Article Text
Abstract
Background: The use of array comparative genome hybridisation (CGH) analyses for investigation of children with mental retardation has led to the identification of a growing number of new microdeletion and microduplication syndromes, some of which have become clinically well characterised and some that await further delineation. This report describes three children with de novo 17p13.1 duplications encompassing the PAFAH1B1 gene, who had similar phenotypic features, including mild to moderate developmental delay, hypotonia and facial dysmorphism, and compares them to the few previously reported cases with this duplication.
Methods: Multiplex ligation-dependent probe amplification (MLPA) or array-CGH was used to diagnose three developmentally delayed children with duplications of 17p13. The duplications were characterised further using Agilent array technology, revealing duplication sizes from 1.8 to 4.0 Mb, with a region of overlap corresponding to 1.8 Mb. Detailed clinical information was obtained from patient files and personal examinations.
Results: The developmental delay and similar clinical features in the three patients were most likely due to a common microduplication of 17p13.
Conclusions: In contrast to patients with deletion of the region (Miller–Dieker syndrome) the patients reported here had mild to moderate retardation and displayed no lissencephaly or gross brain malformations. Further cases with similar duplications are expected to be diagnosed, and will contribute to the delineation of a potential new microduplication syndrome of 17p13.
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With the advent of new technologies such as multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridisation (array CGH) to study small genomic imbalances in patients with mental retardation, a growing number of new microdeletion and microduplication syndromes are emerging.
The short arm of chromosome 17 is particularly prone to submicroscopical rearrangements due to a high density of low copy repeats. Thus, the proximal 17p region harbours regions with microdeletion and reciprocal microduplication syndromes, each caused by non-allelic homologous recombination: CMT1A (Charcot–Marie–Tooth syndrome type 1A), due to a duplication at 17p11.2; HNPP (hereditary neuropathy with liability to pressure palsies), due to a reciprocal deletion, Smith–Magenis syndrome, caused by a deletion at 17p11.2; and the relatively recently described Potocki–Lupski syndrome, due to a reciprocal duplication at 17p11.2.1 2
Deletions in the more distal region 17p13.3, including the PAFAH1B1 gene (encoding LIS1), result in the brain malformation lissencephaly, with reduced gyration of the cerebral surface and increased cortical thickening. Depending on the size of the deletion, the phenotype varies from isolated lissencephaly (ILS) to Miller–Dieker syndrome (MDS); the latter consists of severe grade ILS and additional characteristic dysmorphic features and malformations.3 Deletions in MDS vary in size, from 0.1 to 2.9 Mb. The critical region differentiating ILS from MDS is approximately 400 Kb, and is referred to as the “MDS telemeric critical region”.4
Complete trisomy of 17p is relatively rare and associated with psychomotor delay, prenatal and postnatal growth retardation, hypotonia, microcephaly and craniofacial dysmorphia. Heart malformations may be present and there may be neurological findings suggestive of peripheral neuropathy.5 To date, only a few patients with a microduplication of the Miller–Dieker region have been reported in the literature, including the very recent report of Bi et al of seven patients with small duplications of varying sizes in this region.6 7 8
We report three unrelated patients with overlapping de novo microduplications of varying size from 1.8 to 4.0 Mb at the 17p13.3 region, but all encompassing the PAFAH1B1 gene.
The phenotypes included delayed psychomotor development, hypotonia and similar dysmorphic features.
Case reports
Patient 1
The patient was born at gestational age (GA) 38+4 weeks as the second child of healthy, non-consanguineous parents. There was no family history of mental retardation or congenital disorders.
Medical history
Apart from common middle-ear infections in early childhood, requiring several consequent grommet insertions, the patient has not been hospitalised.
Growth
Starting with normal birth weight and length, the patient’s linear growth velocity increased from 1 year of age and at the age of 2 years his height was +3 SD and weight +1 SD. Head circumference was −1 SD at the age of 2 years (fig 1). At the last examination, aged 14 years, his height was 196 cm (+3.5 SD) and he was showing signs of puberty (Tanner scale III–IV). The patient is predisposed to tall stature, with parental heights of 179 cm (mother) and 194 cm (father).
Physical examination
The patient had a slightly asymmetrical cranium, with the right side of the skull being smaller than the left, and flattening of the occipital region. He had a mildly dysmorphic appearance with frontal bossing, low-set ears, broad nasal bridge, small nose and hypertelorism. He had abnormal body proportions with long limbs, and he had been diagnosed with anisomelia (left leg 2 cm shorter than the right) (fig 2).
Neurological investigations
The patient displayed severe hypotonia from an early age and slightly hyperactive reflexes. A muscle biopsy at 4.5 years of age raised the suspicion of congenital muscle fibre-type disproportion. Type 1 cells were increased in number, and these cells were slightly atrophic. Signs of degeneration were seen under electron microscopy. Creatine kinase level was normal, and an EEG was normal. A brain axial CT scan was performed at the age of 2 years and delayed myelination was suspected. A second axial CT scan at the age of 4 years was normal.
Psychomotor development
The patient was referred for clinical evaluation at 2 years of age because of delayed early developmental milestones. He sat independently at 11 months and walked at 27 months. At 2 years, he used about four words. From 3 years of age, he received language training and attended a special unit in kindergarten. Later he went to a special school for children with learning disabilities. He received physiotherapy, which improved his motor skills considerably. At the last examination at 14 years of age, he had good verbal skills, but was unable to read and had difficulties performing simple arithmetical problems.
Patient 2
This girl is the second child of healthy non-consanguineous parents. The pregnancy and delivery at GA 40 weeks were uneventful.
Medical history
The child had feeding difficulties in the neonatal period. At the age of 3 months, she was referred to the paediatric department due to failure to thrive. Because of serous otitis media, she had a intubation performed at the age of 12 months with subsequent improvement of her speech. On cardiological evaluation, she was found to have a clinically unimportant patent foramen ovale. There was no history of seizures.
Growth
Starting from a normal birth weight, the child’s weight has followed the −2 SD curve and her height is average. At 12 months, the head circumference was −1.5 SD.
Physical examination
The patient had a hypotonic face, with a protruding tongue and marked salivation. Fifth finger clinodactyly was present on the left hand. Dysmorphic features included a slightly triangular cranium with frontal bossing, low-set ears, downslanting palpebral fissures, discrete bilateral epicanthus and hypertelorism. The midface was broad and flat, and the nose was small with a round nose tip. The chin was triangularly shaped and the neck was short (fig 3).
Neurological investigations
The child had general hypotonia and the limbs appeared short with muscle atrophy. No brain imaging was performed.
Psychomotor development
Early developmental milestones were mildly delayed. The child sat independently at 10 months of age, crawled at the age of 15 months and walked without support at 18 months. At 18 months, she used a few words. At 20 months, she started at a normal nursery. At the last examination, she was 28 months old and requiring extra physical and language training.
Patient 3
The patient is the first child of healthy non-consanguineous parents. During the pregnancy, a prenatal ultrasound examination at GA 24 weeks revealed dilation of the lateral ventricles and suspicion of corpus callosum agenesia/atrophy, and at GA 33, oligohydramnios was seen. Chromosome analysis of cultured amniocytes found a normal male karyotype (46,XY). The child was born at GA 38+1 by caesarean section due to suspicion of progressive hydrocephalus. Apgar score was 10/1.
Medical history
Slight equinovalgus of the right foot and left-sided hip luxation were seen in the neonatal period. At the age of 2 months an open reposition of the hip was required. Within the first 6 months of life, the child had recurrent upper airway infections, atopic dermatitis and asthmatic bronchitis. There was no history of seizures.
Growth
Birth weight, length and head circumference were normal, and the child’s weight curve followed the +1 SD curve. His length increased from +1 SD at 13 months to +2 SD at 22 months (time of last examination).
Physical examination
The child had a hypotonic face. Dysmorphic features included frontal bossing, large but normally set ears, hypertelorism, downslanting palpebral fissures, broad flat midface and a round nose tip. The mouth was small and the palate very high. The chin was triangularly shaped and the neck was short. In the right palm, a simian crease was present. The thorax was short with pectus excavatus and increased distance between the nipples, and the child had a micropenis and right-sided inguinal hernia. On eye examination, he was found to have normal vision, but both retinas had an immature appearance.
Neurological examination
General hypotonia, hypermobility and severely delayed motor development were found. The knee and ankle reflexes could not be released.
Brain imaging
Brain MRI revealed hypoplasia of the corpus callosum, a remarkably domed and high forehead, and dilated lateral ventricles. There was a suggestion of thinning of the white matter and possibly increased signal intensity periventricularly. A small pituitary gland and enlargement of the cisterna magna were also seen.
Psychomotor development
Early developmental milestones were delayed. At the last examination, aged 22 months old, he could sit without support, but could stand for only a few seconds, and moved around by pulling his arms. He had no meaningful words. He was attending a special care nursery and required regular physical training.
A summary of the clinical findings is presented in table 1.
Methods
Informed consent was obtained from the parents.
Diagnosis of patients
The patients were diagnosed as part of a clinical diagnostic test for genomic imbalance using array CGH (Cytochip Bacterial Artificial Chromosome Array; BlueGnome Ltd,Great Shelford, Cambridge, UK) (patient 1) and SALSA MLPA P064-B2 Mental Retardation-1 Kit; MRC-Holland, Amsterdam, The Netherlands (patients 2 and 3)). The duplications were further characterised by oligo array CGH (Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark) as described below.9
Oligo array
Oligo array CGH was performed (Human Genome Microarray Kit 244A; Agilent Technologies, Santa Clara, California, USA). Labelling and hybridisation were performed according to the protocol provided by the manufacturer. Briefly, 2 μg of patient DNA and of a sex-matched control were double-digested with AluI and RsaI (Promega, Madison, Wisconsin, USA) for 2 hr at 37°C. The digested DNA was labelled by random priming (Genomic DNA Labelling Kit Plus; Agilent) Patient and control DNA labelled with Cy3-dUTP and Cy5-dUTP, respectively, were purified by filtration (Microcon YM-30 filters; Millipore, Billerica, Massachusetts, USA). Patient and control DNAs were pooled and hybridised with 50 μg of human CotI DNA at 65°C with rotation for 40 hrs. Washing was performed according to the Agilent protocol. Arrays were analysed using a scanner (Genepix 4200A; Axon Instruments, Union City, California, USA) andAgilent Feature Extraction V.9.1software. Results were presented by Agilent CGH Analytics V.3.4 software. DNA sequence information refers to the public UCSC database (Human Genome Browser, March 2006 Assembly (hg18).
Investigations of parents (fluorescent in situ hybridisation, multiplex ligation-dependent probe amplification and real-time PCR)
Blood samples of the parents of patient 1 were investigated with MLPA as described below and with fluorescent in situ hybridisation (FISH) using a probe targeting PAFAH1B1 (Vysis 321-90065; Abbott Laboratories, Abbot Park, Illinois, USA). Blood samples from the parents of patients 2 and 3 were investigated by FISH using a subtelomeric 17p probe (D17S643) and by MLPA.
MLPA in all cases was performed using the P064 SALSA probe mix as previously described.7
Real-time PCR was used for investigation of parental DNA in patient 2 on a sequence detection system (ABI Prism 7000; Applied Biosystems, Foster City, California, USA) according to the manufacturer’s instructions. Primers were designed using Primer Express Version 2.0 (Applied Biosystems).
SYBR Green PCR master mix (Applied Biosystems) was used for quantitative PCR according to the manufacturer’s instructions. Relative copy numbers were measured relative to GAPDH. Two normal controls were included. Each assay was duplicated and evaluated by a comparative method validated by Applied Biosystems with the formula 2−ΔΔCt.
Results
The oligo array CGH analyses are shown in figure 5 and detailed below.
Patient 1
Patient 1 had an interstitial duplication of chromosome 17p13.3p13.3. The duplication was ∼1.8 Mb (distal breakpoint in chr17:1195561–1203082; proximal breakpoint in chr17:2983700–2997328).
Patient 2
Patient 2 had a terminal duplication of chromosome 17p13.3pter. The duplication was ∼3.0 Mb (base position for the start of the most terminal probe on array: chr17:29169; breakpoint in chr17:3005571–3012800; log2 ratio of chr17:1448942–1503503 was 1.0, indicating four copies of this sequence). Triplication was confirmed by real-time PCR indicating a complex rearrangement.
Patient 3
Patient 3 had a terminal duplication of chromosome 17p13.2pter. The duplication was ∼4.0 Mb (breakpoint in chr17:4044849–4052847).
Parental investigations
Investigations of parental samples found that the duplications and the triplication were de novo in all patients. FISH analysis did not indicate balanced rearrangements.
Figure 6 shows the 17p13.3 region in which deletions of varying sizes were found in patients with MDS.
Discussion
Array CGH has led to the identification of new syndromes by facilitating a “reverse” dysmorphology approach, in contrast to the previous “phenotype first” approach. In the present study, we compared three children with de novo duplications encompassing the same region that is deleted in MDS and suggest a common phenotype, comprising delayed psychomotor development, hypotonia and similar craniofacial dysmorphic features.
The clinical characteristics of the patients described here differ significantly from patients with MDS. Patients with MDS are severely mentally retarded, whereas our patients only have mild to moderate psychomotor retardation and discrete dysmorphic features with several similar facial features, including a high forehead with frontal bossing, small nose and small mouth. The seven people with 17p13.3 duplications reported recently by Bi et al have varying degrees of mental retardation ranging from fine motor delay to global developmental delay, and several common facial features including broad nasal bridge and bossing forehead.6 However, only one of these patients had a duplication that is comparable in size and gene content to those of our patients. Furthermore, our patients, like three of the seven patients in the study of Bi et al, were all hypotonic; patient 1 so severely that a muscle biopsy was performed, which suggested a fibre-type disproportion myopathy.
One of our patients (patient 1) had tall stature (196 cm at 14 years of age) and an unusual, almost Marfan-like appearance. The patient was predisposed to tall stature (his target height is 193 cm, based on parental height). Bi et al reported macrosomia in subjects whose duplication included the CRK gene, and hypothesised that the overgrowth may be attributed to the duplication of CRK, which is involved in growth regulation and cell differentiation.6 The CRK gene is included in the duplication of all three of our patients; however patient 2 and 3 had growth parameters within normal range.
A cardinal feature of MDS, lissencephaly, is not present in our patients described. Patient 3 had a dilated ventricular system diagnosed prenatally and confirmed by postnatal MRI scan. Patient 1 was suspected of delayed myelinisation, but a later CT scan was normal. However, his last brain scan was 10 years ago, and a MRI scan was not performed, thus we cannot exclude brain malformation that could have been detected by MRI. Apart from these, no brain abnormalities were found in patients 1 and 3. Patient 2 did not receive any brain scan.
The PAFAH1B1gene, encoding LIS1, was the first lissencephaly gene identified and is vital for the migration of the cortical neurons during embryological development. Deletion or mutation of this gene leads to the severe brain disease lissencephaly. Studies of model organisms have uncovered an evolutionarily conserved pathway that involves LIS1 and cytoplasmic dynein. The pathway involves a complex of proteins important for neuronal proliferation and survival and migration.10 It has recently been shown that transgenic mice overexpressing LIS1 have a possible migration defect and reduced brain volume.6 In the same study, brain imaging of the human subjects with overexpression of LIS1 found subtle structural brain abnormalities, including mild cerebral volume loss. Thus, overexpression of LIS1, resulting from PAFAH1B1 duplication, could contribute to the developmental delay seen in our three patients.
As opposed to isolated lissencephaly, which, in connection with PAFAH1B1, is a single-gene disorder, MDS is considered a contiguous gene-deletion syndrome with variable breakpoints. In a study by Cardoso et al, a 400 kb region telomeric to PAFAH1B1 (the “MDS telomeric critical region”) was identified as responsible for the more severe phenotype compared with isolated lissencephaly.4 The identification of eight genes in this region was the first step to characterise the genes involved in the MDS phenotype in addition to PAFAH1B1. Our patients have duplications of varying sizes with an overlapping area of approximately 1.8 Mb, which contains around 15 genes, including the 8 genes in the MDS critical region.
One of the genes, YWHAE, is consistently deleted in MDS. Mouse models confirm that this gene plays an important role in neuronal migration and mice that are double heterozygotes for mutations in Pafah1b1 and Ywhae have more severe migration defects than those deficient in LIS1 alone, supporting the role of YWAHE in the severe phenotype in MDS.11 12 A recent report has characterised the significance of a duplication of YWHAE further. It seems that people with duplications including YWHAE have distinct facial features, not present in patients whose duplications did not include this gene. Furthermore, duplication of YWHAE was associated with overgrowth, but only if the duplication also included the CRK gene.6
When the duplication includes all three genes (PAFAH1B1, YWHAE and CRK), the clinical picture seems to be complex. Our patients have a large duplication, including the entire MDS critical region. All three have a slightly dysmorphic appearance, but only one has overgrowth. This suggests an interaction between the genes in the MDS critical region and other genes. The patient with the largest duplication (patient 3) seems to have the most severe phenotype of the three, with additional symptoms including micropenis, inguinal hernia, equinovalgus and corpus callosum agenesia, suggesting a phenotypic effect of duplication of the genes centromeric to the PAFAH1B1 gene.
Regarding to the genes located in the region telomeric to the ABR gene, hemizygosity seems to have no phenotypic consequences, indicating a lack of dosage effect.4 Whether this applies to duplications has yet to be elucidated.
Many of the known microdeletion syndromes and their corresponding microduplication syndromes occur on the basis of non-allelic homologous recombination in low copy repeats, including HNPP/CMT and Smith–Magenis/duplication 17p11.2.2 13 In MDS, the variable size of the deletion indicates that other mechanisms may be responsible and the same applies for the duplications we describe. Further studies of the duplication breakpoints are needed to suggest alternative mechanisms.14
In conclusion, our patients with de novo duplication encompassing the region of MDS have several, but somewhat nonspecific, phenotypic characteristics in common: psychomotor retardation, hypotonia and similar facial dysmorphic features. These clinical features and the additional overgrowth found in one of our patients, is in accordance with the features of the few cases of 17p13.3 duplications described to date. The phenotype differs significantly from MDS.
Additional patients will further substantiate the significance of 17p13.3 duplication and contribute to delineation of the clinical spectrum.
Authors's note
After submission of this manuscript, a MRI brain scan was performed on patient 2. Apart from what appeared to be a vascular variant, the scan was normal.
Acknowledgments
We thank the participating families for their cooperation in this study.
REFERENCES
Footnotes
Competing interests None.
Patient consent Parental consent obtained.
Provenance and peer review Not commissioned; externally peer reviewed.