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Phenotypic Heterogeneity in Body Fat Distribution in Patients with Congenital Generalized Lipodystrophy Caused by Mutations in the AGPAT2 or Seipin Genes

Division of Nutrition and Metabolic Diseases, Center for Human Nutrition, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Address all correspondence and requests for reprints to: Abhimanyu Garg, M.D., Chief, Division of Nutrition and Metabolic Diseases University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Y3-222 Dallas, TX 75390-9052. E-mail: abhimanyu.garg{at}utsouthwestern.edu.

	Abstract

Top Abstract Introduction Patients and Methods Results Discussion References

 
Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive syndrome characterized by extreme paucity of adipose tissue since birth, acanthosis nigricans, severe insulin resistance, marked hypertriglyceridemia, and early-onset diabetes mellitus. Recently, we reported mutations in the 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) gene in CGL pedigrees linked to chromosome 9q34 (CGL1 subtype), and mutations in the Seipin gene were reported in pedigrees linked to chromosome 11q13 (CGL2 subtype). Whether the two subtypes have differences in body fat distribution has not been investigated. We, therefore, compared whole-body adipose tissue distribution by magnetic resonance imaging in 10 CGL patients, of whom seven (six females, one male) had CGL1 and three (two males, one female) had CGL2. Both subtypes had marked lack of metabolically active adipose tissue located at most sc, intermuscular, bone marrow, intraabdominal, and intrathoracic regions. Paucity of mechanical adipose tissue in the palms, soles, orbits, scalp, and periarticular regions was noted in CGL2, whereas it was well preserved in CGL1 patients. We conclude that CGL patients with Seipin mutations have a more severe lack of body fat, which affects both metabolically active and mechanical adipose tissue, compared with patients with mutations in the AGPAT2 gene.

	Introduction

Top Abstract Introduction Patients and Methods Results Discussion References

 
CONGENITAL GENERALIZED LIPODYSTROPHY (CGL), or Berardinelli-Seip syndrome, is a rare autosomal recessive disorder [Online Mendelian Inheritance in Man (OMIM) no. 269700] characterized by extreme paucity of adipose tissue since birth, acanthosis nigricans, severe insulin resistance, marked hypertriglyceridemia, and early-onset diabetes mellitus (1, 2). Additional characteristic features of this disorder include accelerated growth during childhood, increased basal metabolic rate and voracious appetite, acromegaloid appearance (enlarged mandible, hands, and feet), hepatosplenomegaly, umbilical hernia, and in women, clitoromegaly and hirsutism (2). Recently, there has been considerable progress in understanding the genetic basis of this disease. We had previously mapped a CGL locus on chromosome 9q34 (3) and have recently reported several different mutations in the gene encoding the enzyme 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) (4) in pedigrees linked to this locus (CGL1 subtype). AGPAT2 belongs to the family of acyltransferases and catalyzes acylation of lysophosphatidic acid to phosphatidic acid, an essential reaction in the biosynthetic pathway of triacylglycerol and phospholipids from glycerol-3-phosphate (5, 6). Magre and coworkers (7) mapped another locus to 11q13 chromosome and reported mutations in the Seipin or Berardinelli-Seip congenital lipodystrophy 2 (BSCL2) gene in patients mostly of Lebanese and Norwegian origin (CGL2 subtype). BSCL2 encodes a protein called seipin, the biological function of which remains unknown. There is no homology between AGPAT2 and seipin proteins. We had previously described a characteristic pattern of body fat distribution by using whole-body magnetic resonance imaging (MRI) in three women with CGL (8). Our recent studies showed that all of them had mutations in the AGPAT2 gene (4). Detailed studies of body fat distribution in patients with CGL2 due to Seipin mutations have not been reported. Therefore, we compared adipose tissue distribution using whole-body MRI to investigate phenotypic differences in patients with CGL1 and CGL2 subtypes.

	Patients and Methods

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients

We studied 11 patients with CGL, seven with CGL1 and four with CGL2, at the University of Texas Southwestern Medical Center, Dallas. The study protocol was approved by the Institutional Review Board, and all subjects provided informed written consent.

Among the seven CGL1 patients, six were female and one was male, whereas among CGL2 patients, two were male and two were female (Table 1). Six of the seven CGL1 patients were of African origin and were found to harbor a splice site mutation, IVS4–2A>G (Q196fsX228), in the AGPAT2 gene. Patients CG400.8 and 8200.3 were homozygous for this mutation, whereas affected patients from pedigrees CG600 and 800 were compound heterozygotes having additional missense and frameshift mutations, 683T>C (L228P) and 377insT (L126fsX146), respectively (4). CG700.4, who was of European origin, was also a compound heterozygote with a 406G>A (G136R) missense mutation and a 504delGA (V167fsX183) frameshift mutation (4). Three of the four CGL2 patients were of Chinese origin. Affected individuals from pedigree CG1300 showed a homozygous IVS6–2A>G (F224Y225-Q271fsX288) splice site mutation in the Seipin gene (9). CG1100.3, also of Chinese origin, had a heterozygous single nucleotide (guanine) insertion at position 1126 in exon 7 (1126insG; G271fsX283), but we were unable to find another mutation in the coding region, splice site junctions, or the 1.5-kb 5' proximal region of the Seipin gene in this subject. We found no substantial variation in the AGPAT2 gene in this subject. CG6800.3, who was of Lebanese origin, was found to be homozygous for a five-nucleotide deletion in exon 4, 659delGTATC (F105fsX111) of the Seipin gene (9). Magre et al. (7) also observed the same mutation in CGL patients ascertained from Lebanon.

Whole-body MRI was also obtained from a healthy 30-yr-old woman and a 12-yr-old boy for comparison.

Anthropometry

Height and body weight were measured by standard procedures. Body volume was measured underwater with a Whitmore Volumeter (Whitmore Enterprises, San Antonio, TX). Residual lung volume was measured by the helium dilution technique, and the proportion of body fat was estimated by Siri’s equation (10).

MRI

MRI was performed using a 1.5-Tesla imaging device (Phillips Medical Systems, Best, The Netherlands). The entire body was surveyed employing contiguous axial 10-mm slices and a relatively T1-weighted spin echo sequence (repetition time/echo time of 580/8 ms or 600/20 ms), a quadrature body coil, 384 x 512 matrix and a 45-cm field of view. The test could not be performed on patient CG1100.3 who was young and had significant mental retardation.

Fat can be easily identified on MRI because of its short T1 and long T2 proton relaxation time compared with other tissues, such as muscle (11), leading to a high signal intensity (increased brightness) on T1-weighted images. Although there are a few other substances that may appear bright on T1-weighted images (i.e. subacute hemorrhage, proteinaceous fluid, and injected paramagnetic contrast material), these would not be expected in the regions of the body where fat is normally located. Hence, in this study there is negligible probability that areas identified as body fat on MRI are composed of any other material.

Biochemical analysis

Fasting blood samples were obtained on at least two consecutive days. Serum glucose was measured by the glucose oxidase method with a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA). Serum leptin levels were determined by RIA using commercial kits (Linco Research, Inc., St. Charles, MO). Serum chemistry was measured as a part of the systematic multichannel analysis by a commercial laboratory (SmithKline Beecham Clinical Laboratories, Dallas, TX).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Table 1 summarizes the anthropometric data and other clinical features of patients with both subtypes of CGL. Physical examination revealed loss of sc fat from the face, extremities, and trunk in all the patients. However, fat on the palms and soles was well preserved in CGL1 patients with AGPAT2 mutation but was markedly deficient in CGL2 patients with the Seipin mutation. Decreased total body fat and low serum leptin levels were noted in all the patients.

Whole-body MRI revealed that all seven patients with CGL1 had a similar, homogenous and unique pattern of body fat distribution. Likewise, the three CGL2 patients had similar body fat distribution. There were many similarities in body fat distribution between the two subtypes, but also significant differences were noted. Extreme paucity of sc fat was noted in both the CGL subtypes (Figs. 1–3). Similarly, both subtypes had a negligible amount of fat in the intraabdominal (omental, mesenteric, and retroperitoneal) (Fig. 2, A and B), and intrathoracic (retrosternal, epicardial, and superior mediastinal) regions. Bone marrow and intermuscular fat was also markedly reduced in all CGL patients compared with normal patients (Fig. 3).

View larger version (119K): [in this window] [in a new window]   FIG. 1. A and D, Axial and sagittal MRI through the orbits in a 31-yr-old female patient with CGL1 (CG800.7) showing normal amounts of fat in the retroorbital region, temporal region, and sc area of the scalp. Interestingly, sc fat in the submental, anterior, and posterior neck regions is nearly absent. B and E, Similar image in 11-yr-old female, a patient with CGL2 (CG1300.5) showing near total lack of retroorbital fat as well as sc fat from the temporal region, scalp, and neck. C and F, Similar images from a healthy 30-yr-old woman showing normal fat distribution in the retroorbital, temporal, scalp, submental, and cervical areas.

View larger version (88K): [in this window] [in a new window]   FIG. 2. A, Axial T1 weighted MRI at the level of kidneys in a 13-yr-old male patient with CGL1 (CG600.6) showing marked loss of sc and intraabdominal (mesenteric, perinephric, and omental) fat. Increased signal intensity in the liver is consistent with hepatic steatosis. B, Corresponding transabdominal MRI in a 17-yr-old male patient with CGL2 (CG1300.3) showing similar features. C, Corresponding transabdominal MRI in a 12-yr-old healthy boy showing normal sc and intraabdominal fat distribution. D, Axial section through the hip joint in the patient CG600.6 showing near complete absence of sc fat except for minimal amounts in the posterior and lateral sc region. Medullary signal intensity is decreased in the femoral heads due to lack of bone marrow fat. Periarticular fat around the hip joint is present in normal amounts posterior and lateral to the femoral heads (P indicates posterior region). E, Corresponding section in patient CG1300.3 showing near total absence of sc and bone marrow fat. Periarticular fat around the hip joint is also markedly reduced. F, Corresponding section through the hip joint in a healthy 12-yr-old boy showing normal sc, intramedullary, and periarticular fat distribution.

View larger version (143K): [in this window] [in a new window]   FIG. 3. Transaxial and sagittal T1 weighted MRI at the level of the thigh, knee joint, and foot in a 31-yr-old female patient with CGL1 (CG800.7) (A, D, and G, respectively), an 11-yr-old female patient with CGL2 (CG1300.5) (B, E, and H, respectively), and a healthy 30-yr-old woman (C, F, and I, respectively). Sections through the thigh reveal decreased sc, intermuscular, and bone marrow fat in both the CGL patients compared with normal. Periarticular fat around the knee joint, including the prefemoral fat anteriorly and popliteal fat posteriorly, seems normal in the CGL1 patient (D) but is reduced in the CGL2 patient (E). Similarly, sc fat in the sole is preserved in the CGL1 patient 1 (G), whereas it is reduced in the CGL2 patient (H).

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
Patients with CGL have a remarkable absence of sc adipose tissue as evident from previous clinical reports (1). This has been confirmed by autopsy and MRI studies that further revealed that fat loss also involved the bone marrow, parathyroid gland, and intraabdominal and intrathoracic regions (8, 12). Nevertheless these studies also revealed that adipose tissue was preserved in certain anatomical regions, including the orbits, crista galli, buccal region, tongue, palms and soles, scalp, perineum, periarticular regions, and epidural areas. We had therefore proposed that two distinct types of adipose tissue may be present in humans: metabolically active adipose tissue, which participates in storage and release of energy in response to various hormones, and mechanical adipose tissue, which is relatively inactive metabolically and may serve mainly supportive or protective functions. For example, orbital adipose tissue protects eyeballs, and adipose tissue in the scalp, palm, and sole as well as around major joints provides cushion and shock absorption. Adipose tissue in these areas serves only a mechanical function, and we had hypothesized previously that the genetic defect causing CGL leads to severe lack of metabolically active adipose tissue, whereas the mechanical adipose tissue is well preserved (8). Since these observations were made, mutations in two genes, AGPAT2 (4) and Seipin (7), have been noted in patients with CGL1 and CGL2, respectively. Previous MRI and autopsy studies were limited to patients with AGPAT2 mutations (4, 8, 12). The pattern of body fat distribution in CGL2 patients with Seipin mutation has not been previously investigated. Therefore, we compared body fat distribution by MRI in the two subtypes of CGL patients to identify phenotypic differences.

Comparison of body fat distribution using MRI studies revealed similarities in the extreme lack of body fat from sites where metabolically active adipose tissue is present, such as most of the sc, intermuscular, bone marrow, intraabdominal, and intrathoracic regions. However, some significant differences were also noted between the two subtypes. CGL2 patients had lost a significant amount of fat from sites where predominantly mechanical adipose tissue is present, such as the scalp, retroorbital region, periarticular regions, palms, and soles, even though the adipose tissue in these sites was well preserved in CGL1 patients. Because the fat loss in CGL patients of both genders and all ethnicities is noted at birth, and is not known to change with age, it is highly unlikely that age, gender, or ethnic differences are responsible for the observed differences between the two CGL subtypes.

The clinical significance of fat loss from sites of mechanical adipose tissue is unclear. Some of the CGL2 patients (CG1300.3, CG6800.3) had severe calluses on their feet, presumably due to loss of fat from the soles. Similar calluses have been noted in patients with acquired generalized lipodystrophy who have also lost adipose tissue from the soles of the feet (13). Whether patients with CGL2 are also predisposed to arthropathy due to loss of periarticular fat and to eye dysfunction due to loss of retroorbital fat needs to be determined by long term follow-up.

Even though two distinct genetic defects are now known to cause CGL, it is still not clear how these mutations cause the various physical and metabolic abnormalities seen in this syndrome. As mentioned earlier, AGPAT2 catalyzes an essential reaction in the biosynthetic pathway of glycerophospholipids and triglycerides in eukaryotes (5, 6). Mutations in the gene encoding for this enzyme presumably affect triglyceride synthesis and storage in the adipocytes. There are five known isoforms of AGPAT encoded by different genes, although only two (AGPAT1 and AGPAT2) are well characterized (5). AGPAT1 mRNA is ubiquitously expressed in human tissues whereas AGPAT2 is more restricted in tissue expression (5, 14, 15). We have previously shown that in the human omental adipose tissue, AGPAT2 mRNA was expressed at least 2-fold more than AGPAT1, whereas the other isoforms, AGPAT3, -4, and -5 were undetectable (4). Relative expression of various AGPAT isoforms in adipose tissue from different anatomic locations has not been studied. Whether preservation of mechanical adipose tissue in CGL1 patients is due to differential expression of AGPAT isoforms in the different fat depots remains to be investigated. Seipin encodes a 398-amino-acid protein that is homologous to the product of a murine guanine nucleotide-binding protein, 3-linked gene. The biological function of seipin remains unknown. On the basis of high expression of Seipin mRNA in the brain and weak expression in the adipose tissue, Magre et al. (7) proposed a primary defect in the hypothalamic-pituitary axis. However, our observations suggest that the defect in Seipin may affect the differentiation of both mechanical and metabolic adipocytes at an early stage, probably at the level of preadipocytes or stem cells (2, 16).

We conclude that CGL2 patients with mutations in the Seipin gene have more severe lack of body fat, which affects both metabolically active and mechanical adipose tissue, compared with CGL1 patients with mutations in the AGPAT2 gene, in whom mechanical adipose tissue is well preserved.

	Acknowledgments

 
We are grateful to Drs. Jonathan Darling and Mark Lipson for patient referral, Dr. Peter Snell for hydrodensitometry, Jerry Payne for MRI studies, Angela Osborn for technical assistance, and the nursing and dietetic services of the General Clinical Research Center for patient care.

	Footnotes

 
This work was supported in part by the National Institutes of Health Grants R01-DK54387 and M01-RR00633 and grants from the Southwest Medical Foundation.

Abbreviations: CGL, Congenital generalized lipodystrophy; MRI, magnetic resonance imaging.

Received May 13, 2003.

Accepted July 30, 2003.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Simha, V. Articles by Garg, A. Search for Related Content PubMed PubMed Citation Articles by Simha, V. Articles by Garg, A.