del Gaudio, D., Fang, P., Scaglia, F., Ward, P. A., Craigen, W. J., Glaze, D. G. et al. Increased MECP2 gene copy number as the result of genomic duplication in neurodevelopmentally delayed males. Genet. Med. 8, 784-792

Article (PDF Available)inGenetics in Medicine 8(12):784-92 · January 2007with60 Reads
DOI: 10.1097/01.gim.0000250502.28516.3c · Source: PubMed
Abstract
Mutations in the MECP2 gene are associated with Rett syndrome, an X-linked mental retardation disorder in females. Mutations also cause variable neurodevelopmental phenotypes in rare affected males. Recent clinical testing for MECP2 gene rearrangements revealed that entire MECP2 gene duplication occurs in some males manifesting a progressive neurodevelopmental syndrome. Clinical testing through quantitative DNA methods and chromosomal microarray analysis in our laboratories identified seven male patients with increased MECP2 gene copy number. Duplication of the entire MECP2 gene was found in six patients, and MECP2 triplication was found in one patient with the most severe phenotype. The Xq28 duplications observed in these males are unique and vary in size from approximately 200 kb to 2.2 Mb. Three of the mothers who were tested were asymptomatic duplication carriers with skewed X-inactivation. In silico analysis of the Xq28 flanking region showed numerous low-copy repeats with potential roles in recombination. These collective data suggest that increased MECP2 gene copy number is mainly responsible for the neurodevelopmental phenotypes in these males. These findings underscore the allelic and phenotypic heterogeneity associated with the MECP2 gene and highlight the value of molecular analysis for patient diagnosis, family members at risk, and genetic counseling.

Figures

Increased
MECP2
gene copy number as the result
of genomic duplication in neurodevelopmentally
delayed males
Daniela del Gaudio, PhD
1
, Ping Fang, PhD
1
, Fernando Scaglia, MD
1
, Patricia A. Ward, MS
1
, William J. Craigen, MD, PhD
1
,
Daniel G. Glaze, MD
2,3
, Jeffrey L. Neul, MD
2
, Ankita Patel, PhD
1
, Jennifer A. Lee, BS
1
, Mira Irons, MD
4
,
Susan A. Berry, MD
5
, Amber A. Pursley, MS
1
, Theresa A. Grebe, MD
6
, Debra Freedenberg, MD
7
, Rick A. Martin, MD
8
,
Gary E. Hsich, MD
9
, Jena R. Khera, MD
9
, Neil R. Friedman, MD
9
, Huda Y. Zoghbi, MD
1,10
, Christine M. Eng, MD
1
,
James R. Lupski, MD, PhD
1,2,11
, Arthur L. Beaudet, MD
1
, Sau Wai Cheung, PhD
1
, and Benjamin B. Roa, PhD
1
Purpose: Mutations in the MECP2 gene are associated with Rett syndrome, an X-linked mental retardation disorder in
females. Mutations also cause variable neurodevelopmental phenotypes in rare affected males. Recent clinical testing
for MECP2 gene rearrangements revealed that entire MECP2 gene duplication occurs in some males manifesting a
progressive neurodevelopmental syndrome. Methods: Clinical testing through quantitative DNA methods and chromo-
somal microarray analysis in our laboratories identified seven male patients with increased MECP2 gene copy number.
Results: Duplication of the entire MECP2 gene was found in six patients, and MECP2 triplication was found in one
patient with the most severe phenotype. The Xq28 duplications observed in these males are unique and vary in size from
approximately 200 kb to 2.2 Mb. Three of the mothers who were tested were asymptomatic duplication carriers with
skewed X-inactivation. In silico analysis of the Xq28 flanking region showed numerous low-copy repeats with potential
roles in recombination. Conclusions: These collective data suggest that increased MECP2 gene copy number is mainly
responsible for the neurodevelopmental phenotypes in these males. These findings underscore the allelic and pheno-
typic heterogeneity associated with the MECP2 gene and highlight the value of molecular analysis for patient diagnosis,
family members at risk, and genetic counseling. Genet Med 2006:8(12):784–792.
The MECP2 gene located on chromosome Xq28 encodes the
methyl-CpG-binding protein 2 (MeCP2), which normally
functions as a transcriptional repressor.
1
The MeCP2 protein
preferentially binds methylated CpG dinucleotides and func-
tions as a co-repressor of target gene transcription.
2
MECP2
encodes two isoforms: MeCP2_e2 (exons 2–4) and MeCP2_e1
isoform (exons 1, 3, and 4), which is more abundant in brain
tissue.
3–5
Mutations in the MECP2 gene cause Rett syndrome
(Online Mendelian Inheritance in Man [database] 312750), a
progressive neurodevelopmental disorder that affects approx-
imately 1 in 10,000 females.
6
Classic Rett syndrome is charac-
terized by apparently normal development in girls 6 to 18
months, followed by regression. Clinical features include loss
of purposeful hand use replaced by stereotypic hand wringing,
loss of speech, acquired microcephaly, mental retardation, au-
tistic features, seizures, ataxia, and breathing dysrhythmias.
7,8
In addition to classic Rett syndrome, MECP2 mutations have
been identified in patients with a broader range of phenotypes
including severe or mild Rett variants, Angelman syndrome-
like phenotype, mental retardation with seizures, autistic phe-
notypes, or mild learning disabilities.
9,10
The clinical pheno-
type in females can be modulated significantly by nonrandom
X-inactivation patterns.
11
Molecular testing currently involves sequencing of the en-
tire MECP2 coding region, which detects the majority of mu-
tations in Rett syndrome. Additional tests for MECP2 gene
rearrangements, such as deletions and duplications, have been
developed to augment overall test sensitivity. Our previous
analysis on a cohort of 216 females with classic Rett syndrome
identified MECP2 mutations in 96% of cases. Sequencing of
MECP2 exons 1 to 4 detected mutations in 86%, and additional
testing by dosage-sensitive Southern analysis identified MECP2
partial gene deletions in another 10% of females with classic Rett
syndrome.
12
Rett syndrome was initially considered to be an X-linked
dominant lethal condition in males. However, MECP2 gene
From the Departments of
1
Molecular and Human Genetics,
2
Pediatrics, and
3
Neurology,
Baylor College of Medicine, Houston, Texas;
4
Division of Genetics, Children’s Hospital Bos-
ton, Massachusetts;
5
Department of Pediatrics, Division of Genetics Metabolism, University
of Minnesota, Minneapolis, Minnesota;
6
Children’s Health Center Phoenix Genetics Pro-
gram, St. Joseph’s Hospital, Phoenix, Arizona;
7
Department of Pediatrics, Division of Medical
Genetics, Vanderbilt Children’s Hospital, Nashville, Tennessee;
8
Department of Pediatrics,
Division of Medical Genetics, Washington University, St. Louis, Missouri;
9
Section of Pedi-
atric Neurology, The Cleveland Clinic, Cleveland, Ohio;
10
Howard Hughes Medical Insti-
tute; and
11
Texas Children’s Hospital, Houston, Texas.
Sau W. Cheung, PhD, MBA, Director, Kleberg Cytogenetics Laboratory, Baylor College of
Medicine, One Baylor Plaza, NAB2015, Houston, TX 77030.
Submitted for publication June 9, 2006.
Accepted for publication August 29, 2006.
DOI: 10.1097/01.gim.0000250502.28516.3c
brief report December 2006 Vol. 8 No. 12
784 Genetics IN Medicine
mutations were identified in males affected with a broader
range of neurodevelopmental phenotypes.
13,14
Hemizygous
MECP2 point mutations that inactivate the protein are associ-
ated with a severe neonatal encephalopathy phenotype in af-
fected males.
15
When such mutations occur in males with so-
matic mosaicism or a 47, XXY karyotype, the presentation is
more consistent with a classic Rett phenotype because of a
compensating normal X-chromosome.
16–18
Hypomorphic
mutations, which barely cause a phenotype in females, cause
mental retardation, tremors, and a variety of neuropsychiatric
features in affected males.
13,19,20
More recent clinical testing for
MECP2 gene rearrangements revealed that entire MECP2 gene
duplications occur in some males manifesting a progressive
neurodevelopmental syndrome.
21–23
Diagnostic studies at the Baylor Medical Genetics Laborato-
ries have identified seven affected males with increased copy-
number of the MECP2 gene. These include two males who
were referred specifically for mutation analysis of the MECP2
gene and five males who were referred for microarray compar-
ative genomic hybridization analysis for deletions or duplica-
tions of multiple genomic regions involved in clinical genomic
disorders.
24,25
Confirmatory testing using complementary
methods were performed in all seven cases.
MATERIALS AND METHODS
Patient material
Peripheral blood samples from patients were submitted for
clinical testing to the Baylor Medical Genetics Laboratories for
either MECP2 deletion/duplication analysis (patients 1 and 2)
or chromosomal microarray analysis (CMA) (patients 3–7).
Patients’ clinical features are summarized in Table 1. Genomic
DNA was extracted from blood leukocytes using the Puregene
DNA isolation kit (Gentra Systems, Minneapolis, MN), and DNA
concentration was measured using the NanoDrop ND-1000
Spectrophotometer (NanoDrop Technologies, Rockland, DE).
MECP2
Southern analysis
Genomic DNA (2
g) was digested with PstIorBanI restric-
tion enzymes (New England Biolabs, Ipswich, MA), electro-
phoresed on a 0.8% agarose gel, and blotted onto a Hybond XL
nylon membrane (Amersham Pharmacia, Buckinghamshire,
UK). Blots were hybridized to
32
P-labeled probes correspond
-
ing to MECP2 exon 2, 3, and 4 polymerase chain reaction
(PCR) products. Dosage differences indicative of MECP2
whole gene duplication by Southern analysis were detected by
visual inspection of autoradiogram band intensities compared
with ethidium bromide-stained agarose gel images.
Quantitative real-time polymerase chain reaction
Quantitative real-time PCR (qRT-PCR) analysis for the
MECP2 gene was performed using 20 ng of genomic DNA, and
PCR primers and Taqman MGB probes were designed using
Primer Express software (Applied Biosystems, Foster City,
CA). Reactions were done in triplicate, wherein each target
region was coamplified with an internal control (RNaseP) us-
ing the ABI 7500 RT-PCR system (Applied Biosystems). Rela-
tive gene copy number was determined by the comparative
threshold cycle method (ddCt).
26
qRT-PCR analysis of multi-
ple loci flanking MECP2 on Xq28 was performed using the
SYBR-Green chemistry, which measured the fluorescent signal at
the end of the elongation phase. Data were normalized against an
endogenous reference gene (GAPDH), and a melting curve anal-
ysis was performed to verify PCR product specificity.
Multiplex ligation-dependent probe amplification
Dosage analysis of the MECP2 gene and flanking loci was
performed by multiplex ligation-dependent probe amplifica-
tion (MLPA) analysis using commercially available reagents
and instructions from MRC-Holland (Amsterdam, The Nether-
lands). Ligation products were amplified using a Gene Amp PCR
System 9700 (Applied Biosystems). Products were resolved on an
ABI-3100 genetic analyzer, and data were analyzed using Genes-
can software (Applied Biosystems). Patient data were normalized
to controls for copy-number differences using the Gene Marker
Software (Softgenetics, State College, PA).
Chromosomal microarray analysis
Microarray-based comparative genomic hybridization for se-
lected genomic regions involved in clinical genomic disorders was
performed at our institution as previously described.
27
Patients 1
and 2 and their respective mothers were analyzed with our version
4 microarray containing 366 fluorescence in situ hybridization
(FISH)-verified bacterial artificial chromosome (BAC) clones
that cover genomic regions involved in more than 40 deletion/
duplication disorders.
27
Subsequent patients 3 to 7 were analyzed
with an expanded microarray version 5 containing 860 FISH-ver-
ified BAC clones, including four clones for MECP2 and flanking
loci (http://www.bcm.edu/cma/). Reciprocal dye reversal experi-
ments were performed for each patient sample, and hybridized
arrays were scanned into 16-bit tiff image files using an Axon
two-color microarray scanner 4000B and quantified by using
GenePix Pro 6.0 software (GenePix 4000B from Axon Instru-
ments, Union City, CA). Data analysis was performed by a web-
based software platform with the capability of displaying the raw,
normalized, and integrated data of all clones to detect genomic
copy-number changes for each patient relative to a sex-matched
control, as previously described.
27
Fluorescence in situ hybridization
FISH experiments on interphase or metaphase nuclei chromo-
some preparations were performed to confirm increased copy-
number changes in the MECP2 or other loci identified by CMA.
Miniprep BAC DNA (100 ng) was labeled with Spectrum Orange-
dUTP or Spectrum Green-dUTP (Vysis, Downers Grove, IL), ac-
cording to the manufacturer’s instructions, and used as probes for
FISH analysis using standard protocols.
28
X-inactivation studies
Skewing of X-inactivation patterns in duplication carrier
mothers was assessed with the androgen receptor methylation
assay.
29
One microgram of genomic DNA was digested at 37°C
MECP2 gene duplications in male patients
December 2006 Vol. 8 No. 12 785
overnight with 10 units of the methylation-sensitive restriction
enzyme HpaII (New England Biolabs, Ipswich, MA) followed
by heat inactivation at 65°C for 20 minutes. Undigested and
digested DNA were used as templates for
32
P-labeled PCR am
-
plification of the Androgen Receptor CAG
n
repeat.
29
Resulting
PCR products were analyzed on a 6% (39:1 acrylamide/bisac-
rylamide) denaturing gel, which was dried and exposed to x-
ray film overnight at 80°C.
RESULTS
In the course of DNA diagnostic testing for MECP2 muta-
tions, our laboratories received referrals for MECP2 deletion/
duplication testing for neurodevelopmentally delayed males.
Of 122 males referred specifically for MECP2 gene rearrange-
ment testing within a 17-month period, two patients tested
positive for duplication of the entire MECP2 gene. Represen-
Table 1
Comparison of clinical findings observed in male patients with increased MECP2 dosage
Clinical features Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Literature cases (N 53)
a
Age at testing 8.5 y 11.5 y 1.1 y 3 mo 4 y 8.3 y 16 y
Developmental delay ⫹⫹⫹ ⫹⫹⫹ 53/53 (100%)
Infantile hypotonia ⫹⫹⫹ ⫹⫹⫹ 47/47 (100%)
Absent speech ⫹⫹⫹ ⫹⫹⫺ 27/32 (84%)
Lack of ambulation ⫺⫺⫹ ⫺⫺⫺ 14/31 (45%)
History of recurrent infections ⫹⫺⫺ ⫹⫺⫹ 40/48 (83%)
Breathing dysrhythmias ⫺⫺⫺ ⫺⫺⫺ 0/53 (0%)
Stereotypic hand movements ⫺⫺⫺ ⫹⫺⫺ 1/53 (2%)
Autistic-like features ⫺⫹⫺ ⫺⫹⫹ 1/53 (2%)
Seizures ⫺⫺⫺ ⫺⫺⫺ 27/48 (56%)
Microcephaly ⫺⫺⫹(Mac)
b
⫺⫺⫺ 19/48 (40%)
Brachycephaly ⫹⫹⫹ ⫹⫹⫺ 5/28 (18%)
Triangular face ⫺⫺⫺ ⫺⫺⫺ 0/28 (0%)
Narrow forehead ⫺⫺⫺ ⫺⫺⫺ 1/28 (4%)
Flat midface ⫹⫹⫺ ⫺⫹⫹ 6/28 (21%)
Large ears ⫺⫺⫺ ⫹⫺⫺ 8/28 (29%)
Broad nasal root ⫺⫹⫺ ⫺⫺⫺ 5/28 (18%)
Anteverted nares ⫺⫹⫺ ⫺⫺⫺ 1/28 (4%)
Prominent nasal bridge ⫺⫺⫺ ⫺⫺⫹ 4/28 (14%)
Upslanting palpebral fissures ⫺⫹⫺ ⫺⫺⫺ 0/28 (0%)
Epicanthal folds ⫺⫹⫺ ⫺⫺⫺ 2/28 (97%)
Ptosis ⫹⫺⫺ ⫺⫺⫺ 0/28 (0%)
Hypertelorism ⫺⫺⫺ ⫺⫹⫺ 5/28 (18%)
Deep-set eyes ⫺⫺⫺ ⫺⫺⫹ 0/28 (0%)
Proptosis ⫺⫺⫺ ⫺⫺⫺ 4/28 (14%)
Widely spaced teeth ⫺⫺⫺ ⫹⫺⫺ 0/28 (0%)
High arched palate ⫺⫺⫺ ⫺⫺⫹ 1/28 (4%)
Bifid uvula ⫺⫺⫺ ⫺⫺⫹ 0/28 (0%)
Digital abnormalities
c
⫹⫹⫹ ⫺⫺⫹ 10/28 (36%)
Genital abnormalities
d
⫺⫹(Cr) ⫺⫺(Hs) ⫺⫺ 19/28 (68%)
Other organ systems
e
⫺⫺⫺(Tr, Hn) DU ⫺⫹(AD) 4/28
f
(14%)
a
Sanlaville et al.,
23
Van Esch et al.,
22
and Friez et al.
35
b
Mac, macrocephaly.
c
Fifth finger clinodactyly, 2–3 toe syndactyly, broad thumbs/great toes, long/slender fingers.
d
Cr (cryptorchidism), Hs (hypospadias).
e
Tr (tracheomalacia), Hn (hydronephrosis), DU (duplex urethra), AD (aortic root dilatation).
f
Hearing loss, peripheral vasomotor disturbance, coarctation of the aorta, tracheomalacia, and laryngomalacia.
del Gaudio et al.
786 Genetics IN Medicine
tative data are shown for patient 1. Our standard test for dele-
tion/duplication consisted of a dosage-sensitive Southern
analysis for MECP2 exons 1 to 4. Figure 1A shows the results of
MECP2 Southern analysis for patient 1 using a genomic re-
striction digestion with the enzyme PstI. The resulting hybrid-
ization pattern consists of a 7.60-kb PstI fragment containing
MECP2 exons 1 and 2, and a second fragment of 2.5 kb con-
taining MECP2 exons 3 and 4. Relative band intensities are
internally consistent within this patient lane. However, the col-
lective band intensities are disproportionately higher com-
pared with a normal male and resemble a normal female with
two X-chromosomes. Comparison of signal intensities on the
PstI autoradiogram (Fig. 1A) versus the corresponding
ethidium bromide gel (Fig. 1B) is consistent with duplication
of MECP2 exons 1 to 4 in male patient 1. These findings were
corroborated by Southern analysis using a second restriction
enzyme, Ban I. Figure 1C shows the autoradiogram of three
BanI fragments of 2.14 kb, 1.74 kb, and 1.2 kb that correspond
to MECP2 exons 3, 2, and 4, respectively. Thus, Southern anal-
yses using two restriction enzymes indicate duplication of the
entire MECP2 gene. Our laboratories performed confirmatory
testing by independent dosage-sensitive methods. MECP2 du-
plication in these patients was verified by MLPA analysis using
commercially available reagents. Figure 1D shows the raw
MLPA data for patient 1, and Figure 1E shows patient 1 data
normalized to a male control, which indicates a twofold in-
creased copy number of probes corresponding to MECP2 ex-
ons 1 to 4, plus flanking loci on Xq28 (SLC6A8, IDH3G,
L1CAM and IRAK1). In addition to dosage-sensitive Southern
and MLPA analyses, we developed qRT-PCR testing for
MECP2 exons 1 to 4 using a Taqman assay. Representative data
are shown in Figure 1F, wherein MECP2 exon 4 is coamplified
with the autosomal RNaseP gene as an endogenous control.
Patient 1 was determined to have a twofold MECP2 gene copy
number relative to a normal male using the comparative cycle
threshold method on qRT-PCR. Thus, duplication of the en-
tire MECP2 gene was established by several DNA methods in
two male patients referred specifically for MECP2 gene rear-
rangement testing.
The Baylor Medical Genetics Laboratories have also been per-
forming a highly multiplexed CMA for genetic disorders associ-
ated with deletions and duplications of specific chromosomal re-
gions. We developed a microarray containing large-insert
genomic DNA clones of clinically relevant regions as targets for
comparative genomic hybridization (array-CGH).
27
The advan-
tages of CMA include increased sensitivity for subchromosomal
deletions or duplications that are likely to go undetected by con-
ventional karyotyping or metaphase FISH, and simultaneous de-
tection of a wide range of duplication and deletion syndromes in
one highly multiplexed assay.
27,30–32
Our CMA microarray was
Fig. 1. Molecular detection of MECP2 gene duplication. A: Southern analysis with PstI-digested genomic DNA from male patient 1 (lane 3), normal male controls (lanes 1 and 2), and
normal female controls (lanes 4 and 5). Hybridization with MECP2 exon 2 probe generates a 7.6-kb fragment containing MECP2 exons 1 and 2 and a 2.5-kb fragment containing MECP2
exons 3 and 4. B: Corresponding ethidium bromide-stained gel of the PstI-digested DNA. C: Southern analysis with BanI-digested genomic DNA from male patient 1 (lane 3), normal male
controls (lanes 1 and 2), and normal female controls (lanes 4 and 5). Hybridization with probes for MECP2 exons 2 to 4 detects three fragments of 2.14 kb (exon 3), 1.74 kb (exon 2), and
1.2 kb (exon 4). D: MLPA data of male patient 1 with MECP2 duplication. E: The same MLPA data normalized to a male control using Gene Marker Software (Softgenetics, State College,
PA). MECP2 duplicated exons appear as outliers (blue) from the data set, as points outside of the normal copy threshold lines. F: qRT-PCR analysis of MECP2 exon 4 (orange) and RNAaseP
control (blue) in a duplex assay. Results are shown for a male control and patient 1 with MECP2 duplication (ddCt ratio 2.00). Rn is plotted on the y-axis, and the cycle number is on
the x-axis.
MECP2 gene duplications in male patients
December 2006 Vol. 8 No. 12 787
designed to include BAC clones for MECP2, based on the obser-
vation that increased MECP2 levels cause progressive neurologic
features in a mouse model.
33,34
CMA detection of MECP2 dupli-
cations was demonstrated on our initial patients 1 and 2, along
with their asymptomatic mothers, using our version 4 microarray
(366 FISH-verified BAC clones).
27
Figure 2A shows representative
CMA data from patient 1, which was cohybridized with a normal
male genome; the results show a twofold increased copy number
of the two Xq28 genomic clones on the CMA version 4 microarray
(i.e., BAC RP11-119A22 including MECP2, and RP11-24410 in-
cluding the flanking L1CAM gene). Corresponding DNA and
CMA studies on the asymptomatic mother of patient 1 were pos-
itive for heterozygous carrier status (Fig. 2B). The magnitude of
her deviation is relatively smaller, consistent with three copies of
MECP2 in the carrier mother versus two copies in the normal
female. Similar results were obtained on the healthy carrier
mother of male patient 2 with duplication (data not shown).
Clinical CMA testing using an updated version 5 microarray
identified five additional male patients with increased MECP2
gene copy number of 1380 cases tested by CMA within a
6-month period. Collectively, increased copy number for BAC
clones RP11-119A22 (containing MECP2) and RP11-24410
(L1CAM) were seen in all seven patients. A third BAC clone on
CMA version 5 (RP11-54120 centromeric to MECP2) was du-
plicated in patients 1, 3, 4, and 5. A fourth BAC clone RP11-
157E12 (more proximally located) was duplicated in patients 3
and 4 (data not shown). Patient 4 had unusual CMA results
that suggested triplication of the MECP2 clone. This unex-
pected finding was confirmed by additional studies, with the
most graphic evidence provided by FISH analysis.
28
Figure 2C
and D show interphase FISH data for patient 4 using clone
RP11-119A22, which clearly demonstrate three signals for
MECP2 (red) versus one signal for the control probe. In con-
trast, Figure 2E shows duplication of clone RP11-24410 flank-
ing the MECP2 gene in the same patient. Patient 4 has a com-
plex rearrangement that involves triplication of the MECP2
gene amid duplication of a flanking region of approximately 2
Mb on Xq28. The detection of increased MECP2 gene copy
number by CMA analysis in these five additional male patients
was also confirmed by quantitative DNA testing methods in
our laboratories. Figure 3 shows a composite duplication map
based on collective data from Southern, MLPA, CMA, and
qRT-PCR studies. Endpoint mapping was facilitated by qRT-
PCR analysis performed on 25 loci flanking the MECP2 gene.
Individual duplications ranged from approximately 2.2 Mb as
the largest to a minimal region of approximately 200 kb con-
Fig. 2. Molecular cytogenetic detection of increased copy number of the MECP2 gene. A: Chromosomal microarray detection of MECP2 duplication in our male patient 1 using CMA
version 4.0 microarray. B: Corresponding CMA data showing heterozygous MECP2 duplication in the mother of patient 1. C and D: FISH detection of MECP2 triplication in male patient
4 using RP11-119A22 clone containing the MECP2 gene. E: FISH detection of a flanking duplication in male patient 4 using the RP11-24410 clone containing the L1CAM gene.
del Gaudio et al.
788 Genetics IN Medicine
taining the L1CAM and MECP2 genes. The genomic rear-
rangements are unique and vary in size among our seven male
patients.
The major clinical features in these patients are summarized
in Table 1. Predominant features include developmental delay,
hypotonia, mild to moderate dysmorphic features, and limited
to absent speech. Some patients presented with autistic fea-
tures, seizures, and progressive loss of ambulation. Four of
seven patients had a history of respiratory infections. Involve-
ment of other organ systems was seen in two of seven patients,
whereas patient 4 had hydronephrosis and patient 7 had mild
aortic dilatation. The most severe, early-onset presentation
was seen in patient 4. This is a white male who was 3 months
old at the time of testing, with dysmorphic facies, moderate
developmental delay, hydronephrosis, tracheomalacia, and re-
current pneumonia. Notably, recurrent respiratory infections
have been reported in a number of males with MECP2 gene
duplication.
35
Our laboratories received samples from several of the patients’
mothers, who were reported to be asymptomatic and tested pos-
itive as heterozygous duplication carriers. Unexpectedly, the
mother of our complex patient 4 has a heterozygous duplication
of the MECP2 gene and the surrounding region of approximately
2 Mb (data not shown); this is in contrast with her affected son
who has triplication of MECP2 and duplication of the flanking
approximately 2-Mb region. These unusual results underscore
the complexity of the genomic rearrangement in this family,
which has yet to be fully characterized. Given the normal clinical
status of these carrier mothers, we performed X-inactivation stud-
ies at the androgen receptor locus. Genomic DNA samples were
treated with and without predigestion with the methylation-sen-
sitive restriction enzyme HpaII, followed by PCR amplification of
the androgen receptor CAG
n
repeat.
29
The X-inactivation results
for three cases wherein sufficient material was available are shown
in Figure 4. In all three cases, one of two alleles predominates after
HpaII digestion. These data are consistent with skewed X-inacti-
vation that presumably inactivates the mutant allele in these un-
affected carrier mothers.
DISCUSSION
Mutations in the MECP2 gene encompass a wide range of
mutation types that include inactivating and hypomorphic
point mutations, as well as loss-of-function deletions
36
and
gain-of-function duplications.
21,22
MECP2 mutations are asso-
ciated with a broad range of neurodevelopmental phenotypes
that extend beyond classic Rett syndrome in females. We de-
scribe seven male patients with increased copy number of the
MECP2 gene and surrounding loci on Xq28 identified by
quantitative molecular testing and CMA in our diagnostic lab-
oratory. The phenotypic severity in these male patients did not
correlate strictly with their duplication sizes, which ranged
from approximately 200 kb to 2.2 Mb. We identified Xq28
duplications that involve MECP2 and flanking genes such as
L1CAM and SLC6A8, which are known to play a role in neu-
ronal cell migration or nonspecific mental retardation.
37,38
Al-
though flanking gene contribution is a theoretic possibility,
these collective data suggest that increased MECP2 gene dosage
Fig. 3. Nonrecurrent duplications involving the MECP2 gene in the Xq28 region among seven affected males determined by quantitative molecular methods. Array CGH BAC clones
mapping in the Xq28 region (blue bars). Locations of MLPA probes (red arrows); Xq28 genes tested by qRT-PCR (loci in black text). The extent of the duplicated region based on combined
molecular and cytogenetic data for each patient (1–7) (colored horizontal lines). The common duplicated region (vertical dotted lines)(200 kb) including the MECP2 and L1CAM genes.
CMA, chromosomal microarray analysis; MLPA, multiplex ligation-dependent probe amplification.
MECP2 gene duplications in male patients
December 2006 Vol. 8 No. 12 789
is specifically responsible for the phenotypes in male patients.
Submicroscopic Xq28 duplications containing the MECP2
gene with corresponding increase in RNA expression were re-
ported in multiple male patients.
21,22
Those include one male
who was duplicated for the entire MECP2 gene but not the
flanking L1CAM gene.
21
In our study, the most severely af-
fected patient is triplicated for the MECP2 gene. Taken to-
gether, the critical duplication region seems to be limited to the
MECP2 gene. These patient findings are consistent with data
from transgenic mouse models overexpressing the wild-type
human MECP2 protein.
33,34
These mice appear clinically nor-
mal at birth but develop Rett-like progressive neurologic prob-
lems including motor dysfunction, hypoactivity, tremors, and
ataxia, and die prematurely.
33
Higher MECP2 protein levels
were found to correlate with more severe phenotypes in these
mice.
33
Duplications within the human genome are increasingly
recognized as a cause of neurodegenerative phenotypes. Such
rearrangements are responsible not only for Mendelian traits
such as Charcot-Marie-Tooth neuropathy type 1A
39
and Pel-
izaeus-Merzbacher leukodystrophy,
40,41
but also for condi-
tions generally thought to be acquired in nature such as
Parkinson
42
and Alzheimer
43
diseases. Recurrent rearrange-
ments in Charcot-Marie-Tooth neuropathy type 1A are medi-
ated by nonallelic homologous recombination,
44
wherein
genome architectural features seem to lead to genomic
instability.
44,45
In a similar manner, nonrecurrent rearrange-
ments may also have predisposing features in the local
genome.
46,47
Segmental duplications or low-copy repeats
(LCRs) have been implicated in the cause of chromosomal
rearrangements that are associated with several genomic
disorders.
24,48
Some of these repeats have the potential to form
cruciform structures that are highly susceptible to double-
strand breaks, which could initiate recombination events lead-
ing to duplications within Xq28. To investigate this possibility
we performed extensive in silico analysis of an approxi-
mately 4-Mb region surrounding the MECP2 gene on Xq28
(149,400,000 –154,824,264, Build 35). Figure 5 shows the pres-
ence of numerous LCRs in both the direct and inverted orien-
tations, which range from approximately 31 kb to 59 kb in size.
Similar analysis was performed by Lee et al.
49
on the region
surrounding the PLP1 locus, wherein duplications are associ-
ated with the X-linked Pelizaeus-Merzbacher disease. Their
studies found a statistically significant association between this
complex genomic architecture and the various duplication
breakpoints. This suggests that the LCRs may stimulate the
genomic rearrangements responsible for the majority of Pel-
izaeus-Merzbacher disease cases and supports an alternative
role of genomic architecture in nonrecurrent rearrangements.
Along these lines, we hypothesize that LCRs in the Xq28 region
may be involved in nonrecurrent rearrangements observed in
our male patients with MECP2 duplication. Nonallelic homol-
ogous recombination
44,48,50
is the usual mechanism for recur-
rent rearrangements with breakpoints clustering in LCRs.
However, nonrecurrent rearrangement breakpoints are also
Fig. 4. X-inactivation studies by methylation analysis of the androgen receptor locus.
Data for individual maternal DNA samples (lanes 1–3). PCR amplifications performed on
template DNA without prior digestion with the methylation-sensitive HpaII enzyme
(lanes 1a–3a) and corresponding results with prior HpaII digestion of template DNA
(lanes 1b–3b). All samples are informative, and the observed predominance of one allele
after HpaII digestion indicates skewing of X-inactivation in the three tested carrier moth-
ers. Data for female controls DNA with skewed X inactivation and random X inactivation
before HpaII digestion (lanes 4a and 5a) and post-HpaII digestion (lanes 4b and 5b).
Fig. 5. In silico analysis using BLAST 2
55
on repeat-masked sequence reveals LCRs in an 4-Mb region surrounding the MECP2 gene. Novel LCRs are displayed centromeric (cen) to
telomeric (tel) and labeled A to O. Those with apparent shared identity are shown graphically in like colors; the directions of the block arrows indicate relative orientations. The largest LCRs
identified include LCR-MECP2 B1 and B2 (49 and 59 kb, 95.9% shared identity), J1 and J2 (40 and 41 kb, 99.4% shared identity and highly complex), L1 and L2 (31 and 37 kb, 99.6%
shared identity), and M2 and M3 (37 and 38 kb, 98.9% shared identity). MECP2 gene (pink circle). Genomic positions (Build 35) are given relative to the MECP2 gene in Megabases.
del Gaudio et al.
790 Genetics IN Medicine
associated with LCRs.
41,51
Sequencing of the actual MECP2
duplication breakpoint junctions could confirm the mecha-
nism of nonhomologous end joining,
41,51
which has been ob-
served for other nonrecurrent rearrangements. Such experi-
ments are challenged by the highly complex and repetitive
nature of this genomic region, and lie beyond the immediate
scope of our present study.
Males carrying hypomorphic mutations in the MECP2 gene
present with a broad and less predictable phenotypic range of
mental retardation and diverse neurologic signs and symptoms.
52
In addition, MECP2 hypomorphic mutations may be a less com-
mon cause of unexplained mental retardation, developmental de-
lay, and associated neurologic features in males than MECP2
duplications,
52,53
and to add further complexity, some of the re-
ported MECP2 hypomorphic mutations have been found to be
normal genetic variants.
54
Table 1 summarizes the clinical findings in our seven male
patients and compares their features with literature reports of
other males with MECP2 gene duplication. Infantile axial hy-
potonia was a finding observed in all of our patients. This neu-
rologic finding was also observed by Van Esch et al.
22
and Friez
et al.
35
; however, our patients did not exhibit the facial hypo-
tonia described by these two groups. The infantile axial hypo-
tonia in this syndrome leads to progressive spasticity later on in
childhood, and this finding was corroborated in our group. A
majority of the patients with MECP2 duplication in our study
exhibited susceptibility to respiratory infections. Frequent
childhood infections, in particular respiratory infections such
as pneumonias, have been reported among males with MECP2
duplication.
22,23,35
Immune deficiency has been postulated as a
possible cause
35
; however, an extensive immunologic evalua-
tion has not been carried out by other groups or our study to
clearly establish immune deficiency as the underlying factor of
frequent infections. Although only one of our patients (Table
1) had absent swallowing with aspiration pneumonias, it is not
inconceivable to speculate that discoordinated swallowing
leading to microaspiration could be responsible for the ob-
served frequent respiratory infections in our group.
The dysmorphic features observed in our patients were het-
erogeneous. However, brachycephaly, midfacial hypoplasia,
and large ears were among the most frequent dysmorphic fea-
tures in our group. Although the clinical data for some of the
patients were not available, Van Esch et al.
22
also mentioned
prominent ears and flat nasal bridge as some of the most com-
monly observed mild dysmorphic features among the patients
they described. A longitudinal clinical study enrolling a larger
number of patients will be required to carefully establish the
most characteristic dysmorphic features observed in this syn-
drome. Multiple congenital anomalies are not commonly
found in this condition. The patient with the MECP2 triplica-
tion (patient 4, Table 1) exhibited hydronephrosis and tra-
cheomalacia, and another patient with MECP2 duplication
presented with mild aortic dilatation (patient 7, Table 1). Two
of our patients had genital abnormalities (cryptorchidism and
hypospadias). None of the cases described by Van Esch et al.
22
presented with hypoplastic genitalia; however, genital abnor-
malities were commonly reported in the cases described by
Sanlaville et al.
23
and in the case reported by Meins et al.,
21
suggesting that they could be part of the clinical spectrum of
this condition. In addition, digital abnormalities such as long,
slender fingers were observed in some of our patients. Digital
abnormalities have also been described in the published liter-
ature of Xq28 disomy.
22,23
Three of our patients (Table 1) exhibited autistic-like fea-
tures, including a limited range of facial expression, gaze
avoidance, and repetitive behaviors. Autism or autistic-like
features have been associated with Rett syndrome
10
. The male
patient with MECP2 duplication described by Meins et al.
21
exhibited autistic features. Autism or autistic-like features were
not described in previous studies; however, it is not known
whether the patients previously described underwent a neuro-
psychologic evaluation.
22,23
The current findings suggest that
autistic-like features could also be found in patients with
MECP2 duplication expanding the neuropsychologic pheno-
type of MECP2-related disorders. Our patients did not exhibit
the breathing dysrhythmias commonly observed in females
with classic Rett syndrome. Only one patient in our study (pa-
tient 5, Table 1) developed stereotypic hand movements; hand
stereotypies were only observed in the study described by
Meins et al.,
21
but not in the studies carried out by other
groups.
22,23
The carrier females with MECP2 duplication did
not manifest any evidence of cognitive dysfunction or abnor-
mal neurologic findings, which could be a reflection of the
skewed X-inactivation pattern.
These data collectively highlight the value of comprehensive
MECP2 clinical testing in both female and male patients with a
diverse range of neurodevelopmental phenotypes, in addition
to classic Rett syndrome. This includes sequence analysis for
the majority of Rett syndrome mutations, plus quantitative
analysis to assess MECP2 deletions and duplications. Although
we have a limited dataset to assess recurrence risks associated
with MECP2 duplications, our findings of three of three
asymptomatic mothers tested to be duplication carriers with
skewed X-inactivation raises important issues for carrier test-
ing and genetic counseling in families with identified abnor-
malities in the MECP2 gene. In conclusion, a large cohort study
of patients with MECP2 duplication will be required to estab-
lish the frequency of this microduplication in males with men-
tal retardation and associated neurologic features, and to de-
termine the variability in the clinical phenotype observed for
this syndrome.
ACKNOWLEDGMENTS
This work was supported in part by National Institutes of
Health Grants P01 HD40301 (to Huda Y. Zogbhi) and HD24064
to the Baylor College of Medicine Mental Retardation and Devel-
opmental Disabilities Research Center.
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del Gaudio et al.
792 Genetics IN Medicine
    • "The striking finding that MECP2 nucleotide mutations or duplica- 71 tions cause Rett syndrome or MECP2 duplication syndrome, respective- 72 ly, suggests that careful regulation of this gene is necessary for correct 73 brain development and function, as both overexpression and reduced 74 expression are associated with neurodevelopmental disorders (Collins 75 et al., 2004; del Gaudio et al., 2006). Intriguingly, a loss of MECP2 "
    [Show abstract] [Hide abstract] ABSTRACT: Epigenetic mechanisms are fundamental for shaping the activity of the central nervous system (CNS). Methyl-CpG binding protein 2 (MECP2) acts as a bridge between methylated DNA and transcriptional effectors responsible for differentiation programs in neurons. The importance of MECP2 dosage in CNS is evident in Rett Syndrome and MECP2 duplication syndrome, which are neurodevelopmental diseases caused by loss-of-function mutations or duplication of the MECP2 gene, respectively. Although many studies have been performed on Rett syndrome models, little is known about the effects of an increase in MECP2 dosage. Herein, we demonstrate that MECP2 overexpression affects neural tube formation, leading to a decrease in neuroblast proliferation in the neural tube ventricular zone. Furthermore, an increase in MECP2 dose provokes premature differentiation of neural precursors accompanied by greater cell death, resulting in loss of neuronal populations. Overall, our data indicate that correct MECP2 expression levels are required for proper nervous system development.
    Full-text · Article · Jul 2014
    • "Although the classical form of RTT is associated with loss-of-function MECP2 mutations, duplication of this gene is responsible for RTT-like neurological phenotypes (Del Gaudio et al., 2006); therefore, a fine-tuning of MeCP2 level might be important for normal development. "
    [Show abstract] [Hide abstract] ABSTRACT: Novel classes of small and long non-coding RNAs (ncRNAs) are increasingly becoming apparent, being engaged in diverse structural, functional and regulatory activities. They take part in target gene silencing, play roles in transcriptional, post-transcriptional and epigenetic processes, such as chromatin remodeling, nuclear reorganization with the formation of silent compartments and fine-tuning of gene recruitment into them. Among their functions, non-coding RNAs are thought to act either as guide or scaffold for epigenetic modifiers that write, erase, and read the epigenetic signature over the genome. Studies on human disorders caused by defects in epigenetic modifiers and involving neurological phenotypes highlight the disruption of diverse classes of non-coding RNAs. Noteworthy, these molecules mediate a wide spectrum of neuronal functions, including brain development, and synaptic plasticity. These findings imply a significant contribution of ncRNAs in pathophysiology of the aforesaid diseases and provide new concepts for potential therapeutic applications.
    Full-text · Article · Feb 2014
    • "Loss-of-function MECP2 mutations had been identified as the cause of Rett syndrome, mainly affecting females [34] and initially thought to be lethal in males [22]. Duplications involving MECP2 have been frequently identified in male patients presenting with a different phenotype from that of Rett syndrome and mainly characterized by severe mental retardation, recurrent respiratory infections, epilepsy, cerebellar degenerative change and progressive encephalopathy [4-6,8,12,14,24]. Whereas the phenotypic effect of MECP2 duplications has already been well documented in males, the clinical implications determined by these rearrangements is still poorly known in females. "
    [Show abstract] [Hide abstract] ABSTRACT: Xq28 duplications, including MECP2 (methyl CpG-binding protein 2; OMIM 300005), have been identified in approximately 140 male patients presenting with hypotonia, severe developmental delay/intellectual disability, limited or absent speech and ambulation, and recurrent respiratory infections. Female patients with Xq28 duplication have been rarely reported and are usually asymptomatic. Altogether, only fifteen symptomatic females with Xq28 duplications including MECP2 have been reported so far: six of them had interstitial duplications while the remaining had a duplication due to an unbalanced X;autosome translocation. Some of these females present with unspecific mild to moderate intellectual disability whereas a more complex phenotype is reported for females with unbalanced X;autosome translocations. Here we report on the clinical features of three other adolescent to adult female patients with Xq28 interstitial duplications of variable size, all including MECP2 gene. Mild to moderate cognitive impairment together with learning difficulties and speech delay were evident in each of our patients. Moreover, early inadequate behavioral patterns followed by persistent difficulties in the social and communication domains, as well as the occurrence of mild psychiatric disturbances, are common features of these three patients.
    Full-text · Article · Jan 2014
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