Molecular and clinical characterization of de novo and familial cases with microduplication 3q29: guidelines for copy number variation case reporting.
ABSTRACT Microdeletions of 3q29 have previously been reported, but the postulated reciprocal microduplication has only recently been observed. Here, cases from four families, two ascertained in Toronto (Canada) and one each from Edinburgh (UK) and Leiden (Netherlands), carrying microduplications of 3q29 are presented. These families have been characterized by cytogenetic and molecular techniques, and all individuals have been further characterized with genome-wide, high density single nucleotide polymorphism (SNP) arrays run at a single centre (The Centre for Applied Genomics, Toronto). In addition to polymorphic copy-number variants (CNV), all carry duplications of 3q29 ranging in size from 1.9 to 2.4 Mb, encompassing multiple genes and defining a minimum region of overlap of about 1.6 Mb bounded by clusters of segmental duplications that is remarkably similar in location to previously reported 3q29 microdeletions. Consistent with other reports, the phenotype is variable, although developmental delay and significant ophthalmological findings were recurrent, suggesting that dosage sensitivity of genes located within 3q29 is important for eye and CNS development. We also consider CNVs found elsewhere in the genome for their contribution to the phenotype. We conclude by providing preliminary guidelines for management and anticipatory care of families with this microduplication, thereby establishing a standard for CNV reporting.
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ABSTRACT: Anophthalmia and microphthalmia (A/M) represent severe developmental ocular malformations. Currently, mutations in known genes explain less than 40% of A/M cases. We performed whole genome copy number variation analysis in sixty patients affected with isolated or syndromic A/M. Pathogenic deletions of 3q26 (SOX2) were identified in four independent patients with syndromic microphthalmia. Other variants of interest included regions with a known role in human disease (likely pathogenic) as well as novel rearrangements (uncertain significance). A 2.2-Mb duplication of 3q29 in a patient with nonsyndromic anophthalmia and an 877-kb duplication of 11p13 (PAX6) and a 1.4-Mb deletion of 17q11.2 (NF1) in two independent probands with syndromic microphthalmia and other ocular defects were identified; while ocular anomalies have been previously associated with 3q29 duplications, PAX6 duplications, and NF1 mutations in some cases, the ocular phenotypes observed here are more severe than previously reported. Three novel regions of possible interest included a 2q14.2 duplication which cosegregated with microphthalmia/microcornea and congenital cataracts in one family, and 2q21 and 15q26 duplications in two additional cases; each of these regions contains genes that are active during vertebrate ocular development. Overall, this study identified causative copy number mutations and regions with a possible role in ocular disease in 17% of A/M cases.Clinical Genetics 05/2013; · 4.25 Impact Factor
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ABSTRACT: The 3q29 microdeletion syndrome is a rare, recurrent genomic disorder, associated with a variable phenotype, despite the same deletion size, consisting in neurodevelopmental features, such as intellectual disability (ID), schizophrenia, autism, bipolar disorder, depression and mild facial morphological anomalies/congenital malformations. A thorough neuropsychiatric evaluation has never been reported in patients with such syndrome. We analyzed the clinical phenotype of four individuals with 3q29 microdeletion syndrome, with special emphasis on the cognitive and behavioral assessment, in order to delineate the neuropsychiatric phenotype related to this condition. We assessed these patients with standardized scales or checklists measuring the cognitive (WISC III or LIPS-R), behavioral (CBCL) and adaptive (VABS) performances. An accurate evaluation in our sample highlights different degrees of ID, variable behavioral disorders, and a preservation of communicative skills among remaining adaptive areas, as the neuropsychiatric hallmark of 3q29 microdeletion syndrome. © 2013 Wiley Periodicals, Inc.American Journal of Medical Genetics Part A 09/2013; · 2.30 Impact Factor
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ABSTRACT: Interstitial microduplication of 3q29 has been recently described. Individuals with this syndrome have widely variable phenotypes. We describe the first clinical case with a 1.607 Mb duplication at 3q29 (chr3: 195,731,956–197,339,329), accompanied by severe intellectual disability, epilepsy, and cerebral palsy. This duplication involves 22 genes; PAK2, DLG1, BDH1, and FBXO45 are implicated in neuronal development and synaptic function and could play an important role in this syndrome. We propose considering genetic studies, particularly array comparative genomic hybridization, in patients with epilepsy and/or cerebral palsy of unknown etiology when dysmorphic features are present. © 2014 Wiley Periodicals, Inc.American Journal of Medical Genetics Part A 05/2014; · 2.30 Impact Factor
Molecular and clinical characterization of de novo and familial cases with microduplication
3q29: guidelines for copy number variation case reporting
Sharan Goobiea,**, Jeroen Knijnenburgb,**, David FitzPatrickc, Anath Christopher Lioneld,e,
Christian R. Marshalld,e, Tara Azamf, Mary Shagog, Karen Chonga,h, Roberto Mendoza-Londonoa,
Freddie H. Sharkeyc,i, Nicolette S. den Hollanderj, Claudia Ruivenkampj, Eddy Maheri, Hans J.
Tankeb, Karoly Szuhaib, Richard F. Wintled,e, Stephen W. Schererd,e*,
a Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, Toronto, Ontario,
b Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the
c Medical and Developmental Genetics Section, Medical Research Council, Human Genetics Unit,
Western General Hospital, Edinburgh, UK, EH4 2XU
d The Centre for Applied Genomics and Program in Genetics and Genomic Biology, The Hospital
for Sick Children, Toronto, Ontario, Canada
e Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
f Regional Molecular Genetics Laboratory, Western General Hospital, Edinburgh, UK EH4 2XU
g Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada
h The Prenatal Diagnosis and Medical Genetics Program, Mount Sinai Hospital, Toronto, Ontario,
i Microarray Facility, Regional Cytogenetics Laboratory, Western General Hospital, Edinburgh, UK
j Center for Human and Clinical Genetics, Department of Clinical Genetics, Leiden University
Medical Center, Leiden, the Netherlands
* Correspondence: firstname.lastname@example.org or email@example.com
** The first two authors contributed equally to this work.
Keywords: microduplication, copy number variant, chromosome 3, 3q29
Running title: Microduplication 3q29
Microdeletions of 3q29 have previously been reported, but the postulated reciprocal
microduplication has only recently been observed. Here, cases from four families, two ascertained
in Toronto (Canada) and one each from Edinburgh (UK) and Leiden (Netherlands), carrying
microduplications of 3q29 are presented. These families have been characterized by cytogenetic and
molecular techniques, and all individuals have been further characterized with genome-wide, high
density single nucleotide polymorphism (SNP) arrays run at a single centre (The Centre for Applied
Genomics, Toronto). In addition to polymorphic copy-number variants (CNV), all carry
duplications of 3q29 ranging in size from 1.9 to 2.4 Mbp, encompassing multiple genes and
defining a minimum region of overlap of about 1.6 Mbp bounded by clusters of segmental
duplications that is remarkably similar in location to previously reported 3q29 microdeletions.
Consistent with other reports, the phenotype is variable, although developmental delay and
significant ophthalmological findings were recurrent, suggesting that dosage sensitivity of genes
located within 3q29 is important for eye and CNS development. We also consider CNVs found
elsewhere in the genome for their contribution to the phenotype. We conclude by providing
preliminary guidelines for management and anticipatory care of families with this microduplication,
thereby establishing a standard for CNV reporting.
Contiguous gene syndromes involving small chromosomal duplications are typically less frequently
reported in comparison to their microdeletion counterparts. Although these rearrangements can both
arise from a common mechanism involving non allelic homologous recombination with region-
specific low copy repeats (Lupski 2004), microduplication syndromes are usually less commonly
recognized, possibly due to ascertainment bias, milder and more variable phenotype, and technical
limitations of cytogenetics and fluorescent in situ hybridization (FISH). Well characterized
chromosomal regions shown to involve these reciprocal duplication and deletion events include
duplication of 17p11.2 causing a phenotype associated with moderate mental retardation and
behavioural disturbances (Potocki et al. 2000), with the reciprocal microdeletion resulting in Smith-
Magenis syndrome; microduplication of 22q11.2 (Ensenauer et al. 2003) having a somewhat
variable phenotype with cardiac malformation and features similar to the classical microdeletion
22q11.2 syndrome; microduplication of 15q11-q13 characterized by developmental delay and
autism, reciprocal to deletions causing Prader-Willi/Angelman syndromes (Dimitropoulos and
Schultz 2007), and microduplication of 7q11.23, which has been related to severe expressive
language delay (Somerville et al. 2005), while the corresponding deletion causes Williams-Beuren
syndrome (Osborne et al. 1996). Most recently, copy number variations (CNVs) in the form of
microdeletions and microduplications of chromosome 16p11.2 have also been observed in autism
spectrum disorder (Kumar et al. 2008; Marshall et al. 2008; Weiss et al. 2008).
With the use of microarray-based techniques, increasing numbers of novel copy number variants are
being discovered both in apparently healthy control individuals (Redon et al. 2006; Pinto et al.
2007), and in patients with genetic disorders such as autism (Autism Genome Project Consortium
2007; Sebat et al. 2007; Marshall et al. 2008) and schizophrenia (Walsh et al. 2008; Xu et al. 2008).
Improved resolution of these microarray platforms is resulting in greater power to detect ever
smaller events, well below the level of resolution of conventional cytogenetic examination (Feuk et
al. 2006; Carter 2007).
A microdeletion syndrome on chromosome 3q29 was originally described in six patients (Willatt et
al. 2005). The common phenotypic features included a long narrow face, short philtrum, high nasal
bridge, developmental and significant speech delay. The microdeletion was approximately 1.5 Mb
in length and was between identical low copy repeat sequences on either side of the deletion
breakpoints. This suggests that this region is susceptible to nonallelic homologous recombination,
which could result in reciprocal exchange events at chromosome 3q29. Two recent reports describe
the apparent reciprocal microduplication event: the first, in the heterozygous state in five
individuals of a three-generation pedigree (Lisi et al. 2008), and the second including 19 cases, five
of which appear to be the reciprocal event with the remainder overlapping this region (Ballif et al.
2008). Here, we describe index cases from four pedigrees (Case 1 apparently de novo, Case 2 a
mother-child inheritance, Case 3 a nuclear family with multiple members carrying the duplication,
and Case 4 an adopted child from whom information about the biological parents is unavailable).
These cases all have microduplication of chromosome 3q29, validated by fluorescent in situ
hybridization (FISH), array-CGH, MLPA and/or high-resolution DNA SNP microarrays.
Regardless of the initial discovery and validation techniques, we have also analyzed these
individuals with genome-wide Affymetrix 500k SNP arrays in order to provide fine-map
duplication breakpoints and ascertain other copy number variation (CNV) events in their genomes.
The clinical phenotypes of these patients are described in detail. Of interest, two have significant
ophthalmological findings and developmental delay was frequent, suggesting that dosage sensitivity
of genes located within 3q29 might be important for eye and cognitive development.
Case 1 (Toronto):
This patient is a 23 month old girl (Figure 1a), who was born to healthy, non-consanguineous
parents. The family history was negative for congenital anomalies (See pedigree, Figure 2a). The
pregnancy was complicated by hyperemesis for the first five months and hypertension for the last
two weeks. There were no known teratogenic exposures. Fetal ultrasounds at 9 and 20 weeks of
gestation were reportedly normal. The patient was born at 36 weeks gestation via spontaneous
vaginal delivery. Labour and delivery were uncomplicated with no neonatal resuscitation required.
Apgars were nine at one and five minutes. The birth weight was 2,580g (50th-75th centile), length
was 50 cm (90th centile), and head circumference was 31.5 cm (25th centile). Multiple congenital
anomalies noted at birth included a large anterior fontanel, a high forehead with bitemporal
narrowing, a downslanting right palpebral fissure, simple low-set ears, a broad nasal root and slit-
like nares, a deeply grooved philtrum, thin upper lip and short neck with redundant nuchal skin
(Figure 1a). She had a U-shaped cleft of the secondary palate. Extensive ophthalmologic
abnormalities included bilateral microphthalmia, a right iris coloboma, right corneal clouding
consistent with a Peter’s anomaly, and a cataract of the left eye. There was a 2 cm umbilical hernia.
The anus was simple and anteriorly displaced. An abdominal ultrasound revealed a cyst of unknown
etiology located at the right crest of the diaphragm. Examination of the extremities revealed partial
2-3 toe syndactyly bilaterally, sandle-gap bilaterally, and camptodactyly of the toes. A skeletal
survey in the newborn period revealed bilateral proximal radial-ulnar synostosis. MRI of the brain
at birth revealed absence of the inferior cerebellar vermis with an enlarged cisterna magna,
consistent with a Dandy-Walker variant. There were also multiple small cystic changes of the
periventricular white matter within the frontal horns of the lateral ventricles. An echocardiogram at
birth was reported as normal; however re-evaluation at approximately one month of age for a
persistent murmur revealed an 8 mm secundum atrial septal defect with left to right shunting, which
has remained asymptomatic since birth.
Abdominal ultrasound at 5 weeks of age further defined her abdominal cyst as arising from the
stomach wall and wrapping around the inferior vena cava. The cyst was resected and she had an
unsuccessful attempt at umbilical hernia repair. She has had severe gastroesophageal disease and
feeding difficulties since birth, requiring multiple high dose antireflux medications. Conductive
hearing loss was detected at 6 months of age and required the insertion of myringotomy tubes. She
underwent a right corneal transplant and left cataract excision at 4 months of age. With the use of a
contact lens in the left eye, her visual acuity was 20/190 in the left eye and 20/960 in the right eye.
A 2.7x2.8 cm subcutaneous mass was noted on the posterior right thigh. CT scan of the mass
suggested that it was likely a hemangioma. No medical intervention was required. Growth
parameters at 8 months of age revealed weight less than the 3rd centile, length at the 25th centile, and
head circumference just below the 50th centile. Her physical features, including microphthalmia,
were similar to her newborn exam. There was central hypotonia. At 18 months of age, the patient’s
first tooth erupted. Tooth shape was normal. A repeat attempt at surgical repair of the umbilical
hernia and her extensive diastasis recti was successful at 20 months of age.
Developmental concerns were noted in the first year of life as she had significant hypotonia and
visual impairment. She was smiling at 3 months of age, reaching and grasping at 8 months of age.
She began rolling over at eight months. Following her surgery at 20 months, she began to sit
independently, crawl and stand with support. She had a formal communication assessment at 15
months of age which indicated that her receptive language abilities were in the 7-9 month old range,
and her expressive skills were in the 5 month old range. She was babbling at 23 months but did not
yet have specific words. She receives occupational therapy, speech therapy, and is enrolled in an
infant development program.
Case 2 (Edinburgh):
This girl was the first child of non-consanguineous parents. She was born by spontaneous vaginal
delivery weighing 3,080g (12th percentile) at 41 weeks of gestation. An increased nuchal
translucency was noted during the pregnancy but no invasive testing or detailed ultrasound
examination was carried out. She was noted to be hypotonic soon after birth and was admitted to the
neonatal intensive care unit. A cardiac ultrasound demonstrated an atrioventricular septal defect.
She was thought to have facial dysmorphism compatible with a diagnosis of Down syndrome but
chromosome analysis revealed a 46,XX apparently normal female karyotype. At this point she was
reviewed by a clinical geneticist (DRF) who noted significant craniofacial dysmorphisms including
upslanting palpebral fissures, large anterior fontanelle, brachycephaly, hypoplastic supraorbital
ridges and a depressed nasal root (Figure 1b). Her eye examination was remarkable with a left sided
iris “coloboma” caused by segmental aniridia with no evidence of an optic fissure closure defect.
Her occipito-frontal cirumference at 1 week of age was 34cm (25th percentile). She had minor
digital dysmorphisms with 5th finger clinodactyly and mild syndactyly of the 2nd and 3rd toes on the
left foot. She had unusual buttock folds. At this point she had further investigations including FISH
for deletion 22q11.2 and Smith-Magenis syndrome, a full skeletal survey, a diasiallotransferrin
assay for congenital disorders of glycosylation, and quantitative plasma amino acid urinary organic
acids analysis, all of which were normal. She had an emergency admission for four different
infective episodes during the first five months of life: bronchiolitis, adenovirus pneumonia,
pneumococcal conjunctivitis and Clostridium difficile. At the age of seven months she had an
elective repair of her AVSD and a secundum atrial septal defect. She had a prolonged recovery in
intensive care and required continuous inotropic support for 38 days following the operation.
She had a Griffiths assessment at the age of 10 months and 24 days which showed global
developmental delay with a developmental age equivelance for locomotor 2.75 mo., personal &
social 3.5 mo., hearing & language 6.5 mo., eye and hand coordination 4.5 mo. and performance 3.5
mo. She was noted to have a mild ataxia and a brain MRI at the age of 4.3 years showed a small
cerebellar vermis. When last reviewed at the age of 8,6 years her height was -3.3SD, weight 21st
percentile and OFC -3.2 SD. She is a very pleasant and friendly girl who was in good general health.
She remains hypotonic and mildly ataxic. She speaks in sentences and has no behavioural problems.
She attends a special educational establishment where she is making progress with all aspects of her
development but she has significant global cognitive impairment.
The proband’s mother and 19-year-old maternal half-sister were both healthy (See pedigree, Figure
2b). The mother had a subsequent pregnancy that resulted in a termination for multiple fetal
anomalies identified on antenatal ultrasound scanning. An autopsy on this fetus showed esophageal
atresia with a tracheoesophageal fistula, a ventricular septal defect, truncus arteriosis, bilateral renal
agenesis, bilateral radial aplasia, bilateral postaxial polydactyly of the feet and bilateral syndactyly
of the 2nd/3rd and 3rd/4th toes. The mother’s full sister, who is healthy, had a child who died as a
result of a complex cardiac defect. The proband’s maternal half uncle had been well until the age of
45 years when he was diagnosed with renal cell carcinoma.
Case 3 (Leiden):
This 16 year old girl was born at term with normal birth weight (Figure 1c). There were no neonatal
feeding problems or hypotonia. Motor development was slightly retarded. She was able to walk at
two years of age. There was a more severe delay in speech development. At 10 years of age her
vocabulary covered 40 words. MRI at 12 years of age showed no brain abnormalities. At 16 years
of age the girl was not toilet-trained. When walking she would easily stumble over. She was obese
and had a small, narrow forehead, straight eyebrows, narrow palpebral fissures, hypotelorism, open
mouth appearance, crowding of the teeth and low posterior hairline. There was profound mental
Both brothers of this girl attended special schools because of learning difficulties (See pedigree,
Figure 2c). The father of the girl lives in an institution. His IQ is 64. He is unable to read or write.
He has straight eyebrows, deep set eyes and narrow palpebral fissures.
Case 4 (Toronto):
The proband is an adopted male who was thirty years of age at the time of last examination. He was
the product of first pregnancy for a then 16 year old mother, who gave him up for adoption soon
after birth. He was born at full term via vaginal delivery in breech presentation with a birth weight
of 2,150 g (below 3rd centile). He was noted at birth to have micrognathia, significant limb
reduction defects of four extremities, congenital right hip dislocation, grade 1 hypospadias and left
cryptorchidism. At 17 months of age his weight was 5.4 kg (well below the 3rd centile), head
circumference 45.5 cm (-2 SD); he had mild dysmorphisms described as a hypoplastic mandible
with overbite as well as mild developmental and significant speech delay. Cardiac evaluation
revealed a grade 2/6 systolic murmur, but his EKG was normal. His hearing was tested at two years
of age and was low-normal, with very mild conductive hearing loss in the left ear. ENT evaluation
at 4 years of age (Figure 1d) revealed a narrow, high vaulted palate with submucous cleft palate and
very mild tongue coordination difficulties. He was assessed by ophthalmology at eight years of age
and was found to have slight nystagmus, visual acuity of 20/20 and no structural eye defects. At age
11 (Figure 1d), he was assessed by the craniofacial service because of severe class II malocclusion
and underwent extensive orthodontic treatment and surgery including LeFort 1 to intrude the
maxilla, mandibular sagittal split advancement and vertical reduction with advancement genioplasty.
At 15 years of age he had left inguinal exploration that revealed an atrophic testis that was removed.
The patient has mild developmental delay and learning disabilities. His milestones were delayed
and he did not sit by himself until 2 years of age. At 34 months of age he was performing at the
level of a 20 month old, with prominent speech delay. The patient received therapy and was able to
attend regular school with additional help due to learning disabilities affecting his reading
comprehension. He finished high school, obtained a college degree, and now lives independently
and works in customer care services.
At last examination at 30 years of age his head circumference was 58 cm (+2SD); his features
include a broad nasal bridge, high arched palate; ears that are normally placed but have simple,
pointed pinnae with a thin upper border. He has increased adipose tissue and has developed multiple
stria in the torso and abdomen. His extremities show significant transverse reduction defects. His
most well developed limb is his upper right arm which includes a normal humeral arm segment and
a partly developed forearm that extends 20 cm below the elbow and ends on a blind stump. The left
arm and both legs consist only of proximal segments (Figure 1d-iv-vii). All extremities have
dermatoglyphic patterns at the tips suggesting at least partial development of the hands and feet.
However, no digits or metacarpals are appreciated.
MATERIALS AND METHODS
Case 1 (Toronto): PHA-stimulated lymphocytes from peripheral blood were cultured for 72 hours
with thymidine synchronization. GTG-banding analysis was performed on peripheral blood
lymphocytes using standard cytogenetic techniques. G-banded karyotypes at 500 band resolution
were prepared for the patient and both of her parents. The de novo change in our patient was further
evaluated using fluorescent in situ hybridization (FISH). FISH was performed on cultured
lymphocytes using the following probes: a chromosome 3q subtelomeric probe (Oncor,
Gaithersburg, MD), and BAC clones RP11-159K3 and RP11-962B7, directly labeled with
Spectrum Orange and Spectrum Green, respectively (Figures 3 and 4). Hybridized metaphase
spreads were analyzed using a Zeiss Axioplan 2 epifluorescence microscope. Images were captured
by an Axiocam MRm Camera (Imaging Associates, Bicester, UK) and analyzed using an imaging
system with MetaSystems Isis Software version 5.1.110 (Boston, MA).
Case 2 (Edinburgh): The 3q29 duplication in the proband was initially discovered with the
BlueGnome CytoChip V1.1 1Mb BAC-CGH array (BlueGnome Ltd., Cambridge, UK), which has
contig coverage of microdeletion regions. BAC array-CGH was performed on the proband and both
parents where genomic DNA from each case was labeled by random priming. Hybridization and
washes were performed on an HS 400TM Pro hybridization station (Tecan Ltd., UK). Each subarray
was prehybridized for 45 minutes at 37° C with 1.5 μg of herring sperm DNA (Sigma-Aldrich, UK)
in 75 μl of hybridization buffer (50% formamide, 7% dextran sulphate, 2x saline sodium citrate
(SSC), 10mM Tris-HCl pH 7.5, & 0.1% (v/v) Tween 20). Test and reference samples were mixed,
co-precipitated, and resuspended in a 75 μl hybridization solution that also contained 2.5 μg/μl Cot-
1 DNA (Invitrogen), denatured at 75° C for 15 minutes, incubated for two hours at 37° C to block
repetitive sequences, and hybridized for 21 hours. Post-hybridization washes were performed using
3 wash cycles in each of PBS/0.05%Tween at 37° C, 0.1x SSC at 54 °C, 1x PBS at 37° C, and a
final wash in PBS/0.05%Tween at 23° C. Slides were dried using high purity nitrogen. Arrays were
scanned using a GenePix Pro 5.0 array scanner (Axon Instruments, UK) and analysed using
BlueFuse for Microarrays analysis software version 3.4 (BlueGnome Ltd, UK).
The proband, an unaffected sister (age 19) and mother, as well as an uncle who has renal cancer at
the age of 49 and a maternal aunt and her child who died with complex congenital heart disease
were also assayed by MLPA. Confirmatory MLPA was performed using both P036B and P070
human telomere assays (MRC Holland, Netherlands), which contain two independent probes for the
3q29 region. The P036B probe is situated in the BDH gene on 3q. The proximal probe sequence
was GCCACCGGGAGGAACTGGGCCAT and the distal probe sequence
TCTAACACCCGTTGCTACCATGCTGGCCACCCGCCTCTCCAGA. The second probe on 3q,
P070, is located in
KIAA0226 and has a proximal probe sequence
CTCTTTCTCCAGGTCACTGCGCTGGAGGACAG and distal probe sequence
ATGTGCCGTCTTGTCCTGCCTGTTTCACATCAGCATAGGATCA. MLPA products were
processed using an ABI 3100 Genetic Analyzer with ABI GeneScan™ ROX500™ size standard.
Quantitative data analysis was obtained using the SoftGenetics® Gene Marker® v1.4 software.
Case 3 (Leiden): Conventional cytogenetic analysis on GTG-banded chromosomes from cultured
lymphocytes of the index case was performed according to standard techniques. Array-CGH was
performed on all five family members using the ~1.0 Mb spaced whole genome large insert clone
arrays, for which the clones were kindly made available by the Wellcome Trust Sanger Institute
(http://www.sanger.ac.uk). The clones were grown, PCR amplified and spotted as previously
described (Knijnenburg et al. 2005; Fiegler et al., 2003). Genomic DNA of the patient was isolated
using standard techniques, and 500 ng was labeled with Cy3-dCTP (GE Healthcare, Diegem,
Belgium) using the BioPrime® DNA Labeling System (Invitrogen, Breda, the Netherlands). As a
reference DNA, 500 ng female human genomic DNA (Promega, Leiden, the Netherlands) was
labeled using Cy5-dCTP. Hybridization and slide washing was performed without prehybridization
on an HS400 hybridization station (Tecan, Giessen, the Netherlands). Arrays were scanned with a
GenePix 4100A scanner (Axon Instruments, Union City, CA) and images were processed with
GenePix Pro 4.1 software. Final analysis of the intensity ratios of the hybridized DNA was as
previously described (Knijnenburg et al. 2005).
Confirmatory MLPA was performed on the index case as described (White et al. 2004). The
selected probes were located in the NCBP2 gene. The proximal sequence was
and the distal sequence was
Quantitative readout was performed with an ABI 3730 DNA analyzer. The accompanying Genescan
3.5 software was used for peak analysis and further downstream normalization and calculations
were performed as described (White et al. 2004). Two-colour interphase FISH confirmation of the
duplication in the proband was performed with clones CTC-196F4 at 3q29, partly overlapping the
DLG1 gene (as in Willatt et al. 2005), and 3p subtelomeric clone GS-1186B18 as a control.
Case 4 (Toronto)
Routine cytogenetic workup was as for Case 1, above. The initial karyotype report of 46, XY was
followed up with chromosomal microarray analysis (Kleberg cytogenetics Laboratory, Baylor
College of Medicine, Houston TX USA; CMV version 5.0).
Affymetrix Genome-Wide SNP Array and Copy Number Analyses
For CNV analysis, we adhered to recommended guidelines (Scherer et al. 2007). In order to
maximize consistency between samples collected at the three sites (Toronto, Leiden and Edinburgh),
all samples were characterized with the Affymetrix 500k array set at The Centre for Applied
Genomics in Toronto. Each sample was genotyped with the GeneChip® Human Mapping NspI and
StyI Arrays (Affymetrix, Inc., Santa Clara, CA) according to the manufacturer’s instructions and as
described previously (Kennedy et al. 2003). For copy number determination, we used three
approaches: DNA Chip Analyzer (dChip) (Li and Wong 2001; Lin et al. 2004; www.dchip.org),
CNAG (Nannya et al. 2005) and GEMCA (Komura et al. 2006). The first two algorithms were
applied separately to each 250k array, and GEMCA was applied to combined 500k array data.
CNVs were scored if they were detected in the same individual either a) on both arrays, or b) by
two of the algorithms. In our hands, these criteria result in high confidence CNV calls that are >95%
likely to be confirmed by an independent technology such as qPCR (Pinto et al. 2007; Marshall et
All phenotype and CNV data are entered in the Database of Chromosomal Imbalance and
Phenotype in Humans using Ensembl Resources (DECIPHER; http://decipher.sanger.ac.uk/).
Case 1 (Toronto)
A subtle cytogenetically detectable difference at 3q29 was detected at a G-band resolution of 500
bands in Case 1. This alteration was not observed in her parents’ chromosomes at the same
resolution (data not shown). The chromosomal difference was determined to be interstitial as the
chromosome 3q subtelomeric probe revealed two normal signals in the correct position in this
patient (Figure 3c). Further investigation using the Affymetrix 250K NspI Array revealed a 2.4 Mb
duplication of 3q29 (Figure 4; Table 2). The duplication was determined to be a de novo event as
neither parent revealed a CNV at this locus (data not shown). The patient also had a 60 kb loss at
6q16.1 and a 407 kb gain at 8p23.1 (Table 2). The 6q16.1 locus contains no known genes and
overlaps numerous known segmental duplications and CNVs, and the 8p23.1 region is a locus of
known copy-number polymorphisms and segmental duplications in the vicinity of the beta-defensin
gene DEFB130. This CNV overlaps with the proximal end of the region of 8p23.1 duplication
reported by Barber et al. 2007 (see Discussion). Interphase FISH analysis of the 3q29 region using
BAC clone probes RP11-159K3 and RP11-962B7 revealed three signals for each probe, confirming
the duplication (Figure 3a). Clone RP11-962B7 is located approximately in the middle of the region
identified as a duplication by microarray, while clone RP11-159K3 is located approximately 600 kb
distal to RP11-962B7, also within the duplicated region in this patient (Figure 3). Co-hybridization
of the two BAC clone probes suggested that the structure of the rearrangement was a tandem, direct
duplication (Figure 3b). The parents of this patient had the normal two signals for each probe,
confirming that the duplication occurred de novo in our patient (data not shown). These same FISH