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RESEARCH ARTICLE
Four Unrelated Patients With Lubs X-Linked MentalRetardation Syndrome and Different Xq28 DuplicationsOliver Bartsch,1* Konstanze Gebauer,1 Stanislav Lechno,1 Hilde van Esch,2 Guy Froyen,2
Michael Bonin,3 J€org Seidel,4 Barbara Thamm-M€ucke,5 Denise Horn,6 Eva Klopocki,6
Christoph Hertzberg,7 Ulrich Zechner,1 and Thomas Haaf1
1Institut f€ur Humangenetik, Universit€atsmedizin der Johannes Gutenberg-Universit€at Mainz, Mainz, Germany2Center for Human Genetics, University Hospital Gasthuisberg, Leuven, Belgium3Institut f€ur Humangenetik, Universit€atsklinikum T€ubingen, Germany4Childrens Hospital, SRH Wald-Klinikum Gera, Germany5Private Practice Ackermann & Reisig, Leipzig, Germany6Institut f€ur Medizinische Genetik, Charit�e Universit€atsmedizin Berlin, Berlin, Germany7Diagnose- und Behandlungszentrum, Vivantes Klinikum Neuk€olln, Berlin, Germany
Received 8 September 2009; Accepted 18 October 2009
The Lubs X-linked mental retardation syndrome (MRXSL) is
caused by small interstitial duplications at distal Xq28 including
the MECP2 gene. Here we report on four novel male patients
with MRXSL and different Xq28 duplications delineated by
microarray-based chromosome analysis. All mothers were
healthy carriers of the duplications. Consistent with an earlier
report [Bauters et al. (2008); Genome Res 18: 847–858], the distal
breakpoints of all four Xq28 duplications were located in regions
containing low-copy repeats (LCRs; J, K, and L groups), which
may facilitate chromosome breakage and reunion events. The
proximal breakpoint regions did not contain known LCRs.
Interestingly, we identified apparent recurrent breakage sites in
the proximal and distal breakpoint regions. Two of the four
patients displayed more complex rearrangements. Patient 2 was
endowed with a quadruplicated segment and a small triplication
within the duplication, whereas patient 3 displayed two tripli-
cated segments within the duplication, supporting that the Fork
Stalling and Template Switching (FoSTeS) model may explain a
subset of the structural rearrangements in Xq28. Clinically,
muscular hypertonia and contractures of large joints may pres-
ent a major problem in children with MRXSL. Because injection
of botulinum toxin (BT-A; Botox) proved to be extremely helpful
for patient 1, we recommend consideration of Botox treatment
in other patients with MRXSL and severe joint contractures.
� 2010 Wiley-Liss, Inc.
Key words: Lubs X-linked mental retardation syndrome; chro-
mosome Xq28; MECP2; microarray-based chromosome analysis;
recurrent breakpoints; Botox; botulinum toxin
INTRODUCTION
Lubs et al. [1999] reported the first family with the Lubs X-linked
mental retardation syndrome (MRXSL; OMIM 300260) and
linkage to the distal 5 cM of Xq28. The underlying duplications at
chromosome Xq28 vary in location and size (�0.1–2.6 Mb), and
the smallest region of overlap contains the methyl CpG binding
protein 2 (MECP2) and interleukin-1 receptor-associated kinase 1
(IRAK1) genes [van Esch et al., 2005; del Gaudio et al., 2006;
Carvalho et al., 2009; Lugtenberg et al., 2009]. An observation of
a patient with a more severe phenotype and a MECP2 triplication
[del Gaudio et al., 2006] supported the view that the mental
retardation (MR) in the patients with MRXSL is caused by
the imbalance of the MECP2 gene [van Esch et al., 2005]. The
Additional supporting information may be found in the online version of
this article.
*Correspondence to:
Dr. Oliver Bartsch, Institute of Human Genetics, Johannes Gutenberg
University Mainz, Langenbeckstraae 1, D-55101 Mainz, Germany.
E-mail: [email protected]
Published online 15 January 2010 in Wiley InterScience
(www.interscience.wiley.com)
DOI 10.1002/ajmg.a.33198
How to Cite this Article:Bartsch O, Gebauer K, Lechno S, van Esch H,
Froyen G, Bonin M, Seidel J, Thamm-M€ucke
B, Horn D, Klopocki E, Hertzberg C, Zechner
U, Haaf T. 2010. Four unrelated patients with
Lubs X-linked mental retardation syndrome
and different Xq28 duplications.
Am J Med Genet Part A 152A:305–312.
� 2010 Wiley-Liss, Inc. 305
duplicated IRAK1 gene may be responsible for the recurrent
infections [Smyk et al., 2008]. Further clinical findings include
hypotonia in infancy later changing to spasticity, very late ambula-
tion, absent or near-absent speech, minor facial, and genital anom-
alies [Lugtenberg et al., 2009].
Low-copy repeats (LCRs) may mediate the formation of recur-
rent chromosome rearrangements (by nonallelic homologous re-
combination, NAHR) and of nonrecurrent rearrangements such as
the Xq28 duplications underlying the MRXSL [Stankiewicz et al.,
2003; Bauters et al., 2008]. A recent array comparative genomic
hybridization (CGH) study of 30 patients with Xq28 duplications
showed that some rearrangements were more complex than simple
tandem duplications and that 83% of the distal breakpoints fell
in two distinct regions of LCRs [Carvalho et al., 2009]. This
coincidence of duplication breakpoints with LCRs suggested that
a particular genomic architecture renders the MECP2 region un-
stable. The DNA-replication-based Fork Stalling and Template
Switching (FoSTeS) mechanism is thought to underlie these com-
plex rearrangements [Lee et al., 2007].
Here we present four patients with different Xq28 duplications
and compare their findings to those in the previously reported
patients with complex rearrangements.
CLINICAL REPORTS
Patient 1
Patient 1 (Fig. 1) was the first child of healthy nonconsanguineous
German parents, a 34-year-old mother and a 36-year-old father,
both with higher education. Apart from a spontaneous abortion,
family history was unremarkable. Third trimester fetal ultrasound
studies showed mild intrauterine growth restriction. Following
spontaneous labor at 41 weeks and cardiac decelerations, he was
born by vacuum extraction with Apgar scores of 7 and 8 at 1 and
5 min. Birth weight was 2,860 g (10th centile), length 49 cm (25th
centile), and occipitofrontal circumference (OFC) 32.5 cm (10th
centile). His mother noted muscular hypotonia and lack of visual
fixation and social smiling. From age 5 months he showed, espe-
cially on days of noisy family visits, unusual movements and attacks
of fluctuating hypertonia lasting some 10–15 sec and occurring
several times per day. Ophthalmologic examination, because of
absent eye contact, disclosed infantile exotropia and hyperopia
(þ3 dpt). He had his first eye contact and social smile at age
9 months, from 1 day to the next, representing a major step forward.
At age 10 months, magnetic resonance imaging (MRI) of the brain
documented symmetrical reduction of the gray and white matter,
FIG. 1. Pedigrees and facial views of patients. Note broad nasal root in all patients and brachycephaly in patients 2–4. (A) Patient 1, age 4 years, note
proptosis. (B) Patient 2, age 16 years, note abnormal posture of hands. (C) Patient 3, age 26/12 years and (D) patient 4, age 12 years, note widely
spaced teeth and full lips. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
306 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
enlarged ventricles, hypoplasia of the frontal parts of the temporal
lobes, and hypoplasia of corpus callosum. An electroencephalogram
(EEG) showed a pathological increase in slow frequency rhythms
but no epileptiform activity. Consecutive EEGs were normal.
He began turning over from the prone to the supine position at
age 12 months and crawled at age 12=12 years. At 19=12 years, length
was 81 cm (10th centile) and OFC 46.5 cm (2nd centile). He had a
mildly abnormal face (Table I), high-arched palate, bifid uvula,
incomplete simian crease on the left, cryptorchidism, and muscular
hypotonia. Muscle strength was normal. At age 2 years he could
sit independently and could stand with much support, and
at 26=12 years he could walk single steps when held firmly by both
hands.
His facial appearance at age 4 years is shown in Figure 1 (see also
Table I). He had recurrent respiratory tract infections, chronic
constipation, and cryptorchidism on the left; and adenoidectomy,
small bowel biopsy, and orchiopexy were performed. Growth
failure, mild obesity, and beginning knee contractures were noticed.
His length was 89.8 cm (<3rd centile) with mildly bent knees
and his OFC was 48.8 cm (10th–25th centile). Muscle tone was
increased. He showed ataxia and lacked free ambulation, but could
walk a number of steps when held by both hands.
TABLE I. Clinical Findings in This Study and in Other Patients With Increased MECP2 Dosage
Clinical findings Patient 1 Patient 2 Patient 3 Patient 4 Literature (n¼ 91)a
MainAge of examination 5 Years 16 Years 2 Years 12 Years n %Mental retardation þ þ þ þ 90/91 (99)Infantile hypotonia þ þ þ þ 78/86 (91)Absent or delayed speech þ þ þ þ 60/66 (91)Spasticity þ þ � þ 29/47 (62)Recurrent infections þ þ þ þ 63/83 (76)Ataxia þ þ � � 10/10 (100)Lack of ambulation þ � þ þ 42/67 (63)Stereotypic hand movements � þ � þ 6/75 (8)Seizures � þ � þ 42/83 (51)MRI abnormalities þ þ þ � 17/20 (85)
Facial abnormalitiesMicrocephaly � � � � 24/71 (34)Macrocephaly � � � � 3/71 (4)Brachycephaly � þ þ þ 16/51 (31)Triangular face � � � � 1/72 (1)Narrow forehead � � � � 1/72 (1)Large ears � þ þ � 24/72 (33)Flat midface � þ � þ 17/72 (24)Broad nasal root þ þ þ þ 15/72 (21)Anteverted nares � � � � 4/72 (6)Prominent nasal bridge þ þ � � 7/72 (10)Upslanting palpebral fissures � � � þ 1/72 (1)Epicanthal folds � þ � � 4/72 (6)Ptosis � � � � 2/72 (3)Hypertelorism � þ � � 8/72 (11)Deep-set eyes � � � � 3/72 (4)Proptosis þ � � � 4/72 (6)Widely spaced teeth � � � þ 3/72 (4)High-arched palate þ � � þ 3/72 (4)Bifid uvula þ � � � 1/72 (1)
Congenital abnormalitiesSwallowing problems � � � � 19/32 (59)Genital abnormalities Cryptb Cryptb � � 26/52 (50)Digital abnormalities � � � Clinoc 22/51 (43)
Other complicationsObesity þ þ � � 1/1 (100)Constipation þ þ þ � 19/21 (91)
aMeins et al. [2005]; Sanlaville et al. [2005]; van Esch et al. [2005]; del Gaudio et al. [2006]; Friez et al. [2006]; Smyk et al. [2008]; Clayton-Smith et al. [2009]; Lugtenberg et al. [2009].bCrypt ¼ cryptorchidism.cClino¼ clinodactyly of fifth fingers.
BARTSCH ET AL. 307
When last seen at the age of 58=12 years he was able to stand and
walk held on one hand only, this was an enormous progress for the
patient and his parents. Three months before, treatment of his knee
contractures with botulinum toxin (Botox�) had been initiated,
enabling him to stand upright with near-unbent legs. His height was
102 cm (<3rd centile; �2.7 SD), weight 22.5 kg (40% overweight
for height), and OFC 50.0 cm (25th centile). He had no speech but
understood his parents and caring persons. He loved eating, looking
at photographs, and swimming in deep warm water with swimming
aids. His younger sister had developed normally and his mother was
expecting her third child, a boy with normal findings after prenatal
diagnosis.
Patient 2Patient 2 (Fig. 1) was the second child of a healthy nonconsangui-
neous Croatian mother and father age 23 and 24 years. Family
history was unremarkable. His older brother was normal. The
mother recalled few fetal movements, and third trimester fetal
ultrasonography showed mild intrauterine growth restriction. He
was born after 37 weeks of gestation by caesarean section because of
large head size, with Apgar scores of 10 at 1 and 5 min. Birth weight
was 4,080 g (>90th centile), length 54 cm (>90th centile), and OFC
35 cm (>90th centile). Two days old, he was transferred to the
childrens’ hospital with icterus and suspected infection. Absence of
visual fixation and social smiling, muscular hypertonus, and de-
velopmental delay were noticed during infancy. Mild opisthotonus
was recorded at age 6 months.
He could sit independently at age 16=12 years and walked at age
24=12 years. At age 25=12 years findings included severe psychomotor
delay and highly abnormal gait with severe flexion at the hip joints
and reduced flexion at the knees and ankles. Muscle tonus was
symmetric but changing between low, normal, and increased.
Muscle reflexes were only mildly increased, not pathological. He
had no nystagmus, tremor, or overt ataxia. A cranial MRI at age 26=12
years was normal apart from a small cyst of the septum pellucidum.
Sedation for the MRI with droperidol (Dehydrobenzperidol�)
provoked an adverse reaction with extrapyramidal motor distur-
bances, which was terminated by intravenous administration of
biperiden (Akineton�). At age 5 years latent hypothyroidism was
diagnosed and treated with L-thyroxine. A follow-up MRI at age 9
years confirmed the cyst of the septum pellicidum and showed
multiple small white matter defects located bilaterally at the poste-
rior horn of the ventricles. At age 11 years, a cartilaginous exostosis
was removed from the lateral distal end of the right femur. At age 13
years, complex partial seizures began. At age 14 years he had a first
generalized tonic/clonic seizure and anticonvulsive treatment with
valproic acid was initiated.
When last seen at age 16 years he had hypertelorism, epicanthus,
flat nasal bridge, small mouth with full lips, open mouth appear-
ance, mildly abnormal ears, proximal placement of thumbs, and
mild truncal obesity. Puberty had progressed normally. He had
contractures; the knees could not be fully stretched. His height was
166.5 cm (10th–25th centile), weight was 57.2 kg (BMI 20.6 kg/m2),
and OFC was 57.4 cm (75th–90th centile). He had increased
tendon reflexes, restless stereotypic motor activity with persistent
swinging of the upper part of the body and frequent jerky move-
ments, broad-based and swaying grossly abnormal gait. Speech was
absent but he could understand his mother and followed simple
requests.
Patient 3Patient 3 (Fig. 1) was the first child of healthy nonconsanguineous
German parents, a 23-year-old mother and a 38-year-old father.
His family history, pregnancy, and birth were unremarkable.
Birth weight was 3,150 g (25th–50th centile), length 50 cm
(50th–75th centile), and postnatal adaptation was normal. Recur-
rent infections and severe developmental delay were noted by age
12 months.
At the age of 18=12 years his developmental age was at 6 months.
Findings at age 26=12 years included brachycephaly, facial hypotonia,
open mouth, large tongue, excessive drooling, and large low-set
ears. Length was 83.5 cm (3rd centile), weight 10.7 kg (3rd centile),
and OFC 50 cm (50th centile). He had muscular hypotonia, muscle
weakness, decreased muscle reflexes, and marked chronic consti-
pation. He was able to sit unsupported for several seconds but could
not walk. He did not speak and showed no understanding of words.
He showed responses to visual stimuli including rapid movements
and vivid colors, but his behavior included autistic traits and eye
contact was limited. Abdominal sonography disclosed an enlarged
colon reflecting chronic constipation and a distended urinary
bladder. Craniospinal MRI showed frontotemporal cortical atro-
phy, enlarged ventricles, and a dilated spinal canal in the lumbar
region. Ophthalmologic findings including fundoscopy were
normal.
Patient 4This patient was first seen by one of us at age 9 months for genetic
evaluation of developmental delay. He was the second child of
nonconsanguineous German parents, a 28-year-old mother and a
25-year-old father. His mother had two miscarriages in the first
trimester. Following an uneventful pregnancy, he was born
at 41 weeks of gestation with normal measurements (weight
3,120 g, length 50 cm, and OFC 35 cm). Apgar scores were 8 and
10 at 1 and 5 min. From birth, feeding difficulties and poor suck
were noted. Muscular hypotonia was apparent at age 6 months. He
had recurrent respiratory tract infections and cystic fibrosis was
excluded. At age 9 months, length was 72 cm (50th centile), weight
7,200 g (3rd centile), and OFC 43.5 cm (3rd centile). He had a flat
occiput, upslanting palpebral fissures, flat nasal bridge, and full
lips, and severe developmental delay. He was able to walk freely at
the age of 46=12 years.
At the age of 106=12 years, he had a severe respiratory tract
infection and developed progressive spasticity. Unsupported walk-
ing ceased and he became wheelchair-bound. He also developed
recurrent tonic–clonic seizures, which responded to anticonvul-
sants. A brain MRI showed no abnormalities.
At the age of 12 years, his height was 147 cm (25th–50th centile),
weight was 33 kg (25th–50th centile), and OFC 52.5 cm
(25th–50th centile). He showed midface hypoplasia, upslanting
palpebral fissures, sparse eyebrows, full lips, widely spaced teeth,
high-arched palate, mildly abnormal ears, bilateral clinodactyly V,
308 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
spasticity of lower limbs, and stereotypic hand movements. He was
not toilet-trained. He could understand simple requests but his
speech was limited to a few words.
METHODS
Cytogenetic AnalysisIn all patients, karyotype analysis was performed on metaphases
from cultured blood lymphocytes using Giemsa banding at
450–550 bands resolution.
Multiplex Ligation-Dependent ProbeAmplification (MLPA)In patients 1–3, the MECP2 duplications were first identified by
MLPA and then confirmed by microarray analyses. Patients 1 and 2,
the mothers of patients 1–3, and the maternal grandmother of
patient 1 were analyzed with the P245-A1 kit (MRC Holland,
Amsterdam, The Netherlands), which is generally used for screen-
ing patients with unexplained developmental delay or MR for
common microdeletion and microduplication syndromes. Patient
3 was analyzed with the P015C kit for Rett syndrome (MRC
Holland) including probes for SLC6A8 (gene for solute carrier
family 6 member 8), IDH3G (gene for isocitrate dehydrogenase 3
gamma), L1CAM (gene for L1 cell adhesion molecule), IRAK1,
MECP2, and SYBL1 (gene for synaptobrevin-like protein 1). Re-
actions were performed according to the manufacturer’s protocols.
Amplification products were analyzed on a CEQ� 8000 Genetic
Analysis System (Beckman Coulter, Krefeld, Germany).
Molecular Chromosome AnalysesIn patient 1, array CGH was performed using a full-coverage
X-chromosome BAC array (Supplemental Table) with a resolution
of approximately 80 kb [van Esch et al., 2005]. In patients 2 and 3,
we used the Affymetrix Genome-Wide Human SNP Array 6.0
(Affymetrix, Santa Clara, CA) with an average distance of 1.3 kb
between neighboring probes. Analyses were performed using the
GeneChip Genome-Wide SNP Sty Assay Kit 5.0/6.0 (Affymetrix)
and the Genotyping Console 3.0.1 (Affymetrix). In patient 4, the
duplication at Xq28 was identified using an Agilent 244A Oligonu-
cleotide array CGH Kit (Agilent Technologies, Santa Clara, CA)
with an 8.9 kb overall median probe spatial resolution. The Feature
Extraction and CGH analytics software (Agilent Technologies) was
used for data analysis. Array CGH experiments were performed
according to the protocols of the manufacturers.
Real-Time Quantitative PCR (qPCR)In patient 1, the partial duplication of BAC RP11-76K17 by array
CGH was further studied by qPCR. Forward (50-GCAGTTGA-
GTCGGGAAAC-30) and reverse (50-GCCTGTGGTAGGAAA-
TGTTG-30) primers amplifying within BAC RP11-76K17 a specific
segment (151,977,395–151,977,477 bp) of the paraneoplastic
cancer–testis–brain antigen (PNMA3) gene were designed using
the AlleleID software (Premier Biosoft International, Palo Alto,
CA). Experiments were performed in triplicate using an ABI 7500
Real Time PCR system (Applied Biosystems, Foster City, CA).
Reaction mixtures (total volume 25ml) contained 1mM of each
primer, 25 ng genomic DNA, and 12.5ml 2� QuantiTect SYBR
Green PCR Master Mix (Qiagen, Hilden, Germany). The nuclear
RNA export factor 5 (NXF5) gene was used as an endogenous
control; the NXF5 forward (50-TGAAGAAGCCAAGAGAAGG-30)and reverse (50-GAGGGAGACTTAGGAATGG-30) primers ampli-
fy a segment (100,982,735–100,982,816 bp) from Xq22. To dem-
onstrate the specificity of the PCR products, a melting curve analysis
was performed for each primer set. Absolute quantification of copy
numbers was accomplished using the standard curve method and
normalization against a normal diploid genome by calculating the
Multiple of Median (MoM) for each amplicon [Unger et al.,
2008].
Genomic DNAs of patient 4 and his mother were obtained from
EDTA blood. qPCR was performed with primer sets for ALB,
NXF5, MECP2, MECP2-50, TKTL_ex12, FLNA_30, FLNA_int14,
FLNA_int1, and EMD_30UTR [Meins et al., 2005; van Esch et al.,
2005]. PCR reactions were performed on an ABI Prism� 7500
Sequence Detection System, as described above. Samples were run
in triplicates in separate tubes to permit quantification of the target
sequences. The albumin (ALB) gene was used for normalization.
PCR conditions were according to manufacturer’s protocol. An
initial denaturation step of 95�C for 8 min was followed by 40 cycles
with denaturation at 95�C for 15 sec and a combined annealing/
elongation step at 60�C for 1 min. Gene copy numbers were
estimated using the DDCT method for relative quantification of
real-time, qPCR data. As a control, we determined the copy number
of the coagulation factor VIII (F8) gene at Xq28 with respect to ALB.
X-Inactivation Studies in Female CarriersThe polymorphic CAG repeats in the human androgen receptor
(HUMARA) gene were amplified from peripheral blood DNA.
Their methylation status on the active versus inactive X chromo-
somes was determined using HpaII restriction digestion and sub-
sequent PCR amplification as reported elsewhere [Allen et al.,
1992]. Amplification products were analyzed on a CEQ� 8000
Genetic Analysis System (Beckman Coulter).
RESULTS
All patients had a normal male karyotype. MLPA identified Xq28
microduplications in patients 1–3, in their mothers, and the
maternal grandmother of patient 1. In patients 1 and 2, the P245
MLPA kit yielded abnormal ratios (1.88–2.01) at all three probes for
the MECP2 gene, 3409-L02797 (exon 1), 9310-L09999 (exon 4), and
9311-L10002 (exon 4). In patient 3, the P015C kit gave normal
ratios of �1.0 for all probes of the SLC6A8, IDH3G, L1CAM, and
SYBL1 genes and abnormal ratios of �2.0 for the IRAK1 and
MECP2 probes. X-inactivation studies were performed in the
healthy mothers of patients 1–3, who had been identified as
asymptomatic carriers by MLPA. They all showed a complete
(100%) skewing of X-inactivation at the HUMARA locus. Most
likely, the allele that was preferentially inactivated in the mother is
the one that was transmitted to her son.
BARTSCH ET AL. 309
Different array CGH protocols were used to confirm the micro-
duplication in patients 1–3 and to identify the microduplication in
patient 4. In patient 1, BAC array CGH showed a duplication
spanning approximately 1,445 kb from clone RP11-403G10 to
clone RP5-1087L9 (see supporting information Table II which
may be found in the online version of this article), including the
segment from MAGEA1 (gene for melanoma-associated antigen 1)
to the 30-end of IKBKG (gene for inhibitor of kappa light poly-
peptide gene enhancer in B-cells, kinase gamma). In addition,
the data indicated a partial duplication of BAC RP11-76K17, which
is located directly proximal to clone RP11-403G10 (Fig. 2, and
see supporting information Table II which may be found in the
online version of this article). The qPCR detected only one copy of
PNMA3 and therefore, the duplicated segment of patient 1 con-
tained the MR-related genes SLC6A8, L1CAM, MECP2, FLNA, and
GDI1 (gene for guanosine diphosphate dissociation inhibitor 1),
karyotype 46,XY.mlpa Xq28(P245)x2.arr Xq28(152,102,622–153,
547,872x2) mat.
In patient 2, microarray analysis (Affymetrix 6.0) identified a
462 kb segmental imbalance at Xq28, comprising the genes from
AVPR2 (gene for arginine vasopressin receptor 2) to EMD (gene for
emerin). Most data points in this region showed ratios of �2.0,
indicative of a duplication. However, a 32 kb segment within
TKTL1 (gene for transketolase-like protein) showed ratios of
�4, consistent with a quadruplication, and a 3 kb segment showed
ratios of �3 indicating a triplication (Fig. 2). The duplication
interval included the MR-related genes MECP2 and FLNA (gene
for filamin A), karyotype 46,XY.mlpa Xq28 (P245)x2.arr Xq28-
(152,815,231–153,061,160x2,153,177,486–153,209,762x4,153,211,
153,209,762x4,153,211, 354–153,269,633x2,153,274,201–153,277,
230x3) mat.
In patient 3, the Affymetrix 6.0 array demonstrated a 457 kb
segmental imbalance from AVPR2 to EMD, including two dupli-
cated segments of 240 and 34 kb, and two triplicated segments of
62 and 14 kb. The proximal triplication contained the 50-end of
the TEX28 gene and the entire TKTL1 gene, whereas the distal
triplication contained the 30-end of the EMD gene. The 30-end of
TEX28 and the 50-end of EMD could not be evaluated, because they
were not covered by the array. The duplication contained MECP2
and FLNA as MR-associated genes, karyotype 46,XY.mlpa
Xq28(SLC6A8,IDH3G,L1CAM)x1,(IRAK1,MECP2)x2,(SYBL1)-
x1.arr Xq28(152,820,356–153,061,160x2,153,158,642–153,220,
801x3,153,225,755–153,259,446x2,153,263,028–153,277,230x3)
mat.
In patient 4, the microduplication was identified using a 244K
Agilent array indicating a 1,128 kb duplication from MAGEA1 to
TKTL1. Using primer sets for NXF5, MECP2, TKTL_ex12, FLNA-
_30, FLNA_int14, FLNA_int1, and EMD_30UTR, qPCR was per-
formed in the patient and his mother. Compared to normal male
controls, MECP2 and TKTL_ex12 displayed a �2� increased
dosage in the patient and a �1.5� increased dosage in his mother.
Primer sets for NXF5, FLNA, and EMD showed normal copy
numbers. Thus, qPCR confirmed the array CGH data and demon-
strated that the duplication was maternally inherited. The duplica-
tion included the MR-related genes SLC6A8, L1CAM, and MECP2,
karyotype 46,XY.arr Xq28(152,089,094–153,217,424x2) mat.
FIG. 2. Schematic representation of the Xq28 duplications in the four patients. Upper part: Positions of genes on chromosome Xq28 (based on Ensembl,
release 52, December 2008); positions of BAC clones and qPCR products used for delineating the duplication in patient 1; shaded BAC indicates
partial duplication, gray BAC indicates duplication, white BAC indicates normal result. Lower part: Positions of LCRs at the distal breakpoints and
overview of the duplication sizes and complex rearrangements identified in this study. The predicted proximal breakpoint of the duplication in patient
1 is located within the dotted line. Gray boxes represent duplications, striped gray boxes indicate triplications, and black box indicates
quadruplication.
310 AMERICAN JOURNAL OF MEDICAL GENETICS PART A
DISCUSSION
We report on four male patients with the MRXSL syndrome and
different Xq28 duplications (sized 457, 462, 1,128, and 1,445 kb), all
maternally inherited, containing MECP2 among other genes. The
patients were at age 23=12, 58=12, 12, and 16 years, respectively, and
presented with typical clinical findings. Contractures of the knees
and other large joints may present a major problem in children
with MRXSL. An effective treatment has not been described yet;
however, it is well known that intramuscular injections of Botox can
very efficiently reduce lower limb spasticity and improve functional
mobility in children with cerebral palsy [Ubhi et al., 2000]. At the
age of 5 years, patient 1 still was unable to walk independently and
could stand only supporting both hands. Botox treatment of the
knee joint contractures enabled him to stand upright with almost
unbent knees and one hand free. Patient 2, age 16 years, could walk
independently despite severe contractures of knees and hips and
a grossly abnormal broad-based, swaying gait. Patient 3 was too
young to develop contractures. Patient 4 was wheelchair-bound
since his severe respiratory tract infection. Despite our limited
experience, we recommend Botox treatment in MRXSL patients
suffering from similar clinical problems as patient 1.
In their study of 16 MRXSL patients, Bauters et al. [2008] first
reported a coincidence of seven distal breakpoints with specific
LCRs in Xq28. Therefore, we analyzed the present patients with
array CGH. The distal breakpoints (Fig. 2) are consistent with
Carvalho et al. [2009] who reported that 83% (25 out of 30) of the
distal duplication breakpoints fall within two distinct LCR regions
(Fig. 2). The more proximal 215 kb region, comprising LCR J and K
groups (JA, JB, JC, K1, K2) lay 47 kb telomeric to MECP2 and
included 23 (77%) breakpoints. The more distal 93 kb region,
comprising LCR L1 and L2 groups was located >400 kb telomeric
to MECP2 and included 2 (6%) breakpoints [del Gaudio et al., 2006;
Carvalho et al., 2009]. All four distal breakpoints in this study
occurred within these two distinct regions, confirming a role for
genomic architecture in the origin of MECP2 duplications. Patients
2–4 had their breakpoints in LCRs K2 and K1, respectively, of the
215 kb region and patient 1 in LCR L1 of the 93 kb region.
Patients 2 and 3 presented with duplications from AVPR2 to
EMD (Fig. 2). Among the 30 patients reported by Carvalho et al.
[2009] we found another case (patient B2772) with duplication
from AVPR2 to EMD. In addition, Carvalho et al. [2009] described
two patients (B2771 and B2800) with very similar duplications from
L1CAM to the 30-end of TEX28. Thus, altogether there are five
MRXSL cases with almost identical distal breakpoints, all located in
the 215 kb LCR region. Three patients (2, 3, and B2772) had distal
breakpoints in LCR K2 and two (B2771 and B2800) in LCR JC.
The proximal breakpoints of individuals 2, 3, and B2772 were
located in the 152,815,000–152,820,000 bp interval near the 50-end
of the AVPR2 gene and the proximal breakpoints of individuals
B2771, B2796, and B2800 clustered near the 30-end of L1CAM
between 152,629,017 and 152,637,719 bp. These proximal break-
point regions contain no LCRs and no other segmental duplications
[UCSC Genome Bioinformatics; http://genome.ucsc.edu/]. Col-
lectively, these data suggest that the apparent recurrent MECP2
duplications account for a significant proportion of Xq28 dupli-
cations associated with MRXSL.
This study confirms the presence of complex Xq28 rearrange-
ments (including triplications or quadruplications, or interrupted
by stretches of nonduplicated sequences) in approximately 25%
(12 in 50) of MRXSL patients [Bauters et al., 2008; Carvalho et al.,
2009]. Similarly, complex duplications of PLP1 (gene for proteo-
lipid protein 1) with interspersed sequences of normal, triplicated,
or quadruplicated copy numbers have been reported at Xq22,
causing Pelizaeus-Merzbacher disease [Lee et al., 2007]. In the first
report on complex Xq28 rearrangements, Bauters et al. [2008]
identified two patients with a duplicated region on telomeric Xq28,
which was inserted between the duplicated MECP2 genes. Carvalho
et al. [2009] reported duplications interrupted by triplications in six
patients and by nonduplicated stretches in two patients. Here
we describe two further complex rearrangements. Patient 2 had
a duplication interrupted by a quadruplication of 32.3 kb and a
triplication of 3 kb, and discussion on the complex patient 3 had
two duplicated segments of 240 and 34 kb interrupted by triplicated
segments of 62 and 14 kb (Fig. 2). There has been no previous report
of two triplicated segments or a quadruplication in patients with
MRXSL.
Most of the recurrent genomic rearrangements are explained by
NAHR, but the nonrecurrent, different-sized, and complex rear-
rangements on Xq28 can be better explained by other mechanisms
[Bauters et al., 2008; Gu et al., 2008; Carvalho et al., 2009].
Nonhomologous end-joining (NHEJ) is a recombination-based
mechanism that does not depend on sequence similarity between
breakpoint regions [Stankiewicz et al., 2003; Gu et al., 2008].
NAHR, NHEJ, and notably the DNA-replication-based mechanism
of FoSTeS, which can generate genomic duplications of essentially
all sizes and complexities [Lee et al., 2007; Zhang et al., 2009], could
possibly explain the structural rearrangements in Xq28 [Bauters
et al., 2008; Carvalho et al., 2009; this report]. According to the
FoSTeS model, the active DNA replication fork stalls and switches
templates using microhomology of the complementary template for
annealing and priming DNA replication. FoSTeS is thought to occur
between breakpoint regions that are closely juxtaposed in the repli-
cating interphase nucleus, both in germ cells and in somatic cells.
At least three genes (TEX28, TKTL1, and EMD) lie in the
triplicated and quadruplicated segments of patients 2 and 3. Copy
number gains at TEX28 have been associated with MYP1 X-linked
myopia phenotypes [Metlapally et al., 2009] and loss of function
mutations in the EMD gene are known to cause Emery-Dreifuss
muscular dystrophy (OMIM 310300), but patients 2 and 3 showed
no myopia and no muscular dystrophy. Variants of TKTL1 (gene
for transketolase 1) have been associated with the Wernicke-Kor-
sakoff syndrome (OMIM 277730), an acute encephalopathy fol-
lowed by chronic impairment of short-term memory that can be
stabilized by high doses of thiamine. Encephalopathy and ataxia
are overlapping phenotypic manifestations of MRXSL and the
Wernicke-Korsakoff syndrome, but this study provides no evidence
for a clinical relevance of the TEX28, TKTL1, and EMD copy
number gains.
ACKNOWLEDGMENTS
We thank the patients and their parents whose help and participa-
tion made this work possible, and Cornelia Wetzig, Institut f€ur
BARTSCH ET AL. 311
Humangenetik at Mainz, as well as Fabienne Trotier, Institut
f€ur Medizinische Genetik at Berlin, for excellent technical
assistance.
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