• array CGH;
  • epilepsy;
  • CNV;
  • intellectual disability;
  • autism


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

The clinical significance of chromosomal microdeletions and microduplications was predicted based on their gene content, de novo or familial inheritance and accumulated knowledge recorded on public databases. A patient group comprised of 247 cases with epilepsy and its common co-morbidities of developmental delay, intellectual disability, autism spectrum disorders, and congenital abnormalities was reviewed prospectively in a diagnostic setting using a standardized oligo-array CGH platform. Seventy-three (29.6%) had copy number variations (CNVs) and of these 73 cases, 27 (37.0%) had CNVs that were likely causative. These 27 cases comprised 10.9% of the 247 cases reviewed. The range of pathogenic CNVs associated with seizures was consistent with the existence of many genetic determinants for epilepsy. © 2012 Wiley Periodicals, Inc.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

Molecular approaches continue to find causes for epilepsy beyond those well-established monogenic epilepsies with known genes [Mulley and Dibbens, 2011]. Array comparative genome hybridization (CGH) for the detection of copy number variants (CNVs) in epilepsy [Sharp et al., 2008; Helbig et al., 2009; Heinzen et al., 2010; Mefford et al., 2010, 2011; de Kovel et al., 2010; Mulley and Mefford, 2011; Talkowski et al., 2011; Bartnik et al., 2012; Galizia et al., 2012; Striano et al., 2012] and exome sequencing for the detection of DNA sequence variants [Corbett et al., 2010, 2011; Bamshad et al., 2011; Najmabadi et al., 2011] are powerful investigative platforms with considerable research and diagnostic application for the epilepsies. These technologies interrogate the entire genome without needing the very detailed clinical descriptions to focus the laboratory on the appropriate test directed at a specific region of the genome. Fluorescence in situ hybridization (FISH) [Trask, 1991] and multiplex ligation-dependent probe amplification (MLPA) [Schouten et al., 2002; Heron et al., 2007; Wang et al., 2008; Marini et al., 2009; Mei et al., 2010] improved cytogenetic resolution for the determination of the location of losses and gains of submicroscopic chromosomal segments in the epilepsies but unless present in a subtelomeric portion of the genome these technologies require prior clinical diagnosis of an established microdeletion–microduplication syndrome. Exome sequencing and array CGH, both heavily reliant on bioinformatics for interpretation, have now enabled the transition to a high resolution whole genome scan with far greater sensitivity than conventional cytogenetics. These new technologies are leading to fewer and cheaper diagnostic platforms with the transition on the horizon for CNV detection using the same platform as for the detection of DNA sequence variations.

Retrospective analyses of large patient cohorts with epilepsy by array CGH have indicated substantial causative contributions by CNVs. These vary from the rare or novel highly penetrant disease-related CNVs to recurrent susceptibility CNVs with incomplete penetrance and variable expression. Recurrent susceptibility genetic variants arising from non-allelic homologous recombination mediated by regional DNA sequence architectures have been documented [Dibbens et al., 2009; Helbig et al., 2009; Heinzen et al., 2010; Mefford and Mulley, 2010; Mefford et al., 2010, 2011; de Kovel et al., 2010; Sisodiya and Mefford, 2011] but many, especially those where their effects are small, remain undiscovered. The extent to which variations responsible for seizures affect other systems such as cognitive functioning or autistic features can be explained by pleiotropy, even for disorders with complex inheritance [Sivakumaran et al., 2011], or because a given CNV can affect many different genes within and close to the microdeletion or microduplication. For monogenic or CNV associated epilepsies where affected individuals can also exhibit cognitive or autistic effects [Strømme et al., 2002; Dibbens et al., 2008; Sharp et al., 2008; Auvin et al., 2009; Corbett et al., 2010; Heinzen et al., 2010; Mefford et al., 2010], mutations in the same gene and even the same mutation in the same gene can be expressed with considerable phenotypic or clinical variation. Whether the inheritance is monogenic or complex, or the etiology genetic or environmental, approximately 20% of individuals with intellectual disability (ID) are known to have associated seizures [Airaksinen et al., 2000].

Necessary criteria for referral for diagnostic array CGH in Australian cytogenetics laboratories are either developmental delay (DD), ID or autism spectrum disorders (ASD), or presence of at least two congenital abnormalities (CAs). Array CGH has progressively replaced the combination of routine cytogenetics and subtelomere MLPA in genetic pathology. Patients with the clinically and genetically heterogeneous DDs, IDs, ASDs, and CAs, often presenting in combination, are commonly referred to the laboratory for investigation from a variety of clinics. Inclusion of additional clinical features such as epilepsy on the test request form can assist the laboratory with interpretation of the significance of detected CNVs involving genes where dosage sensitivity might be responsible. This patient group provided the opportunity to prospectively assess the contribution of array CGH to diagnosis in the subset of cases where epilepsy was a feature of this highly heterogeneous group of disorders. Testing was carried out in an accredited diagnostic laboratory environment with formal reporting of test results. The aim was to determine for patients with seizures associated with any of referral criteria of DD, ID, ASD, and CA what proportion carry CNVs that will likely explain the patient's condition, could help with the patient's prognosis and will enable the family to be informed through genetic counseling of potential risk to other family members.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information


The patient group comprised 247 index cases referred prospectively with one or more of the phenotypes of ID, DD, ASD, or CA. All had epilepsy. The information available to the laboratory was the clinical summary provided by the referring clinician on the test requisition form. Cases were referred from both the public and private sector clinics throughout South Australia over the 18-month period between May 2010 and November 2011. This timeline began with the introduction of Medicare reimbursement for the test, and hence a flow of patients who could not previously access this technology. DD was provisionally diagnosed in cases up to 4 years of age pending formal IQ tests to determine if the DD progressed to ID. Epilepsy as stated on the request form was unprovoked seizures of unknown cause witnessed more than once.

Array Comparative Genome Hybridization (Array CGH)

DNA was hybridized to the BlueGnome Cytochip ISCA (4080-5) 8 plex 60K array [Human Mar 2006 (hg18) assembly]. This oligo-microarray provided average backbone coverage of 35 kb and covered all known genes associated with DD or ID. Selected non-coding sequences cover gene deserts and large introns within characterized genes. Genes known to be associated with DD and ID are covered more densely than average, at about 5–10 kb intervals. Post-processing, slides were flooded with nitrogen gas and posted to the Australian Genome Research Facility (AGRF) for scanning and image capture. Images were then processed and results obtained using BlueFuse multi software.

Three or more contiguous probe displacements were considered to represent a potential CNV. Analysis was carried out using an in-house database of accumulated cases, Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources (DECIPHER) [Firth et al., 2009;], Database of Genomic Variants (DGV) ( and The Children's Hospital of Philadelphia Database (CHOP) []. CNVs detected by array CGH were confirmed by FISH, MLPA or repeat array where the CNVs were too small for FISH or where genes were not covered by commercially available MLPA kits.

We manually checked cases with low numbers of probe displacements (down to three contiguous probe displacements) which needed to pass the more stringent threshold of log-2 ratio of −0.45. Displacements not meeting this quality threshold were eliminated from further consideration. The analysis software allows direct access to the raw data for assessment to determine spurious results. Derivative Log-Ratio Spread (DLRS) indicate quality of non-normalized data and DRLs >0.2 rejected that experiment. DRLs accepted were mostly in the range 0.12–0.16. The analysis software contains within the algorithm the capability for a minimum of three oligo calls to pass data that needs to be five standard deviations away from the autosome mean. This represents a high and robust threshold call designed for diagnostic application.

Fluorescence In Situ Hybridization (FISH)

FISH was used to confirm CNVs detected by array CGH, as a cost effective approach to parental studies and additional cascade testing, and to detect rare insertional translocations of gains into regions of the genome remote from their point of origin [Kang et al., 2010]. Microdeletions <200 kb and microduplications <400 kb or CNVs with no known pathogenic genes were generally excluded from FISH follow-up. Parental studies to distinguish between inherited and de novo CNV were carried out where parents were available, unless the CNV clearly fitted with a well-established clinical phenotype and contiguous gene syndrome, in which case parental studies were not required.

FISH probes were selected by positioning the array CGH change using the Ensembl browser linked to BlueGnome software, choosing the corresponding bacterial artificial chromosome (BAC) probes from the 32K probe set and picking the DNA from the library stock. DNA was amplified using a Qiagen Repli-G kit and fluorescently labeled using a Vysis Nick Translation kit.

Multiplex Ligation-Dependent Probe Amplification (MLPA)

MLPA was carried out according to the manufacturer's instructions and in house detail as previously described when applied to epilepsy [Heron et al., 2007]. MLPA kits were sourced from MRC Holland, Amsterdam, The Netherlands. Those relevant to cases in this report were P015 for MECP2, P049 for L1CAM, and P104 for ATP7A.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

In contrast with the present study, epilepsy cohorts examined in genome-wide surveys are generally assessed retrospectively or structured differently for the mix of epilepsy associated phenotypes [Heinzen et al., 2010; Mefford et al., 2010, 2011; Bartnik et al., 2012; Galizia et al., 2012; Striano et al., 2012]. Developmental abnormalities in conjunction with epilepsy can include short stature, dysmorphic features, or various other CAs such as microcephaly and digital abnormalities. Screening infants first by array CGH at an early stage of their clinical workup can lead to an earlier cause for their condition. Revisiting older patients who previously had a normal karyotype and applying current array CGH technology can resolve longer term cases of unknown cause.

Results from array CGH were interpreted and categorized as presented in Table I. Cases within each category and their frequency in the cohort were summarized in Table II and are discussed below. Of the 73 positive cases (29.6% of the cohort), most would not have been detected by conventional cytogenetics where resolution varies depending upon the density and size of G-bands where the CNV is located. The key clinical features are summarized in Table I for each patient with additional features, such as CAs where these occur, described in the text. These clinical features can assist the laboratory with interpretation of the significance of detected CNVs where dosage sensitivity might be responsible.

Table I. CNVs Detected From 247 Cases of Epilepsy With Cognitive or Autism Spectrum Disorders
CaseAge (years)SexCNV(s), start end (bp) [Oligosd]Size(s), FISH probe(s)Genes involvedaOverlapping phenotypesbFamily studyc
  • a

    Primary gene candidate stated where known, otherwise total number of genes within the lesion(s) is stated. HGNC refers to genes recognized by the Hugo Gene Nomenclature Committee and OMIM refers to genes recognized by Online Mendelian Inheritance in Man.

  • b

    ASD is autism spectrum disorders, E is epilepsy, IS is infantile spasms, DD is developmental delay, ID is intellectual disability, FS is febrile seizures. See text for presence of various congenital abnormalities, where present.

  • c

    NA is not applicable, Fam is familial, FamX is familial on the X chromosome, del is deletion, DN is de novo, IT is insertional translocation, ? means one or both parents not available for testing, or not yet responded to request for testing.

  • d

    Number of consecutive oligonucleotides displaced.

  • e

    With ataxia and microcephaly.

  • f

    With encephalopathy.

Established pathogenic microdeletion/microduplication syndromes (4 cases)
 122Mdel(1)(p36.23-p36.32), 2,684,617-7,853,163 [99]5.1 Mb, BAC RP11-391P21See MIM 607872E, ID?
 250Fdel(2)(q22.3-q23.3), 146,519,777–150,763,806 [55]4.2 Mb, BAC RP11-804F22MBD5E, IDDN
 320Fdup(7)(q11.23), 72,404,279–73,831,310 [71]1.4 Mb, Vysis WilliamsSee MIM 609757E, IDDN
 465Fdup(22)(q11.21), 16,977,255–19,770,485 [153]2.79 Mb, Vysis TUPLEISee MIM 608363E, ID?
Predicted pathogenic CNVs (12 cases)
 511Fdel(X)(p22.13), 18,197,641–19,037,887 [84]840 kb, BAC RP11-762N22CDKL5E, IDDN
 635Mdup(X)(q28), 152,784,105–152,801,608 [21]16 kb, N.A. (MLPA)L1CAME, IDFam
   dup(X)(q28), 152,825,772–153,231,083 [39]382 kb, N.A. (MLPA)MECP2 Fam
 716Mdel(1)(p13.2-p13.3), 108,727,865–111,774,485 [46]2.89 Mb, BAC RP11-260A246734E, DDIT
   del(4)(q26), 117,646,387–119,257,623 [17]1.37 Mb, BAC RP11-346E10    
 88Mdup(2)(q24.3-q31.1), 163,531,266–174,842,325 [252]11.19 Mb, BAC RP11-181J06SCN1A, SCN2AE, DDDN
   dup(15)(q26.3), 97,917,098–98,484,131 [7]430 kb, PAC CTD-2288D0342 Fam
 93Fdel(2)(q32.1-q33.2), 184,228,343–204,139,009 [340]19.9 Mb, BAC RP11-720C149855E, DDDN
 101Mdel(3)(q25.32-q26.1), 158,488,866–168,358,770 [138]9.8 Mb, BAC RP11-343L173019E, DDDN
   dup(11)(q13.4), 71,978,187–72,643,014 [11]665 kb, BAC RP11-475M2073  
 1120Mdel(6)(p22.3-p24.1), 12,637,216–17,041,150 [60]4.3 Mb, N.A.1610E, ID?
 1223Mdup(7)(p21.3-p22.3), pter-7,481,603 [137]7.2 Mb, PAC 164D187440E, IDDN
   del(10)(q26.3), 132,358,382-qter [80]2.8 Mb, PAC 137E243014  
 1315Fdup(7)(p22.1), 5,823,784–7,025,338 [15]1.0 Mb, BAC RP11-437P05228E, IDDN
   dup(17)(q12), 31,930,198–33,323,002 [45]1.39 Mb, BAC RP11-383B12149  
 1417Mdel(14)(q23.3), 66,421,411–66,613,015 [4]191 kb, BAC RP11-182D06GPHNE, ID, ASDDN
 151Mdel(14)(q32.31-q32.33), 101,635,698–106,358,521 [112]4.7 Mb, N.A.14837E, ASDDN
   del(15)(q15.3), 41,710,685–41,735,609 [18]99 kb, N.A.See below Fam
 169Mdel(16)(p13.3), 2,317,072–2,662,080 [6]345 kb, N.A.TBC1D24E, IDDN
Established pathogenic disease susceptibility CNVs (11 cases)
 171Mdup(1)(q21.1), 143,324,038–144,610,726 [42]380 kb, N.A.

Cooper et al. [2011

 182Fdup(1)(q21.1), 143,324,038–144,680,159 [42]484 kb, BAC RP11-293J20

Cooper et al. [2011

 1920Mdel(2)(p16.3), 50,790,978–50,835,647 [7]45 kb, BAC RP11-463P12NRXN1E, IDFam
 2051Fdel(2)(p16.3), 50,735,529–50,801,204 [9]55 kb, BAC RP11-631J24NRXN1E, ID?
 2114Fdel(2)(p16.3), 50,824,196–50,903,179 [11]79 kb, BAC RP11-463P12NRXN1eEFam
   del(6)(q22.2), 117,884,042–118,309,669 [7]426 kb, BAC RP11-446J1632  
 2216Fdel(2)(q13), 111,265,433–112,926,973 [21]1.66 Mb, BAC RP11-809E17

Yu et al. [2012

E, DDFam
 2345Mdel(15)(q13.2-q13.3), 28,278,735–30,297,189 [60]2.02 Mb, BAC RP11-463D21

Sharp et al. [2008

E, ID?
 249Mdup(16)(p11.2), 29,500,314–30,240,052 [8]517 kb, PAC CTD-2215A12

Weiss et al. [2008

E, DDFam
 2533Mdel(16)(p11.2), 29,500,314–30,240,052 [9]598 kb, N.A.

Weiss et al. [2008

E, ID?
   del(12)(q14.3), 64,025,314–64,319,713 [3]117 kb, N.A.

Shinawi et al. [2010

 265Mdel(16)(p12.1-p12.2), 21,375,203–22,530,130 [17]1.15 Mb, BAC RP11-101E07

Girirajan et al. [2010

 2724Mdel(16)(p12.1-p12.2), 21,561,935–21,745,023 [3]183 kb, N.A.

Girirajan et al. [2010

   dup(2)(q24.3), 168,392,938–169381,670 [15]989 kb, N.A.43  
CNVs of unknown clinical significance (37 cases)
 283Fdup(X)(p22.33), 1,287,588–2,871,414 [150]1.6 Mb, N.A.1012E, DD?
   dup(16)(q24.1), 83,650,168–84,236,052 [10]586 kb, N.A.41  
 2951Fdup(X)(p22.31), 8,601,794–8,867,513 [15]266 kb, N.A.11E, ID?
   del(11)(q21-q22.1), 95,249,257–96,729,244 [22]1.5 Mb, N.A.21  
 3024Mdup(X)(q21.1), 76,992,097–77,014,080 [3]22 kb, N.A.MAGT1E, ID, ASDFamX
 3119Mdel(X)(q22.3), 106,759,975–106,764,030 [7]4 kb, N.A.PRPS1E, IDFamX
 3271Mdup(X)(q22.3), 104,410,352–104,918,601 [8]508 kb, N.A.IL1RAPL2E, ID?
 3311Mdup(X)(q25), 122,871,991–123,111,228 [7]240 kb, N.A.22E, ID?
 346Mdel(X)(q27.2), 140,180,787–140,566,110 [5]385 kb, N.A.24E, ASD?
 3518Mdup(1)(q24.3), 169,165,514–169,665,535 [7]500 kb, N.A.94E, ID?
   dup(5)(q23.2), 126,240,674–126,730,543 [9]489 kb, N.A.20  
 363Mdel(4)(q13.1), 59,642,541–63,676,747 [59]4.03 Mb, BAC RP11-640N0710E, DD?
 3716Mdel(15)(q22.1-q22.2), 56,937,892–60,627,614 [59]3.7 Mb, N.A.1712E, ID 
 386Fdel(X)(p22.11), 22,819,146–23,031,999 [5]213 kb, BAC RP11-646K0820EFam
 3910Mdup(X)(p21.1), 33,000,964–33,022,056 [3]21 kb, N.A.11E, ID, ASD?
 404Fdel(X)(p11.3), 47,215,186-47,219,609 [3]4.4 kb, N.A.ZNF41E, DD?
   dup(9)(p24.3), 312,720-490,555 [11]178 kb, N.A.?KANK1  
 4111Fdup(1)(p36.33), 1,643,869-2,271,102 [28]627 kb, N.A.GABRDE, DD?
   dup(1)(p36.21-p36.22), 12,276,149-12,960,333 [15]684 kb, N.A.151  
 4223Fdup(1)(p36.22), 11,226,010-11,424,781 [4]199 kb, N.A.22E, ID?
   del(4)(q21.21), 80,882,820-81,053,724 [3]171 kb, N.A.11  
 431Mdup(1)(q25.1), 173,408,043-174,216,759 [13]808 kb, BAC RP11-542L1432IS, DDFam
   del(10)(q11.22), 48,499,353-48,932,383 [3]433 kb, BAC RP11-342C2480  
 442Mdup(2)(p25.1), 9,955,349-10,193,350 [5]238 kb, BAC RP11-338K0354FS, DDFam
 451Fdup(3)(p26.3), 1,358,649-2,252,884 [36]894 kb, BAC RP11-275P2121E, DDFam
 468Fdel(3)(p26.2), 3,729,575-4,236,190 [9]507 kb, BAC RP11-629L2421E, DDFam
   dup(15)(q14), 32,824,321-32,966,154 [4]142 kb, BAC RP11-814P0533  
 471Mdup(3)(p14.2), 59,697,820-60,968,093 [20]1.27 Mb, N.A.11fE, ID?
 485Mdel(4)(q28.3), 135,674,915-136,850,922 [17]1.18 Mb, BAC RP11-03N2400E, IDFam
   del(18)(q12.2), 32,207,392-32,939,065 [12]732 kb, BAC RP11-713H1031  
 4921Mdel(6)(q15), 88,942,300-89,473,045 [8]530 kb, BAC RP11-205L1611E, ID?
 502Fdel(7)(q22.1), 103,291,846-103,307,744 [3]7.2 kb, N.A.11E, DD?
 514Fdup(8)(q24.21), 129,948,597-131,064, 141 [17]1.1 Mb, N.A.21E, DD?
 5266Mdup (8)(q24.2), 146,106,030-146,250,795 [27]145 kb, N.A.31E, ID?
 5349Mdup(10)(q21.1), 57,577,697-58,432,767 [12]855 kb, BAC RP11-724N1111E, IDFam
 5442Fdup(10)(q24.32), 103,274,279-103,328,637 [5]27.5 kb, N.A.11E, ID?
 554Mdup(11)(p13), 334,322,342-35,300,442 [15]978 kb, RP11-805H0786E, DD?
 5650Fdel(11)(p11.2), 44,258,155-44,259,604 [3]1.5 kb, N.A.11E, DD?
 5764Mdel(11)(q14.1), 80,494,232-81,502,050 [13]830 kb, BAC RP11-104B1700E, ID?
 5826Mdup(11)(q14.3), 88,657,952-88,986,450 [6]328 kb, N.A.?TYRE, DD?
   del(15)q15.3), 41,638,870-41,737,986 [22]99 kb, N.A.54  
 591Mdup(12)(p13.33), 2,663,919-2,743,908 [24]24 kb, N.A.11FS, DD?
 606Mdup(13)(q21.32), 65,185,994-66,113,705 [80]908 kb, BAC RP11-453H2311E, ID, ASDFam
 611Fdel(13)(q31.3), 90,863,691-91,088,202 [27]225 kb, BAC RP11-487A0211E, IS, DDFam
   dup(13)(q33.1), 102,354,168-102,565,165 [5]211 kb, BAC RP11-11L0811  
 623Fdup(15)(q15.3), 41,638,870-41,736,835 [21]98 kb, BAC RP11-263I19See belowfE, DDFam
 6312Mdup(17)(p13.3), 1,235,294-1,245,589 [3]5.1 kb, N.A.?YWHAEE, ID?
 643Mdup(17)(q25.3), 77,956,577-78.462,809 [9]506 kb, BAC RP11-388C12117E, DD?
Incidental findings of potentially pathogenic CNVs (9 cases)
 651Mdel(X)(q21.1), 77,135,260-77,143,328 [4]8 kb, N.A. (MLPA)ATP7AE, DD?
 6647Mdel(6)(q26), 162,504,320-162,853,117 [6]348 kb, N.A.PARK2E, IDFam
 6745Mdel(15)(q15.3), 41,638,870-41,735,609 [20]99 kb, N.A.CATSPERS2E, IDFam
 6819Mdel(15)(q15.3), 41,710,685-41,735,609 [18]99 kb, N.A.CATSPERS2E, IDFam
 699Mdel(15)(q15.3), 41,638,411-41,736,834 [20]99 kb, N.A.CATSPERS2, STRCE, DD?
 7015Fdel(15)(q15.3), 41,710,685-41,737,986 [20]99 kb, N.A.CATSPERS2, STRCE, IDFam
 7120Mdel(15)(q15.3), 41,638,411-41,738,566 [22]99 kb, N.A.CATSPERS2, STRCE, DD?
 7211Mdel(15)(q15.3), 41,638,411-41,835,623 [23]99 kb, N.A.CATSPERS2, STRCE, ASD?
 7317Mdel(15)(q15.3), 41,638,870-41,738,566 [23]99 kb, N.A.CATSPERS2, STRCE, ID, ASDFam 
Table II. Number and Frequency of CNVs Detected in Each Category of Cases Examined During the Study Period
Category of cases delineated by clinical significanceNumber of casesFreq% of cohortCumulative no.Cumulative freq %Freq% out of positive cases
  1. Key numbers and frequencies are highlighted in bold.

Established pathogenic microchromosomal syndromes41.6  5.5
Predicted pathogenic CNVs124.9166.516.4
Established pathogenic disease susceptibility CNVs114.52710.915.1
Unknown significance3714.96415.050.7
Incidental findings of potentially pathogenic CNVs93.67329.612.3
Predicted pathogenic among positive cases for any CNV27  37.0 
Cases with no detectable CNV of potential significance17470.4   
Total number of cases247    

Established Pathogenic Microdeletion/Microduplication Syndromes

Four cases with epilepsy and ID fell within this category of well-established microdeletion and microduplication syndromes (Table I). Case 1 was a male patient with a 5.1 Mb interstitial 1p36.23-p36.32 microdeletion diagnostic for 1p36 microdeletion syndrome (MIM 607872). Case 2 was a female with epilepsy and ID showing a de novo 4.2 Mb microdeletion at 2q22.3-q23.3. The deleted region encompassed nine genes, including methyl-CpG binding domain protein 5 (MBD5) and EPC2. Haploinsufficiency of this region has previously been documented in association with seizures and severe DD [Williams et al., 2010] and recently MBD5 was implicated as the single causal locus for ID, epilepsy and ASD [Talkowski et al., 2011]. Case 3 was a female with de novo 1.4 Mb interstitial 7q11.23 microduplication, which is the reciprocal to the 7q11.23 microdeletion causing Williams–Beuren syndrome. This microduplication is known as the Williams–Beuren region microduplication syndrome (MIM 609757). Case 4 was a female with a 2.79 Mb interstitial 22q11.21 microduplication indicative of 22q11.2 microduplication syndrome (MIM 608363). These four cases comprised 1.6% of the total cohort and 5.5% of positive cases detected (Table II).

Predicted Pathogenic CNVs

Predicted pathogenic CNVs are determined by a combination of the following factors: de novo status, gene content including presence of more than one gene which might affect the phenotype described in the clinical summary and especially the involvement of a gene known to cause epilepsy, DD, or MR. However, these changes are not recurrent and therefore cannot be recognized as pre-existing microdeletion/microduplication syndromes.

Twelve cases with rare microdeletion or microduplication CNVs were predicted as pathogenic (Table I), in addition to the well-established microdeletion and microduplication syndromes described in the previous section. These rare cases predicted as pathogenic comprised 4.9% of the entire cohort and 16.4% of the positive ascertainment (Table II). Clinical features are described in greater detail for these cases to illustrate the diversity of cases routinely referred for cytogenetic testing and to reinforce the unique nature of positive cases that comprise this classification.

Case 5 was a female with epilepsy and ID with a de novo 840 kb microdeletion at Xp22.13. It contains CDKL5 which when mutated causes X-linked dominant infantile spasms in females (MIM 300672). Deletion of RS1 confers carrier status for retinoschisis (MIM 312700). She presented with infantile spasms at age 6 weeks. She continues to have seizure disorder with variable seizure types, including myoclonic jerks and absence seizures. Speech was absent with low muscle tone and inability to walk, lack of eye contact, gastro-esophageal reflux, small cold feet and hands and very limited hand skills, suggesting CDKL5 mutations or atypical Rett syndrome but sequencing MECP2 and CDKL5 failed to identify a mutation. Thus, a CNV containing CDKL5 was not unexpected.

Case 6 was a male with epilepsy, severe ID, dysmorphic craniofacial features, inguinal hernia treated by herniotomy, left lower lobe bronchiectasis treated by lobectomy, long standing drooling, and incontinence with deteriorating gait since 30 years of age. Brain MRI suggested bifrontal cortical dysplasia. A brother was similarly affected. Two non-contiguous microduplications within Xq28 of 16 and 382 kb affecting MECP2 associated with MECP2 duplication syndrome [Van Esch, 2012] and L1CAM (associated with ID and associated disabilities) were inherited from his healthy mother. Microduplications of both genes were confirmed by MLPA.

Case 7 was a male who works in a sheltered workshop who has epilepsy and DD with a 2.89 Mb interstitial microdeletion at 1p13.2-p13.3 and a 1.37 Mb interstitial microdeletion at 4q26. The clinical description includes the mild dysmorphic facial features of upward-slanting palpebral fissures, prominent medial epicanthi, anteverted nares, smooth philtrum, thin upper lip, and small chin. This was accompanied by mild thoracic scoliosis. FISH study of the father using the BAC probe RP11-260A24 for 1p13.3 showed one signal on one chromosome 1 and one signal on the short arm of chromosome 4. This indicated that there was an insertional translocation between chromosomes 1 and 4 in the father. The BAC probe RP11-346E10 for 4q26 showed a microdeletion on the long arm of chromosome 4, the same homologue as the insertion on the short arm. The microdeletion of 1p13.2-p13.3 is likely pathogenic based on its size and number of affected genes (Table I). As this microdeletion was derived from malsegregation of a paternal insertional translocation involving chromosomes 1 and 4, the recurrence risk is high.

Case 8 was a boy with neonatal seizures and DD with a 11.19 Mb de novo microduplication 2q24.3-q31.1 which involved the known epilepsy-related genes SCN1A and SCN2A. Microduplications affecting each were associated with ID [Marini et al., 2009; Heron et al., 2010]. The paternally inherited 430 kb 15q26.3 microduplication was considered likely non-pathogenic as it was inherited from his healthy father.

Case 9 was a female with epilepsy and DD who had a de novo 19.9 Mb microdeletion of 2q32.1-q33.2 involving many genes. Additional clinical features included micrognathia, U-shaped cleft palate, mildly flat mid-face noted at birth, left hip dysplasia treated with splinting, feeding difficulties, gastroesophageal reflux, very prominent rounded forehead, scaphocephaly, nephrocalcinosis, chronic serous otitis media, sparse hair on scalp, prominent smooth philtrum, small mouth, and downward slanting palpebral fissures. The size of the microdeletion and number of genes affected was consistent with pathogenicity affecting multiple systems.

Case 10 was a male with infantile spasms and global ID with a de novo 9.8 Mb interstitial 3q25.32-q26.1 microdeletion which involved many neuronally expressed genes. Despite the number of genes deleted, neurological examination was normal apart from infantile spasms and global ID and there were no dysmorphic features. He also had a maternally inherited 665 kb interstitial microduplication at 11q13.4 which was considered non-pathogenic as it was inherited from his healthy mother.

Case 11 was a boy with epilepsy, ID and gynaecomastia caused by a 4.3 Mb microdeletion at 6p22.3-p24.1. Overlapping deletions have been reported in patients with DD, brain, heart and kidney defects, eye abnormalities, short neck, craniofacial malformations, hypotonia, and clinodactyly or syndactyly [Bremer et al., 2009]. Epilepsy susceptibility may now need to be considered as part of the group of conditions affected by genes in this region.

Case 12 (male) has epilepsy and ID with a terminal microduplication of 7.2 Mb at 7p21.3-p22.3 and a terminal 2.8 Mb microdeletion of 10q26.3 resulting from malsegregation of a paternal t(7;10). Additional clinical features included dysmorphism with hypertelorism, a divergent squint of the right eye and a large forehead.

Case 13 (female) has epilepsy and ID with a with a 1.0 Mb microduplication at 7p22.1. and a 1.39 Mb interstitial microduplication at 17q12. The microduplication at 17q12 was known to be associated with cognitive impairment and epilepsy with incomplete penetrance [Mefford et al., 2007; Nagamani et al., 2010; Cooper et al., 2011]. Additional clinical features detected were restricted to mildly depressed but symmetrical tendon reflexes.

Case 14 was a male with epilepsy, ID, and autism with a de novo 191 kb 14q23.3 microdeletion. This removed multiple exons of Gephyrin (GPHN) known to be involved in GABAA and glycine receptor subunit assembly at post-synaptic inhibitory synapses. He has frequent seizures averaging one generalized tonic clonic seizure per week and three to five absence seizures per day. Seizure pattern is refractory to treatment with large daily doses of anticonvulsants consisting of Epilim, clonazepam, and Keppra required to reduce seizures to the level described, but with associated drowsiness. This case may have serendipitously implicated haploinsufficiency of GPHN as causative for one or both of the seizure pattern and ID.

Case 15 is a boy with early onset seizures which have evolved into infantile spasms, ASD, and dysmorphism with a prior diagnosis from conventional cytogenetics of Ring 14. The terminal 4.7 Mb 14q32.31-q32.33 microdeletion affected many genes which likely accounts for the multiple clinical abnormalities. These included intrauterine growth restriction, microcephaly, dysmorphic facial features (synophrys, medial flaring of the eyebrows, telecanthus, hypertelorism, short palpebral fissures, epicanthic folds, minor epicanthus versus, broad nasal tip, anteverted nares, micrognathia and small mouth), hypospadias, congenital heart disease (atrial septal defect, left pulmonary artery narrowing, and possible left pulmonary artery sling), and short stature. This patient also had a 15q15.3 microdeletion of 99 kb as an incidental finding, and was a carrier for deafness (see further discussion of this CNV under Incidental Findings of Potentially Pathogenic CNVs, below).

Case 16 was a male with epilepsy and ID and a de novo 345 kb interstitial microdeletion at 16p13.3. Interestingly, this region contained TBC1D24 which when mutated is responsible for a rare autosomal recessive focal epilepsy with ID [Corbett et al., 2010]. The TBC1D24 sequence from the undeleted homolog and the possibility of mild haploinsufficiency acting alone or in concert with other deleted genes within this CNV will be of interest.

Established Pathogenic Disease Susceptibility CNVs

Phenotype can be determined by gene content of CNVs and their interaction with the genetic background and environmental triggers, which can be unique to each case. Eleven cases were diagnosed with CNVs known to have had incomplete penetrance and variable phenotype (Table I). This is an expanding category as cases continue to accumulate in the literature and on public databases. The initial recognition of susceptibility regions can be complicated by the unpredictable expression of the associated CNV. However, once recognized and validated as a susceptibility region, usually by retrospective analysis of very large research cohorts, knowledge of susceptibility CNVs prospectively enable diagnosis of significant numbers of new cases as they present to the clinic and are diagnosed by laboratory testing. Prenatal diagnosis for susceptibility lesions remains problematic because the phenotype cannot be predicted given that the CNV needs to be conceptualized as just one component of a polygenic profile for the range of conditions that it may be associated with.

Two cases presented with microduplications in 1q21.1. Case 17 was a male infant with infantile spasms with a 380 kb microduplication. A recent study demonstrated a doubling of risk for DD and ID in carriers of this microduplication [Cooper et al., 2011]. This likely contributed to a polygenic causation of the severe phenotype in this patient, with the other causative genetic determinants remaining cryptic. Case 18 was a female infant with febrile seizures and DD who had 484 kb microduplication within 1q21.1.

Cases 19–21 presented with interstitial microdeletions at 2p16.3. Case 19 was a male with epilepsy and ID with an inherited a 45 kb microdeletion from his unaffected mother. Case 20 was a female, with epilepsy and ID, with a 55 kb microdeletion which also included the (NRXN1) gene. Both deletions were in intron 5 and disrupted a minor isoform of neurexin 1 (NRXN1) [Ching et al., 2010]. There is mounting evidence that missense mutations and deletions of NRXN1 exons are associated with a wide spectrum of developmental disorders such as autism, schizophrenia, ID, language delay, and hypotonia [Ching et al., 2010; Wisniowiecka-Kowalnik et al., 2010]. Presentation with epilepsy now means that this condition needs to be considered as an additional disorder within the accepted constellation of phenotypes associated with this region, pending acceptance of these intronic NRXN1 CNVs (as well as exonic NRXN1 CNVs) as causative.

Case 21 was a female with a 79 kb microdeletion at 2p16.3, together with another 426 kb microdeletion at 6q22.2. The phenotype included ataxia and microcephaly in addition to epilepsy. We speculated that the extended phenotype could be the result of the larger 2p16.3 deletion incorporating additional genes or the cumulative effect of microdeletions at both 2p16.3 and 6q22.2. The 2p16.3 microdeletion was inherited from the unaffected father. The 6q22.2 microdeletion was transmitted from her mother who has mild ID and epilepsy.

Case 22 was a female with epilepsy, DD and a 1.66 Mb microdeletion at 2q13 inherited from an unaffected mother. This microdeletion is a likely risk factor for DD and dysmorphism with incomplete penetrance [Cooper et al., 2011; Yu et al., 2012]. Epilepsy must now also be considered as a possible associated phenotype.

Case 23 was a male with epilepsy and ID who had a 2.02 Mb microdeletion at 15q13.2-q13.3 larger than but incorporating the lesion originally and generally implicated in ID with seizures [Sharp et al., 2008]. Subsequently, the recurrent 15q13.3 CNV proved to be the most common cause of genetic generalized epilepsy [Dibbens et al., 2009; Helbig et al., 2009] accounting for approximately 1% of all such cases. The recurrent detection of this lesion in the population created the possibility of homozygosity, which has been observed [Lepichon et al., 2010] and seen to be responsible for visual impairment, hypotonia, profound ID, and refractory seizures. Recurrent susceptibility lesions at 15q11.2 and 16p13.11 also mediated by flanking segmental duplications were almost as common as the 15q13.3 lesion among large patient cohorts with genetic generalized epilepsy, ID, autism, and schizophrenia [Mulley and Mefford, 2011] but by chance neither was detected in the present study.

The next two cases, Cases 24 and 25, carry lesions at 16p11.2. Case 24 with a 517 kb paternally derived microduplication, had epilepsy and DD. Recurrent microduplication at this site was associated with autism [Weiss et al., 2008], seizures [Bedoyan et al., 2010], and a broader range of neurodevelopmental conditions [Shinawi et al., 2010]. The reciprocal microdeletion was also pathogenic and well characterized [Weiss et al., 2008; Shinawi et al., 2010] and present in Case 25 (598 kb), who had epilepsy and ID. Case 9 also had a 117 kb microdeletion at 12q14.3 of unknown clinical significance.

The last two cases had an interstitial microdeletion at 16p12.1-p12.2. Case 26 was a male with febrile seizures, autism, and DD with a 1.15 Mb microdeletion. This microdeletion overlaps the recurrent 520 kb microdeletion at 16p12.1, which is a risk factor for ID and DD [Girirajan et al., 2010]. Autism and febrile convulsions may now need to be considered as disorders associated with this microdeletion; however, these phenotypes could conceivably be associated with the additional genes located in this larger lesion extending beyond the proximal end of the usual recurrent microdeletion at 16p12.1. One of these genes, OTOA, caused congenital deafness when homozygous for pathogenic mutations [Shahin et al., 2010].

Case 27 was another male with a smaller 183 kb microdeletion at 16p12.1-p12.2 associated with epilepsy and autism. This case also carried a 989 kb microduplication at 2q24.3 of unknown clinical significance.

These known susceptibility regions accounted for 4.5% of cases in the entire cohort and 15.1% of the positive ascertainment (Table II). Likely, a proportion of cases currently in the unknown clinical significance category (see below) belong with cases just discussed within the disease susceptibility category; however, current evidence was insufficient to support a more definitive classification and the gathering of the evidence required will likely require ongoing analyses in a research context of much larger patient cohorts where effects to be detected are small. Therefore, in this patient group with epilepsy we established a baseline frequency of 10.9% of cases with likely pathogenic CNVs, where epilepsy is associated with either DD, ID, ASD, or CA. This was comparable to the figure of 11.8% from Mefford et al. [2010], 14.2% from Cooper et al. [2011] based on a range of clinical referrals where it was noted that the frequency among cases with the general diagnosis of epilepsy was less than the 14.2% average, and 15.6% from Galizia et al. [2012].

CNVs of Unknown Clinical Significance

CNVs in this group could not be categorized as established pathogenic microdeletion/microduplication syndromes, predicted pathogenic CNVs, or established pathogenic disease susceptibility CNVs. They are classified as being of Unknown Clinical Significance due to one or more of the following reasons: (1) CNVs irrespective of size involveing genes of unknown clinical significance; (2) CNVs within an intron of a known pathogenic gene; (3) CNVs involving a known haploinsufficiency pathogenic gene but as a duplication rather than a deletion; and (4) CNVs for which their origin could not be demonstrated due to the unavailability of either or both parents. Typically, these CNVs were neither recorded in public patient databases (e.g., DECIPHER) or in the published literature as pathogenic nor recorded in public normal control databases (e.g., DGV and CHOP) as a normal variant. CNVs on the X chromosome are particularly challenging. Where maternally transmitted X-linked pathogenic CNVs can be carried in a phenotypically unaffected mother, they may be pathogenic when uncovered in the hemizygous male or pathogenic in a female with skewed X-inactivation.

This group of 37 cases of unknown clinical significance (Table I) highlights the uncertainty of interpretation of novel findings in a diagnostic setting. Since these cases inflated the detection frequency well beyond the 14.2% rate likely to be realistic based on a very large study [Cooper et al., 2011], very few of these additional cases described in this section could be expected to be truly pathogenic. A proportion of these could include as yet unrecognized disease susceptibility regions; however, such a conclusion cannot be substantiated without additional observations from large research cohorts incorporating cases affecting the same microchromosomal region. These cases, where a clear determination could not be made with the desired degree of confidence, even with in silico analysis of gene content, are briefly described in Supplementary Table I.

The distinction between likely pathogenic CNVs and rare benign variations remains challenging when novel CNVs were identified. The availability of public databases such as DECIPHER [Firth et al., 2009], Database of Genomic Variants (DGV), and Children's Hospital of Philadelphia (CHOP) represented the best approach for documenting and assembling like cases, and sometime in the future drawing conclusions about their clinical effects. Many laboratories also have access to the non-public International Standards for Cytogenomic Arrays (ISCA) Consortium database. The positions of CNVs of unknown clinical significance were recorded by coordinates expressed as base-pair position on the chromosome (Table I) to enable these cases to be revisited if the significance of their associated CNVs can be clarified in the future. Realistically, very few of the CNVs described in Supplementary Table I are likely to be relevant, either as highly penetrant likely pathogenic CNVs or as susceptibility CNVs.

Incidental Findings of Potentially Pathogenic CNVs

The most common finding in this group is the carrier status of an autosomal recessive condition (deafness and male infertility, OMIM #611102). The CNVs in this group were either unlikely to be the cause of the patient's condition or there was insufficient information based on current knowledge for the CNV to be reportable as a possible cause (Table I). However, results were definitive in terms of detecting cryptic genetic defects that can have well known implications for other family members.

Case 65 was a female with seizures, DD, and microcephaly who had an interstitial 8 kb Xq21.1 microdeletion. Deletion of exons 5 and 6 of the ATP7A gene within this CNV was confirmed by MLPA. Disruption of this gene was causal for Menkes syndrome (MIM 309400) which had implications for carrier status in female relatives.

A FISH confirmed interstitial 348 kb 6q26 microdeletion in Case 66 with epilepsy and ID affected the Parkin 2 (PARK2) gene, which identified this individual as a carrier of autosomal recessive early-onset Parkinson disease (OMIM 600116).

In another seven cases (Cases 67–73) with epilepsy and various combinations of DD, ID, and ASD, an interstitial 15q15.3 microdeletion of 99 kb involving CATSPER2 gene was identified. When homozygous for mutations, CATSPER2 is associated with male infertility. This CNV was relatively common in the population at a frequency of about 1.6% [Zhang et al., 2007]. These deletions also involved three additional genes (HISPPD2A, CKMT1B, and STRC), but none were considered with any certainty to be responsible for the condition present in the patient. STRC was, however, associated with an autosomal recessive deafness when homozygous [Zhang et al., 2007]. These incidental findings were present in 3.6% of the cohort and represented 12.3% of the positive findings (Table II).

The Challenges in Array Results Interpretation and Genetic Counselling

While array CGH provided more positive diagnoses than possible by routine cytogenetics, the number of novel diagnoses and variants of uncertain significance provided a challenge to both the reporting laboratory and referring clinician.

Identification of well-established chromosome microdeletion and microduplication syndromes provided a firm diagnosis and, while some have only been described recently, the published literature and databases were usually sufficient to provide families with useful information about additional clinical assessments to do at the time of diagnosis, surveillance for late onset features, and prognosis. Diagnosis also allowed recurrence risk counseling and options for prenatal testing in future pregnancies.

Determining the clinical significance of novel microdeletions and microduplications that are likely to be pathogenic was more difficult. Factors taken into account when determining pathogenicity included whether the CNV occurred de novo, whether it contained genes associated with specific disorders/phenotypes and whether it had been reported previously in databases. Even when it had been reported previously, the number of similar cases may have been small, the size of the CNVs and the breakpoint coordinates in the cases may have varied significantly, phenotypic information may have been limited and the phenotypes of the reported cases with overlapping CNVs might have been variable. Deletions containing known disease genes were easier to interpret, as it can be assumed that there is haploinsufficiency for the genes they contain, especially when the disease gene was known to be pathogenic when haploinsufficient. Interpretation of duplications, even when they involved disease-related genes, was more difficult, as there was often little or no information about the likely functional effects of having three rather than two copies of the gene in question. These cases relied heavily on family studies to assist with interpretation.

An increasing number of CNVs have been recognized to create susceptibility to particular phenotypes rather than being the only, or even main, factor in pathogenicity. These CNVs typically show variable penetrance and variable expressivity. It was difficult to explain to the parents of an affected child how it is that their child has the disorder, while one of them, who has the same CNV, was only mildly affected, or was unaffected at the level of sensitivity of clinical detection. The challenges for genetic counseling were even greater. While the chance of a future child inheriting the CNV was clear, variability of penetrance and expressivity created impossible difficulties for parents who were left trying to make sense of complex and uncertain probabilistic information. They were in most cases willing to accept a risk of having a child with a mild phenotype but wished to avoid having a child with a serious phenotype. They also had to grapple with whether it is appropriate to use prenatal diagnosis when there was such uncertainty about the meaning of a test result which might show that the fetus has inherited the CNV. Further research will likely provide better founded risk figures and a greater understanding of the other genetic factors that determine penetrance and expressivity of specific CNVs.

Other scenarios that created diagnostic difficulty included the detection of duplications that may, or may not, disrupt the gene in question, CNVs identified on the X-chromosome in females and when it was not possible to obtain DNA from both parents to determine whether a CNV occurred de novo or was inherited.

Finally, array-CGH identified CNVs that provided information incidental to the original diagnostic question. For example, that the intellectually disabled child was a carrier of a recessive disorder (e.g., Deafness-Male infertility or Menkes disease), or was at risk of a dominant disorder (e.g., von Hippel Lindau disease or Brugada syndrome). In cases where there may have been implications for the child this created an obligation to recommend cascade testing.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

New CNVs are continuously injected into the population through mutations [Hastings et al., 2009; Itsara et al., 2009]. However, the cost to the population is a CNV load where the deleterious CNVs have public health implications. These are detectable using array CGH for patients with a range of clinical conditions, including those patients who present to the clinic with epilepsy as part of their clinical picture. Precise characterization of causative molecular defects can be extremely helpful to the health professional in counselling the family. Sensitivity of Array CGH is significantly higher than conventional cytogenetic testing often ending the family's diagnostic odyssey. That avoids further testing that might otherwise have been ordered in an ongoing search for a cause. Whilst the application of array CGH to diagnosis can often raise unanswered questions about the significance of a previously hidden CNV, it solves many more cases than was previously possible.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

We thank the Blood Team in Cytogenetics who triaged incoming cases (Trudy Hocking, Sarah Higgins, Janaky Samuel, Rebecca Buchheim, Alex Marks, Cathy Derwas, Rachel Coates, and Lucy Teicher), Sin Lay Kang in Molecular Genetics for DNA extraction, Kathy Cox, Louisa Sanchez, and Linda Burrows in Molecular Genetics for MLPA confirmation of deletions involving MECP2, L1CAM, and ATP7A and the many South Australian clinicians who referred cases to Cytogenetics for Array CGH.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  7. Acknowledgements
  9. Supporting Information

Additional supporting information may be found in the online version of this article.

ajmg_32114_sm_SuppTab1.doc50KSupplementary Table 1. CNVs of unknown clinical significance

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