Gene xxx (2013) xxx–xxx

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Array CGH analysis of a cohort of Russian patients withintellectual disability

Anna A. Kashevarova a,⁎, Lyudmila P. Nazarenko a, Nikolay A. Skryabin a, Olga A. Salyukova a,Nataliya N. Chechetkina a, EkaterinaN. Tolmacheva a, Elena A. Sazhenova a, PamelaMagini b, ClaudioGraziano b,Giovanni Romeo b, Vaidutis Kučinskas c, Igor N. Lebedev a

a Institute of Medical Genetics, Tomsk, Russiab University of Bologna, Bologna, Italyc Vilnius University, Department of Human and Medical Genetics, Vilnius, Lithuania

Abbreviations: ABLIM3, actin binding LIM protein famactin filament associated protein 1-like 1; AGA, aspartylgrelated cysteine peptidase; CGH, comparative genomic hycontactin 6; CNTN6, contactin 6; CNV, copy number variahumans using ensembl resources; EEG, electroencephalocrest derivatives expressed 2; HFE, hemochromatosis; HFE2; ING2, inhibitor of growth family,member 2; IQ, intelligeclass A domain containing 3;METTL4, methyltransferase lik(membrane-inserted); MRI, magnetic resonance imagingVIII-like 3 (E. coli); NEIL3, nei endonuclease VIII-like 3 (E.of central nervous system; PON1, paraoxonase 1; PON2, pabinding protein; SCRG1, stimulator of chondrogenesis(sodium/choline cotransporter), member 7; SLC7A7, sochromosomes flexible hinge domain containing 1; SnoN70 kDa subunit; SWAP70, SWAP switching B-cell comWAGR, Wilms tumor, aniridia, genitourinary anomalie⁎ Corresponding author at: Laboratory of Cytogen

Tel.: +7 3822 51 31 46; fax: +7 3822 51 37 44.E-mail address: anna.kashevarova@medgenetics.ru (A

0378-1119/$ – see front matter © 2013 Elsevier B.V. All rhttp://dx.doi.org/10.1016/j.gene.2013.11.029

Please cite this article as: Kashevarova, A.A.,http://dx.doi.org/10.1016/j.gene.2013.11.029

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 1 November 2013Available online xxxx

Keywords:Intellectual disabilityArray comparative genomic hybridization(array CGH)Copy number variation (CNV)

The use of array comparative genomic hybridization (array CGH) as a diagnostic tool in molecular genetics hasfacilitated the identification of many new microdeletion/microduplication syndromes (MMSs). Furthermore,this method has allowed for the identification of copy number variations (CNVs) whose pathogenic rolehas yet to be uncovered. Here, we report on our application of array CGH for the identification of pathogenicCNVs in 79 Russian children with intellectual disability (ID). Twenty-six pathogenic or likely pathogenic changesin copy number were detected in 22 patients (28%): 8 CNVs corresponded to known MMSs, and 17 were notassociated with previously described syndromes. In this report, we describe our findings and comment ongenes potentially associated with ID that are located within the CNV regions.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Genetic disorders underlie a significant number of cases of intellec-tual disability (ID). However, conventional karyotype analysis isnot able to detect small or cryptic aberrations less than 5–10 Mb insize, which may be clinically relevant. Overall, the detection rate ofchromosomal aberrations in patients with ID and/or congenitalmalformations using conventional karyotyping is approximately 9.5%(van Karnebeek et al., 2005). High-resolution genome-wide microarray

ily, member 3; ACAD10, acyl-CoA dehlucosaminidase; ASTN1, astrotactinbridization; CHERISH, grant of Euroption; DDX10, DEAD (Asp-Glu-Ala-Aspgram; EU, European Union; FJX1, fou, hemochromatosis; ID, intellectual dnce quotient; IRF1, interferon regulate 4;METTL4,methyltransferase like 4; NDC80, NDC80 kinetochore complecoli); NO, nitrogen oxide; OMIM, onliraoxonase 2; PON3, paraoxonase 3; qP1; SLC1A2, solute carrier family 1 (lute carrier family 7 (amino acid tr, Ski-related novel protein N; SUFUplex 70 kDa subunit; TGFβ, transfos and mental retardation syndromeetics, Institute of Medical Genetic

.A. Kashevarova).

ights reserved.

et al., Array CGH analysis of

technologies have been shown to increase the diagnostic output in IDpatients. Genome alterations that may cause the disabilities are detect-ed in up to 25% of patients with idiopathic syndromic ID (Hochstenbachet al., 2011; Rosenberg et al., 2006). However, CNVs are also frequentlyfound in apparently healthy individuals. Databases of normal and path-ogenic genome variations are available on the web and are extremelyvaluable tools for interpreting CNVs identified in patients (Databaseof Genomic Variants; Database of Chromosomal Imbalance andPhenotype in Humans Using Ensembl Resources). To our knowledge,

ydrogenase family, member 10; ADHD, attention-deficit hyperactivity disorder; AFAP1L1,1; CASP3, caspase 3, apoptosis-related cysteine peptidase; CASP3, caspase 3, apoptosis-ean Community's Seventh Framework Programme; CNS, central nervous system; CNTN6,) box polypeptide 10; DECIPHER, database of chromosomal imbalance and phenotype inr jointed box 1 (Drosophila); GLRA3, glycine receptor, alpha 3; HAND2, heart and neuralisability; IFN, interferon; IL17B, interleukin 17B; ING2, inhibitor of growth family, memberory factor 1; IRF2, interferon regulatory factor 2; LDLRAD3, lowdensity lipoprotein receptor;MMSs,microdeletion/microduplication syndromes;MMP14, matrixmetallopeptidase 14x component; NDC80, NDC80 kinetochore complex component; NEIL3, nei endonucleasenemendelian inheritance inman; PCR, polymerase chain reaction; PL CNS, perinatal lesionCR, quantitative PCR; SBF, SET domain binding factor; SCGN, secretagogin, EF-hand calciumglial high affinity glutamate transporter), member 2; SLC5A7, solute carrier family 5ansporter light chain, y + L system), member 7; SMCHD1, structural maintenance of, suppressor of fused homolog (Drosophila); SWAP70, SWAP switching B-cell complexrming growth factor beta; TNR, tenascin R; TRIM44, tripartite motif containing 44;.s, Russian Academy of Medical Sciences, 10 Nab. Ushaiki, 634050, Tomsk, Russia.

a cohort of Russian patients with intellectual disability, Gene (2013),

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267 different genomic loci have been associated with well-describedmicrodeletion/microduplication syndromes (MMSs) (Weise et al.,2012), but these figures are constantly updated.

The EU Seventh Framework Programme project “ImprovingDiagnoses of Mental Retardation in Children in Central EasternEurope and Central Asia through Genetic Characterisation andBioinformatics/-Statistics” (CHERISH)was specifically aimed at clinical-ly characterizing 1457 patients with ID and identify the underlyinggenetic etiologies. The Institute of Medical Genetics (Tomsk, Russia)

Table 1Patients with intellectual disability and potentially pathogenic CNVs.

Case # Age IQ Clinical features and brain examination results

1 5.8y 57 ADHD, speech delay, hyperdynamia.2 2.9y – Delay in motor milestones, motor stereotypy, abse

ADHD, autistic signs, convergent strabismus (paralateral nystagmus, hypermetropic astigmatism, lopear-shaped nose, narrow face, atactic gait.

3 3y – Speech delay, sporadic sounds, aggression, auto-aautistic signs, hydrocephalic skull, large ears, Cupiclinodactyly of the 5th finger, joint hypermobilitypoor posture, hypomyotonia, overweight, recurrenhydrocephalus.

4 8y 45 Motor stereotypy, dysarthria, aggression, ADHD, alarge ears, wide nasal bridge, big teeth, wide-spac

5 16y – Delay in motor milestones, motor stereotypy, systunderdevelopment of speech, anxiety, aggressiondolichocephaly, hypertensive hydrocephalus synddivergent strabismus, horizontal nystagmus, partiatrophy, large, dysplastic ears, large nose, slight faopened mouth, clinodactyly of 5th finger, scoliosisbrain malformation, the picture of idiopathic hypopost-parietal and occipital cortical areas.

6 7y 60 Delay in motor milestones, mumbled speech, hydhypertelorism, wide teeth, poor posture.

7 9y 47 Delay in motor milestones, motor skills deficiencyADHD, dolichocephalic skull, mongoloid slant, divprotruding ears, hypoplastic antihelix and antitragshort philtrum, large irregular teeth, large and smoperated hypospadias at the left, scoliosis, slouch,hypotonia; EEG: hydrocephalus; MRI: hypertensiv

8 10y 55 Dysarthria, ADHD, tower skull, frontal bossing, anepicanthus, wide nasal bridge, low-set ears, clinodhypertrichosis. MRI: lateral ventricles asymmetryfrom left ventricles, neurodystrophic focuses due

9 6y 50 Vocalization, pronounces single syllables, food fasautistic signs, hydrocephalus, abnormal hair growears, wide-spaced nipples, hypertrichosis, perinatawith disseminated microneurotic symptomatolog

10 4y – Aggressiveness, antimongoloid slant, small joint hthorax and spine deformation.

11 8y 50 Speech delay, aggressiveness, auto-aggressivenessmicrocephaly, small ears, gothic palate, irregular tabdominal obesity, undue fatigability.

12 5y 54 Delay in motor milestones, motor stereotypy, deladevelopment, dysarthria, echolalia, tower skull, coepicanthus, large protruding ears, gothic palate, smclinodactyly of the 5th finger, flat valgus, joint hypscoliosis, enuresis, recurrent infections.

13 10y 68 Perinatal hypotrophy of 2nd degree, hydrocephaliphiltrum, hypogonadism, abdominal obesity, sym

14 12y 45 Delay in motor milestones, absent speech, certainauto-aggressiveness, autistic signs, ADHD, protrudfeatures, triangular face, seizures.

15 6y 49 Delay in motor milestones, brachycephalic skull, mhypertensive-hydrocephalic syndrome, exophthalantimongoloid slant, astigmatism, bilateral diverglow-set ears, wide nasal bridge, maxillofacial dysomicrognathia, carp mouth, gothic palate, small teegrowth of the teeth, clinodactyly of the 5th fingerplano-valgus foot, cleft foot, hypotonia in the limbmicropenis, kyphoscoliosis, barrel chest, winged sseptal defect, atrial septal defect, moderate hepatochanges in liver, symptomatic focal epilepsy.

Footnote. Potentially pathogenic CNVs are shown in bold.

Please cite this article as: Kashevarova, A.A., et al., Array CGH analysis ofhttp://dx.doi.org/10.1016/j.gene.2013.11.029

became involved with this project with the selection of 206 Russianchildren with ID, who underwent clinical examination at the ClinicalGenetics service of the Institute. Array CGH was performed in a totalof 378 patients in the project, including 79 Russian patients.

Here, we summarize the CNVs identified in these Russianchildren with ID, describe potentially pathogenic variants not previ-ously associated with known microdeletion/microduplication syn-dromes, and provide details on candidate pathogenic genes withinthese regions.

Array CGH results (ISCN (2013) (Shaffer et al., 2013))

arr[hg18]11p13(35,137,000-36,292,000) × 1nce of speech,lytic strabismus),w-set ears,

arr[hg18]11q22.3107,970,000–108,140,000) × 1,21q21.1(17,060,000–17,309,000) × 1

ggression, ADHD,d's face, short neck,, nipple hypertelorism,t infections; EEG:

arr[hg18]11p15.4(9,718,000–9,967,000) × 1 dn,11p15.5(522,000–614,000) × 3

utism signs,ed nipples.

arr[hg18]18p11.32(2,322,000–2,672,000) × 3 pat

emic, mood swings,rome, ptosis,al optic nervecial asymmetry,; MRI: congenitaltrophy of the

arr[hg18]8p11.23p11.22(39,378,051–39,505,315) × 1,12q24.12q24.13(110,668,504–110,811,179) × 3 mat,15q11.2(18,741,716–20,010,618) × 1

rocephalus, arr[hg18]10q24.32(104,147,000–104,668,000) × 1

, fine dysarthria,ergent left strabismus,us, large mouth,all joint hypermobility,hinge-like gait,e syndrome.

arr[hg18]3p26.3(701,645–1,467,721) × 3 pat,8p11.23p11.22(39,378,051–39,505,315) × 1,15q11.2(18,741,716–20,010,618) × 1

timongoloid slant,actyly, hair nevus,as a result of outflowto hypoxia.

arr[hg18]3p26.3(1,164,000–1,533,000) × 1

tidiousness,th, large protrudingl encephalopathyy.

arr[hg18]2q37.3(242,579,000–242,596,000) × 1,6p22.2(25,677,000–26,393,000) × 3

ypermobility, arr[hg18]5q33.1(148,620,000–148,735,000) × 3 mat

, mood swings,eeth, hypogonadism,

arr[hg18]8p11.23p11.22(39,378,051–39,505,315) × 1,14q11.2(20,767,632–22,722,130) × 3 dn

yed psychoverbalarse hair,all irregular teeth,ermobility,

arr[hg18]1q25.1q25.2(172,269,000–178,409,000) × 3 pat

c skull, shortptomatic focal epilepsy.

arr[hg18]7q21.3(94,769,000–94,901,000) × 3

sounds, aggressiveness,ing ears, thin facial

arr[hg18]2q12.3(107,971,000–108,435,000) × 1

icrocephaly,mos, eye hypertelorism,ent strabismus,stosis, mandibularth, irregular, single palmar crease,s, shawl scrotum,capula, ventricularmegaly, diffuse

arr[hg18]4q34.1q34.3(173,195,432–179,098,955) × 3,4q34.3q35.1(179,503,640–185,418,489) × 4,4q35.1q35.2(185,485,228–191,133,668) × 1,6p21.32(32,593,151–32,673,042) × 3,15q11.2(chr15:18,741,716–20,010,618) × 3

a cohort of Russian patients with intellectual disability, Gene (2013),

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2. Materials and methods

2.1. Patient data

Patients were examined according to the standardized criteriaagreed upon by the CHERISH consortiummembers. Descriptions of clin-ical findings are presented in Table 1 and Supplementary Tables 1 and 2.Seventy-nine patients were selected to undergo array CGH analysisusing the following criteria: the absence of large chromosomal rear-rangements, metabolic disorders, and some known monogenic syn-dromes causative of ID; and the presence of congenital malformations.Eleven of these patients were 3–5 years old and showed developmentaldelay; the other 68 patients were older than 5 years of age and had IDdefined as an IQ lower than 70. For the segregation analysis, peripheralblood of both parents was available for 30 patients, of either themotheror father for 26 patients, and of neither parent for 23 patients.

The following real-time quantitative PCR was performed for 15patients, in whom the likely pathogenic CNVs not associated withknown microdeletion/microduplication syndromes were identified.

2.2. Array CGH

Array CGH analysis was performed using the Agilent 44 K and 60 Karrays with the resolutions of about 75 kb and 41 kb, respectively,(Human Genome CGH Microarray, Agilent Technologies, Santa Clara,CA, USA) according to themanufacturer's recommendations. Data anal-ysis was performed using Cytogenomics Software (v. 2.0.6.0) (AgilentTechnologies, USA) and the publicly available Database of GenomicVariants and Database of Chromosomal Imbalance and Phenotype inHumans Using Ensembl Resources. The functions of genes located inthe regions of genomic imbalances were retrieved from databasessuch as NCBI Gene Database, GeneCards and OMIM.

2.3. Real-time quantitative PCR

Specific target sequences were selected for real-time quantitativePCR (qPCR) using Primer 3 software Primer3 (v. 0.4.0). Two primerpairs were created for each pathogenic genomic region.

3. Results and discussion

Samples from 79 children with intellectual disability and congenitalmalformations were analyzed using Agilent 44 K and 60 K arrays.Examples of array CGH profiles are shown in the figures. Clinicaldescriptions of patients and array CGH results are presented in Table 1and Supplementary Tables 1 and 2.

Array CGH analysis did not identify any unbalanced chromosomalaberrations in 35 of the patients (44%) (Supplementary Table 1).Copy number variations that were observed in the remaining 44patients were first classified using the Database of Genomic Variantsand Gene Database. Twenty-two children carried only benign CNVs(Supplementary Table 1). A total of 26 pathogenic or likely pathogenicCNVs were detected in the other 22 affected children (28%). Amongthese CNVs, microdeletions associated with known syndromes wereidentified in seven patients, allowing for a definite diagnosis in 9% ofthe patients in this study. These diagnoses were Cri du Chat syndrome,15q24 microdeletion syndrome (two cases), 16p11.2 microdeletionsyndrome (two cases), 16p12.2 microdeletion syndrome, and 22q11.2microdeletion syndrome (Supplementary Table 2).

In 15 patients, 17 CNVs that had not previously been associatedwithany known syndrome were detected. Below, we will focus on these 15cases and the candidate genes located in the regions of copy numbervariation that may lead to pathology. Potentially pathogenic candidategenes were selected based on the role of genes in various processes,such as brain development and synaptic function, as listed in GeneDatabase (Table 1).

Please cite this article as: Kashevarova, A.A., et al., Array CGH analysis ofhttp://dx.doi.org/10.1016/j.gene.2013.11.029

Case #1

A boy with a 1.155 Mb microdeletion at 11p13 (SupplementaryFig. 1). Although the deleted region seems to correspond to the locationindicated inWilms tumor, aniridia, genitourinary anomalies andmentalretardation syndrome (WAGR) 11p13 deletion syndrome, the preciselocation of the microdeletion in this patient does not overlapwith the deletion given in DECIPHER (11:35180424–36335424 vs.11:31806339–32457087 — hg19). Thus, it is not surprising that theboy does not have the typical WAGR symptoms. However, the probandis still mentally retarded, and there are several genes within the regionof the microdeletion that may be associated with intellectual disability.For example, the SLC1A2 protein is responsible for glutamate transport.The accumulation of extracellular glutamate causes a dysfunction in cal-cium homeostasis, increased production of NO, free radicals, and cyto-toxic transcription factors, and activation of proteases. These factorscan result in neuronal damage, leading to neurodegenerative disease,inflammation, or ischemia development (Kim et al., 2011). The functionof FJXI, the product of another gene in this region, remains unknown inhumans; however, this gene product has been shown to regulate den-drite extension in rodents (Ashery-Padan et al., 1999). The product ofa third gene in this region, TRIM44,may play a role in neuronal differen-tiation andmaturation [GeneCards]. The product of a fourth gene in thisregion, LDLRAD3, participates in the proteolysis of an amyloid precursorprotein, leading to beta amyloid formation; this fiber form is the prima-ry component of amyloid plaques found in the brain of Alzheimer's dis-ease patients (Ranganathan et al., 2011). Themicrodeletion observed inthis patient was confirmed by real-time PCR. Parental DNA was notavailable for analysis.

Case #2

A boy with a 170 kb microdeletion at 11q22.3 (SupplementaryFig. 2). The only gene located within this region is DDX10. This gene en-codes aDEADboxprotein,which are putative RNAhelicases that are im-plicated in a number of cellular processes involving the alteration ofRNA secondary structure such as translation initiation, nuclear and mi-tochondrial splicing, and ribosome and spliceosome assembly. Based ontheir distribution patterns, somemembers of this family are believed tobe involved in embryogenesis, spermatogenesis, cellular growth, andcell division [NCBI Gene Database]. The microdeletion was confirmedby real-time PCR. Parental DNA was not available for analysis.

Case #3

A boy with a 249 kb microdeletion at 11p15.4. This region overlapswith the locus indicated in Charcot–Marie-Tooth disease, type 4B2.Among the genes located within the deletion region, SWAP70 andSBF2 are potentially relevant. SWAP70 is the switching B-cell complex70 kDa subunit that has been shown to be involved in pathogenicmechanisms of multiple sclerosis, a chronic inflammatory demyelinat-ing and neurodegenerative disease of the central nervous system(Erdağ et al., 2012). Mutations in the SBF2 gene have been associatedwith Charcot–Marie-Tooth disease, type 4B2: a type of demyelinatinghereditary motor and sensory neuropathy characterized by abnormalfolding of myelin sheaths (OMIM #604563) [Online MendelianInheritance in Man]. Real-time PCR confirmed the de novo origin ofthis microdeletion.

Case #4

A boy with a 351 kb microduplication at 18p11.32. There are threeinteresting genes located within this region: METTL4, NDC80, andSMCHD1. METTL4 is a probable methyltransferase whose function isnot yet fully described. However, methylation is an important andwidespread epigenetic mechanism; thus, this gene may regulate the

a cohort of Russian patients with intellectual disability, Gene (2013),

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expression of other pathogenic genes. The other two genes in this regionparticipate in the maintenance of genome integrity such that copynumber changes in these genes may lead to genome instability, whichis known to be associated with brain disorders (Faggioli et al.,2011; Bosshard et al., 2012). NDC80 encodes a component of theNDC80 kinetochore complex, which functions to organize and stabi-lize microtubule–kinetochore interactions and is required for properchromosome segregation [NCBI Gene Database]. SMCHD1 encodes aprotein that contains a hinge region domain found in members ofthe SMC (structural maintenance of chromosomes) protein family[NCBI Gene Database]. The microduplication was inherited from anapparently healthy father.

Case #5

A boy with a 142 kb microduplication at 12q24.12–q24.13. The im-portant gene in this region is ACAD10, which encodes a mitochondrialdehydrogenase. This gene is highly expressed in the fetal brain andmay be essential for central nervous systemdevelopment at early stagesof ontogenesis (He et al., 2011). The microduplication was inheritedfrom an apparently healthy mother.

Case #6

A girl with a 521 kb microdeletion at 10q24.32. The gene of interestin this region is SUFU. This gene encodes a negative regulator of theSonic hedgehog signaling pathway, which controls cellular prolifera-tion. Mutations in this gene are associated with brain tumors [NCBIGene Database]. The microdeletion was confirmed by real-time PCR.The mother's karyotype contained two copies of the examined locusand therefore did not have the deletion. Paternal DNAwas not availablefor analysis.

Case #7

Aboywith a 766 kbmicroduplication at 3p26.3. The only gene locat-edwithin this region is CNTN6, which encodes contactin 6. Significantly,contactin 6 is a neuronal membrane protein that functions as a cell ad-hesionmolecule andmay take part in the formation of axon connectionsin the developing nervous system. CNTN6 and other members ofthis family have been identified as potentially pathogenic genes inneurodevelopmental disorders. These genes have been suggested toparticipate in pathways important for correct brain development(Zuko et al., 2011). The microduplication was inherited from an appar-ently healthy father. In contrast to Case #7, a 369 kb microdeletion at3p26.3 overlapping with CNTN6 was also identified in Case #8. Themicrodeletion was confirmed by real-time PCR. Parental DNA was notavailable for analysis. Although the chromosomal aberrations in thesecases were the reverse of one another, both patients had moderateintellectual disability, dysarthria, and attention-deficit hyperactivitydisorder (ADHD).

Case #9

A boy with a 716 kb microduplication at 6p22.2 (SupplementaryFig. 3). One of the genes within this region that has gained attentionin neuroscience is HFE, which regulates ion homeostasis. Muta-tions in this gene are commonly associated with hereditaryhemochromatosis — an autosomal recessive disorder of ionmetabolism (OMIM #235200) [Online Mendelian Inheritance inMan]. It has been recently shown that the H67D polymorphismin HFE in mice impacts brain ion homeostasis, creates an environ-ment favorable for oxidative stress, and predisposes mice to neu-rodegenerative disorders (Nandar and Connor, 2011; Nandaret al., 2013). The other gene of interest in this region is SCGN,which encodes secretagonin. SCGN is expressed in neurons in

Please cite this article as: Kashevarova, A.A., et al., Array CGH analysis ofhttp://dx.doi.org/10.1016/j.gene.2013.11.029

the embryonic nervous system and is confined to differentiatedcells in the adult brain. Secretagonin may be implicated in thecontrol of neuronal turnover and differentiation. This protein isalso re-expressed in brain tumors. In addition, secretagonin mayfunction as a Ca2+ sensor regulating the exocytosis of neuro-transmitters, neuropeptides, and hormones (Alpár et al., 2012).This microdeletion was confirmed by real-time PCR. ParentalDNA was not available for analysis.

Case #10

A boy with a 115 kb microduplication at 5q33.1 (SupplementaryFig. 4). The genes of interest located within this region are ABLIM3,AFAP1L1, and IL17B. The ABLIM3 gene is a member of the actin-bindingLIM protein family, which plays important roles in biological processessuch as embryonic development, cell lineage determination, and cancerdifferentiation. This gene is expressed at high levels in muscle and neu-ronal tissue (Barrientos et al., 2007). AFAP1L1, actin filament-associatedprotein 1-like 1, is expressed in the granule cell layer, glial cells,and Purkinje cells (Snyder et al., 2011). IL17B is also highly expressedin the central nervous system, where it has been suggested toparticipate in the local inflammatory response (Moore et al., 2012).Themicroduplicationwas inherited fromanapparently healthymother.

Case #11

A boy with a 1.95 Mb microduplication at 14q11.2 (SupplementaryFig. 5). There are two genes attracting particular attention in this region:SLC7A7 and MMP14. The SLC7A7 protein transfers cationic and largeneutral amino acids from the cell to the extracellular space. Mutationsin the SLC7A7 gene are found in patients with lysinuric protein intoler-ance (OMIM #222700) [Online Mendelian Inheritance in Man], amulti-organ diseasewith a variety of clinical symptoms, includingmod-erate intellectual disability (Font-Llitjos et al., 2009). IncreasedMMP14 expression has been observed in different cancers, includingbrain tumors [GeneCards]. Real-time PCR confirmed the de novo ori-gin of this microduplication.

Case #12

A boy with a 6.14 Mb duplication at 1q25.1–q25.2 (SupplementaryFig. 6). The TNR gene locatedwithin this region is an extracellularmatrixprotein expressed primarily in the central nervous system. This proteinregulates cell proliferation and migration, fate determination, axonalpathfinding, myelination, and synaptic plasticity. TnR expressionis upregulated after spinal cord injury in the lesion area, thus implicat-ing TnR in inhibiting axonal re-growth after injury (Jakovcevskiet al., 2013). The other important gene in this region is ASTN1, which en-codes astrotactin. Astrotactin is a neuronal adhesion molecule requiredfor the glial-guided migration of young postmitotic neuroblasts incortical regions of the developing brain, including the cerebrum, hippo-campus, cerebellum, and olfactory bulb [NCBI Gene Database]. Themicroduplication was inherited from an apparently healthy father.

Case #13

A boy with a 132 kb microduplication at 7q21.3 (SupplementaryFig. 7). This region contains a cluster of three related paraoxonasegenes: PON1, PON2, and PON3. Polymorphisms in the PON1 gene havebeen shown to be a risk factor in coronary artery disease [NCBI GeneDatabase]. Mutations in the PON2 genemay be associatedwith vasculardisease and diabetes [NCBI Gene Database]. The microduplication wasconfirmed by real-time PCR. There were two copies of the studiedlocus in the mother's karyotype, corresponding to the norm. PaternalDNA was not available for analysis.

a cohort of Russian patients with intellectual disability, Gene (2013),

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Case #14

A boywith a 464 kbmicrodeletion at 2q12.3 (Supplementary Fig. 8).One gene in this region, SLC5A7, is an Na(+)- and Cl(−)-dependenthigh-affinity transporter that mediates the uptake of choline for acetyl-choline synthesis. Acetylcholine is a neurotransmitter in the central andperipheral nervous system that regulates a variety of autonomic, cogni-tive, and motor functions. This gene was shown to be associated withADHD (English et al., 2009). This microdeletion was confirmed byreal-time PCR. Parental DNA was not available for analysis.

Case #15

A boy with a 5.9 Mb duplication at 4q34.1–q34.3, a 5.9 Mbtriplication at 4q34.3–q35.1, and a 5.6 Mb deletion at 4q35.1–q35.2(Supplementary Fig. 9). The identified rearrangements are veryextensive and include several amplifications and deletions. Thegenes of particular interest localized within the duplicated regionsare SCRG1, HAND2, GLRA3, NEIL3, AGA, and ING2. The SCRG1 gene isassociated with neurodegenerative changes observed in transmissi-ble spongiform encephalopathies. SCRG1 is abundantly expressedin the central nervous system of human adults. This gene is also dif-ferentially expressed in schizophrenia patients in the postmortemdorsolateral prefrontal cortex (Vawter et al., 2006). The HAND2gene controls cardiac morphogenesis and probably mediates acongenital heart defect. GLRA3 encodes the alpha-3 subunit ofthe neuronal glycine receptor. NEIL3 is a glycosylase initiatingthe first step in base excision repair, which is the major pathwayfor the removal of oxidative DNA lesions. Neil3-knockout miceshowed impaired proliferation of neural stem cells and reducedDNA repair activity (Regnell et al., 2012). The AGA gene encodes anaspartylglucosaminidase that is the key enzyme in the catabolismof N-linked oligosaccharides on glycoproteins. Mutations in thisgene lead to aspartylglucosaminuria, a lysosomal storage diseasecharacterized by motor and mental retardation (OMIM #208400)[Online Mendelian Inheritance in Man]. ING2 is a chromatin remod-eling protein that promotes TGFβ-induced transcription and cellcycle arrest. Through its interaction with SnoN, this protein may beimportant for postmitotic neuron functioning (Bonni and Bonni,2012).

The deletion at 4q35.1–q35.2 contains the following genes ofinterest: IRF2 and CASP3. The IRF2 gene encodes interferon regulato-ry factor 2, which, together with IRF1, controls interferon responsesand is involved in oncogenesis in fibroblasts. Park and colleaguesshowed that these proteins may participate in the regulation ofIFN-beta and IFN-inducible genes and that IRF2 may function as anactivator and repressor in CNS-derived cells (Park et al., 1998).Caspase-3 is a death-mediating protease that has been implicatedin neurodegenerative processes. In addition, CASP3 has been sug-gested to take part in the modulation of synaptic function (Snigdhaet al., 2012). The duplication, triplication, and deletion were allconfirmed by real-time PCR. In the mother's genome, the numberof copies of the examined loci did not differ from the norm, i.e., shedid not have these aberrations. Paternal DNA was not availablefor analysis.

In conclusion, the detection rate of chromosomal aberrations in ourgroupof patients appeared to be 28%,which is in agreementwith the re-ported 25% (Rosenberg et al., 2006). There were 7 patients with CNVscorresponding to known syndromes and 15 patients with 17 CNVsnot previously associated with any microdeletion/microduplicationsyndrome, which are described in this paper. One microdeletion andone microduplication were shown to be de novo. The other fivemicroduplications were inherited either from an apparently healthymother or father. For the remaining eight patients, it was not possibleto perform the segregation analysis due to incomplete families ororphanhood. That CNV is de novo or inherited is of particular importance

Please cite this article as: Kashevarova, A.A., et al., Array CGH analysis ofhttp://dx.doi.org/10.1016/j.gene.2013.11.029

for the detection of the diagnostic and prognostic significance of therearrangement, i.e., the de novo CNV is likely to be pathogenic, whilethe inherited CNV might require additional factors (genetic orenvironmental) to cause a disease, or it could be benign.

Here, we have also identified potentially pathogenic genes locatedwithin the 17 determined rearrangements. The genes were selectedaccording to their function and implications in different processes inthe brain, using publically available databases. Further determiningthese genes' contributions to intellectual disability could be achievedby sequencing them and by searching for mutations in other patientswith ID.

More detailed descriptions of patients and identification of poten-tially causative CNVs and genes in the CNV regions will be necessaryto identify new syndromes and improve diagnoses of intellectualdisabilities.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.gene.2013.11.029.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This study was supported by the European Community's SeventhFramework Programme, CHERISH (project 223692), and by the FederalProgram of theMinistry of Education and Science of Russian Federation(grant 8727).

Web resources

• Database of Chromosomal Imbalance and Phenotype in HumansUsing Ensembl Resources (http://decipher.sanger.ac.uk)

• Database of Genomic Variants (http://projects.tcag.ca/variation/)• GeneCards (http://www.genecards.org/)• NCBI Gene Database (http://www.ncbi.nlm.nih.gov/gene)• Online Mendelian Inheritance in Man (http://www.omim.org/)• Primer3 (v. 0.4.0) (http://frodo.wi.mit.edu/primer3/)

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