Novel mutations of the BSCL2 and AGPAT2 genes in 10 families with Berardinelli-Seip congenital generalized lipodystrophy syndrome.

Abstract

Congenital generalized lipodystrophy, or Berardinelli-Seip syndrome, is a rare autosomal recessive disease caused by mutations in either the BSCL2 or AGPAT2 genes. This syndrome is characterized by an almost complete loss of adipose tissue usually diagnosed at birth or early infancy resulting in apparent muscle hypertrophy. Common clinical features are acanthosis nigricans, hepatomegaly with or without splenomegaly and high stature. Acromegaloid features, cardiomyopathy and mental retardation can also be present.
We investigated 11 kindreds from different geographical areas of Brazil (northeast and southeast). All coding regions as well as flanking intronic regions of both genes were examined. Polymerase chain reaction (PCR) amplifications were performed using primers described previously and PCR products were sequenced directly.
Four AGPAT2 and two BSCL2 families harboured the same set of mutations. BSCL2 gene mutations were found in the homozygous form in four kindreds (c.412C>T c.464T>C, c.518-519insA, IVS5-2A>G), and in two kindreds compound mutations were found (c.1363C>T, c.424A>G). In the other four families, one mutation of the AGPAT2 gene was found (IVS3-1G>C and c.299G>A).
We have demonstrated four novel mutations of the BSCL2 and AGPAT2 genes responsible for Berardinelli-Seip syndrome and Brunzell syndrome (AGPAT2-related syndrome).

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O R I G I N A L A R T I C L E
Novel mutations of the BSCL2 and AGPAT2 genes in
10 families with Berardinelli–Seip congenital generalized
lipodystrophy syndrome
Debora M. Miranda*, Bernardo L. Wajchenberg†, Maria R. Calsolari‡, Marcos J. Aguiar*,
Jose ´ M. C. L. Silva§, Marcia G. Ribeiro–, Cristina Fonseca–, Daniela Amaral–, Wolfanga L. Boson**,
Bruna A. Resende** and Luiz De Marco**
Departments of *Paediatrics and **Pharmacology, Universidade Federal de Minas Gerais, Belo Horizonte, †Department of
Medicine, Universidade de Sa ˜o Paulo, ‡Santa Casa de Miserico ´rdia de Belo Horizonte, §Department of Medicine, Universidade
Federal do Piaui, Teresina and –Department of Genetics, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Summary
Context Congenital generalized lipodystrophy, or Berardinelli–
Seip syndrome, is a rare autosomal recessive disease caused by
mutations in either the BSCL2 or AGPAT2 genes. This syndrome is
characterized by an almost complete loss of adipose tissue usually
diagnosed at birth or early infancy resulting in apparent muscle
hypertrophy. Common clinical features are acanthosis nigricans,
hepatomegaly with or without splenomegaly and high stature.
Acromegaloid features, cardiomyopathy and mental retardation
canalso bepresent.
Design We investigated 11 kindreds from different geographical
areas of Brazil (northeast and southeast). All coding regions as well
as flanking intronic regions of both genes were examined. Polymerase
chain reaction (PCR) amplifications were performed using primers
describedpreviouslyandPCRproductsweresequenceddirectly.
Results Four AGPAT2 and two BSCL2 families harboured the same
set of mutations. BSCL2 gene mutations were found in the homo-
zygous form in four kindreds (c.412C>T c.464T>C, c.518–519insA,
IVS5-2A>G), and in two kindreds compound mutations were found
(c.1363C>T, c.424A>G). In the other four families, one mutation of
theAGPAT2genewasfound(IVS3-1G>Candc.299G>A).
Conclusions We have demonstrated four novel mutations of the
BSCL2 and AGPAT2 genes responsible for Berardinelli–Seip
syndrome andBrunzellsyndrome(AGPAT2-relatedsyndrome).
(Received 1 August 2008; returned for revision 22 August 2008;
finallyrevised8December2008;accepted9December2008)
Introduction
Congenital generalized lipodystrophy (CGL), also known as
Berardinelli–Seipsyndromeor
lipodystrophy (BSCL, MIM 269700), is a rare disease characterized
by near absence of adipose tissue from birth or early infancy
associated with severe insulin resistance.1–3Affected patients also
present other clinical and biochemical features such as acanthosis
nigricans, muscular hypertrophy, hepatomegaly, hyperandrogenism,
altered glucose tolerance or diabetes mellitus, and hypertriglyceri-
daemia. For reasons as yet unexplained, a subset of affected subjects
frequently develops hypertrophic cardiomyopathy, which can lead to
deathdue to cardiacfailure4,5orcysticangiomatosis.6
The high incidence of parental consanguinity and high recurrence
rate among siblings has long suggested that BSCL was an autosomal
recessive condition. First, a locus in 9q34 was identified7and then a
second locus in 11q13 was characterized in a set of families from
Lebanon and Norway,8
rapidly followed by identification of
corresponding disease-causing mutations in a gene of unknown
function (BSCL2). The gene was named BSCL2 for Berardinelli–Seip
congenital lipodystrophy 2 or seipin (MIM 606158), and its open-
reading frame encodes a 462-amino-acid protein that is highly
expressed in brain and testis. Eventually, the 9q34 gene (BSCL1, MIM
603100) was defined as AGPAT2.9The 278-amino-acid AGPAT2
protein belongs to the family of acyltransferases and catalyses an
essential reaction in the biosynthetic pathway of glycerophospholipids
andtriacylglycerolineukaryotes.
Recently, a mutation in the seipin gene in patients with distal
hereditary motor neuropathy and Silver syndrome (MIM 182960
and 270685, respectively) was described.10Furthermore, in a kindred
with generalized lipodystrophy associated with cystic angiomatosis
of the bones and polycystic ovaries,6also known as Brunzell
syndrome11(MIM 269700), a mutation of the AGPAT2 gene was
demonstrated. More recently, patients with a phenotype compatible
with BSCL (although atypical) had mutations in a third gene
(Cav1)12that awaitsconfirmation.
Berardinelli–Seip congenital
Correspondence: Prof. L. De Marco, Department of Pharmacology,
Universidade Federal de Minas Gerais, Avenue Antonio Carlos 6627,
Belo Horizonte 31270-901, Brazil. Tel.: +55 31 3409 9135;
Fax: +55 31 3409 2983; E-mail: ldemarco@ufmg.br
Clinical Endocrinology (2009) 71, 512–517doi: 10.1111/j.1365-2265.2009.03532.x
512
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In this report, we examined 11 patients from 10 families with
Berardinelli–Seip syndrome. Novel mutations of the seipin gene were
found in four kindreds. In the remaining families we also detected
two previously described mutations, one in the AGPAT2 gene and
anotherintheseipingene.
Materials and methods
Subjects
This study, which involved 10 Brazilian families with 11 affected
patients, was performed with written and signed informed consent
of all subjects and with approval of the Institutional Ethics committee.
Consanguineous marriages were informed by all but one family. The
pedigrees of all families are shown in Fig. 1. All patients had the
cardinal signs of Berardinelli–Seip syndrome, such as lipoatrophy,
hyperlipidaemia, hypertriglyceridaemia, hepatomegaly, acromega-
loid facies and insulin resistance. Clinical and biochemical data for
all the families are summarized in Table 1 and Fig. 2. In addition,
the propositus from kindred #2 (III:17) had generalized cystic
angiomatosis (Fig. 3). One family of 146 subjects with 11 affected
members (eight males, three females) was from northeast Brazil
(state of Piaui) and had relatives in southeastern Brazilian states. All
of the other families were assumed to be from three southeastern
states. The patient from the state of Piaui was referred as having
Brunzell syndrome because of the presence of cystic angiomatosis
(Fig. 3), also present in her affected father (II:14) and two cousins
(III:2, III:3). In addition, Fig. 4 shows T1 and T2-weighted magnetic
resonance imaging (MRI) of her knee and foot, demonstrating lack
ofadiposetissue.
Molecular methods
DNA was extracted from peripheral blood leucocytes using standard
protocols. All coding and flanking regions of the six exons of the
AGPAT2 gene and 11 exons of the BSCL2 gene were amplified by
polymerase chain reaction (PCR) using sets of primers and reaction
conditions described previously8(and A. Garg, personal communi-
cation). Both genes were investigated in all affected patients. PCR
products were verified for correct size on 6Æ5% polyacrylamide gel
after silver staining. Thereafter, all PCR products were purified using
aGFXPCRDNAandGelBandPurificationKit(AmershamBiosciences,
Piscataway, NJ, USA) and sequenced in ABI 3130 using the ABI
Prism Dye Terminator Sequencing Kit (Applied Biosystems, Foster
City, CA, USA). Sequences were performed in sense and antisense
directions,bothinduplicateandusingseparateDNAextractions.
Fig. 1 Pedigrees of kindreds #1 to #10. Each propositus/a is indicated by an arrow. Shaded squares or circles denote clinically affected individuals.
BSCL2 and AGPAT2 genes in Berardinelli–Seip syndrome
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Results
The molecular alterations found in the AGPAT2 and BSCL2 (seipin)
genes in all 11 families are summarized in Table 2. Figure 5 shows
chromatogramsofallthenovelmolecularalterations.
AGPAT2 gene
Analysis of the AGPAT2 gene in patients from kindreds #3 to #6,
thought to be from geographically distinct regions, showed the pres-
ence of previously described mutations in Brazil. DNA analyses
revealed a homozygous mutation, an IVS3-1G>C in exon 2,6and,
in addition to the IVS3-1G>C alteration, kindreds #2 and #3 were
found to have a homozygous c.299G>A (not shown). Of note, the
propositus (III:17) as well as three other relatives in kindred #2 also
had generalized cystic angiomatosis, a phenotype not present in the
patientsdescribedpreviouslybyFu etal.6
Seipin gene
Sequencing of the coding region of the seipin gene in probands
from families #1 and #6 to #10 identified four novel molecular
alterations(Fig.5)andonepreviouslyreportedmutation.8
The patient II:2 from kindred #1 was homozygous for the
IVS5-2A>G mutation (Fig. 5a). Patient II:1 from family #6 was
homozygous for the c.518–519insA mutation whereas his mother
(I:2) was heterozygous (Fig. 5d,e). DNA sequencing analysis from
both affected brothers (II:1 and II:2) in kindred #7 demonstrated a
Table 1. Clinical and biochemicaldata for patients with Berardinelli–Seip syndrome
Patient
(kindred no.) Sex
Age at diagnosis
(years)
Acanthosis
nigricans HepatomegalyCardiomyopathy
Bone
cysts
BMI
(kg/m2)
Fat mass*
(% BMI)
Insulin
(lU/ml)
TG
(mg/dl)
Leptin
(ng/dl)
II:2 (1)
III:17 (2)
II:2 (3)
IV:1 (4)
III:2 (5)
II:1 (6)
II:1 (7)
II:2 (7)
III:1 (8)
II:2 (9)
II:1 (10)
F
F
F
M
F
M
M
M
F
M
F
14
21
15
2
Yes
Yes
Yes
Yes
Yes
No?
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
No
Yes
No
Yes
Yes
No
No
No
No
Yes
No
No
No
No
No
No
No
No
No
17Æ9
19Æ7
23Æ2
20Æ0
16Æ5
18Æ5
18Æ7
21Æ7
15Æ1
14Æ4
18Æ1
5Æ3
4Æ5
6Æ4
3Æ0
NA
3Æ0
NA
NA
5Æ3
6Æ3
NA
68Æ4
9Æ7
NA
NA
20Æ0
52Æ0
44Æ0
32Æ5
35Æ0
7Æ1
63Æ6
98< 0Æ2
NA
< 0Æ2
< 0Æ2
1Æ0
0Æ3
0Æ4
0Æ5
< 0Æ2
0Æ4
0Æ3
132
1454
649
0Æ3
0Æ3
2Æ7
0Æ2
6
0Æ4
6Æ2
No
NA
Yes
Yes
No
No
Yes
73
572
217
292
73
164
189
BMI, body mass index; TG, triglycerides; NA, not available.
*By dual energy X-ray absorptiometry (DXA) scan.
Fig. 2 (a) Acanthosis nigricans in proband II:1 from kindred #7. (b) Muscle hypertrophyand acromegaloid facies in proband III:17 from kindred #2.
514
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homozygous c.464T>C mutation in exon 4 (L155P, CTG>CCG)
(Fig. 5f). Their mother was heterozygous for the c.464T>C mutation
(Fig. 5g). In addition, both patients and their mother had a polymor-
phism at positionc.1137A>G(E379) (datanot shown).
Bothpatientsfromkindreds#8(III:1)and#9(II:2)wereheterozygous
forthec.424A>G(ACC>GCC;T142A)andc.1363C>T(Q455X) altera-
tions (Fig. 5b,c). Patient II:1 from kindred #10 harboured a previously
describedc.412C>T(R138X)mutation(datanorshown).
Discussion
Three genes, AGPAT2 located on 9q34,9BSCL2 located on 11q13,8
and caveolin-1 (CAV1) located on 7q31,12have been implicated in
the pathogenesis of this rare syndrome. To date, 24 and 28 nonsense
or frameshift alterations of the BSCL2 and AGPAT2 genes,
respectively, have been reported. Mutations N88S and S90L of the
Fig. 3 X-rays of the proposita (III:17) from kindred #3: (a) multiple cystic
lesions of the right elbow; (b) cystic lesions of the right hand.
Fig. 4 T-weighted MRI at the level of the knee (a) and foot (b) of the proposita (III:17)from kindred #3. Note the presence of hypo signal showing marked
loss of subcutaneous fat and cystic areas throughout, resembling multiple fractures.
Table 2. Molecular alterations in six families with Berardinelli–Seip
syndrome
Family no.SeipinAmino acid
Novel
mutation
Homozygous
or heterozygous
1
6
7
8 and 9
IVS5-2A>G
c.518–519insA
c.464T>C
c.424A>G
c.1363C>T
c.412C>T
Yes
Yes
Yes
Yes
Homozygous
Homozygous
Homozygous
Heterozygous
p.E177X
p.L155P
p.T142A
p.Q455X
p.R138X 10No Homozygous
Mutations of the BSCL2 gene are based on the additional 44 residues
(NM_032667).
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BSCL2 gene have also been shown to cause Silver syndrome.10,13The
vast majority of patients with Berardinelli–Seip syndrome harbour
nonsense frameshift or mutations and heterozygous carriers do not
present diseasephenotypes.
In our study, four families with BSCL1 shared the same molecular
alterations (i.e. IVS3–1G>C); in addition, two of these kindreds also
harboured a c.299G>A (S100N) mutation, both previously reported
in affected patients of European/Argentinean extraction.9The
IVS3-1G>C most probably causes downstream exon skipping because
an EGTR motif is present in exon 4. In the report by Agarwal et al.,9
both mutations were present in the homozygous form and the
authors suggested that IVS3-1G>C was the most important disease-
causing mutation. Fu et al.6reported an affected patient with a
heterozygous compound mutation, IVS3-1G>C and Phe189X, and
Fig. 5 Chromatogramsshowingsevendifferent germline seipinmutationsinfourunrelated kindreds: (a)ahomozygousmutationIVS5-2A>G,whicheradicates
aconsensussplicesite,inthepropositusinkindred#1;(b)aheterozygous mutation(c.424A>G,p.T142A)ofthe propositusIII:1fromkindred #8; (c) a
heterozygousmutation(c.1363C>T,p.Q455X)inproband III:1fromkindred#8;(d)ahomozygousmutation(c.518–519insA,p.E177X)inprobandII:1from
kindred#6;(e)aframeshift mutation(c.518–519insA) inpatientI:2fromkindred #6; (f)ahomozygousmutation(c.464T>C,p.L1555P)inprobandII:1
fromkindred #7;and(g)aheterozygousmutation(c.464T>C) inpatientI:2fromkindred#7. Thearrowsindicatemolecularalterations.
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here we have described the presence of homozygous IVS3-1G>C and
c.299G>A mutations. Taken together, these data suggest that all three
molecular alterations are disease-causing mutations. Whether they
cause more subtle phenotypes remains to be established. Of note,
the propositus also had severe cystic angiomatosis, a clinical feature
present in Brunzell syndrome, which is thought to be a subset of
Berardinelli–Seip syndrome. Radiological evidence of bone cysts
have been reported in 10 patients with AGPAT2 mutations. Fu et al.6
reported bone cysts in two sisters but not their brother, all carrying
homozygous IVS4-2A>G mutation. In our study, the propositus
with the IVS3-1G>C and three relatives, including her father, have
bone cysts, suggesting that other genes and/or environmental effects
havearoletoplayinsuchdivergent clinicalfeatures.
In the remainingsix families, we found fournoveland one recurrent
BSCL2mutation.Therecurrentmutationc.412C>T(R138X)wefound
is reported for the first time in a Brazilian family and was previously
reportedinPortugal,Belgium,France,MoroccoandTurkey,suggesting
a founder effect. The novel homozygous IVS5-2A>G mutation present
in proband II:2 from kindred #1 affects a nucleotide in the consensus
sequence for the acceptor site in intron 5 and causes exon skipping.
The propositus from kindred #6 presented with a homozygous
mutation in exon 4 (c.324–325insA), which generates a TGA termina-
tioncodon,predictingatruncated113-amino-acidprotein.
In kindred #7, we identified a homozygous T to C transition at
nucleotide 464, resulting in a leucine (hydrophobic amino acid) to
proline (nonpolar amino acid) substitution at codon 155, a change
compatible with a drastic effect on the tertiary structure and
function of the protein, crucial for the conformation of the expressed
protein.Theproband’smotherwasheterozygousforthe L155P.
Two families had similar mutations. Both affected patients had a
heterozygous C to T transition in codon 142 (exon 3) leading to a
tyrosine for adenine substitution and the Q455X mutation.
Congenital generalized lipodystrophy is an autosomal recessive
disease and the number of males to females would be expected to
be similar. Van Maldergem et al.5showed an excess of females with
AGPAT2 mutations and an excess of males with seipin mutations.
They attributed the discrepancy for the AGPAT2 mutations to
ascertainment referral bias and that of the seipin mutations to the
fact that females tend to have a more severe disease and earlier
mortality, resulting in greater referral of males. Our data show a
preponderance of males to females (8 : 3) in kindred #2, who carry
an AGPAT2 mutation, supporting the idea of referral bias. Of note,
it has been reported that BSCL2 is a disease predominantly found
in Caucasians andina few Asian families.8,14–16However, themajority
ofourpatients withBSCL2areofAfricanextraction.
In conclusion, we describe four novel mutations of the AGPAT2
and BSCL2 genes in 10 Brazilian families with Berardinelli–Seip
syndrome, a rare autosomal recessive disease that appears to be
prevalent in Brazilian families. Our results could not define a clear
genotype–phenotypeassociationandfurtherstudiesareneeded.
Acknowledgements
We thank the patients and their families for participation in our
study. This work was supported in part by FAPEMIG, CNPq and
Instituto Nacional de Medicina Molecular (Brazil).
Competing interests/financial disclosure
Nothing to declare.
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