genotype and early development in rett syndrome: the value of international data
TRANSCRIPT
Original article
Genotype and early development in Rett syndrome:
The value of international data
Helen Leonarda,*, Hannah Moorea, Mary Careya, Susan Fyfeb, Sonj Hallc, Laila Robertsona,
Xi Ru Wud, Xinhua Baod, Hong Pand, John Christodouloue,f,
Sarah Williamsone,f, Nick de Klerka
aTelethon Institute for Child Health Research, Centre for Child Health Research, The University of Western Australia, Perth, Western AustraliabCurtin University of Technology, Perth, Western Australia
cSchool of Population Health, The University of Western Australia, Perth, Western AustraliadPeking University First Hospital, Beijing, China
eDiscipline of Paediatrics and Child Health, University of Sydney, New South Wales, AustraliafWestern Sydney Genetics Program, Children’s Hospital at Westmead, New South Wales, Australia
Received 17 August 2004; received in revised form 1 October 2004; accepted 5 March 2005
Abstract
Background: Rett syndrome is a neurodevelopmental disorder mostly affecting females and caused by mutations in the MECP2 gene.
Originally the syndrome was characterised as having a normal prenatal and perinatal period with later regression. Previous work has speculated
that the girl with Rett syndrome may not be normal at birth. Aims: to examine whether early development between birth and ten months varies
by genotype in Rett syndrome. Methods: cases were sourced from two databases, the Australian Rett Syndrome Database (est. 1993) and the
newly formed InterRett - IRSA Rett Phenotype Database. Data available on 320 cases included information provided by parents on perinatal
problems, early developmental behaviour and mobility. Problem scores, mobility scores and a total composite score for each mutation were
generated and compared. Results: overall, 58% of respondents noted unusual behaviour during the first six months and 70.6% from the period
between 6 and 10 months of life. Statistically significant differences were detected between some of the common mutations. Infants with R294X
(PZ0.05) and R133C (PZ0.03) were less likely than those with R255X to have problems in the perinatal period. The most severe profile
overall for early development was associated with mutations R255X and R270X. Conclusion: This is the largest study to date examining the
effects of individual mutations in Rett syndrome. With the ongoing case ascertainment and expansion of InterRett, sample size will increase
rapidly and provide improved statistical power for future analyses. Results from this study will contribute to understanding the mechanism of
early development in Rett syndrome and determining if and at which time(s) early intervention might be feasible.
q 2005 Elsevier B.V. All rights reserved.
Keywords: Rett syndrome; MECP2; Phenotype; Early development; International; Internet; Database
1. Introduction
The clinical criteria for Rett syndrome identified by
Hagberg et al. [1] and then revised by Trevarthan and Moser
[2] characterised Rett syndrome as a neurodevelopmental
disorder with a normal prenatal and perinatal period and
early development in the normal range with a later
0387-7604/$ - see front matter q 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.braindev.2005.03.023
* Corresponding author. Tel.: C61 8 9489 7790; fax: C61 8 9489 7700.
E-mail address: [email protected] (H. Leonard).
regression period. However, it has become apparent that
girls with Rett syndrome may not be normal at birth [3,4]
and that there are subtle differences which indicate
developmental delay early in life [5,6]. Early hypotonia
[7–9], motor problems [10,11] including early jerky
incoordination [7], placidity and perinatal difficulties
requiring admission to a special care nursery [3] have
been noted in the pre-regression period.
Since the association between mutations in MECP2 and
Rett syndrome was recognized [12], many studies have tried
to characterise the relationship between genotype and
clinical severity. In a sample of 56 girls Amir et al. [13]
found respiratory dysfunction was more frequently
Brain & Development 27 (2005) S59–S68
www.elsevier.com/locate/braindev
H. Leonard et al. / Brain & Development 27 (2005) S59–S68S60
associated with truncating and scoliosis with missense
mutations. Other studies have found a more severe clinical
phenotype in truncating when compared with missense
mutations [14–17]. Where investigations have taken into
account mutation location as well as mutation type a more
refined picture appears. Cheadle et al. [14] showed that early
truncating mutations were likely to be more severe than late
truncating mutations. Similarly, Hoffbuhr et al. [18] found
that missense mutations in the MBD and mutations
truncating the entire TRD had a more severe clinical
phenotype than missense and nonsense mutations in the
TRD and C-terminal. Huppke et al. [16] found that
mutations in the TRD NLS were associated with a more
severe phenotype than those in the MBD and in the rest of
the TRD. Characterisation of phenotypic severity using a
large population by Leonard et al. [19] and Colvin et al. [20]
has shown differential phenotypic severity between the
common mutations including confirmation that, in com-
parison with other MECP2 mutations, the R133C mutation
has a milder clinical presentation. This was the first clear
and clinically meaningful description of the association
between genotype and clinical phenotype. The present study
aimed to investigate the relationship between early
development and individual mutations using both Austra-
lian population based data and international data.
Our previous work showed that, compared with singleton
births of a similar gestation, a greater proportion of Rett
syndrome cases had birthweights !3500 g and lower birth
head circumference; 41% of families reported some
perinatal problem and nearly half (46.5%) of families had
reported their child’s behaviour to be unusual in the first 6
months of life [3]. This led us to speculate that the girl with
Rett syndrome may not be normal at birth. The purpose of
this current study has been to examine whether any of these
early indicators of reduced optimality at birth and in the
early months vary by genotype.
2. Methods
2.1. Case ascertainment
Cases were sourced from two databases, the Australian
Rett Syndrome Database (ARSD) and InterRett, the IRSA
Rett Phenotype Database. Established in 1993, the ARSD is
a population-based database with ongoing ascertainment of
Australian Rett syndrome cases born since 1976 [21]. It
contains demographic, clinical, behavioural, developmen-
tal, functional and genetic information [22]. InterRett is an
international phenotype database, established in 2003 with
funding from the International Rett Syndrome Association
and managed from the Telethon Institute for Child Health
Research in Western Australia [23]. Data contributions to
InterRett cases can be made individually or as multiple
submissions from international collaborating centres.
Both databases collect data from families and clinicians.
The InterRett family questionnaire administered on case
enrolment is similar to and has been developed from the
original Australian version providing comparability
between the two datasets.
As at 31st October 2003, 251 cases had been reported to
ARSD and verified according to the clinical criteria for
classical or atypical Rett syndrome [3]. Of the 251 verified
cases, data from families have been provided in 235 and in
16, information was currently only available from the
clinician. Data provision to InterRett commenced in January
2003. Cases were included in this analysis either if they had
a valid pathogenic mutation or if the diagnosis of Rett
syndrome was considered by the child’s doctor to be
‘definite’. As at June 2003, 88 InterRett cases meeting this
study definition had been reported. Three of these cases
were duplicates of ARSD cases and so were excluded from
this analysis. Thus a total of 235 ARSD (including 130 cases
from the original cohort [3]) and 85 InterRett cases with
family information were available for this study. Ethical
approval for the study was granted by the local institutional
ethics committee.
2.2. Clinical data collection
This report focuses on information relating to early
development provided by the family. As previously
described, data were collected by paper-based question-
naires for ARSD cases [21] and by paper-based or Internet
questionnaires for InterRett cases [23]. Questions were
asked about problems in the first week of life, abnormal
behaviours and development in the first 6 months and from
after 6–10 months. Open responses were coded according to
type of problem as identified by the family e.g. placid,
sleeping problems, gastro-intestinal, developmental
problems.
For each question, cases were generally allocated a score
of 0 if there were no problems identified and a score of 1 for
each problem type identified. However for the questions on
admission to special nursery and for help with breathing at
birth, responses were simply scored as 0 or 1. A composite
perinatal score was derived from the three questions
involving the perinatal period. The maximum number of
problems coded for any of the three problem questions was
3. Thus each case could receive a score of between 0 and 5
for the perinatal period and a score of between 0 and 3 for
the other two time periods and thus a total problem score
ranging from 0 to 11. Additionally, cases were scored
according to their reported mobility at 10 months with the
lowest score of 0 allocated to those performing best (i.e.
walking independently) and the highest score of 12 to those
who could not move around. A score of 10 was allocated for
rolling, 8 for commando crawling, 6 for bottom shuffling, 4
for crawling, and 2 for moving around by furniture walking
or knee walking. A total composite (problem and mobility)
score was computed with a possible range from 0 to 23.
Cases with missing data were not allocated scores.
Table 1
Distribution of cases by year of birth, country of birth and age at diagnosis
and data source
Category Data source
ARSD
(NZ235) n(%)
InterRett
(NZ85) n(%)
All cases*
(NZ320) n(%)
Year of birth
1962–1970 – 6(7.1) 6(1.9)
1971–1975 – 2(2.4) 2(0.6)
1976–1980 33(14.0) 9(10.6) 42(13.1)
1981–1985 49(20.9) 10(11.8) 59(18.4)
1986–1990 59(25.1) 13(15.3) 72(22.5)
1991–1995 58(24.7) 17(20.0) 75(23.4)
1996–2000 35(14.9) 28(32.9) 63(19.7)
2001-present 1(0.4) – 1(0.3)
Country of birth
Argentina – 1(1.2) 1(0.3)
Australia 222(94.5) 2(2.4) 224(70.0)
Canada – 2(2.4) 2(0.6)
China – 15(17.6) 15(4.7)
Hong Kong 1(0.4) – 1(0.3)
India – 1(1.2) 1(0.3)
Jordan 1(0.4) – 1(0.3)
Mexico – 1(1.2) 1(0.3)
New Zealand 3(1.3) 1(1.2) 4(1.3)
Philippines 1(0.4) – 1(0.3)
Romania 1(0.4) – 1(0.3)
United Kingdom 3(1.3) 1(1.2) 4(1.3)
United States 1(0.4) 61(71.8) 62(19.4)
Age at diagnosis (yrs)
0!6 132(76.3) 57(68.7) 189(73.8)
6!11 28(16.2) 14(16.9) 42(16.4)
11!16 11(6.4) 6(7.2) 17(6.6)
16!21 2(1.2) 4(4.8) 6(2.3)
21 and above – 2(2.40) 2(0.8)
*Denominator used taking into account missing values for individual
questions.
H. Leonard et al. / Brain & Development 27 (2005) S59–S68 S61
2.3. Mutational analyses
Mutation testing has been carried out on 197/235
(83.8%) ARSD and on 66/85 (77.6%) InterRett cases with
pathogenic mutations identified in 136/197 (69.0%) and
45/66 (68.2%), respectively. This analysis concentrates
particularly on those cases where a pathogenic MECP2
mutation was identified. Pathogenicity was verified as
previously [19,20] by laboratory report for ARSD cases.
For InterRett cases, multiple sources (clinician and family
questionnaires) were used to confirm mutation status and
pathogenicity was validated using the mutation database,
RettBASE [24]. For the purposes of this study any
ambiguous mutation data were not treated as pathogenic.
Cases with any pathogenic mutation were identified and
then cases with any of the seven most common pathogenic
mutations (R133C, T158M, R168X, R255X, R270X,
R294X and R306C) were grouped together and according
to their specific mutation. Other groupings were cases with
other pathogenic mutations and cases who had either not
been tested or in whom a mutation had not been found. The
ARSD and InterRett data were stored in Filemaker Pro 6
databases and transferred into SPSS Version 10.0 for
analysis.
2.4. Data analysis
For each group the proportions reporting any or a specific
problem for the three time periods, perinatal, 0–6, O6–10
months were presented. In all cases the denominators were
adjusted to account for missing values on individual
questions. The chi-square test was used to determine the
presence of associations between categorical variables [25].
Means were calculated for individual and total problem
scores, the mobility score and the composite score
[including problems and mobility]. One-way analysis of
variance [Kruskal Wallis for non-normally distributed
variables] was used to compare differences in means
according to mutation categories. P values of less than 0.1
were considered indicative of important differences and no
adjustments were made for multiple testing [26].
3. Results
The distribution of cases by year, country of birth and age
at diagnosis by data source is shown in Table 1. Of those
262 (81.9%) had results from molecular testing. In 181 of
those tested, a valid pathogenic mutation as defined by study
criteria was present. For each of the mutations R133C,
T158M, R168X, R255X, R270X, R294X and R306C
there were eleven or more cases. This group accounted for
122/181 (67.4%) of those cases with pathogenic mutations.
Over a third of respondents reported that their child had
experienced a problem in the first week of life, with almost
the same proportion (39.3%) in the group with pathogenic
mutations. Within the seven common mutations the
proportions ranged from 3/11 (27.3%) in those with
R133C to 9/16 (56.3%) in those with R255X (PZ0.14,
Table 2). Feeding problems were the most common
individual problem reported in 24 (13.5%) of those with
valid mutations and in 18 (15%) of those with the seven
common mutations. Parents were also asked whether the
infant needed help breathing. Again there was variation with
no such reports in the 11 with R133C compared with 4/15
(26.7%) in those with R255X (PZ0.06). Only 1/16 (6.3%)
of R294X cases had been admitted to a special nursery
compared with 5/16 (31.3%) of those with R255X (PZ0.07). Overall 81/180 (45.0%) of those with pathogenic
mutations were reported to have some perinatal difficulty:
either help to start breathing, admission to a special nursery
or a problem in the first week of life.
Overall 184/317 (58%) of respondents reported that they
had noted unusual behaviour or development during the first
6 months of their daughter’s life (Table 3). Nineteen reports
included in the analysis were ambiguous in that respondents
provided a response to the open but not the closed question.
Table 2
Presence and nature of perinatal problems by mutation type
Observation/problem R133C
(NZ11)
n (%)
R294X
(NZ17)
n (%)
R168X
(NZ22)
n (%)
T158M
(NZ28)
n (%)
R255X
(NZ16)
n (%)
R270X
(NZ16)
n (%)
R306C
(NZ12)
n (%)
Seven common
mutations (NZ122)
n (%)
Other patho-
genic mutations
(NZ59) n (%)
All pathogenic
mutations
(NZ181) n (%)
No Mutation or
Not tested
(NZ139) n (%)
All cases*
(NZ320)
n (%)
Problems in the 1st week
of life
3(27.3) 6(35.3) 9(45.0) 13(46.4) 9(56.3) 6(37.5) 6(50.0) 52(43.3) 18(31.0) 70(39.3) 63(46.3) 123(38.8)
Feeding – 2(11.1) 2(10.0) 7(25.0) 3(18.8) 1(6.3) 3(25.0) 18(15.0) 6(10.3) 24(13.5) 27(19.8) 51(16.1)
Jaundice – 1(5.6) 4(20.0) 2(7.1) – 1(6.3) 2(16.7) 10(8.3) 7(12.1) 17(9.6) 5(3.7) 22(7.0)
Gastrointestinal 1(9.1) 1(5.6) – 2(7.1) 1(6.3) 2(12.6) 1(8.3) 8(6.7) 2(3.4) 10(5.6) 6(4.4) 16(5.1)
Breathing problems – – 1(5.0) 3(10.7) 2(12.5) 1(6.3) 2(16.7) 9(7.5) 1(1.7) 10(5.6) 4(2.9) 14(4.4)
Sleep – – – 2(7.1) 1(6.3) 3(18.8) 6(5.0) 2(3.4) 8(4.5) 4(2.9) 12(3.8)
Hypoglycaemia – – 2(10.0) – – – 2(1.7) 2(3.4) 4(2.2) – 4(1.3)
Hypothermia – – – 1(3.6) – – 1(1.0) 1(1.7) 2(1.1) – 2(0.6)
Hypotonia – – – 1(3.6) 1(6.3) – 2(1.7) – 2(1.1) – 2(0.6)
Birth defect – – – – 1(6.3) – 1(1.0) – – 1(0.3)
Other 2(18.2) 2(11.1) – 2(7.1) 2(12.5) – 2(16.7) 10(8.3) 2(3.4) 12(6.7) 11(8.1) 23(7.3)
Special nursery 2 (8.2) 1(6.3) 5(22.7) 4(15.4) 5(31.3) – 2(16.7) 19(16.0) 8(13.8) 27(15.3) 24(17.8) 51(16.8)
Help breathing – 1(6.7) – 6(23.1) 4(26.7) 1(6.7) 2(16.7) 14(8.7) 5(8.6) 19(11.0) 22(16.4) 41(13.4)
Any perinatal difficulty 4(36.4) 6(35.3) 10(45.5) 13(46.4) 10(62.5) 7(43.8) 7(58.3) 57(46.7) 24(41.4) 81(45.0) 64(46.4) 145(45.6)
*Denominator used taking into account missing values for individual questions.
Table 3
Presence and nature of abnormal development or behaviour in first 6 months of life by mutation type
Observation/problem R133C
(NZ11)
n (%)
R294X
(NZ17)
n (%)
R168X
(NZ22)
n (%)
T158M
(NZ28)
n (%)
R255X
(NZ16)
n (%)
R270X
(NZ16)
n (%)
R306C
(NZ12)
n (%)
Seven com-
mon
mutations
(NZ122)
n (%)
Other
pathogenic
mutations
(NZ59)
n (%)
All patho-
genic
mutations
(NZ181)
n (%)
No
mutation or
not tested
(NZ139)
n (%)
All cases*
(NZ320)
n (%)
Unusual behaviour in
the 1st 6 months
4(36.4) 9(52.9) 15(68.2) 19(67.9) 11(68.8) 9(56.3) 6(50.0) 73(59.8) 33(56.9) 106(58.9) 78(56.9) 184(58.0)
Placid – 3(17.6) 3(13.6) 4(14.3) 4(25.0) 2(12.5) 4(33.3) 20(16.4) 12(20.7) 32(17.8) 26(19.0) 58(18.3)
Feeding – – 3(13.6) 5(17.9) 1(6.3) 4(25.0) 1(8.3) 14(11.5) 8(13.8) 22(12.2) 17(12.4) 39(12.3)
Developmental – 1(5.9) 1(4.5) 5(17.9) 3(18.8) 3(18.8) – 12(9.8) 5(8.6) 17(9.4) 18(13.1) 35(11.0)
Floppy – – 5(22.7) 5(17.9) 1(6.3) 2(12.5) – 13(10.7) 5(8.6) 18(10.0) 14(10.2) 32(10.1)
Gastrointestinal 1(9.1) 3(17.6) 1(4.5) 2(7.1) 2(12.5) 2(12.5) – 11(9.0) 1(1.7) 12(6.7) 9(6.6) 21(6.6)
Eyes – – 1(4.5) 1(3.6) 1(6.3) – 1(8.3) 4(3.3) – 4(2.2) 11(8.0) 15(4.7)
Sleep 1(9.1) 1(5.9) – – – – – 2(1.6) 4(6.9) 6(3.3) 3(2.2) 9(2.8)
Strange behaviour – 1(5.9) – 2(7.1) – – – 3(2.5) 1(1.7) 4(2.2) 4(2.9) 8(2.5)
Hand behaviour – – – – – – – – – – 5(3.6) 5(1.6)
Other 3(27.3) 2(11.8) 1(4.5) 1(3.6) 1(6.3) – 1(8.3) 9(7.4) 2(3.4) 11(6.1) 1(0.7) 12(3.8)
*Denominator used taking into account missing values for individual questions.
H.
Leo
na
rdet
al.
/B
rain
&D
evelopm
ent
27
(20
05
)S
59
–S
68
S6
2
Box 1. Examples of family expression of unusual behaviour from the first 6 months of life
Code: Placid
‘Unusually passive—happy always! Never crying not even when hungry—or when had chicken pox at 6 months’
‘Was very placid and slept a lot compared to friends babies’
‘She was always ‘too good’ a baby. Very placid, fed well (breast fed)’
‘Over placid. Did not cry much’
‘She was ‘too good’. Extremely placid with no acknowledgement of the world around her’
‘Extremely placid, very good sleeper’
‘Very placid, easily satisfied’
‘Low tone, slow to smile, very placid, sleepy’
‘She was a placid baby with a voracious appetite and a good feeder’
Code: Floppy
‘Poor feeder. Very floppy. Cried a lot’
‘She was floppy compared to other children’
‘Until 6 months was unable to support her own head—hypotonic—floppy’
‘She always seemed to have a very floppy tone compared with my first child and did not seem to be making her
milestones’
‘Her doctor as the time classed her as a ‘floppy baby’’
‘**** was a very ‘floppy’ baby—however in the first 6 months we just saw this as her being a very cuddly baby’
‘Kind of ‘floppy’ tone’
‘Lots of reflux, otherwise a very content ‘floppy’ baby’
‘She was a little floppy/soft’
H. Leonard et al. / Brain & Development 27 (2005) S59–S68 S63
A very similar proportion of unusual behaviour was
reported in those cases with pathogenic mutations. For the
seven specific mutations examined, the proportion varied
from 4/11 (36.4%) in cases with R133C to 11/16 (68.8%) in
cases with R255X mutations (PZ0.10, Table 3). When the
open responses were coded into specific categories the most
commonly reported category was placid reported in 18.3%
of all cases and in 16.4% of those with the common
mutations. Some examples of how the families expressed
this are shown in Box 1. Other high rankings were reports of
a floppy baby in 10.1% of all cases and 10.7% of those with
common mutations and of developmental problems in
11.0% of all cases and 9.8% of those with common
mutations.
Participants were then asked whether they had noticed
anything unusual about their child’s development or
behaviour in the period from after six to ten months of
age. Overall 221 of the 313 (70.6%) reported in the
affirmative (7 cases with ambiguous responses in that they
provided a response to the open but not the closed question
were also included) and 122/180 (67.8%) of those cases
with a valid mutation (Table 4). Thus the proportion
reporting in the affirmative among those who did not have a
mutation was marginally higher than among those who did
(74.4 vs 67.8%, PZ0.20). For the seven specific mutations
the proportions varied from 9/17 (52.9%) in those with
R294X to 13/16 (81.3%) in those with R270X. The
comparison between these two approached significance at
PZ0.08. Respondents’ most predominant recollection
within this time period, was concern specifically about
their child’s development (Box 2). This was mentioned in
nearly half (45.7%) of all cases and in 42.6% of those with
common mutations. Other categories were much less
frequently reported and generally in less than 10% of all
cases (Table 4).
For 311 cases a description of the child’s mobility at 10
months was available. In 62/311 (19.9%) of all cases and in
15.0% of those with the common mutations the child was
reported not to be moving around at 10 months. In 88/311
(28.3%) of all cases and a quarter (25.8%) of those with the
common mutations the child was reported to be moving
around by rolling. The child was commando crawling in
29/311 (9.3%) of all cases and in 7.5% of those with the
common mutations and bottom shuffling in 40/311 (12.9%)
and 15.8%, respectively. Sixty one (19.6%) of all cases and
nearly a quarter (24.2%) of those with the common
mutations were crawling at this time. Whilst 2.3 and
3.3%, were said to be able to walk independently, a further
8.0% of all cases and 8.3% of those with the common
mutations were using other forms of mobility such as
furniture or knee walking.
The mobility and other scores for each of the seven
common mutations are shown in Fig. 1. For mobility the
highest score indicating the least mobility at 10 months was
for R270X (meanZ8.25) and the lowest mean scores
indicating the best mobility were for R306C and R294X
(5.17, 5.76). After R294X and R306C the next most mobile
were the R133C and T158M cases, followed by R168X, and
R255X. However the only statistically significant differ-
ences were between R306C and both R255X and R270X
Tab
le4
Pre
sen
cean
dn
ature
of
abn
orm
ald
evel
op
men
to
rb
ehav
iou
rfr
om
6to
10
mon
ths
of
life
by
mu
tati
on
typ
e
Ob
serv
atio
n/p
rob
lem
R1
33
C
(NZ
11
)n
(%)
R2
94
X
(NZ
17
)n
(%)
R1
68
X
(NZ
22
)n
(%)
T1
58
M
(NZ
28
)n
(%)
R2
55
X
(NZ
16
)n
(%)
R2
70
X
(NZ
16
)n
(%)
R3
06
C
(NZ
12
)n
(%)
Sev
enco
mm
on
mu
tati
on
s(N
Z1
22
)n
(%)
Oth
er
pat
ho
gen
ic
mu
tati
on
s
(NZ
59
)
All
pat
ho
-
gen
ic
mu
tati
on
s
(NZ
18
1)
No
mu
tati
on
or
no
tte
sted
(NZ
13
9)
All
case
s*
(NZ
32
0)
Un
usu
al
beh
av
iou
rO
6–
10
mo
nth
s
6(5
4.5
)9
(52
.9)
15
(68.2
)1
7(6
0.7
)1
2(7
5.0
)1
3(8
1.3
)9
(75
.0)
81
(66.4
)4
1(7
0.7
)1
22
(67
.8)
99
(74.4
)2
21
(70
.6)
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H. Leonard et al. / Brain & Development 27 (2005) S59–S68S64
(PZ0.04, 0.02) and between R294X and R270X (PZ0.04).
For the combined perinatal score, which included data from
three questions, R255X had the highest mean score of 1.27
followed by R306C (1.17) and T158M (1.08) and the lowest
scores were for R294X (0.43) and R133C (0.45). The
differences between R255X and R133C and between
R255X and R294X were significant (PZ0.05, 0.03).
Additionally the mean of the total problem scores for each
mutation are also shown (Fig. 1). R255X, T158M and
R306C had the highest mean scores (3.00, 2.84 and 2.75)
and R294X and R133C had the lowest (1.50, 1.64). The
differences between the lowest scoring mutation R294X and
the highest two R255X and T158M were just significant
(PZ0.05). However when the mobility and problem scores
were combined the most severely affected was R255X
(meanZ10.73) closely followed by R270X (meanZ10.60)
with R294X and R306C the least severe (meanZ7.50,7.91).
The differences between R294X and R255X (PZ0.06) and
R270X (PZ0.07) approached significance.
4. Discussion
This study has found that, in general, cases with R255X
and R306C mutations were slightly more likely to have
problems at birth with appreciable differences between
R255X and both R294X and R133C. For the period after
birth up to 10 months cases with R255X and R270X
mutations were more likely to have problems. Cases with
R270X were likely to be least and cases with R306C most
mobile at 10 months. When all the scores were combined
R255X and R270X were the most severe and R294X the
least severe. R306C had the least consistent pattern overall,
with relatively high combined perinatal problems but
particularly good mobility and thus scoring overall on the
mild side.
This is the largest study to date examining the effects of
individual mutations in numbers of this magnitude. This has
only been possible because we have been able to combine
cases from our Australian population-based registry of
juvenile Rett syndrome ascertained over a 10 year period
[21,22] with cases from the international phenotype
database InterRett ascertained so far over only a 6 month
period [23]. Rett syndrome is fortunately a rare disorder
with an incidence of 1:10,000 females such that in Australia
we only expect 12 cases per birth year [21]. Given that
T158M, the commonest mutation, only represents less than
10% of Australian Rett Syndrome cases we would only
expect to ascertain approximately one case per birth year
with the frequency for the other common mutations being
even less. With the introduction of online-based ques-
tionnaires [23,27] we are able to reach a wider international
audience with minimal costs. Therefore, this form of data
collection utilised by InterRett provides an efficient
mechanism to maximise case numbers for genotype-
phenotype studies in a rare condition, which has multiple
Box 2. Examples of family expression of unusual behaviour from 6 to 10 months of life
Code: Development
‘Her development was slower than my other two children’
‘Did not sit up and very slow to roll over’
‘No crawling or weight bearing/standing’
‘No attempts to crawl despite being able to sit well from 5.5 months’
‘In between those months rolling around on the floor became less frequent, and moving around in a baby walker began to
stop. Also, no crawling or sitting independently eventuated’
H. Leonard et al. / Brain & Development 27 (2005) S59–S68 S65
different mutations. An additional strength of this and
further studies that will follow is that it provides the
opportunity for collaboration with other centres such as has
already occurred with the Chinese group (XW, XB, HP).
The advantage of studies using the ARSD over other
studies has always been the fact that it is population-based
and has shown to be effective in representing juvenile Rett
syndrome in Australia (population approximately
20 million) [21]. InterRett has the capacity to provide
much larger numbers and although we hope that in the
future it will contain representative data for many countries
it will never fully represent its target population—the world.
Similarly, although for our Australian study [3] we were
able to use state and national population norms for
comparing birthweight and head circumference, this
becomes much more difficult in an international multi-
ethnic sample and for this reason we did not include such an
analysis here.
We acknowledge that our research relies on retrospective
parental recall. Nevertheless with a rare condition such as
this a prospective epidemiological study involving the
period prior to regression is not logistically feasible and
hence for information on early development one is
dependent on a study design, which uses retrospective
0 2 4
Problem Perinatal
Problem 0-6mth
Problem >6-10mth
Total Problem
Mobility
Total Composite
Me
Fig. 1. Mean score for problem and mobility and problem/m
report. In studies of this nature there is always concern about
whether the information is recalled accurately but more
importantly about whether differential recall between
groups is biasing the results [28]. Our experience of
collecting data from both clinicians and families is that
the information provided by families is often more detailed
and comprehensive than that provided by clinicians. Also
when information is requested of them we note that
clinicians will often return to the families to supplement
or validate their own data. With respect to differential recall
we do not have any evidence to suggest that this is likely to
be systematically different between families of children
with Rett syndrome whose subsequent outcome is more or
less severe because of their mutation type. Whilst the
primary purpose of the present study was to compare early
development in different categories of children with Rett
syndrome, Majnemer and Rosenblatt [29] found little
evidence of bias in a case control study of parental recall
of developmental milestones in high risk and healthy
newborns. In our study we encouraged parents to use the
child’s baby book to help answer some of the questions. As
this record was made at the time there should be little
chance that later outcome would affect their recording of
this event and thus their reporting.
6 8 10 12an Score
R306CR270XR255XT158MR168XR294XR133C
obility composite scores for seven common mutations.
H. Leonard et al. / Brain & Development 27 (2005) S59–S68S66
The data collection methods have not been entirely
uniform for the whole dataset in that all the Australian data
were provided by paper-based questionnaires while for the
InterRett cohort, the emphasis was on online data collection
using the Internet. In future research we plan to investigate
differences in data produced by the two approaches but this
is beyond the scope of this paper. A proportion (40%) of the
records used were previously presented [3] but the early
analysis did not examine the effect of genotype. In other
reports [22] we have used four scales – Kerr, Percy, Pineda
and WeeFIM to help characterise phenotype. One of the
twenty items contributing to the Kerr scale did relate to
early development, whilst the Percy scale did seek
information about crawling and the Pineda scale included
an item on age at sitting. Age at sitting was not used in this
analysis. The nature of our data makes our information on
crawling incomplete and so in this previous analysis we had
to use surrogate measures [22]. However, in both cases (for
the Kerr and Percy scores) these developmental items only
contributed w5–6% of the total score.
In our previous reports [19,20] we have included X
inactivation data and have found no protective effect on the
phenotype by the presence of skewed X inactivation when
specific mutations have been examined. However more
recently, we demonstrated that skewed X inactivation was
associated with an overall protective effect (including in
cases where no mutation was found) in particular reference
to health service utilisation and hospital admissions [30]. In
this present study X inactivation data were only available
for the Australian cohort and therefore were not included.
Apparently normal pre- and perinatal period and
apparently normal psychomotor development through the
first six months of life are two of the diagnostic criteria for
Rett syndrome set out by Trevathan et al. in 1988 [2].
Despite this the issue of early normality in Rett syndrome
has been questioned for many years in case reports [5,7,8,
11,31]. These clinical observations were corroborated by
our epidemiological studies where we compared birth
weight and head circumference in Rett syndrome with
population norms and reported on parental perceptions of
the perinatal period and early months of life [3]. Much more
recently and independently Huppke et al. [4] also reported
that birth weight, length and head circumference in a group
of 120 mutation positive Rett syndrome cases were
significantly lower than in the general population. In 2002
it was suggested that the clinical criteria be revised such that
early normality was no longer mandatory [32].
The results of the present analysis with respect to
parental recall of the perinatal period and first 6 months of
life confirm our previous findings [3]. The proportion
reporting any kind of perinatal abnormality (46.4%) in this
study was very similar to the 41% reported in the previous
study. However a higher proportion, nearly 60% of
respondents indicated that they had concerns about their
daughter’s behaviour or development in the first 6 months
compared with just under half in the original study.
Considering that the 108 Australian cases ascertained
since 1995 had a younger average age at ascertainment
than the original cohort, this could be due to better recall
when parents did not have access to baby books.. This age
differentiation was a phenomenon noted by Charman et al.
[6] who also found that normal pre-regression development
was the exception and occurred in only 30% cases. Our
findings are also corroborated by the recent work of Burford
et al. [33] and Einspieler et al. [34]. In Burford’s study
health visitors were asked to review home videos taken
during the first year of life in girls who were later diagnosed
with Rett syndrome and in a comparison group without
developmental problems at 5 years. The nurses were
significantly more likely to be alerted to signs of
developmental deviation in the Rett syndrome cases again
suggesting that the neurodevelopmental aberration in Rett
syndrome is present at a much earlier age than has typically
been recognised. Einspeler et al. on the other hand
conducted a detailed video analysis of movements, posture
and behaviour of 22 cases of Rett syndrome during the first
6 months of life. They found that there was an abnormal
quality of general movements in all cases and in over half
the quality of tongue protrusion, postural stiffness, eye and
finger movements were abnormal. Thus our research adds to
the accumulating and complementary body of literature
which is scrutinising early development in Rett syndrome
through a range of methodologies.
One of the four case reports cited by Huppke et al. [4] to
illustrate the phenotypic spectrum in Rett syndrome (a child
with a C terminal deletion who achieved few milestones)
exemplifies well how abnormal early development may be.
However, none of these or any studies, of which we are
aware, has attempted to examine early development in
the context of specific mutations. In fact other than our own
[19,20], there has been little analysis of the effects of
specific mutations, although Huppke et al. also demon-
strated the severity of truncating mutations involving the
TRD-NLS [16]. We acknowledge that even with a sample
size of 320 cases the statistical power to discriminate
between individual mutations, when even the commonest of
these will not be responsible for more than one sixth of
mutation positive cases (and !9% of all cases in this study),
needs to be improved. Yet the present analysis does show
that two of the three mutations with the lowest problem and
mobility scores, namely R133C and R294X, were those
we had previously identified as having a mild phenotype
[19,20]. Conversely the mutations with the highest scores
were the R255X and R270X. Thus the pattern we [20] and
Huppke et al. [16] had seen in regard to general functioning
of individuals with mutations truncating the TRD-NLS was
also seen in terms of early development. Also particularly
striking was the better mobility with the R294X mutation
which was the aspect of the associated phenotype which
most contributed to its low composite score. As mentioned
the pattern with R306C, a missense mutation located in the
TRD after the NLS was more variable with apparently more
H. Leonard et al. / Brain & Development 27 (2005) S59–S68 S67
problems at birth but good mobility at 10 months.
Previously we would have graded R306C as being neither
particularly severe or mild although we did find that the
onset of regression tended to be a little later on average [20].
The results of this study have important implications
from a variety of perspectives. Our findings support the
increasing body of literature showing that in general the
onset of this disorder is much earlier than originally
appreciated. It now becomes evident that those children
previously coined congenital Rett syndrome [35], and those
subsequently described by Huppke et al.[4] and by ourselves
[36] and those diagnosed as newborn encephalopathy
[37–39] are a clearly defined part of the phenotypic
spectrum. We have now been able to show that the
manifestation of this early developmental aberration does
seem to be associated with the underlying genotype. These
findings also have major implications for the diagnosis and
recognition of this disorder. It is clear we have to reverse the
entrenched clinical dogma that a diagnosis of Rett syndrome
should not be considered unless the child’s early develop-
ment is normal. Previously and especially before the
identification of the association with the MECP2 gene,
such children may have remained underdiagnosed. More-
over the information is particularly relevant to under-
standing the mechanism of early development in Rett
syndrome and determining if and at which time(s) early
intervention might be feasible.
This is the first report to have made use of this new
source of international Rett syndrome data. However,
InterRett is still in its infancy with data collection only
commencing in 2003. Despite having comparatively large
numbers of subjects, statistical power will be further
improved as InterRett expands and population data from
countries such as Israel, Spain, Italy, Netherlands, Belgium,
Turkey and France are included. Current negotiations with
these countries for data contributions will yield in excess of
1000 cases.
Acknowledgements
The authors would like to acknowledge the International
Rett Syndrome Association for their funding of InterRett
and also for their ongoing support and encouragement. The
National Institute of Child Health and Human Development
is also acknowledged for its current funding of the
Australian Rett Syndrome Database under NIH grant
number 1 R01 HD43100-01A1 PI and the Financial Markets
Foundation for Children and the Rett Syndrome Australian
Research Fund for previous funding. HL is part funded by
NHMRC program grant 003209. The Sydney research
group was also in part funded by NHMRC project grant
185202. We also acknowledge Neil Leonard and the
information technology team at the Telethon Institute for
Child Health Research for their information technology
expertise and assistance. Finally we would also like to
express our gratitude to all the families who have
participated in the study; the Australian Paediatric
Surveillance Unit and the Rett Syndrome Association of
Australia who facilitated case ascertainment in Australia;
and those members of the InterRett International Reference
panel who helped with the piloting of InterRett.
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