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Familial Partial Lipodystrophy Phenotype Resulting from a Single-Base Mutation in Deoxyribonucleic Acid-Binding Domain of Peroxisome Proliferator-Activated Receptor-

Department of Vascular Medicine (H.M., E.S.) and Radiology (M.M.), Academic Medical Center, 1150 AZ Amsterdam, The Netherlands; Department of Pathology (L.Z., G.L., T.L.), Center for Integrative Metabolic and Endocrine Research, Wayne State University School of Medicine, Detroit, Michigan 48201; Department of Metabolic and Endocrine Diseases (E.H.J., E.K.), University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands; Robarts Research Institute and Schulich School of Medicine (H.C., R.A.H.), London, Ontario, Canada N6A 5K8; and Department of Internal Medicine (C.B.B.), Onze Lieve Vrouwe Gasthuis, 1090 HM Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Houshang Monajemi, Academic Medical Center, Department of Vascular Medicine, Meibergdreef 9, 1105AZ Amsterdam, The Netherlands. E-mail: H.monajemi{at}amc.uva.nl; or Robert A. Hegele, Robarts Research Institute, London, Ontario, Canada N6A 5K8. E-mail: hegele{at}robarts.ca.

	Abstract

Top Abstract Introduction Subject and Methods Results Discussion References

 
Context: Familial partial lipodystrophy (FPLD) results from coding sequence mutations either in LMNA, encoding nuclear lamin A/C, or in PPARG, encoding peroxisome proliferator-activated receptor- (PPAR). The LMNA form is called FPLD2 (MIM 151660) and the PPARG form is called FPLD3 (MIM 604367).

Objective: Our objective was to investigate whether the clinical phenotype of this proband is due to mutation(s) in PPAR.

Design: This is a case report.

Patient and Setting: A 31-yr-old female with the clinical phenotype of FPLD3, i.e. lipodystrophy and early childhood diabetes with extreme insulin resistance and hypertriglyceridemia leading to recurrent pancreatitis, was assessed at an academic medical center.

Results: The proband was heterozygous for a novel CT mutation in the PPARG gene that led to the substitution of arginine 194 in PPAR2 isoform, a conserved residue located in the zinc finger structure involved in DNA binding, by tryptophan (R194W). The mutation was absent from the genomes of 100 healthy Caucasians. In vitro analysis of the mutated protein showed that R194W (and R166W in PPAR1 isoform) could not bind to DNA and had no transcriptional activity. Furthermore, R194W had no dominant-negative activity.

Conclusions: The R194W mutation in PPARG disrupts its DNA binding activity and through haploinsufficiency leads to clinical manifestation of FPLD3 and the associated metabolic disturbances.

	Introduction

Top Abstract Introduction Subject and Methods Results Discussion References

 
DUNNIGAN-TYPE FAMILIAL PARTIAL lipodystrophy (FPLD) results from rare coding sequence mutations either in LMNA, encoding nuclear lamin A/C, or in PPARG, encoding peroxisome proliferator-activated receptor- (PPAR) (1, 2). The LMNA and PPARG forms are called FPLD2 (MIM 151660) and FPLD3 (MIM 604367), respectively. These mutations underlie profound redistribution of fat stores, characterized by lipoatrophy of the extremities and gluteal region in combination with lipohypertrophy in face, neck, trunk, and central adipose stores. This redistribution can be accompanied by a variety of clinical characteristics, including severe insulin resistance, often with acanthosis nigricans, and hypertriglyceridemia, sometimes associated with pancreatitis and eruptive xanthomata (3). The core clinical phenotype is fat loss with subsequent development of secondary metabolic disturbances that are characteristic of the insulin resistance syndrome.

The presence of lipodystrophy in subjects with dysfunctional PPARG missense mutations, such as R425C, F388L, E138fsAATG, V290M, P467L, and Y355X (4, 5, 6, 7, 8, 9) and in PPAR-deficient murine models (10, 11) has confirmed the central role of PPAR in adipogenesis. PPAR interacts with retinoid X receptor (RXR), binds DNA as a heterodimer, and subsequently regulates transcription of PPAR-responsive genes. Heterozygous loss of function or haploinsufficiency is clinically important when gene dosage is strictly regulated. Here, we show that a heterozygous mutation of a conserved arginine residue into tryptophan in the PPAR (referred to as R166W in PPAR1 and R194W in PPAR2 isoform) zinc finger II region disrupts DNA binding and transcriptional activity and thus underlies FPLD3.

	Subject and Methods

Top Abstract Introduction Subject and Methods Results Discussion References

 
Study subject

The study was approved by the University of Western Ontario Ethics Review Panel (protocol 07920E), and the subject gave informed consent to participate.

Magnetic resonance imaging (MRI)

MRI was performed using a 1.5 Tesla, Signa scanner (GE Medical Systems, Milwaukee, WI) with a neck coil and body coil. Axial and sagittal T1 weighted images of the cervical spine and axial T1 weighted images of the abdomen and the lower legs were acquired according to the procedure described earlier (12).

DNA sequence analysis

After DNA sequencing showed no mutation in LMNA, we amplified and sequenced the six exons of PPARG plus more than 100 bp at intron-exon boundaries and approximately 700 bp of the promoter (5, 7). The R194W mutation was genotyped by scoring the electropherogram tracing of exon 4 sequences from the Applied Biosystems 3730 automated DNA sequence analyzer (ABI, Mississauga, Ontario, Canada). Genomic DNA from 100 healthy Caucasian subjects was studied, permitting 70% power to exclude a mutation with frequency greater than 2% in the healthy population (two-tailed < 0.05).

PPAR clones

A cDNA encoding full-length human PPAR1 was cloned into the pTRE-shuttle2 eukaryotic expression vector (Clontech, Palo Alto, CA). A double-FLAG epitope tag (MDYKDHDGDYKDHD) was added to the N terminus of the clone. The pCDNA3-PPAR1 and pCDNA3-PPAR2 constructs were kind gifts from Dr. V. K. K. Chatterjee. The R194W mutation was introduced using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and verified by sequencing.

EMSA

EMSA experiments were performed as described (13). In short, a radiolabeled double-stranded DNA oligomer, containing the PPRE from the rat acyltransferase-coenzyme A oxidase promoter, was incubated with in vitro-translated PPAR [wild-type (WT) or mutants] and/or in vitro-translated RXR proteins. For supershift experiments, 1 µg -RXR (sc-553; Santa Cruz Biotechnology, Santa Cruz, CA), -PPAR (sc-7273), or -Gal4 (sc-510) antibodies were added. Receptor-DNA complexes were separated from unbound DNA on native gels and visualized by autoradiography. At least three independent experiments were performed. The complete probe sequences used for binding and competition analysis were as follows: PPRE wild-type, 5'-CCG GGG ACC AGG ACA AAG GTC ACG AAG CT-3', and PPRE-mutant, 5'-CCG GGG GAC CAG CAC AAA GCA CAC GAA GCT-3'. Western blot analyses of the different in vitro-translated PPAR proteins was performed as described (14). -PPAR antibody (sc-7196) was used to probe for PPAR protein and enhanced chemiluminescence (Amersham Biosciences) was used for detection.

Cell culture, reporter assays, and dominant-negative assays

NIH 3T3 mouse fibroblasts and human U2OS osteosarcoma cells were maintained in DMEM Glutamax (Dulbecco) containing 10% fetal calf serum (Life Technologies, Inc., Ottawa, Ontario, Canada), 100 µg penicillin/ml, and 100 µg streptomycin/ml (Life Technologies). NIH 3T3 mouse fibroblasts were grown in 24-well plates (1.0 x 10⁵ cells per well) in DMEM plus 10% fetal calf serum. Cells were transfected with 25 ng WT or R194W expression plasmid, 6 ng pTET-off, 25 ng pRXR, 2 ng of a ß-galactosidase control plasmid, and 200 ng of the PPAR-dependent luciferase reporter pFATP-Luc (i.e. three copies of the mouse FATP gene PPRE inserted upstream of the minimal thymidine kinase promoter). Cells were transfected for 4 h with Lipofectamine-plus and then treated with DMSO or increasing doses of rosiglitazone for 16 h. Transfections were performed in triplicate. Mixing experiments examining dominant-negative activity (see Fig. 4E) were conducted as described above except with the amounts of PPAR plasmids; NIH 3T3 cells that were transfected with the combination of 5 ng WT PPAR and increasing amounts of WT, the R194W mutant, or the dominant-negative mutant P467L (5, 10, or 20 ng; indicated in the figure as 1:1, 1:2, and 1:4 respectively), in the presence or absence of rosiglitazone (see Fig. 4, E and F).

View larger version (34K): [in this window] [in a new window]   FIG. 4. PPARG R194W mutation is transcriptionally inactive. A, In vitro-translated RXR and/or PPAR1 (wt or R166W) proteins were incubated with 32P-labeled probe in the absence or presence of 10X unlabeled probe as indicated. Protein-DNA complexes were separated from unbound DNA on native gels and visualized by autoradiography of dried gels. To analyze the specificity of the binding, the same experiment was performed in the presence of antibodies against PPAR or RXR or an irrelevant antibody (-Gal4) as indicated (right panel). Expression of the different PPAR proteins was confirmed by Western blot analysis using an antibody directed against PPAR. B, Competition and supershift EMSA as described in A was performed for PPAR2 isoform using in vitro-translated PPAR2 (wt or R194W) proteins. C, The R194W mutant does not respond to rosiglitazone. NIH 3T3 cells were transfected with WT, R194W mutant, or empty vector, together with a PPAR-responsive reporter construct and a ß-galactosidase reference plasmid. After transfection, NIH3T3 cells were treated with the indicated dose of rosiglitazone for 16 h. Data are presented as a percentage of the maximal level of transcription achieved in the rosiglitazone curve (10 µM). Zero percent was defined separately for each curve as the lowest level of transcription for that curve. The data points are means ± SD (n = 3). D, U2OS cells were transfected with expression vectors encoding PPAR1 (WT or mutant) or PPAR2 (WT or mutant) and a 3xPPRE-tk-luc reporter. Activation of the luciferase reporter, in the absence or presence of 1 µM rosiglitazone, is expressed as fold induction over that with empty vector (pCDNA) in the absence of ligand, after normalization for Renilla luciferase activity. Results are averages of at least three independent experiments performed in duplicate ± SE of the means. E, R194W has no dominant-negative activity. Increasing amounts of R194W or P467L mutant receptors were cotransfected with a fixed amount of WT PPAR. Data are normalized to the transcriptional activity of WT receptor alone (1:0) and are presented as means ± SD (n = 3). The legend indicates the molar ratios of the two transfected receptors (WT+WT or WT+mutant). *, P < 0.05; , P < 0.01 relative to the WT receptor alone (1:0). F, The same experiment (as described in E) was performed in the presence of a saturating amount (20 µM) of rosiglitazone. Under this condition, P467L had no dominant-negative activity as was published in the original article (4 ).

	Results

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Patient medical history and clinical evaluation

The proband was a 31-yr-old Turkish female living in The Netherlands. Menarche occurred at age 11, followed by regular menstrual cycles. At age 15, she was diagnosed with diabetes with severe insulin resistance. Despite insulin therapy, she developed severe hypertriglyceridemia, with plasma concentration more than 50 mmol/liter, leading to eruptive xanthomas on her trunk and extremities. At age 17, her menstrual cycle became irregular and her extremities and face developed excessive hair growth, leading to the diagnosis of polycystic ovarian syndrome. At age 19, she became pregnant after in vitro fertilization and gave birth to a healthy son. Subsequently, she was hospitalized twice more for pancreatitis at ages 20 and 22. During outpatient follow-up, her insulin dose was increased more than 300 U/d. At the end of 2005, she was referred to the Academic Medical Center, Amsterdam, for management of refractory hypertriglyceridemia despite fibrate and insulin treatment. On examination, she was mildly obese (weight, 68 kg; height, 167 cm; and body mass index, 25 kg/m²). Her resting blood pressure was 130/70 mm Hg. She had excess sc fat on the face, neck, trunk, and abdomen, with lack of sc fat on the gluteal region and extremities (Fig. 1). This was confirmed with MRI (Fig. 2), which showed excessive and relatively symmetrical deposition of sc fat on the face, neck, and upper trunk, with disproportionate depletion of sc fat in the lower body. Furthermore, she had acanthosis nigricans on her feet, axillae, and neck. She was also hirsute. Measurements from fasting plasma were glucose, 8.8 mmol/liter; HbA1c, 8.2%: insulin, 1074 pmol/liter (reference, 34–172); C-peptide, 950 pmol/liter (reference, 176–664); total cholesterol, 9.42 mmol/liter; high-density lipoprotein cholesterol, 1.33 mmol/liter; and triglyceride, 35.0 mmol/liter. APOE genotype was E3/E3. Lipoprotein lipase (LPL) activity was normal, and no genomic DNA sequence changes were seen in the LPL gene (data not shown). The free androgen index was 134 (normal ratio, 0–8). At the time of these measurements, she was being treated with multiple daily insulin injections totaling 300 U/d, 100 mg ciprofibrate, and 50 mg cyproterone daily.

View larger version (119K): [in this window] [in a new window]   FIG. 1. Clinical aspects of the proband. A and B, Photos showing the masculine appearance with a clear trunk-sparing lipodystrophy; C–E, acanthosis nigricans on her neck, axilla, and feet; F, eruptive xanthoma.

View larger version (100K): [in this window] [in a new window]   FIG. 2. MRI scans. T1 weighted images were obtained. A and B, Scans of the neck showing a layer of sc fat measuring 2.52 cm; C, cross-section at the abdomen, showing a symmetrical layer of sc fat measuring 1.09 cm; D, cross-section at the gluteal region, showing sc fat measuring 0.80 cm; E, cross-section at the level of upper leg region, showing a dorsal layer of sc fat measuring 0.64 cm; F, cross-section at the level of lower leg region, showing a dorsal layer of sc fat measuring 0.56 cm.

DNA sequence analysis

In the genome of the proband, we found a heterozygous nucleotide substitution CT at position 1762 in the PPAR isoform 4 (Fig. 3). All other regions analyzed were free of DNA sequence changes. This mutation was absent from the genomes of 100 normal Caucasian controls. This mutation causes an amino acid substitution R194W in PPAR isoform 2 (R166W in PPAR isoform 1).

View larger version (16K): [in this window] [in a new window]   FIG. 3. Reported PPARG mutations in FPLD3 and genomic DNA sequence electropherograms of heterozygous R194W mutation. Schematic genomic map of PPARG, showing the positions of known mutations along with the disease mechanism. Coding exons are shown with Arabic numerals, whereas noncoding exons are designated by letters. The start of transcription for 1, 2, 3, and 4 isoforms is shown. The electropherogram tracing shows both alleles from proband (R194W) compared with corresponding genomic DNA sequence from a healthy subject. The position of the mutation is indicated by the arrow. Normal nucleotide and amino acid sequence is shown above the WT electropherogram tracing. Note that the mutation numbers refer to 1 isoform in a few and to 2 isoform in other subjects.

R194W mutant PPAR does not bind DNA and is transcriptionally inactive

The location of the mutation within the DNA binding domain of PPAR suggested that it might influence DNA binding. To investigate this possibility, the binding of the R166W as well as R194W mutant to a standard PPRE sequence was assessed using an EMSA. Although WT PPAR in the presence of RXR was capable of binding to PPAR response element, the R166W mutant had no detectable DNA binding activity (Fig. 4A). As expected, lack of DNA binding was also observed in the PPAR2 isoform (Fig. 4B).

The transcriptional activity of the R194W mutant PPAR was assessed by transient transfection of PPAR expression plasmids into NIH 3T3 cells and analysis of luciferase activity from a PPAR-responsive reporter. The R194W mutant receptor was inactive at all doses of the ligand rosiglitazone (Fig. 4C). In addition, U2OS cells were transfected with PPAR1 (WT and R166W) or PPAR2 isoform (WT and R194W). Whereas both WT isoforms had a slight basal expression level that was highly induced by rosiglitazone, both mutant isoforms displayed no transcriptional activity in the absence or presence of exogenous ligand (Fig. 4D).

R194W mutant PPAR displays no dominant-negative activity

To investigate whether the R194W receptor had dominant-negative activity against WT PPAR, a mixing experiment was performed in which an increasing amount of mutant or WT receptor was mixed with a fixed amount of WT PPAR (Fig. 4E). Although simply increasing the amount of the WT receptor caused a significant increase in transcriptional activity (WT+WT), the addition of increasing amounts of R194W PPAR to a fixed amount of WT receptor resulted in no change in total PPAR transcriptional activity (WT+R194W). For comparison, the same experiment was conducted with the P467L that has dominant-negative activity (4). Increasing amounts of P467L PPAR caused a dose-dependent decrease in WT PPAR transcriptional activity (WT+P467L). When the cells were treated with a high concentration of rosiglitazone (Fig. 4F), the dominant-negative activity of P467L was abolished as described earlier (4). Together, these findings indicate that the R194W mutant does not possess any dominant-negative activity against the WT PPAR receptor.

	Discussion

Top Abstract Introduction Subject and Methods Results Discussion References

 
The principal findings of this study are: 1) association of a novel heterozygous PPAR missense mutation, R194W (R166W in 1 isoform), with FPLD3, including fat redistribution, severe insulin resistance, hypertriglyceridemia, hirsutism, and acanthosis nigricans; and 2) functional analysis showing that the R194W mutant is transcriptionally inactive, independent of PPAR isoform (1 and 2) and cell type (NIH-3T3 and U2OS).

The substitution of a hydrophilic arginine to a hydrophobic tryptophan within an -helix would predict disrupted structure and decreased DNA binding, as was seen with EMSA. The importance of the conserved arginine residue is underscored by natural mutations in other nuclear receptors causing hormone resistance. For instance, an R614H mutation and deletion of this amino acid (614) in the androgen receptor (AR) have been reported in two patients with complete androgen insensitivity (15). Furthermore, mutation of the analogous residue (R477H) in the glucocorticoid receptor (GR) was detected in a patient with primary cortisol resistance (16). In addition, mutation of this conserved arginine residue in the photoreceptor-specific nuclear receptor PNR into tryptophan (R104W) (17) or glutamine (R104Q) (18) were found in patients with enhanced S-cone syndrome. DNA binding of the AR 614 and R614H mutants and the GR R477H mutant was impaired (15, 16), analogous to the PPAR R194W mutant, emphasizing the importance of this conserved arginine residue in nuclear receptor signaling.

R194W brings the number of reported PPARG mutations associated with clinical phenotypes to 14. Only the PPAR2 P115Q mutation was not associated with FPLD3 (19). Two PPAR missense mutations (P467L and V290M), along with the recently published subjects by Agostini et al. (20) (C114R, C131Y, C162W, FS315X, and R357X) act via a dominant-negative mechanism, whereas five (–14AG, F388L, E138fsAATG, Y355X, and R194W) caused FPLD3 through haploinsufficiency (5, 7, 12) (Fig. 3). The R425C mutation (9) also lacks dominant-negative activity (21). All patients with PPARG haploinsufficiency mutations were ascertained based upon a diagnosis of FPLD; almost every patient with a PPARG mutation had partial lipodystrophy as a core phenotype. FPLD3 has proven to be a useful and appropriate clinical designation; the term acknowledges the centrality of lipodystrophy while concurrently distinguishing FPLD3 from phenotypically similar but molecularly distinct forms of lipodystrophy, such as FPLD2 due to LMNA mutations.

Mutations can lead to disease through 1) loss of function, 2) gain of function, or 3) dominant-negative activity. According to the classical dominant-negative hypothesis, the mutant allele eliminates the WT function by direct interference. For instance, in the case of nuclear receptors, the mutant receptor competes with the WT for binding DNA. However, there is some evidence that nuclear receptors can also have indirect dominant-negative activity by affecting the bioavailability of other components of the transcriptional machinery, such as coactivators, and hence could interfere with the WT allele. We have shown that R194W has neither direct (Fig. 4A) nor indirect (Fig. 4E) dominant-negative activity under our experimental conditions. With haploinsufficiency, 50% reduced gene expression results from one nonfunctional allele, whereas dominant-negative mutations induce even greater reduction in gene expression. How do these two mechanisms underlie the same phenotype? One possibility is that subjects with either mutation type might have slightly different clinical phenotypes that are not easily discerned using current methods. For instance, hypertension in human subjects with dominant-negative mutations seems to be more severe than in subjects with haploinsufficiency mutations (22). Additional pedigrees with PPARG would allow for better comparisons of these two mechanisms in vivo.

Since the first publication on FPLD by Dunnigan et al. (23), awareness of this condition by clinicians has increased. Several mutations both in LMNA and PPARG have been described, yet many such patients are probably overlooked, because of clinical similarities with the common obesity-related metabolic syndrome that currently is endemic to westernized societies, largely due to lifestyle changes. Careful physical examination of patients with insulin resistance and hypertriglyceridemia could help identify partial lipodystrophy. In summary, in a proband with FPLD3, we found a novel PPARG mutation that fails to bind DNA and is transcriptionally inactive. Human PPARG mutations will improve our understanding of mechanisms involved in lipodystrophy and insulin resistance.

	Acknowledgments

 
We thank Dr. V. K. K. Chatterjee for plasmid constructs.

	Footnotes

 
This work was supported by operating grants from the Heart and Stroke Foundation of Ontario, the Ontario Research and Development Challenge Fund (Project 0507), Genome Canada, the Canadian Institutes of Health Research (MT14030), the Blackburn Group, and the American Diabetes Association (to T.L.). R.A.H. is a Career Investigator of the Heart and Stroke Foundation of Ontario and holds the Edith Schulich Vinet Canada Research Chair (Tier I) in Human Genetics and the Jacob J. Wolfe Distinguished Medical Research Chair. E.K. is supported by a fellowship from the Royal Netherlands Academy of Arts and Sciences.

The authors have no conflicts to disclose.

First Published Online February 13, 2007

¹ L.Z., G.L., and E.H.J. contributed equally.

Abbreviations: FPLD, Familial partial lipodystrophy; MRI, magnetic resonance imaging; PPAR; peroxisome proliferator-activated receptor-; RXR, retinoid X receptor; WT, wild type.

Received August 17, 2006.

Accepted February 7, 2007.

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