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Page 1: CTNND2--a candidate gene for reading problems and mild intellectual disability

ORIGINAL ARTICLE

CTNND2—a candidate gene for reading problemsand mild intellectual disabilityWolfgang Hofmeister,1,2 Daniel Nilsson,1,2,3,4 Alexandra Topa,5

Britt-Marie Anderlid,1,2,3 Fahimeh Darki,6 Hans Matsson,7 Isabel Tapia Páez,7

Torkel Klingberg,6 Lena Samuelsson,5 Valtteri Wirta,8 Francesco Vezzi,9 Juha Kere,7,10

Magnus Nordenskjöld,1,2,3 Elisabeth Syk Lundberg,1,2,3 Anna Lindstrand1,2,3

▸ Additional material ispublished online only. To viewplease visit the journal online(http://dx.doi.org/10.1136/jmedgenet-2014-102757).

For numbered affiliations seeend of article.

Correspondence toDr Anna Lindstrand,Department of MolecularMedicine and Surgery,Karolinska Institutet,Stockholm S-171 76, Sweden;[email protected]

Received 17 September 2014Revised 31 October 2014Accepted 12 November 2014

To cite: Hofmeister W,Nilsson D, Topa A, et al. JMed Genet Published OnlineFirst: [please include DayMonth Year] doi:10.1136/jmedgenet-2014-102757

ABSTRACTBackground Cytogenetically visible chromosomaltranslocations are highly informative as they can pinpointstrong effect genes even in complex genetic disorders.Methods and results Here, we report a mother anddaughter, both with borderline intelligence and learningproblems within the dyslexia spectrum, and twoapparently balanced reciprocal translocations: t(1;8)(p22;q24) and t(5;18)(p15;q11). By low coverage mate-pairwhole-genome sequencing, we were able to pinpoint thegenomic breakpoints to 2 kb intervals. By directsequencing, we then located the chromosome 5pbreakpoint to intron 9 of CTNND2. An additional casewith a 163 kb microdeletion exclusively involvingCTNND2 was identified with genome-wide arraycomparative genomic hybridisation. This microdeletion at5p15.2 is also present in mosaic state in the patient’smother but absent from the healthy siblings. We theninvestigated the effect of CTNND2 polymorphisms onnormal variability and identified a polymorphism(rs2561622) with significant effect on phonologicalability and white matter volume in the left frontal lobe,close to cortical regions previously associated withphonological processing. Finally, given the potential roleof CTNND2 in neuron motility, we used morpholinoknockdown in zebrafish embryos to assess its effects onneuronal migration in vivo. Analysis of the zebrafishforebrain revealed a subpopulation of neurons misplacedbetween the diencephalon and telencephalon.Conclusions Taken together, our human genetic andin vivo data suggest that defective migration ofsubpopulations of neuronal cells due tohaploinsufficiency of CTNND2 contribute to the cognitivedysfunction in our patients.

INTRODUCTIONIn a small subset of patients, apparently balancedstructural chromosome rearrangements may associ-ate with neurocognitive problems ranging frommild (attention deficit hyperactivity disorder, dys-lexia) to severe (within the autism spectrum disor-ders and intellectual disability),1 suggestingpositions for susceptibility genes resulting fromcryptic abnormalities in the vicinity of the break-points, gene disruption at the breakpoint or pos-ition effects.2 Fine mapping of chromosomerearrangements has often resulted in the identifica-tion of novel candidate genes associated with differ-ent neurodevelopmental symptoms,3 4 and two of

the dyslexia candidate genes, DYX1C1 and ROBO1,were identified by this approach.5 6

The armadillo repeat family member CTNND2(delta catenin) is expressed almost exclusively in thebrain7 and is a component of the adherens junctioncomplex.8 9 The disregulation of cell–cell interac-tions at these junctions by cadherins andcadherin-associated proteins has been shown to beinvolved in a wide spectrum of genetic disordersaffecting various organ systems from the skin to thecentral nervous system (CNS). In the CNS, deletionof several cadherin and cadherin-related genes hasbeen linked to various cognitive disorders, such asautism, schizophrenia and bipolar disorder (for areview, see El-Amraoui and Petit10). Most recently,the protocadherins, FAT4 and DCHS1, have beenlinked to Van Maldergems syndrome,11 an auto-somal disorder with intellectual disability andimpaired cortical development as one of thecharacteristics. In humans, CTNND2 is localised onchromosome 5p15.2, in a genome region associatedwith the mental retardation syndrome, Cri-du-Chat,and recently, Asadollahi et al12 reported one patientand retrieved a further four from the literature andonline databases with intragenic heterozygousexonic deletions of CTNND2 and mild intellectualdisability. The importance of CTNND2 in neuro-cognitive functions has previously been demon-strated by multiple in vivo and in vitro studies.Knockout mice show impaired spatial learningbehaviour13 despite normal basal levels of synaptictransmission and synaptic ultrastructure. At birth,these mice had normal dendritic complexity, spinedensity and cortical responsiveness, but they rapidlyexperienced progressive dendritic retraction, areduction in spine density and stability, and 5 weeksafter birth, a reduced cortical responsiveness.14 15

More recently, transient palmitoylation of Ctnnd2has been shown to be increased following enhancedsynaptic plasticity, increasing its interaction withcadherin at the synapse.16 Knockout mice also showan increase in spine and synapse density.17 Whileother studies have suggested a potential role forCTNND2 in neuronal migration based on both thedynamic expression pattern of Ctnnd2 in the mousecortex14 and in vitro studies where overexpressionled to an increase in motile cell behaviour.9 18

Here, we use a combination of low coveragemassive parallel whole-genome sequencing andmicroarray analysis to identify loss-of-function

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mutations in CTNND2 in three individuals from two unrelatedfamilies who show borderline to moderate intellectual dysfunc-tion and specific problems with reading. We also demonstratethat a common CTNND2 polymorphism affects both phono-logical ability and white matter volume in normally developingchildren and young adults. Finally, transient knockdown in zeb-rafish embryos result in ectopic neurons in the zebrafishforebrain.

MATERIALS AND METHODSParticipantsWritten informed consent was obtained for all participants, andall protocols were approved by the local ethical boards inStockholm (Sweden).

Patients 1 and 2Patient 1, a woman, was the first and only child to non-relatedparents (figure 2). Her mother (patient 2) had previously hadone early miscarriage and experienced an intrauterine death inweek 38 of her gestation. No karyotyping had been performed.The maternal grandmother had had two early miscarriages andcommitted suicide. Some psychiatric problems were alsodescribed in a sister of the maternal grandmother, but other-wise, the family history was uneventful. Prenatal karyotyping inpatient 1 revealed two apparently balanced translocations, alsopresent in the mother (patient 2). The translocations were notpresent in the maternal grandfather or the maternal aunt;however, the maternal grandmother was not available for ana-lysis. Phenotypical data are summarised in table 1.

Table 1 Clinical symptoms of patients with structural abnormalities affecting CTNND2

Patient 1 Patient 2 Patient 3

Decipher ID N/A N/A 275504Gender F F MAge at lastevaluation

29 years 68 years 14 years

Measurements atlast evaluation

L: 166 cm (−0.25 SD) L: 163 cm (−0.75 SD) L: 175 cm (+1 SD)W: 60 kg (+0.8 SD)OFC: 58 cm (+2 SD)

CTNND2 mutation Translocation breakpoint Translocation breakpoint Deletion of exons 12–18; out of frame (hg19)Inheritance Maternal No information Maternal

(mother mosaic)Weeks of gestation Term No information TermBirth measurements BW: 3520 g (M)

BL: 52 cm (+1 SD)No information No information

Facial features None None Long and narrow face with flat malar regionsTelecanthusDown-slanted palpebral fissuresBilateral ptosisMildly everted lower eyelidsMicroretrognathiaLarge and beaked nose with deviated septumSmall mouth with prominent lower lipHigh palateCrowded teeth

Developmentalmilestones

Normal motor milestones.Late language development. Speech therapy wasstarted at 3 years of age.

Normal motor milestones. No information

IQ 13 years of school (went to special class grades4–9).Self-reported difficulties with reading.WISC-III at age 17 years gave an uneven profilewith the verbal result age appropriate and thenon-verbal well below average (at the level of a9-year-old).

Spent 2 years in 4th grade and3 years in high school.Self-reported difficulties withreading.Good at learning to speak newlanguages

Mild intellectual disability.Mainstream school from age 11–14 years with need ofpersonal assistant.Self reported difficulties with reading.Learned to speak Swedish fluently after moving to Swedenat age 11 years.WISC-IV at age 14 years 10 m gave an uneven profile withthe lowest performance on non-verbal tests (at the level ofan 8.5 year-old).

Occupation At age 29 years, she works as a nanny at a daycare centre, and plans to go back to university tobecome a preschool teacher.

Retired. Worked for 40 years asa receptionist and handling ofthe cash register.

N/A

Neurologicalmanifestations

Attention deficit None Pervasive developmental disorder with difficulties withsocial-emotional behaviour and impulse control. Does notfulfil criteria for autism or autism spectrum disorder.

MRI N/A N/A NormalOther features Myopia (−3.75/−4; Glasses from grade one)

Normal body proportionsMyopia (−1.5/−1.5)Normal body proportions

MyopiaMarfanoid habitusBroad hands with prominent metacarpo-phalangeal jointsProximal inserted thumbsLong fingers with joint laxityHyperextended proximal interphalangeal jointsCamptodactyly of distal interphalangeal jointsBroad feet with short and broad big toes

BL, birth length; BW, birth weight; L, length; OFC, occipital frontal circumference; W, weight; WISC, Wechsler Intelligence Scale for children.

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Patient 3Patient 3 is a boy of central Asian origin, possibly with distantlyrelated parents originating from a small community of 5000individuals. He is the fourth child and he has five healthy sib-lings (four brothers and one sister). There are no other familialcases with a similar phenotype. The family moved to Swedenwhen the boy was 11 years old. The mother and the siblingswere not available for detailed clinical assessment, but they arereported to be healthy and with a normal cognitive develop-ment. Phenotypical data are summarised in table 1.

The Nynäshamn cohortA cohort of 76 (41 males and 35 females) typically developingchildren and young adults aged 6–25 years from the populationregistry in Nynäshamn, Sweden, were included in our behav-ioural imaging longitudinal study. The sample recruitment wasdone randomly with equal gender distribution in nine agegroups (6, 8, 10, 12, 14, 16, 18, 20 and 25 years). The exclu-sion criteria were: first language other than Swedish, vision orhearing impairment and neurological or neuropsychiatric disor-ders with exception of dyslexia or ADHD. Based on availablereports of parents, in 88.7% of the cases, both the participantsand their parents were born in Sweden, 9.3% had at least oneparent born outside Sweden but within Europe and the remain-ing 2% had one or both parents born outside Europe. Informedconsent was provided by the participants or by the parents forchildren younger than 18 years of age. The study was approvedby the local ethics committee of the Karolinska UniversityHospital, Stockholm, Sweden. The longitudinal data collectionsfor both behavioural and imaging assessments were conductedon three time-points, each 2 years apart.

Mate-pair sequencingTo pinpoint the exact positions of the chromosome breaks, weused whole-genome sequencing of the index patient. Librarieswere prepared using Illumina’s Nextera Mate-Pair SamplePreparation Kit according to the manufacturer’s instruction fora gel-free preparation of 2 kb effective insert size library (sizedistribution mode 2 kb). The libraries were sequenced on anIllumina 2500 sequencer (2×100 bp) resulting in a total of113 137 889 sequence pairs and 226 275 778 reads, giving anaverage raw coverage depth of 5×. Raw sequence reads werebase-called using CASAVA RTA 1.8. Following Illumina guide-lines for mate-pair postprocessing (http://res.illumina.com/documents/products/technotes/technote_nextera_matepair_data_processing.pdf), adapter sequences were removed usingTrimmomatic V.0.32. The remaining pairs were aligned to thehg19 human reference genome sequence using bwa 0.7.4-r385resulting in a 3× mapping coverage. After linker removal, 62%of total reads remain (n=140 133 690/226 275 778) and 58%of total reads were mapped (n=131 856 943/226 275 778).Read mapping was processed using in-house software (https://github.com/vezzi/FindTranslocations) implementing a slidingwindow analogue of a previously published procedure.19

Samtools20 and custom awk scripts were used to process cover-age tracks for visualisation with Circos.21 We previouslymapped the four chromosomal breakpoints with FISH to 81–218 kb windows,22 and this information was helpful in optimis-ing the bioinformatic analysis.

Breakpoint junction PCRBreakpoint PCR was performed by standard methods usingphusion taq (Thermo Scientific, Pittsburgh, Pennsylvania, USA)

and run out on an agarose gel. Specific products not present incontrol samples were Sanger sequenced at the KI gene corefacility. Primers are given in online supplementary table S1.

Affymetrix SNP arrayThe CNV detection in patient 3 was performed with theAffymetrix (Santa Clara, California, USA) whole-genome humanSNP 6.0 array containing 1.8 million probes with an averagespacing of 3.8 kb. All experiments were performed according tothe manufacturer’s recommendations with minor modifications.The data analysis was performed using the Genotyping Consoleand the chromosome analysis suite software.

Genotyping of the Nynäshamn cohortExtraction of genotyped SNPs overlapping with the genomicregion of CTNND2 (RefSeq annotation, hg19) from whole-genome human SNP 6.0 array (Affymetrix) data, pruning andfiltering was performed using PLINK V.1.07.23 To removeredundant markers, we selected the SNPs overlapping CTNND2that also fulfilled the criteria of Hardy–Weinberg equilibrium>0.001, minor allele frequency >10% and pair-wise linkagedisequilibrium of r2<0.3. The pruning process in PLINK wasmade using the command–indep–pairwise with a pairwise geno-typical correlation of SNPs in a sliding window of 50 SNPsshifting five SNPs in every step. After pruning, 37 out of 267SNPs remained.

Image acquisition and processingT1-weighted spin echo scans were collected using a three-dimensional (3D) magnetisation prepared rapid gradient echosequence with repetition time (TR)=2300 ms, echo time (TE)=2.92 ms, field of view of 256×256 mm2, 256×256 matrixsize, 176 sagittal slices and 1 mm3 isotropic voxel size.Generalised autocalibrating partially parallel acquisition withacceleration factor of 2 was used to speed up the acquisition.

Scanning was repeated three times with the same parameters.Voxel-based morphometry was performed on the structural datacollected across all three rounds of data using statistical paramet-ric mapping (SPM8), Diffeomorphic Anatomical RegistrationThrough Exponentiated Lie Algebra toolbox. This method seg-mented the brain into grey matter, white matter and cerebro-spinal fluid. The white matter-segmented images were thensmoothed with an 8 mm Gaussian kernel to be used in SNPassociation assessments.

SNP association analysesAs the underlying cause of the difficulties in dyslexia often canbe traced to phonological processes,24 25 we assessed the effectof genotype on performance in a phonological choice task. Thistask involves non-word and word reading (and the child shoulddecide which was the word), which requires phonologicaldecoding of the non-words. The test is adjusted for age groupsof 6–10 years and ≥12 years, respectively. Basic association ana-lysis was initially performed on the first assessment of perform-ance data using PLINK V.1.0723 with sex and handedness ascovariates. Three SNPs, rs2561622 (for group 6–10 years)together with rs16901339 and rs26149 (both ≥12 years) werefound in significant association with phonological choice taskand were selected for a subsequent association analysis using thetotal available data with phonological choice task performanceassessed three times, 2 years apart.

Whole-brain exploratory analysis was then performed onwhite matter-segmented images to find the white matter-specificregions associated with the variations in the CTNND2 SNP

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associated with the phonological processing. The SNP wasentered as a main factor in a flexible factorial designsecond-level SPM analysis (http://www.fil.ion.ucl.ac.uk/spm/software/spm8) that modelled whether the main effects werefrom the same or different participants by including subject andtesting round as factors. The model included the participantswith and without repeated measures. Age, sex, handedness andtotal white matter volume were entered as covariates. SNP inter-actions with age and sex were also added to the model.

Fish maintenance and lines usedIn order to analyse a subpopulation of forebrain neurons, an avail-able isl:GFP line was used. For in situ hybridisations, wildtype TLlines were used. All fish were maintained on a 14 h day/night cycleat the Karolinska Institute zebrafish core facility. Embryos wereproduced via light-induced spawning and raised at 28°C.

Morpholino knockdown of CTNND2 orthologuesSplice-blocking morpholinos (MO) were used to knockdownctnnd2a and ctnnd2b. For each of the duplicate copies, twonon-overlapping splice blockers were used to control for specifi-city of the observed phenotype. For ctnnd2a, MOs were used totarget exon4/intron4 (ctnnd2a MO1; see online supplementarytable S3) or exon2/intron2 (ctnnd2a MO2; online supplemen-tary table S3). For ctnnd2b, MOs were designed against exon3/intron3 (MO1; online supplementary table S3) or intron2/exon3 (ctnnd2b MO2; online supplementary table S3). A stand-ard control MO (cont MO) that has been shown previously tohave no toxic effects in zebrafish was used as a control for MOdose (Genetools, Std cont MO; online supplementary table S2).MOs were diluted to desired concentrations and injected intonewly fertilised embryos at the one-cell stage following standardmethods26 and raised to the desired stage.27 All injections wererepeated at least twice. The efficacy of the MOs was confirmedby sequencing PCR products amplified from cDNA samples ofMO injected and uninjected wild-type Tupfel longfin zebrafishembryos (online supplementary figure S2).

Confirmation of splice blockersIn order to confirm mis-splicing of ctnnd2a and ctnnd2b, RNAwas extracted from fish at 2 dpf using Trizol as per manufac-turer’s (Invitrogen) instructions. Random hexamer-primedcDNAwas synthesised using Superscript III First strand synthesiskit (Invitrogen) and used in PCR reactions. Primers are given inonline supplementary table S1.

ImmunofluorescenceEmbryos were fixed in 4% (w/v) paraformaldehyde (PFA) over-night at 4°C or for 2 h at RT, and brains were dissected usingfine forceps. The brains were subsequently processed forimmunofluorescence as described previously28 using ananti-HuC antibody (cat #A21271 life technologies, Carlsbad,California, USA) or antiacetylated tubulin (cat# T6793; Sigma)and a goat antimouse Alexa 594 secondary antibody (cat #A11032 Life Technologies, Carlsbad, California, USA), mountedin Gelvatol and imaged.

In situ hybridisationEight hundred and sixty base pairs of CTNND2b were ampli-fied from zebrafish cDNA using the specific primers (see onlinesupplementary table S1), and products were subsequently gel-purified and cloned into pGEMT-easy (Promega). PCR tem-plates were amplified from pGEMT-easy clones using T7 andSp6 primers to facilitate incorporation of RNA polymerase

binding sites. The resulting PCR products were then used astemplates in a Dioxigenin-labelling reaction (F Hoffman-LaRoche, Basel, Switzerland) to synthesise RNA antisense andsense control probes using a T7 or SP6 RNA polymerase.Whole-mount in situ hybridisation was carried out as previouslydescribed.29

Confocal imagingEmbryos fixed in 4% PFA were mounted rostrally in gelvatolmounting medium and imaged using an inverted OlympusFV1000 CSLM. Acquired Z-stacks were compiled using Image Jand analysed for the presence of ectopic isl:GFP neurons. Thesignificance in difference of phenotypical penetrance was ana-lysed using the χ2 test both in the separate experimental groupsand in the pooled samples. Ectopic isl:GFP cells between thetelencephalon and diencephalon at the region of the optic recesswere subsequently counted and compared using a standard ttest; 3D reconstruction of images was carried out using IMARISsoftware.

RESULTSIdentification of two balanced translocations in a motherand daughter, both with borderline intellectual disabilitiesThis study was initiated when a prenatally identified carrier oftwo balanced reciprocal chromosomal translocations, t(1;8)(p22;q24) and t(5;18)(p15;q11) (patient 1; figures 1A, B and2A), turned out to have learning problems within the dyslexiaspectrum, attention deficit, speech delay, myopia, normalgrowth and recurrent upper airway infections. This prompted usto ask whether one of the chromosomal breakpoints had dis-rupted a locus important for cognitive development. Both trans-locations were inherited from the mother (patient 2) whopresented with a similar phenotype, including learning difficul-ties, normal growth, no obvious dysmorphic features and refrac-tion error (figure 2A).

Whole-genome mate-pair sequencing and breakpoint PCRTo pinpoint the exact positions of the chromosomal breaks, weused low-coverage mate-pair whole-genome sequencing of patient1. The average coverage was that the two chromosomal transloca-tions were supported by multiple hybrid reads in the whole-genome sequencing data (n=40 for t(1;8) and n=63 for t(5;18);figure 1C). The breakpoint regions were narrowed down to205 bp (chr 1), 70 bp (chr 18), 150 bp (chr 5) and 315 bp (chr 8).We then designed primers and performed PCR spanning the 1/8and 5/18 translocation junctions (online supplementary table S1).The exact genomic coordinates for the breakpoints werechr1:78931219–78931227 (+ strand) and chr8:137813394–137813393 (− strand) for t(1;8) and chr5:11291109–11291112(+ strand) and chr18:32849314–32849311 (− strand) for t(5;18)(figure 1D, I, all coordinates in hg19).

The detailed analysis of the junction fragment signaturesreported here revealed that in the t(1;8) (p22;q24) junction,two stretches of four nucleotide tandem repeats were present(TCCT and AAGG), and seven nucleotides were deleted onchromosome 1 directly adjacent to the break. In t(5;18)(p15;q11) the junction sequence may be interpreted in two ways:(1) there was a one-nucleotide microhomology (G) at the break-point and a potentially novel point mutation (G to T) waspresent on chromosome 18; one-nucleotide from the junction,or (2) a 2 bp duplication has occurred on chromosome5 (chr5:11291110–11291111) masking a deletion of 2 bp onchromosome 18 (chr18:32849313–32849314) (figure 1D, I).

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The reciprocal translocations disrupt candidate lociNext, we turned our attention to potential candidate genesaffected by the chromosomal breakpoints. No genes wereaffected on chromosome 1 or chromosome 8. The 5p break-point was located in intron 9 of the CTNND2 gene (MIM604275; NM_001332; figure 3), and the 18q break affected anuncharacterised gene, ZSCAN30 (syn.ZNF917, ZNF397OS). Asthe genes were fused in opposite orientation, both were consid-ered to be loss-of-function mutations. A thorough literaturesearch revealed little information on ZSCAN30; there was noevidence of brain expression.30 However, in situ hybridisationin mouse showed transcript expression in postnatal (P56)mice (Allen mouse brain atlas; http://mouse.brain-map.org/experiment/show?id=695309). By contrast, the armadillorepeat family member CTNND2 (delta catenin) is expressed

almost exclusively in the brain7 and is a component of the adhe-rens junction complex.8 9 Given these findings, we focused ourefforts on CTNND2.

Identification of a third patient with cognitive difficultiesand CTNND2 deletionWe then browsed the Database of Chromosome Imbalance andPhenotype in Humans using Ensembl Resources (DECIPHER;http://decipher.sanger.ac.uk) and identified an additional individ-ual with similar phenotypical presentation and a maternallyinherited microdeletion exclusively involving CTNND2 (Patient3; table 1; figures 2B and 3). This boy had more pronouncedlearning disabilities and, specifically, difficulties in reading sug-gestive of dyslexia. Some dysmorphic features were present aswell as joint laxity in the fingers and a marfanoid habitus.

Figure 1 Illumina Nextera mate-pairwhole-genome sequencing of a patientwith two reciprocal chromosomaltranslocations. (A) and (B) Twochromosomal breakpoints werecharacterised that result in twobalanced translocations, t(1;8)(p22;q24), t(5;18)(p15;q11). The karyotypeof the first translocation is shown in(A), and the second in (B) with theaberrant chromosomes to the right.(C) The circle diagram illustrates themultiple reads supporting the locationof the genomic breakpoints. Thelocations of CTNND2 and ZSCAN30 areshown. (D) Direct sequencing of thet(1;8) junction revealed multiplesequence homologies (bold text).Additionally, 7 bp were deleted fromchromosome 1 directly adjacent to thebreakpoint. The 1/8 junction sequenceis marked in blue and 8/1 in green.Correlations with chromosome 1p22(top) and chromosome 8q24 (bottom)are indicated by vertical lines. Sangersequencing traces are shown in(E) (1/8) and (F) (8/1). (G) The t(5;18)chromosomal junction as in (A).The sequence may be interpreted intwo different ways: (1) a 1 bpmicrohomology is present at thejunction (pink) and at chr18:32849313a novel single nucleotide variants(SNV) is found in the patient (purple)or (2) a 2 bp duplication has occurredon chromosome 5 (chr5:11291110-11291111) and a deletion of 2 bp onchromosome 18 (chr18:32849313-32849314) framed in black. The toptrack is chromosome 5p15, the middlethe junction sequence and the bottomtrack chromosome 18q11 as above.Sanger sequencing traces are shown inH (5/18) and I (18/5), the novel SNV isannotated with a red star.

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The heterozygous deletion was identified by chromosomalmicroarray analysis. A total of 162 probes were deleted onchromosome 5p15.2, located 11055920 to 11218488 bp fromthe p telomere, affecting exons 12–18 of the CTNND2 gene(online supplementary figure S1). The deletion was confirmedby qPCR using two TaqMan copy number (CN) assays per-formed according to the manufacturer’s recommendations(hs06067454_cn and hs06128465_cn). Segregation analysis inthe family using the same qPCR assays showed that the motherwas mosaic for the 5p15.2 microdeletion (CNCalculated=1.63; minCN=1.47 maxCN=1.83; online supple-mentary figure S1), and the microdeletion was absent in thepatient’s healthy siblings (online supplementary figure 2B, datanot shown).

Taken together, the segregation of the disruptive chromosome5/8 translocation in Family 1 and the intragenic deletion inFamily 2 strengthen the hypothesis that loss-of-function muta-tions of CTNND2 are involved in the development of learningdifficulties and reading disabilities in these patients.

Association of a CTNND2 SNP with changes in white mattervolume and reading performanceSince all three patients reported here presented with clearreading difficulties, we decided to further evaluate the effect ofCTNND2 on reading performance and variability of whitematter volume. We used longitudinal data from a cohort31 of 76normally developing children and young adults aged 6–25 yearsand studied associations to polymorphisms in CTNND2.Interestingly, using sex and handedness as covariates and consid-ering three repeated measures, we found rs2561622 to be sig-nificantly associated with performance in non-word reading inchildren of age 6–10 years (p=1.54×10−4; n=28; AA: 11, AG:13, GG: 4). The association remained significant after adjust-ment for multiple testing (Bonferroni correction for 37 SNPs;p<0.0013), with the G allele of rs2561622 associated with abetter test performance. By contrast, the rs2561622 associationwith phonological choice tasks for individuals aged 12–14 years(p=0.816; n=18; AA: 8, AG: 6, GG: 4) and above 16 years(p=0.018; n=28; AA: 10, AG: 16, GG: 2) did not survive the

Figure 2 Pedigrees of the families with CTNND2 mutations. (A) and (B) Pedigrees of Family 1 and Family 2. Squares denote males and circlesdenote females, small black circles indicate a spontaneous miscarriage. The large blackened symbols represent a severely affected stillborn child, andthe small blackened circles are first-trimester spontaneous abortions. Grey symbols indicate learning difficulties. Individual identification numbers aregiven below the symbols. Karyotypes are given for those individuals in Family 1 where a chromosome analysis has been performed and deletioncarrier status is annotated in Family 2 as +, − or ± (mosaic carrier).

Figure 3 A schematic illustration ofCTNND2. The exon–intron structure ofCTNND2 gene (A). Exons are denotedby black boxes and introns by lines.Black stars indicate the genomicposition of the TaqMan copy numberassays (hs06067454_cn andhs06128465_cn). The breakpoint inintron 9 is indicated as a dashed linevertical red line, and the microdeletionof exons 12–18 in patient 3 is shownas a solid red horizontal bar in (A) and(B). The predicted protein structure isshown in (B) compared with itsorthologues in several commonly usedmodel organisms.

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multiple comparison correction. The allele frequencies for all37 SNPs are shown in online supplementary table S2.

Finally, we used the SPM8 software to test genetic associationsto white matter structure by a flexible factorial model consideringthree different time-points of our longitudinal MRI data. Imageacquisition and analyses were performed as described previ-ously.31 Briefly, the SNPs were entered separately as main factor,and the model was corrected for age, sex and handedness. Thegene interactions by age and sex were also entered as covariates.The SNP rs2561622 showed significant effect on white mattervolume in the left frontal lobe (peak coordinate: −47, 25, 22,p=1.28×10−5; corrected at the cluster level Pfamilywise error,

FWE<0.01) with higher white matter volume for GG allele car-riers (figure 4A, B). The significant cluster partly overlapped withthe arcuate fasciculus, superior longitudinal fasciculus and theinferior fronto-occipital fasciculus in the left white matter area.Moreover, the boundary of this significant cluster extended togrey matter mainly in the left inferior frontal gyrus and partiallyto the middle frontal cortex (figure 4C).

Knockdown of ctnnd2b in zebrafish results in misplaced isl:GFP cellsThe zebrafish genome contains two orthologues of humanCTNND2, named ctnnd2a and ctnnd2b, (figure 3B). To knockdown these two orthologues, we used non-overlapping MOstargeting two different splice junctions in either of the twogenes (Genetools, Philomoth, USA; online supplementary tableS2). Initial independent injections of the two morpholinosagainst ctnnd2a in isl:GFP embryos up to a dose of 10 ng didnot result in any gross morphological defects at 48–54 hpf(ctnnd2a MO1; n=104, ctnnd2a MO2 n=36), or in the mis-placement of isl:GFP cells in the forebrain or hindbrain (figure5H and data not shown). By contrast, injection of ctnnd2bMO1 or ctnnd2b MO2 resulted in a small percent of embryoswith gross abnormalities (10 ng ctnnd2b MO1: 11% n=13/118,5 ng ctnnd2b MO2: 14% n=6/43; online supplementary figureS3D) compared with control MO-injected embryos (5% n=3/60). The rest of the embryos were indistinguishable from unin-jected or control MO-injected embryos (online supplementaryfigure S3C). Additionally, similarly to ctnnd2a MO-injectedembryos, analysis of isl:GFP expressing hindbrain motoneuronsshowed no significant defects in migration (data not shown).

We then analysed a subpopulation of neurons in the rostral fore-brain in morphologically normal embryos at 48 and 54 hpf andnoted the presence of ectopic isl:GFP cells between the

telencephalon and diencephalon at both 48 and 54 hpf when com-pared with control MO-injected embryos (figure 5). Expression ofctnnd2b in this region at 48 hpf was confirmed via FISH onwhole-mount brains, as previously described32 (see online supple-mentary figure S3A,B). Misplaced neurons were observed directlyadjacent to the optic recess, which defines the frontal horn of theforebrain ventricle. At 54 hpf, a total of 42% (n=13/31) and 48%(12/25) of embryos injected with either ctnnd2b MO1 or ctnnd2bMO2 showed this misplacement of cells. The number of ectopicisl:GFP cells around the optic recess proved to be significantlyincreased in affected ctnnd2b knockdown embryos compared withcontrol MO-injected embryos (10 ng ctnnd2b MO1; mean: 9.1,SE of mean (SEM): 0.82 and 5 ng ctnnd2b MO2; mean: 8.6,SEM: 0.76) compared with controls (control MO; mean: 2.4,SEM 0.40; figure 5).

As the transcription factor islet-1 (isl) has been previouslyshown to be expressed specifically in a subpopulation of earlyneuron progenitors in the zebrafish telencephalon and dienceph-alon,33 we wanted to investigate whether a more general misplace-ment of neuronal cells was observed. In order to analyse allneurons at 48 hpf–54 hpf, embryos injected with ctnnd2b MO1or MO2 were stained with the postmitotic neuronal marker,HUC.34 No significant difference in the positioning of the grossneuronal population was observed (figure 6A–C). Additionally,analysis at an earlier stage in 30 hpf embryos injected with eitherof the two ctnnd2b MOs showed normal specification of isl:GFPcells at that stage and normal formation of the forebrain commis-sures, indicating normal patterning of the forebrain (see onlinesupplementary figure S3E,F). Specific analysis of single slice con-focal sections 0.50 μm of the identified mispositioned isl:GFP cellsin proximity to the forebrain ventricle (figure 6D–D00) revealedthat the majority of these cells did not overlap with Huc C/Dexpression or showed only weak onset of expression in contrastwith isl:GFP cells that had integrated into the diencephalic cluster(figure 6D).

DISCUSSIONIn the present study, we show loss-of-function mutations inCTNND2 in three individuals from two unrelated families, all pre-senting with mild cognitive dysfunction and reading disabilities.

Mechanisms underlying the two balanced translocationsin Family 1The fundamental mechanisms underlying the formation ofchromosomal translocations are still incompletely understood.

Figure 4 White matter structure is influenced by CTNND2 polymorphisms. (A) Main effect of CTNND2 polymorphisms (rs2561622) on whitematter structure shown in sagittal section. The significant cluster partly overlaps with the left arcuate fasciculus and the inferior fronto-occipitalfasciculus. (B) Distribution of residuals of white matter volume in the cluster associated with rs2561622 across different genotypes after correctionfor age, sex and handedness (error bars: ±1 SEM). (C) The extension of the significant cluster to its adjacent cortical regions: left inferior frontalgyrus and the middle frontal cortex.

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Even though approximately 1 out of 500 individuals are carriersof a balanced structural chromosome rearrangement, the occur-rence and cotransmission of two reciprocal translocations in afamily are very rare. From our breakpoint data, it is clear thatthe two translocations are separate rearrangements. Segregationdid not reveal any indication of mosaicism, as both mother anddaughter carried both translocations in all analysed cells. Fromthe family history, we suspected that the maternal grandmotherwas a carrier of at least one translocation (two miscarriages andpsychiatric illness resulting in suicide), but no DNA was avail-able to test this hypothesis and no other family members arecarriers (figure 2A).

By characterising the translocation breakpoints at the DNAlevel, we had an opportunity to study potential mechanisms under-lying the translocational events. Most previous studies of break-point features of genomic structural variants have focused oneither genomic CNVs (deletions and duplications)35 or acquired

somatic rearrangements associated with human malignancies.36

The dominating mechanistic models hypothesised to explain suchalterations, particularly non-recurrent events, are non-homologousend-joining (NHEJ) and replicative mechanisms (such as fork stal-ling and template switching/microhomology-mediated break-induced replication, FoSTeS/MMBIR).35 37 Not many balancedconstitutional reciprocal translocations have been studied at thebreakpoint level yet, but in a large study using massive parallelsequencing, Chiang et al38 suggested NHEJ as the predominantmechanism. The microarchitecture at the DNA breakpointsreported here suggests that the two translocations have beenformed separately, but precludes us from decisively determiningthe exact nature of the underlying mechanism of formation. Forinstance, the observed junction signatures were similar to CNVbreakpoints generated by replication-based repair mechanisms.Detailed studies of breakpoints of duplications have shown thatthe combination of microhomology, a few base-pair deletions,

Figure 5 Knockdown of zebrafish ctnnd2b results in misplaced isl:GFP (isl-1) neurons. Confocal z-stack images of whole-mount zebrafish embryosare shown in (A)–(D) rostral facing with dorsal to the top. Three-dimensional projections of (C) and (D) are shown in (F), (F0) and (G), (G0),respectively, with (F), (G) being rostral projections and (F0), (G0) lateral. Embryos were injected with 10 ng of standard control MO (cont MO), 10 ngctnnd2a MO1, 10 ng ctnnd2a MO2, 10 ng ctnnd2b MO1 or 5 ng ctnnd2b MO2. Morpholino penetrance is shown in H. Injection of eithermorpholino targeting ctnnd2b resulted in the presence of ectopic isl:GFP cells between the telencephalon and diencephalon at both 48 hpf (B) and54 hpf (red rectangle D, yellow cells in G and G0) compared with injection of a control MO (red rectangle A, yellow cells in F and F0) or ctnnd2a MO(not shown). The number of embryos presenting this phenotype was significant for both ctnnd2b MO1 (**p<0.01) and ctnnd2b MO2 (*p<0.05)injected embryos when compared with cont. MO injected embryos. The number of ectopic neurons demarcated by the red square in C-D werecounted (E) and shown to be significantly increased compared with controls (***p<0.001). The χ2 test was used to compare phenotype penetrance.For comparison of the number of ectopic isl:GFP cells, the t test was used. Error bars denote SE of the mean.

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insertions and novel point mutations flanking the junctions mayindicate FoSTeS/MMBIR.39–41 However, since FoSTeS/MMBIRgenerates non-reciprocal rather than reciprocal events, furtherstudies are needed to determine whether replicative mechanismshave a role in reciprocal translocations. Therefore, our resultssupport NHEJ as the underlying mechanism of formation of thetranslocation events that resulted in the disruption of CTNND2.

CTNND2 haploinsufficiency and learning disabilityHaploinsufficiency of CTNND2 was recently linked to mildintellectual disability with or without autistic traits.12 Here, wereport two additional cases with heterozygous structural variantsresulting in CTNND2 loss-of-function mutations with lownormal intelligence and learning disabilities borderlining onintellectual disability (table 1) and a third patient with mildintellectual disability. Some similarities with the patientsreported by Asadollahi et al can be observed possibly outlyingthe CTNND2 haploinsufficiency phenotype. Wechsler intelli-gence scale for children tests (WISC) performed on Patient 1(WISC-III) and patient 3 (WISC-IV) gave uneven profiles similarto the WISC result of Patient 1 in the report by Asadollahi et al,with a better verbal than non-verbal result for both. Both,Patients 1 and 2 in our report, had significant problems through-out middle school (grades 4–9); were moved to special class andrepeated a grade, respectively, but then caught up and were ableto finish normal high school. Hence, compared with Asadollahiet al, these two patients present with a milder phenotype withIQ in the low normal range, and thus, fit a type 4 classificationas set out recently by Beaudet.42 However, the cognitive profileand neurological manifestations of patient 3 reported here, andpatient 2 in Asadollahi’s report,12 both present with low IQ, dis-tinct facial features and hyperextensibility of the finger joints.These cases may represent the more severe end of the CTNND2haploinsufficiency phenotypical spectrum.

Hemizygous loss of CTNND2 in Cri-du-chat patients corre-lates with an increase in the severity of the syndrome.43

However, the region deleted in Cri-du-chat contains many othergenes, including genes coding for axon guidance molecules,such as semaphorin, suggesting that the clinical pathology is aresult of various perturbed developmental processes actingeither synergistically in the same pathway or parallel in an addi-tive manner. Further supporting an involvement of CTNND2 inthe Cri-du-chat-cognitive phenotypes is provided by a recentreport describing a Cri-du-chat patient with a complexCTNND2 rearrangement and a milder cognitive phenotypeincluding an improvement in reading.44

To date, nine dyslexia susceptibility loci (DYX1-DYX9) havebeen proposed, and four strong-effect candidate genes have beenidentified: DYX1C1 (DYX1),45 DCDC2 (DYX2),46 KIAA0319(DYX2)47 and ROBO1 (DYX5).6 We have previously demon-strated that SNPs from the DYX1C1, DCDC2 and KIAA0319genes influence white matter volume.31 Here, we show a similarresult for a common SNP in CTNND2 and rs2561622, which issignificantly associated with both performance in non-wordreading and white matter volume. The significant white mattercluster is located in the left frontal lobe and extends over to greymatter mainly in the left inferior frontal gyrus and partially tothe middle frontal cortex (figure 4C). These findings support theargument that CTNND2 is involved in reading, as the inferiorfrontal gyrus includes Broca’s area, a key region in phonologicalprocessing.48 49 Further, dyslexic readers have previously beenreported to have lower activity in the left inferior frontal gyrusduring reading and phonological processing.50 51

The polymorphism reported here, rs2561622, is a non-conserved common SNP of yet unknown functional significanceand located in a Long interspersed nuclear element (LINE) inan intron of the gene CTNND2 on chromosome 5p15.2. To thebest of our knowledge, this SNP does not overlap with previous

Figure 6 Ectopic isl:GFP (isl1) cells fail to express the postmitotic neuronal marker HUC. Single optical slices (0.50 mm) at the level of the opticrecess are shown in (A)–(D00) with rostral facing and dorsal to the top. isl:GFP cells are shown in green and cells labelled with the pan-neuronalmarker anti-HUC in red. A magnified view of the orange rectangle in B is shown in (D)–(D00); where D’ shows Huc-stained cells, D00 isl:GFP cells andD the overlay. Knockdown of ctnnd2b does not affect the general neuron population (red labelled cells in B and C) compared with controls (A),migrating isl:GFP cells that have not yet integrated into the diencephalon can be observed near the ventricles (yellow rectangle in B) many of whichhave not yet started expressing HUC (filled arrow heads D) or show weak expression (unfilled arrowhead D) in contrast with cells that haveintegrated into the diencephalon (blue rectangle in D).

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replicated linkage regions for developmental dyslexia (for anoverview, see Kere.52). Furthermore, it is not found significantlyassociated with reading and writing in recently publishedgenome-wide association analysis using quantitative measures ofreading or writing skills.53–55 The community sample used herefor assessing the role of rs2561622 in phonological processingis of limited size, and further, targeted studies in larger inde-pendent sample sets are needed to verify the role of rs2561622alleles on susceptibility to developmental dyslexia.

Many dyslexia candidate genes, such as DYX1C1, DCDC2,KIAA0319, KIAA0319 L, S100B and ROBO1 have been linkedto defects in neuronal migration both in vivo and in vitro.56–63

In particular, disruption of Dyx1c1 in adult rodent brains viaRNAi was shown to cause neuronal migration defects,59 and invitro perturbation of Dyx1c1 altered the expression of genesinvolved in cell migration and CNS development in vitro.64

Even more conclusive evidence for abnormal neuronal migra-tion, as one potential neurodevelopmental mechanism of dys-lexia, stems from the postmortem examination and brainimaging studies of human patients. Neuroanatomical examina-tions of four male and three female patients revealed variousabnormalities, including cerebral asymmetries, focal architec-tonic dysplasias and neuronal ectopias, the latter of which isindicative of neuronal migration defects.65 66 More recentresults come from studies using structural MRI in conjunctionwith neuropsychological testing in patients with periventricularnodular heterotopia (PNH), anatomically defined as the pres-ence of misplaced grey matter nodules along the ventricularwall, due to a disruption of radial neuronal migration from theventricular zone to the cortical plate. Most patients with PNHhave average intelligence despite the rather severe anatomicalpresentation, but suffer from epilepsy and frequently have aform of dyslexia specifically affecting reading fluency.67

Here, we show that knockdown of one of the two Ctnnd2orthologues resulted in the presence of ectopic isl1 neurons inthe zebrafish forebrain suggestive of potential defects in neur-onal migration. Recently, an orthologous subpopulation hasbeen shown to differentiate into cholinergic neurons inmouse.68 Additionally, a somatic loss of CTNND2 in humanglioblastomas has been shown to lead to a mesenchymal trans-formation associated with an invasive progression and a moreaggressive prognosis69 suggesting an in vivo role of CTNND2 incell motility. Interestingly, the misplaced neurons reported hereshow no or only weak expression of the postmitotic neuronalmarker, HUC, suggesting that the ectopic cells are not yet fullydifferentiated (figure 6). Further work is required to assesswhether this lack of differentiation is the cause of the misplacedneurons, as shown previously in cases of periventricular neur-onal heterotopias, due to loss of cadherin receptor ligand pairs,DCHS1 and FAT4,11 or an after effect, due to inappropriateintegration into the neuroepithelium.

In summary, we show loss-of-function mutations in CTNND2and learning difficulties within the dyslexia spectrum in threeindividuals from two unrelated families. The gene was identifiedby low-coverage massive parallel whole-genome sequencing of apatient with learning difficulties within the dyslexia spectrumand two balanced reciprocal translocations. This illustrates thecapability of whole-genome end sequencing to detect balancedstructural variation,70 which is re-emerging as part of a compre-hensive whole-genome test in leading clinical testing laborator-ies.71 It serves as a complement to balanced CNV analysis.72

CTNND2 function was further evaluated first in a communitycohort of normal developing children where a CTNND2polymorphism was significantly associated with phonological

processing and white matter volume in the left inferior frontallobe, close to Broca’s area, a region previously linked to phono-logical processing and developmental dyslexia. Second, we per-formed in vivo studies in zebrafish embryos and revealed thepresence of ectopic neurons in the zebrafish forebrain after MOknockdown of ctnnd2. The combined data suggest that loss ofCTNND2 is involved in learning difficulties possibly throughdefects in neuronal migration.

Author affiliations1Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm,Sweden2Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden3Department of Clinical Genetics, Karolinska University Hospital, Stockholm, Sweden4Science for Life Laboratory, Karolinska Institutet Science Park, Solna, Sweden5Department of Clinical Genetics, Sahlgrenska University Hospital, Gothenburg,Sweden6Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden7Department of Biosciences and Nutrition, Center for Innovative Medicine,Karolinska Institutet, Huddinge, Sweden8SciLifeLab, School of Biotechnology, KTH Royal Institute of Technology, Stockholm,Sweden9SciLifeLab, Department of Biochemistry and Biophysics, Stockholm University,Stockholm, Sweden10Molecular Neurology Research Program, University of Helsinki, and FolkhälsanInstitute of Genetics, Helsinki, Finland

Acknowledgements We are grateful to the patients and their families for theircooperation and enthusiasm during this study. We also gratefully acknowledge theuse of computer infrastructure resources at UPPMAX, Project b2011157, and theKnut and Alice Wallenberg foundation for funding the CLICK facility at theKarolinska Institutet. We would also like to thank the zebrafish core facility formaintenance of our fish, Satish Kitambi for use of his islet:GFP fish stock, MiriamArmenio for her technical assistance and Dr Myriam Peyrard-Janvid for genotyping ofBrainChild samples.

Contributors WH: conceived and designed the experiments, performed theexperiments, analysed the data, contributed to the writing of the manuscript. DN:conceived and designed the experiments, analysed the data, contributed to thewriting of the manuscript. AT and B-MA: analysed the data, contributed to thewriting of the manuscript, contributed reagents/materials/analysis tools. FD and HM:performed the experiments, analysed the data, contributed to the writing of themanuscript. ITP: conceived and designed the experiments, contributed to the writingof the manuscript. TK and VW: conceived and designed the experiments,contributed reagents/materials/analysis tools, contributed to the writing of themanuscript. LS: performed the experiments, analysed the data. FV: performed theexperiments, analysed the data, contributed to the writing of the manuscript. JK:conceived and designed the experiments, contributed reagents/materials/analysistools, contributed to the writing of the manuscript. MN: conceived and designed theexperiments, contributed reagents/materials/analysis tools, contributed to the writingof the manuscript. ESL: conceived and designed the experiments, contributed to thewriting of the manuscript. AL: conceived and designed the experiments, analysedthe data, contributed reagents/materials/analysis tools, contributed to the writing ofthe manuscript.

Funding This work was funded by the Swedish Brain Foundation, the Harald ochGreta Jeanssons Foundation, the Nilsson-Ehle Foundation, Erik RönnbergsFoundation and the Swedish Research Council grant 2012-1526 to AL, 2010-3286to ESL, 2012-2279 to JK and 2011-4592 to MN.

Competing interests None.

Patient consent Obtained.

Ethics approval The local ethical boards in Stockholm (Sweden).

Provenance and peer review Not commissioned; externally peer reviewed.

Data sharing statement All the data is reported and no unpublished data isavailable in the current study.

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12 Hofmeister W, et al. J Med Genet 2014;0:1–12. doi:10.1136/jmedgenet-2014-102757

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problems and mild intellectual disabilitya candidate gene for reading−−CTNND2

Magnus Nordenskjöld, Elisabeth Syk Lundberg and Anna LindstrandKlingberg, Lena Samuelsson, Valtteri Wirta, Francesco Vezzi, Juha Kere,Anderlid, Fahimeh Darki, Hans Matsson, Isabel Tapia Páez, Torkel Wolfgang Hofmeister, Daniel Nilsson, Alexandra Topa, Britt-Marie

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