alexa A Genome Wide Copy Number Variations Analysis in Autism Spectrum Disorder (Asd) and Intellectual Disability (Id) in Italian Families | OMICS International
ISSN: 2157-7412
Journal of Genetic Syndromes & Gene Therapy

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A Genome Wide Copy Number Variations Analysis in Autism Spectrum Disorder (Asd) and Intellectual Disability (Id) in Italian Families

Mucciolo Mafalda1, Chiara Di Marco1,3, Roberto Canitano2, Sabrina Buoni2, Elisa Frullanti1, Maria Antonietta Mencarelli13, Bizzarri Veronica1, Sonia Amabile1, Lucia Radice2, Margherita Baldassarri1,3, Caterina Lo Rizzo13, Ilaria Meloni1, Joussef Hayek2, Alessandra Renieri1,3 and Francesca Mari1,3

1Medical Genetics-Department of Biotechnology, University of Siena-Policlinico S. Maria alle Scotte, 53100 Siena, Italy

2Department of Child Neuropsychiatry, University Hospital of Siena, 53100 Siena, Italy

3Department of Medical Genetics, Azienda Ospedaliera Universitaria Senese, Siena, Italy

*Corresponding Author:
Alessandra Renieri, MD, PhD
Department of Medical Biotechnology
Medical Genetics Unit University of Siena Viale Bracci
253100 – Siena, Italy
Tel: +39 0577 233303
Fax: +39 0577 233325
E-mail: [email protected]

Received date August 08, 2016; Accepted date September 10, 2016; Published date September 19, 2016

Citation: Mafalda M, Marco CD, Canitano R, Buoni S, Frullanti E, et al. (2016) A Genome Wide Copy Number Variations Analysis in Autism Spectrum Disorder (ASD) and Intellectual Disability (ID) in Italian Families. J Genet Syndr Gene Ther 7:307. doi: 10.4172/2157-7412.1000307

Copyright: © 2016 Mafalda M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Background: Autism Spectrum Disorders (ASD) and Intellectual Disability (ID) represent lifelong conditions with severe impact on behavior and lifestyle of patients and their families. Array comparative genomic hybridization (array-CGH) has clarified the underlying genetic causes of ASD and ID by CNVs identification in several chromosomal regions with susceptibility to different levels of severity of ASD or ID in up to 1% of patients. Methods: Using oligo array-CGH we analyzed 476 unrelated subjects with ASD or ID, thoroughly investigated by both child neuropsychiatrists and clinical geneticists. The inheritance of the CNV were tested in the majority of cases (82% of positive cases). Results: A total of 198 rearrangements was identified in 154 cases. CNVs were classified in three groups: i- CNVs previously known to be associated with ASD or ID (28/198, 14%), including 16p11.2, 15q13.3, 17p12 and 17q12; ii- CNVs including genes known to be associated with either ASD or ID (9/198, 4.5%); iii- CNVs of unknown significance (161/198, 81.3%). Conclusions: Our study confirmed that array-CGH analysis is able to detect the underlying genetic cause in about 18% of ASD or ID patients, highlighting it as an essential diagnostic tool for patients assessment. Overall, a prevalence of duplications with respect to deletions was observed (62% and 38% respectively) but among the deleted cases an enrichment of microdeletions in ASD cases (p=0.03) is present. Furthermore, we shown a prevalence of multiple CNVs in ASD cases compared to ID (p=0.05), pointing out the complex nature of ASD.


Array-CGH; CNVs; Autism spectrum disorders; Intellectual disability; Genomic disorders


ASD: Autism Spectrum Disorders; ID: Intellectual Disability; DSM-IV: Diagnostic and Statistical Manual 4th Edition; ADI-R: Autism Diagnostic Interview-Revised; ADOS-G: Autism Diagnostic Observation Schedule Generic; Array-CGH: Array Comparative Genomic Hybridization; CNV: Copy Number Variation


Autism Spectrum Disorders (ASDs) and intellectual disability (ID) are the most common development disorders in humans representing an important health burden in the population [1]. ASDs are a spectrum of psychological conditions characterized by impairments in communication, dysfunctional reciprocal social interaction and the presence of restricted, repetitive and stereotyped patterns of behavior. In addition to Autism, ASDs include Asperger syndrome and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) [2]. ASDs present complex and heterogeneous etiology with a strong evidence of genetic involvement placing it among the most heritable neurodevelopmental disorders. ID is a condition characterized by below average intellectual functioning (IQ<70) together with significant limitation in both intellectual and adaptive functioning [3]. ASD is associated in 70% of individuals with intellectual disability. To date, no single gene has been shown to account for a majority of ASD or ID susceptibility [4]. The complex heterogeneity of both conditions, possibly resulting from the interaction of several genes and environmental factors, makes the identification of contributory genes extremely difficult.

Comparative genomic hybridization (CGH) technology has been widely used in research studies and in clinical practice of ASDs in order to detect copy number variants (CNVs) throughout the genome. CNVs represent a significant source of genetic variability and are responsible of disease susceptibility for several neurobehavioral phenotypes. Several studies revealed clinical relevant CNVs detection rate variable from a 10–20% [5,6] depending on the resolution of the applied array platform and by clinical selection of patients on the basis of family history and associated anomalies. Overall, array-CGH analysis increased the diagnostic yield in ASDs and ID, allowing the identification of new genetic causes. In addition to the well-known recurrent pathogenic rearrangements, several new microdeletions and microduplications have been identified in ASDs and ID patients as potentially pathogenic [5-7].

Here, we report the results of array-CGH analysis performed on a panel of 476 ASD or ID patients for which accurate clinical assessment and genetic counseling were performed.


A cohort of 476 unrelated patients referring to the Medical Genetics Unit of Siena (Italy) and classified as Autism Spectrum Disorder (ASD) (267/476; 56%) or intellectual disability without ASD (ID ) (206/476; 44%), was selected for this study. Among these, 333 were males and 143 were females (M:F=2.3:1). Considering the 2 classes, the M:F ratio is 3.8:1 for the ASD group, in accordance with literature data, and 1.3:1 for the ID group. A cognitive evaluation for all subjects was carried out by the team of the Neuropsychiatric Unit of Siena based on standardized Diagnostic and Statistical Manual 4th Edition (DSM-IV) criteria using Autism Diagnostic Interview-Revised (ADI-R) and/or Autism Diagnostic Observation Schedule Generic (ADOS-G) standards. The patients were therefore classified based on the severity of intellectual disability: mild, moderate, severe. The patients were classified based on the severity of intellectual disability. Among the ASD group, 141 out of 267 cases also presented ID and were classified as follows: 34 patients were severe cases, 71 were moderate and 36 were mild. In ID group, the majority of patients were moderate or mild (60 and 50 respectively) and 24 were severe (Table 1).

All patients also underwent a comprehensive evaluation by a clinical geneticist (FM or MAM) who excluded a diagnosis of a recognizable syndrome. All families had genetic counseling and biological samples of parents were available in the majority of cases (82%). All patients, except those with microcephaly, have been tested for FMR1 gene expansion and resulted negative.

Biological samples of these patients and their parents, whenever available, were collected after obtainment of informed consent by patients’ guardians/parents and stored in the “Cell Lines and DNA Bank of Rett syndrome, X linked mental retardation and other genetic diseases”, member of the Telethon Network of Genetic Biobanks.

Array-CGH analysis

Genomic DNA of the patients was isolated from an EDTA peripheral blood sample using the QIAamp DNA Blood Kit according to the manufacturer’s protocol (Qiagen, Valencia, CA, USA). Genomic DNA of normal male and female controls was obtained from Agilent (Agilent Technologies, Santa Clara, CA, USA). One microgram of genomic DNA from the patient (test sample) and the control (reference sample) were labeled with Cy5 and Cy3 fluorocrome, respectively.

Array-CGH analysis was performed using commercially available oligonucleotide microarrays containing about 60.000 60-mer probes (Human Genome CGH Microarray 60 K Kit, Agilent Technologies, Santa Clara, CA, USA) according to manufacturer’s standard protocol. The median functional resolution was 45 Kb. Copy number variations (CNVs) were considered significant if they were defined by three or more oligonucleotides, and were not present in the Database of Genomic Variants ( Confirmation of results was performed by a second independent experiment. Segregation analysis of the identified rearrangements was performed with the same technique. Map positions are based on hg19 GRCh build 37 (Feb 2009) and listed based on ISCN 2013.


CNVs discovery

Our cohort included 476 patients with ASD (56%) or ID without ASD (44%) (Table 1).

Among the 476 patients, 154 exhibited at least one rearrangement. In these 154 patients, a total of 198 rearrangements were identified: 76 deletions and 122 duplications. CNVs were classified in three groups: i) CNVs (28/198, 14%) previously known to be associated with ASD or ID, including 16p11.2, 15q13.3, 17p12 and 17q12 (Table 2); ii) CNVs (9/198, 4.5%) including genes known to be associated with either ASD or ID (Table 3); iii) CNVs (161/198, 81.3%) of unknown significance (Table 4 and Figure 1). The size of rearrangements ranged from 1 Kb to 13 Mb with a mean size of 868 Kb. Observing the size of the CNVs in the three classification groups we noted a prevalence of large CNVs in group i) and ii) for both ASD and ID cases. In the group of variant of unknown significance we observed instead a prevalence of small CNVs (Figure 2).

Out of 154 patients, 122 (79.2%) patients had only one rearrangement and 32 (20.8%) had two or more rearrangements (four in one case) (Table 2). Among the 32 patients with more than one rearrangement, 21 (65.6%) were classified as ASD, while the remaining 11 (34.4%) were in the ID group (Figure 3). The data show a prevalence of multiple CNVs in ASD cases compared with ID cases (p=0.05). In addition, we noted that the deletions are more represented in ASD than in ID patients (49 and 27, respectively), while the duplications are similarly represented in ASD and ID (65 and 58 respectively) (Table 2 and Figure 4). These results suggest a stronger association between the presence of a microdeletion and the ASD phenotype (p=0.03).

  ASD n=267 (56)  ID n=209 (44) 
  Positive n=83 (31) Negative n=184 (69) Tot n=267 Positive n=71 (34) Negative n=138 (66) Tot n=209
Male 66 (79.5) 146 (79.4) 212 (79.4) 40 (56.3) 81 (58.7) 121 (57.9)
Female 17 (20.5) 38 (20.6) 55 (20.6) 31 (43.7) 57 (41.3) 88 (42.1)
Cognitive impairment            
Severe 11 (13.2) 23 (12.5) 34 (12.7) 8 (11.3) 16 (11.6) 24 (11.5)
Moderate 17 (20.5) 54 (29.3) 71 (26.6) 17 (23.9) 43 (31.1) 60 (28.7)
Mild 10 (12.0) 26 (14.1) 36 (13.5) 19 (27.8) 31 (22.5) 50 (23.9)
n.a. 45 (54.2) 81 (44) 126 (47.2) 27 (38) 48 (34.8) 75 (35.9)
EEG and/or MRI abnormality            
present 17 (20.5) 39 (21.2) 56 (21.0) 28 (39.4) 59 (42.7) 87 (41.6)
absent 12 (14.5) 32 (17.4) 44 (16.5) 13 (18.3) 19 (13.8) 32 (15.3)
n.a. 54 (65) 113 (61.4) 167 (62.5) 30 (42.2) 60 (43.5) 90 (43.1)

Table 1: Characteristics of samples.

ID # Sex Phenotype Chr Cytoband CNV start CNV stop CNV Size Inheritance References
2476 M ASD 1 1q21.1 145,632,334 145,747,214 Loss 115 Kb Mat Pinto et al. [5]
326* M ASD 1 1q21.1 145,632,334 145,747,214 Loss 115 Kb Mat Pinto et al. [5]
2117 M ID 1 1q21.1 145,632,334 145,747,214 Loss 115 Kb Mat Pinto et al. [5]
180 M ASD 2 2q31.1q33.2 180,306,799 193,335,172 Loss 13 Mb De novo Cocchella et al. [33]
168 M ID 3 3p14.1p14.3 54,452,525 65,609,348 Loss 11 Mb De novo Okumura et al. [36], De la Hoz et al. [37]
387 F ID 4 4p16.3 51,413 2,079,430 Loss 2,03 Mb De novo Battaglia et al. [40]
762 M ASD 7 7q11.23 72,420,745 74,139,390 Gain 1,72 Mb De novo Velleman and Mervis [41]
803 F ID 9 9q31.1q32 107,970,048 114,341,159 Loss 6.37 Mb De novo Mucciolo et al. [42]
952 F ID 14 14q32.31 101,697,865 107,258,824 Loss 5,6 Mb De novo Engels et al. [43]
2720 M ASD 15 15q11.2q13 20,849,110 28,525,401 Gain 7,7 Mb De novo Thomas et al. [26]
174 M ID 15 15q11.2q13 23,739,358 28,525,460 Loss 4,8 Mb Unknown Thomas et al. [26]
221  M ASD 15 15q13.3 32,021,733 32,510,804 Loss 489 kb Unknown Bacchelli et al. [27]
846 M ID 15 15q13.3 32,021,733 32,438,943 Gain 417 kb Mat Szafranski et al. [28]
278 F ASD 15 15q26.3 98,612,748 101,320,461 Gain 2,7 Mb Pat Tatton-Brown et al. [44]
1537 M ASD 16 16p13.11 14,944,560 15,960,084 Gain 1 Mb Mat Ullmann et al. [45]
1383 F ID 16 16p13.11 14,944,560 16,305,677 Gain 1,36 Mb Pat Ullmann et al. [45]
7* M ASD 16 16p11.2 29,673,954 30,198,553 Loss 524 kb De novo Jacquemont et al. [25]
518 M ASD 16 16p11.2 29,673,954 30,197,341 Loss 523 kb De novo Jacquemont et al. [25]
 41 M ID 16 16p11.2 29,673,954 30,119,712 Loss 446 kb Unknown Jacquemont et al. [25]
1573 M ASD 16 16p11.2 29,673,954 30,198,553 Loss 525 kb Pat Jacquemont et al. [25]
778 M ID 16 16p11.2 29,673,954 30,198,600 Gain 525 kb Mat Jacquemont et al. [25]
936 F ASD 17 17p11.2 16,603,130 20,434,018 Gain 3,8 Mb De novo Potocki et al. [46]
27 F ID 17 17p11.2 16,892,401 20,193,196 Gain 3,3 Mb De novo Potocki et al. [46]
1 M ASD 17 17q12 34,851,537 36,473,234 Gain 1,62Mb Pat Nagamani et al. [47]
1129 M ASD 22 22q11.2 18,896,972 21,379,958 Gain 2,48 Mb Mat Wentzel et al. [48]
66* M ASD X Xp22.31 7,555,292 8,266,181 Gain 710 kb   Mat Esplin et al. [49]
1046 F ID X Xp22.31 6,457,403 8,266,240 Gain 1,80 Mb Pat Esplin et al. [49]
656 F ID X Xq28 152,764,591 154,841,455 Gain 2,08 Mb De novo Bijlsma et al. [50]

Table 2: CNVs affecting known deleterious regions.

ID # Sex Phenotype Chr Cytoband CNV start CNV stop CNV Size Inheritance Genes References
1070 * M ID 2 2q13 110,841,715 110,980,342 Gain 138 Kb Pat NPHP1 Yasuda et al. [8]
1740 M ASD 7 7q32.3q33 131,948,767 133,002,068 Gain 1,05 Mb De novo PLXNA4 Suda et al. [9]
1302 * M ASD 9 9p24.3 611,628 762,947 Gain 151 kb Pat KANK1 Vanzo et al. [10]
302 M ID 9 9q22.32 97,843,040 98,659,815 Gain 817 kb Mat PTCH1 Izumi et al. [11]
1986 * F ID 12 12p12.1p12.2 20,038,565 25,826,850 Loss 5,8 Mb         De novo SOX5 Lee et al. [12]
2256 F ASD 16 16q24.2 87,340,135 87,420,919 Gain 80 kb Pat FBXO31 Handrigan et al. [13]
681 M ASD 20 20p12.1 14,824,372 15,268,002 Loss 443 kb Mat MACROD2 Jones et al. [14]
36 M ID X Xp22.11 22,836,324 23,411,163 Loss 575 kb Mat PTCHD1 Chaudhry et al. [17]
1420 M ID Xp11.22
432 kb; 649 kb Mat
Fieremans et al. [15], Matsumoto et al. [16]

Table 3: CNVs affecting known deleterious regions.

CNVs affecting known deleterious regions

To identify specific CNVs which may contribute to ASD or ID phenotype, we first looked for CNVs in well-known ASD/ID associated region (Table 3). Among the validated CNVs, we identified a common hotspot at 16p11.2 (four deletions and one duplication), whose pathogenicity in ASD has been longtime established. In order of frequency in our cohort, we found three maternally inherited deletion in 1q21, two 15q13.3 duplication, two duplications in 17p11.2, two Xp22.31 duplication, a deletion and a duplication in 16p13.11 and a deletion and a duplication in 15q11.2q13. Additional CNVs occurring in single cases are listed in Table 2.

The 46.4% of these diseases associated CNVs are inherited (28.6% maternal and 17.8% paternal) while the 42.8% occurring de novo.

CNVs affecting ASD/ID associated genes

We evaluated our data looking also for CNVs involving genes already known to be involved in ASD or ID (Table 4). We identified the following rearrangements: a duplication of NPHP1 gene in 2q13 [8]; a de novo 7q32.3 gain involving PLXNA4 [9]; a 9p24.3 duplication involving KANK1 gene [10]; a duplication of PTCH1 in 9q22 [11]; a 5.8 Mb deletion on 12p12.2p12.1 including SOX5 [12]; a duplication of the 16q24.2 region [13]; an inherited 20p12.1 deletion interrupting the MACROD2 gene [14]; a Xp11.22 duplication involving KDM5C and IQSEC2 [15]; a duplication in Xp22.12 including the RPS6KA3 gene [16]; and the deletion of PTCHD1 in Xp22.11 [17].

The 72.7% of these CNVs are inherited (45.5% maternal and 27.2% paternal) while the 18.2% occurring de novo.

ID # Sex Phenotype Chr Cytoband CNV start CNV stop CNV Size Inheritance
1151 M ASD 1 1p31.1 78,470,596 79,383,315 Gain 912 kb Pat
853 M ASD 1 1q43 236,631,519 236,748,164 Gain 117 kb Pat
2327 M ID 1 1q44 247,136,811 247,348,787 Gain 212 kb Unknown
445 M ASD 2 2q36.3 229930692 230824760 Gain 894 kb Unknown
460 M ASD 2
72 kb                    121 kb Pat
2449 F ID 2 2q14.2 120,126,884 120,567,392 Gain 440 kb Unknown
122 F ID 2 2q14.2 120126884 120567451 Gain 440 kb Pat
364 M ASD 3
2,2 Mb                 629 kb Mat
314 M ASD 3
60 kb                    4,4 Mb                339 kb Mat
2329 F ASD 3 3q25.33q26.1 160,576,359 160,734,451 Loss 158 kb Pat
1519 M ASD 3 3p25.3 11,296,962 11,301,588 Gain 4,6 kb Pat
1757 F ID 3 3q11.2 94,798,716 95,275,664 Gain 477 kb Mat
2868 M ID 3
173 kb             94 kb              303 kb Mat
318 M ASD 4 4p16.3 72,447 113,524 Gain 41 kb Pat
159 F ASD 4 4q12 53,483,283 54,424,231 Loss 941 kb Mat
100 M ASD 4
1,29 Mb         94 kb Pat
886 M ASD 4
212 kb            73 kb Mat
224 M ASD 4 4p16.1 10,077,404 10,141,989 Gain 65 kb Pat
1842 M ID 4 4p16.3 72,447 113,524 Gain 41 kb Pat
197 M ASD 5 5q21.1 100,191,817 100,373,993 Gain 182 kb Pat
352 F ASD 5 5q11.2 56,471,611 56,538,065 Loss 66 kb De novo
1621 M ASD 5 5q12.3 64,085,869 64,217,093 Loss 131 kb Mat
1734 M ASD 5 5q15 93,728,464 93,918,134 Loss 190 kb Mat
1877 M ID 5 5p15.33 435,961 548,072 Gain 112 kb De novo
2798 M ID 5 5q23.1 115,551,734 115,628,469 Gain 76 kb Unknown
1302** M ASD 6 6q14.1 82,840,207 83,335,748 Gain 495 kb Pat
283   ASD 6 6q27 168,343,841 168,776,873 Gain 433 kb Pat
420 F ASD 6 6q27 170,228,674 170,890,108 Loss 661 kb De novo
1494 F ID 6p11.2 57,467,120 58,014,473 Gain 547 kb Pat
7* M ASD 6 6q22.31 123,539,625 124,166,602 Gain Mat 627 kb
2658 M ASD 7 7q21.13 89,621,308 89,626,894 Loss 5,6 kb Mat
2553 M ASD 7 7q21.13 88,956,630 89,445,171 Gain 488 kb Unknown
274 F ASD 7 7q34 142,429,334 142,487,095 Gain 58 kb De novo
41 M ASD 7
108 kb            36 kb              147 kb Mat
57 M ID 7
93 kb                    125 kb Mat
303 M ASD 8 8p23.1 9,895,739 9,992,928 Loss 97 kb Pat
857 F ID 8 8q12.1 56,428,093 56,922,601 Gain 494 kb Unknown
183 M ASD 9 9p13.2p13.1 37,857,269 38,050,719 Loss 193 kb Mat
1022 M ASD 9 9p24.1 6,328,511 6,331,140 Loss 2 kb Mat
1704 M ASD 9 9p22.1 18,882,921 19,047,891 Gain 165 kb Pat
1082 F ID 9 9q22.33 99,795,072 99,799,469 Gain 4,4 kb Mat
1986** F ID 10
46 kb
86 kb
235 M ASD 10 10q21.1 60,274,699 61,008,293 Gain 733 kb Mat
193 F ASD 10 10q24.2 99,379,380 99,508,437 Gain 129 kb Pat
2759 M ID 10 10q22.2 75,004,669 75,010,521 Gain 6 kb Pat
1560 M ASD 11
48 kb
678 kb
1071 M ID 11 11p11.2 44,151,640 44,228,368 Gain 77 kb Pat
452 F ID 11 11p15.2 12,848,840 12,923,522 Loss 75 kb Mat
1157 M ID 11 11q14.2 86,317,846 86,374,738 Gain 569 kb Unknown
815 M ASD 12
390 kb        494 kb Mat
405 M ASD 12 12q23.2 102,541,996 102,591,550 Loss 49 kb Pat
1650 M ASD 12 12q24.33 132,421,843 132,564,931 Gain 143 kb Pat
2173   ASD 12
1,13 Mb
76 kb        
De novo
2818 M ASD 12 12q24.33 133,589,041 133,767,927 Loss 179 kb Pat
2292 F ID 12  12q24.21 114,336,264 114,397,847 Gain 62 kb Mat
1061 F ID 13 13q12.11 20,797,139 21,059,910 Gain 260 kb Unknown
598 M ID 13 13q13.2 35,619,499 36,124,728 Gain 505 kb Unknown
1 F ID 13 13q21.33 73,255,663 73,330,259 Gain 75 kb Unknown
1061 F ID 13 13q12.11 20,797,139 21,059,910 Gain 260 kb Unknown
1871 M ASD 14 14q21.1 41,067,302 41,374,628 Gain 307kb Pat
384 M ASD 14 14q21.1 41,018,728 41,310,931 Loss 292 kb Pat
798 M ASD 15 15q15.3 43,888,927 44,043,043 Loss 154 kb Pat
1070** M ID 16 16p13.11 15,131,723 15,154,746 Loss 23 kb Mat
1536 M   ASD 16
99,7 kb                 19 kb De novo
2260 M ASD 16 16p13.3 2,636,803 3,187,007 Gain 550 kb Pat
613 F ID 16 16p11.2 28335078 28601225 Gain 266 kb Unknown
865 F ID 16 16p12.2 21,599,687 21,837,551 Loss 238 kb Pat
1594 M ID 16 16p13.2 8,846,009 8,958,889 Gain 113 kb Pat
1041 M ID 16 16p13.2p13.3 6,427,764 6,755,045 Loss 327 kb Unknown
1900 F ID 16 16q23.2 80,518,009 80,913,149 Gain 395 kb Unknown
345 F ID 16
286 kb          149 kb Unknown
807 M ASD 17
51 kb 2,25 Mb         1,59 Mb   731 kb  Unknown
1501 M ASD 17
51 kb            296 kb Mat
2840 M ASD 17 17q23.3 61,947,138 61,998,256 Loss 51 kb De novo
2073 M ID 17 17p11.2 21,320,065 21,501,883 Gain 182 kb De novo
1315 F ID 17 17q24.3q25.1 70,879,778 71,425,811 Loss 546 kb Mat
1511 M ID 17
350 kb
1,07 Mb
66 * M ASD 17 17p13.3 1,009,242 1,184,475 Gain 175 kb Mat
326 * M ASD 19
72,5 kb                 227 kb               Unknown
1035 F ASD 19 19q13.42 54,754,462 54,845,360 Gain 91 kb Pat
8 F ID 19 19p13.3 2,890,901 2,918,258 Loss 27 kb Mat
1614 M ID 19 19q13.42 53,882,840 53,954,264 Loss 71 kb Mat
277 M ASD 21 21q21.3 29,912,404 30,026,553 Loss 114 kb Pat
1778 F ASD 21 21q21.2 24,493,619 24,622,114 Loss 128 kb Pat
1839 M ID 21 21q22.3 45,673,257 45,835,771 Gain 162 kb Pat
1090 M ID 21 21q22.3 45,877,333 46,001,538 Loss 124 kb Pat
1178 M ASD 22
840 kb
80 kb
De novo
2567 M ID 22 22q11.22 22,323,105 22,569,822 Loss 247 kb Unknown
2452 M ASD X Xp22.2 10,845,239 11,135,472 Gain 290 kb Mat
142 M ASD X Xq12 65,815,490 65,895,015 Loss 79 kb De novo
940 M ASD X Xq22.3 106,351,712 106,629,060 Gain 277 kb Mat
1681 M ASD X Xq26.2 130,571,153 130,960,558 Gain 389 kb Mat
36 M ID X Xp22.11 22,836,324 23,411,163 Loss 575 kb Mat
2159 M ID X Xp22.31 8,498,107 8,759,709 Gain 262 kb Mat
375 F ID X Xp22.31 8,498,107 9,082,705 Gain 585 kb Pat
1240 F ID X Xq23 115,591,058 115,864,916 Gain 274 kb Mat
135 M ID Y
170 kb
58 kb
94 M ASD 6 6p25.3 1,580,622 1,663,440 Gain 80 kb Unknown
1478 M ASD 9 9q34.3 140,707,451 141,008,863 Loss 300 kb De novo
296 F ID 6 6q16.1 95,958,454 96,238,765 Loss 280 kb Pat
1346 M ASD 2 2q11.2 100,625,292 100,722,540 Gain 100 kb Mat
177 M ASD 19 19q13.41 53,424,222 53,569,529 Gain 150 kb De novo
331 F ASD 16 16p12.2 21,475,060 21,806,299 Gain 330 kb Mat
313 M ID 3 3p22.2 38,502,327 38,830,481 Gain 330 kb Mat
  M ID 6 6q25.1 152,201,766 152,367,137 Gain 170 kb Unknown
1258 M ASD 3 3q12.2 100,354,612 100,451,345 Amp 97 kb Pat
2695 M ID 10 10q11.21 45,872,003 46,017,634 Gain 150 kb Mat
1366 F ID 11 11p11.2 48,081,727 48,466,844 Gain 390 kb Unknown
172 M ASD 1 1q21.1 145,413,388 145,609,172 Loss 200 kb De novo
535 M ASD 2 2q32.2 189,865,631 189,925,490 Loss 60 kb Mat
2740 M ID 5  5p13.2 37,142,520 37,516,603 Loss 370 kb Unknown
315 F ID 7  7q35q36.3 148,255,537 153,360,454 Loss 5.1 Mb De novo
746 M ASD 15 15q11.2 22,784,523 23,085,096 Loss 300 kb Mat
1275 M ID 6 6q22.31 123,539,625 124,166,602 Gain 630 kb Unknown
107 F ASD 5 5p13.2 37,516,603 37,697,730 Gain 180 kb Mat
2578 M ASD 5 5q35.3 177,756,155 178,507,278 Gain 750 kb Mat
116 M ID X Xq25 124,439,330 127,799,936 Loss 3.36 Mb Mat
850 F ASD 17 17q11.2q12 31,917,720 32,858,733 Gain 940 kb Unknown
556 F ID 8 8q22.2q22.3 101,947,980 103,870,397 Loss 1.92 Mb De novo
271 M ASD 2 2q32.2 189,307,596 189,682,921 Gain 380 kb Mat
956 F ASD 16 16q24.3 89,849,284 89,909,419 Gain 60 kb Unknown
1852 M ASD 2
290 kb
690 kb
De novo
1475 M ASD 3
230 kb
1.1 Mb
1505 F ASD 1
30 kb
140 kb
605 F ASD 21
4.3 Mb
170 kb
De novo
De novo
268 M ID 3
170 kb
90 kb
300 kb
400 M ID 10
410 kb
1 Mb
3.03 Mb

Table 4: CNVs of unknown significance.


Figure 1: Graphical representation of positive and negative cases in ASD and ID group according to the three groups: CNVs previously known to be associated with ASD or ID (green); CNVs including ASD or ID associated genes (red); CNVs of unknown significance (blue).

CNVs with potential clinical impact

In addition to the already reported ASD/ID associated CNVs or genes, we identified several CNVs never reported in the literature and thus classified with unknown significance (Table 5). In this group of variants, we found 6 potentially pathogenic CNVs involving genes playing a role in the nervous system. Among these we identified two Xp22.31 duplications encompasses the KAL1 (Kallmann syndrome 1 sequence) gene, a gene involved in embryonic development of the kidney and human central nervous system, including the spinal cord, olfactory bulbs, olfactory nerves and retina [18]. Loss-of-function mutations of the KAL1 gene are a known cause of Kallmann syndrome, but neither complete nor partial duplications of KAL1 have been associated to specific clinical symptoms [19]. Sowi?ska-Seidler et al. [19] reported a patient manifesting hyperosmia and ectrodactyly accompanied by mild intellectual disability, unilateral hearing loss, genital anomalies and facial dysmorphism. Hemizygous tandem duplication on Xp22.31, encompassing the promoter region and the first two exons of KAL1, has been identified in that patient. We also identified two duplications in 2q14.2 including DBI (diazepam binding inhibitor) gene. This gene encodes an acyl-CoA binding protein that is an allosteric binder of GABA receptors involved in lipid metabolism and in signal transduction at type A gamma-aminobutyric acid receptors located in brain synapses [20]. Due to the prior evidence of the importance of GABAergic genes in autism, it is possible that duplication of DBI could affect signaling and lead to deficits in neuronal function [21]. We found a 2q36.3 duplication involving Delta/Notch-like EGF-related receptor (DNER) gene. DNER is a trans-membrane ligand for Notch that is specifically expressed in the somatodendritic domain in CNS neurons and is essential for precise cerebellar development [22,23]. Finally, we found a deletion in 4q12 including the USP46 gene. Huo et al. [24] recently reported USP46 as the deubiquitinating enzymes specific for Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs) both in vitro and in vivo. AMPARs have been shown to regulate neuronal development and mediate the excitatory synaptic transmission in the brain. Knockdown of USP46, due to siRNAs or shRNAs, leads to an amount of AMPAR ubiquitination and consequently to a reduction in AMPAR protein amount in neurons [24].

  Positive ASD
Positive ID
  CNVs in known deleterious regions CNVs involving ASD/ID genes CNVs on unknown significance CNVs in known deleterious regions CNVs involving ASD/ID genes CNVs on unknown significance
CNV orientation            
Deletions 6 1 42 6 2 19
Duplications 7 4 54 7 4 47
No. of CNV            
Single CNV 11 3 53 12 4 44
2 CNVs 3 1 14 - 2 6
3 CNVs - - 2 - - 3
4 CNVs - - 1 - - -

Table 5: Characteristics of CNVs in ASD and ID group.


Figure 2: Distribution of CNVs in ASD and ID group based on rearrangement size. Large CNVs (>500 kb) enriched in first and second group


Figure 3: Frequency of single (left panel) and multiple (right panel) CNVs in ASD (blue) and ID (red) group. A prevalence of multiple CNVs is observed in ASD cases compared with ID cases.

The 68.9% of the unknown CNVs are inherited (50% maternal and 50% paternal) while the 11.2% occurring de novo.


Figure 4: Duplication and deletion distribution in ASD (blue) and ID (red) group. A prevalence of deletion is observed in ASD cases compared with ID cases.


In the present study, we report the investigation of CNVs in a cohort of 476 patients classified as ASD and ID without ASD. We identified clinical relevant CNVs in 28 cases, which correspond to about 18.2% of the positive cohort, falling within the range of detection rate reported in literature [6].

Among the rearrangements in regions already associated to ASD, alterations in 16p11.2 are the most frequent occurring in our population (5/154, 3.2%). Deletions of this region have been associated with ASD while the reciprocal duplication with schizophrenia [25]. The 15q11.2q13 duplication is considered one of the common genomic causes for autism, occurring in 1-3% of cases [26]. However in our cohort it was less frequent than the CNV at 16p11.2 (we found a duplication and a deletion in two patient classified as ASD and ID, respectively). In our cohort we detected also a 15q13.3 duplication encompassing CHRNA7 in two cases. Chromosome 15q13.3 recurrent microdeletions are causally associated with a wide range of phenotypes, including ASD, ID, seizures, and other psychiatric conditions [27]. The pathogenicity of the reciprocal microduplication is less certain. Recently, Szafranski et al. suggested that the CHRNA7 duplication may confer a predisposition to neurodevelopmental and neuropsychiatric phenotypes, including ASD, possibly in association with other genetic modifiers [28]. Three maternally inherited losses including PDZK1 (1q21) were identified in two ASD patients and in one patient with ID. Casey et al identified ASD-specific risk haplotypes at 1q21.1 in three different population cluster and PDZK1 was the only gene including in the genetic region shared by all three haplotypes [29]. Furthermore, Bernier et al. recently reported an increased prevalence of macrocephaly and increased ASD symptom severity in patients currying the 1q21 duplication [30].

We also found 2q31.1q33.2 deletion of 13 Mb and “de novo” origin in a patient with intellectual disability, macrocephaly, behavioural disturbance, speech defect and facial dysmorphisms, in accordance with previously reported patients [31-33]. Current literature provides more than 30 patients with interstitial deletions in chromosome 2q31q33. The critical region points to a few genes, namely NEUROD1, ZNF804A, PDE1A and ITGA4, which are good candidates to explain the cognitive and behavioural phenotype, as well as the severe speech impairment associated with the this deletion and are included in our CNV [33]. Finally we found a large deletion of 11 Mb, in region 3p14.1p14.3, of “de novo” origin. Interstitial 3p deletions have been very rarely reported and the phenotype-genotype correlation is not well understood. Previous reports have documented a chromosome 3p14 deletion in several patients with global developmental delay, intellectual disability, language impairment and autistic features, but without any other major malformations and only mild facial dysmorphism [34-37]. The region deleted in our patients includes 78 genes of those FEZF2, CADPS, SYNPR, ATXN7, PRICKLE, and MAGI1, are known or presumed to have a role in neurodevelopment.

We also reported here a number of CNVs involving genes previously implicated in ASD.

We identified a paternally inherited duplication in 9p24.3 involving KANK1 gene. Deletions of KANK1 have been associated with neurodevelopmental disease including congenital cerebral palsy, hypotonia, quadriplegia and ID. Since a random monoallelic expression has been suggested for this gene [10], a tight regulation of the expression of this gene is hypothesized and thus we cannot exclude a contributing role of KANK1 duplication in the phenotype of our patient. A 1.05 Mb de novo gain involving PLXNA4 was identified in an ASD case. Decreased expression of axon-guidance proteins, as PLXNA4, was found in the brains of people with ASD, suggesting that dysfunctional axon-guidance protein expression may play an important role in the pathophysiology of autism [9]. Handrigan data highlight 16q24.2 as a region of interest for ASD, ID and congenital renal malformations. These conditions are associated, albeit without complete penetrance, with deletions affecting C16orf95, ZCCHC14, MAP1LC3B and FBXO31. The function of each gene in development and disease warrants further investigation [13].

In one patient an inherited 20p12.1 deletion of nearly 450 Kb interrupting the MACROD2 gene (previously known as C20orf133) has been identified. This gene is a strong positional candidate risk factor for autistic-like traits in the general population [14]. A maternally inherited duplication of band 9q22 was found in an ID case. The rearrangement is associated with growth retardation, mild ID and mild facial dysmorphisms. Based on the described functions of duplicated genes, PTCH1 represents a candidate gene that may be responsible for the phenotypic findings [11]. Deletions or loss-offunction mutations of PTCH1 gene result in basal cell nevus syndrome (Gorlin syndrome). We also found de novo 5.8 Mb deletions on 12p12.2p12.1 involving SOX5. The SOX5 gene encodes a transcription factor involved in the regulation of nervous system development and chondrogenesis. Deletion involving this gene is reported to be associated with global developmental delay, intellectual disability, expressive language delay, mild motor impairment, distinct features and multiorgan involvement [12]. KDM5C and IQSEC2 are located adjacent to each other at the Xp11.22 locus. Deletion and mutations in either of these genes are associated with severe ID in males while female carriers are mostly unaffected [15]. Here, we identified a maternally inherited duplication in a male patient who also presented duplication in X22.12 including the RPS6KA3 gene. This gene is responsible for Coffin-Lowry syndrome (CLS), which is characterized by ID and facial and bony abnormalities but also affects non-syndromic X-linked ID [16]. In another male we identified a maternally inherited Xp22.11 deletion. Hemizygous PTCHD1 loss of function is known to cause an X-linked neurodevelopmental disorder with variable degrees of ID and prominent behavioral issues [17]. Thus, also in this case, the use of array-CGH technique has enabled the detection of a clinically relevant rearrangement.

Within the unknown significance group we speculate about the potential pathogenicity of six CNVs, mainly basing on gene content: two duplications in Xp22.31, encompassing the KAL1 gene, two duplications in 2q14.2 involving the DBI gene, duplication in region 2q36.3 including the DNER gene and a deletion in 4q12 involving the USP46 gene. All these three genes were particularly interesting because of their expression and function in the nervous system.

The KAL1 gene encodes a protein, anosmin-1, that is known to directly stimulate tyrosine kinase activity of the fibroblast growth factor receptor 1 (FGFR1), an important signaling molecule involved in a wide range of developmental processes. Tole et al. demonstrated that FGF signaling is required for generating telencephalic midline structures, in particular septal and glial cell types and all three cerebral commissures [38]. The DBI gene encodes a protein that is involved in lipid metabolism and the displacement of beta-carbolines and benzodiazepines, which modulate signal transduction at GABAa receptors located in brain synapses. Experiments in vivo, demonstrated that DBI can promote neurogenesis in the subventricular zone, counteracting the inhibitory effect of GABA, while the DBI gene product acted as positive allosteric modulators of GABAa receptors in prolonging the duration of IPSCs in reticular nucleus, so it could be endogenously effective by modulating seizure susceptibility [39]. DNER gene is strongly expressed in Purkinje cells in the cerebellum; it contributes to the morphological and functional maturation of Bergmann glia via the Notch signaling pathway, and is essential for cerebellar development. However, with the exception of KAL1, complete or partial duplications of the other two genes have not been reported in the literature. Thus, clinical symptoms associated with duplications and/or increased gene expression remains unknown. These VOUS might still deserve further investigations for any possible association with neurodevelopmental disorders. We also identified a deletion in 4q12 which involves the USP46 gene. USP46 encodes for a deubiquitinating enzyme that plays a role in behavior, possibly by regulating GABA action. Huo et al. identified USP46 as the deubiquitinating enzyme for AMPARs (Alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid receptor) that are the primary mediators for inter-neuronal communication and play a crucial role in higher brain functions including learning and memory [24].

However, our findings also underline that the majority of identified CNVs are still of unknown significance highlighting the challenging role of the clinical geneticists in interpreting correctly the role of these CNVs and estimating the exact recurrence risk. The pathogenicity of these unknown variants can be determined based on different characteristic also highlighted in this study: the orientation (deletion or duplication) of the CNV, the size and gene content.

In conclusion, our study confirmed that array-CGH analysis is able to detect the underlying genetic susceptibility factors in a consistent number of ASD and ID patients, strongly indicating that it has became an essential diagnostic tool for assessing these patients. Moreover our data shown a general amount of duplications in the cohort of positive patients, but no differences are detectable among the ASD and the ID group. Otherwise when the deletion and duplication are considered as single classes, we note a strong association among the presence of a microdeletion and the ASD phenotype. This association confirms once again observation that deletions have a stronger effect than their reciprocal duplications. The variability in genetic susceptibility to ASD from one subject to another one is today well established. In some cases a single de novo rearrangement is sufficient to cause ASD while in other cases a combination of multiple CNVs is reported. In our study we found that the majority of cases carrying more than one rearrangement are classified as ASD (65% of cases) with a prevalence of small CNVs. These data confirm that a synergic effect of multiple CNVs occur in ASD phenotype and further highlight the multifactorial nature of ASD already reported in literature. We also noted that the majority of the CNVs in the entire three groups are inherited. These data should not lead on to a wrong interpretation of the CNVs. The phenotypic differences among proband and a carrier parent may in fact be associated to subtle phenotypic signs, different chromosome rupture point or other independent factors as epigenetic and environment. It is therefore important to carefully consider all these aspects in the interpretation of CNVs, particularly for those of unknown significance.

Further studies in sufficiently large cohort of ASD and/or ID cases are however required not only to refine our understanding of previously isolated genes and regions in ASD and ID, but also to identify novel molecular pathways involved in the etiology of autism and other neurodevelopmental disorders.

Competing Interests

The authors declare that they have no competing interests.


This work was supported by Ricerca Finalizzata 2007 Ministero della Salute to AR and by PAR 2006 University of Siena to FM. We acknowledge “Cell Lines and DNA Bank of Rett syndrome and X linked mental retardation” (Medical Genetics-Siena), member of the Telethon Network of Genetic Biobanks (Project No. GTB07001C to AR)”, funded by Telethon Italy.


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