-old Caucasian woman who had one sister and both father and mother were alive (Fig. 1A). Other family members were not available for this study. Subject II.2 presented with acute onset of pancreatitis after a fatty meal. Physical examination revealed signs of partial lipodystrophy, including loss of sc fat from the extremities; additional fat in the area of face, chin, and trunk; and muscular hypertrophy on the lower limbs (Fig. 1B). She had no polycystic ovary disease and a normal menstrual cycle and presented with hirsutism and acanthosis nigricans on axillae, neck, and inguinal region. We examined her family members and found that her 36-yr-old sister (subject II.1) and the 60-yr-old father (subject I.1) had clinical features of partial lipodystrophy, whereas her mother (subject I.2) appeared unaffected. Subject II.1 had the same pattern of distribution of sc fat with loss of adipose tissue from the extremities and additional fat on face and chin. She also presented with muscular hypertrophy of the legs (Fig. 1C). She had normal menstrual cycles, no polycystic ovaries, slight hirsutism, and no acanthosis nigricans. Subject I.1 had a less striking lipodystrophic phenotype (Fig. 1D). Muscular hypertrophy and additional fat around face and chin were not remarkable, and the triceps-skinfold thickness was within the 10th to 25th percentile when compared with a normal population (38). Due to a stroke at 45 yr of age, he suffered from left hemiparesis. All three subjects had arterial hypertension.

View larger version (80K): [in this window] [in a new window]   FIG. 1. Pedigree and photographs of subjects in a family with partial lipodystrophy. A, Pedigree of the family. The index patient is marked with an arrow. Black symbols indicate affected subjects; unaffected subject is marked by unfilled symbol. B, Index patient (II.2) with loss of fat and muscular hypertrophy, especially of the legs and accumulation of sc fat on the trunk. C, The index patient’s sister (II.1) with similar clinical features. D, Their father (I.1), suffering from hemiparesis on the left side after a stroke at age 45 yr. The loss of sc fat as well as the muscular hypertrophy are less severe than in the female subjects.

Because of the clinical presentation of partial lipodystrophy, we sequenced the candidate genes LMNA and PPARG. All three subjects had a heterozygous thymine to adenine transversion at nucleotide 568 in exon 3, leading to a substitution of cysteine to serine (C190S) at residue 190 in the encoded protein (amino acid nomenclature refers to PPAR isoform 2). The clinically unaffected subject I.2 had the WT sequence and was homozygous for thymine at nucleotide 568 (Fig. 2A). The mutation was verified by restriction fragment length polymorphism analysis, which also confirmed that subject I.2 was unaffected (data not shown). This mutation was absent from 124 healthy controls of the same ethic background as well as 25 subjects with partial lipodystrophy due to LMNA mutations, excluding that it is a rare polymorphism. In addition, the mutated cysteine is highly conserved in PPAR in different species and also in other human nuclear receptors (Fig. 2B).

View larger version (30K): [in this window] [in a new window]   FIG. 2. Identification of the PPARG C190S mutation. A, DNA sequencing chromatogram of a portion of the PPARG gene, illustrating the thymine to adenine transversion at nucleotide 568, leading to heterozygous substitution of cysteine by serine at residue 190 in subject II.2. Shown below is the DNA sequencing chromatogram of the index patient’s unaffected mother, lacking the mutation. B, Part of the sequence of the DNA binding domain, including the cysteine that is changed to serine in the PPARG C190S mutation (red box). Alignment of the DNA sequences of different nuclear receptors (above) and different species (below) reflects conservation in the area of zinc-finger 2, which contains the C190S mutation.

The C190S PPAR has decreased transcriptional activity

To examine transcriptional activity of WT and C190S PPAR, 293T cells were transiently transfected with cDNA encoding WT PPAR, C190S PPAR, or the empty expression vector together with the reporter gene acyl-CoAx3-TK-LUC and ß-galactosidase. In the absence of rosiglitazone, reporter gene activity in cells expressing C190S PPAR was 42.4 ± 3.9% (mean ± SD) and therefore significantly lower, compared with the reporter gene activity in cells expressing WT PPAR (P < 0.05). Addition of rosiglitazone enhanced the difference between cells expressing mutant PPAR and those expressing the WT protein (Fig. 3A), indicating a loss of function of C190S PPAR. Whereas the difference between WT plus DMSO and WT plus rosiglitazone was significant, the difference between C190S plus DMSO and C190S plus rosiglitazone was not significant. Transfection with equal amounts of WT and mutant PPAR cDNA resulted in transcriptional activity similar to transfection with WT PPAR cDNA in absence as well as presence of rosiglitazone (Fig. 3A). Hence, the C190S mutant does not interfere with WT PPAR. To confirm this observation, cells were transfected with WT PPAR cDNA and an equal amount of either empty vector, WT, or C190S PPAR cDNA. Transcriptional activity of cells transfected with WT and C190S PPAR cDNA was not decreased when compared with cells transfected with WT PPAR cDNA only, so that dominant-negative activity of the C190S mutant against the WT PPAR receptor could be excluded (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   FIG. 3. Protein expression and transcriptional activity in transfected 293T cells expressing WT and C190S PPAR. A, Cells were transfected with equal amounts of empty vector or plasmids encoding WT and C190S PPAR, either individually or in combination and were treated with either vehicle (DMSO) or 10 µM rosiglitazone (Rosi) for 24 h. Relative luciferase activity was measured in extracts and normalized to ß-galactosidase activities. Background activity (of cells transfected with empty vector) was subtracted from results, and relative transcriptional activity was calculated as a percentage of the maximum activity achieved by WT PPAR in the absence of rosiglitazone. Values are means ± SD for three independent experiments. Asterisks indicate statistically significant differences (P < 0.05). B, 293T cells were transfected with 2.4 µg empty vector (pSVK3) or 1.2 µg WT PPAR cDNA plus 1.2 µg empty vector, WT, or C190S PPAR cDNA. Cells were treated with either vehicle or 10 µM rosiglitazone. Transcriptional activity was measured, and results are shown as a percentage of the maximum activity measured in cells transfected with empty vector and treated with vehicle. C, Rosiglitazone dose-response curves for cells transfected with plasmids encoding WT and C190S PPAR cDNA as well as empty vector. 293T cells were transfected with 1 µg plasmid and treated with increasing concentrations of rosiglitazone. Results were normalized to ß-galactosidase activity and calculated as a percentage of the WT treated with vehicle. Data are presented as mean ± SD (n = 3). D, Immunoblot analysis of FLAG-tagged WT PPAR, C190S PPAR, and the combination of WT and C190S PPAR. Cells were transfected with empty vector (lane 1), plasmids encoding WT PPAR (lane 2), C190S PPAR (lane 3), or equal amounts of both plasmids (lane 4). Proteins were fused to a FLAG epitope, which was detected with an anti-FLAG antibody. One representative blot of three experiments is shown.

DNA binding capacity of C190S PPAR is abrogated

The substituted cysteine in C190S PPAR represents one of the four zinc-binding residues in zinc-finger 2 of the protein’s DBD. To establish the position of the mutated cysteine in the heterodimer PPAR/RXR in complex with DNA, we created a model of the three-dimensional structure of the DBD of PPAR, showing the protein after heterodimerization with RXR and in complex with its cognate DNA sequence (Fig. 4A). PPAR has been shown to occupy the 5' half-site of the consensus DNA sequence, with RXR occupying the 3' half-site (39). The second zinc-finger of PPAR is involved in heterodimerization as well as DNA binding. We therefore examined the DNA binding capacity of C190S PPAR for different PPREs, including acyl-CoA, which is the sequence used in the reporter gene experiments. For all tested PPREs, DNA binding capacity of C190S PPAR was negligible when compared with WT PPAR (Fig. 4B).

View larger version (28K): [in this window] [in a new window]   FIG. 4. DNA binding capacity of WT and C190S PPAR. A, Model of the three-dimensional structure of a PPAR/RXR heterodimer bound to DNA (marked in purple). Zinc molecules are yellow; cysteines binding zinc in zinc-finger 1 are also yellow; cysteines binding zinc in zinc-finger 2 are orange. The cysteine corresponding to the mutation C190S is marked in red. B, DNA binding was determined by EMSA. Labeled probe incubated without protein served as negative control. RXR and WT PPAR alone did not bind to the probe, whereas the combination of RXR and WT PPAR shows DNA binding. DNA binding capacity is abrogated or negligible in all tested PPREs when RXR is combined with C190S PPAR.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

Seven other mutations in PPARG are associated with partial lipodystrophy. Of these mutations, six are located in the LBD and one, PPARG-14A>G, was identified in the promoter region of isoform 4. The LBD mutation R425C has not been assessed concerning protein function (9). Among the other mutations, only two, P467L and V260M, are reported to exert a dominant-negative effect on the WT protein (7). In all other cases, including the PPARG C190S mutation, haploinsufficiency with an in vitro loss of function of the mutant protein equal to or larger than 50% appears to be the pathophysiological mechanism that causes the lipodystrophic phenotype without dominant-negative activity (8, 11, 12). Mutations in the DBD and LBD affect all four PPAR isoforms, whereas the mutation in the promoter region of PPAR4 leads to a selective deficiency of isoform 4 (10). Although the other PPAR isoforms are normally expressed, subjects lacking PPAR4 suffer from partial lipodystrophy.

Lipodystrophic symptoms in all subjects with PPARG mutations include diabetes mellitus, hypertriglyceridemia, and hypertension. In contrast, another PPARG mutation was reported to lead to severe insulin resistance only if subjects were doubly heterozygous for a second mutation in an unrelated gene (41). Symptoms of partial lipodystrophy such as loss of fat or muscular hypertrophy were not reported in these subjects, indicating that, possibly depending on the genetic background, symptoms in subjects with PPARG mutations can vary extensively. Due to the small number of subjects described so far, a meaningful genotype-phenotype correlation is not yet possible.

Partial lipodystrophy may occur as a component of several hereditary diseases (42). Mutations in PPARG and LMNA cause partial lipodystrophy as the major abnormality with the typical maldistribution of adipose tissue and metabolic derangements. The main difference between subjects with partial lipodystrophy caused by mutations in these two genes appears to be the prevalence of arterial hypertension, which has been reported in most of the subjects with PPARG mutations but only in some subjects with LMNA mutations (7, 8, 9, 10, 11, 12, 43, 44). In general, loss of sc fat seems to be less distinct in subjects with PPARG mutations, whereas diabetes mellitus tends to be more severe and occur at a younger age. The similar phenotypes in subjects with partial lipodystrophy caused by PPARG and LMNA mutations suggest that PPAR and A-type lamins might be involved in the same mechanisms or pathways leading to loss of sc fat and metabolic consequences. Studies showing that mutations in LMNA and PPARG are associated with partial lipodystrophy suggest that polymorphisms in LMNA or PPARG could potentially explain the development of different forms of obesity, diabetes mellitus, and the susceptibility of drug-induced lipodystrophy in the broad population.

Since we submitted this manuscript, five mutations within the DBD of PPAR have been reported, demonstrating loss of function and abrogated DNA binding. Interference with WT protein function was observed for three of these five mutants (45, 46). The increasing number of mutations reported during a relatively short period indicates that partial lipodystrophy due to PPARG mutations is presumably more common than originally thought and in many cases still undiagnosed.

	Acknowledgments

 
We thank Dr. M. Agostini for her advice on the EMSA.

	Footnotes

 
This work was supported by a fellowship from the Deutsche Forschungsgemeinschaft (DFG LU 1206/1-1; to A.L.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online March 13, 2007

¹ H.H.-J.S. and H.J.W. contributed equally to this work.

Abbreviations: acyl-CoA, Acyl coenzyme A; DBD, DNA binding domain; DEXA, dual-energy x-ray absorptiometry; DMSO, dimethylsulfoxide; HbA1c, hemoglobin A1c; HDL, high-density lipoprotein; LBD, ligand binding domain; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR-responsive element; RXR, retinoid X receptor; WT, wild type.

Received December 5, 2005.

Accepted March 7, 2007.

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