Original article

Identication and characterization of novel sequence variations

in MECP2 gene in Rett syndrome patients
Leila Schuindt Monnerat
a
, Aline dos Santos Moreira
b
, Maria Carolina Viana Alves
a
,
Cibele Rodrigues Bonvicino
a,c
, Fernando Regla Vargas
a,d,
*
a
Programa de Gene tica, Instituto Nacional de Cancer (INCA), Rio de Janeiro, Brazil
b
Laborato rio de Genomica Funcional e Bioinforma tica, Instituto Oswaldo Cruz, FIOCRUZ, Plataforma Genomica,
Sequenciamento de DNA PDTIS/FIOCRUZ, Brazil
c
Laborato rio de Biologia e Parasitologia de Mamferos Silvestres Reservato rios, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil
d
Departamento de Gene tica e Biologia Molecular, Universidade Federal do Estado do Rio de Janeiro (UniRio), Rio de Janeiro, Brazil
Received 13 July 2009; received in revised form 6 October 2009; accepted 21 November 2009
Abstract
Rett syndrome (RS) is a neurodevelopmental disorder caused by mutations in MECP2 gene. Exons 2, 3, and 4, in addition to
intronic and 3
0
UTR adjacent regions, were sequenced in 80 patients with RS. Twenty-nine sequence variations were detected in 49
patients, 34 (69.4%) patients with the classic form of RS, and 15 (30.6%) patients with atypical forms of RS. Thirteen of the 29
detected mutations represent novel sequence variations. Missense mutation T158M was the most commonly observed mutation,
detected in nine patients (11.2%). Six hotspot pathogenic mutations (R133C, T158M, R168X, R255X, R270X, and R294X) were
responsible for the phenotype in 26/80 patients (32.5%).
2009 Elsevier B.V. All rights reserved.
Keywords: MECP2; Rett syndrome; Mutation screening; X-linked dominant disease
1. Introduction
Rett syndrome (RS, OMIM 312750) was rst
described in 1966 [1]. It is an X-linked dominant neuro-
developmental disorder characterized by progressive
neurologic and motor impairment following a period
of apparently normal development that varies from 6
to 18 months of age. Speech and independent walking
are either never achieved or gradually lost. Purposeful
use of the hands is also lost; it is replaced by stereotyped
movements of the hands described as hand washing or
clapping hands [2]. It aects 1/10.000 to 15.000 new-
born females [3]. Clinically the disease is present in
either the classical form, which has four well-dened
stages, or variant forms, which represent approximately
25% of RS patients [4].
De novo mutations in MECP2 (Methyl-CpG Binding
Protein 2) gene (OMIM 300005), allocated to Xq28
[5,6], are responsible for 8590% of RS cases [7]. The
gene has four exons [8] and codes for MeCP2 protein,
which has two isoforms: MeCP2_e1, composed by exons
1, 3, and 4 [9]; and MeCP2_e2, composed by part of
exon 2 and by exons 3 and 4. MeCP2 acts on epigenetic
control of gene transcription, through DNA methyla-
tion and remodelling of chromatin [10]. It has two func-
tional domains: MBD (methyl-CpG binding domain),
capable of recognizing and physically associate to meth-
ylated CpG islands, known to be abundant in gene pro-
moter regions; and TRD (transcription repression
0387-7604/$ - see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.braindev.2009.11.007
*
Corresponding author. Address: Genetics Division, Instituto Nac-
ional de Cancer, Rua Andre Cavalcanti, 37, 4 Andar, 20231-050 Rio
de Janeiro, Brazil. Tel.: +55 21 32331466; fax: +55 21 32331423.
E-mail address: fvargas@inca.gov.br (F.R. Vargas).
www.elsevier.com/locate/braindev
Brain & Development 32 (2010) 843848
domain), responsible for the interaction with co-repres-
sors Sin3A and BRM, and for the recruitment of histone
desacetylases (HDACs) [11]. Besides its role in epige-
netic control of transcription, it is recognized that
MeCP2 acts on the regulation of splicing [12], and is
apparently capable of repressing transcription through
binding to unmethylated DNA [13]. It is known that
MECP2 gene expression in the central nervous system
is greater during the period of neuronal maturation
[14], and the expression of isoform MeCP2_e1 is 10-fold
as high as isoform MeCP2_e2 in the brain [9]. Also,
studies in animal models have demonstrated that either
constitutive or selective neuron cell mutations result in
RS-like phenotype [15]. These data reinforce the idea
that MeCP2 has a fundamental role in the control of
neuronal gene expression. In fact, dierent studies have
identied several MeCP2 target genes with relevant roles
in neuronal maturation [1618].
RS has been described in all major ethnic groups and is
consideredone of the maincauses of mental retardationin
females. In the present study we characterize the muta-
tional spectrum of MECP2 in a sample of 80 Brazilian
RS patients. Genotype/phenotype correlation is
discussed.
2. Materials and methods
Eighty patients with classical or atypical RS according
to diagnostic criteria reviewed by Hagberg et al. [19] were
studied, including a pair of aected monozygotic twins
(cases 4 and 13). Peripheral blood genomic DNA was iso-
lated from patients and available parents according to
Miller et al. [20]. Six primer pairs were used for amplica-
tion of exons 2, 3, and 4 of MECP2 gene, and PCR condi-
tions were used according to Buyse et al. [21]. Amplied
PCR products were puried with GFXe PCR DNA and
Gel BandPuricationKit (GEHealthcare) followingman-
ufacturers instructions, and used in the sequencing reac-
tions (DYEnamic ET Terminator Sequencing Kit GE
Healthcare or BigDye Terminator v3.1 Cycle Sequencing
Kit Applied Biosystems). Samples were submitted to
direct sequencing (ABI Prism377 or ABI 3730 DNAAna-
lyzer Applied Biosystems) [22], and the obtained
sequences were compared with the reference sequence
deposited in GenBank (RefSeq AF_030876). Editing and
alignment were accomplished through use of the following
softwares: Sequenchere 4.05 Demo Version (Gene
Codes Corporation), Chromas Lite 2.01 (Tecnnelysium
Pty Ltd.), and BioEdit Sequence Alignment Editor
7.0.5.3 (TomHall). The literature searchfor the sequence
variations detected in this study was performed with the
following databases: HGMD (http://www.hgmd.org)
and RettBASE (http://mecp2.chw.edu.au/).
Every sequence variation detected was conrmed by a
new sequencing reaction performed from a newly ampli-
ed sample. Estimation of their prevalence in the popu-
lation was accomplished analysing a sample of 50
control females (100 chromosomes). For genetic coun-
seling purposes, analysis of maternal DNA sample
(when available) was performed whenever a sequence
variation was observed. Characterization of detected
frameshift mutations was obtained through cloning the
amplied fragments followed by new sequencing reac-
tion. Competent DH5a Escherichia coli cells were trans-
formed using Blunt-Ended PCR Cloning Kit (GE
Healthcare), and ligation reactions were performed
according to manufacturers instructions.
3. Results
Twenty-nine MECP2 sequence variations were
detected in 49/80 (61.2%) patients. The distribution of
detected sequence variations along the gene is shown in
Fig. 1. Eleven of these variations were not found in the
consulted databases (Table 1). Detected variations,
patients clinical classication and presence or absence of
these variations in maternal DNA are listed in Table 1.
Variations were detected in 34 (69.4%) patients aected
by classic form of RS and in 15 (30.6%) aected by atyp-
ical forms of RS. The 11 previously undescribed varia-
tions include one silent mutation (T338T), two intronic
variations (c.377+28A>G and c.37814G>A) seven
pathogenic mutations (one intronic variation at position
+1, ve frameshift mutations, and one in-frame deletion)
(Table 1). Six variations previously described as polymor-
phisms were also detected: two located on intron 3
(g.66179A>G and g.66214C>T), three silent mutations
(F142F, S194S, and S411S), and one previously described
non-synonymous variation, T203M [23]. Main clinical
ndings in patients with previously undescribed muta-
tions are shown in Table 2.
In one patient (19) the observed frameshift mutation
was associated with a described silent polymorphism
and a described intronic variation, the latter detected
also in her mother. To further characterize the varia-
tions observed in this patient the amplied fragment
(part of exon 4) was cloned and sequenced, conrming
the four variations were located in cis (Fig. 2).
Conceptual translation was used to determine the
potential eect of 10 not previously described variations
(ve frameshift mutations, one in-frame deletion, and
four intronic variations). Mutation g.65480dupA(patient
39) results in a truncated protein (Q110Afs) containing
the N-terminal region and the 32 rst residues of MBD.
Mutation g.66476dupG(patient 33) results in a truncated
protein (R190Tfs). Mutation g.66810_66818del (patient
7) results in an in-frame deletion of three aminoacids
located within TRD (L301_I303del). Mutation
g.67070_67076del (patient 17) results in a 405 amino acid
truncated protein. Mutations g.67071_67079del and
g.67091_67101del, detected in cis in patient 19, result in
a 396 amino acid truncated protein.
844 L.S. Monnerat et al. / Brain & Development 32 (2010) 843848
Population frequency of non-described MECP2 vari-
ations was veried analyzing 50 control samples, and
ve polymorphisms were identied: g.66687C>T
(dbSNP rs1042870), g.65553C>G (dbSNP rs2075597),
g.66214C>T (dbSNP rs2071569), g.67143C>T (dbSNP
rs3027928), and g.66179A>G (dbSNP rs3850326). The
last three were also identied in patients samples. In
addition, one novel variation was detected among con-
trol samples: g.66513G>A. Sequence variation
g.66271delT was detected in one control sample, and
also in patient 19 and her mother.
4. Discussion
Inthis study we found29sequence variations inMeCP2
in 49/80 girls with RS. Eleven non-described variations,
Fig. 2. Electropherograms of patient 19 indicating mutations [g.67071_67079del + g.67088C>T + g.67091_67101del] detected in cis in patient 19.
(A) Cloned fragment correspondent to wild-type allele. (B) Cloned fragment correspondent to mutant allele. Rectangles I and II indicate deletions of
9 and 11 bp. Large arrow indicates mutation g.67088C>T.
Fig. 1. Illustration showing type and location of detected variations in study patients. (A) Previously described variations; (B) undescribed
variations.
L.S. Monnerat et al. / Brain & Development 32 (2010) 843848 845
present in11patients (Tables 1 and2), were observed. They
include four variations on intron 3, ve frameshift muta-
tions, one in-frame deletion, two silent mutations located
on C-terminal region, and one 3
0
UTR variation.
Mutation Q110Afs has, probably, signicant biolog-
ical eect, because the truncated protein lacks most part
of MBD. Mutation R190Tfs results in a truncated pro-
tein conserving MBD, but lacking most of the interdo-
main region (including NLS) and all TRD. Mutation
P387Lfs results in a truncated protein encompassing
the highly conserved C-terminal region, which is impor-
tant to MeCP2-nucleosome interaction [11]. In spite of a
three aminoacid deletion within TRD, eect of mutation
L301_I303del should be milder, because the downstream
sequence of MeCP2 is kept intact. In fact, this patient
(7) has an atypical (forme fruste) form of disease. Muta-
tions c.326dupA, c.566dupG, c.900_908del, and
c.1159_1165del have been the only detected mutations
in four of the patients (39, 33, 7, and 17, respectively),
and probably corroborate the hypothesis that these vari-
ations are pathogenic.
Mutations c.1161_1169del and c.1181_1191del were
detected in a patient (19) with classical RS. These 9
and 11 base pair deletions in exon 4 were separated by
a C>T transition, and both were in cis. Conceptual
translation of the protein suggests these mutations
would generate a truncated MeCP2 protein, compatible
with the classical phenotyope observed in this patient.
We detected variations located on non-coding regions
of MECP2. While c.377+1G>A is expected to be path-
Table 1
List of detected variations, localization in the gene, correspondent variation on protein sequence, clinical form, number of patients with the variation,
mothers status, and reference.
Variation
a
Region Protein Mutation
classication
Clinical
form
Patient Mothers
DNA
Reference
65443G>T E3 D97Y PM C 43 [26]
65480dupA E3 Q110Afs PM C 39 This study
65532G>A I3 IV/PM C 58 This study
65559A>G I3 IV C 9 This study
66179A>G I3 IV A, A 42, 67 U, GenBank
66214C>T I3 IV C, C, C,
C, C, A, C
26, 27,
30, 36,
48
+/+
,
59, 64
U, U, U,
, U, ,
RettBASE: IRSF
66271delT I3 IV C 19 + [37]
66274G>A I3 IV C 29 U This study
66307C>T E4 R133C PM C, A, C 5, 15, 60 , , [7]
66330delG E4 Y141Tfs PM C 79 This study
66336C>T E4 F142F SP A 73 [26]
66365C>G E4 P152R PM A, C 21, 32 , U [38]
66383C>T E4 T158M PM C, C, A, C,
A, C, A, A, C
29, 38,
46, 49,
50, 54,
55, 59, 71
U, , U,
, U, , , ,
[7]
66412C>T E4 R168X PM C, C, A 57, 65, 70 , , [24]
66476dupG E4 R190Tfs PM C 33 This study
66492C>T E4 S194S SP A 21 + [7]
66518C>T E4 T203M NSP A 73 [23]
66606delC E4 K233Rfs PM C 11 [29]
66673C>T E4 R255X PM C, C, A,
C, A, C
4, 13, 24,
47, 61, 69
, , ,
, , U
[7]
66718C>T E4 R270X PM C, C 8, 41 , [38]
66790C>T E4 R294X PM A, C, A 18, 34, 63 , , [38]
66810_66818del E4 L301_I303del PM A 7 This study
66924C>T E4 T338T SM C 9 This study
67043C>G E4 A378G PM C 47 [27]
67067_67107del E4 L386Hfs PM C 35 [39]
67070_67076del E4 P387Lfs PM C 17 U This study
67071_67079del E4 P389Efs PM C 19 This study
67088C>T E4 P393P PM C 19 This study
67091_67101del E4 E394Gfs PM C 19 This study
67143C>T E4 S411S SP C, C, C 19, 20, 34 , U, [21]
67385G>A 3
0
UTR ND A 50 U This study
Abbreviations: A atypical; C classical; E exon; I intron; IV intronic variation; NSP non-synonymous polymorphism; PM pathogenic
(disease-causing) mutation; SM silent mutation; SP silent polymorphism; U unavailable.
a
Ref Seq AF030876.
846 L.S. Monnerat et al. / Brain & Development 32 (2010) 843848
ogenic, because it alters an invariably conserved splice
site, c.378-17delT and c.378-14G>A aect the -17 and
-14 nucleotides in 5
0
splicing site of exon 4, respectively,
and their potential impact on the protein level is
unknown.
Intronic variation g.65559A>G and two silent muta-
tions (T338T and S411S) would apparently not have
inuence on the clinical features of patients. 3
0
UTR var-
iation g.67385G>A was detected in one patient (50) who
also carried one previously described pathogenic muta-
tion (Table 1). T158M was the most frequent mutation,
being observed in nine patients (29, 38, 46, 49, 50, 54, 55,
59, and 71) with either clinical classical or atypical
presentations.
Six previously described pathogenic missense muta-
tions were detected, ve located within MBD and one
at the C-terminal region. Six other pathogenic variations
were detected, four nonsense mutations, and two frame-
shift mutations. Six of these 11 pathogenic mutations
(R133C, T158M, R168X, R255X, R270X, and R294X)
are considered hotspots [24], and were observed in 26/
80 patients (32.5%). The other ve pathogenic mutations
(D97Y, P152R, K233Rfs, A378G, and L386Hfs),
reported in low frequency in the literature, were detected
in 6 patients (7.5%).
Various studies point to a possible genotypepheno-
type correlation in some recurrent variations detected
in this study. Missense mutations D97Y and A378G
are associated with classical RS [2528]. Mutations
R133C, P152R, and T158M are associated either classi-
cal [7,29] and atypical forms of RS [3032]. Nonsense
mutation R168X was associated only with classical RS
[24], but R255X, R270X, and R294X are associated
either with classical or atypical forms [23,29,31].
R270X is also associated with progressive neonatal
encephalopathy [33].
Silent mutations S194S and S411S were observed, not
only in sporadic mental retardation and autism cases,
respectively [23,34], but also in classical RS and other
neurological disorders [7,35,36].
In the present study, sequencing of coding and intro-
nic anking regions of MECP2 was able to detect vari-
ations in approximately 70% of classical RS patients.
Further molecular studies may be necessary, including
screening for large gene rearrangements in MECP2, in
order to characterize the full spectrum mutation in
MECP2 gene in RS patients.
Inclusion of patients in the study, and collection of bio-
logical samples from patients and available parents were
performed following signature of informed consent by
the parents under a protocol approved by the local Institu-
tional Review Board (CEP/CONEP 219/05, 12.12.2005).
Acknowledgements
L.S. Monnerat was a recipient of a Instituto
Oswaldo Cruz (IOC/FIOCRUZ) master fellowship.
C.R. Bonvicino is recipient of a CNPq research fellow
grant.
Genome Sequencing Platform PDTIS/FIOCRUZ.
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Table 2
Main clinical ndings in patients with previously undescribed mutations.
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