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Charman et al. Genotype-phenotype associations in Rett syndrome
Supplementary data
Methods
Mutation screening
MECP2 mutation screening was carried out for this study on 62 patients. Multiple tissue
samples (blood, buccal and hair) were collected from patients, and from their mothers, fathers and
siblings over 11 years old where available.
DNA extractions from blood were performed at the Department of Medical Genetics,
University of Glasgow, UK under NHS medical genetics QC conditions. DNA was extracted from
approximately 7ml of EDTA blood by standard kit-based procedures. Buccal brushings were also
taken (1 brush per individual). Buccal brushes (Epicentre Technologies; Madison, WI) were used to
swab inner cheeks, and left to air dry for a period of 2 hours before packaging under sterile
conditions and transportation to the lab. On arrival, brushes were stored at -20˚C before processing.
Buccal DNA was extracted using the Qiagen QIAamp DNA Blood mini kit (Qiagen Ltd; Crawley,
UK), as per manufacturer’s instructions.
Amplification of exonic fragments of MECP2 from genomic DNA was carried out as
follows. Exons 2, 3 and 4 were amplified along with splice sites and small amounts of flanking
intron. Exon 4 was amplified as 5 separate, overlapping fragments. Primer sequences are given in
Supplementary Table ST1 below.
Exon 4 fragment ‘a’ (exon 4a) was amplified in some cases using primers exon 4a1F and
4a2R. Amplification was generally carried out for 30 cycles under standard ‘step-down’ conditions
(similar to conditions given in Buyse et al. (ref. 35): 10-25l reactions with 20-50ng genomic DNA
and employing AB Gene’s (ABgene Ltd; Epsom, UK) PCR MasterMix (final annealing
temperatures are available from the authors).
S1
Supplementary Table ST1
PCR primers used to screen MECP2 in Glasgow
Exon Primer Name Primer Sequence
2
3
4a1
4a2
4b
4c/d
4e
Reference
2-For
5’-TAAGCTGGGAAATAGCCTAGTAC-3’
Buyse et al. 200034
2-Rev
5’-TTATATGGCACAGTTTGGCACAG-3’
Buyse et al. 2000
3-For
5’-AGGACATCAAGATCTGAGTGTAT-3’
Buyse et al. 2000
3-Rev
5’-GGTCATTTCAAGCACACCTG-3’
Buyse et al. 2000
4a-For.2
5’-CGCTCTGCCCTATCTCTGA-3’
Buyse et al. 2000
exon 4a1R
5’-GTGGCCGCCTTGGGTCTC-3’
This study
exon 4a2 F
5’- AGAGCAGAAACCACCTAAGAAG -3’
This study
exon 4a2 R
5’- ACAATCGGATAGAAGACTCCTT -3’
This study
4b-For.3
5’- GGCAGGAAGCGAAAAGCTGAG -3’
Buyse et al. 2000
4b-Rev.3
5’- TGAGTGGTGGTGATGGTGGTGG -3’
Buyse et al. 2000
4c/d-cFor
5’- GGAAAGGACTGAAGACCTGTAAG -3’ Buyse et al. 2000
HZR4
5’- CTCCCTCCCCTCGGTGTTTG -3’
Buyse et al. 2000
4e-For
5’- GGAGAAGATGCCCAGAGGAG -3’
Buyse et al. 2000
4e-Rev
5’- CGGTAAGAAAAACATCCCCAA -3’
Buyse et al. 2000
Sequences of each fragment were ascertained by automated fluorescent sequencing of PCR
products in most cases. Exon 2 fragments could only be successfully sequenced in the forward
direction due to the presence of mononucleotide tracts close to the R primer. In some cases, PCR
products were cloned using T-vector kits and multiple clones were sequenced and aligned using
ABI Autoassembler 2.0 (Applied Biosystems; Foster City, CA) to detect variant sites. Exon 4a and
4b fragments were initially screened for the presence of several common mutations that generate or
abolish restriction sites. All point mutations and variants observed were confirmed either by two
methods (restriction digestion and sequencing), or by sequencing two separate fragments (e.g. exon
4a and 4b), or by sequencing fragments generated in independent PCR reactions in the forward and
reverse directions, depending on the identity and location of the mutation/variant. Small insertions
and deletions were confirmed by gel electrophoresis of the fragment containing the alteration
followed by sequencing in the forward and reverse directions. Larger deletions were confirmed by
S2
gel electrophoresis of amplified fragments (in some cases, larger fragments were amplified for this
purpose by combining primers from different amplicons) and sequencing of gel-purified normal and
deletion allele products.
MECP2 mutations identified were categorised into 3 classes – missense, early truncating
and late truncating. The late truncating class included all mutations involving small (1-60bp)
deletions and in/dels in the region encoding the C-terminal portion of MECP2, as well as a small
number of larger deletions of 100-200bp. All these mutations alter the reading frame and are thus
expected to cause premature truncation of the protein at stop codons within the new reading frame.
As they are all located in the final exon, they are not expected to lead to nonsense-mediated mRNA
decay, and should thus lead to expression of a truncated protein that is presumed to enter the
nucleus and be capable of DNA-binding and possibly other aspects of the normal function of
MECP2. All nonsense mutations located downstream of the TRD-NLS (amino acids 255-271 of the
MECP2e2 isoform) were also included in this class. The early truncating class consisted of
nonsense mutations up to and including the TRD-NLS, all larger deletions removing this region and
all DNA ‘null’ mutations involving deletions spanning large parts of the gene. The TRD-NLS was
chosen as the cut-off, since proteins that do not incorporate it are likely to be excluded from the
nucleus and are therefore unlikely to be able to perform the major functions of MECP2 as they are
currently understood. Since this effect is probably functionally equivalent to that of large deletions
that result in ‘protein null’ phenotypes, the latter mutations were included in this class. The
missense mutations were treated as a single group, as their phenotypic effects are not expected to
vary consistently with position in the protein’s primary structure. For analyses of the phenotypic
effects of the commonly occurring mutations, we have combined R306C and R306H, on the
assumption that the phenotype arises from loss of a function provided by R306, and not from
separate gains of function. This conclusion is supported by the fact that the substituted residues are
of different functional categories, and H306 should have a positive charge (as does R306) in the
nuclear environment whereas C306 does not.
X chromosome inactivation analysis
XCI analysis was performed using the HUMARA test. Briefly, genomic DNA extracted
from blood and from buccal samples from female subjects was subjected to PCR amplification at
the AR locus with and without prior HpaII digestion and the products were resolved and analysed
by ABI GeneScan (Applied Biosystems, Foster City, CA) gel technology. XCI ratios in AR
heterozygotes were calculated from peak areas for each allele36 and were expressed as the
proportion of the allele with the greater peak area in the sample (i.e. 50-100%). A few additional
datapoints were generated by similar analysis of the FMR1 promoter (CGG)n polymorphism
S3
according to ref. [37], in patients homozygous at AR. Where available, the XCI ratio was calculated
as the mean of replicate experiments performed on blood lymphocyte DNA. In a few cases, only
buccal DNA was available, and values are given as means of replicate experiments using these
samples. Direction of skewing relative to the MECP2 mutant allele has not yet been ascertained.
Results
Details of all mutations and mean XCI ratios present in cases analysed in this study are
presented below.
Table ST2 shows all mutations present in cases analysed in this study, ordered by their
position from 5’- to 3’- in the gene (amino acid position is given relative to the initiator methionine
of the MECP2e2 isoform, as used in most previous reports). Mutations found in Glasgow as part of
this study are listed separately. References for mutations reported in other studies are given where
appropriate. In addition to the data in the Table, a further 9 patients without identified mutations in
MECP2 have been included in previously published datasets (four in ref. 22; three in ref. 23; one in
ref. 38; one in both ref. 25 and ref. 24 [in both of these papers, the patient was reported as carrying
p.R168X, but further analysis indicated that no mutation could be confirmed]). ## indicates
patients reported previously as ‘No mutation’ in whom mutations were later discovered. Mutations
in bold are those common mutations that were analysed singly (see text). The large deletions were
found using either the MLPA procedure31 or a quantitative PCR procedure30. Note that several cases
had two separate mutations. In these cases, the mutation predicted to have the more severe effect
(early truncating or missense, in comparison with late truncating, for example, or bigger deletion vs.
smaller deletion/indel) was used in the analysis. All cases with more than one mutation will be
described in other publications. For those mutations observed in more than one case, their
proportion in the mutation-positive set is given (%). The bold line below p.R270X represents the
split between the Early truncating mutation category and the Late truncating category for
nonsense/frameshifting mutations. CR indicates cases with classic Rett syndrome together with a
small number of cases who have a typical but incomplete classic profile, usually due to their age
(incCR). RnonC indicates atypical forms of Rett syndrome. * indicates case (female) was mosaic
for mutation. ** indicates a small-medium truncating deletion or in/del mutation in the region of
MECP2 encoding the C-terminal portion of the protein. Y indicates that the mutation is novel and
reported for the first time here. Informative XCI ratio mean values (expressed as %, in the range 50100%) are given for each informative case for each mutation. ‡ indicates buccal sample only was
available for XCI ratio testing. XCI ratio values in bold are moderately skewed (75%) and those in
bold italic are extremely skewed (>90%).
7
S4
Supplementary Table ST2
Mutation (DNA)
Mutation (Protein)
All
CR
All Glasgow Glasgow % of cases
Novel No. previously XCI ratio
RnonC CR
RnonC
with
this study
reported
(%)
mutation
(and ref.)
1
2
0
3.7
N/A
2 [23]
53, 58, 65,
1 [25]##
85, 95
0
81
g.Large deletion, multiple exons
Null
4
c.91delG
p.V31fsX30
1
c.139C>T
p.Q47X
1
0
83
c.302C>T
p.P101L
1
0
85
c.316C>T
p.R106W
3
1
1
0
g.IVS3 -3C>G
Null
0
1
0
0
c.382C>T
p.Q128X
1
c.397C>T
p.R133C
4
c.423C>G
p.Y141X
1
0
c.455C>G
p.P152R
6
5
4.4
c.473C>T
p.T158M
13
8
9.6
c.481_987del507 + 481_482ins8
p.G161-G329delfsX8
1
0
c.495_1163del669
p.S166-P388del223
1
c.502C>T
p.R168X
14
2
4
0
c.502C>T + 1136_1142del7
p.R168X + other
0
1
0
0
c.622C>T
p.Q208X
1
0
c.730C>T
p.Q244X
2
1
c.748_749insC
p.R250fsX257
1
0
c.753delC
p.G252fsX287
2
2
c.763C>T
p.R255X
7
c.784C>T
p.Q262X
1
0
c.792_804del13
p.I264fsX283
1
1
c.806delG
p.G269fsX287
1
0
c.808C>T
p.R270X
9
6
c.808C>T*
p.R270X*
0
1*
0
0
c.880C>T
p.R294X
3
1
1
0
c.905C>G
p.P302R
2
1
1.5
c.905C>T
p.P302L
2
0
1.5
c.910A>C
p.K304Q
1
c.916C>T
p.R306C
8
c.917G>A
p.R306H
2
1
c.1054_1259del206**
p.K352fsX366
1
1
Y
96
c.1101_1201del101**
p.H367fsX369
1
1
Y
66
c.1116_1201del86**
p.H372fsX374
1
0
c.1126_1159del34ins39**
p.P376fsX400
1
1
c.1152_1192del41**
p.P385fsX390
1
1
c.1152_1195del44**
p.P385fsX389
1
0
c.1154_1197del44**
p.P385fsX402
1
0
c.1157_1196del40**
p.L386fsX394
1
1
3.0
60, 75, 77
0
5
2
0
6.7
2
1 [22,25]
1 [22;25##]
1
1
3 [22]
1 [25]
51, 52, 64,
65, 66‡, 68,
77
57‡, 58, 60,
63, 82
51, 56, 62,
68, 68, 69,
74, 78, 80
80
Y
0
11.9
2 [24]
1 [25]
50, 57, 63,
64, 73, 74,
78‡
1 [25]
62‡
1.5
79, 98
1.5
64
5.9
1 [22,25]
1[22,24,25]
1 [25]
59, 68, 72,
72, 77, 91
54
Y
6.7
1 [22]
3.0
63, 67, 70,
70, 74, 75,
91
51
1 [22]
0
2
S5
6
1
7.4
1 [22; 25##]
1.5
54, 58, 64,
66, 66, 67,
72, 72, 84
54, 63
Y
52‡
1 [22, 25]
67
50
Y
88
c.1157_1197del41**
p.L386fsX389
3
c.1157_1200del44**
p.L386fsX388
4
1
c.1157_1200del44**, +c.862G>A
1
0
c.1157_1202del46**
p.L386fsX388,
+p.V288M
p.L386fsX392
1
0
c.1158_1201del44**
p.P387fsX388
1
c.1159_1202del44**, +1098_1100del3
0
1
0
0
83
c.1163_1206del44**
p.P387fsX389
+p.H367del
p.P388fsX486
0
1
0
0
72
c.1164_1207del44**
p.P389fsX388
2
0
c.1224_1267del44 [=c.1160_1203del44**?] p.S409fsX481?
1
0
c.1398_1428del31**?
1
0
p.M466fsX470
Totals
1
1
0
3.0
1 [22]
58, 59
3.0
1 [25]
60, 61, 71
74
0
N=116 N=19
N=51
1.5
N=1
77.3%
55
6 Novel
Mutation screening
A summary of the results for Set A (patients screened specifically for this study in Glasgow)
is set out here: of the 62 RS patients, both classic (n=57) and atypical (n=5), specifically screened
for this study, mutations were identified in 52, including two cases with large deletions of the region
of MECP2 containing exons 3 and 4, identified using the MLPA assay. Ten patients had no
identifiable mutations in exons 2, 3 or 4 by sequencing, seven of whom were screened using the
MLPA assay or by qPCR - no copy number anomalies were found in these patients.
Of the 57 classic RS patients in Set A, 51 (89.5%) were found to have MECP2 mutations; 2
of the 6 classic cases without identified mutations remain to be checked by appropriate dosagesensitive methods. One of the 6 atypical Rett patients was found to have a mutation (p.R306C).
The following statistics are presented for the entire dataset reported in this paper (set A and
set B): 77% of cases with identified mutations carried a ‘recurrent’ mutation (observed in more than
1 case in the dataset). 19% of classic CR cases (22/116) and 16% of atypical cases (3/19) with
identified mutations in our dataset carried small-medium truncating deletions or in/dels in the
region encoding the C-terminal portion of MECP2 (indicated by ** in Table ST2). 85% of classic
CR cases with mutations carried ‘recurrent’ mutations, including the C-terminal region deletions
and in/dels.
XCI ratio analysis
Of the 135 patients with identified mutations, we were able to generate or gather existing
XCI ratio data for 110 (81%). Of these, only 85 were informative, the rest being homozygous at the
AR and FMR1 repeat loci genotyped or for whom the assay failed. We also generated or gathered
XCI ratio data for 39 cases with no identified mutation, of which 33 were informative (data not
shown).
S6
N=85
References
35
Buyse IM, Fang P, Hoon KT, Amir RE, Zoghbi HY, Roa BB. Diagnostic testing for Rett
syndrome by DHPLC and direct sequencing analysis of the MECP2 gene: Identification of several
novel mutations and polymorphisms. Am J Hum Genet 2000; 67: 1428-36
36
Pegoraro E, Schimke RN, Arahara K, et al. Detection of new paternal dystrophin gene
mutations in isolated cases of dystrophinopathy in females. Am J Hum Genet 1994; 54: 989-1003
37
Carrel L, Willard HF. An assay for X inactivation based on differential methylation at the
fragile X locus, FMR1. Am J Med Genet 1996; 64: 27-30
38
Borg I, Freude K, Kübart S, Hoffmann K, Menzel C, Laccone F, Firth H, Ferguson-Smith
MA, Tommerup N, Ropers H-H, Sargan D, Kalscheuer VM. Disruption of Netrin G1 by a balanced
chromosome translocation in a girl with Rett syndrome. Eur J Hum Genet; AOP
doi:10.1038/sj.ejhg.5201429
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