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Human Molecular Genetics, 2000, Vol. 9, No. 9 1377–1384
MECP2 mutations account for most cases of typical
forms of Rett syndrome
Thierry Bienvenu, Alain Carrié, Nicolas de Roux1, Marie-Claude Vinet, Philippe Jonveaux2,
Philippe Couvert, Laurent Villard3, Alexis Arzimanoglou4, Cherif Beldjord, Michel Fontes3,
Marc Tardieu5 and Jamel Chelly+
Laboratoire de Génétique et Physiopathologie des retards mentaux—ICGM, Faculté de Médecine Cochin, 24 rue du
Faubourg Saint Jacques, 75014 Paris, France, 1Laboratoire d’Hormonologie et de Biologie Moléculaire, CHU Bicêtre,
78 rue du général Leclerc, 94275 Le Kremlin Bicêtre, France, 2Laboratoire de Génétique, Hôpitaux de Brabois, rue du
Morvan, 54511 Vandoeuvre Les Nancy, France, 3INSERM U406, Faculté de la Timone, 27 boulevard Jean Moulin,
13358 Marseille Cedex, France, 4Service de Neurologie Pédiatrique, Hôpital Robert Debré, 48 boulevard Sérurier,
75019 Paris, France and 5Département de Pédiatrie, Service de Neurologie, CHU Bicêtre, 78 rue du général Leclerc,
94275 Le Kremlin Bicêtre, France
Received 25 January 2000; Revised and Accepted 23 March 2000
Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively
females, with an estimated prevalence of approximately one in 10 000–15 000 female births. Most cases
are sporadic, but several reports about familial recurrence support X-linked dominant inheritance with
male lethality. The gene responsible for this disorder,
MECP2, was recently identified by candidate gene
strategy. Mutations were detected in <25% of RTT
cases in this first report. To characterize the spectrum
of mutations in the MECP2 gene in RTT patients, we
selected 46 typical RTT patients and performed mutation screening by denaturing gradient gel electrophoresis combined with direct sequencing. We
identified 30 mutations, accounting for 65% of RTT
patients. They include 12 novel mutations (11 located
in exon 3 and one in exon 2). Mutations, such as
R270X and frameshift deletions in a (CCACC)n rich
region, have been found with multiple recurrences.
Most of the mutations were de novo, except in one
family where the non-affected transmitter mother
exhibited a bias of X inactivation. Although this study
showed that MECP2 mutations account for most
cases of typical forms of RTT (65%) and mutations in
non-coding regions cannot be excluded for the
remaining cases, an alternative hypothesis that takes
into account the homogeneous phenotype and exclusive involvement of females, could be the implication
in RTT of a putative second X-linked gene.
INTRODUCTION
Rett syndrome (RTT) (MIM 312750) is a progressive encephalopathy which appears to affect females only. It was first
described by Rett in 1966 (1,2). After normal development up
+To
to the age of 7–18 months, developmental stagnation occurred,
followed by rapid deterioration of higher brain functions. RTT
is characterized by severe mental retardation, autism, gait
apraxia, hypotonia, disturbance of sleep and breathing,
seizures, stereotypical hand movements and deceleration of
head growth. Its prevalence is estimated at 1:10 000–15 000
female births. More than 95% of cases are sporadic, but rare
reports of familial recurrence have been made. Previous exclusion mapping studies using the rare RTT families mapped the
locus to Xq28 (3). Xq28 is a very gene-rich region and more
than one syndrome with mental handicap and neurological
signs and symptoms has already been identified within it.
However, using a systematic gene screening approach, Zoghbi
and colleagues (4) have identified mutations in the gene MECP2
encoding X-linked methyl-CpG-binding protein 2 as the cause of
some cases of RTT (5/21 sporadic patients and 1/8 familial
patients). More recently, they reported further data showing
that MECP2 accounts for 50% of RTT (5).
MeCP2 is an abundant chromosome-binding protein that
selectively binds 5-methylcytosine residues in symmetrically
positioned CpG dinucleotides in mammalian genomes (6).
MeCP2 is rich in the basic residues lysine and arginine
(22.5%) and in proline (11%) and serine (10.5%). MeCP2
contains two functional domains, an 85 amino acid methylCpG-binding domain (MBD), essential for its binding to 5methylcytosine, and a 104 amino acid transcriptional repression domain (TRD) which interacts with histone deacetylase
and the transcriptional corepressor Sin3A. Interactions
between this transcription repressor complex and chromatinbound MeCP2 leads to deacetylation of core histones, which in
turn leads to transcriptional repression.
In the present study we have analysed the entire coding
sequence of the MECP2 gene in a sample of 46 typical RTT
sporadic cases. We have used the denaturing gradient gel electrophoresis (DGGE) assay combined with direct DNA
sequencing and characterized 12 novel mutations. The
sequence differences that were found clustered in the third
whom correspondence should be addressed. Tel: +33 1 44 41 24 10; Fax: +33 1 44 41 24 21; Email: [email protected]
1378 Human Molecular Genetics, 2000, Vol. 9, No. 9
exon include recurrent nonsense, frameshift and missense
mutations. Although the number of RTT patients investigated
is not sufficient for statistical analysis, genotype–phenotype
correlations suggest the presence of a different frequency of
some relevant symptoms, such as epilepsy, between the group
of RTT patients with a mutated allele and the group of RTT
patients with no mutation.
RESULTS
RTT patients and mutation screening of the MECP2 gene
In this study we investigated MECP2 gene in 46 patients,
exclusively girls, with a uniform and typical RTT according to
the international criteria (see Materials and Methods) (1,7).
To carry out mutation screening by DGGE, we have
designed appropriate primers to analyse the three exons of the
MECP2 gene, and for each amplified segment we determined
the optimal position of the chemical clamps (8). The theoretical melting analysis of each fragment was determined by the
computer program developed by Lerman and colleagues (9).
Exon 2 was analysed in three PCR fragments named 2.1, 2.2
and 2.3, and exon 3 was analysed in five PCR fragments
named 3A, 3B, 3C, 3D and 3E (Table 1). Amplification by
PCR and DGGE analysis followed by direct sequencing of
fragments exhibiting abnormal migration profiles were
performed in 38 typical sporadic cases of RTT and their
parents. In the remaining eight patients, mutation analysis was
performed by direct sequencing of PCR products. In total, this
investigation of the nine fragments covering the coding part of
MECP2 gene identified in 30 unrelated RTT patients the presence of 17 different mutations mainly clustered in exon 3, and
some of them appeared with multiple recurrences. Most of
these mutations, which account for 65% of typical RTT cases,
are novel and only five mutations have been described previously.
Analysis of exon 1 of the MECP2 gene revealed the presence
in one RTT sporadic case of an abnormal migration pattern of
the PCR fragment corresponding to this exon. The sequence of
the PCR product showed a C→T substitution at cDNA position
–15 upstream of the AUG initiation codon. This change was
also identified in her unaffected mother. This RTT patient also
presents a stop mutation in exon 3 of the MECP2 gene,
suggesting that this change (C→T at position –15) is a nonpathogenic variant.
Exon 2 revealed the presence in one typical RTT case of an
abnormal migration pattern of the PCR fragment 2.2, which
covers part of exon 2 (Fig. 1B). The sequence of the PCR
Human Molecular Genetics, 2000, Vol. 9, No. 9 1379
Figure 1. (A) DGGE results corresponding to the fragment 3A of the MECP2 gene. Lane 1, T158M; lanes 2–4, normal; lane 5, R168X. (B) Novel missense
MECP2 mutations in typical sporadic RTT patients. Portions of the displayed electrophoregrams illustrate three mutations in RTT patients: P302R, R106Q and
1461A→C. The underlined nucleotides and arrows indicate mutated nucleotides for each patient. (C) Novel MECP2 nonsense and frameshift mutations in typical
sporadic RTT patients. Portions of the displayed electrophoregrams illustrate five mutations found in RTT patients: R198X, R270X, R294X, 677insA and
1156del17. Deletion is indicated by bold type. The underlined nucleotides and arrows indicate mutated nucleotides for each patient.
product of exon 2 revealed a C→T substitution at position 317.
This mutation, R106Q, is the second missense mutation identified in the first part of the MBD. Another mutation R106W
was found previously in the same codon (4).
In contrast to these rare events occurring in exons 1 and 2, DGGE
screening of exon 3 of the MECP2 gene revealed the presence of
different abnormal migration patterns of PCR fragments 3A, 3B, 3C
and 3D corresponding to exon 3 (Fig. 1A). The sequences of the
PCR products corresponding to fragments 3A–3D revealed five
nonsense mutations [R168X (n = 3), R198X (n = 1), R255X (n = 2),
R270X (n = 5) and R294X (n = 3)] (Fig. 1C), three missense mutations [T158M (n = 3), P302R (n = 1) and R306C (n = 1)] (Fig. 1B),
one insertion [677insA (n = 1)], four deletions [1156del17 (n = 1),
1158del10 (n = 1), 1163del26 (n = 1) and 1164del26+1165A→T
1380 Human Molecular Genetics, 2000, Vol. 9, No. 9
Table 1. Parameters for amplification of the MECP2 gene fragments and for DGGE conditions
Fragment
Sequences of primers
Length (bp)
Annealing temp (°C)
Gradient (%)
Exon 1
1F: 5'-Pso-tttctttgttttaggctcca-3'
190
55
20–70
6.8
170
55
20–70
6.2
150
48
20–70
5.6
200
55
40–90
7.8
182
58
30–80
6.5
240
58
40–90
9.3
344
55
40–90
10.6
244
56
40–90
9.3
200
55
30–80
7.8
Running time (h) at 160 V
1R: 5'-ggccaaaccaggacatatac-3'
Exon 2.2
2.2F: 5'-atgtatgatgaccccaccct-3'
2.2R: 5'-Pso-ctgtagagataggagttgct-3'
Exon 2.3
2.3F: 5'-gtgatacttacatacttgtt-3'
2.3R: 5'-Pso-ggctcagcagagtggtgggc-3'
Exon 2.1
2.1F: 5'-Pso-gagcccgtgcagccatcagc-3'
2.1R: 5'-cgtgtccagccttcaggcag-3'
Exon 3A
3AF: 5'-Pso-tgtgtctttctgtttgtccc-3'
3AR: 5'-gatttgggcttcttaggtgg-3'
Exon 3B
3BF: 5'-Pso-cctcccggcgagagcagaaa-3'
3BR: 5'-tgacctgggtggatgtggtg-3'
Exon 3C
3CF: 5'-tgccttttcaaacttcgcca-3'
3CR: 5'-Pso-tgaggaggcgctgctgctgc-3'
Exon 3D
3DF: 5'-gcagcagcagcgcctcctca-3'
3DR: 5'-Pso-tggcaaccgcgggctgagtca-3'
Exon 3E
3EF: 5'-Pso-tgccccaaggagccagctaa-3'
3ER: 5'-gctttgcaatccgctccgtg-3'
Pso, psoralen–TA
(n = 1)] (Fig. 1B and C) and one silent polymorphism (S194S). The
nonsense mutations were due most frequently (four out of five
cases) to C→T transitions occurring in CpG dinucleotides. All the
genomic deletions resulted in a shift of the translation reading frame
leading to a premature termination of MeCP2 synthesis. The
screening of the PCR fragment 3E revealed the presence of an
abnormal migration pattern of exon 3 in only one RTT sporadic
case. The sequence revealed an A→C substitution changing the
normal TGA stop codon of the MECP2 gene to a TGC cysteine
codon (Fig. 1B). This base-pair substitution was predicted to
generate a MeCP2 protein of 513 amino acids, 27 amino acids
longer than the normal protein. If the mutation destroys a unique
restriction site, this event was used to study the segregation of the
base substitution in the family of the patient (Table 2). In addition to
these mutations revealed by DGGE screening, analysis of the
MECP2 gene by direct sequencing in eight patients showed five
additional mutations located also in exon 3 (R168X, R255X,
R306C, P322A and 1194insT).
We analysed DNA samples from both parents of all individuals with a MECP2 mutation and none of the parents’ samples
showed any abnormalities by DGGE or restriction analysis,
demonstrating that these are de novo mutations, except in one
case. In one family with a RTT girl bearing the T158M mutation, we also found the same mutation in the unaffected
mother, but not in the normal brother. This mutation has
already been described by Amir et al. (4). This change may
disrupt the structure of the MBD, thereby interfering with its
function. The crucial role of this domain is suggested by the
fact that deletion of residues 157–162 from MeCP2, which
corresponds to most of the hairpin loop, resulted in a total loss
of methyl-CpG-binding activity (10). To clarify the discrep-
ancy between the phenotype and the genotype in the mother,
we analysed the X-inactivation pattern in this family. We used
DNA prepared from peripheral blood leukocytes and assessed
the X-chromosome inactivation pattern as described by Allen
et al. (11) using PCR analysis of the androgen receptor gene,
which contains two methylation-sensitive sites (HpaII and
HhaI) flanking a polymorphic trinucleotide repeat in the first
exon. Interestingly, we found that the patient’s mother
presented a totally skewed pattern of X inactivation (data not
shown). In the affected girl, the analysis was not conclusive
because the trinucleotide repeat marker was not informative.
Although these analyses did not allow unambiguous demonstration that this mutation is deleterious and assessment of
whether the mutated allele lies on the inactive X chromosome,
it is reasonable to propose the skewed pattern of X inactivation
as the likely event involved in the rescue of the mother phenotype.
Genotype–phenotype correlations
We first focused on the group of patients with mutations in
MECP2 and looked for phenotype–genotype correlation,
taking into account for the genotype the type of mutation
(missense, nonsense or frameshift) and its position with respect
to the functional domains and the 3' end of the open reading
frame (ORF). This analysis did not show any significant correlation.
We next compared the 30 patients (mean age ± 1 SD: 14.6 ±
5 years) who had a mutation in the MECP2 gene, with the 16
patients (mean age ± 1 SD: 13.5 ± 5 years; mutated versus nonmutated NS) who had no detected mutation for different clinical items obtained before the onset of the genetic study, as
Human Molecular Genetics, 2000, Vol. 9, No. 9 1381
Table 2. Types of MECP2 mutation detected in RTT individuals
Patient no.
Base change
Mutation
Sibling investigation methods
1
808C→T
R270X
NlaIV (–)
2
317G→A
R106Q
DGGE
3
808C→T
R270X
DGGE; NlaIV (–)
7
502C→T
R168X
HhpI (+)
8
473C→T
T158M
NlaIII (+)
9
502C→T
R168X
HphI (+)
10
880C→T
R294X
DGGE
12
916C→T
R306C
HhaI (–)
13
473C→T
T158M
NlaIII (+)
1165del26
DGGE; MnlI (–)
14
15
592A→T
R198X
MaeII (+)
16
502C→T
R168X
HphI (+)
17
916C→T
R306C
HhaI (–)
1194insT
BspWI (+)
18
23
473C→T
T158M
NlaIII (+)
25
763C→T
R255X
DGGE
1156del17
DGGE; MnlI (–)
27
502C→T
R168X
HphI (+)
28
905C→G
P302R
SfaNI (+)
29
1038C→G
P322A
DraII (+)
26
30
808C→T
R270X
DGGE; NlaIV (–)
33
808C→T
R270X
DGGE; NlaIV (–)
34
1461A→C
X487C
Fnu4HI (+)
36
808C→T
R270X
NlaIV (–)
37
38
880C→T
40
677insA
DGGE
R294X
DGGE
1163del26
DGGE; MnlI (–)
41
880C→T
R294X
DGGE
43
763C→T
R255X
DGGE
1158del10
DGGE; MnlI (–)
44
Mutations either create (+) or destroy (–) the restriction site for the enzyme
shown in the right column.
described in Materials and Methods (2,7). The general characteristics of the families were similar in the two groups with a
mean of 2.8 (mutated patients) and 2.3 (non-mutated) children
per family. The repartition of sex among brothers and sisters
was identical in the two groups but the female:male ratio in
families with more than one child (including the propositus)
was slightly skewed towards girls in both cases (62 versus 76%
girls, mutated versus non-mutated). As expected in these
patients pre-selected for having a typical case of RTT, the
frequency of the most characteristic symptoms of RTT (normal
initial development, acquired microcephaly, stereotypic hand
movement, phase of social withdrawal, breathing dysfunction)
were identical in both groups. Although statistically not significant, it is worth mentioning that the patients with detected
mutation lost more frequently acquired purposeful hand skills
(71 versus 50%, mutated versus non-mutated; P = 0.15), had
more frequent peripheral vasomotor disturbances (77 versus
50%; P = 0.33) and epilepsy (41 versus 23%; P = 0.25) while
they were more frequently able to walk (21 versus 55%; P =
0.24).
DISCUSSION
In order to evaluate the prevalence of RTT related to MECP2
mutations, we have carried out a systematic analysis of the
MECP2 gene in 46 typical RTT patients and screened by
DGGE (n = 38) and by direct sequencing (n = 8) the whole
coding sequence of this gene. Upon analysis by DGGE of exon
1 to exon 3, and sequencing of PCR fragments exhibiting
abnormal DGGE migration profiles, we have identified 25/38
(66%). Direct sequencing of the whole coding sequence
revealed five of eight mutations (62.5%). Altogether, these
analyses allowed identification of disease-causing MECP2
mutations in 65% (30/46) of typical RTT patients. Of these
mutations, 12 were novel and clustered in the third exon of the
MECP2 gene. Though all patients included in this study have
homogeneous clinical phenotype and fulfilled the same diagnosis criteria, and the frequency of mutations in this gene is
very high, no mutation was identified in 35% of the screened
patients. Although investigations reported in this study cannot
exclude the presence of mutations that might lie the 3'-UTR,
promoter or intronic sequences, an alternative hypothesis,
which takes into account the exclusive involvement of females,
could be the involvement in RTT of a putative second X-linked
gene. MeCP2 acts as a molecular link by binding to 5-methylcytosine with its MBD domain and to the corepressor Sin3A
via its TRD, thus recruiting histone deacetylases and other
proteins to the silencing complex. Therefore, X-linked genes
encoding the different components of the histone deacetylase
complex could be considered as reasonable potential candidate
genes for RTT.
These studies identified in 30/46 unrelated families 17
different mutations with independent de novo recurrences of
most of them (Fig. 2; Table 2), five of these different mutations
have already been reported by Amir et al. (4) and Wan et al.
(5), and 12 are novel. Although the spectrum of mutations is
very heterogeneous, occurrence of mutations mainly in exon 3,
and the multiple recurrences of R270X (five times) and R168X
(four times), R255X (twice) and R294X (twice), points to true
mutational hotspots that could influence molecular diagnosis
strategies of RTT. Including the data reported by Amir et al.
(4) and Wan et al. (5), the spectrum of mutations now encompasses eight missense and 15 nonsense or frameshift mutations, including four small deletions, ranging from 10 to 26 bp
and localized in the region 1150–1200 of the coding sequence
of the cDNA (Fig. 2). As deletion events in human genes
appear to be, at least in part, related to local DNA sequence
environment (11), we examined carefully the sequence environment of the short deletions and identified four CCACC
direct repeat sequences distributed over the region of interest
which is also a very C-rich sequence (Fig. 3). This observation
could be coherent with the previously reported model of
slipped mispairing (12) as molecular basis for the occurrence
of deletions.
Among the molecular defects reported in this work, 11 are
nonsense or frameshift mutations (R168X, R198X, 677insA,
1382 Human Molecular Genetics, 2000, Vol. 9, No. 9
Figure 2. Distribution of the mutations in the MECP2 gene along the coding sequence. Top, mutations identified in this study. Bottom, mutations described previously (4,11). Novel mutations described in this study are underlined.
Figure 3. Sequence environment of the small deletions identified in the MECP2 gene. Deleted nucleotides are indicated by bold type. The CCACC direct repeats
flanking and/or overlapping the MECP2 gene deletions are underlined.
R255X, R270X, R294X, 1156del17, 1158del10, 1163del26,
1165del26 and 1194insT) leading to premature polypeptide
chain termination. In contrast to conventional wisdom,
mRNAs that contain these mutations (also referred to as
nonsense mRNAs and chain-termination mutations, respectively) rarely produce truncated proteins. Most nonsense
mRNAs are highly unstable because they are degraded by a
decay pathway called nonsense-mediated mRNA decay
(NMD) (13,14). This process, whereby mRNAs are monitored
for errors that arise during gene expression, has been found in
several species, including human (15,16). Typically, chaintermination mutations that reduce mRNA abundance by
reducing the half-life of mRNA behave like loss-of-function
alleles, except in some cases (i.e. mutations near the 3' end of
the ORF), where the RNA surveillance system is bypassed.
Although most of the mutations described here are located in
the first half of the coding region and should in theory trigger
the NMD process, investigation of transcripts and proteins
resulting from the diverse panel of mutations is still a relevant
issue that might provide additional information about MecP2
functional domains.
During this study, we identified three novel mutations
causing amino acid substitutions. These three missense mutations, R106Q, P302R and P322A, are drastic amino acid
changes at the protein level. Moreover, all these amino acids
are conserved in human, mouse and Xenopus laevis (4). The
R106Q mutation is located in the MBD of the protein. A mutation identified previously in this domain is located at the same
codon (R106W) (4). These two amino acid substitutions may
reduce or abolish methyl-CpG binding. The P322A mutation is
located in a conserved C-terminus and the other missense
mutation P302R is located at the end of the TRD. Replacement
of proline by arginine or alanine may cause abnormal folding
of the protein. All these base substitutions were absent in >100
Human Molecular Genetics, 2000, Vol. 9, No. 9 1383
normal X chromosomes. Altogether, these data suggest that
these DNA variants are disease-causing mutations rather than
polymorphisms. In addition, we identified an original mutation
in the termination codon. The 1461A→C substitution was
predicted to generate a MeCP2 protein of 513 amino acids (27
amino acids longer than the normal one). It has been reported
previously that the abnormal mRNA translation due to a mutation in the termination codon is associated with decreased
mRNA stability such that no mRNA or protein synthesis from
the mutant allele can be detected in cells (17).
Although mutations identified are heterogeneous (17
different mutations in this study), nearly 65% of typical RTT
individuals were found to have changes within the coding
region of exon 3. In our population, the novel R270X nonsense
mutation accounts for 16% of our RTT chromosomes. This
finding suggests that initial analysis of this exon would provide
the most efficient approach in a mutation detection protocol.
Concerning the remaining 35% of typical RTT with no mutation in the coding sequence of the MECP2 gene, our further
investigation will focus on the study of the 5'- and 3'-UTR
combined with quantitative studies of MECP2 mRNA. In addition, we will search for X-linked candidate genes required for
methyl-CpG-binding protein complexes.
MATERIALS AND METHODS
Patients
The 46 sporadic cases of RTT investigated in this study were
issued from the French register established in 1993. To validate the diagnosis, we used the international criteria adopted
by the Rett Syndrome Diagnostic Criteria Work Group (1,7).
Briefly, for all patients, after normal general and psychomotor
development up to the age of 7–18 months, development stagnation occurred, followed by rapid deterioration of higher
brain functions. This deterioration led to severe dementia,
autism, loss of purposeful use of the hands, jerky truncal ataxia
and acquired microcephaly. Additional insidious neurological
abnormalities, such as spastic parapareses, vasomotor disturbances of the lower limbs and epilepsy were also observed.
For each case, the clinical status was first reviewed by a
skilled and experienced neuropaediatrician and the diagnosis
was graded as ‘established’, ‘probable’ or ‘possible’. Independently, a questionnaire evaluating 50 items, including the necessary, supportive and exclusion criteria of the Rett Syndrome
Diagnostic Criteria Work Group was completed by two
specially trained investigators. At the same time, a blood sample
of the patient and her family was obtained and a lymphoblastoid
cell line was established with family consent. For different
genetic studies (and well before the initiation of the present
study), we selected patients who: (i) had an ‘established’ clinical
diagnosis of RTT; (ii) fulfilled more than seven of the nine
necessary criteria in the questionnaire, at least one of the
supportive criteria and none of the exclusion criteria; (iii) were
>8 years old. These patients were tested in the present study. The
χ2 test was used to analyse the comparative frequency of the
most characteristic symptoms of RTT in patients with detected
mutation and in patients with no detected mutation.
Mutation analysis
Denaturing gradient gel electrophoresis (DGGE). DNA was
extracted from peripheral blood leukocytes or lymphoblastoid
cells, and the three exons and the flanking intronic sequences
of the MECP2 gene were separately PCR-amplified from
genomic DNA using primers listed in Table 1, with psoralen
clamps. DGGE conditions were chosen according to the
Meltmap program, kindly provided by L.Lerman and
colleagues (9). The denaturants were 7 M and 40% formamide,
and gels were run at 60°C (8,9). PCR products were subjected
to electrophoresis as described in Table 1. Formal consents
were obtained from the families for mutation screening.
Mutation identification. PCR products showing an abnormal
migration pattern on DGGE analysis were sequenced directly
on an automated sequencer (ABI 373; Perkin Elmer, Foster
City, CA) using the Dye Terminator method. Every sequence
variation was checked by restriction analysis of genomic DNA.
In eight typical RTT patients, screening of the whole coding
sequence of the MECP2 gene has been performed by direct
sequencing.
ACKNOWLEDGEMENTS
We thank the patients and the families for their contribution in
this study. We also thank Genethon bank for providing DNA
samples, and colleagues from the Société Française de
Neuropédiatrie who participated to the diagnosis evaluation
and allowed us to study their patients. This work was
supported mainly by the Association Française du syndrome
de Rett (ASFR). This work was also supported by grants from
Institut National de la Santé et de la Recherche Médicale
(INSERM), the Association Française contre les Myopathies
(AFM), the Fondation Jerome Lejeune (FJL) and the Fondation pour la Recherche Médicale.
REFERENCES
1. Rett, A. (1966) Ueber ein eigenartiges hirnatrophisches syndrom bei
hyperammoniamie in kindesalter. Wien. Med. Wsch., 116, 723–738.
2. Hagberg, B., Aicardi, J., Dias, K. and Ramos, O. (1983) A progressive
syndrome of autism, dementia, ataxia and loss of purposeful hand use in
girls: Rett’s syndrome: report of 35 cases. Ann. Neurol., 14, 471–479.
3. Webb, T., Clarke, A., Hanefeld, F., Pereira, J.L., Rosenbloom, L. and
Woods, C.G. (1998) Linkage analysis in Rett syndrome families suggests
that there may be a critical region at Xq28. J. Med. Genet., 33, 997–1003.
4. Amir, R.E., Van den Veyver, I.B., Wan, M., Tran, C.Q., Francke, U. and
Zoghbi, H.Y. (1999) Rett syndrome is caused by mutations in X-linked
MECP2, encoding methyl-CpG-binding protein 2. Nature Genet., 23,
185–188
5. Wan, M., Sung Jae Lee, S., Zhang, X., Houwink-Manville, I., Song, H.R.,
Amir, R.E., Budden, S., Naidu, S., Pereira, J.L.P. et al. (1999). Rett syndrome and beyond: recurrent spontaneous and familial MeCP2 mutations
at CpG hotspots. Am. J. Hum. Genet., 65, 1520–1529.
6. Lewis, J.D., Meehan, R.R., Henzen, W.J., Maurer-Fogy, I., Jeppesen, P.,
Klein, P. and Bird, A. (1992) Purification, sequence and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell,
69, 905–914.
7. The Rett Syndrome Diagnostic Criteria Work Group (1988) Diagnostic
criteria for Rett syndrome. Ann. Neurol., 23, 425–428.
8. Bienvenu, T., Cazeneuve, C., Kaplan, J.C. and Beldjord, C. (1995) Mutation heterogeneity of cystic fibrosis in France: screening by denaturing
gradient gel electrophoresis using psoralen-modified oligonucleotide.
Hum. Mutat., 6, 23–29.
1384 Human Molecular Genetics, 2000, Vol. 9, No. 9
9. Myers, R.M., Maniatis, T. and Lerman, L.S. (1987) Detection and localisation of single base changes by denaturing gradient gel electrophoresis.
Methods Enzymol., 155, 501–527.
10. Nan, X., Meehan, R.R. and Bird, A. (1993) Dissection of the methyl-CpG
binding domain from the chromosomal protein MeCP2. Nucleic Acids
Res., 21, 4886–4892.
11. Allen, R.C., Zoghbi, H.Y., Moseley, A.B., Rosenblatt, H.M. and Belmont,
J.W. (1992) Methylation of HpaII and HhaI sites near the polymorphic
CAG repeat in the human androgen-receptor gene correlates with X chromosome inactivation. Am. J. Hum. Genet., 51, 1229–1239.
12. Cooper, D.N. and Krawczak, M. (1993) Human Gene Mutation. BIOS
Scientific Publishers Limited, Oxford, UK.
13. Leeds, P., Peltz, S.W., Jacobson, A. and Culbertson, M.R. (1992) The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev., 5, 2303–2314.
14. Leeds, P., Wood, J.M., Lee, B.S. and Culbertson, M.R. (1992) Gene products that promote mRNA turnover in Saccharomyces cerevisiae. Mol.
Cell. Biol., 12, 2165–2177.
15. Applequist, S.E., Selg, M., Raman, C. and Jack, H.M. (1996) Cloning and
characterization of hUPF1, a human homolog of the Saccharomyces cerevisiae non-sense mRNA-reducing UPF1 protein. Nucleic Acids Res., 25,
814–821.
16. Sun, X., Perlick, H.A., Dietz, H.C. and Maquat, L.E. (1998) A mutated
human homologue to yeast Upf1 protein has a dominant-negative effect
on the decay of non-sense-containing mRNAs in mammalian cells. Proc.
Natl Acad. Sci. USA, 95, 10009–10014.
17. Hunt, D.M., Higgs, D.R., Winichagoon, P., Clegg, J.B. and Weatherall,
D.J. (1982) Haemoglobin constant Spring has an unstable a chain messanger RNA. Br. J. Haematol., 51, 405–413.