 
|
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Journal of the New Zealand Medical Association,
05-June-2009, Vol 122 No 1296
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
Spectrum of MECP2 mutations in New Zealand Rett
syndrome patients
Anthony M Raizis, Mohammed Saleem, Richard MacKay, Peter M
George
Classical Rett syndrome (OMIM 312750) is a
neurodevelopmental disorder, one of the most common causes of mental retardation
in females and is usually due to mutations in the methyl-CpG binding protein 2
(MECP2) gene.1
MECP2-related disorders also include variant or atypical
Rett syndrome, mild learning disabilities in females, and neonatal
encephalopathy and mental retardation syndromes in males. Thus the variability
of the clinical features is a significant problem in the diagnosis of Rett
syndrome, and many clinicians face a difficulty in deciding when to request
genetic testing.
Atypical Rett syndrome, in which ~50% of patients have MECP2
mutations,2 is identified in patients
previously classified as having autism, mild learning difficulties, or
Angelman's syndrome, adding to the complexity of diagnosis. In addition, many
clinical manifestations of Rett syndrome only occur after the age of 3
years,2 rendering younger infants difficult to
diagnose.
The threshold of necessary clinical criteria to justify
testing is difficult to define due to the variable phenotype of variant Rett
syndrome. In the study described here, we examine a cohort of 75 patients who
were referred for MECP2 gene testing in order to determine the spectrum of
phenotypic features observed by clinicians before referral.
Materials and MethodsThe patients—The group consisted
of 74 patients (71 female and 3 male) aged between 1 and 31 years, who were
referred for testing as part of the investigation of global developmental delay
and mental retardation. Where appropriate fragile X and Angelman’s
syndrome were excluded by specific mutation analyses, particularly when the
clinical features did not strongly support classical Rett syndrome.
Patient clinical information was derived from medical
records after obtaining consent from the legal guardians in accordance with the
conditions set out by the Multi-Regional Ethics Committee. Not all clinical
features were available. Nine “necessary” clinical features were
recorded which are a prerequisite for classical Rett syndrome, while for variant
Rett there are six “main” criteria (Table 1).
Table 1 Diagnostic features of classical and
atypical Rett
(also available at http://www.genetests.org/query?dz=rett)
Isolation of DNA—Genomic DNA was
extracted from 5 ml of EDTA blood3 yielding
approximately 80–100 μg. For PCR, the DNA was diluted to 20
ng/μl and 5 μl was used in a 50 μl PCR reaction.
PCR amplification and DNA
sequencing—The PCR primers for MeCP2 gene amplification and PCR
amplification conditions were done as described
previously.4 Coding regions of the MECP2 were
amplified by PCR and sequenced by automated fluorescent sequencing using the ABI
Big Dye terminator kit version 3.1. Sequencing products were separated by
capillary electrophoresis on an ABI 3130 genetic analyser. DNA sequence data was
compared to the reference GenBank sequence AF030876.
MLPA analysis—MLPA was performed
following the general directions provided by MRC-Holland (www.mlpa.com), using a probe set to cover the
entire MECP2 gene. Amplification products were analysed with an ABI 3100 genetic
analyzer (ABI). Electropherograms were analysed by GeneMapper version 3.5 (ABI),
and peak height data were exported to an Excel spreadsheet
(http://www.ngrl.org.uk/Manchester/Informaticspubs.htm)
and quantified.
ResultsOf 74 patients analysed for MECP2 mutations, 15 were found
to have mutations, as summarised in Table 2. Four novel mutations were
identified including a 44 bp deletion (c.1158_1201del44) and a single base
insertion (c.695dupG ).
A complex insertion/deletion was also identified by DNA
sequencing (AF030876:g.22631_22614conAL078639:g.94544_94611). MLPA analysis
revealed a large deletion, which was subsequently characterised and found to
span exons 3 and 4 of the MECP2 gene.
Table 2. Summary of MECP2 mutations identified
in New Zealand cases of Rett syndrome
Note: Cytosine to thymidine transition
mutations are listed as CpG or CpT; NA=Not applicable.
Eleven patients had known point mutations in the MECP2
coding sequence and of these 8 (89%) were due to cytosine to thymidine
transition mutations within a CpG dinucleotide.
The age distribution of the 74 patients analysed is shown in
Figure 1, with most referrals from paediatric services within New Zealand. Only
8 (11%) of this cohort were under the age of three and one of these had a known
mutation p.R106W. As the majority of patients were over 3 years old, most would
be expected to have sufficient diagnostic manifestations to confirm a clinical
diagnosis of either classical or variant Rett syndrome.
Figure 1. Observed age frequency in a New
Zealand cohort of patients referred for Rett gene testing
 Approximately 24% of the patients referred for gene testing
via paediatric services had MECP2 mutations compared to 14% referred via genetic
services.
Polymorphic sequence variants were also identified. The
coding sequence polymorphisms identified included c.1189G>A and
c.31GGA[6]+GGA[5], while non-coding intronic variants found were
c.377+266C>T, c.378-241C>T, c.377+242C>T, c.378-19delT, c.378-74C>T,
and c.378-109A>G.
Table 3 shows the clinical
characteristics observed in a subset (18/74) of referred patients who were
distributed throughout New Zealand and for whom legal guardian consent was
obtained to examine clinical notes. Of these, 7/18 had identifiable mutations in
the MECP2 gene. Only three of the seven having mutations displayed the full set
of necessary clinical criteria associated with classical Rett syndrome.
Of the 11 patients not having MECP2 mutations, patient A9
was subsequently found to have a balanced translocation of unknown significance
involving chromosomes 6 and 2 (Table 3) and at least 7/9 of the necessary Rett
criteria. Patients A3, A5 and A7 displayed only 1/6 of the main criteria, and no
MECP2 mutations were identified in these patients.
DiscussionWe have analysed the MECP2 gene from the samples of 74
patients with Rett syndrome or Rett-like features. Of these patients, only 20%
had MECP2 mutations. In other series, mutations were detected in 60 to 88% of
those with classical Rett5–7 and ~50% of
those with atypical Rett syndrome.2,8 In
another study, patients were selected with mental retardation as the main
diagnostic feature, and of these only 0.25% had MECP2
mutations.9
In our series, the patients not having MECP2 mutations (80%)
are probably clinically heterogeneous. Some cases may be due to mutations in
either non-coding regions of MECP2 or other gene(s) e.g.
CDKL510 giving rise to Rett-like features, but
others probably have acquired causes.
The mutations identified in this series are all clearly
pathogenic and the high frequency of cytosine to thymidine transitions suggests
that deamination of methylated cytosines is a common cause of Rett syndrome.
Spontaneous deamination is likely to contribute to the high frequency of
methyl-cytosine transitions to thymidine, but a number of factors have been
found to accelerate deamination—e.g. cytosine protonation in response to
aberrant base-pair formation or base
modification.11
Diagnosis under the age of three is difficult for Rett
syndrome since many diagnostic features do not manifest until this age. Only 11%
of our patients analysed fell into this category, so the majority were old
enough to manifest the diagnostic features required to diagnose classical or
atypical Rett syndrome.
Even when strict criteria are used Rett diagnosis can be
difficult. A score of 4-8 out of the 9 necessary criteria (Table 1) has been
previously observed in Japanese patients having MECP2
mutations.2 However, similar scores were
observed in these patients with and without detectable mutations in MECP2. These
observations illustrate the difficulties associated with interpreting diagnostic
criteria.
We found that paediatric services were more likely to
identify a patient having a MECP2 mutation (24%) when compared to genetic
services (14%). This probably reflects the referral patterns, with most patients
with mental disability disorders of this type being more likely to be seen by
paediatric services before genetic services. Furthermore the higher frequency of
<3 year olds seen be genetic services increases the probability of
misdiagnosis.
A high proportion of referrals fell short of classical Rett
syndrome ~83%. Patients A3, A5 and A7 displayed only 1/6 of the main criteria
(Table 3), with no identifiable MECP2 mutations. While the criteria supporting
Rett for these patients were weak and perhaps insufficient to warrant MECP2
testing, collection of DNA samples from such patients should still be considered
of value as they will facilitate the identification of other genes associated
with mental disability disorders.
Only a handful of other genes have been linked to Rett
syndrome—e.g. CDKL5 and NTNG1.12,13 These
genes are not considered a common cause of Rett. Our finding of a balanced
translocation (6p11-12;2p25) in a patient with Rett-like features further
supports the existence of other Rett-like genes. This patient had at least 7/9
necessary criteria with a translocation involving a chromosomal region not
previously associated with Rett syndrome suggesting that a novel gene might be
at this locus.
Very few of the patients in this cohort not having MECP2
mutations had been examined for high resolution karyotypic analysis. Where
possible, cytogenetic analysis should be requested.
In New Zealand there is no formal policy to record and
report all available necessary diagnostic criteria before referral. Currently,
diagnostic criteria are recorded ad hoc and are difficult and time consuming to
obtain from medical records. However, more detailed clinical information
supplied to the testing laboratory may be useful for phenotypic classification
of patients.
With respect to Rett-like mental disability disorders, we
recommend that (where possible) clinicians in New Zealand should adopt a
standardised approach to recording the nine necessary diagnostic criteria and
that this information is provided with diagnostic test requests, in order to
direct appropriate testing.
Competing interests: None known.
Author information: Anthony M Raizis,
Scientific Officer, Department of Molecular Pathology, Canterbury Health
Laboratories, Christchurch; Richard MacKay, Chemical Pathologist , Clinical
Biochemistry Unit, Christchurch Hospital, Christchurch; Mohammed Saleem,
Chemical Pathology Registrar, Clinical Biochemistry Unit, Christchurch Hospital,
Christchurch; Peter M George, Professor and Chemical Pathologist, Department of
Molecular Pathology, Canterbury Health Laboratories, Christchurch
Acknowledgements: We thank all the
clinicians from across New Zealand who referred samples for genetic
testing.
Correspondence: Professor Peter George,
Department of Molecular Pathology, Canterbury Health Laboratories, PO Box 151,
Christchurch, New Zealand. Fax: +64 (0)3 3640545; email: peter.george@cdhb.govt.nz
References:
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| | Current
issue | Search journal |
Archived issues | Classifieds
| Hotline (free ads) Subscribe | Contribute | Advertise | Contact Us | Copyright | Other Journals |
|