Clinical and Molecular Characterization of a Novel PLIN1 Frameshift Mutation Identified in Patients With Familial Partial Lipodystrophy

Adipocytes within white adipose tissue are uniquely adapted to storing large quantities of triglyceride in a single unilocular lipid droplet, providing a highly economical mechanism for surplus energy storage. Perilipin 1 is the most abundant phosphoprotein in adipocytes, where it constitutively associates with the phospholipid surface monolayer of the lipid droplet (1). Here it regulates lipases, particularly adipose tissue triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). ATGL and HSL catalyze the sequential hydrolysis of tri- and then diacylglycerol, releasing fatty acids and monoacylglycerol, which undergoes a final hydrolytic cleavage step by monoacylglycerol lipase (MGL) (2). Precise regulation of this process is a major factor in determining the efficacy of adipose tissue as a lipid buffer in the fed state and then subsequently as a source of lipid fuel in the fasting state or during exercise.

The importance of perilipin 1 function in human metabolism was very recently highlighted by the discovery of two heterozygous PLIN1 frameshift mutations affecting the COOH-terminus of the protein in patients with FPLD4 (3). Both mutations cosegregated with partial lipodystrophy, severe insulin resistance, hepatic steatosis, and severe dyslipidemia in three French families. Molecular characterization of these mutations demonstrated the inability of both mutants to inhibit basal lipolysis. In each case, this was attributable to the failure of the proteins to effectively prevent ABHD5 (αβ-hydrolase domain containing 5), a coactivator of ATGL, from activating ATGL and thus increasing basal lipolysis (4). Here, we report the identification, clinical characterization, and functional analysis of a third PLIN1 heterozygous mutation.

The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the U.K. National Health Service Research Ethics Committee. Each participant, or a parent in the case of minors, provided written informed consent; minors provided oral consent.

Genomic DNA was isolated from peripheral blood leukocytes. After excluding mutations in the coding regions and splice junctions of LMNA and PPARG, the coding regions and splice junctions of PLIN1 were amplified by PCR and sequenced as described previously (3).

Blood samples for biochemical assays were taken after an overnight fast. Patients underwent skin-fold measurements using calipers; results were expressed as the mean of two independent measurements and compared with previously reported reference ranges (5). Dual-energy X-ray absorptiometry (DEXA) (Lunar Prodigy, software version 12.20) was used to evaluate body composition.

Site-directed mutagenesis was performed on pCR-Blunt II-TOPO PLIN1 WT (3) using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) to generate the PLIN1 p.Pro439ValfsX125 (from here onwards referred to as PLIN1 439fs) mutant using the following set of primers 5′-CGGAGCGCAGAGCGTCGGGCGCCGTCCGCCGG-3′ and 5′-CCGGCGGACGGCGCCCGACGCTCTGCGCTCCG-3′. For the generation of stable cell lines, a N-terminally Myc-tagged PLIN1 439fs mutant sequence was subcloned using SalI restriction enzyme sites into the retroviral expression vector pBABE-puro (Clontech) harboring a puromycin resistance cassette. For transient transfections, the same sequence was subcloned into pcDNA3.1 vector using EcoRV-XbaI restriction sites. For bimolecular fluorescence complementation (BiFC) assays, pcDNA3.1 mycPLIN1-439fs-Yc was cloned in frame with the COOH-terminal Yc fragment in previously generated pcDNA3.1-Yc using EcoRV-BlpI (4). The following constructs pcDNA3.1-Yc, pcDNA3.1-Yn, pcDNA3.1-Yn-hABHD5, pcDNA3.1-hATGL(S47A)-Yc, and pcDNA3.1-MycPLIN1-398fs-Yc were previously generated as described by Gandotra and colleagues (3,4).

Stable cell lines were generated and maintained as described by Gandotra et al. (3). In order to study the effects of PLIN1 439fs in the presence of WT perilipin 1, a previously described 3T3-L1 preadipocyte stable cell line expressing pLXSN-Flag-PLIN1-WT (3) was subjected to a second round of retrovirus transduction with pBABE-puro myc-PLIN1 WT or mutants.

Quantitative real-time PCR mRNA was harvested and extracted using a RNeasy Mini Kit (Qiagen). cDNA synthesis was performed using the SuperScript II Reverse Transcriptase enzyme (Invitrogen). Quantitative real-time PCR was performed using an Applied Biosystems TaqMan Master Mix according to the manufacturer’s protocols. The following primer sets were used: hPLIN1 (forward, 5′-CCCCCTGAAAAGATTGCTTCT-3′; reverse, 5′-GGAACGCTGATGCTGTTTCTG-3′; probe, 5′FAM-CATCTCCACCCGCCTCCGCA TAMRA-3′) and PPIA (forward, 5′TTCCTCCTTTCACAGAATTATTCCA-3′; reverse, 5′-CCGCCAGTGCCATTATGG-3′; probe, 5′FAM-6ATTCATGTGCCAGGGTGGTGACTTTACAC-TAMRA-3′). Results were analyzed on an ABI PRISM 7900HT (Applied Biosystems) and normalized to housekeeping gene (Cyclophilin A) expression levels.

For patient adipose tissue protein expression analysis, tissue was homogenized in liquid nitrogen and directly lysed in RIPA buffer (Sigma-Aldrich) containing protease and phosphatase inhibitors (Roche). Protein was quantified using Bio-Rad DC protein quantification kit and 10 µg of protein lysate was diluted in NuPAGE 4X LDS sample buffer (Invitrogen) containing 0.05% β-mercaptoethanol and subjected to SDS-PAGE, following transfer onto a nitrocellulose membrane. Following transfer, membranes were washed in Tris-buffered saline containing 0.1% TWEEN-20 (Sigma-Aldrich), then blocked for 1 h at room temperature in 3% bovine serum albumin (BSA) or 5% powdered skimmed milk diluted in TWEEN-20. Membranes were further incubated with appropriate primary antibody diluted in blocking buffer for 16 h at 4°C. The following antibodies were used: perilipin 1 N-terminal antibody (GP29, Progen), perilipin 1 COOH-terminal antibody (Abcam), perilipin 2 (GP40, Progen), Myc (Millipore), Flag (Sigma-Aldrich), ABHD5 (Abnova), Calnexin (Abcam), or β-actin (Abcam). For cellular protein extracts, 50 µg of lysate was subjected to SDS-PAGE.

3T3-L1 preadipocytes stably expressing either WT or 439fs perilipin 1 were grown until confluency and treated with either DMSO as a control or 100 µg/mL cycloheximide, 10 μmol/L MG132, and 25 mmol/L NH₄Cl for indicated times before lysis.

For microscopy studies, cells were seeded into 12-well plates containing slides previously rinsed in 70% ethanol. Cells were harvested by rinsing three times in PBS and fixing with 4% formaldehyde diluted in PBS for 15 min at room temperature. Membrane permeabilization was achieved with 0.5% Saponin for 10 min, followed by blocking in 3% BSA, 0.05% TWEEN-20 PBS for 1 h. Afterward, cells were incubated with primary antibody diluted in blocking buffer for 16 h at 4°C. Then cells were washed three times in 0.1% BSA-PBS and incubated with fluorescent probe conjugated secondary antibody for 1 h at room temperature wrapped in foil. Where necessary, cells were stained for neutral lipid by rinsing three more times with 0.1% BSA-PBS and adding LipidTOX Deep Red reagent (Invitrogen) diluted in PBS (1:1,000) for 20 min at room temperature. Afterward, cells were rinsed in PBS and slides mounted using ProLong Gold antifade mounting reagent with DAPI (Invitrogen) for nuclear staining.

3T3-L1 preadipocytes were treated with 400 μmol/L oleic acid (Sigma-Aldrich) for 48 h and harvested for microscopy studies as described above. LipidTOX Deep Red was used to stain neutral lipids within lipid droplets, and anti-Myc primary antibody (1:500) dilution in 3% BSA, followed by goat anti-mouse Alexa Fluor 488 fluorescent secondary antibody, was used for perilipin 1 staining. Cell imaging was performed on a Zeiss LSM 510 META confocal microscope using the Zen software package (Carl Zeiss Microscopy GmbH). On average 10 Z-stack images were taken per sample using the ×63 objective, and lipid droplet volume was measured using Volocity 5 software (PerkinElmer).

Lipolysis experiments were performed as described by Gandotra et al. (4) in the presence of 6 μmol/L Triacsin C in order to prevent fatty acid re-esterification. Lipolysis was expressed as a ratio of the amount of radioactivity released into the medium over the total amount of incorporated radioactivity.

BiFC experiments and signal quantification were performed in COS-7 cells as described by Gandotra et al. (4) and Patel et al. (6). In cells expressing perilipin 1 (WT or mutants), ATGL (S47A)-Yc, and ABHD5-Yn, total yellow fluorescent protein (YFP) signal was normalized to control cells only expressing ATGL(S47A)-Yc and ABHD5-Yn. All components were normalized for equal protein expression in these experiments.

Quantitative data are presented as mean ± SEM. One-way or two-way ANOVA with post hoc Bonferroni analyses were performed on data at a minimum P < 0.05.

Proband A, a 41-year-old Caucasian woman (Fig. 1A, I.1), was referred to specialist physicians in Sydney in 1998 with a 10-year history of type 2 diabetes, complicated by diabetic retinopathy, extreme hypertriglyceridemia leading to recurrent pancreatitis, nonalcoholic fatty liver disease (NAFLD; visualized on ultrasound and computerized tomography), and hypertension. She also had new-onset exercise intolerance (exertional dyspnea without chest pain) and muscle cramping. Her diabetes was suboptimally controlled despite high-dose insulin therapy (>500 units daily). Insulin resistance was confirmed by a hyperinsulinemic-euglycemic clamp (glucose infusion rate 10.6 µm/min/kg fat-free mass). The patient had no history of polycystic ovarian syndrome (PCOS) and no difficulty conceiving as is apparent from four successful pregnancies. Her first pregnancy was uncomplicated, but her second child was delivered early because of large size for gestational age. The diagnosis of diabetes was made after her third pregnancy.

Her BMI was 31.2 kg/m² and waist circumference 93 cm. Lipoatrophy was most notable in the femorogluteal depot and lower limbs, with abdominal fat largely preserved (Supplementary Fig. 1). Skin-fold measurements (Supplementary Fig. 2) confirmed excess subcutaneous truncal fat and reduced peripheral fat. In addition, DEXA scan confirmed disproportionate fat loss on the limbs (Table 1). Her cardiovascular and respiratory examinations were normal. There were no dysmorphic features.

Biochemical investigations revealed severe hypertriglyceridemia and suboptimal glycemic control (Table 1). Her lipoprotein-a level was elevated at 680 mg/L (<300). and her leptin level was low (Table 1). An echocardiogram revealed a reduced ejection fraction of 30%, and a coronary angiogram showed an occluded right coronary artery. Nerve conduction studies and electromyography were reported as normal, with only occasional small atrophic fibers. A deltoid muscle biopsy was normal.

The patient’s eldest daughter, aged 38 years (Fig. 1A, II.1), was diagnosed with PCOS, dyslipidemia, fatty liver disease (ultrasound), hypertension, and type 2 diabetes at age 24 years. She was initially managed with metformin but required >500 units/day of insulin during the later stages of her first pregnancy, during which she weighed up to 120 kg (BMI 40.1 kg/m²). She has had four pregnancies, all of which were complicated by hypertension, necessitating hospital admission in two pregnancies. All four children are well and have not been tested for diabetes.

The proband’s oldest son, aged 37 years (Fig. 1A, II.2), has a lipodystrophic phenotype and a BMI of 29.1 kg/m² but has not been genetically tested or assessed for diabetes or dyslipidemia. Her middle son (Fig. 1A, II.3), aged 33 years, is centrally obese with a BMI of 39 kg/m², diabetes, and mild hypertriglyceridemia. Her youngest son, aged 18 years (Fig. 1A, II.4), has type 2 diabetes managed with oral medication and a BMI of 29.1 kg/m². The proband’s brother, aged 55 years (Fig. 1A, I.2), has a BMI of 25.3 kg/m², diabetes, a fat mass ratio (% fat trunk to % fat lower limb ratio) of 2.2, and skin-fold measurements consistent with a lipodystrophic phenotype.

In 2005, proband A underwent Roux-en-Y gastric bypass surgery in an effort to improve her glycemic control and hypertriglyceridemia. This resulted in a 20-kg weight loss, substantial reduction in insulin requirements (from 500 to 100 units/day), improved lipid profile, and reversal of cardiomyopathy on echocardiography. The patient’s daughter also underwent Roux-en-Y bypass surgery, resulting in a 55-kg loss of weight (BMI 22.4 kg/m²), cessation of insulin, and a subsequent twin birth and two further successful uncomplicated pregnancies. In the past 2 years, the proband has been diagnosed with a popliteal artery aneurysm, requiring surgical repair and vascular stenting. Her diabetes is currently treated with a lower dose of insulin and a very low−fat diet. Hypertriglyceridemia is controlled with gemfibrozil and maxepa. Her echocardiogram is now within normal limits, with an ejection fraction increasing from 30 to 59%.

Proband B, an unrelated 15-year-old boy of Caucasian origin, presented with acanthosis nigricans, hepatomegaly, and NAFLD (Fig. 1A). His BMI was 25.1 kg/m² (1.51 SD score) with central adiposity (waist to height ratio 0.54, normal <0.5) and a striking paucity of limb fat. Biochemical investigations showed severe hypertriglyceridemia with normal total cholesterol and low HDL cholesterol levels (0.4 mmol/L). An oral glucose tolerance test confirmed the presence of extreme insulin resistance. He had impaired glucose tolerance with a 2-h glucose level of 10.7 mmol/L and an astonishing peak insulin level of 12,396 pmol/L. His leptin level was low (Table 1). Liver biopsy confirmed significant hepatic steatosis.

The patient’s mother had been diagnosed with PCOS, treated with metformin since age 25 years, and required in vitro fertilization for her first pregnancy at age 28 years. She was subsequently diagnosed with type 2 diabetes requiring >200 units of insulin daily during pregnancy. She continued to have poor glycemic control, microalbuminuria, and NAFLD complicated by cirrhosis (on liver biopsy). Lipoatrophy was first recognized clinically in her early 40s affecting her limbs and buttocks, with mild central adiposity and prominent deltoid, gluteal, and calf muscles. She had coarse facial features and androgenic alopecia. She was lean (BMI 21.8 kg/m²) and her leptin level was low (Table 1). The patient's father was obese (BMI 37 kg/m²) with associated dyslipidemia but did not have features of severe insulin resistance (Fig. 1A).

Both probands were wild type (WT) for PPARG and LMNA gene sequencing. Candidate gene sequencing of all exons and splicing regions of the PLIN1 gene revealed a heterozygous deletion of two adjacent nucleotides (thymidine and guanine) within exon 9 (c.1298_1299delTG), resulting in a translational frameshift (Fig. 1B). The mutation is predicted to lead to the incorporation of 125 aberrant amino acids from amino acid position 439 onwards, thus producing an elongated protein (563 amino acids rather than 522) (Fig. 2A). The same mutation was present in both probands, although they are not known to be related. The mutation cosegregated with partial lipodystrophy, severe insulin resistance, severe dyslipidemia, and NAFLD in both kindreds (Fig. 1A). Gestational hypertension was prominent in affected women.

In order to determine whether the frameshift perilipin 1 mutant protein is expressed in vivo, samples of both visceral abdominal and subcutaneous adipose tissue were obtained from proband A (at the time of a medically indicated surgical procedure) and subjected to SDS-PAGE alongside subcutaneous fat samples from sex-matched control subjects (Fig. 2B). Both the WT and 439fs copy of perilipin 1 are detectable with an N-terminal epitope-targeted antibody; however, the higher molecular weight band corresponding to the 439fs protein was not detected by an antibody that targets a COOH-terminal epitope. Notably, the levels of WT and particularly the 439fs protein were reduced in the patient compared with control subjects.

In addition, in both murine and cell models, a decrease in perilipin 1 expression levels in adipocytes is typically associated with upregulation of perilipin 2 expression (7). In accordance with this, proband A manifested an increase in perilipin 2 expression in both visceral and subcutaneous adipose tissue when compared with control subjects (Fig. 2B).

In order to functionally characterize the effects of the PLIN1 439fs mutation, 3T3-L1 preadipocytes were stably transfected with retroviral vectors expressing pBABE-puro PLIN1 WT or the PLIN1 398fs or 439fs mutants, all bearing a N-terminal Myc tag. 3T3-L1 preadipocytes do not express endogenous perilipin 1 enabling us to directly compare their function in the absence of the potentially confounding influence of endogenous perilipin 1. The PLIN1 398fs mutation was characterized in previous work and served as a negative control in these studies (3). All of the above mutant proteins are predicted to have a disordered COOH-terminus following the frameshift. In keeping with the patient biopsy findings, expression levels of the 439fs mutant were lower than those of the WT protein despite equivalent mRNA expression levels (Fig. 3A and B). Expression of the 398fs mutant was even lower than that of the 439fs. In the presence of cycloheximide, the half-life of the 439fs protein was reduced compared with WT perilipin, which is known to be particularly stable when associated with lipid droplets (Fig. 3C and D) (8–10). The fact that coincubating the stable cell lines exposed to cycloheximide with a proteasomal inhibitor (MG132) modestly increased 439fs expression whereas the addition of a lysosomal inhibitor (NH₄CL) had no discernible affect (Fig. 3E) suggests that the 439fs perilipin 1 mutant is probably degraded via the proteasome. We also noted that the addition of MG132 increased endogenous perilipin 2 expression, which has been previously shown to be degraded via the proteasome (Fig. 3E), providing useful corroborative evidence for the efficacy and specificity of MG132 as a proteasomal inhibitor in these cells.

As perilipin 1 is critical for lipid accumulation in adipocytes (11), we assessed the impact of expression of the 439fs mutant on lipid droplet size in 3T3-L1 fibroblasts. After 48 h of exposure to medium containing oleic acid, perilipin 1 WT−overexpressing cells accumulated several large lipid droplets, whereas cells expressing the perilipin 1 439fs mutant contained a more heterogeneous population of significantly smaller lipid droplets (Fig. 4A and B). There was no difference in the ability of COOH-terminal perilipin 1 mutants to target to the lipid droplets (Fig. 4A). Perilipin 1 has previously been shown to exert its effect on lipid accumulation primarily through the inhibition of basal lipolysis rather than an increase in triglyceride synthesis (12). We thus hypothesized that the 439fs mutant may facilitate higher rates of basal lipolysis. In order to test this hypothesis, cells were incubated with oleic acid overnight, promoting lipid droplet accumulation, followed by a 4-h chase period in the presence of Triacsin C to inhibit free fatty acid (FFA) re-esterification. WT perilipin 1 significantly suppressed FFA release, whereas the 439fs perilipin 1 mutant did so less effectively, albeit to a greater extent than the 398fs perilipin 1 protein (Fig. 4C).

In an effort to reveal the molecular mechanisms underpinning the 439fs mutant’s failure to fully suppress basal lipolysis, we used BiFC assays to assess the interaction of the perilipin 1 439fs with ABHD5 (Fig. 5). As both the 439fs and 398fs mutants are expressed at lower levels than the WT protein, expression plasmids were titrated in order to achieve equal protein expression. YFP signal was reconstituted when ABHD5-Yn was expressed with either the WT or 439fs mutant, but not in the presence of the 398fs mutant, implying that 439fs is still able to interact with ABHD5. This is in agreement with previous studies where amino acids 382–429, equivalent to amino acids 380–427 in the human sequence of perilipin 1, were shown to be crucial for ABHD5 binding (13).

One could hypothesize that the conformational change induced by the COOH-terminal frameshift would allow the 439fs mutant to bind ABHD5 but might not necessarily prevent the subsequent interaction between ABHD5 and ATGL, as occurs with WT perilipin 1. We tested this hypothesis by assessing the ability of the perilipin 1 439fs mutant to inhibit direct interaction between ABHD5 and ATGL. In this assay, expression of ATGL (S47A)-Yc and ABHD5-Yn results in bright YFP fluorescence, which is reduced by ∼70% when either WT or the 439fs perilipin 1 mutant is expressed (Fig. 6). In contrast, expression of the 398fs mutant has no effect on the interaction between ATGL and ABHD5.

Finally, in an effort to mimic the in vivo consequences of the heterozygous mutation, the 439fs mutant was coexpressed with flag-tagged WT perilipin 1. 439fs protein levels were stabilized to some extent by the expression of a WT copy of perilipin 1 (Fig. 7A). The results suggest that increasing the amount of WT perilipin 1 protein increases lipid droplet volume (Fig. 7C and D), whereas coexpression of either the 439fs or 398fs mutants fails to further increase lipid droplet volume. In accordance with these data, measurement of basal lipolysis in these cell lines showed less inhibition of basal lipolysis by the 439fs perilipin 1 mutant than WT perilipin 1 even in the presence of WT perilipin 1 (Fig. 7E).

Lipodystophic syndromes are a rare but fascinating cause of insulin-resistant type 2 diabetes. The archetypal feature of all lipodystrophies is a lack of adipose tissue, which can either be partial or generalized. Nevertheless, the consequences of lipodystrophy, which include dyslipidemia, NAFLD, hyperandrogenism, PCOS in women, and accelerated cardiovascular disease, are remarkably similar to those usually associated with obesity as part of the metabolic syndrome (14). This observation in itself constitutes a key piece of evidence underpinning the so-called lipid overflow hypothesis, which posits that the capacity of mammalian adipose tissue to accommodate surplus lipid is finite, and that when this capacity is exceeded, lipids accumulate in ectopic sites, such as the liver and skeletal muscle, where they are instrumental in causing insulin resistance (15).

Within the last 15 years, several monogenic defects have been causally linked to human lipodystrophic syndromes, collectively advancing the understanding of human adipogenesis and adipocyte function and the systemic metabolic consequences of adipocyte dysfunction (16,17). More recently, our laboratory has reported the consequences of loss-of-function mutations in proteins implicated in the formation and functional regulation of adipocyte lipid droplets (3,18). Interestingly, CIDEC and PLIN1 encode lipid droplet proteins almost exclusively expressed in white adipocytes, emphasizing first their importance in determining the unique properties of adipocyte lipid droplets and second the whole-organism consequences of dysfunction of proteins primarily expressed in adipocytes (perilipin 1 is also expressed in adrenal cells, and both proteins can be expressed in steatotic hepatocytes).

The original description of patients with FPLD4 due to PLIN1 mutations was based on a description of six affected subjects. In order to better understand this novel phenotype, we have attempted to identify more affected subjects. These efforts enabled us to identify another two probands and four affected relatives with a different PLIN1 mutation (439fs) to those previously reported. The key features of patients with the 439fs mutant include lack of peripheral fat depots, severe insulin resistance, and extreme hypertriglyceridemia. These features are consistent with what was observed in patients with either the PLIN1 398fs or 404fs mutations. The effect of bariatric surgery on the metabolic phenotype of proband A was particularly striking. She experienced a 20-kg weight loss, with an 80% reduction in insulin requirements, improved lipid profile, reversal of cardiomyopathy, and improvement in muscle strength. Her daughter exhibited a similar improvement in metabolic profile. These metabolic improvements are consistent with case reports describing the response to bariatric surgery in other forms of partial lipodystrophy (19–21) and highlight the importance of relieving the energetic stress imposed upon their dysfunctional adipocytes. The beneficial symptomatic and functional effect of bariatric surgery on cardiac function in obese patients with cardiomyopathy has been described in a limited number of reports but not yet in patients with lipodystrophy (22).

Functional studies of the 439fs mutant suggest that it is associated with smaller lipid droplets and higher basal lipolytic rates. These data are consistent with previously reported studies by Garcia et al. (11), who observed less triglyceride accumulation in cells overexpressing an artificially generated perilipin 1 truncation mutant that truncates the protein at an amino acid very close to the site of our 439fs mutant, namely E427 stop. However, understanding why the 439fs mutant is associated with higher basal lipolytic rates is more complex than was the case for the previously reported human mutants (398fs and 404fs) as those fail to bind and stabilize ABHD5, whereas our studies suggest that the 439fs mutant does bind ABHD5, stabilize its expression, and sequester it from ATGL. One possible explanation might be the strikingly reduced expression of the 439fs mutant, which was observed in tissue samples from proband A and in transfected cells. The aberrant COOH-terminus of 439fs mutant protein is likely to be disordered, which would account for its propensity to degradation as occurs with many mutant proteins (23). Interestingly, even partial loss of perilipin 1 is sufficient to reduce fat mass as observed in Plin1 heterozygous knockout mice (7). Absent perilipin 1 expression in Plin1 null mice was also associated with concomitant upregulation of perilipin 2 (7,24). Thus, the observed upregulation of perilipin 2 expression in fat biopsies from proband A provides further evidence for reduced total perilipin 1 expression in vivo. We cautiously conclude that reduced perilipin 1 expression is likely to be an important factor in the phenotype observed in patients with the 439fs PLIN1 mutation. It is also very likely that the COOH-terminus of perilipin 1 has additional, as yet incompletely understood, biological functions that are likely to be impaired by this mutation.

In summary, we report the clinical and molecular characterization of a novel PLIN1 frameshift mutation in two kindreds with FPLD4. The severity of metabolic disease seen in affected patients further highlights the importance of precise lipolytic regulation in adipose tissue in human health. Finally, the highly beneficial response of two patients with this disorder to bariatric surgery emphasizes the importance of weight loss in these patients. Surgery lessens ectopic fat deposition and offers at least one useful management option for an otherwise devastating metabolic illness.

Acknowledgments. The authors are very grateful to the patients who participated in these studies and to the medical staff who assisted in their management. This work was supported by grants from the Wellcome Trust (D.B.S., S.O., I.B.), the U.K. National Institute for Health Research Cambridge Biomedical Research Centre, the Medical Research Council Centre for Obesity and Related Metabolic Diseases, and a Biotechnology and Biological Sciences Research Council CASE studentship (K.K.).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. K.K. performed functional studies. K.K., V.H.M.T., S.P., J.R.G., L.V.C., and D.B.S. contributed to data analysis and discussion and wrote the manuscript. V.H.M.T., Y.-H.C., S.S., and L.V.C. undertook clinical studies. K.K., W.B., and I.I. performed genotyping. S.G., M.M., K.L., and S.P. assisted with functional studies. V.S. provided functional insight into the expected consequences of the mutation. D.B.S. designed the study in collaboration with S.O. and I.B. All authors reviewed and edited the manuscript. D.B.S. and L.V.C. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.