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Battaglia 2013
Battaglia 2013
Original article
Agatino Battaglia a,*, Viola Doccini a, Laura Bernardini b, Antonio Novelli b, Sara Loddo b,
Anna Capalbo b, Tiziana Filippi a, John C. Carey c
a
Stella Maris Clinical Research Institute for Child and Adolescent Neuropsychiatry, via dei Giacinti, 2, 56128 Calambrone, Pisa, Italy
b
Mendel Laboratory, IRCCS Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo (FG), Italy
c
Division of Medical Genetics, Dept. of Pediatrics, University of Utah Health Sciences Center, Salt Lake City, UT, USA
Article history: Background and objectives: Submicroscopic chromosomal rearrangements are the most
Received 30 October 2012 common identifiable causes of intellectual disability and autism spectrum disorders
Received in revised form associated with dysmorphic features. Chromosomal microarray (CMA) can detect copy
28 February 2013 number variants <1 Mb and identifies size and presence of known genes. The aim of this
Accepted 28 April 2013 study was to demonstrate the usefulness of CMA, as a first-tier tool in detecting the eti-
ology of unexplained intellectual disability/autism spectrum disorders (ID/ASDs) associ-
Keywords: ated with dysmorphic features in a large cohort of pediatric patients.
Chromosomal microarray (CMA) Patients and methods: We studied 349 individuals; 223 males, 126 females, aged 5 months-19
Array-CGH years. Blood samples were analyzed with CMA at a resolution ranging from 1 Mb to 40 Kb.
Developmental delay The imbalance was confirmed by FISH or qPCR. We considered copy number variants (CNVs)
Intellectual disability causative if the variant was responsible for a known syndrome, encompassed gene/s of
Neurodevelopmental disorders known function, occurred de novo or, if inherited, the parent was variably affected, and/or the
Autism spectrum disorders involved gene/s had been reported in association with ID/ASDs in dedicated databases.
Dysmorphic features Results: 91 CNVs were detected in 77 (22.06%) patients: 5 (6.49%) of those presenting with
borderline cognitive impairment, 54 (70.13%) with a variable degree of DD/ID, and 18/77
(23.38%) with ID of variable degree and ASDs. 16/77 (20.8%) patients had two different
rearrangements. Deletions exceeded duplications (58 versus 33); 45.05% (41/91) of the
detected CNVs were de novo, 45.05% (41/91) inherited, and 9.9% (9/91) unknown. The CNVs
Abbreviations: ASDs, autism spectrum disorders; CMA, chromosomal microarray; DD, developmental delay; ID, intellectual disability.
5
Whats known on this subject: Recommendations in medical genetics practice call for the use of chromosomal microarray as a first-
tier clinical diagnostic test for individuals with developmental disabilities or congenital anomalies. The recommendations were based
on reports dealing with heterogeneous populations studied with a variety of protocols.
55
What this study adds: After comprehensive phenotypic characterization at one tertiary center including a standardized psycho-
pathological assessment for Developmental Delay/Intellectual Disability/Autism Spectrum Disorders, we detected a high diagnostic
yield of Chromosomal Microarray in children with unexplained Intellectual Disability/Autism Spectrum Disorders/dysmorphic features,
confirming its value as first-tier tool in the pediatric setting.
* Corresponding author. Tel.: 39 (0)50 886248.
E-mail address: agatino.battaglia@inpe.unipi.it (A. Battaglia).
1090-3798/$ e see front matter 2013 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ejpn.2013.04.010
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
2 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
caused the phenotype in 57/77 (74%) patients; 12/57 (21.05%) had ASDs/ID, and 45/57
(78.95%) had DD/ID.
Conclusions: Our study provides further evidence of the high diagnostic yield of CMA for
genetic testing in children with unexplained ID/ASDs who had dysmorphic features. We
confirm the value of CMA as the first-tier tool in the assessment of those conditions in the
pediatric setting.
2013 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights
reserved.
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 3
180 K; Agilent technologies, Waldbronn, Germany) and/or SNP- suggested by Miller et al., [2010]17 and successively by the
array platform (6.0 Chip; Affymetrix, Palo Alto, CA) were used American College of Medical Genetics.23
for CMA analysis; manufacturers suggestions were used for All samples analyzed with SNP-array and negative to CNVs
experiments. Sex-matched pooled DNAs (Promega, Madison, analysis were evaluated for LSH (Long Stretches of Homozy-
WI) were used as reference in BAC- and oligonucleotide-array gosity) scattered along the genome. This analysis can even-
analysis, while the 270 International HapMap Project control tually detect a rate of homozygosity higher than expected as a
samples were used as a reference model in SNP-array marker of ancestral homozygosity or parental consanguinity,
experiments. with an increased risk for recessive diseases, or uniparental
All copy number changes were confirmed by FISH or disomy in relevant imprinted regions. The LSH analysis was
quantitative PCR. FISH analysis was carried out using com- performed as suggested by Kearney and colleagues [2011],24
mercial probes (Vysis Inc., Downers Grove, IL) or BACs clones with minor modifications. The rate of homozygosity was
selected from a genomic library (32 K library; BACPAC Re- calculated as the sum of autosomal LSH >1 Mb in relation with
sources, Oakland, CA). DNA was extracted by Quantum Prep the autosomal genome covered by SNPs. In particular, Gen-
MiniPrep Kit (BioRad, Hercules, CA) and SpectrumGreen-dUTP eChip 6.0 platform covers about 2,781,532 kbp (hg19 release).
or SpectrumOrange-dUTP labeled using the Nick Translation
kit (Vysis Inc.), according to the manufacturers protocol.
Metaphase spreads were obtained following standard pro- 3. Results
cedures and FISH analysis was performed as described in
Bernardini et al., [2007].21 Quantitative-PCR (qPCR) was per- We identified 91 CNVs in 77/349 (22.06%) patients; 53/223
formed using an ABI 7000 Sequence Detection System (23.76%) males and 24/126 (19.04%) females. Five of 77 (6.49%)
(Applied Biosystem, Foster City, CA) and DNA-binding dye patients showed a borderline cognitive impairment, 54/77
SYBR Green (Invitrogen Corporation, Carlsbad, CA) as (70.13%) variable degrees of DD/ID, and. 18 of 77 (23.38%) pa-
described by Carbone et al., [2008].22 Primers were designed by tients had an ASD associated with ID of variable degree (Table
Primer Express 2.0 software (Applied Biosystems) and an 1). A causative CNV was found in 57/77 (74.02%) children, 45
amplified fragment of the TERT locus was used as a reference. (79%) of whom presenting only ID of variable degree, and 12
After the confirmation test and the inheritance assess- (21%) also ASDs (Table 2). Sixteen of 77 (20.78%) patients had
ment, CNVs were classified as pathogenic, benign or with an two chromosome rearrangements (3 of whom with ASDs/ID),
unknown clinical significance based on the guidelines in 10 of them on different chromosomes (Table 3aec).
Table 1 e Flow diagram showing the number of patients studied and the number and characteristics of the detected CNVs.
Males: 4/21 Female: 1/13 Males: 34/142 Females: 20/95 Males: 15/60 Females: 3/18
(19%) (7,69%) (23.94%) (21.05%) (25%) (16.67%)
In 1 (33.3%) CNV the origin was unknown In 4 (7.8%) CNVs the origin was unknown In 2 (18.2%) CNVs the origin was unknown
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
4 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
Table 2 e Percentage of borderline ID, mixed ID, and ID/ASDs patients in whom different CNVs categories were found.
CNVs causative CNVs probably CNVs of uncertain CNVs non-causative of the
of the phenotipe causative of the significance phenotipe
phenotipe
Borderline ID: 5 patients 2/5 (40%) 1/5 (20%) 0/5 (0.0%) 2/5 (40%)
Mixed ID: 54 patients 43/54 (79.63%) 2/54 (3.70%) 3/54 (5.56%) 6/54 (11.11%)
ID and ASDs: 18 patients 12/18 (66.67%) 3/18 (16.66%) 2/18 (11.11%) 1/18 (5.56%)
In 8/77 (10.38%) patients the rearrangement involved a sex defined by a high-resolution technology in a CNV database for
chromosome. In 5/77 (6.49%) patients with an abnormal patients with DD/ID/ASDs/dysmorphic features, contained
standard karyotype/FISH or FRAXA-E, CMA was pursued morbid OMIM genes, was gene rich, was a deletion, was a
because the clinical phenotype did not correlate well with the homozygous deletion (Table 3aec). The 8.8% (8/91) (Table 4)
reported chromosome aberration (47,XYY; del 1p36; del 1q43- was considered probably causative because the CNVs,
qter; FRAXA premutation allele, Robertsonian 13; 14 trans- although paternally or maternally inherited, encompassed
location at amniocentesis) (Table 3aec). genes, such as CNTNAP2 (involved in synapse formation and
Of the 91 detected CNVs, 65/91 (71.4%) were classified as maintenance),26 SOS2 (possible RAS activator)27,28 in patient 45
causative; 13/91 (14.3%) non causative; 5/91 (5.5%) uncertain; with a phenotype reminiscent of a Noonan-CFC syndrome, or
and 8/91 (8.8%) probably causative (Tables 1 and 4). Fifty-eight had already been associated with ID. The 5.5% (5/91) (Table 4)
were deletions and 33 duplications. was considered uncertain because the CNVs, although
The percentage for the different CNVs categories found in parentally segregated and being either duplications (pat. 78;
DD/ID patients versus DD/ID/ASDs ones are reported in Table 4. Table 3), or deletions (pat. 32, 33; Table 3), encompassed genes
Forty five and zero five percent (41/91) of the identified involved in synapse and dendritic spine formation, such as
CNVs were inherited; 45.05% (41/91) de novo; while in 9.9% (9/ NLGN4X (associated with ASDs),29 and IL1RAPL2 (located,
91) the origin was unknown. Twenty (48.8%) of the inherited together with IL1RAPL1, to a region on the X chromosome,
CNVs were deletions, and 21 (51.2%) duplications. Thirty-two associated with X-linked non-syndromic MR)30 (Table 3aec).
(78.1%) of the de novo CNVs were deletions, and 9 (21.9%) du- The 14.3% (13/91) (Table 4) was considered non-causative
plications. Seventeen of forty-one (41.5%) inherited CNVs because the CNV was identical to that inherited from a
were causative (Table 1). healthy parent, was completely contained within genomic
Thirty-seven of 91 CNVs (40.6%) were <1 Mb in size, 28/91 imbalance defined by a high-resolution technology in a CNV
(30.7%) were 1e5 Mb, 12/91 (13.2%) were 5e10 Mb and 14/91 database of healthy individuals, was gene poor, was a dupli-
(15.5%) were >10 Mb. 7/37 CNVs < 1 Mb in size were de novo; 10 cation containing not known dosage-sensitive genes, was
of the 14 CNVs > 10 Mb in size were de novo. devoid of known regulatory elements.17,23 The data related to
The largest CNV identified in our study sized 21 Mb, while parentally segregated CNVs should be interpreted with
the smallest was 92 Kb. Most CNVs were detected by CMA at caution, because such imbalances could also contribute to the
250 Kb resolution. probands phenotype through variable penetrance or expres-
The evaluation of LSH in the 20 patients evaluated by SNP- sivity, or both; through epigenetic effects, or by uncovering a
array and negative to CNVs analysis disclosed in one sample a recessive mutation on the non-deleted allele.
rate of homozygosity of 25%, indicating a first-degree parental As previously reported,31 we observed a greater number of
relationship. All other patients displayed an average rate of deletions than duplications (58 versus 33). This can have a
2.6%, consistent with the expected total length of LSH of the dual explanation, both technical and biological. Technically,
general population.25 there is a greater chance that some duplications may be
missed. Biologically, duplications generally cause a milder
phenotype, possibly leading to a selection bias. In addition,
4. Discussion the frequency of random duplications in the human genome
may be lower then that of deletions.32
We identified chromosomal rearrangements in 22.06% of our Sixteen patients had two different chromosome rear-
patients; this figure is in keeping with literature data.17 rangements, in 10 of whom on different chromosomes (Table
Although the study sample was represented by more males 3aec). This could explain the complexity of their clinical pic-
than females, the percentage with causative CNVs was almost ture, and is in keeping with the two-hit model, in which one
the same in the two sex groups, suggesting that other sex- CNV predisposes to neuropsychiatric phenotypes as a single
linked factors, could contribute to the higher male frequency event and exacerbates neuro-developmental phenotypes in
amongst DD/ID/ASDs individuals. association with the second one.33
The 71.4% (65/91) (Table 4) of aberrations was considered In some cases the identified anomalies involved known genes
causative to the phenotype, because in each situation the CNV (Table 3aec), some of these related to well defined neuropsychi-
was de novo, responsible for a known syndrome, expanded or atric disorders such as RAI1 (SmitheMagenis syndrome), NF1
altered a CNV inherited from a parent, was identical to a CNV (neurofibromatosis 1), EDNRB (Waardenburg syndrome).
inherited from an affected parent, was similar to a CNV in an Although the diagnostic yield among the patients with
affected relative, was a CNV overlapping a genomic imbalance severe-profound ID would be expected to be higher than
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 5
Table 3 e a, b, c. Summary of the clinical and molecular cytogenetic data of 77 individuals with CNVs.
Case Sex Age at Previously normal Previously Severity of Epilepsy Autism Dysmorphic
diagnosis karyotype/fish normal DD/ID features
fraxa/E
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
6 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
Table 3 e (continued )
Case Sex Age at Previously normal Previously Severity of Epilepsy Autism Dysmorphic
diagnosis karyotype/fish normal DD/ID features
fraxa/E
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 7
Table 3 e (continued )
Case Method and CNV Size Origin/
effective resolution (ISCN 2009)a segregation
array-CGH
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
8 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
Table 3 e (continued )
Case Genes OMIM
morbid
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 9
Table 3 e (continued )
Case Genes OMIM
morbid
Legend yrs: years; ms: months; segr.: segregation; N.i.: no information available; N.D.: not determined; o-array: oligonucleotide-array.
a Based on Release hg18.
b Patients n. 3 and 4 are dyzigotic twins; patients n. 32 and 33 are brothers; a: premutation allele at the FRAXA locus (61-62 triplets).
c Chromosome Y genes were not considered as pathogenic in our study.
among those with borderline/mild/moderate ID6 we observed and the standardized psychopathological assessment for
a higher yield in the latter group (65/77 versus 12/77). DD/ID/ASDs.
Our study results are inherently limited by the different Our study provides further evidence of the high diagnostic
CMA platforms used and the varying knowledge of the yield of CMA for genetic testing of individuals with unex-
parental phenotype. There are, however, several strengths plained DD/ID/ASDs and dysmorphic features, confirming its
represented by the comprehensive phenotypic character- first-tier usefulness in such conditions. This technology ap-
ization by a single pair of clinicians (A.B. and J.C.C.) at a pears to be capable of finding the cause of DD/ID/ASDs in a
single tertiary center (Stella Maris Clinical Research Insti- much greater number of affected individuals (w23%) as is
tute), the prior exhaustive evaluation including high reso- conventional cytogenetic analysis (w3%, excluding Down
lution banding, molecular analysis at the FRAXA/E loci, syndrome and other recognizable chromosomal syndromes).17
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
10 e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1
Table 4 e Percentage for the different CNVs categories found in the entire sample, in the borderline ID, in the mixed ID, and
in the DD/ID/ASDs patients.
Entire sample Borderline ID Mixed ID ID/ASDs patients
Causative CNVs 65/91 (71.4%) 3/7 (42.8%) 51/64 (79.69%) 11/20 (55%)
Probably causative CNVs 8/91 (8.8%) 2/7 (28.6%) 2/64 (3.11%) 4/20 (20%)
Uncertain CNVs 5/91 (5.5%) 0/7 (0.0%) 3/64 (4.7%) 2/20 (10%)
Non causative CNVs 13/91 (14.3%) 2/7 (28.6%) 8/64 (12.5%) 3/20 (15%)
High resolution screening using microarray not only detects Laura Bernardini PhD: Dr. Bernardini carried out the CMA,
submicroscopic chromosomal imbalances, but also allows ac- confirmed its results, revised the manuscript, and approved
curate delineation of the duplicated or deleted chromosomal the final manuscript as submitted.
segment. This is crucial for phenotypeegenotype correlation Antonio Novelli PhD: Dr. Novelli coordinated and super-
and for identifying candidate genes involved in the develop- vised data collection at the Mendel institute, revised the
ment of certain anomalies and of ID/ASDs. manuscript, and approved the final manuscript as submitted.
The additional CNVs detected in 5/77 (6.49%) patients with Sara Loddo PhD: Dr. Loddo carried out the CMA, confirmed
an already known chromosomal disorder, support the rele- its results, revised the manuscript, and approved the final
vance of the clinical observation even in the era of CMA. manuscript as submitted.
We were able to analyze only a small group of patients with Anna Capalbo PhD: Dr. Capalbo carried out the CMA,
SNP-array platform, due to its very recent introduction as a confirmed its results, revised the manuscript, and approved
clinical tool. Anyway, the opportunity to evaluate the rate of the final manuscript as submitted.
homozigosity throughout the genome appears promising to Tiziana Filippi MD: Dr. Filippi carried out the initial anal-
unmask recessive form of ID/ASDs and identify new AR dis- ysis, revised the manuscript, and approved the final manu-
ease-genes. script as submitted.
John C. Carey MD: Dr. Carey contributed to design the
study, critically reviewed the manuscript, and approved the
5. Conclusions final manuscript as submitted.
Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010
e u r o p e a n j o u r n a l o f p a e d i a t r i c n e u r o l o g y x x x ( 2 0 1 3 ) 1 e1 1 11
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Please cite this article in press as: Battaglia A, et al., Confirmation of chromosomal microarray as a first-tier clinical diagnostic
test for individuals with developmental delay, intellectual disability, autism spectrum disorders and dysmorphic features,
European Journal of Paediatric Neurology (2013), http://dx.doi.org/10.1016/j.ejpn.2013.04.010