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CASE REPORT |
Departments of 1 Endocrinology and Metabolism and 2 Pediatric Institute, Children's Hospital of Fudan University, Shanghai 200032, China3 Department of Pediatrics, Hospital for Children and Adolescence, University of Leipzig, Leipzig, Wieland Kiess D-04317, Germany and4 Department of Pathology, Children's Hospital of Fudan University, Shanghai 200032, China
(Correspondence should be addressed to F Luo who is now at Department of Pediatric Endocrinology and Metabolism, Children's Hospital of Fudan University, 183 Feng Lin Road, Shanghai 200032, China; Email: doctorlfh{at}yahoo.com.cn)
Abstract
Context: Congenital generalized lipodystrophy (CGL) is a rare and heterogeneous disease of autosomal recessive inheritance. Until now, no genetic findings had been reported in Chinese patients with CGL.
Objective: To analyze Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) and 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2) gene variation in a Chinese boy with CGL and his family.
Design, setting, and participants: All exons of BSCL2 and AGPAT2 with adjacent intron–exon junctions were analyzed using direct sequencing.
Main outcome measures: Sequences of each exon and nearby intron of the BSCL2 and AGPAT2 genes of the family members were compared with the gene bank genomic sequences.
Results: DNA sequence analysis of the entire coding regions and surrounding uncoding regions disclosed a novel homozygous G
T mutation at nucleotide 909 in exon 5 of the BSCL2 gene in the affected child. A heterozygous state of the GT mutation of the BSCL2 gene was also found in other family members. This mutation predicts the substitution of glutamic acid at codon 189 by the stop codon (Glu189X or E189X). No variation was found in the AGPAT2 gene.
Conclusion: E189X is a novel BSCL2 gene mutation that contributes to CGL formation in a family of Chinese origin.
Introduction
Congenital generalized lipodystrophy (CGL; Online Mendelian Inheritance In Man (OMIM) 269700 [OMIM] ), originally described in Brazil by Berardinelli in 1954 and reviewed by Seip in 1959, became known as Berardinelli–Seip syndrome. It is a rare autosomal disease characterized by near-complete absence of adipose tissue already apparent at birth or in early infancy, resulting in striking muscular appearance (1). The pattern of body fat loss is unique, i.e., near-total absence of metabolically active adipose tissue in subcutaneous, intra-abdominal, intra-thoracic, and bone marrow regions, but preservation of mechanical fat in the orbits, palms, soles, scalp, perineum, and periarticular regions (2). Even during infancy, patients show extreme hyperinsulinemia, hypertriglyceridemia, and hepatomegaly because of hepatic steatosis. More clinical features include widespread acanthosis nigricans, acromegaloid appearance, umbilical hernia, and, in women, clitoromegaly, hirsutism, oligo/amenorrhea, and polycystic ovaries. Diabetes mellitus develops mostly during pubertal years and is ketosis resistant (3). Other reported features involve mild mental retardation, hypertrophic cardiomyopathy, nephropathy, and nubecula (4, 5).
Recent studies have reported mutations in two unrelated genes – BSCL2 and AGPAT2 – in patients with CGL linked to human chromosomes 11q13 and 9q34 respectively (3, 6, 7). Other genes (or a single gene) might be inferred from genetic studies of a few families where the disease is not linked to the two identified loci (4, 8). The first gene, AGPAT2, scanned in 1999, encodes 1-acylglycerol-3-phosphate O-acyltransferase 2 that catalyzes acylation of lysophosphatidic acid to phosphatidic acid during the biosynthesis of glycerophospholipids and triglyceride from glycerol-3-phosphate (9, 10). The other affected gene, BSCL2, first identified in 2001, encodes seipin, a 398 amino acid protein (11). Although it might be understood how AGPAT2 mutations result in a lack of body fat (10), the mechanisms by which BSCL2 gene defects cause lipodystrophy remain obscure.
BSCL2 mutations were found in patients from Europe, the Middle East, and Japan (12). AGPAT2 mutations were found predominantly in African ancestry. However, no information is available about putative alterations of these genes in Chinese subjects with CGL. The present study is the first report on gene analysis of CGL in a Chinese family.
Subjects and methods
Study subjects
A 7-year-old Chinese CGL boy was the third child of consanguineous parents from an eastern Chinese province. The first two children had been stillborn. There was no other particular birth history except low birth weight with 2.25 kg after 40-week gestation. Five healthy unrelated children (two males, three females, body mass index (BMI) 16.3–22.3 kg/m2) were recruited as controls. The patient presented with complete lack of generalized subcutaneus fat at birth, accelerated growth, voracious appetite, hypertrophy of all limb muscles, acanthosis nigricans in the neck, groin and oxter, enlarged hands and feet, hirsutism in the back, hepatosplenomegaly, crassitude of the penis, nubecula, a cardiac murmur, and mild mental retardation (Fig. 1A–E). He was admitted for gum bleeding repeatedly. The height was 130.5 cm (90–97 percentile) and the weight was 28.0 kg (>90 percentile) with BMI 16.4 kg/m2 at the current presentation. Laboratory examinations showed evidence of liver dysfunction (glutamate-pyruvate transaminase 64 U/l and glutamic oxaloacetic transaminase enzyme 73 U/l), high levels of bilirubin (total bilirubin 26.6 µmol/l and direct bilirubin 20.7 µmol/l), dyslipidemia with slight increased triglyceride (1.79 mmol/l) but normal cholesterol and lipoprotein. The patient was diagnosed with diabetes mellitus (fasting blood glucose 8.9 mmol/l and haemoglobin A1c 12.5%) and hyperinsulinemia (fasting insulin 153.0 mU/l and fasting C-peptide 11.6 ng/ml). The patient had a normal 46 XY karyotype. Upon echocardiography, a left ventricle hypertrophy was detected and histologic examination of a liver biopsy specimen suggested hepatocirrhosis. Serum leptin levels were not determined. Parents and grandparents were all healthy with no CGL phenotype, their BMI ranging from 20.2 to 21.5 kg/m2.
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Materials and methods
Venous blood samples from the subjects were collected and genomic DNA was extracted using the proteinase K and phenol method (13). Eleven primers were designed (Shanghai Shinegene Molecular Biotechnology Co., LTD, Shanghai, China) using online software (http://scitools.idtdna.com/Primerquest/) to specifically amplify exons 1–11 and the surrounding intronic sequences of the BSCL2 gene (NM_632667). PCR was performed at 94 °C for 3 min, followed by 35 cycles of 94 °C for 45 s, and 52–55 °C for 45 s, 72 °C for 45 s, and a final extension at 72 °C for 5 min. The annealing temperature for amplification of each exon is listed in Table 1. Six exons and exon–intron boundaries of AGPAT2 gene (NM_006412 [GenBank] ) were amplified as reported earlier (14). Each PCR product was sequenced thrice independently (Shanghai Shinegene Molecular Biotechnology Co., LTD).
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DNA sequence analysis of the entire coding regions and surrounding uncoding regions disclosed a novel homozygous GT mutation at nucleotide 909 in exon 5 of the BSCL2 gene in the patient (Fig. 2) (4, 7, 15). This mutation predicts a substitution of glutamic acid at codon 189 with a stop codon (Glu189X or E189X). We also found the same mutation in the heterozygous state in the child's parents and grandparents. This is in accordance with the Mendelian autosomal recessive pattern of inheritance of CGL. Because the mutation found by us is predicted to cause a severe alteration of the predicted protein sequence, it is highly likely that this novel mutation is causative for the disease. No alterations were found in the healthy control subjects in the BSCL2 gene. There was no AGPAT2 gene mutation in the family affected by CGL nor in the control subjects.
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Since Berardinelli first described CGL in 1954, more than 200 patients have been reported. The major clinical characteristics of this rare syndrome include severe insulin resistance and lipodystrophy at birth or in early infancy. The well-documented high prevalence of parental consanguinity in affected individuals is suggestive of autosomal recessive transmission.
CGL is a rare autosomal recessive disorder for which mutations in two genes, AGPAT2 on 9q34 and BSCL2 on 11q13, have been implicated to play a role in the pathogenesis. Other as yet unknown genes may also be involved in the pathogenesis of the disease. Seipin, coded by BSCL2, is homologous to the protein product of the mouse Gng3lg gene (localized on mouse chromosome 19) that is orthologous to human 11q (7, 16, 17). It is a predicted protein with 398 amino acids and more than two hydrophobic amino acid stretches, suggesting that it should be a transmembrane protein. Seipin has no similarity with other known proteins except the product of mouse Gng3lg that could be predictive for its function. Therefore, the mechanisms by which BSCL2 mutations cause lipodystrophy still remain unclear. BSCL2 mRNA is expressed in most tissues, with the highest expression in the brain and low abundance in adipose tissue. Based on the high expression of BSCL2 mRNA in the brain, it was proposed that the lack of body fat in patients with BSCL2 mutations might be due to hypothalamo-pituitary dysfunction (11). In addition, omental adipose tissue was found to express greater levels of BSCL2 mRNA than liver and skeletal muscle (18). Thus, an alternate hypothesis is that lipodystrophy is due to the direct disruption of adipocyte differentiation or function. The differentiation of preadipocytes in humans to white adipocytes can potentially proceed towards either metabolically active or mechanical adipocytes (1). Although the exact gene expression pattern of these two types of adipocytes is unknown, it seems logical to speculate that BSCL2 gene defects could affect the process of differentiation more proximally compared with AGPAT2 gene defects, which mainly affect triglyceride and/or glycerophospholipid biosynthesis, which is probably a late event in adipocyte differentiation and function (11).
According to Agarwal's analysis of the phenotypic and genetic heterogeneity in CGL from 45 pedigrees, all subtypes had extreme paucity of subcutaneous adipose tissue already at birth and markedly decreased serum leptin levels (14). Other characteristic features of CGL such as acanthosis nigricans and umbilical prominence were also noted in almost all subjects. There was a uniformly high prevalence of diabetes mellitus and hypertriglyceridemia in all individuals with CGL. A higher proportion of female subjects with AGPAT2 mutations has also been reported. However, patients with BSCL2 mutations had an earlier onset of diabetes compared with those with AGPAT2 mutations. Although half of the patients with BSCL2 mutations were documented to have mental retardation, none with AGPAT2 mutations were noted to have impaired mental functions. The prevalence of cardiomyopathy was also more than three times higher in patients with BSCL2 mutations compared to those with AGPAT2 mutations. Serum leptin levels were lower in patients with BSCL2 mutations compared to those with AGPAT2 mutations. Simha et al. report that CGL patients with BSCL2 mutations had a more severe lack of body fat, which affected both metabolically active and mechanical adipose tissue, as compared with patients with mutations in the AGPAT2 gene (19). In summary, the genetic heterogeneity in CGL patients is accompanied by phenotypic heterogeneity. These observations suggest that the protein product of the BSCL2 gene may turn out to have less restricted functions and/or a less limited expression pattern than that of the AGPAT2 gene product. The findings also emphasize the importance of attempting to make a molecular diagnosis of the syndromes of CGL as this may aid in both prognostic and genetic counseling.
As reported, patients with CGL have either homozygous or compound-heterozygous mutations of the BSCL2 gene, most of which are nonsense or frameshift mutations that are expected to cause loss of function of the protein. Individuals that are heterozygous for these mutations, such as the parents and siblings of the affected CGL subjects, do not reveal any phenotype. Our studies found that the Chinese patient with the homozygous 909 GC mutation showed typical clinical signs of CGL and metabolic abnormalities including insulin resistance and dyslipidemia. Early onset of diabetes, cardiomyopathy, and mild mental retardation were characteristic in our subject. This is in accordance with findings reported in CGL subjects with BSCL2 mutations from other ethnic groups. BSCL2 mutations, in the meantime, have also been identified in American–Taiwanese (CG 1100), American–Chinese (CG 1300), and Japanese patients (6, 12). We did not find any AGPAT2 mutations in our Chinese patient, suggesting that AGPAT2 has a minor causative role, if any.
In summary, CGL is one of the human diseases to be caused by a dysfunction of adipogenesis or adipocyte differentiation. It has prompted interest in physiology and pathophysiology of adipose tissue disorders in the human. Future functional studies of BSCL2 and AGPAT2 and their protein products will provide greater insights into adipose tissue biology, and the clinical spectrum of obesity and lipodystrophy.
Acknowledgements
The authors thank the members of the affected family and the control children for their invaluable contribution to this project. We are grateful that Professor Abhimanyu Garg, Division of Nutrition and Metabolic Diseases Endowed, Human Nutrition Research UT Southwestern Medical Center at Dallas, kindly provided the APGAT2 gene primer sequence and PCR running parameters. W K is supported by Deutsche Forschungsgemeinschaft, DFG, Bonn, Germany, ‘KFO 152 Atherobesity’, and the European Union, 6th framework program, PIONEER. This work was supported by the Science and Technology Commission of Shanghai Municipality (064119514). The authors have nothing to disclose.
References
3 subunit gene and a divergently transcribed novel gene, gng3lg. Genomics 1998; 53: 220–230.[CrossRef][Web of Science][Medline]17. Inoue S, Sano H & Ohta M. Growth suppression of Escherichia coli by induction of expression of mammalian genes with transmembrane or ATPase domain. Biochemical and Biophysical Research Communications 2000; 268: 553–561.[CrossRef][Web of Science][Medline]18. Agarwal AK & Garg A. Genetic basis of lipodystrophies and management of metabolic complications. Annual Review of Medicine 2006; 57: 297–311.[CrossRef][Web of Science][Medline]19. Simha V & Garg A. Phenotypic heterogeneity in body fat distribution in patients with congenital generalized lipodystrophy caused by mutations in the AGPAT2 or Seipin genes. Journal of Clinical Endocrinology and Metabolism 2003; 88: 5433–5437.This article has been cited by other articles:
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  D. Ito and N. Suzuki Seipinopathy: a novel endoplasmic reticulum stress-associated disease Brain, January 1, 2009; 132(1): 8 - 15. [Abstract] [Full Text] [PDF]
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