Download X chromosome inactivation failed to explain normal phenotype Clin

Clin Genet 2008: 73: 257–261
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# 2008 The Authors
Journal compilation # 2008 Blackwell Munksgaard
CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2007.00944.x
Short Report
Skewed X chromosome inactivation failed to
explain the normal phenotype of a carrier
female with MECP2 mutation resulting in
Rett syndrome
Takahashi S, Ohinata J, Makita Y, Suzuki N, Araki A, Sasaki A,
Murono K, Tanaka H, Fujieda K. Skewed X chromosome inactivation
failed to explain the normal phenotype of a carrier female with MECP2
mutation resulting in Rett syndrome.
Clin Genet 2008: 73: 257–261. # Blackwell Munksgaard, 2008
Mutations in the X-linked MECP2 gene cause Rett syndrome,
a neurodevelopmental disorder that exclusively affects girls. Females
with the MECP2 mutations exhibit a broad spectrum of clinical
presentations ranging from classical Rett syndrome to asymptomatic
carriers, which can be explained by differences in X chromosome
inactivation (XCI). Here, we report a family with a girl with Rett
syndrome in whom a novel missense mutation in the MECP2 gene was
transmitted through the maternal germ line. The carrier mother was
asymptomatic and presented non-random XCI in the peripheral blood
cells, which resulted in the X chromosome harboring the mutant allele
that was predominantly active. Thus, the presence of non-random XCI in
the peripheral blood cells did not provide an explanation for the normal
phenotype of the carrier mother. This result suggests that mechanisms
other than XCI may contribute to the phenotypic heterogeneity
associated with MECP2 mutations.
Rett syndrome is a neurodevelopmental disorder
that exclusively affects girls. Currently, it is considered an X-linked dominant disorder because
of mutations in the MECP2 gene encoding
methyl-CpG binding protein 2 (MECP2) (1).
Most cases of Rett syndrome are sporadic
(approximately 99.5% of all cases), with the
majority being caused by a de novo mutation in
the paternal copy of the MECP2 gene (2). Simultaneous incidence of Rett syndrome in sisters
has been reported in families with mothers who
are either germ-line mosaics for the MECP2
mutation or heterozygotes with favorably
skewed X chromosome inactivation (XCI) patterns (1, 3, 4). Thus, with regard to females with
MECP2 mutations, a broad spectrum of clinical
S Takahashia, J Ohinataa,
Y Makitaa, N Suzukia, A Arakia,
A Sasakib, K Muronob, H Tanakaa
and K Fujiedaa
a
Department of Pediatrics, Asahikawa
Medical College, Asahikawa, Japan,
and bDepartment of Pediatrics,
Nayoro City Hospital, Nayoro, Japan
Key words: MECP2 – mutation – Rett
syndrome – X chromosome inactivation
Corresponding author: Satoru Takahashi,
MD, PhD, Department of Pediatrics,
Asahikawa Medical College, 2-1-1-1
Midorigaoka-Higashi, Asahikawa,
Hokkaido 078-8510, Japan.
Tel.: 181 166 68 2483;
fax: 181 166 68 2489;
e-mail: [email protected]
Received 26 September 2007,
revised and accepted for publication
5 November 2007
presentations ranging from classical Rett syndrome to asymptomatic carriers has been explained by differences in the XCI pattern.
However, such an idea was not supported by the
presence of asymptomatic carrier females exhibiting a random XCI pattern (5). Here, we report
a family with a girl with Rett syndrome; the
MECP2 mutation was inherited through the
maternal germ line. The obligate carrier mother
was asymptomatic and possessed a non-random
XCI pattern, which resulted in the X chromosome harboring the mutant allele that was
predominantly active. This suggests that mechanisms other than XCI may play a role in modifying the clinical phenotype associated with the
MECP2 mutation.
257
Takahashi et al.
Materials and methods
Case report
The patient, a girl aged 1 year and 6 months,
was the first child of healthy unrelated parents.
The patient was born at full term after an
uneventful pregnancy. Her weight was 3146 g
with a head circumference (34.0 cm) at the 75th
percentile. Her parents were of normal intelligence. The mother did not present any of the
clinical symptoms observed in Rett syndrome.
Early motor development of the patient was normal; she was able to hold her head at 4 months
and could sit unaided at 7 months. Developmental abnormalities were first noted at the age of
10 months when she showed inconsolable crying and stopped smiling. The parents also noticed
that her face lacked expression. At 11 months of
age, she lost her ability to sit unaided and became
less interested in her toys. Physical examination
showed hypotonia, strabismus, and intention
tremor of her upper limbs. Her head circumference at the age of 18 months was 43.7 cm (third
percentile), indicating that microcephaly appeared
postnatally. At 13 months of age, she developed
her first seizures characterized by tonic movement of the upper limbs and loss of consciousness. At that time, electroencephalography and
brain magnetic resonance imaging did not reveal
any abnormalities. Thus, she was diagnosed with
Rett syndrome, which was further confirmed by
a mutation in the MECP2 gene (Fig. 1). At this
point, the parents requested genetic testing to
assess the risk of having more affected children.
The father and mother provided their informed
consent for this study; all the studies were performed in compliance with the guidelines of Asahikawa Medical College.
Mutation analysis of the MECP2 gene
Genomic DNA was extracted from the peripheral blood leukocytes of the patient and her
parents and used as the template for polymerase
chain reaction (PCR). Appropriate primers were
used to yield DNA fragments spanning the
entire MECP2 coding region and intron–exon
boundaries (1). The PCR fragments were analyzed using automated sequencing. To further
confirm the mutation identified by direct DNA
sequencing, the DNA fragment encompassing
the mutation site was amplified by PCR using
the following primers: forward, 5#-GGAAAAGCAAGGAGAGCAGC-3#, and reverse, 5#-TCGGGAAGCTTTGTCAGAGC–3#; subsequently, it
was digested with restriction endonuclease Eag I.
The reaction products were then visualized by
258
ethidium bromide staining following electrophoresis on a 2% agarose gel.
X chromosome inactivation
XCI patterns were determined using the method
of Allen (6), as previously described. Briefly,
aliquots of DNA obtained from the peripheral
blood leukocytes were digested overnight using
the methylation-sensitive restriction endonuclease Hpa II. PCR was used to amplify 100 ng of
either digested or undigested DNA using fluorescent PCR primers (7). The amplified PCR fragment contains an Hpa II site, as well as the
highly polymorphic trinucleotide repeat of the
androgen receptor gene. As the Hpa II sites on
the inactive X chromosome are methylated, only
this allele is amplified when the Hpa II-digested
genomic DNA is used as the template. Further,
because the methylation site is adjacent to the
polymorphic trinucleotide repeat, the alleles
amplified by PCR can be distinguished by their
length, which allows determination of the relative ratio of XCI. The allele peak areas were
analyzed using an ABI 310 automated sequencer
and GENESCAN software (Applied Biosystems,
Foster City, CA).
RNA isolation and RT-PCR
Total RNA was extracted from the peripheral
blood cells using the PAXgene Blood RNA Kit
(QIAGEN GmbH, Hilden, Germany). Reverse
transcription (RT) was performed using the
SuperScript First-Strand Synthesis System (Invitrogen Corporation, Carlsbad, CA) for generation of cDNA using 1 mg of total RNA in a
20 ml reaction. To examine the expression level
of the mutant MECP2, RT-PCR was first performed with the same primers used in the PCRrestriction digestion analysis of DNA; subsequently,
the RT-PCR products were digested with restriction endonuclease Eag I. The reaction products
were then separated on 2% agarose gels containing ethidium bromide. As an internal control,
glyceraldehyde-3-phosphate dehydrogenase was
used as described (8).
Results
Identification of a mutation in the MECP2 gene
A missense mutation, i.e. a G-to-A nucleotide
change resulting in p.A447T, was detected in
the girl with Rett syndrome and her obligate
A carrier female with MECP2 mutation
Fig. 1. Maternal origin of a MECP2 mutation associated with Rett syndrome. Automated DNA sequencing using the
polymerase chain reaction (PCR) product from the girl with Rett syndrome and her asymptomatic mother exhibits a G-to-A
transition at nucleotide 1506 (c.1506G.A, numbering according to Genbank accession no. NM_004992) in exon 3 that results
in an alanine-to-threonine substitution at amino acid position 447 (p.A447T), as indicated by the arrows (a). Eag I-digestion
of the PCR product encompassing the mutation site shows an additional fragment (525 bp) in the patient and her mother,
which resulted from the G-to-A transition eliminating an Eag I restriction site (b). This additional fragment is observed
together with the wild-type fragments (304 bp and 221 bp), confirming the heterozygous mutation. Amino acid residue at
position 447 in MECP2 is conserved among different mammalian MECP2 (c).
mother (Fig. 1a). This missense mutation eliminated an Eag I restriction site. Consequently, PCRrestriction digestion analysis of DNA obtained
from the patient and her mother revealed a novel
fragment in addition to the estimated wild-type
fragments, further confirming the presence of
this heterozygous mutation (Fig. 1b). This
mutation has never been reported in Rett syndrome patients (IRSA MECP2 variation database, http://mecp2.chw.edu.au/). We also found
that this mutation was absent from 200 unrelated X chromosomes, thus ruling out the possibility that it might represent a common
polymorphism. Amino acid sequence alignments
showed the changed amino acid (p.A447) is con-
served among four different mammalian
MECP2 including human, cat, rabbit and mouse
(Fig. 1c).
Analysis of XCI
The mother was a carrier with the p.A447T
mutation that affected her daughter. Non-random
XCI has been implicated in reducing the severity
of Rett syndrome phenotype because of preferential inactivation of the X chromosome that
harbors the mutant allele (3, 9, 10). Therefore,
we examined if a favorably skewed XCI pattern
could explain the normal phenotype of the
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Takahashi et al.
carrier mother. In order to assess the XCI pattern of the mother and her affected daughter, we
assayed the methylation status of the androgen
receptor alleles (Fig. 2a). In the carrier mother,
a non-random XCI pattern was discovered; the
ratio of the two alleles was 75:25. However, the
more active X chromosome was transmitted to
her daughter with Rett syndrome. Thus, in the
carrier mother, the X chromosome harboring
the mutant allele was predominantly active,
which was expected to lead to a severe disease
phenotype. Therefore, the difference in the XCI
patterns did not provide an explanation for the
normal phenotype of this carrier mother. We
could not determine the XCI pattern in the girl
with Rett syndrome because she was homozygous at the androgen receptor locus.
Expression of mutant MECP2 in the carrier mother
A meiotic crossover may have taken place
between two X chromosomes of the mother,
which results in separation of the androgen
receptor alleles from the MECP2 alleles.
To exclude this possibility, we examined the
expression of mutant MECP2 in the peripheral
blood cells of the carrier mother. Eag I-digestion
of the RT-PCR product revealed the predominant expression of mutant MECP2 in the carrier
mother (Fig. 2b). This result was compatible
with that of the XCI assay at the androgen
receptor locus.
Discussion
We described a family with a girl with Rett syndrome in whom a novel missense mutation in the
MECP2 gene was transmitted through the
maternal germ line. The carrier mother was clinically asymptomatic and presented non-random
XCI, resulting in the X chromosome harboring
the mutant allele that was predominantly active.
The p.A447T mutation may be a rare variant
that does not cause the disease. Although the
changed amino acid is conserved among different mammalian MECP2, further functional studies may be required to make the association clear
between this mutation and Rett syndrome. Our
data revealed that the carrier mother expresses
the mutated MECP2 in the peripheral blood
cells. If this variant is a disease-causing mutation, the non-random XCI pattern in the peripheral blood cells does not provide an explanation
for the normal phenotype of this carrier mother.
Notably, the XCI patterns in the peripheral tissues do not necessarily reflect the status of the
brain that would be most likely the major determinant of the phenotypic severity (11–13). Considering the possibility of tissue-specific XCI
variation, this mother may have an inverted XCI
Fig. 2. Non-random X chromosome inactivation (XCI) resulting in predominant expression of mutant MECP2 in the carrier
mother. Patterns of XCI were determined in the peripheral blood cells (a). The polymorphic repeated sequence at the
androgen receptor locus was amplified by polymerase chain reaction (PCR) using the Hpa II undigested or digested genomic
DNA as the template. After digestion with the methylation-sensitive restriction endonuclease Hpa II, a non-random pattern
of XCI is observed in the asymptomatic carrier mother in whom the X chromosome harboring the mutant allele is
predominantly active. The Rett syndrome patient is homozygous at the androgen receptor locus. The expression of mutant
MECP2 was examined by RT-PCR on RNA extracted from the peripheral blood cells of each individual in this family (b).
Eag I-digestion of the RT-PCR products reveals the predominant expression of mutant MECP2 (525 bp) in the peripheral
blood cells of the carrier mother. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. Negative
controls without reverse transcription for each PCR reaction were performed and showed no expression.
260
A carrier female with MECP2 mutation
pattern in the brain that favors the expression of
the normal allele. There is another possible explanation for the normal phenotype of this carrier
mother. Other genes may influence the biological
consequences of a mutation in the MECP2 gene.
This idea is supported by the identification of
identical MECP2 mutations that have been
found in males with X-linked recessive mental
retardation and in females with Rett syndrome
and their asymptomatic mothers with a random
XCI pattern (14). Thus, a different genetic background possibly modifies the clinical phenotype
associated with the MECP2 mutation, although
such a second gene remains to be identified. In
conclusion, it is important for genetic counseling
to note that non-random XCI in the peripheral
blood cells does not necessarily provide an explanation for the normal phenotype of the carrier
mothers of patients with MECP2 mutations.
4.
5.
6.
7.
8.
9.
Acknowledgements
We sincerely thank the members of the family described here,
whose help and participation made this work possible.
10.
11.
References
1. Amir RE, Van den Veyver IB, Wan M, Tran CQ, Francke
U, Zoghbi HY. Rett syndrome is caused by mutations in
X-linked MECP2, encoding methyl-CpG-binding protein 2.
Nat Genet 1999: 23: 185–188.
2. Trappe R, Laccone F, Cobilanschi J et al. MECP2
mutations in sporadic cases of Rett syndrome are almost
exclusively of paternal origin. Am J Hum Genet 2001: 68:
1093–1101.
3. Villard L, Kpebe A, Cardoso C, Chelly PJ, Tardieu PM,
Fontes M. Two affected boys in a Rett syndrome family:
12.
13.
14.
clinical and molecular findings. Neurology 2000: 55: 1188–
1193.
Villard L, Levy N, Xiang F et al. Segregation of a totally
skewed pattern of X chromosome inactivation in four
familial cases of Rett syndrome without MECP2 mutation:
implications for the disease. J Med Genet 2001: 38: 435–442.
Meloni I, Bruttini M, Longo I et al. A mutation in the rett
syndrome gene, MECP2, causes X-linked mental retardation and progressive spasticity in males. Am J Hum Genet
2000: 67: 982–985.
Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM,
Belmont JW. Methylation of HpaII and HhaI sites near the
polymorphic CAG repeat in the human androgen-receptor
gene correlates with X chromosome inactivation. Am J
Hum Genet 1992: 51: 1229–1239.
Hickey T, Chandy A, Norman RJ. The androgen receptor
CAG repeat polymorphism and X-chromosome inactivation in Australian Caucasian women with infertility related
to polycystic ovary syndrome. J Clin Endocrinol Metab
2002: 87: 161–165.
Lehmann J, Retz M, Harder J et al. Expression of human
beta-defensins 1 and 2 in kidneys with chronic bacterial
infection. BMC Infect Dis 2002: 2: 20–29.
Ishii T, Makita Y, Ogawa A et al. The role of different Xinactivation pattern on the variable clinical phenotype with
Rett syndrome. Brain Dev 2001: 23 (Suppl. 1): S161–S164.
Huppke P, Maier EM, Warnke A, Brendel C, Laccone F,
Gartner J. Very mild cases of Rett syndrome with skewed X
inactivation. J Med Genet 2006: 43: 814–816.
Sharp A, Robinson D, Jacobs P. Age- and tissue-specific
variation of X chromosome inactivation ratios in normal
women. Hum Genet 2000: 107: 343–349.
Zoghbi HY, Percy AK, Schultz RJ, Fill C. Patterns of X
chromosome inactivation in the Rett syndrome. Brain Dev
1990: 12: 131–135.
Gibson JH, Williamson SL, Arbuckle S, Christodoulou J.
X chromosome inactivation patterns in brain in Rett
syndrome: implications for the disease phenotype. Brain
Dev 2005: 27: 266–270.
Renieri A, Meloni I, Longo I et al. Rett syndrome: the
complex nature of a monogenic disease. J Mol Med 2003:
81: 346–354.
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