provides a summary of the cytogenetic and phenotypic characteristics on five female patients with a MECP2 duplication. All patients showed a normal karyotype by G-banding.
3.1. Case reports
Patient 1 is a 10-year old girl, first born to a 24-year old mother and a 25-year old father. Parents are healthy and non-consanguineous. The maternal grandfather developed epilepsy around the age of 40 years, otherwise the family history is unremarkable.
The patient was born at term with a birth weight of 3040 g (−1 SDS). Apgar scores were 8 and 10 at 1 and 5 min, respectively.
It was noted early on that her neuromotor development was slow (rolling over at 10 months of age, standing and walking a few steps (both with support) at the age of 3.5 years). She developed no speech. In the first year of life she made little contact, but this improved gradually and she became a friendly, sociable girl. She disliked changes in daily routine and loved spinning toys.
Her health is severely compromised. She had noisy breathing from birth on and laryngoscopy at the age of 2.5 years showed laryngomalacia. In infancy she was repeatedly admitted because of recurrent infections (at 7 years of age she had had several episodes of pneumonia (13 times), otitis media (five times), and pyelonephritis (twice)). She had surgery for reimplantation of the right urether. Her deciduous teeth did not fall out spontaneously, resulting in a double row of teeth, and had to be removed surgically.
At the age of 8 years and 2 months she was operated because of a luxation of the right hip and a subluxation of the left hip. Afterwards her condition deteriorated, she lost the ability to crawl and to walk with support. At the age of 8.5 years she developed febrile convulsions, followed by seizures and she was diagnosed with Lennox–Gastaut type of epilepsy, with a poor response to medical treatment. She has severe constipation, sleeping problems (refusing to go back to sleep after waking up in the middle of the night), and feeding problems (unable to eat solid food). She had a nasogastric tube for fluid, extra feeding and medication. At the age of 10 years she needed a PEG tube. Placement was complicated and prolonged due to an aberrant position of the liver (ventral to the stomach).
At 8 years of age she was able to communicate using pictures, she has limited understanding of sign language. After the hip surgery and development of epilepsy she lost most of these abilities. Also from that time on, parents noted loss of appetite, loss of energy and reported her to be increasingly lethargic. She showed a delayed reaction to stimuli. She was hypersensitive to loud noise and sunlight.
IQ testing using Kent Infant Development Scale confirmed severe developmental delay and regression (at the ages of 18, 32, 68, and 108 months, her development was conform 7, 8, 12, and 9 months, respectively).
When first assessed at the age of 17 months, her head circumference was 50.5 cm (+2.3 SDS). She showed mild dysmorphic features: prominent broad and high forehead, thin curly hair, telecanthus and epicanthus, thin eyebrows, small nose, full cheeks, open mouth with everted lower lip, broad alveolar ridges, narrow palate, widely spaced teeth, slightly pointed ears, short neck and inverted nipples (a).
Fig. 1 Phenotypical characteristics of individuals with an Xq28 duplication including MECP2. a-c. patient 1, aged 1 year and 5 months (a), 3 years and 7 months (b) and 7 years (c). Note facial hypotonia, large mouth, and widely spaced teeth. d,e. patient 2, (more ...)
On examination at the age of 3 years and 7 months her height was 98 cm (−0.8 SDS), her weight 16.4 kg, (+0.7 SDS for height) and her head circumference 52.5 cm (+1.7 SDS). Most facial features had become more obvious, however her forehead had grown normal and her philtrum was short and prominent (b). She drooled constantly. Neurological examination showed axial and peripheral hypotonia and brisk tendon reflexes. She had no apparent breathing abnormalities.
On follow up at the age of 7 years (c) she could pull herself up to her knees and stand with support. She made stereotypic movements with her hands, with preserved hand function.
On follow up at the age of 9 years her height was 128 cm (−1.5 SDS), her weight was 25 kg (−0.5 SDS for height) and her head circumference 54.5 cm (+1.4 SDS). She had difficulty to sit unsupported. She had no scoliosis. She had a limitation of 20° in the extension of right hip and knee, for which she wears nightly splints.
A brain MRI at the age of 10 months showed mildly enlarged ventricles and prominent sulci, suggestive of mild brain atrophy. MRI was repeated at 4 years of age and showed a Dandy-–Walker malformation and demyelination.
MLPA analysis of the MECP2 gene showed a duplication of this gene. Subsequent SNP array analysis identified a 279 kb duplication on chromosome Xq28 and a 207 kb duplication on chromosome 3q25.33q26.1. The duplication on Xq28 encompasses eight protein coding genes including MECP2, OPN1LW, TEX28P2, OPN1NW, TEX28P1, TEX28, OPN1NW2 and TKTL1. Parental analysis showed that both rearrangements occurred de novo. X-inactivation analysis unravelled a random-methylation (60:40). Additional FISH analysis showed that the Xq28 duplication has been inserted into chromosome 3, adjacent to the 3q25.33q26.1 duplication (a).
Fig. 2 Illustration of Xq duplication of the MECP2 region. a. FISH analysis in patient 1. The Xq duplication (RP11-333O06; red) is inserted in the long arm of chromosome 3 (black arrow), coinciding with the 3q duplication (RP11-67F24; green). Control probes: (more ...)
Patient 2 is a currently 7-year old girl, born at term after an uneventful pregnancy with a birth weight of 2940 g (−1 SDS). Postnatally, she was diagnosed with laryngomalacia at 3 days of age, which improved with time.
She presented at the age of 2 years and 8 months with failure to thrive despite a good appetite (height 81 cm, −3.5 SDS; weight 10.6 kg, −2.4 SDS, −0.2 SDS for height), microcephaly (OFC 45.5 cm, −2.2 SDS), and developmental delay. She has had recurrent urinary tract infections, with no underlying abnormalities.
Her development was moderately delayed, with sitting at the age of 14 months and crawling at 22 months. She had no speech, although her level of understanding was thought to be better than her expressed speech. She had some repetitive behaviour (hair pulling, hand flapping, biting herself). She had sleeping problems (refused to go back to sleep after waking up in the middle of the night).
On examination she was microcephalic (head circumference 45.6 cm (−2.1 SDS). She had no apparent dysmorphic features, apart from prominent infraorbital fullness (the appearance is of very prominent ‘bags’ under her eyes) (d). She had repeated chest infections and a persistent cough.
She was seen for follow up at the age of 4 years and 6 months (e). She had started to walk at the age of 3 years and 9 months, she walked with a wide-based gait. She was smiling a lot and she might use ‘mama, dada, and bye–bye’ appropriately, but had no other words. Her repetitive behaviour was getting less as her communicating skills had increased. Her sleeping problems had not responded to melatonin. She was about to start at a normal school, with full time personal help.
Apart from a marginally elevated TSH, additional investigations were normal, including an EEG and a brain MRI.
Array CGH analysis showed a 1.69 Mb duplication on chromosome Xq28 and a 1.54 Mb deletion on chromosome 21q22.3. Confirmatory FISH experiments using tiling path BAC clones, showed that BAC clones RP11-119A22, RP11-103M23, and RP11-402H20 from the Xq28 region have been translocated to the distal long arm of one of the chromosomes 21 homologues. FISH analysis with specific clones from the distal 21q region, RP11-162F19 and RP11-71A7, confirmed the telomeric deletion of one of the chromosome 21 homologues. Consequently, this patient carries a cryptic unbalanced translocation between chromosomes X and 21, der(21)t(X; 21)(q28; q22.3). Studies of parental chromosomes showed that the rearrangement is de novo and occurred on the paternal chromosomes. X-inactivation analysis showed random-methylation (45:55).
Patient 3 is a 6.5-year old girl, first child of non-consanguineous and healthy Caucasian parents. She was born at term after an uneventful pregnancy by caesarian section, due to breech presentation. At birth, weight was 2320 g (−1.5 SDS), length 47 cm (−1.0 SDS) and head circumference 33.5 cm (−0.5 SDS). Apgar scores were 8 and 9 at 1 and 5 min, respectively.
Her development was delayed: sitting at the age of 10 months, walking with ataxic gait at 24 months, babbling at 4 years. She had feeding difficulties and growth deficit. In infancy, she suffered from constipation. She had several episodes of upper airway infections (bronchitis, pneumonia, and tonsillitis) since the age of 7 months, as well as nocturnal apnea.
She was evaluated at the age of 5 years. She was moderately interactive and she had a sociable behaviour. On follow up at the age 6 years and 5 months she showed little interest in people. She had developed dystonic movements. On physical examination her height was 112 cm (−1.0 SDS) and weight 23 kg (+0.7 SDS). She was microcephalic (head circumference 48.1 cm, −2.0 SDS), with a round face, up-slanting palpebral fissures, severe bilateral ptosis of eyelids (also noted in her paternal grandmother), bilateral epicanthic folds, flat and wide nasal bridge, small and widely spaced teeth, normal hand and foot length, bilateral hallux valgus, and cutis marmorata. She walked with ataxic gait, did not speak and she made repetitive movements with her head (stretching upward).
A cerebral CT recorded at the age of 7 months showed a cyst of the septum pellucidum and cavum vergae. A brain MRI at 2 years and 7 months of age showed an immature aspect of cortical white matter, mainly in the frontal lobes. An EEG, recorded at the age of 4 years and 10 months showed slowing down of background rhythm. Endocrinological evaluation performed for her growth deficit showed hypothyroidism. CD4/CD8 ratio and the study of T-lymphocyte subpopulations were normal.
Array CGH analysis identified a chromosome X subtelomeric duplication of about 2.10 Mb (b). Confirmatory FISH experiments showed that the Xq28 region has been translocated to the short arm of one of the chromosomes 22 homologues. Studies of parental chromosomes showed that the rearrangement is de novo. The karyotype is 46,XX.ish der(22),t(X; 22)(q28; p13)(VAMP7+)dn.
is a member of an X-linked mental retardation family. The index patient in this family (V-1; f) was included in a Spanish collaborative XLMR study. The duplication was identified by an X-chromosome array-CGH and previously published (case 5, pedigree E in 
). Subsequent attempts to study the complete family were in vain. The family consists of three known affected males in three generations (). Three other males died a few days after birth and were probably also affected. Only one branch of the family could be studied (III-6; IV-2; IV-3 and the index patient V-1). The aforementioned paper included few clinical details 
. A detailed description of the index is provided in the Supplementary data
, clinical details on his mother and grandmother are presented here. Clinical data from other family members are scarce, but confirm X-linked inheritance of severe mental retardation.
Fig. 3 Pedigree of X-linked mental retardation family (previously published as pedigree E in Madrigal et al., 2007). 49 y, age at last examination; d 15 y, died at the age of 15 years. (For interpretation of the references to colour in this (more ...) Patient 4
, mother of the index patient (IV-3, ) was born to healthy non-consanguineous parents. Pregnancy and delivery were uneventful. She was born at 39 weeks of gestation, birth weight 2850 g (−1.1 SDS), length 48 cm (−1.2 SDS), and OFC 32 cm (−1.7 SDS). During early childhood she did not show signs of developmental delay, speech development was normal. She had autistic features, interaction both with her mother and others was poor. She presented with learning difficulties at school and she could not finish primary school. Before having her son, psychiatric symptoms had become apparent (depression, compulsions). She is currently rather impulsive and has a strong and difficult character when she is upset. Her cognitive level is below average (IQ 84) and below the level in her family (see Suppl. Table 1
for Wechsler Adult Intelligence Scale (WAIS-III) results). She shows little interest in other people and she is unemployed. She lives with her mother.
On physical examination at 26 years of age her height was 162 cm (−0.3 SDS), weight was 68 kg (+1.2 SDS), and OFC 56.5 cm (+0.7 SDS). Apart from fifth finger clinodactyly she had no apparent dysmorphic features (g).
Patient 4's mother, grandmother of the index case (III-6) is healthy, with a cognitive level within the normal range (see Suppl. Table 1
for the results of formal testing). She takes care of her daughter and grandson. She reported not to be very skillful with her hands and to suffer from a panic disorder. On physical examination weight and height were within normal limits, OFC is 56.5 cm (+0.7 SDS). She had large ears. Apart from clinodactyly of fifth fingers and hyperextensible interphalangeal joints, no dysmorphic features were present (h).
Her son (IV-4) suffered from meningitis when he was 9 days of age. Subsequently he presented with severe mental retardation, he never developed speech although he was able to understand simple tasks. He walked independently at the age of 3 years despite ataxic gait. He presented progressive epilepsy at 11 years of age, had recurrent episodes of severe pneumonia and died when he was 15 years old due to a complicated respiratory infection.
MLPA confirmed the Xq duplication in the index patient (c) and identified the same duplication in his mother (patient 4, IV-3) and grandmother (III-6, c). X-inactivation studies in the mother, patient 4, showed random X-inactivation (63.3:36.7), whereas it showed skewed X-inactivation in the grandmother (88.2:11.8).
Patient 5 is an 8-year old girl, first born to 29-year old parents. Maternal hypertension developed at 36 weeks of gestation. The girl was born at 38 weeks of gestation, weighing 3430 g (0 SDS), length 53.3 cm (+1.7 SDS) and with normal Apgar scores.
Apart from eczema, no health problems were noted in the neonatal period. At 6 months of age hypotonia and developmental delay became apparent. Neurological evaluation at the age of 18 months left this unexplained. Brain MRI and EEG were normal.
Her motor development was delayed (walking around 22 months of age), as was her speech development (first words around 3 years of age). At the age of 7 years she could speak in 5-word sentences, though she was at times difficult to understand. She was in a special needs class. She had difficulties with social interaction and with changes in her environment or routine. Formal assessment for autism could not diagnose autism spectrum disorder.
When first assessed in a genetics clinic at the age of 22 months, she had a history of recurrent otitis media. On physical examination, length was 82.2 cm (−0.5 SDS), weight 10.8 kg (−1.0 SDS), and head circumference 47.7 cm (+0.5 SDS). Apart from slight epicanthal folds, there were no dysmorphic features. The skin revealed scattered patches of eczema. There was muscular hypotonia.
On follow up at the age of 7 years, she had a history of chronic constipation and chronic ear infections. On examination, her height was 121.8 cm (−0.5 SDS), her weight was 23 kg (−0.2 SDS), and her head circumference was 53.3 cm (+1.0 SDS). She had no apparent dysmorphic features.
Family history is unremarkable, her younger sister is doing well without concern for any delays.
SNP array analysis detected a 107.5 kb duplication on Xq28 and an 824.5 kb duplication on chromosome 2q23.1–q23.2. FISH experiments with BAC clone RP11-119A22 confirmed the duplication on the X chromosome. X inactivation studies showed skewing, though not complete (84:16). Parental analyses showed that the phenotypically normal father carried the 2q23 duplication, the Xq28 rearrangement is de novo.
In summary, all females described in this study had mental retardation or learning difficulties (). Accompanying features were mixed, but speech delay and recurrent infections were frequent symptoms. Notably, patient 1 had the most features as usually seen in affected boys.
Array CGH data are summarized in and .
Summary of array results in patients with Xq28 duplications, including MECP2.
Fig. 4 Schematic representation of part of the Xq28 region. The location of the duplications of our five patients and five previously reported females is depicted. Due to missing data, the patient described by Kirk et al.  could not be included. (more ...)