A New Missense Mutation in the Leptin Gene Causes Mild Obesity

ORIGINAL E n d o c r i n e

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R e p o r t

A New Missense Mutation in the Leptin Gene Causes Mild Obesity and Hypogonadism without Affecting T Cell Responsiveness Pamela Fischer-Posovszky,* Julia von Schnurbein,* Barbara Moepps, Georgia Lahr, Gudrun Strauss, Thomas F. Barth, Jan Kassubek, Hannes Mu¨hleder, Peter Mo¨ller, Klaus-Michael Debatin, Peter Gierschik, and Martin Wabitsch Division of Pediatric Endocrinology and Diabetes (P.F.-P., J.v.S., M.W.), Department of Pediatrics and Adolescent Medicine (G.L., G.S., K.-M.D.), University of Ulm, 89075 Ulm, Germany; Institute of Pharmacology and Toxicology (B.M., P.G.), Departments of Pathology (T.F.B., P.M.) and Neurology (J.K.), University of Ulm, 89081 Ulm, Germany; and Children’s Hospital Wels (H.M.), 4600 Wels, Austria Objective: Leptin, a protein product of adipocytes, plays a critical role in the regulation of body weight, immune function, pubertal development, and fertility. So far, only three homozygous mutations in the leptin gene in a total of 13 individuals have been found leading to a phenotype of extreme obesity with marked hyperphagia and impaired immune function. Design: Serum leptin was measured by ELISA. The leptin gene (OB) was sequenced in patient DNA. The effect of the identified novel mutation was assessed using HEK293 cells. Results: We describe a 14-yr-old child of nonobese Austrian parents without known consanguinity. She had a body mass index of 31.5 kg/m2 (⫹2.46 SD score) and undetectable leptin serum levels. Sequencing of the leptin gene revealed a hitherto unknown homozygous transition (TTA to TCA) in exon 3 of the LEP gene resulting in a L72S replacement in the leptin protein. RT-PCR, Western blot, and immunohistochemical analysis indicated that the mutant leptin was expressed in the patient’s adipose tissue but retained within the cell. Using a heterologous cell system, we confirmed this finding and demonstrated that the side chain of Leu72 is crucial for intracellular leptin trafficking. Our patient showed signs of a hypogonadotropic hypogonadism. However, in contrast to the literature, she showed only mild obesity and a normal T cell responsiveness. Conclusions: These findings shed a new light on the clinical consequences of leptin deficiency. Congenital leptin deficiency should be considered possible in pediatric patients with mild obesity even if parents are lean and unrelated. (J Clin Endocrinol Metab 95: 2836 –2840, 2010)

ongenital leptin deficiency is a very rare cause of severe early-onset obesity (1). In 1997, it was first described in two severely obese cousins from a highly consanguineous family of Pakistani origin (2). Both children had undetectable levels of serum leptin due to a homozygous frameshift mutation in the LEP gene (⌬133G), which resulted in a truncated, unsecreted protein. Five further affected individuals homozygous for the same mutation have since been identified, all of Pakistani origin (1, 3, 4).

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Furthermore, a R105W substitution was described in a Turkish family (5–7). Recently, a N103K substitution has been reported in an obese Egyptian patient (8). Typical features of congenital leptin deficiency include morbid obesity, severe hyperphagia, hyperinsulinemia or type 2 diabetes, hypogonadotropic hypogonadism, T cell hyporesponsiveness, and neuroendocrine/metabolic dysfunction (1– 6, 8 –10). A hormone replacement therapy with recombinant leptin had enormous effects on food

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2010 by The Endocrine Society doi: 10.1210/jc.2009-2466 Received November 18, 2009. Accepted March 11, 2010. First Published Online April 9, 2010 * P.F.-P. and J.v.S. contributed equally to this work.

Abbreviation: BMI, Body mass index.

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Materials and Methods

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Pakistani patients Austrian patient

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All methods applied in this study are described in detail in the Supplemental Methods (published on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).

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Sequencing of leptin genomic DNA LEP sequencing was performed on genomic DNA using standard protocols. 6

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Histology and immunohistochemistry Immunohistochemistry was performed on paraffin-embedded tissue sections using a rabbit antihuman leptin antibody (Biovendor, Heidelberg, Germany).

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Ile Leu Thr Ser Ser Lys ATC CTG ACC TCA TCC AAG

C/C, congenital leptin deficiency T/C, heterozygous carrier deceased, genotype unknown

Patient

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Body fat percentage and abdominal fat volume Body fat percentage was assessed by dualenergy x-ray absorptiometry. Abdominal fat volumes of sc and visceral fat were assessed by a whole body magnetic resonance imaging scan.

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Leptin measurement in serum samples and medium supernatants Leptin RIA was performed using a commercially available kit (Linco/Millipore, Schwalbach, Germany).

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Wild-type Ile Leu Thr Leu ATC CTG ACC TTA

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Accelerometry Physical activity was measured on two consecutive weekdays using an ActiTrainer accelerometer (ActiGraph, LLC., Pensacola, FL).

Adiponectin

Indirect calorimetry FIG. 1. A 14-yr old girl with congenital leptin deficiency with a new mutation in the leptin gene. A, Photograph of the patient at the age of 14 yr. (B) The weight curve of the patient was compared with normal percentiles for girls and data from affected Pakistani patients extrapolated from the literature. (C) The complete LEP gene was sequenced. A homozygous missense mutation in codon 72 in exon 3 of the LEP gene was identified resulting in a L72S replacement. (D) Family pedigree. Symbols indicate the genotype: circles ⫽ female; squares ⫽ male, white ⫽ wild-type leptin sequence, black ⫽ mutant leptin sequence, black⫹white ⫽ heterozygous subjects. (E) Adipose tissue samples from the affected patient (top) and a control subject (bottom) were subjected to immunohistochemical analyis using a leptin antibody. Scale bar, 50 ␮m. (F) Serum of the patient with congenital leptin deficiency and a control subject was immunoprecipitated using a specific leptin antibody followed by Western blot analysis using a leptin antibody. As a control, supernatants were probed for adiponectin. The result of one representative experiment of 2 performed is shown.

intake, body weight, and fat mass as well as metabolic/ endocrine functions (3, 9, 10). We report here a 14-yr-old girl carrying a new homozygous mutation in the leptin gene (L72S) leading to undetectable leptin serum levels. In contrast to published patients, this patient showed only a mild obesity and a normal T cell responsiveness.

Resting energy expenditure was measured after a resting and fasting period by assessing the respiratory ratio via indirect calorimetry.

Test meal A mixed-diet breakfast was offered to the patient after an overnight fast. Total calories consumed were assessed.

Cold pressor test Blood pressure was taken after immersion of the right arm (hand up to elbow) in cold water (4 C).

Transfection studies and Western blot analysis Wild-type and mutant leptin cDNA were cloned and transiently transfected into HEK293 cells. Point mutations were introduced by site-directed mutagenesis according to the manufacturer’s instructions (Stratagene, La Jolla, CA). Protein lysates (50 ␮g) or undiluted media supernatants (20 ␮l) were subjected to Western blot analysis.

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Three-dimensional molecular structure The three-dimensional structure of wild-type leptin was analyzed and visualized using the PyMOL software (http:// www.pymol.org).

Proliferation assays and determination of cytokine concentrations Peripheral blood lymphocytes from the patient and controls were treated with IL-2, CD3/28, phytohemagglutinin, phorbol myristate acetate/ionomycin, or candidin. Medium supernatants were analyzed simultaneously by Bio-Plex technology. Incorporation of [methyl-3H]thymidine was measured on a ␤-counter.

Results Case history We report a 14-yr-old girl with a body mass index (BMI) of 31.5 kg/m2 (2.46 SD score) (Fig. 1A) and serum levels of leptin that were at the limit of detection of two different assays (Table 1). Absence of leptin was confirmed by immunoprecipitation (Fig. 1F). Our patient is the first child of two healthy, nonobese Austrians without known consanguinity. She was born with normal size and TABLE 1. Anthropometric, endocrinological, and metabolic characteristics of the patient Reference range

Variable Age (yr) Weight (kg) Height (cm) BMI (kg/m2) Body fat (%) Waist circumference (cm) Visceral abdominal fat (cm3) Subcutaneous abdominal fat (cm3 ) Leptin (ng/ml) Adiponectin (ng/ml) Total cholesterol (mmol/liter) Triglycerides (mmol/liter) ALT (U/liter) AST (U/liter) Thyrotropin (U/liter) Free thyroxine (ng/dl) Cortisol at 0900 h (␮g/dl) Corticotrophin at 0900 h (pg/ml) GnRH stimulation test Basal serum FSH (U/liter) Peak serum FSH (U/liter) Basal serum LH (U/liter) Peak serum LH (U/liter) Arginine test Maximal stimulated GH levels (ng/ml) Oral glucose tolerance test Fasting glucose (mg/dl) Fasting insulin (mU/liter) 2-h glucose (mg/dl) 2-h insulin (mU/liter)

13.75 88.9 168.0 31.5 50.1 98.7 4132 11943 0.4 6.5 208 255 72 35 1.54 0.85 10.7 20.0 ⬍0.1 0.22 0.27 1.08 1.25 95 45.8 124 312.6

2.0 –5.6 5.0 –7.5 ⬍200 ⬍203 ⬍35 ⬍50 0.53–3.59 0.9 –1.6 6.2–19.4 7.2– 63.3 ⬍11.9 2.1–11.1 ⬎8.0 74 –100 2.6 –24.9 ⬍140 ⬍50.0

ALT, Alanine aminotransferase; AST, aspartate aminotransferase.


weight (3440 g) but gained weight rapidly thereafter leading to obesity at the age of 3 months. The rapid weight gain continued despite a low-caloric diet started at the age of 9 months (Fig. 1B). Genetics Sequencing of the LEP gene revealed an unknown homozygous transition (TTA to TCA) in exon 3 of the LEP gene resulting in a L72S replacement in the leptin protein (Fig. 1C). Both parents and the sister were heterozygous carriers (Fig. 1D). The mutation was not detected in 720 alleles of 360 healthy schoolchildren from Ulm. Immunohistochemical analysis revealed expression of the LEP gene product (Fig. 1E). RT-PCR and Western blot analysis further confirmed this finding (Supplemental Fig. 1). Endocrine and metabolic changes The patient showed signs of hypogonadotropic hypogonadism with an arrest of pubertal development at Tanner stage PH3 and B3 after a spontaneous onset of puberty and a blunted response in a GnRH stimulation test. Nevertheless, her bone age matched her chronological age. Her response to a GH stimulation test was blunted, despite her growing within the upper range of her familial target height. All other aspects of her hypothalamic-pituitary axis were normal apart from slightly lowered free T4 levels. She showed a hyperinsulinemia, increased transaminases, and dyslipidemia, but her blood pressure lay within the normal range (Table 1). In a cold pressor test, the patient showed a suppressed reaction with no changes in systolic or diastolic blood pressure and an increase in heart rate of only nine beats per minute. Eating behavior Starting from early infancy, the patient had been put on a low-caloric diet, which she adhered to until today. Her current recorded average caloric input per day lay between 1000 and 1300 kcal/d as assessed by a 3-d weighed-food protocol under which the patient reported no feelings of hunger. However, when the patient was offered a single test meal and allowed to eat ad libitum, she rapidly consumed 1600 kcal. Daily activity and energy expenditure Compared with a control group (11), the daily activity of the patient assessed by mean counts per minute lay within the average (405 counts per minute). Her resting metabolic rate (4.3 kJ/d and 35 kcal/kg fat-free mass/d) was not reduced in comparison with a control group (12). Immune function Our patient had no history of recurrent infections and no changes in T lymphocyte counts (1587/␮l) or in T lym-



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creted into the circulation. Here we show that the secretory behavior of the patient’s leptin protein was not depenMedium Leptin Leptin dent on the ␥-hydroxyl group of Ser72 Medium and that leptin secretion seems to Lysate Leptin Leptin Lysate depend on side-chain hydrophobicity β-Actin β-Actin rather than side-chain size of the position 72 residue. FIG. 2. Secretion of mutant leptin is impaired. A, Wild-type or mutant leptin cDNAs were Despite leptin deficiency, our patient cloned into pcDNA3.1. HEK293 cells were transiently transfected with empty vector, or vector was remarkably less obese than all deencoding wild-type (wt, Leu72) or mutant leptin (mutant, Ser72), or left untreated. After 48 h, culture media were collected and protein lysates were prepared. Leptin immunoreactivity was scribed patients [31.5 vs. ⬎35 kg/m2 in examined by Western blot in media (top) and cell lysates (bottom). ␤-Actin served as a children and ⬎50 kg/m2 in adults (1–7); loading control. (B) To address the functional role of Leu72 side chain in the wild-type see also Supplemental Fig. 4]. Our paprotein, the residue was replaced by other amino acids, alanine (Ala), valine (Val), isoleucine (Ile), and threonine (Thr). Leptin immunoreactivity was examined by Western blot in media tient showed an extremely low energy (top) and cell lysates (bottom). ␤-Actin served as a loading control. intake in everyday life despite an in⫹ ⫹ creased consumption of calories in a phocyte subpopulation ratios (CD4 /CD8 ratio, 1.952; reference range, 1.0 –2.6). Her lymphocytes showed nor- test meal. Even if one takes into account a substantial mal proliferation and secretion of Th1 and Th2 cytokines underreporting, this observation suggests that despite lepin response to antigen-specific and polyclonal stimuli tin deficiency, it is possible to control energy intake and (Supplemental Fig. 2) compared with lymphocytes from thus to prevent extreme obesity. The question remains healthy age- and BMI-matched controls. how our patient was able to restrict her caloric input in contrast to other patients, among whom daily energy inLeptin cDNA cloning and expression take lay well above 2000 kcal/d (7, 10). Despite a residual Wild-type and mutant leptin cDNA were cloned and activity of leptin, one possible explanation is that the patransiently expressed in HEK293 cells. After 48 h, the tient’s parents provided a favorable environment by conwild-type but not the mutant protein was readily detected trolling the patient’s eating behavior vigorously from early in the culture medium by ELISA (wild type, 184 ⫾ 3 ng/ml; infancy onward, where future feeding habits are estabmutant, ⬍0.5 ng/ml) and Western blotting (Fig. 2A). In lished (15). A further explanation might be related to the contrast, both wild-type and mutant leptin was detected different genetic backgrounds of different patients, which in cell lysates. Furthermore, the mutant protein was could render the current patient more resistant to the effects much more abundant in the lysates than its wild-type of leptin deficiency, as indicated by experiments in mice (16, counterpart. Biological activity of the mutant protein 17). has not been tested so far. Leu72 is highly conserved Nevertheless, the patient was still too obese for the reamong species (13, 14). To address the functional role ported energy intake, indicating that energy expenditure of Leu72 side chain, we replaced it by other aliphatic might also play a role. However, in accordance to Farooqi amino acids, isoleucine, valine, and alanine, and by the et al. (3), we did not find a reduced resting energy expensecond hydroxyl residue, threonine. Figure 2B shows diture in our patient. that the Ile72 and Val72 variants were secreted into the Besides its function in the regulation of body weight, medium. However, although the Ile72 mutant was inleptin plays an important role in immune and inflammadistinguishable from the wild-type Leu72 protein, more tory responses. Ob/ob mice show an impaired T-cell imof the Val72 protein was retained in the transfected cells munity (18). Likewise, leptin-deficient children have an compared with the more hydrophobic Leu72 and Ile72 increased risk for infections, reduced lymphocyte counts variants. Both the Ala72 and the Thr72 mutant were (3, 6), altered T lymphocyte subpopulation ratios, and very similar to the Ser72 but clearly distinct from the severely impaired lymphocyte function (3). Val72 variant in terms of their secretory behavior. A This immunophenotype, however, was reported only three-dimensional structure of wild-type leptin and furin children (3, 6). Thus, we assume a so far undescribed age ther information is given in Supplemental Fig. 3. dependency of this phenotype. There are no data in the literature addressing lymphocyte function of adult patients with leptin deficiency. In accordance with our preDiscussion sumption, lymphocyte counts and subpopulations were As described for the first known leptin mutation (2), the normal among the adult patients (3). Furthermore, adult mutated leptin of our patient was expressed but not se- patients with severe congenital lipodystrophy, character-

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ized by a paucity of leptin-synthesizing fat cells, show no reduction in T lymphocyte subpopulations or stimulated T cell proliferation (19). Distinct differences exist between adults and children with leptin deficiency concerning thyroid function (2, 6). In addition, whereas there are no signs of puberty in young adult patients, one patient spontaneously entered puberty at an age of 36 yr (10). Unsurprisingly, our adolescent patient showed an arrest of pubertal development but, in contrast to the adult patients (5), also hardly any response to stimulation of LH and FSH. So apparently, our patient combines features of the pediatric and the adult phenotype showing alterations in thyroid function and pubertal development but none of the typical immunological changes. This casts a new light on the natural course of leptin deficiency in humans. In conclusion, we show here a child of lean parents without known consanguinity with congenital leptin deficiency with only moderate obesity. Leptin is a wellknown regulator of energy balance and body weight. However, the history of our patient suggests that overweight and fat mass might be regulated by early behavioral training and programming, even despite an unfavorable genetic background like leptin deficiency.

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Acknowledgments We thank A. Killian, S. Horenburg, and A. Moss for excellent technical assistance; S. Helm and S. Niedersu¨ss-Markgraf for indirect calorimetry; V. Aigner and H. Mayr for providing blood samples from age- and BMI-matched control patients; T. Illig and P. Singmann for MALDITOF and TaqMan analysis; and H.-P. Mu¨ller for assistance with magnetic resonance imaging. Address all correspondence and requests for reprints to: Prof. Dr. Martin Wabitsch, Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics and Adolescent Medicine, University of Ulm, Eythstrasse 24, 89075 Ulm, Germany. E-mail: [email protected]. This work was supported by the German Federal Ministry of Education and Research (National Genome Research Network, BMBF 01GS0824 and BMBF 01GI0851) and the Centre of Excellence Baden-Wuerttemberg “Metabolic Disorders,” financed by the State Baden-Wuerttemberg to M.W. This project is supported by the European Social Fund and by the Ministry of Science, Research, and Arts Baden-Wuerttemberg (to P.F.-P.). Disclosure Summary: The authors have nothing to disclose.

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