© 2000 Oxford University Press
Human Molecular Genetics, 2000, Vol. 9, No. 9 1369–1375
Rett syndrome: analysis of MECP2 and clinical
characterization of 31 patients
P. Huppke+,§, F. Laccone1,§, N. Krämer, W. Engel1 and F. Hanefeld
Abteilung Kinderheilkunde, Schwerpunkt Neuropädiatrie and 1Institut für Humangenetik, Georg-August-Universität
Göttingen, Robert Koch Straße 40, D-37075, Göttingen, Germany
Received 24 January 2000; Revised and Accepted 27 March 2000
Only recently have mutations in MECP2 been found
to be a cause of Rett Syndrome (RTT), a neurodevelopmental disorder characterized by mental
retardation, loss of expressive speech, deceleration
of head growth and loss of acquired skills that almost
exclusively affects females. We analysed the MECP2
gene in 31 patients diagnosed with RTT. Sequencing
of the coding region and the splice sites revealed
mutations in 24 females (77.40%). However, no
abnormalities were detected in any of the parents
that were available for investigation. Eleven mutations have not been described previously.
Confirming two earlier studies, we found that most
mutations are truncating and only a few of them are
missense mutations. Several females carrying the
same mutation display different phenotypes indicating that factors other than the type or position of
mutations influence the severity of RTT. Four
females with RTT variants were included in the study.
Three of these presented with preserved speech
while the fourth patient with congenital RTT lacked
the initial period of normal development. Detection of
mutations in these cases reveals that they are indeed
variants of RTT. They represent the mild and the
severe extremes of RTT. Conclusions: mutations in
MECP2 seem to be the main cause for RTT and can
be expected to be found in ~77% of patients that fulfil
the criteria for RTT. Therefore analysis of MECP2
should be performed if RTT is suspected. Three
mutation hotspots (T158M, R168X and R255X) were
confirmed and a further one (R270X) newly identified.
We recommend screening for these mutations before
analysing the coding region.
INTRODUCTION
Rett Syndrome (RTT) was first described in 1966 by Andreas
Rett (1) but international recognition as a distinct disorder was
only achieved after Hagberg et al. (2) described 35 cases in
1983. Subsequently it was found to be one of the most common
causes of severe mental retardation in females with an inci+To
dence of 1/10 000 to 1/15 000 (3). After a period of normal
development females with RTT show a regression of motor
and mental abilities. Stereotypic hand movements appear with
the loss of purposeful manual skills. In most patients head
circumference growth decelerates. Despite the severity of the
clinical picture metabolic and morphological examinations fail
to reveal any gross abnormalities (4,5).
Last year the chromosomal location was narrowed down to
Xq28 (6–8) and, more recently, Amir et al. were able to describe
mutations in MECP2, encoding methyl-CpG-binding protein
(MeCP2) (9,10). It was an unexpected finding that mutations
were discovered in a gene that is expressed in all tissues (11,12)
because RTT was thought to be primarily a neurodevelopmental
disorder. On the other hand, there is an enhanced expression of
MECP2 during the differentiation and maturation of the hippocampus (13). MeCP2 is involved in the long-term silencing of
genes in mammalian cells (14). It binds specifically to methylated CpGs and interacts with the Sin3A–histone co-repressor
complex and histone deacetylases (15,16). By deacetylating
core histones the chromatin structure is converted into an inactive state (17). Four functionally important domains have been
identified: (i) the methyl-CpG-binding domain (MBD) (18);
(ii) the transcriptional repression domain (TRD) interacts with
the co-repressor Sin3A and the histone deacetylases (19);
(iii) the nuclear localization signal (NLS) mediates the transport
of MeCP2 into the nucleus (20); (iv) only recently has it been
recognized that the C-terminal segment of MeCP2 facilitates the
binding to the nucleosome core (21). Defects in MeCP2 would
presumably lead to an overexpression of genes (22). Experiments in mice using mutated male embryonic stem cells showed
that MeCP2 is dispensable in stem cells but essential for
embryonic development (23). Chimeras with strong mutant cell
contribution showed a severely retarded development and an
abnormal appearance.
So far 10 different mutations in MECP2 present in 18 patients
with RTT have been described (9,24). They are located predominantly in exon 3 and encompass five missense and three
nonsense mutations as well a single nucleotide deletion and a
single nucleotide insertion both causing a frame shift.
We studied the MECP2 gene in 31 RTT females to answer
the following questions: (i) are mutations in MECP2 the
primary cause of RTT? (ii) what is the spectrum of mutations
in MECP2 that causes RTT? (iii) is there is a genotype–phenotype correlation in RTT with MECP2 mutations?
whom correspondence should be addressed. Tel: +49 551 396210; Fax: + 49 551 396252; Email: [email protected]
authors contributed equally to this work
§These
1370 Human Molecular Genetics, 2000, Vol. 9, No. 9
Table 1. Mutations in MECP2
Patient
Domain
Nucleotide changea
Amino acid change
Restriction site (+/–)b
Reference
32
MBD
316C→T
R106W
NlaIII (+)
(9)
26
MBD
397C→T
R133C
none
(9)
27
MBD
401C→G
S134C
none
25
MBD
455C→G
P152R
NlaIV (–)
6, 16
MBD
473C→T
T158M
NlaIII (+)
(9)
502C→T
R168X
HphI (+)
(24)
(9)
5, 11, 15
12, 24, 29
TRD/NLS
763C→T
R255X
none
3, 4, 19
TRD/NLS
808C→T
R270X
NlaIV (+)
10
TRD
880C→T
R294X
none
30
TRD
904C→G
P302A
none
13
TRD
916C→T
R306C
HhaI (–)
1
965C→T
P322L
AluI (+)
2
258–259delCA
none
9
378–2 a→g splice site
NlaIV (+)
28
CTS
1157–1200del
22
CTS
1163–1197del
18
CTS
1364–1365insC
(24)
none
aNumbered
from the ATG initiator codon.
abolished; +, generated.
CTS, C-terminal segment.
b–,
RESULTS
Analysis of the MECP2 gene in 31 patients diagnosed with
RTT revealed mutations in 24 of them (77.40%) (Table 1).
Eleven mutations have not been described previously. Four of
them are missense mutations that affect highly conserved
amino acids. The 455C→G, 904C→G and the 965C→T transitions lead to the substitution of the amino acid proline, which
might affect the structure of the protein. The 401C→G and the
455C→G transversions are located in the MBD and might
therefore affect the binding of MeCP2 to methylated CpGs.
The 904C→G transversion is located in the TRD while the
965C→G transversion does not affect any known domain. In
patients 3, 4 and 19 an 808C→T transition was identified that
changes an arginine residue to a stop codon in position 270.
The truncation of the protein affects the TRD and the NLS. The
880C→T transition identified in patient 10 is localized in the
TRD as well and leads to a stop codon at position 294.
Two mutations that we describe here for the first time can be
expected to cause a complete loss of function of MeCP2. At
position 258–259 a deletion of 2 bp was identified in patient 2. It
is localized immediately upstream of the MBD and leads after
two missense amino acids to a stop codon at position 89 of the
protein. In patient 9 a 378–2 a→g splice site mutation was identified. This mutation will affect all four important domains.
The finding of two deletions within the same area and of
similar length was surprising. The 1157–1200del and the
1163–1197del were detected by direct sequencing and
confirmed by cloning and sequencing of the corresponding
PCR products. The 1364–1365insC, identified in patient 18,
causes a frame shift that leads to a stop codon after 31 missense
amino acid changes. All three mutations are located in the Cterminal segment of MeCP2, a region important for the binding
to the nucleosome core (21).
In 13 patients mutations that have already been described were
detected (9,24). The sequencing of the coding region and the
splice sites of seven patients did not detect any abnormalities. In
two patients a silent single nucleotide polymorphism was
detected: a 582C→T substitution in patient 7 that has been
described previously (9) and a 999G→T transversion in patient 1.
In 20 of the patients that carry a mutation in MECP2, DNA
of both parents was available. Analysis of the region of
MECP2 that showed a mutation in their daughter revealed no
abnormalities. The parental DNA of four patients was not
available (patients 3, 4, 15 and 24). The mutations that were
found in these patients were either truncating or have been
described previously.
Sequence tracings of 11 newly described mutations are
shown in Figure 1. Clinical data are summarized in Table 2.
The comparison of the severity scores for truncating and
non-truncating mutations failed to reach statistical significance
(P = 0.2246).
DISCUSSION
Are mutations in MECP2 the primary cause of RTT?
In our study, mutations in MECP2 were found in 77.40% of
RTT patients diagnosed according to the Vienna criteria as
Human Molecular Genetics, 2000, Vol. 9, No. 9 1371
Figure 1. Sequence tracings of 11 mutations that are newly described in this article.
defined by Hagberg et al. (25). Preliminary data from ongoing
investigations on other patients (unpublished data) support this
percentage. Mutations were found in the MECP2 gene of four
patients that were considered to be RTT variants (patients 5, 26
and 30, preserved speech variants; patient 18, congenital RTT).
Our study therefore strongly indicates that mutations in
MECP2 are the main cause of RTT. One can only speculate
about the higher rate of detected mutations in our study in
comparison with the other published studies. One explanation
might be our strict selection process of patients according to the
inclusion and exclusion criteria. Because the clinical data of the
patients in previous studies were not described in detail, a direct
comparison between the two groups is not possible. Another
possibility is that in previous studies some mutations were not
discovered for methodological reasons. We analysed our
patients by direct sequencing of only two PCR products and
found two new, large deletions in the 3′ region of the coding
sequence. It seems that this might be a hotspot for deletions.
Finally, ethnic difference could explain the discrepancy between
the detected rate of mutations. Our data questions the possibility
of the large 3′ UTR region as an important mutation spot.
In seven patients no mutations were found. Five of them fulfil
the criteria for classical RTT; patient 7 presented without a
deceleration of head growth and developed an RTT phenotype
following an acute epileptic encephalopathy. Patient 14 was
considered to be an atypical RTT because she had preserved
hand function. The RTT in the classical RTT patients could be
caused by other defects that affect the same system of gene inactivation, e.g. the co-repressor complex. The rate of mutations in
classical RTT and RTT variants was similar in this study.
What is the spectrum of mutations in MECP2 that causes
RTT?
In our study 17 different mutations were detected, 11 of which
have not been described previously. Two of those, 808C→T
1372 Human Molecular Genetics, 2000, Vol. 9, No. 9
Table 2. Summary of the clinical data
Patient Nucleotide changea
Psychomotor Deceleration of Stereotypical
Mental
EpilepsyMicrocephaly Loss of ability to
regression
head growth hand movements retardation
sit
walk
speak
Severity score
2
258–259delCA
+
+
+
+
+
+
–
+
+
2
31
316C →T
+
+
+
+
+
+
+
+
+
3
9
378–2 a→g splice site
+
+
+
+
–
+
–
+
+
2
26
397C →T
+
+
+
+
+
–
–
–
–
0
27
401C →G
+
+
+
+
+
+
–
+
+
2
25
455C →G
+
+
+
+
+
–
–
–
+
1
6
473C →T
+
+
+
+
+
+
–
–
+
1
16
473C →T
+
+
+
+
–
–
–
–
+
1
11
502C →T
+
+
+
+
+
+
+
+
+
3
15
502C →T
+
+
+
+
–
+
–
–
+
1
5
502C →T
+
+
+
+
–
–
–
–
–
0
12
763C →T
+
+
+
+
–
+
+
+
+
3
24
763C →T
+
+
+
+
–
–
–
–
+
1
29
763C →T
+
+
+
+
+
+
–
+
+
2
19
808C →T
+
+
+
+
+
+
–
+
+
2
3
808C →T
+
+
+
+
+
–
–
+
+
2
4
808C →T
+
+
+
+
+
+
+
+
+
3
10
880C →T
+
+
+
+
+
+
–
+
+
2
30
904C →G
+
+
+
+
–
+
–
–
–
0
13
916C →T
+
+
+
+
+
–
–
–
+
1
1
965C →T
+
+
+
+
+
–
+
+
+
3
28
1157–1200del
+
+
+
+
+
+
–
–
+
1
22
1163–1197del
+
+
+
+
+
+
–
–
+
1
18
1364–1365ins C
+
+
+
+
+
+
+
+
+
3
7
none
+
–
+
+
+
–
–
–
+
1
8
none
+
+
+
+
–
+
+
+
+
3
14
none
+
+
+
+
+
–
–
–
+
1
17
none
+
+
+
+
+
+
–
+
+
2
20
none
+
+
+
+
+
–
+
+
+
3
21
none
+
+
+
+
–
+
–
+
+
2
23
none
+
+
+
+
+
+
–
+
+
2
aNumbered
from the ATG initiator signal.
and 880C→t, were located at hotspots of mutation at CpG
dinucleotides that were predicted by Wan et al. (24). The
R270X mutation was found three times and therefore seems to
represent one of the common mutations. In patient 9 the first
splice site mutation responsible for causing RTT was identified
in MECP2. Whether this mutation leads to the skipping of
exon 3 is the subject of further investigation. Two deletions of
34 and 43 bp were identified in almost the same position. There
is a short homologous sequence at the 5′ and the 3′ ends
(C1149–C1154 and C1195–C1200) that might be involved in
the mutagenesis. The other new mutations include one dele-
tion, 258–259delCA, one insertion, 1364–1365insC and three
missense mutations, 455C→G, 904C→G and 956C→T. As in
other studies, a surprisingly large number of mutations were
identified that lead to a truncation of the protein while only a
few missense mutations were found. Possibly some missense
mutations cause a quite different phenotype that is not identified as being RTT. Further investigations of patients with a
broader phenotype (i.e. autism and microcephaly) may answer
this question.
Four mutations were detected more then once (T158M,
R168X, R255X and R270X). Three of them abolish or create a
Human Molecular Genetics, 2000, Vol. 9, No. 9 1373
restriction site (T158M, R168X and R270X). It is therefore an
appropriate approach to test for these mutations before
sequencing the coding region.
Is there a genotype–phenotype correlation in RTT with
MECP2 mutations?
To assess genotype–phenotype correlation in RTT we have
listed features that are typical for RTT but not consistent in all
patients and might therefore reflect the severity of the
syndrome (Table 2): microcephaly, epilepsy, inability to sit,
walk and speak. If there is a genotype–phenotype correlation,
females who carry mutations that cause a loss of function
would be expected to give the most severe phenotype. The
258–259delCA (patient 2) and the 378-2 a→g splice site mutation (patient 9) affect all known domains. Both these patients
were never able to walk and have no expressive speech; they
are among the more severely affected group of females. At the
milder end of the spectrum one would expect to find patients
who carry missense mutations that lie outside known functionally important domains. Patient 1 (965C–T transition) fulfils
these criteria. She is, however, also severely affected; unable to
sit or walk unsupported.
To further support a correlation between genotype and
phenotype it would be expected that females who carry the
same mutation would present with a similar clinical picture. In
our study, however, this is not the case. The 502C→T transition was found in patients 5 and 11. Patient 5 can sit and walk
unsupported and speaks a few words, while patient 11 at a
similar age is unable to sit, walk or speak. To be able to
compare the genotype of the truncating with the non-truncating
mutations we created a severity score based on the loss of
ability to sit, walk or speak. The exact Wilcoxon–Mann–
Whitney test did not deliver a significant P value due to the
small number of patients, but indicates that truncating mutations are associated with a more severe phenotype than nontruncating mutations. It seems that factors other than the type
of mutation influence the phenotype in a major way. Wan et al.
(24) supposed that one important factor might be the X-inactivation pattern. They presented the mother of a female with
RTT who carried the same mutation. Obviously due to a
skewed XCI pattern the mother developed only minor symptoms while the daughter had classical RTT. However, several
studies on XCI in RTT have been performed using white blood
cells and in one study brain tissue (26–32) but only minor
abnormalities with partial preferential inactivation of the
paternal X chromosome have been described.
Description of RTT variants has been hindered by the
problem that no biological marker was available to make the
diagnosis. Many features of RTT are seen in other forms of
mental retardation and a mild form, e.g. RTT with preserved
speech or severe forms like the congenital RTT could have
represented separate entities. In our study we included three
females with RTT and preserved speech (patients 5, 26 and
30). In all of them mutations in MECP2 were found. They
seem to represent the mild form of RTT because all three of
them are able to sit and walk. In patient 18 analysis of the
MECP2 gene revealed an insertion of a guanine, which causes
a frame shift and a stop codon immediately upstream of the
natural stop codon. She presented as a congenital RTT, never
learned to sit or walk and developed a severe epilepsy at 10
months of age. This patient seems to represent the other end of
the spectrum of RTT in females.
A study similar to ours has been published by Cheadle et al.
(33). Their results regarding the mutation frequency, the type
and localization of mutations and the genotype–phenotype
correlation are similar to ours indicating that there is no influence from the ethnic background.
The phenotype of RTT is expanding. MECP2 mutations
have been identified in females with only minor neurological
deficits and in males with severe encephalopathy (20). There
are no good clinical criteria for those extreme variants so far
and nothing can be said about the rate of mutation in those
groups of patients. In German patients that do fulfil the criteria
for RTT, mutations are found in 77.40% and in this group of
patients screening for mutations in MECP2 should be
performed before other investigations.
MATERIALS AND METHODS
All patients are of German origin. They were diagnosed
according to the criteria for RTT defined by Hagberg et al. (25)
which includes psychomotor regression after a period of
normal development, severe mental retardation, deceleration
of head growth and loss of purposeful hand skills with appearance of stereotypical hand movements. To establish whether
there is any genotype–phenotype correlation we selected
features that are typical for RTT but not present in all patients
and which therefore might reflect the severity of the syndrome:
epilepsy, microcephaly (hc < 3rd centile) and loss of ability to
sit, walk or speak.
To facilitate a direct comparison of truncating mutations and
non-truncating mutations we created a severity score ranging
from 0 to 3 in which one point was added for the inability to sit,
walk or speak at age 5 years. The severity scores for truncating
mutations were compared with those for non-truncating mutations using the exact Wilcoxon–Mann–Whitney test (Statxact/
Cytel) because of the ordinality of the scores.
Patients 5, 26 and 30 possessed some speech ability and were
therefore classified as preserved speech variants.
Patient 18 presented as a congenital RTT. She lacked a
period of normal motor development and was unable to sit or
walk. Initially her hand function was good and she spoke two
words until regression began and both abilities were lost at 12
months of age.
Mutation detection
Total genomic DNA was prepared from peripheral blood
leukocytes according to standard procedures (32). Primer pairs
were designed to amplify the coding region and the splice sites
of MECP2 using the genomic sequence (GenBank accession
no. AF030876). The following primer pair was used to amplify
exon 1: forward Rett-EX-1F 5′-GCAGCTCAATGGGGGCTTTCAACTT-3′ and reverse Rett-EX-1R 5′-GGCACAGTTATGTCTTTAGTCTTTGG-3′. PCR was performed using
HSTaq MasterMix (Quiagen, Hilden, Germany) in a volume of
25 µl and 100 ng DNA under the following conditions: denaturation/polymerase activation at 97°C for 15 min followed by
30 cycles with a denaturation at 96°C for 20 s, annealing at
60°C for 20 s and extension at 72°C for 30 s in a Primus Cycler
(MWG, Ebersberg, Germany). Exons 2 and 3 were amplified
1374 Human Molecular Genetics, 2000, Vol. 9, No. 9
with a long range Protocol using the LA PCR Kit (TaKaRa,
Tokyo, Japan) in a two-round PCR. For the first PCR primers:
forward RettEX2-F1 5′ctggggccttgcatgtggtggggg-3′ and
reverse RettEX3-R1 5′caactactcccaccctgaagccacg-3′ were
used. The reaction mix contained 20 pmol of each primer and
100 ng of genomic DNA according to the manufacturer’s
instructions using GC buffer I. After an initial denaturation at
96°C for 2 min, 20 cycles at 94°C for 20 s, 60°C for 30 s and
72°C for 3 min were performed followed by 15 cycles under
the same conditions but with a decrease in the denaturation
temperature of 0.3°C per cycle and an increase of the elongation time of 15 s per cycle. The second round of PCR was
carried out with 1 µl of the first round PCR product in a volume
of 100 µl and following primers: RettEX2-F2 CTGCTCACTTGTTCTGCAGACTGG-3′ and RettEX3-R2 5′ GTCAGAGCCCTACCCATAAGGAGA-3′. The PCR setting and PCR
conditions were the same (except for the volume reaction) as
for the first PCR. After purification the PCR product was
analysed by direct sequencing on an ABI 377 automatic
sequencer using the DYEnemic ET Terminator cycle
sequencing Kit (Amersham Pharmacia Biotech, Uppsala,
Sweden ) according to the manufacturer’s instructions. The
sequencing primers were following: RettEX1-F1, RettEX1R1, RettEX2-F2, Rett EX3-R2, Rett EX2-R1B 5′-GTTCCCCCCGACCCC-3′, RettEX3-F1 5′-CTCTGACATTGCTATGGAGAGCC-3′, RettEX3-F2 5′-GTGGCAGCCGCTGCCGCCGAGGCC-3′, RettEX3-R3 GATGGGGAGTACGGTCTCCTGCAC-3′.
Whenever a mutation created or abolished a cleavage site it
was confirmed by restriction enzyme digestion: the 316C→T
and 473C→T transitions create a cleavage site for NlaIII. The
455C→G transversion abolishes a restriction site for NlaIV
while the 808C→T and the 378–2 a→g substitutions create
one. 502C→T creates a restriction site for HphI, 965C→T
generates one for AluI and 916C abolishes a restriction site for
HhaI. The enzymes were used under the recommended conditions (New England Biolabs, Beverly, MA).
In the case of frame-shift causing mutations, 7 µl of PCR
product were cloned into the pGEMT-easy (Promega,
Madison, WI) cloning vector according to the manufacturer’s
instructions. Several colonies were picked and analysed after
DNA isolation by direct sequencing.
ACKNOWLEDGEMENTS
We thank the RTT patients and their parents for their participation, S. Buth, M. Hausmann, U. Lenz and K. Rüker for their
technical contribution and are grateful to the Elternhilfe für
Kinder mit Rett Syndrom for their support.
REFERENCES
1. Rett, A. (1966) Über ein eigenartiges hirnatrophisches Syndrom bei Hyperamonaemie im Kindesalter. Wien Med. Wochenschr., 116, 723–728.
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, 405–408.
3. Hagberg, B. (1985) Rett’s syndrome: prevalence and impact on progressive
severe mental retardation in girls. Acta Paediatr. Scand., 74, 405–408.
4. Hanefeld, F., Hagberg, B. and Percy, A. (1994) Molecular and neurobiology aspects of Rett syndrome. Neuropediatrics, 26, 60–61.
5. Hagberg, B. (1995) Rett syndrome: clinical peculiarities and biological
mysteries. Acta Paediatr., 84, 971–976.
6. 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., 35, 997–1003.
7. Sirianni, N., Naidu, S., Pereira, J., Pillotto, R.F. and Hoffman, E.P. (1998)
Rett syndrome: confirmation of X-linked dominant inheritance, and localization of the gene to Xq28. Am. J. Hum. Genet., 63, 1552–1558.
8. Xiang, F., Zhang, Z., Clarke, A., Joseluiz, P., Sakkubai, N., Sarojini, B.,
Delozier-Blanchet, C.D. et al. (1998) Chromosome mapping of Rett syndrome: a likely candidate region on the telomere of Xq. J. Med. Genet.,
35, 297–300.
9. 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.
10. Lewis, J.D., Meehan, R.R., Henzel, W.J., Maurer-Fogy, I., Jeppesen, P.,
Klein, F. and Bird, A. (1992) Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell,
69, 905–914.
11. D’Esposito, M., Quaderi, N.A., Ciccodicola, A., Bruni, P., Esposito, T.,
D’Urso, M. and Brown, S.D. (1996) Isolation, physical mapping, and
northern analysis of the X-linked human gene encoding methyl CpG-binding protein, MECP2. Mamm. Genome, 7, 533–535.
12. Meehan, R.R., Lewis, J.D. and Bird, A.P. (1992) Characterization of
MeCP2, a vertebrate DNA binding protein with affinity for methylated
DNA. Nucleic Acids Res., 20, 5085–5092.
13. Coy, J.F., Sedlacek, Z., Bachner, D., Delius, H. and Poustka, A. (1999) A
complex pattern of evolutionary conservation and alternative polyadenylation within the long 3′-untranslated region of the methyl-CpG-binding
protein 2 gene (MeCP2) suggests a regulatory role in gene expression.
Hum. Mol. Genet., 8, 1253–1262.
14. Ng, H.H., Zhang, Y., Hendrich, B., Johnson, C.A., Turner, B.M., Erdjument-Bromage, H., Tempst, P. et al. (1999) MBD2 is a transcriptional
repressor belonging to the MeCP1 histone deacetylase complex. Nature
Genet., 23, 58–61.
15. Jones, P.L., Veenstra, G.J., Wade, P.A., Vermaak, D., Kass, S.U., Landsberger, N., Strouboulis, J. et al. (1998) Methylated DNA and MeCP2
recruit histone deacetylase to repress transcription. Nature Genet., 19,
187–191.
16. Wade, P.A., Gegonne, A., Jones, P.L., Ballestar, E., Aubry, F. and Wolffe,
A.P. (1999) Mi-2 complex couples DNA methylation to chromatin remodelling and histone deacetylation. Nature Genet., 23, 62–66.
17. Wade, P.A., Jones, P.L., Vermaak, D., Veenstra, G.J., Imhof, A., Sera, T.,
Tse, C., Ge, H., Shi, Y.B., Hansen, J.C. and Wolffe, A.P.(1998) Histone
deacetylase directs the dominant silencing of transcription in chromatin:
association with MeCP2 and the Mi-2 chromodomain SWI/SNF ATPase.
Cold Spring Harbor Symp. Quant. Biol., 63, 435–445.
18. 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.
19. Nan, X., Campoy, F.J. and Bird, A. (1997) MeCP2 is a transcriptional
repressor with abundant binding sites in genomic chromatin. Cell, 88,
471–481.
20. Nan, X., Tate, P., Li, E. and Bird, A. (1996) DNA methylation specifies
chromosomal localization of MeCP2. Mol. Cell. Biol., 16, 414–421.
21. Chandler, S.P., Guschin, D., Landsberger, N. and Wolffe, A.P. (1999) The
methyl-CpG binding transcriptional repressor MeCP2 stably associates
with nucleosomal DNA. Biochemistry, 38, 7008–7018.
22. Willard, H.F. and Hendrich, B.D. (1999) Breaking the silence in Rett syndrome [news, comment]. Nature Genet., 23, 127–128.
23. Tate, P., Skarnes, W. and Bird, A. (1996) The methyl-CpG binding protein MeCP2 is essential for embryonic development in the mouse. Nature
Genet., 12, 205–208.
24. Wan, M., Lee, S.S., Zhang, X., Houwink-Manville, I., Song, H.R., Amir,
R.E., Budden, S., Naidu, S., Pereira, J.L., Lo, I.F. et al. (1999) Rett syndrome and beyond: recurrent spontaneous and familial MECP2 mutations
at CpG hotspots. Am. J. Hum. Genet., 65, 1520–1529.
25. Hagberg, B., Goutieres, F., Hanefeld, F., Rett, A. and Wilson, J. (1985)
Rett syndrome: criteria for inclusion and exclusion. Brain Dev., 7, 372–
373.
26. Camus, P., Abbadi, N. and Gilgenkrantz, S (1994) X inactivation in Rett
syndrome: a preliminary study showing partial preferential inactivation of
paternal X with the M27 beta probe. Am. J. Med. Genet., 50, 307–308.
Human Molecular Genetics, 2000, Vol. 9, No. 9 1375
27. Kormann-Bortolotto, M.H., Woods, C.G., Green, S.H. and Webb, T.
(1992) X-inactivation in girls with Rett syndrome. Clin. Genet., 42, 296–
301.
28. Migeon, B.R., Dunn, M.A., Thomas, G., Schmeckpeper, B.J. and Naidu,
S. (1995) Studies of X inactivation and isodisomy in twins provide further
evidence that the X chromosome is not involved in Rett syndrome. Am. J.
Hum. Genet., 56, 647–653.
29. Webb, T. and Watkiss, E. (1996) A comparative study of X-inactivation in
Rett syndrome probands and control subjects. Clin. Genet., 49, 189–195.
30. Zoghbi, H.Y., Percy, A.K., Schultz, R.J. and Fill, C. (1990) Patterns of X
chromosome inactivation in the Rett syndrome. Brain Dev., 12, 131–532.
31. Anvret, M. and Wahlstrom, J. (1994) Rett syndrome: random X chromosome inactivation [letter]. Clin. Genet., 45, 274–275.
32. Miller, S.A., Dykes, D.D. and Polesky, H.F. (1988) A simple salting out
procedure for extracting DNA from human nucleated cells. Nucleic Acids
Res., 16, 1215.
33. Cheadle, J.P., Gill, H., Fleming, N., Maynard, J., Kerr, A., Leonard, H.,
Krawczak, M. et al. (2000) Long-read sequence analysis of the MECP2
gene in Rett syndrome patients: correlation of disease severity with mutation type and location. Hum. Mol. Genet., 9, 1119–11229.
1376 Human Molecular Genetics, 2000, Vol. 9, No. 9