Download Influence of Mutation Type and Location on Phenotype

P. Huppke1
M. Held1
F. Hanefeld1
W. Engel2
F. Laccone2
Influence of Mutation Type and
Location on Phenotype
in 123 Patients with Rett Syndrome
Introduction
Rett syndrome (RTT) is a neurodevelopmental disorder that almost exclusively affects girls. It is caused by mutations in the
MECP2 gene that encodes the methyl-CpG-binding protein 2
(MeCP2). In this study we correlated mutation type and location
with the severity of the phenotype in 123 girls with RTT. The
ability to sit, walk, speak, hand function, head growth, occurrence of epilepsy and a combined severity score were assessed
in all girls at 5 years of age and then statistically correlated with
the results of the molecular genetic tests. We found that patients
who carry either missense mutations or deletions located within
the hotspot for deletions, an area between the base pairs (bp)
1030 and 1207 of the MECP2 gene, present with a milder phenotype than other patients. We correlated the location of the mutations with the phenotype and found that all mutations that lead
to either a complete or partial truncation of the region coding for
the nuclear localisation signal (NLS) are associated with a more
severe phenotype than other truncating mutations (p = 0.001).
We did not find a significant difference between the patients
with mutations in the methyl-CpG-binding domain (MBD) and
those with mutations in the transcriptional repression domain
(TRD). We conclude that mutation type and location correlate
with the phenotype in Rett syndrome. All mutations that impair
the nuclear localisation signal (NLS) are associated with more severe phenotypes.
Rett syndrome (RTT) is, with an incidence of 1/10 000 to 1/
15 000, one of the most common causes of severe mental retardation in females [14]. It is characterized by a period of normal
development that lasts 8 to 18 months followed by neurologic
regression affecting both motor and mental abilities. Coinciding
with the loss of purposeful use of the hands is the appearance of
stereotypic hand movements. In most girls head growth decelerates long before the onset of other symptoms [15].
Key words
Rett Syndrome ± Genotype and Phenotype Correlation ± RTT Severity Score
1
In 1999 Amir et al were the first to describe mutations in the
MECP2 gene as a cause of RTT [2]. The MECP2 gene encodes the
methyl-CpG binding protein 2 (MeCP2), one of four methyl-CpG
binding proteins that are known so far [12, 22, 23]. MeCP2 binds
to methylated CpG dinucleotides and interacts with the Sin3 A/
histone co-repressor complex and histone deacetylases [19, 34].
Deacetylation of core histones converts the chromatin structure
into an inactive state [33]. MeCP2 is involved in the long-term silencing of genes in mammalian cells [28]. Two functional domains have been identified:1) the methyl-CpG-binding domain
(MBD) that binds to the methylated DNA, and 2) the transcriptional repression domain (TRD) that interacts with the corepressor Sin3 A and the histone deacetylases [26, 27]. A nuclear localisation signal (NLS) that mediates the transport of MeCP2 into
the nucleus is located within the TRD (27). It has been found that
mutations in MECP2 account for 70 ± 80 % of RTT cases [1, 3, 5 ±
8,10,17,18, 21, 29, 31, 35, 37].
Two different genetic factors are likely to influence the phenotype in RTT: X inactivation and the type and location of the mutations. In three cases of familial RTT, skewed X inactivation was
Affiliation
Abteilung Kinderheilkunde, Schwerpunkt Neuropädiatrie, Georg-August-Universität Göttingen,
Göttingen, Germany
2
Institut für Humangenetik, Georg-August-Universität Göttingen, Göttingen, Germany
Correspondence
Dr. P. Huppke ´ Department of Neuropaediatrics, Georg-August-University of Göttingen ´
Robert-Koch-Straûe 40 ´ 37075 Göttingen ´ Germany ´ E-mail: [email protected]
Received: November 8, 2001 ´ Accepted after Revision: January 25, 2002
Bibliography
Neuropediatrics 2002; 33: 63 ± 68 Georg Thieme Verlag Stuttgart ´ New York ´ ISSN 0174-304X
Original Article
Abstract
63
found on maternal investigation. The mothers were either clinically normal or exhibited only minor neurological symptoms
[32, 35]. Amir et al described one patient with skewed X inactivation and a milder phenotype, while the skewed X inactivation
found by Auranen et al in two females did not contribute to the
severity of the phenotype [3, 5]. Furthermore, several studies
have demonstrated normal X inactivation patterns in the majority of RTT cases [3 ± 5, 9, 20, 24, 36, 38].
Original Article
64
Five studies have been performed so far addressing the influence
of mutation type and location. Amir et al included 57 patients
and compared 13 clinical characteristics, electrophysiological
studies and cerebrospinal fluid neurochemistry between patients with missense mutations and patients carrying truncating
mutations [3]. They found that awake respiratory dysfunction
was more frequent in patients with truncating mutations and
that missense mutations were associated with the presence of
scoliosis. However, no correlation was seen between a Composite
Clinical Severity Score and the type of mutation. Cheadle et al included 44 patients in their study [10]. They used a severity score
that assessed the abilities of hand use, speech, and walking. They
found that truncating mutations were associated with more severe phenotypes than missense mutations and that late truncating mutations produced milder phenotypes than early truncating mutations. Huppke et al also assessed the ability to sit, walk
and speak at age 5 years in 31 patients with RTT and found no
correlation between severity of the phenotype and mutation
type or location [18]. Auranen et al and Nielsen et al have performed the most recent studies on 39 patients and 26 patients
respectively [5, 29]; both found no significant correlation between the clinical course and the mutation type.
This study was performed to analyse genotype/phenotype correlation with respect to type and location of mutations. We did not
study X inactivation because other studies have shown that it is
only an important factor in a minority of patients.
Methods
Subjects and clinical evaluation
We evaluated the clinical data of 123 females with the diagnosis
of RTT and mutations in MECP2. Half of the patients (n = 67) were
examined by us in the Neuropaediatric Department in Göttingen.
For the remaining 68 females the data were obtained by questionnaire and complemented by telephone interviews.
Because the clinical pattern of regression seen in Rett syndrome
occurs over a variable time period [15], we decided to evaluate
the clinical status at 5 years of age in order to obtain comparable
data. At this age most girls with Rett syndrome are in a relatively
stable phase (pseudostationary stage) [15]. The following clinical
features were included in our study: ability to sit, walk and talk
(more than 10 words), hand function (being able to finger feed),
Sitting
Walking
Speech
Hand function
Head growth
Epilepsy
Sitting unsupported at age 5 years
1
Loss of ability to sit
2
Never learned to sit
3
Walking unsupported at age 5 years
1
Loss of ability to walk
2
Never learned to walk
3
More than ten words at age 5 years
1
Loss of ability to speak
2
Never spoken
3
Good hand function at age 5 years
1
Loss of hand function
2
Never had good hand function
3
Normal head growth at age 5 years
1
Deceleration of head growth
2
Microcephaly
3
Epilepsy at age 5 years
1
No epilepsy at age 5 years
2
head growth and epilepsy at age 5. We tried to keep the severity
grading of the selected features as simple as possible to avoid unreliable results (Table 1). Each clinical feature was graded from 1
to 3 except for epilepsy, which was only graded out of 2 reflecting
the yes/no recording of this finding. A cumulative total of the 6
grades was calculated to create a clinical severity score ranging
from 6, representing the mildest phenotype, to 17 representing
the most severe phenotype.
The data gathered concerning some features of RTT (breathing
abnormalities, stereotypic hand movements, autistic behaviour,
onset of first symptoms, onset of regression) were not reliable
enough to be included in this study. Girls with RTT are difficult
to examine because they often react negatively to an unfamiliar
environment. Furthermore, data cannot be reliably obtained
from non-medical persons.
Genotype-phenotype correlation analysis
The type and location of the mutations were correlated with the
clinical data. The types of mutations were divided into three different groups: 1) Truncating mutations that include nonsense
mutations, deletions (excluding patients with deletions between
bp 1030 and 1207 of the genes), insertions and splice site mutations; 2) Missense mutations; 3) Deletions between bp 1030 and
1207 of the gene. This third group of patients represents deletions within the hotspot for deletions at the 3¢ end of the gene
and was considered separately from other patients with truncating mutations because we observed on neurological examination
that these patients are distinctly more mildly affected than most
other patients.
The locations of the mutations were also divided into three
groups: 1) mutations in the MBD, 2) mutations in the TRD, and
3) mutations in the NLS. The NLS is located within the TRD.
Females with mutations in the NLS were not part of the TRD
group.
Huppke P et al. Influence of Mutation ¼ Neuropediatrics 2002; 33: 63 ± 68
Mutation analysis
The entire coding region and flanking intron sequences were sequenced using the method described earlier [18]. A detailed report on the results of the mutation analysis has been published
elsewhere [18, 21]. The sequence analysis of all patients were
performed in the laboratory of one of us (F.L.).
Table 1 Severity grading of the clinical features
The association between each group and the individual characteristics as well as the severity score was calculated using the
Mann-Whitney U-test. A p value below 0.05 was considered significant.
Phenotype
In 8 % of all patients no deceleration of head growth occurred.
39% showed a deceleration of head growth but no microcephaly
and 53% were microcephalic at age 5. Epilepsy was present in
40 % of all patients at 5 years of age. 85% of the girls were still
able to sit unsupported while 7 % had lost this ability and 8%
had never achieved this milestone. 50 % were walking unsupported at age 5, 3 % had lost the ability and 47 % had never learned
to walk. 15 % showed preserved hand function, 65 % had lost their
hand function and 20 % have never had good hand function. Assessment of the ability to speak showed that 12 % were able to
say more than 10 words, 53% had lost this ability and 35% have
never had it.
Genotype
The detailed results of the mutational analysis have been published earlier by Huppke et al (11 patients) and Laccone et al (94
patients) [18, 21]. 18 new patients were included in this study
(Table 2). Among the 123 girls we found 19 deletions, 2 splice site
mutations, 2 insertion, 50 missense mutations and 50 nonsense
mutations. 40 girls carry mutations in the MBD, 29 in the TRD
and 23 in the NLS (Fig. 1).
Patients
(n)
Domain
Mutation
Nucleotide
change
1
MBD
R106 W
316 C > T
[3]
2
MBD
D156E
468 C > G
[17]
2
MBD
T158 M
473 C > T
[3]
1
Reference
531 delA
1
TRD
3
TRD
1
TRD
1
NLS
1
TRD
635 ± 655 del
P225 R
674 C > G
[17]
Original Article
Results
Table 2 Results of the molecular genetic analysis of 18 patients that
were not presented previously. Nucleotides numbered
from the ATG initiator codon
753 delC
R270 X
808 C > T
[17]
880 ± 884 del
1
1098 ± 1117 del
1
1121 ± 1191 del
1
1157 ± 1188 del
1
1163 ± 1188 del
1
1167 ± 1200 del
16
14
Patients (n)
Because we found that patients who carry mutations in the NLS
are more severely affected than the others, we performed a second analysis to see if other mutations that lead to a truncated
protein of the NLS lead to more severe phenotypes as well. We
therefore divided the patients into two groups: 1) girls with
truncating mutations located either before or within the NLS,
2) all other patients with truncating mutations.
12
10
8
6
4
2
0
0
200
400
600
800
1000
1200
1400 bp
Transcriptional repression domain TRD
Nuclear localisation signal NLS
Methyl CpG binding domain MBD
Huppke P et al. Influence of Mutation ¼ Neuropediatrics 2002; 33: 63 ± 68
Genotype-phenotype correlation
Type of mutation: Patients with truncating mutations are more
likely to exhibit decelerated head growth (p = 0.00 003) and be
unable to walk at age 5 (p = 0.031) than patients who carry missense mutations. No significant difference was found when we
tested for epilepsy (p = 0.3413), hand function (p = 0.928) and
ability to sit (p = 0.624) and speak (p = 0.596) at 5 years of age.
Patients with truncating mutations are more likely to present
with a deceleration of head growth (p = 0.007), walk less frequently (p = 0.006) and are more likely to lose their hand function (p = 0.008) than those who carry deletions between bp
1030 and 1207 (the hotspot for deletions). The ability to sit
(p = 0.134) and speak (p = 0.795) and the occurrence of epilepsy
(p = 0.277) were not significantly different between these
groups.
Comparison of patients with missense mutations with those carrying deletions within the hotspot showed that the patients with
deletions were more likely to have preserved hand function
(p = 0.008). No significant difference was found when we tested
for deceleration of head growth (p = 0.434), epilepsy (p = 0.584)
Fig. 1 Type and location of the mutations that were detected in the
RTT patients. Squares represent missense mutations, triangles deletions in the deletion hot spot and diamonds the other truncating mutations.
and ability to sit (p = 0.192), walk (p = 0.113) and speak
(p = 0.992) at age 5.
Patients with truncating mutations (mean score = 11.8) had higher severity scores than both patients with missense mutations
(mean score = 10.3) (p = 0.002) and patients with deletions
within the hotspot (mean score = 9.7) (p = 0.004). The difference
between patients with missense mutations and deletions within
the hotspot was not significant (p = 0.331).
Location of mutations: Patients who carry mutations in the TRD
do not differ significantly from the group with mutations in the
MBD.
The group with mutations in the NLS were more likely to present
with deceleration of head growth (p = 0.018), an inability to walk
65
Table 3 Results of the statistical analysis. The groups with the higher scores are placed on the left. P values under 0.05 were considered significant
Groups
Sitting
p (n)
Walking
p (n)
Hand function
p (n)
Speech
p (n)
Epilepsy
p (n)
Head growth
p (n)
Severity score
p (n)
Truncating/missense
0.624 (62/50)
0.031 (62/50)
0.928 (62/49)
0.596 (60/51)
0.341 (60/50)
0.00003 (62/50)
0.002 (58/50)
Truncating/deletions
0.133 (62/11)
0.006 (62/11)
0.007 (62/11)
0.794 (60/11)
0.277 (60/11)
0.007 (62/11)
0.004 (58/11)
Missense/deletions
0.192 (50/11)
0.113 (50/11)
0.018 (49/11)
0.992 (51/11)
0.584 (50/11)
0.436 (50/11)
0.330 (50/11)
MBD/TRD
0.067 (40/29)
0.312 (40/29)
0.0004 (40/28)
0.394 (40/29)
0.971 (40/29)
0.363 (40/29)
0.454 (40/28)
NLS/TRD
0.063 (23/29)
0.049 (23/29)
0.005 (23/28)
0.424 (22/29)
0.741 (21/29)
0.018 (23/29)
0.004 (21/28)
Original Article
NLS/MBD
0.974 (23/40)
0.225 (23/40)
0.650 (23/40)
0.881 (22/40)
0.749 (21/40)
0.002 (23/40)
0.034 (21/40)
Truncating NLS/
Truncating after NLS
0.010 (48/25)
0.012 (48/25)
0.0001 (48/23)
0.945 (47/24)
0.872 (46/25)
0.0006 (48/25)
0.001 (45/23)
Truncating NLS/rest TRD
0.012 (48/24)
0.021 (48/24)
0.0001 (48/23)
0.332 (47/24)
0.718 (46/24)
0.0003 (48/24)
0.0006 (46/22)
(p = 0.049) and loss of hand function (p = 0.005) than patients
who carry mutations in the TRD. No significant difference was
found when comparing sitting (p = 0.063), speech (p = 0.424)
and occurrence of epilepsy (p = 0.741). The only significant difference between patients with mutations in the NLS and those with
mutations in the MBD is that they were more likely to present
with a deceleration of head growth (p = 0.002).
Patients with mutations in the NLS (mean score = 12) had higher
severity scores than both those with mutations in the MBD
(mean score = 10.8) (p = 0.034) and those with mutations in the
TRD (mean score = 10.1) (p = 0.004), while the MBD and the TRD
group did not differ significantly.
66
Discussion
Phenotype
We present the largest study published to date correlating the
phenotype of RTT patients with type and location of mutations
in the MECP2 gene. We assessed 6 clinical features of RTT patients at the age of 5 years, during the pseudostationary period,
in order to obtain comparable data [15]. We found that the majority was microcephalic at the age of 5 years with only 8% exhibiting no deceleration of head growth. Epilepsy was present in
40 % of the patients. This figure is less than recently reported,
Genotype-phenotype correlation
In the studies published so far, conflicting results have been
found as to whether the type of mutation influences the phenotype. Amir et al found no correlation between their Composite
Clinical Severity Score and the type of mutation [3]. The same result was found by Huppke et al, Auranen et al and Nielsen et al
[5,18, 29]. However, Cheadle et al in their study of 44 patients
found that girls with truncating mutations present with higher
severity scores [10]. They excluded 2 patients with late truncating mutations from their study because they represented outliers
in terms of their clinical presentation. We also found that the
group of girls with deletions within the deletion hotspot (bp
1030 to 1207) of the MECP2 gene present with a milder phenotype and therefore decided to place them in an individual group.
Our clinical impression was confirmed by statistical analysis. The
group with deletions within the hotspot for deletions had significantly lower severity scores than the group that included all other patients with truncating mutations. We also found that the
group with missense mutations had lower severity scores than
the patients with truncating mutations. Most likely Amir et al,
Huppke et al, Auranen et al and Nielsen et al did not find this correlation because the patients with deletions within the hotspot
region were included in the truncating group [3, 5,18, 29].
Huppke P et al. Influence of Mutation ¼ Neuropediatrics 2002; 33: 63 ± 68
Mutations truncating the NLS: When compared with other truncating mutations, we found that mutations that lead to a truncated NLS were more likely to present clinically with deceleration of head growth (p = 0.0006), inability to walk (p = 0.012)
and loss of hand function (p = 0.0001). The severity scores found
in the group with mutations that lead to a partial or complete
truncation of the NLS were significantly higher than in the group
with truncating mutations localised after the NLS (p = 0.001).
The group with mutations that lead to a partial or complete truncation of the NLS also had higher severity scores than those patients with mutations in the remaining segment of the TRD
downstream of the NLS (p = 0.0006). The frequency of deceleration of head growth (p = 0.0003), inability to sit (p = 0.012) and
walk (p = 0.021) and loss of hand function (p = 0.0001) was higher (Table 3).
most likely because the first presentation of epilepsy can occur
later in life [30]. The majority (85%) of all patients learned to sit
unsupported which is in accordance with the fact that this developmental milestone is often acquired before the appearance of
the rapid phase of regression which normally presents between
12 to 18 months. Walking unsupported was only achieved and
maintained by 50 % of the girls while 47% never learned to walk.
Because only very few (3%) of the children lost the ability to walk
the determining factor might be whether or not they had
achieved this milestone before the onset of regression. Loss of
hand function is one of the diagnostic criteria in RTT [14]. However, we found that 20 % of our patients never had normal hand
function. 65% of the patients learned to speak but only 12 %
maintained this ability to a certain extent. It seems that abilities
that require higher brain functions like language and hand function are more prone to be lost during the phase of regression
than gross motor skills like walking and sitting which once
learned appear to be maintained.
Type and location of mutations do not influence all features of
RTT in the same way. We found no significant correlation between the occurrence of epilepsy and the ability to speak. Hand
function in patients with deletion within the hotspot was significantly more preserved than in other patients. Head growth, on
the other hand, was more likely to be decelerated in patients
with truncating mutations than in those with missense mutations while the latter group did not differ from the patients with
deletions within the hotspot region. Too little is known about the
function of MeCP2 to interpret these findings.
Huppke P et al. Influence of Mutation ¼ Neuropediatrics 2002; 33: 63 ± 68
Genotype/phenotype correlations provide valuable information
necessary for genetic counselling. We think our study contributes useful information towards this purpose. Finally, it is important to mention that although a severity score might reflect the
biological severity of the disease it does not necessarily reflect
the social severity. We found that very mildly affected girls often
create problems within their families, which more severely affected ones do not. They are more mobile, restless and some of
them exhibit aggressive behaviour. This must be kept in mind
during consultations with the parents concerning the future development of their children.
Acknowledgments
The authors would like to thank those families who participated
in our study. We also acknowledge the help of the Elternhilfe für
Kinder mit Rett-Syndrom (Parent Support Group for Children
with RTT) and the physicians who answered the questionnaires.
We thank N. Dankenbrink, S. Herlt, K. Rücker and U. Lenz for their
technical help.
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Original Article
Cheadle et al, Huppke et al and Amano et al studied the influence
of the location of the mutations on the phenotype [1,10,18].
Cheadle et al found that early truncating mutations were associated with a more severe phenotype than late truncating mutations and that there was no difference in severity between missense mutations in the TRD and the MBD [10]. Amano et al found
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