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A variant Cri du Chat phenotype and autism

spectrum disorder in a subject with de novo
cryptic microdeletions involving 5p15.2 and
3p24.3-25 detected using whole genomic array
CGH
Article in Clinical Genetics May 2005
DOI: 10.1111/j.1399-0004.2005.00406.x Source: PubMed

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Clin Genet 2005: 67: 341351

Printed in Singapore. All rights reserved

Copyright # Blackwell Munksgaard 2005

CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2005.00406.x

Short Report

A variant Cri du Chat phenotype and autism

spectrum disorder in a subject with de novo
cryptic microdeletions involving 5p15.2 and
3p24.3-25 detected using whole genomic
array CGH
Harvard C, Malenfant P, Koochek M, Creighton S, Mickelson ECR,
Holden JJA, Lewis MES, Rajcan-Separovic E. A variant Cri du Chat
phenotype and autism spectrum disorder in a subject with de novo
cryptic microdeletions involving 5p15.2 and 3p24.3-25 detected using
whole genomic array CGH.
Clin Genet 2005: 67: 341351. # Blackwell Munksgaard, 2005
Cri du Chat syndrome (CdCs) is a well-defined clinical entity, with an
incidence of 1/15,000 to 1/50,000. The critical region for CdCs has been
mapped to 5p15, with the hallmark cat-like cry sublocalized to 5p15.3
and the remaining clinical features to 5p15.2. We report findings in a
subject with a de novo t(5;7)(p15.2;p12.2) and an inv(3)(p24q24), who
was found to have a cryptic microdeletion in the critical region for CdCs
detected using a 1-Mb genomic microarray. In addition to 5p deletion,
the proband had a de novo single clone loss at the 3p breakpoint of
inv(3)(p24q24) and a familial single clone deletion at 18q12. Deletions
were confirmed using microsatellite analysis and fluorescence in situ
hybridization. The 5p deletion encompasses approximately 3 Mb,
mapping to the border between bands 5p15.2 and 5p15.31. The single
clone deletion on chromosome 3 maps to 3p24.3-3p25, for which there is
no known phenotype. The clinical features of our proband differ from
the characteristic CdC phenotype, which may reflect the combined effect
of the two de novo microdeletions and/or may further refine the critical
region for CdCs. Typical features of CdCs that are present in the
proband include moderate intellectual disability, speech, and motor
delay as well as dysmorphic features (e.g. broad and high nasal root,
hypertelorism, and coarse facies). Expected CdCs features that are not
present are growth delay, microcephaly, round facies, micrognathia,
epicanthal folds, and the signature high-pitched cry. Behavioral traits in
this subject included autism spectrum disorder, attention-deficit
hyperactivity disorder, and unmanageable behavior including
aggression, tantrums, irritability, and self-destructive behavior. Several
of these behaviors have been previously reported in patients with 5p
deletion syndrome. Although most agree on the cat-cry critical region
(5p15.3), there is discrepancy in the precise location and size of the
region associated with the more severe manifestations of CdCs. The
clinical description of this proband and the characterization of his 5p
deletion may help to further refine the phenotypegenotype associations
in CdCs and autism spectrum disorder.

Cri du Chat syndrome (CdCs) is a well-recognized

contiguous gene disorder resulting from partial interstitial deletion of chromosome 5p. The incidence of

C Harvarda,g, P Malenfantd,g,
M Koochekb,g, S Creightonb,g,
ECR Mickelsonc,g,
JJA Holdend,e,f,g MES Lewisb,g
and E Rajcan-Separovica,g
a
Department of Pathology, bDepartment
of Medical Genetics, cDepartment of
Pediatrics, University of British Columbia,
Vancouver, British Columbia,
d
Department of Physiology, eDepartment
of Psychiatry, Queens University, fAutism
Research Program, Ongwanada,
Kingston, Ontario, Canada, and gThe
Autism Spectrum Disorders Canadian
American Research Consortium
(www.autismresearch.ca)

Key words: array comparative genomic

hybridization autism spectrum disorder
Cri du Chat syndrome cytogenetics
microarray microdeletions in de novo
rearrangements microsatellite analysis
phenotypegenotype correlation
polymorphisms
Corresponding author: Dr Evica
Rajcan-Separovic, Assistant Professor,
University of British Columbia,
Department of Pathology & Laboratory
Medicine, Cytogenetics Laboratory,
Childrens and Womens Health Center
of BC, 4480 Oak Street, Vancouver,
British Columbia, Canada V6H 3V4.
Tel.: 1 604 875 3121;
fax: 1 604 875 3601;
e-mail: eseparovic@cw.bc.ca
Received 23 September 2004, revised
and accepted for publication 6
December 2004

CdCs is between 1/15,000 and 1/50,000 live births,

and among the intellectually disabled population,
the prevalence may be as high as 1/350 (1).
341

Harvard et al.

Approximately 90% of cases are the result of a de

novo deletion, while the majority of the remaining cases are associated with translocations (2).
Parent-of-origin studies have indicated that the
majority of deletions are paternally derived (3,
4). Hallmark clinical features of CdCs include
microcephaly, round facies, hypertelorism, micrognathia, epicanthal folds, hypotonia, prominent
nasal bridge, and severe psychomotor and intellectual disability. Most affected infants also
exhibit the signature high-pitched, cat-like monochromatic cry considered diagnostic of the syndrome. In addition, the broader phenotype of 5p
deletion syndrome includes several behavioral
abnormalities including self-injurious behavior,
self mutilation, hyperactivity, irritability, tantrums,
aggressive, violent and destructive behaviors, and
attention-deficit hyperactivity disorder (2, 5).
Detailed karyotype analysis of 35 patients with
5p deletions by Niebuhr (1) revealed the absence
of the 5p15.1-5p15.3 chromosomal segment in
all patients with CdCs. Later studies refined this
critical region to 5p15.2-p15.3 (6). The critical
region was further divided based on the
phenotypes of individuals with CdCs, with the
proximal portion of 5p15.3 associated with
speech delay and the cat-like cry (3, 6). A deletion
within 5p15.2 is associated with intellectual
disability and the dysmorphic features observed in
CdCs (3, 6). However, uncertainty exists regarding
the precise location of this latter region due to
slightly different maps proposed by two groups,
one of which lies more distal (7) than the other (8).
Here, we report the clinical findings in a
proband with autism spectrum disorder (ASD),
moderate intellectual disability, and some facial
features of CdCs, who was found to have multiple
microdeletions in three chromosomal regions
including 5p15.2-5p15.31, 3p24.3-3p25, and
18q12 using the 1-Mb resolution array CGH.
The de novo deletion in 5p extends over approximately 3 Mb of the distal segment of 15.2 and
maps within the critical region of CdCs. The
clinical features and natural history of this proband are compared to those typically seen in
patients with CdCs.

Subjects and methods

Clinical description

Subject 1
The proband is a 13-year-old male with ASD and
with a complex karyotype with two chromosomal
rearrangements. He was the fourth of four pregnancies, born at term by induced vaginal delivery
with a birth weight of 4.2 kg. Maternal and pater342

nal ages were 33 years, and the pregnancy was

uncomplicated. There was no history of exposures during pregnancy. The proband has a
15-year-old sister with ASD (Subject 2, described
below) as well as a healthy older brother and
sister. His parents both have post-secondary education and are healthy. The family history is also
significant for a maternal aunt with schizophrenia
and a maternal uncle with Down syndrome.
Despite normal early gross motor milestones
(sitting at 6 months and cruising at 9 months),
by 17 months the probands unsteady gait, poor
balance, and coordination were apparent. This
unsteadiness, combined with oral motor apraxia,
raised concerns of a developmental motor/language delay. Fragile X DNA analysis was normal.
Audiologic testing showed a mild conductive
hearing loss due to frequent otitis media requiring
bilateral myringotomy and tube insertion.
The proband was initially assessed at 2 years
8 months by a developmental pediatrician and
child psychiatrist for global developmental
delay, reduced attention span, tantrums, and
nighttime awakenings. Developmental history
was negative for stereotypic mannerisms such as
rocking or spinning. On observation, he played
appropriately with cars and made some sounds
playing with toy animals. He visually searched
and located other toys in the examining room,
but responded inconsistently to sound. Communication was by eye gaze and high-pitched vocalizations (not cat-like in nature). His eye contact
was good with family members, but fleeting with
strangers. Physical examination at that time
showed that his weight was at the 90%, height
at 50%, and OFC at 98% centiles. He had mild
hypotonia and walked with a wide-based gait. He
was able to kick a large ball and throw a smaller
one. The child psychiatrists impression at that
time was of mild autistic features (pervasive
developmental disorder, PDD). The developmental pediatrician and child psychiatrist commented
on a complex neurodevelopmental profile with
severe language delay, apraxia, and concurrent
concerns regarding attention deficit, distractibility,
and delays in adaptive functioning. Developmental
testing showed that visual reasoning skills were
in the low-average range. He was too young for
formal intellectual testing at initial presentation.
A developmental interdisciplinary team assessment was conducted at age 4 years 2 months. Persistent hypotonia, phonological delay with oral
motor apraxia, and a moderate-to-severe delay
in language were present. Receptive language
was at a 2.53 year level and expressive language
weaker at 1821 months. Cognitive testing was
done using the StanfordBinet, and overall scores

Cryptic microdeletions in a child with CdCs and ASD

were in the borderline range (2.5 year level), with

better performance on visually based tasks up to
the 3 year level (low-average range). Adaptive
skills were more affected as measured by the
Vineland Adaptive Behavior Scale (a parent
report measure that looks at the childs communication, daily living skills, socialization, and
motor skills). Overall composite score on the
Vineland was equivalent to 1 year 9 months or in
the range of mild mental handicap. Autistic features were present but felt to be at subthreshold
for a diagnosis of ASD.
At age 6 years, the proband was prescribed Ritalin,
and at 7 years 3 months, he showed improved
attention and the ability to focus. He continued
to show significant developmental delay in all
areas. His behavior was a continuing problem
with extreme aggression that was difficult to
manage. At this time, a psychologist diagnosed
the proband with ASD based on multiple
tests and scales, including DSM-IV criteria, the
Childhood Autism Rating Scale (CARS), and the
Autism Diagnostic Interview-Revised (ADI-R).
The proband was seen at the age of 13 years
7 months. Since age 9 years, he had been
prescribed Respiradol for behavioral management
and Celexa since age 1011 years. He had a history
of one generalized seizure at 9 years of age
with a prolonged postictal episode. An electroencephalogram at that time was abnormal with
the background dysrhythmic and asymmetric,
compatible with an area of irritability in the
right occipital area. Cranial imaging was normal
and antiepileptic therapy was not initiated.
There had been no subsequent seizures. Upon
examination by a clinical geneticist, weight was
at 95%, height at 98%, and OFC significantly
above 98% centiles. His facies was coarse with
mild dysmorphisms noted to include mild frontal
bossing, prominence of the supraorbital ridges,
hypertelorism, and large ears with thickened
scaphohelices bilaterally (Fig. 1). The nasal root
was broad and high. He had a high-arched palate

Fig. 1. The proband (Subject 1). Frontal and left lateral craniofacial
views.

and clefted chin. There was a single occipital hair

whorl, the hair being coarse textured with an
anterior widows peak. He had bilateral tight heel
cords and mild equinovarus deformity. There were
no neurocutaneous markings. There were no cardiopulmonary, GI, or GU concerns. Neurologically,
there were no focal findings apart from bilateral
toe walking and consistent gaze aversion.
The proband was re-assessed for ASD at this time
using the Autism Diagnostic Observation Scale
(ADOS), module 3 (an observational measure
assessing an individuals social and communication
abilities), and he scores above the cut-off for ASD.
His greatest difficulties were not in communication,
but rather problems with reciprocal social interactions including abnormal eye contact with
non-family members, lack of insight, awkwardness
in social settings, self-stimulatory (mild body
rocking), and atypical behaviors (e.g. holding
objects close for visual inspection).
Subject 2
The probands older autistic sister, was the third
of four pregnancies. She was born at full-term by
vaginal delivery with a birth weight of 3.8 kg.
There was no history of maternal exposures
during pregnancy. Early infancy was unremarkable. Subject 2 was curious about the world
around her, slept well, and had several words by
1 year of age. By age 2 years, however, sleep
disturbances had emerged; she withdrew from
family affection, had a high threshold to
pain, and marked preoccupation with fantasy
(she would interact with her family and interpret
the world around her according to cartoon
characters). Self-stimulatory behaviors appeared
(spinning and major temper tantrums) associated
with some speech regression, echolalia, and
diminished eye contact. The emotional lability
and aggressive behaviors (biting her own hands)
led the family to seek further developmental
evaluations. Subject 2 was diagnosed with ASD
by an interdisciplinary developmental team at age
4 years 6 months. At developmental assessment,
she demonstrated immediate and delayed echolalia, decreased eye contact, monotone voice,
distress when unable to find a toy figurine, and
repetitive singing/humming based on songs from
movies and cartoons. The Wechsler Preschool
and Primary Scales of Intelligence (WPPSI-r)
showed verbal scores to be in the moderately
handicapped range (age 3 years at chronological
age of 4 years 11 months). Performance scores, in
contrast, ranged from below average to high average. Subject 2 met the criteria for ASD based on
the CARS, DSM-IIIR criteria (impaired ability
343

Harvard et al.

to make friends, abnormal non-verbal communication, no social smile, variable eye contact,
limited facial expression, and monotone voice),
and clinical evaluation. Speech therapy evaluation at this time confirmed a language disorder,
with expressive vocabulary at 4.6 years and with
receptive vocabulary at 2.53 years. Pragmatic
language was weak. Findings were therefore
consistent with a language disorder.
She was recently assessed by a clinical geneticist
at age 15 years. Her eye examination and hearing
tests were normal. She had hyperacusis and
tactile defensiveness as well as diminished pain
sense and minor self-abusive behaviors, which
were predominant when she was younger. Fragile
X DNA analysis was negative. On physical examination, her weight was at 75%, height at 50%,
and OFC at 98% centiles. She had mild frontal
bossing, medial eyebrow flaring, with upslanting
palpebral fissures, and high and broad nasal root
(Fig. 2). The ears were large with thickened
scapha helices. The remainder of her physical
examination was normal.
Repeat assessment for ASD at this time
(age 15 years) showed significant changes. On the
ADOS, module 4, Subject 2 scored well below
threshold for ASD on the reciprocal social interactions measure, yet in the autistic range on communication based on difficulties in communication
(empathy and emotional gestures).
Cytogenetic analysis

Chromosome analysis G-banding was performed

on metaphases from peripheral blood using standard cytogenetic techniques: for the proband,
Subject 1, in 1993 (400 band level) and in 1994
(550-band level); and Subject 2 in 1993 (550-band
level). The karyotype for the proband reported in
1993 was 46,XY,inv(3), t(5;7)(p15.1;p13) de novo
and in 1994 46,XY,inv(3)(p24q24),t(5;7)(p15.1;
p12.2), ?del (7) (p12.3) de novo. Subject 2 had a
normal female karyotype.

Fig. 2. The affected sister of the proband (Subject 2). Frontal

and right lateral craniofacial views.

344

Array CGH. Genomic DNA was extracted

from peripheral blood using a PUREGENE1
DNA Isolation Kit (Gentra, Minneapolis, MN).
Normal female or male genomic DNA (Promega,
Madison, WI) to match the sex of the patient was
used as a reference control DNA. Subject and
control DNA samples were sonicated using the
Braun-Sonic 1510 sonicator at 50 W for 20 s to
produce a homogeneous smear DNA extending
from 500 bp to 2 kb. DNA samples were then
purified using a DNA Clean and ConcentratorTM
-5 kit (Zymo Research, Orange, CA). Labeling and
hybridization were performed according to the
manufacturers protocol for Spectrals Genomic
(G) ChipTM (Spectral Genomics, Houston, TX).
Spectrals Genomic (G) ChipTM contains roughly
2600 BAC clones spotted in duplicate. Both forward
and reverse labeling experiments were done for
each patient. Following hybridization, slides
were scanned on a GENEPIX 4000B scanner
(Axon Instruments, Union City, CA) and the
16-bit TIFF images captured using GENEPIX Pro
4.0 software. The images were analyzed using
TM
SPECTRALWARE
BAC Array Analysis Software
v 2.0 (Spectral Genomics) as described by Gunn
et al. (9).

Molecular markers

Microsatellites within or in the vicinity of

unbalanced clones were selected based on their
location and their high heterogeneity using
NCBI Map Viewer (http://www.ncbi.nih.gov/
mapview/). Polymerase chain reaction (PCR)
reactions were carried out with 5 ng of DNA in
a total reaction volume of 5 mL. All PCRs were
performed on a dyad MJ Research DNA Engine
using the conditions and the primer sequences
available from the Genome Database website
(http://www.gdb.org/). Briefly, an initial denaturation 95  C for 5 min was followed by 30 cycles of
95  C for 30 s, 55  C (markers D3S1286,
D5S2081, D5S817, D5S1987, D5S667, D18S57,
and D5S2095) or 60  C (D5S807, D5S2064, and
D5S1486) for 30 s, and 72  C for 45 s. The final
extension was at 72  C for 10 min. In all cases,
primer concentrations were 0.50 mM, and MgCl2
concentration was 1.5 mM, except for marker
D5S2095 (2.0 mM). The products were labeled
with a-32P and separated on a 4 or 6% acrylamide
gels for PCR products larger than 160 bp and
smaller than 160 bp, respectively. The genotypes
at each locus were examined in the subjects and
compared to the parental genotypes to determine
whether there was non-Mendelian segregation and an apparent deletion (i.e. absence of a

Cryptic microdeletions in a child with CdCs and ASD

maternal or paternal allele). A marker was deemed

informative for a subject if, based on the alleles
transmitted from the parents, a deletion could be
ruled out and the subject was deemed normal at a
locus when heterozygous, with an allele inherited
from each parent.
Fluorescence in situ hybridization (FISH)

BAC DNA clones that were identified as deleted

by array CGH were obtained from Spectral
Genomics, labeled indirectly with biotin using the
BioPrime Random Priming Kit (Invitrogen
Canada, Burlington, ON) and hybridized to
metaphase chromosomes (10). FISH signals were
detected using the avidin/biotinylated anti-avidin
system (Vector Laboratories, Burlingame, CA).
Slides were viewed on a Zeiss Axioplan 2 fluorescence microscope and images captured using
MACPROBE software (Applied Imaging, Santa
Clara, CA). For each FISH probe, at least 10
metaphase cells were analyzed.
Results

Chromosome analysis of the probands (Subject 1)

G-banded karyotype at 550-band level resolution
was reported as 46,XY,inv(3)(p24q24),t(5;7)
(p15.1;p12.2),?del (7)(p12.3). The t(5;7) and inv(3)
are shown in Fig. 3. The probands sister had a
normal karyotype by G-banding.
Array CGH was performed on both the proband and his sister to search for cryptic genomic
imbalances not apparent on G-banding. Analysis
of the ratio profiles for the sister, Subject 2, did
not reveal any unbalanced clones. Analysis of the
ratio profiles for the affected brother, Subject 1,
revealed three chromosomal areas showing
microdeletions: a single clone deletion of RP11255O19 on the short arm of chromosome 3, a
contiguous deletion of four clones on the short
arm of chromosome 5 involving clones RP11-

79G1, RP11-72C10, RP11-145B1, and RP1191M12, and a single clone deletion of clone
RP11-90B5 on the long arm of chromosome 18
(Fig. 4). The single deleted clone RP11-255O19
on chromosome 3 is located at 3p24.3-3p25, and
the single deleted clone RP11-90B5 maps to
18q12. Three of the clones (RP11-79G1, RP1172C10 and RP11145B1) from the deletion on
chromosome 5 are located in the sub-band
5p15.2 and the fourth (RP11-91M12) extends
into the proximal end of 5p15.31. These four
clones span a region of at least 1.5 Mb. The distance between the balanced clones on either side
of the deletion range is 5.4 Mb (Fig. 5). Based on
array findings only, the deletion ranges from 1.5
to 5.4 Mb. Information regarding position of
clones and molecular markers as well as subband designations was obtained from the UCSC
database (Human Genome, May 2004 assembly).
Based on the array result, the karyotype of patient
1 was revised to 46,XY,inv(3)(p24q24), t(5;7)(p15.2;
p12.2).ish del (3)(p24.3p24.3)(RP11-255O19-), del(5)
(p15.2p15.31)(RP11-79G1-,RP11-91M12-), del(18)
(q12q12)(RP11-90B1-). The uncertainty regarding
the 7p deletion at the translocation breakpoints
indicated in the karyotype from 1994 was therefore resolved and deletion at the breakpoint of
7p excluded at the 1-Mb resolution level.
Instead, the array analysis showed that the
chromosomal material thought to be missing
from the translocation breakpoints was in fact
of chromosome 5 origin.
Molecular markers were used to confirm the
microdeletions detected by array CGH (Fig. 6).
The deletion on chromosome 3 was tested using
marker D3S1286 which is located within the
unbalanced clone RP11-255O19. The proband
inherited only the maternal allele; however, as
the father had only one allele at this locus, it
was not possible to determine whether the father
also had the deletion or was homozygous for this
marker. The deletion on chromosome 5 was

(a)

(b)
inv(3)(p24q24)
p24

Fig. 3. G-band analysis at the 550band level resolution showed an

apparently balanced inversion of
chromosome 3 (a) and a translocation
between 5p and 7p (b) in the proband
(Subject 1).

t(5;7)(p15.1;p12.2)
p12.2

p15.1

q24
5
inv(3)

der(7)
t(5;7)

der(5)t(5;7)

345

Harvard et al.
(a)
p

Clone position
100

50

Chromosome: 3
200

150

4.50

3p deletion

3.50
3.00
2.50
2.00

0.22
0.29
0.33
0.40
0.50

1.50

0.67

Ratio

RP11-255O19

1.00

1.00

0.67

1.50

0.50
0.40
0.33
0.29
0.22

2.00
2.50
3.00
3.50
4.50

(b)
p
5p deletion

20

40

Clone position
80
100

60

120

140

Chromosome: 5
160
180

4.50
3.50
3.00
2.50
2.00
1.50

RP11-79G1

0.22
0.29
0.33
0.40
0.50
0.67

Ratio

RP11-72C10
RP11-145B1
RP11-91M12

1.00

1.00

0.67
0.50
0.40
0.33
0.29
0.22

1.50
2.00
2.50
3.00
3.50
4.50

(c)
p
18q deletion

10

20

4.50
3.50
3.00
2.50
2.00
1.50

Ratio

RP11-90B5

30

Clone position
40

50

60

Chromosome: 18
70

q
0.22
0.29
0.33
0.40
0.50
0.67

1.00

1.00

0.67

1.50
2.00
2.50
3.00
3.50
4.50

0.50
0.40
0.33
0.29
0.22

Fig. 4. Ratio plots from array data for chromosomes 3, 5, and 18 for the proband (Subject 1). Each ratio plot is comprised by
normalizing data from two independent arrays. Normalized data from the array in which the test sample was labeled with Cy3 is
shown in red while the normalized data from the array in which the test sample was labeled with Cy5 is shown in blue. Individual
spots along the ratio plot represent the normalized ratio of individual clones in linear order with the left-most clone being consistent
with the p-arm terminus, while the right-most clone is consistent with the q-arm terminus. As the normalized Cy5 : Cy3 ratio is
computed for both arrays, a loss of a particular clone is manifested as the simultaneous deviation of ratio plots from the modal value
of 1.0, with the red ratio plot showing a positive deviation (upward), while the blue ratio plot shows a negative deviation (downward)
for the same clone. Conversely, DNA copy number gains show the opposite pattern. (a) Chromosome 3 shows a single clone deletion
(RP11-255O19) on the p-arm. (b) Chromosome 5 has a four-clone deletion (RP11-91M12, RP11-145B1, RP11-72C10, and RP1179G1) on the p-arm. (c) Chromosome 18 shows a single clone deletion (RP11-90B5) on the q-arm.

tested using eight markers spanning the deleted

clones and adjacent clones (Figs 5 and 6b). The
proband inherited only a maternal allele for
marker D5S1486. As the father was heterozygous
at this locus, this finding shows that the 5p deletion in the proband was de novo, occurring on the
paternal chromosome. Based on the findings for
all eight markers, the size of the deletion could
be further refined to approximately 2.44.1 Mb
(Fig. 5). The deletion on chromosome 18 was
confirmed using marker D18S57 located 80 kb
proximal to the unbalanced clone RP11-90B5
(Fig. 6c). The proband inherited only a maternal
346

allele at this locus, and the father had a single

allele making him either homozygous or hemizygous for this allele. Microsatellite analysis of
probands sister, Subject 2, using markers from
chromosome 3 (D3S1286), 18 (D18S57), and 5
(D5S1486) showed that she inherited both maternal and paternal alleles for all three markers, i.e.
was not deleted for these markers (Fig. 6).
FISH analysis of metaphase chromosomes
from Subjects 1 and 2, and their parents, was
used to determine the respective origin of the
deletions found in the proband. The deletion of
single clones from 3p and 18q (Fig 7a,c), and for

Cryptic microdeletions in a child with CdCs and ASD

9M

Speech delay

RP11-91E7

RP11-91M12

D5S2095 Deleted
D5S2064 Deleted
Deleted

RP11-145B1

D5S1486 Deleted
Deleted

p15.3
Cat-like cry

Cat-like cry

Dysmorphism
and severe
MR (CdCCR)

D5S713
(~11.6Mb)

11M

RP11-72C10
RP11-79G1

Deleted
Deleted
D5S667 Non-informative
D5S1987 Non-informative
D5S817

D5S817
(~11.8Mb)

p15.2

CdCCR
severe
MR and
facial
features

Adult facial
dysmorphic
features
and severe
mental
retardation
(MR)

10M

Moderate
MR and
some
facial
features

CdCCR
severe
MR and
L28349 facial
(10.8Mb) features

Childhood
facial
dysmorphic
features
and moderate
mental
retardation

D5S2095
(~9.3Mb)

D5S1880
(~9.3Mb)

Balanced
D5S807 Normal

Deleted

12M

13M

D5S1623E
(14.8Mb)

D5S2081 Normal
14M

Mild Mental
Retardation

p15.1
RP11-81P9

No Symptoms

Balanced

Harvard et al, 2005

Marinescu et al ( 7)

Church et al (8)

Overhauser et al (6)

Church et al (3)

15M

Modified from Maniardi et al (4)

Fig. 5. Deletion of the 5p critical region in the proband (Subject 1). Based on the mapped position for BACs showing a deletion on the
array (blue) and location of tested markers (red lines pointing to markers), the deletion spans from 9.3 to 11.7 Mb and is at least
2.4 Mb in size. The broken arrow indicates that the more proximal end of the deletion is unknown and can span to the beginning of
the normal marker D5S2081. In green are the balanced BACs on the array, which flank the deletion. The summary of clinical features
associated with microdeletions within the 5p critical region is shown to the left of the ideogram (4). The 5p region responsible for
severe MR and CdCs typical facial features mapped by two different groups is also shown. Church et al. (8) mapped it between
markers L28349 and D5S1623E, while Marinescu et al. (7) mapped it more distally between markers D5S1880 and D5S713. The
position of markers was determined using the University of California Santa Cruz genome browser, May 2004 version. Markers
D5S1880 and D5S713 were not available on the browser; however, their positions were determined using specific primer sequences as
published in Goodart et al. (11).

the two clones on either side of the four-clone

deletion on 5p (RP11-91M12 and RP11-79G1)
were confirmed in the proband. FISH was also
performed for Subject 2 and both parents using
single clones from 3p and 18q, and revealed a
normal copy number for these clones in the sister
and mother. The father did not show evidence
for a deletion of the 3p clone (Fig 7b); however,
he had a single FISH signal for the 18q clone in
all 10 metaphase cells examined (Fig 7d), indicating that he too had the 18q deletion found in the
proband. Finally, FISH was preformed on both
parents for the 5p deletion. Neither parent
showed evidence of a deletion of the most distal

and most proximal 5p clones in 10 metaphases.

FISH analysis for chromosome 5 clones was not
performed for the sister due to a normal array
profile and no deletion of marker D5S1486 as
detected by microsatellite analysis (Fig. 6b).
In summary, FISH studies combined with
microsatellite analysis showed that the proband
had a de novo microdeletion on 3p, which
occurred on the paternal chromosome; de novo
microdeletion of 5p, at least 2.4 Mb in size, which
occurred on the paternal chromosome 5; and a
microdeletion on 18q inherited from the father.
His sister, Subject 2, did not have microdeletions
of any of the clones deleted in her brother.
347

126-126

126-126

126-154

Brother

154-

126-126

126-154

Sister

D3S1286

Father

Mother

Chromosome 3p

Subject 1

(a)

Subject 2

Harvard et al.

154

126-

Subject 2

Subject 1
Subject 1

238-238

Subject 2

238-254

Brother

D5S2064

Sister

Mother

Chromosome 5p

Father

(b)

254-

238254

284-288

276-284

276-288

Mother

D5S1486
276-280

280-284

Brother

238-238

Sister

238-254

Father

238-254

288284280276-

288

Fig. 6. Confirmation of microdeletions

in the proband (Subject 1) using
molecular markers. (a) Chromosome 3
marker D3S1286 lies within the
unbalanced clone (RP11-255O19).
Subject 1 inherited a maternal allele but
no paternal allele. (b) Chromosome 5
marker D5S1486 lies between two
unbalanced clones (RP11-91M12 and
RP11-145B1), whereas marker
D5S2064 lies distal to the most distal
unbalanced clone (RP11-91M12, see
Fig. 5 for detailed marker position of
these two and additional markers
tested). Subject 1 inherited a maternal
allele, but no paternal allele for both
markers. Results from marker D5S1486
demonstrate that the deletion occurred
de novo, as the father has two alleles for
that marker. (c) Chromosome 18 marker
D18S57 lies approximately 80 kb
proximal to the unbalanced clone
(RP11-90B5). Subject 1 inherited a
maternal allele, but no paternal allele.
As the father had only one allele for this
marker, it was not possible to determine
whether the deletion is de novo or
paternal in origin.

103-105

113-113

Subject 1

Brother

Sister

D18S57

Father

Mother

Chromosome 18q

Subject 2

(c)

113105103-

103-113

105-113

103-113

105

Discussion

Here, we report our findings in a proband (Subject

1) with multiple microdeletions at 5p15.2-5p15.31,
3p24.3-3p25, and 18q12 detected using a 1-Mb
resolution CGH array. The deletion on 5p extends
over a genomic region of approximately 2.4
4.1 Mb and overlaps with the critical region of
CdCs (Fig. 5). The clinical features and natural
history of the proband include moderate intellectual disability, speech delay, dysmorphic features,
hypertelorism, coarse facies, hypotonia, and mild
motor delay. Classic somatic characteristics of
CdCs that were absent include microcephaly,
growth delay, round facies, micrognathia,
epicanthal folds, and the signature monochromatic
cry. His behavioral phenotype includes ASD with
inattention and externalizing behaviors including
aggression, tantrums, and emotional lability.
Developmentally, he showed moderate-to-severe
language delay, severe phonological disorder with
apraxic features, and delayed adaptive skills. To
348

our knowledge, there has been only one other case

of ASD reported to involve a deletion at 5pter5p15.3 (12). Stathopulu et al. (13), using lower
resolution subtelomeric deletion testing, identified
a presumably more distal deletion from the CdCs
locus at 5p15.33, in a patient with ASD and features suggestive of the typically X-linked Lujan
Fryns syndrome.
The probands diagnosis of ASD is shared with
his sister, Subject 2, who does not have these
deletions. It is possible that ASD in the proband
and his sister has the same etiology with additional independent genomic changes of microdeletions, exacerbating the phenotypic expression in
the proband. On the other hand, the ASD phenotype in both siblings may have a different etiology, and in the proband is due to the de novo
microdeletions at either 5p15.2-15.31 or 3p24.325. The normal array findings in the sister do not
exclude subtle rearrangements, imprinting, or
transcriptional alterations in the regions affected

Cryptic microdeletions in a child with CdCs and ASD

(a)

(b)

(c)

(d)

Fig. 7. Confirmation of multiple microdeletions in the proband

(Subject 1) by FISH (ad). (a) In subject 1, only one homolog of
chromosome 3 shows a signal (arrow) when probed with the 3p
clone RP11-255O19. This deletion was de novo in origin, as
it was not inherited from the father (b), and the maternal
allele was present based on microsatellite analysis (Fig. 6a).
For chromosome 18-specific clone RP11-90B5, only one
of the homologs shows a signal in the proband (c, arrow)
and his father (d, arrow). The deletion is therefore familial in
origin.

in her brother or elsewhere in the genome. It is

reasonable to suspect, however, that her genetic
liability to ASD is much less than that of her
brother as her phenotype is milder. Subject 2 is
able to function in a regular school program, has
well-modulated eye contact, facial expression,
and social reciprocity. She can readily engage in
a conversation and, only with close observation,
do the lack of emotional gestures accompanying
speech and diminished use of descriptive gestures
become apparent. Dense microsatellite marker
analysis of the regions deleted in her brother
may be a useful next step in providing clues as
to her underlying autism.
Heterogeneity in both the severity of intellectual
disability and the extent of dysmorphic features of
5p deletion syndrome has been previously reported
and has contributed to the difficulty in mapping
the exact region within 5p15.2 that is associated
with these different phenotypes (4, 7, 14) (Fig. 5).
This may be due, in part, to the fact that the
phenotype of individuals with CdCs changes with
age, thus making a diagnosis difficult in patients
who do not present with the classic features, such
as the cat-like cry, at an early age.
The phenotypic differences in the proband compared to others with CdCs may also be due to

differences in the genetic backgrounds, allelic

variations, and the unmasking of recessive alleles
on the intact homolog. This point is best supported by the reported cases of familial del(5p)
in which there are often variable phenotypes
among individuals with the same deletion (3, 7).
In addition, chromosomal rearrangements such
as the translocation identified in the proband
may disrupt regulatory sequences in genes located
at the breakpoints and/or may result in position
effects (15). DNA structure has been shown to
affect transcription of neighboring genes, and
the translocation of repeat-rich DNA may downregulate the expression of genes in the breakpoint
region.
The presence of the additional microdeletion at
3p24.3-3p25 may also contribute to the probands
phenotype. The genetic map of this region contains coding sequences for two genes (AB002377
and AK126888) with as of yet unknown function,
both of which are expressed in the brain (16). The
deletion on chromosome 18q12 is presumed to be
a benign polymorphism with no recognizable
clinical effect on the phenotype, as the unaffected
father also carries the deletion (Fig. 7d) and is
phenotypically normal. It is interesting to note
that the 18q clone also contains coding sequences
for two genes (AB051482 and AK128053) with
unknown function, both of which are also expressed
in the brain (16). One possibility is that the 18q12
deletion has no effect in the father because of
imprinting, i.e. the phenotypic effects only occur
when transmitted by the father, and perhaps the
father inherited this from his mother. Although
all the 10 analyzed cells from the father had the
deletion 18q, the possibilities of low-level mosaicism
and differential tissue distribution cannot be
excluded. Interestingly, all microdeletions in the
proband occurred on the paternal chromosomes.
The microarray-based comparative genomic
hybridization procedure used to study the chromosomal rearrangements in the proband allowed the
identification of deletions at two of four chromosomal breakpoints, which is in keeping with the
previously reported frequency of deletions at
breakpoints of apparently balanced chromosomal
rearrangements in nine of 15 patients with intellectual disability and congenital anomalies assessed
by FISH and DNA analysis (17). Whole-genome
screening for subtle microdeletions and microduplications at a resolution 10 times higher
than routine cytogenetic analysis has been shown
to be useful in identifying cryptic chromosomal
rearrangements in individuals with apparently
balanced (18) or normal karyotypes (19, 20,
21), but also in characterizing the extent of cytogenetically visible imbalances. Using a 1.4 Mb
349

Harvard et al.

resolution array, Klein et al. (22) identified an

approximately 7 Mb deletion from the CdCs
critical region and a 813 Mb deletion of the
PraderWilli syndrome (PWS) critical region in
a subject with an unbalanced karyotype due to a
der(5)t(5;15)(p15.2;q13). This subjects clinical
features were described to be most consistent
with PWS (developmental delay and bilateral
cryptorchidism), although features such as
microcephaly, micrognathia, epicanthal folds,
and low-set ears, typically seen in CdCs, were
also present. It was suggested that the extended
PraderWilli phenotype in the subject was the
result of the 5p deletion.
In summary, we describe a subject with ASD and
variant CdCs, showing microdeletions at 5p15.2
and 3p24.3-p25. These microdeletions were located
at the chromosomal breakpoints of de novo
rearrangements, including a t(5;7) translocation
and chromosome 3 inversion. At 550 G-band
level resolution, no loss was seen at the chromosome 3 breakpoints, while a loss of chromosomal
material from 7p was suspected to have resulted
from the t(5;7). With a more extensive use of
whole-genome arrays, relevant phenotype and
genotype correlations will become more precise
and may ultimately help our understanding of the
causes of variant phenotypes that could result from
multiple microdeletion syndromes.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

Acknowledgements
We thank both the proband and his sister and their family for
their enthusiastic support of this study. This work was supported by grants from the Canadian Institutes for Health
Research (RT-64217, MESL, principal investigator), a Hospital
for Sick Children Research Foundation grant to ERS, and
a CIHR Interdisciplinary Health Research Team grant
(RT-43820) to the Autism Spectrum Disorders Canadian
American Research Consortium (ASD-CARC, http://www.au
tismresearch.ca) (JJAH, principal investigator); the Ontario
Mental Health Foundation (to JJAH); and a Scottish Rite
Charitable Foundation of Canada graduate student scholarship,
a Queens University Frank Carrel studentship and an Autism
Society Ontario stimulus grant to PM. M. Koochek and
P. Malenfant are trainees with the CIHR/NAAR STIHR
Inter-Institute Autism Spectrum Disorders Training Program
(PI:JJAH).

References
1. Niebuhr E. The Cri du Chat syndrome: epidemiology, cytogenetics, and clinical features. Hum Genet 1978: 44 (3):
227275.
2. Dykens EM, Clarke DJ. Correlates of maladaptive behaviour in individuals with 5p-(cri du chat) syndrome. Dev Med
Child Neurol 1997: 39 (11): 752756.
3. Church DM, Bengtsson U, Nielsen KV et al. Molecular
definition of deletions of different segments of distal 5p

350

14.

15.
16.

17.

18.

19.

20.

that result in distinct phenotypic features. Am J Hum

Genet 1995: 56 (5): 11621172.
Mainardi PC, Perfumo C, Cali A et al. Clinical and molecular
characterisation of 80 patients with 5p deletion: genotype
phenotype correlation. J Med Genet 2001: 38 (3): 151158.
Collins MS, Cornish K. A survey of the prevalence of
stereotypy, self-injury and aggression in children and
young adults with Cri du Chat syndrome. J Intellect Disabil
Res 2002: 46 (2): 133140.
Overhauser J, Huang X, Gersh M et al. Molecular and
phenotypic mapping of the short arm of chromosome 5:
sublocalization of the critical region for the cri-du-chat
syndrome. Hum Mol Genet 1994: 3 (2): 247252.
Marinescu RC, Mamunes P, Kline AD et al. Variability in a
family with an insertion involving 5p. Am J Med Genet
1999: 86 (3): 258263.
Church DM, Yang J, Bocian M et al. A high-resolution
physical and transcript map of the Cri du chat region of
human chromosome 5p. Genome Res 1997: 7 (8): 787801.
Gunn SR, Mohammed M, Reveles XT et al. Molecular
characterization of a patient with central nervous system
dysmyelination and cryptic unbalanced translocation
between chromosomes 4q and 18q. Am J Med Genet 2003:
120A (1): 127135.
Rajcan-Separovic E, Mahadevan MS, Lefebvre C et al.
FISH detection of chromosome polymorphism and
deletions in the spinal muscular atrophy (SMA) region of
5q13. Cytogenet Cell Genet 1996: 75 (4): 243247.
Goodart SA, Simmons AD, Grady D et al. A yeast artificial
chromosome contig of the critical region for cri-du-chat
syndrome. Genomics 1994: 24 (1): 6368.
Vostanis P, Harrington R, Prendergast M et al. Case reports
of autism with interstitial deletion of chromosome 17
(p11.2p11.2) and monosomy of chromosome 5 (5pter>5
p15.3). Psychiatr Genet 1994: 4 (2): 109111.
Stathopulu E, Ogilvie M, Flinter FA. Terminal deletion of
chromosome 5p in a patient with phenotypical features
of LujanFryns syndrome. Am J Med Genet 2003: 119A:
363366.
Wilkins LE, Bron JA, Nance WE, Wolf B. Clinical heterogeneity in 80 home reared children with Cri du Chat syndrome. J Pediatrics 1983: 102: 528533.
Kleinjan D-J, van Heyningen V. Position effect in human
disease. Hum Mol Genet 1998: 7: 16111618.
Nagase T, Ishikawa K, Nakajima D et al. Prediction of the
coding sequences of unidentified human genes. VII. The
complete sequences 100 new cDNA clones from brain
which can code for large proteins in vitro. DNA Res 1997:
4 (2): 141150.
Astbury C, Christ L, Aughton D et al. Detection of deletions in de novo balanced chromosome rearrangements:
further evidence for their role in phenotypic abnormalities.
Genet Med 2004: 6: 8189.
Tyson C, McGillivray B, Chiewa C, Rajcan-Separovic E.
Elucidation of a cryptic 7q31.1 deletion in a patient with a
language disorder and mild mental retardation. Am J Med
Genet 2004: 129A (3): 254260.
Veltman JA, Jonkers Y, Nuijten I et al. Definition of a
critical region on chromosome 18 for congenital aural
atresia by array CGH. Am J Hum Genet 2003: 72 (6):
15781584.
Shaw-Smith C, Redon R, Rickman L et al. Microarray
based comparative genomic hybridisation (array-CGH)
detects submicroscopic chromosomal deletions and duplications in patients with learning disability/mental retardation and dysmorphic features. J Med Genet 2004: 41 (4):
241248.

Cryptic microdeletions in a child with CdCs and ASD

21. Vissers LE, de Vries BB, Osoegawa K et al. Array-based
comparative genomic hybridization for the genome-wide
detection of submicroscopic chromosomal abnormalities.
Am J Hum Genet 2003: 73 (6): 12611270.

22. Klein OD, Cotter PD, Albertson DG et al. PraderWilli

syndrome resulting from an unbalanced translocation: characterization by array comparative genomic hybridization.
Clin Genet 2004: 65 (6): 477482.

351