J Autism Dev Disord (2018) 48:442–449
DOI 10.1007/s10803-017-3329-4
ORIGINAL PAPER
Array-CGH Analysis in a Cohort of Phenotypically Well-
Characterized Individuals with “Essential” Autism Spectrum
Disorders
Eleonora Napoli1 · Serena Russo2 · Laura Casula1 · Viola Alesi2 ·
Filomena Alessandra Amendola1 · Adriano Angioni2 · Antonio Novelli2 ·
Giovanni Valeri1 · Deny Menghini1 · Stefano Vicari1 
Published online: 12 October 2017
© Springer Science+Business Media, LLC 2017
Abstract  Copy-number variants (CNVs) are associated
with susceptibility to autism spectrum disorder (ASD).
To detect the presence of CNVs, we conducted an array-
comparative genomic hybridization (array-CGH) analysis
in 133 children with “essential” ASD phenotype. Genetic
analyses documented that 12 children had causative CNVs
(C-CNVs), 29 children had non-causative CNVs (NC-
CNVs) and 92 children without CNVs (W-CNVs). Results
on clinical evaluation showed no differences in cognitive
abilities among the three groups, and a higher number of
ASD symptoms and of non-verbal children in the C-CNVs
group compared to the W-CNVs and NC-CNVs groups. Our
results highlighted the importance of the array-CGH analy-
ses and showed that the presence of specific CNVs may dif-
ferentiate clinical outputs in children with ASD.
Keywords  ASD · Cognitive development · Clinical
phenotype · Children · Genetic investigation · CNVs
Introduction
Autism spectrum disorders (ASD) are a set of heterogeneous
neurodevelopmental conditions, characterized by early-onset
Electronic supplementary material  The online version of this
article (doi:10.1007/s10803-017-3329-4) contains supplementary
material, which is available to authorized users.
* Stefano Vicari
stefano.vicari@opbg.net
1
Department of Neurosciences, Child Neuropsychiatry Unit,
Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy
2
Laboratory of Medical Genetics, Bambino Gesù Children’s
Hospital, IRCCS, Rome, Italy
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difficulties in social communication and unusually restricted,
repetitive behaviors and interests (APA 2013). The world-
wide population prevalence is about 1%. About 45% of
individuals with ASD have intellectual disability, and 32%
have regression, i.e. loss of previously acquired skills (Lai
et al. 2014; Postorino et al. 2016). Males are more frequently
affected than females, and comorbidity (more than 70% of
patients with ASD have concurrent conditions) is commonly
found with epilepsy and genetic syndromes, such as fragile
X syndrome, Rett syndrome, Down’s syndrome, Phenylke-
tonuria, CHARGE syndrome, Angelman syndrome, Timothy
syndrome and Joubert syndrome (Miles 2011).
Genetics plays a key role in the etiology of ASD, also
in conjunction with environmental factors. Twin stud-
ies suggested a high heritability of this condition, which
was estimated at 60–90% in monozygotic twins, 0–10% in
dizygotic twins (Posthuma and Polderman 2013; Oikon-
omakis et  al. 2016; Ronald and Hoekstra 2011), and
18.7% in siblings (Ozonoff et al. 2011). Several potential
genes and pathways were identified by linkage associa-
tion and next generations sequencing studies, including
chromatin remodeling, metabolism, mRNA translation
and synaptic function (Merikangas et  al. 2015; Pinto
et al. 2010). The array-comparative genomic hybridiza-
tion (array-CGH) analysis emerged as a powerful tool to
identify genomic copy number variations (CNVs) asso-
ciated with a wide range of developmental disorders,
including ASD (Battaglia et al. 2013; Shen et al. 2010).
The description of specific CNVs in ASD-affected chil-
dren has led to a rapid increase in the discovery of novel
microdeletion and microduplication syndromes asso-
ciated with ASD (Beaudet 2013, 2014; Carreira et  al.
2015; Coe et al. 2014). Array-CGH analysis, due to its
high detection rate (20–25%) in identifying causative
genomic variants (microdeletions/microduplications), is
J Autism Dev Disord (2018) 48:442–449 443
now recommended by Italian and international guidelines
as a first-tier clinical diagnostic test for idiopathic intel-
lectual disability and congenital malformations (Beaudet
2013; Italian Society of Human Genetics http://www.sigu.
net; Miller et al. 2010; Al-Mamari et al. 2015). Disease-
causing CNVs are described in 8–21% of individuals with
ASD and they are more frequent in the severe pheno-
type (Beaudet 2013; Jeste and Geschwind 2014; Schaefer
et al. 2013). The most frequently reported CNVs include
2q37, 5p14, multiple locations on chromosome 7, 11q25,
15q11-q13, 16q22.3, 18q21.1, 18q23, 22q11.2, 22q13.3
and Xp22.2-p22.3 (Oikonomakis et al. 2016; Ozgen et al.
2009). Previous studies indicated that individuals with
ASD and dysmorphology show 27.5% of causative CNVs
(Jacquemont et al. 2006), and that individuals with non-
syndromic ASD and without intellectual disability have
a lower number of pathogenic CNVs than individuals
with syndromic ASD or with intellectual disability (Jac-
quemont et al. 2006; Bremer et al. 2011). Other stud-
ies showed that the presence of pathogenic CNVs has a
stronger correlation with communication and language
deficits (Merikangas et al. 2015).
Despite the improvement of genetic studies (Murdoch
and State 2013), the link between genetic and clinical
phenotypes is still unclear due to the extreme heterogene-
ity of this condition. An accurate characterization of indi-
viduals with ASD is mandatory given the wide variety of
clinical features. In order to overcome the heterogeneity
of this condition, several studies attempted to subgroup
ASD. Ingram et al. (2008) defined seven subgroups based
on multiple behavioral, cognitive and physical measures.
Their results indicated that it is taxometrically valid to
classify patients in three subgroups, based on social inter-
action/communication, intelligence, and essential/com-
plex paradigms. By “essential” ASD, Miles et al. (2005)
meant children with ASD without physical dysmorphol-
ogy and/or microcephaly, while by “complex” ASD, they
meant children with six or more minor anomalies and/or
systemic congenital malformations, such as congenital
heart defects (Ingram et al. 2008). In order to limit het-
erogeneity, our study examined only children with ASD
with an “essential” phenotype.
The first aim of our study was to identify the frequency
of CNVs in children with ASD. CNVs were categorized
as causative CNVs (C-CNVs) and non-causative CNVs
(NC-CNVs) to provide evidence of the diagnostic yield
of array-CGH analysis.
The second aim was to describe the cognitive profile
(intelligence level, ASD severity, language skills) of chil-
dren with ASD by identifying different clinical pheno-
types, based on the presence or absence of CNVs and on
CNV specific subtypes.
Methods
Participants
One hundred thirty-three children were recruited for the
study between June 2009 and December 2014 at the Child
Neuropsychiatry Unit of a Children’s Hospital. They were
part of a larger pool of children attending our Children’s
Hospital for clinical and rehabilitative follow-ups.
Inclusion criteria were as follows:
1. clinical diagnosis of pervasive developmental disorders
(PDD) and of ASD, according respectively to the crite-
ria of the Diagnostic and Statistical Manual of Mental
Disorders, Fourth Edition-Text Revision (DSM-IV-TR,
APA 2000), and to the criteria of the Diagnostic and
Statistical Manual of Mental Disorders, Fifth Edition
(DSM-5, APA 2013);
2. clinical genetic examination of each participant, pin-
pointing the number of dysmorphic features: only
children with less than six dysmorphic features were
included (Eapen et al. 2013; Ingram et al. 2008; Miles
et al. 2005);
3. genetic investigation for GTG-banding, karyotyping
chromosomes, X-fragile, and array-CGH analyses.
Exclusion criteria were as follows:
1. genetic investigation for GTG-banding, karyotyping
chromosomes, X-fragile, and array-CGH analyses with-
out PDD or ASD diagnosis;
2. genetic investigation and PDD or ASD diagnosis with-
out clinical assessment of cognitive abilities and ASD
symptoms;
3. “complex” physical phenotypes with six or more minor
anomalies and/or systemic congenital malformations,
such as congenital heart defects.
Each participants had the genetic examination and the
clinical evaluation of the cognitive abilities and the ASD
symptoms. The mean chronological age was 6.7 years (± 3
SD). Eighty-five percent (n = 113) of participants were
males. The male–female ratio (6.7:1) may be considered
as representative of the general population with ASD. The
thirty-two percent (n = 43) of the participants were non-
verbal children.
Clinical genetic examination assured that only partici-
pants with “essential” phenotypes were included.
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444
Procedure
Genetic Investigation
Culturing, harvesting of peripheral blood cells, and chromo-
some banding were performed according to standard meth-
ods. GTG-banded and karyotype chromosomes were studied
and interpreted according to the International System for
Human Cytogenetic Nomenclature (Shaffer et al. 2013).
DNA was extracted by Qiagen blood extraction kit (http://
www.qiagen.com/) and sample quality was assessed both by
1% agarose gel and by NanoDrop 8000 Spectrophotometers
quantification (ThermoFisher Scientific, http://www.ther-
mofisher.com). X-fragile test was performed using two diag-
nostic kits for X-fragile syndrome (Asuragen Inc. Austin, TX
USA and Abott Chicago, IL, USA) following manufacturer’s
indications. Array-CGH analyses were performed by using
the Agilent Human Genome 4 × 180K CGH microarrays
(Agilent Technologies, Santa Clara, California, USA), fol-
lowing the Agilent Oligonucleotide Array-Based CGH pro-
tocol for Genomic DNA analysis (Version 6.2.1, February
2010). This platform is an oligonucleotide-based microarray
allowing genome-wide survey and molecular profiling of
genomic aberrations with a mean resolution of about 75Kb.
The array images were scanned using the Agilent scanner
and elaborated by Feature Extraction software v.10.5 and
Agilent Genomic Workbench Lite Edition v.7.4 (UCSC
Human Genome Browser hg19). Chromosome aberrations
were calculated by ADM2 algorithm. To evaluate whether
the CNVs detected were benign or potentially related with
the disease, bioinformatics analyses were carried out using
freely available Genome Browsers and scientific databases,
such as Database of Genomic Variants (http://dgv.tcag.
ca/dgv/app/home), OMIM (http://www.ncbi.nlm.nih.gov/
omim), Dechiper (https://decipher.sanger.ac.uk/), Pubmed
(http://www.ncbi.nlm.nih.gov/pubmed).
Array‑CGH Data Analysis and CNVs Classification
CNVs may be involved in the development of several dis-
eases, are the susceptibility loci for their onset (Stankiewicz
and Lupski 2010), and represent one of the most important
sources of interspecies physiological variability (Conrad
et al. 2010; Pang et al. 2010; Kearney et al. 2011). Distin-
guishing between potentially pathogenic deletions/duplica-
tions and non-causative or benign familiar variants is chal-
lenging and requires both the operators’ experience, and the
careful consultation of public and private databases and of
the scientific literature. On the basis of our array platform
resolution and on the guidelines of the Italian Society of
Human Genetics—SIGU—(http://www.sigu.net/) for CNVs
interpretation in diagnostic settings, we have only considered
aberrations larger than 100 Kb in size and involving at least
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J Autism Dev Disord (2018) 48:442–449
one known gene. Even if desert regions can also lead to
pathological phenotypes if implicated in critical gene regula-
tion, the current knowledge about non-coding DNA is still
too defective to be helpful for diagnostic purposes, so we
decided not to consider desert regions.
In the present study, it has been comprised in the C-CNVs
classification all the CNVs described in the OMIM database
(http://www.ncbi.nlm.nih.gov/omim) in association with a
known pathological phenotype, and all the karyotypically
visible differences in size and in gene content, suggesting
their pathogenic role. Further, in the C-CNVs classifica-
tion it has been comprised unknown but potentially causa-
tive CNVs having a more uncertain role in the disease, but
previously described in literature of ASD and considered
as potentially pathogenic. In the NC-CNVs classification,
it has been comprised CNVs which: have not ever been
described in literature (unknown CNVs) or have not ever
been described in association with ASD; not involve genes
correlated to autism susceptibility; have been described as
probably benign variants in more than three different studies
in the public available Database of Genomic Variants report-
ing all the imbalances detected in healthy controls (http://
dgv.tcag.ca/dgv/app/home). When CNVs were not detected,
participants were included in the group named as without
CNVs (W-CNVs).
When parental blood was available, the evaluation test
was extended to patients’ parents by Real-Time PCR in order
to determine whether the micro rearrangements were inher-
ited or arose de novo (Sanders et al. 2012).
Clinical Examination
Cognitive or developmental level was assessed by the brief
intelligence quotient (IQ) from Leiter-R (Roid and Miller
1997) or by the general quotient (GQ) from Griffiths Men-
tal Development Scales Extended Revised for ages 2–8,
GMDS-ER 2–8 (Luiz et al. 2006). Leiter-R was proposed
to all the children and only when a child failed to complete
the test because of his or her reduced attentional resources,
then GMDS-ER was administered. Intellectual disability
or developmental delay was considered when, respectively,
children had an IQ or GQ score lower than 70.
Symptoms of ASD in children were evaluated using the
two “gold-standard” tests: the Autism Diagnostic Interview-
Revised, ADI-R (Lord et al. 1994) and the Autism Diag-
nostic Observation Schedule-Generic, ADOS (Lord et al.
2000). The ADI-R (Lord et al. 1994) is a parent-report semi-
structured interview for establishing a clinical diagnosis of
autism. It is organized following the DSM-IV-TR diagnostic
criteria for autism in children with a mental age of 18 months
and above. The ADI-R generates algorithm scores for each
of the three subdomains of autistic symptoms: (a) qualita-
tive impairments in reciprocal social behavior, (b) delays in
J Autism Dev Disord (2018) 48:442–449 445
language development, (c) restricted range of interests and/
or stereotypic behaviors. The ADOS (Lord et al. 2000) is a
semi-structured direct assessment of communication, social
interaction, and play or imaginative use of materials for indi-
viduals suspected of having autism. The ADOS consists of
four modules designed for children and adults with different
language levels, ranging from non-verbal to verbally fluent.
ADOS was administered and scored by licensed clinicians
who have reached clinical reliability on the instrument. The
calibrated severity score (CSS) index (Gotham et al. 2009)
was also calculated. CSS allows to quantify autism symp-
toms independently from patients’ individual characteristics,
such as age and language level. Gotham et al. (2009) mapped
raw ADOS totals onto 10-point severity metric and divided
them in three classes: “nonspectrum ADOS class”, when
severity scores range 1–3; “ASD class”, when severity scores
range 4–5; and “autism class”, with severity scores ranging
6–10. The presence or absence of language production was
assessed by the clinician with direct observation. Children
were considered as non-verbal when they used less than five
words.
Statistical Analysis
Data were analyzed using the Statistical Program for Win-
dows, Version 13.1 (StatSoft, Inc., Tulsa, OK, USA).
Children with C-CNVs and NC-CNVs and children
W-CNVs were compared on IQ or GQ by using one-way
analysis of variance (ANOVA) to verify whether their cog-
nitive abilities were affected by the presence/absence of
CNVs, and by the specific CNV subtype. The assumption
of homogeneity of the variance was monitored with Lev-
ene’s test.
Statistical analyses on CSS, gender, and the presence/
absence of language were conducted using Chi-Square met-
rics (χ2). When the overall Chi square test was significant,
post-hoc pairwise Chi square tests were conducted and Bon-
ferroni adjustment (α/k = α/[row!/2!(row − 1)!] × [column!/2
!(column − 1)!]) adopted to correct for Type I error inflation.
Results
Genetic Investigation
All patients were karyotyped and tested for molecular analy-
sis of FMR1 gene. GTG-banding identified only an abnor-
mal result, consisting of a huge duplication of the short arm
of chromosome 4 (Appendix 1, child #40). Data obtained
from Fragile-X analyses documented that no participants
were affected by Fragile-X. Forty-one patients out of 133
(37 males and 4 females) tested positive for CNVs (30.8%).
Thirty-three participants showed a single CNV, 7 showed 2
CNVs, and only 1 participant showed 3 or more CNVs. A
total number of 50 imbalances were detected (19 deletions
and 31 duplications). Only for 26 imbalances, it was pos-
sible to test the parental origin: 11 were maternally inher-
ited, 8 were paternally inherited, and 7 arose de novo. Two
duplications, involving chromosome 4 and 15 (respectively,
4p16.3p15.1 and 15q11.2q13.1), arose de novo, and were
classified as pathogenic (4%). Ten CNVs (6 duplications and
4 deletions) were included in the unknown but potentially
causative rearrangements (20%), and 38 (23 duplications and
15 deletions) were considered as NC-CNVs (76%). All the
CNVs were confirmed by Real-Time PCR.
Clinical Evaluation
The mean chronological age of whole group was 6.7 years
(± 3 SD) and the mean IQ or GQ was 73.7 (± 24.3 SD).
Out of one hundred thirty-three participants, the percentage
of children with IQ or GQ lower than 70 was 48.9%. The
median of the calibrated severity score—CSS (Gotham et al.
2009) was 7 (range 3–10). The percentage of non-verbal
children with intellectual disability or developmental delay
are 81% (n = 35). Table 1 shows demographic data, mean IQ
or GQ, and median CSS for the three groups.
Out of 12 children with C-CNVs, 7 children (58.3%) had
IQ or GQ lower than 70 and 6 (85.7%) of them were also
non-verbal. Out of 29 children with NC-CNVs, 16 children
(55.2%) had IQ or GQ lower than 70 and 3 (18.6%) of them
were also non-verbal. Finally, out of 92 children W-CNVs,
43 children (46.7%) had IQ or GQ lower than 70 and 26
(60.5%) were also non-verbal.
Results on cognitive abilities showed no significant dif-
ferences in IQ or GQ among children with C-CNVs, NC-
CNVs, and W-CNVs ­(F2,130 = 1.14, p = 0.32).
For each group (C-CNVs, NC-CNVs and W-CNVs), the
percentage of participants in each CSS class was calculated.
A significant difference was found among groups (𝜒42 = 31.2,
p = 0.000003). In the post-hoc pairwise Chi square tests, the
alfa level considered after Bonferroni adjustment was 0.02.
Groups did not differ (𝜒22 = 1.27, p = 0.53) for the percent-
age of children included in the “autism class” (100% for
C-CNVs, 90% for NC-CNVs, and 85% for W-CNVs). How-
ever, the W-CNVs group differed from the other two groups
(𝜒22 = 18.17, p = 0.0001) for the higher percentage of children
included in the “ASD class” (0% for C-CNVs, 3% for NC-
CNVs, and 14% for W-CNVs). Regarding the “nonspectrum
ADOS class”, the group with NC-CNVs differed from the
other two groups (𝜒22 = 9.7, p = 0.008) for the higher percent-
age of children included in this class (0% for C-CNVs, 7%
for NC-CNVs, and 1% for W-CNVs).
As regards gender, no difference among the three groups
were found (𝜒22 = 1.92, p = 0.38).
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446
Table 1  Demographic data and
clinical results
Participants
Males
Females
IQ or GQ mean (SD)
CSS median (range)
Verbal participants
Non-verbal participants
CNVs copy number variations, C-CNVs causative copy number variations, NC-CNVs non-causative copy
number variations, W-CNVs without copy number variations, IQ intelligence quotient, GQ general quo-
tient, CSS calibrated severity score from ADOS
a
 Chi-Square test (χ2)
b
 One-way analysis of variance (ANOVA)
For each group (C-CNVs, NC-CNVs, W-CNVs), the
percentage of verbal and non-verbal participants was cal-
culated. In the post-hoc pairwise Chi square tests, the alfa
level considered after Bonferroni adjustment was 0.03. A
significant difference was found among groups (𝜒22 = 29.64,
p = 0.00001), with NC-CNVs and W-CNVs groups having
the highest percentage of verbal children (respectively, 86
and 64%), and the C-CNVs group differing from the other
two groups (𝜒22 = 19.97, p = 0.00005) for the highest per-
centage of non-verbal children (50% for C-CNVs, 14% for
NC-CNVs, and 36% for W-CNVs).
Discussion
The first aim of the present study was to identify the fre-
quency of CNVs in children with ASD to provide evidences
of the diagnostic yield of array-CGH analysis. Results docu-
mented that 41 patients tested positive for CNVs (30.8%).
Specifically, C-CNVs were identified in 12 participants (9%)
in accordance with previous studies showing C-CNVs in
about 10% of patients with ASD (Schaefer 2016; Schaefer
et al. 2010, 2013). Furthermore, a total number of 50 imbal-
ances were detected: two duplications were classified as
pathogenic (4%), 10 CNVs (6 duplications and 4 deletions)
were included in the unknown but potentially causative rear-
rangements (20%), and 38 CNVs (23 duplications and 15
deletions) were considered as NC-CNVs (76%). Both the
pathogenic CNVs (duplications) involving chromosome
4 and chromosome 15 (4p16.3p15.1 and 15q11.2q13.1)
arose de novo. Child #40 presented a 28.3  Mb duplica-
tion involving the distal region of the short arm of chro-
mosome 4, (4p16.3p15.1). The deletion of 4p16.3 leads to
Wolf-Hirschhorn syndrome, a contiguous gene syndrome
characterized by severe growth retardation and intellectual
disability, microcephaly, ‘Greek helmet’ facies, and closure
defects. On the contrary, the duplication of the same region
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J Autism Dev Disord (2018) 48:442–449
TOTAL C-CNVs NC-CNVs W-CNVs p Value
133 12 29 92
113 10 27 76 0.38a
20 2 2 16
73.7 (24.3) 63.8 (27.2) 73.4 (25.6) 75.1 (23.5) 0.32b
7 (3–10) 7 (6–10) 6 (3–10) 7 (3–10) 0.000003a
90 (68%) 6 (50%) 25 (86%) 59 (64%) 0.00001a
43 (32%) 6 (50%) 4 (14%) 33 (36%)
is not associated with a well-defined phenotype but the size
of the aberration and its gene content suggested a patho-
genic role. Moreover, the above duplication had already
been described in the literature in a patient affected by ASD
(Tabet et al. 2015), and in another patient included in a study
conducted by the International Standards for Cytogenomic
Arrays consortium, on 15,749 individuals with unexplained
developmental delay, intellectual disability, dysmorphic
features, multiple congenital anomalies, ASD, or clinical
features suggestive of a chromosomal syndrome (Kaminsky
et al. 2011). Similarly to these previous results, our patient
with 4p16.3p15.1 duplication showed severe deficits, such
as marked language delay, moderate symptoms of autism
(CSS: 5/10), marked deficits in social interaction. Child
#32 carried a 4.8 Mb microduplication on the long arm of
chromosome 15 (15q11.2q13.1), involving the 15q11-q13
duplication syndrome (OMIM ID #608,636). The deletion
of this chromosomic region causes PraderWilli/Angelman
syndrome, an autosomal dominant condition associated
with severe intellectual disability, developmental delay and
typical dimorphisms. However, the duplication of the same
region is associated with incomplete penetrance and vari-
able phenotypes, which frequently include ASD (Sorte et al.
2013), intellectual disability, ataxia, seizures, developmen-
tal delays, and behavioral problems. This region comprises
several OMIM genes, including GABRB3 (gamma-amin-
obutyric acid type A receptor subunit beta-3), and UBE3A
(ubiquitin protein ligase E3A). GABRB3 gene is involved in
GABAergic neurotransmission, and Buxbaum et al. (2002)
hypothesized that genetic variants within the GABA recep-
tor gene complex in 15q11-q13 may play a role in ASD. As
regards, in a study on animal models, Hogart et al. (2010)
hypothesized that increased gene dosage of Ube3a could
contribute to some manifestations of ASD, such as atypical
social interaction, impaired communication, and augmented
restricted and repetitive behaviors. These results support the
hypothesis that increased UBE3A gene dosage may result
J Autism Dev Disord (2018) 48:442–449 447
in reduced excitatory synaptic transmission, and suggest
that UBE3A gene dosage could contribute to some traits of
ASD in individuals with 15q11-13 duplication (Hogart et al.
2010). Thus, it is plausible that duplication of these genes
(GABRB3, and UBE3A) contributed to ASD symptoms in
child #32. In support of this hypothesis, this child showed
increased restricted and repetitive behaviors with impaired
communication and severe autistic symptoms (CSS: 7/10).
CNVs classified as unknown but potentially causative
have no certain association with a clinical phenotype, but,
based on their gene content and previous data on patients
with ASD in the literature, they were considered as having
a potential role in the pathological phenotype. The dupli-
cations involved the chromosome regions 20p12.3p12.2,
Xp22.33, 7p21.3, Xp11.3, 4q25, while deletions were
detected in 22q13.1, 7q36.1, 18q12.3, 12p13.33. All of
these regions involve one or more genes for which the dos-
age effect is considered as potentially associated with the
clinical phenotype. Two of them, PLCB1 (20p12.3) and
NXPH1 (7p21.3) are implicated in cellular signal transduc-
tion; ASMT (Xp22.33) is involved in melatonin synthesis;
CXORF36 (Xp11.3) plays a role as exocytosis regulator;
APOBEC3A, APOBEC3C (22q13.1), KMT2C (7q36.1), and
KDM5A (12p13.33) are implicated in post-transcriptional
modifications. Details about the function and evidences
in the literature of these candidate genes are reported in
Appendix 1.
For 38 out of 50 CNVs (76%), it was not possible to spec-
ulate whether they may play a role in the autistic phenotype
expression, or they simply represented physiological human
variability without clinical consequences. They have not
been reported in the literature or in public databases neither
as pathogenic nor as benign variants, and they have been
classified as unknown and useless for diagnostic purposes.
CNVs can probably be considered as a genetic component
conferring an increased risk of developing ASD. ASD are
well known to be a set of multifactorial and heterogeneous
diseases in which genetic (CNVs and/or point mutations),
epigenetic and environmental factors play a role. The onset
mechanisms are mostly still unknown and nowadays, they
are the subject of several scientific studies.
The second aim of the present study was to describe the
cognitive profile (intelligence level, ASD severity, language
skills) of children with ASD by identifying different clinical
phenotypes based on the presence or absence of CNVs and
on the specific subtype of CNVs. Considering their demo-
graphic characteristics, our children may be considered as
representative of the general population with ASD. Indeed,
our group showed male–female ratio and prevalence of intel-
lectual disability or developmental delay corresponding to
those reported in previous studies on ASD (Fombonne et al.
2011; Lai et al. 2014; Postorino et al. 2016). Moreover, CSS
obtained by ADOS, indicated that participants had a level of
severity ranging from mild to severe, which corresponds to
the typical clinical heterogeneity of ASD.
No difference was found in IQ or GQ among the three
groups. These data were in contrast with Qiao et al. (2009),
who found increased percentages of C-CNVs in individuals
with associated intellectual disability, and no C-CNVs in
patients without intellectual disability. This result could be
explained by the fact that our study included only partici-
pants with the “essential” phenotype. Our data, however, are
consistent with Merikangas et al. (2015), who did not find
any significant differences in performance IQ or GQ.
Conversely, different percentages of CSS class partici-
pants, and different percentages of non-verbal children were
found among groups. In general, most of the children with
C-CNVs were included in the “autism class”, thus showing
the highest number of ASD symptoms, while the major-
ity of W-CNVs and NC-CNVs children showed less severe
ASD symptoms, and were respectively included in the “ASD
class” and “nonspectrum ADOS class”. As regards language
skills, we found that in the C-CNVs group, the percentage
of non-verbal children was higher than that of the other two
groups. Our results confirmed previous findings demonstrat-
ing that patients with causative CNVs have more deficits
in social engagement, communication and language skills
(Merikangas et al. 2015).
A possible limitation of our study is the relatively small
number of participants. To support and confirm our results,
a larger group of participants should be enrolled in future
studies. Moreover, we were able to conduct the array-CGH
analysis of parental samples on 26 participants (out of 41),
which limited the interpretation of data on the origin and
family history of CNVs. For the other 15 participants, it
was not possible to determine whether CNVs arose de novo.
However, there are some points of strength characterizing
our study: all participating children were diagnosed by a
multidisciplinary team, which allowed to obtain a multidi-
mensional assessment (intelligence level, ASD severity, lan-
guage skills), and included genetic examination to exclude
“complex” physical phenotypes. Furthermore, we used
restricted criteria for CNVs characterization by only con-
sidering aberrations larger than 100 Kb in size and involving
at least one known gene. Lastly, we accurately classified
CNVs according to the likelihood of their association with
the clinical phenotype, based on gene content and data in
the literature.
In conclusion, our results highlight the importance to
conduct array-CGH analysis in individuals with ASD with
“complex” as well as “essential” phenotypes. Considering
the wide heterogeneity of ASD, both in terms of behavioral
and genetic features, we believe it should be recommended
to better categorize and phenotype each individual with this
disorder. Furthermore, our results suggest that the pres-
ence of C-CNVs may have a significant impact on some
13

448
phenotypic features of ASD, such as symptom severity and
presence/absence of language. For this reason, future stud-
ies should necessarily be conducted in tight cooperation
between neuropsychiatry divisions and medical genetics
laboratories.
Acknowledgments  We thank the families that participated in this
study. The authors would like to thank Paola Giovanna Volpi for her
help with the manuscript.
Funding  There was no funding for this paper.
Authors’ Contributions  EN participated the design of the study and
coordination, performed the measurement and drafted the manuscript;
SR conceived of the study, participated in its design and coordination
and drafted the manuscript; LC participated in the design and coordi-
nation of the study, performed the measurement and helped to draft
the manuscript; VA performed the measurement and participated in
interpretation of the data; FAA performed the measurement; AA par-
ticipated the design of the study, revised critically the manuscript and
participated in interpretation of the data; AN participated the design
of the study and interpretation of the data; GV participated the design
of the study and coordination, interpretation of the data and helped
to draft the manuscript; DM participated in the design, interpretation
of the data, performed the statistical analysis and helped to draft the
manuscript; SV participated the design of the study and coordina-
tion, interpretation of the data and helped to draft the manuscript. All
authors read and approved the final manuscript.
Compliance with Ethical Standards  doi:10.1038/nature08516.
Conflict of interest  The authors declare that they have no conflict
of interest.
Ethical Approval  All procedures performed in studies involving
human participants were in accordance with the ethical standards of
the institutional and/or national research committee and with the 1964
Helsinki declaration and its later amendments or comparable ethical
standards.
Informed Consent  Informed consent was obtained from all indi-
vidual participants included in the study.
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