The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 357-361
Copyright © 2004 by The Endocrine Society
Mutations in the Seipin and AGPAT2 Genes Clustering in Consanguineous Families with Berardinelli-Seip Congenital Lipodystrophy from Two Separate Geographical Regions of Brazil
Karina Braga Gomes,
Ana Paula Fernandes,
Alessandro Clayton Souza Ferreira,
Hermes Pardini,
Abhimanyu Garg,
Jocelyne Magré and
Victor Cavalcanti Pardini
Department of Human Genetics (K.B.G., A.C.S.F., H.P., V.C.P.), Instituto de Patologia Clinica Hermes Pardini, Belo Horizonte, Minas Gerais, 30140-070 Brazil; Faculdade de Farmácia (K.B.G., A.P.F.), Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 30180-112 Brazil; Division of Nutrition and Metabolic Diseases, Department of Internal Medicine (A.G.), University of Texas Southwestern Medical Center, Dallas, Texas 75390-9052; and Institut National de la Santé et de la Recherche Médicale Unité 402 (J.M.), Saint-Antoine Faculty of Medicine, University of Pierre and Marie Curie, Paris, 75012 France
Address all correspondence and requests for reprints to: Victor Cavalcanti Pardini, M.D., Department of Human Genetics, Instituto de Patologia Clinica Hermes Pardini, Rua Aimorés, 33, Funcionários, Belo Horizonte, Minas Gerais, 30140-070 Brazil. E-mail: vpardini{at}labhpardini.com.br.
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Abstract
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Berardinelli-Seip congenital lipodystrophy (BSCL) is characterized by a near total congenital absence of fat and predisposition to develop diabetes mellitus. In this study, we investigated the presence of mutations in the Seipin and 1-acylglycerol phosphate acyltransferase 2 (AGPAT2) genes in 32 affected subjects with BSCL from 17 consanguineous pedigrees living in two separate geographical regions, the northeastern and southeastern regions, of Brazil. All, except one, of the 22 BSCL subjects from 15 families living in the northeastern region were found to have a homozygous 669insA mutation in the Seipin gene. In contrast, all 10 BSCL subjects from two families living in the southeastern region were found to a have a homozygous 1036-bp deletion including exons 3 and 4 of AGPAT2. These results support genetic heterogeneity among BSCL patients in Brazil. Our finding of a single mutation in the Seipin and AGPAT2 genes in the pedigrees from the northeastern and southeastern regions, respectively, will be useful in genetic counseling of subjects from these large pedigrees from Brazil.
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Introduction
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BERARDINELLI-SEIP CONGENITAL lipodystrophy (BSCL) is characterized by near total absence of adipose tissue since birth. Other features include hypertriglyceridemia, hepatomegaly caused by hepatic steatosis (which can lead to cirrhosis), muscle hypertrophy, and acanthosis nigricans (1). The syndrome was first reported by Waldemar Berardinelli, from Rio de Janeiro, Brazil, in 1954 (2). BSCL is a rare autosomal recessive disorder that seems to be distributed worldwide because, besides Brazil, family clusters were also detected in Europe, Lebanon, and Oman, among others (3, 4). In Brazil, the disease occurs in at least two different regions, with a huge cluster of at least 15 families from Rio Grande do Norte state, located in the northeastern region, and two extended families living in Minas Gerais state, in the southeastern region.
Using a genome-wide linkage analysis, Garg et al. (5) found evidence of linkage of BSCL with the locus 9q34 (BSCL1). Agarwal et al. (6) recently identified the 1-acylglycerol phosphate acyltransferase 2 (AGPAT2) gene in this region, which may cause BSCL by inhibiting triacylglycerol synthesis in the adipocytes. AGPAT2 [also known as lysophosphatidic acid acyltransferase-ß] catalyzes acylation of lysophosphatidic acid to phosphatidic acid, an essential reaction in the biosynthesis of triacylglycerol and phospholipids in the eukaryotes. Magré et al. (3) identified another disease locus, BSCL2, on chromosome 11q13. Mutations in the Seipin gene localized in this region were identified in families from Lebanon, Turkey, India, South Africa, Brazil, and Europe. The Seipin gene encodes a protein of unknown function, and, thus, how mutations in Seipin cause BSCL is unknown. Thirteen different mutations were detected in the Seipin gene in the pedigrees linked to 11q13. Most of the detected alterations were frameshift or nonsense mutations, including an AA insertion on position 645, in exon 4, in a family from the state of São Paulo, Brazil. Recent studies reveal that the disease segregates with either the 9q34 or 11q13 locus, or possibly with a third unidentified locus (4, 7, 8). A genotype-phenotype analysis in subjects with mutations in the Seipin gene or cosegregating at 9q34 locus showed higher prevalence of intellectual impairment and an increased risk of premature death in the group with mutations in the Seipin gene (8, 9). Thus, BSCL seems to be a phenotypically and genetically heterogeneous disorder.
In this study, we investigated the presence of mutations in the Seipin and AGPAT2 genes in two clusters in Brazil—one in the northeastern, Seridó region, Rio Grande do Norte state, and the other in the southeastern, Sabinopolis city, Minas Gerais state—in 32 affected subjects with BSCL from 17 consanguineous pedigrees.
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Patients and Methods
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Patients
We studied 32 BSCL subjects, their families, and 50 unaffected and unrelated control individuals. All affected subjects presented generalized absence of adipose tissue, diabetes mellitus with marked insulin resistance, acromegaloid features, acanthosis nigricans, hepatomegaly, hirsutism, hypertriglyceridemia, and muscle hypertrophy. Biochemical analysis of some of these subjects showed that they had significantly decreased insulin sensitivity and plasma leptin levels, when compared with control subjects (10). The pedigrees of the families are shown in Fig. 1
. In the northeastern region, we found 22 subjects with BSCL (13 females and nine males) from 15 different families (families 1–15) (Fig. 1). Twenty-four parents and 16 unaffected siblings of these subjects were also investigated. In the southeastern region, we found 10 subjects with BSCL (four females and six males) from two different families (families 16 and 17) (Fig. 1). Eighteen unaffected relatives of these subjects were also investigated in these families. Almost all families were consanguineous.
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FIG. 1. Pedigrees of BSCL patients. The numbers below the symbols indicate the subjects who were studied. The filled symbols correspond with affected homozygous subjects for mutation 669insA in the Seipin gene (families 1–15) or del31–588 in the AGPAT2 gene (families 16 and 17). The parents and siblings heterozygous for the mutations are indicated by half-filled symbols. The number inside the symbol indicates the number of the unaffected siblings who were not studied. The affected subject (indicated by an arrow) from family 1 is the only one heterozygous for the 669insA mutation. The asterisks indicate the same individual in family 17. The triangles indicate affected individuals who were not studied. The slashes across the symbols indicate deceased subjects.
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All patients and their families gave their informed consent for genetic studies, which were approved by institutional review committees from Universidade Federal de Minas Gerais and Santa Casa. Written and informed consent for genetic studies were also obtained from Rio Grande do Norte state Berardinelli Syndrome Association.
Methods
Mutation screening of the Seipin gene and AGPAT2 was performed as described previously (3, 6, 7). PCR products were sequenced directly with an ABI 310 automated sequencer (Applied Biosystems, Foster City, CA). To search for the specific A insertion in exon 4 (cDNA position 669) in the Seipin gene, we developed a rapid method of restriction fragment length polymorphism. After PCR with specific primers flanking exon 4 (3), the PCR product was digested with the restriction enzyme Hpa I at 37 C for 8 h. In the presence of mutation, digestion with Hpa I results in two fragments (179 and 110 bp) in the affected subjects. Only one fragment (289 bp) is seen in the subjects without this mutation. In the heterozygous subjects, all three fragments are seen. The resultant fragments were analyzed by 6% acrylamide gel electrophoresis (Fig. 2A).
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FIG. 2. Rapid detection of mutations in BSCL patients. A, Detection of the 669insA mutation in exon 4 of the Seipin gene in families 1 and 2 using HpaI restriction fragment length polymorphism. The digested PCR products were analyzed by 6% acrylamide gel electrophoresis. HpaI digestion of the wild-type allele reveals a 289-bp band (lane 3), whereas digestion of the mutated allele generates two fragments of 179 and 110 bp (lanes 5, 6, and 9). In heterozygous individuals, the profile includes the three fragments (289, 179, and 110 bp) that are seen in one patient (lane 1) and in parents or unaffected siblings (lanes 2, 4, 7, 8, and 10). B, Detection by PCR of the specific AGPAT2 1036-bp deletion in exons 3 and 4, followed by a migration onto a 2% agarose gel. The absence of mutation (wild type) presents with a 467-bp fragment, as shown in lane 4. Affected BSCL subjects reveal a 276-bp fragment, as shown in lanes 2, 3, 5, and 7. Heterozygous subjects show both fragments, as seen in lanes 1 and 6.
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The specific AGPAT2 1036-bp deletion encompassing nucleotide 50 of exon 3 to nucleotide +534 of intron 4 was searched using a triplet of primers, as described previously (7), followed by electrophoresis onto a 2% agarose gel. In the absence of mutations (wild type), the PCR results in a 467-bp fragment, whereas in BSCL subjects, a 276-bp fragment is observed. In the heterozygous subjects, both fragments are seen (Fig. 2B
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Results
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We found in the BSCL subjects from the northeastern region an A insertion on position 669, in Seipin gene exon 4, which introduces a premature stop codon, Val108fsX113. By the restriction fragment length polymorphism analysis, this mutation was found in the homozygous state in all affected subjects and in the heterozygous state in their parents and in the 11 of 16 unaffected siblings, in accordance with the autosomal recessive pattern of inheritance of this disease (Table 1 and Figs. 1 and 2A). In only one affected subject, the mutation was detected in the heterozygous state. Because mutations in the AGPAT2 gene were not identified in this subject by direct sequencing of the exons and splice sites, it is likely that this subject harbors another mutation not yet identified in the Seipin gene. Mutations in exon 4 of the Seipin gene were not found in five BSCL subjects from the Brazilian southeastern region.
The southeastern families were then studied for mutations in the AGPAT2 gene. Amplification of the six exons of AGPAT2 in two probands from families 16 and 17 revealed a homozygous deletion, spanning exons 3 and 4. At the gene level, this is a 1036-bp deletion that encompasses the 50th nucleotide of exon 3 to nucleotide +534 within intron 4 (6). At the cDNA level, both exons 3 and 4 are skipped inducing the deletion of nucleotides 317–588, which results in a frameshift mutation and premature termination codon, Gly106fsX188 (6). All 10 BSCL subjects from the southeastern region harbored this mutation in the homozygous state, and 15 of the 18 unaffected relatives were heterozygous (Table 1 and Figs. 1 and 2B).
No alterations were found in the 50 unaffected and unrelated control individuals studied for the 669insA in Seipin gene and exons 3 and 4 deletion mutation in the AGPAT2 gene.
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Discussion
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BSCL is a rare autosomal recessive disorder with severe adverse metabolic consequences. Two loci for BSCL have been identified recently on chromosome 9 (AGPAT2) and chromosome 11 (Seipin). We investigated the presence of mutations in the Seipin and AGPAT2 genes in two clusters of BSCL subjects from Brazil. We first screened the Seipin gene for the presence of mutations, because an AA insertion at position 645, in exon 4, was previously reported in an affected subject from São Paulo state (3). Another mutation, 669insA, in exon 4 of the Seipin gene was found in the homozygous state in all, except one, BSCL subjects from the northeastern region. The one affected subject with this mutation in the heterozygous state probably harbors another mutation in the Seipin gene.
Curiously, all the families from the northeastern cluster had the 669insA Seipin mutation, as has also been reported in a pedigree from Portugal, in the South African subjects analyzed by Magré et al. (3), and in a Canadian subject studied by Bhayana et al. (11), who were also of Portuguese origin. Historical records indicate that the small region where the northeastern BSCL cluster lives in Rio Grande do Norte was colonized, as the majority of other regions in Brazil, by Portuguese people. Based on that, we tested the hypothesis of a founder effect for BSCL in this cluster. We performed haplotype analysis using several microsatellite markers spanning the locus interval (D11S986, D11S4191, D11S4076, D11S1765, and CA10) (3). The results showed that our Brazilian families are certainly related, because the patients are homozygous for the same allele at several contiguous markers (data not shown). Furthermore, the mutation is accompanied by the same allele at marker CA10 located within intron 3 of the Seipin gene, as presented by the South African BSCL subjects. Therefore, it is likely that these families are related to those of Portuguese origin living in South Africa and that this mutation could have arisen from the same mutational event.
In all 10 BSCL subjects from the southeastern region, we found a homozygous deletion spanning exons 3 and 4 on the AGPAT2 gene. This mutation was previously reported by Agarwal et al. (6) in a large consanguineous pedigree from Portugal and has been subsequently found in other pedigrees of Portuguese origin (7).
Thus, we found homozygous mutations in Brazilian subjects with BSCL in two different genes. Three other mutations have been reported previously in affected patients from three other Brazilian families, including the 645insAA mutation in exon 4 (Portuguese origin) in the Seipin gene (3) and the splice site mutations IVS4-2AG (African origin) and IVS3-1GC (German origin) in the AGPAT2 gene (7). These findings clearly suggest a heterogeneous origin of BSCL in different regions of Brazil.
The Brazilian northeastern and southeastern clusters live in poor and far regions and in relative isolation, increasing the probability for a consanguineous marriage and keeping this mutation among generations. This explains the high frequency of the two BSCL mutations reported by us in these clusters in Brazil.
BSCL can be accompanied by intellectual deficit, which can vary greatly among affected individuals. A variable intellectual deficit was also detected in the BSCL subjects from the northeastern cluster with the Seipin mutation, as assessed by questionnaire and analyses of school class performance (data not shown). According to the criteria described by Van Maldergem et al. (9), among these subjects the intellectual deficit varied from not detected in eight, moderate impairment in 10, and mild impairment in four BSCL subjects. Intellectual deficit was not detected in BSCL subjects from the southeastern region who had mutations in the AGPAT2 gene. These findings are in agreement with those described previously (8, 9), which show that affected subjects with BSCL harboring Seipin mutations often present intellectual deficit, whereas those with AGPAT2 mutations rarely show intellectual impairment.
In these poor populations from the northeastern and southeastern regions of Brazil, BSCL is associated with insulin-resistant diabetes and other important phenotypic and physiological alterations that have profound medical, psychological, and social consequences in the affected patients. In this study, although we gathered evidence that more than one gene and mutations in these genes are involved in BSCL in Brazil, the fact that two common mutations (which can be detected easily by the rapid methods used here) were found in the BSCL clusters will certainly be useful in genetic counseling for affected subjects and unaffected relatives of this population.
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Acknowledgments
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We thank all subjects who participated in these studies and the Rio Grande do Norte State Berardinelli Syndrome Association. We also thank M. Delépine and M. Lathrop for sequencing data.
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Footnotes
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This work was supported in part by National Institutes of Health Grant R01-DK54387 (to A.G.) and by the Association de Langue Française pour l’Etude du Diabète et des Maladies Métaboliques and Ministère de la Recherche et de la Technologie.
Abbreviations: AGPAT2, 1-Acylglycerol phosphate acyltransferase 2; BSCL, Berardinelli-Seip congenital lipodystrophy.
Received March 10, 2003.
Accepted September 15, 2003.
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References
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Abstract
Introduction
