11q24.2-25 micro-rearrangements in autism spectrum disorders: Relation to brain structures.

Anna Maruani, Guillaume Huguet, Anita Beggiato, Monique Elmaleh, Roberto Toro, Claire S Leblond, Mathieu Alexandre, Frederique Amsellem, Nathalie Lemière, Alain Verloes, et al.

► To cite this version:

Anna Maruani, Guillaume Huguet, Anita Beggiato, Monique Elmaleh, Roberto Toro, et al. 11q24.2-25 micro-rearrangements in autism spectrum disorders: Relation to brain structures. American Journal of Medical Genetics Part A, 2015, 167 (12), pp.3019-30. 10.1002/ajmg.a.37345. pasteur-01579742

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11q24.2-25 Micro-rearrangements in Autism Spectrum Disorders:

Relation to Brain Structures

Anna Maruani,¹,²,³ Guillaume Huguet,¹,² Anita Beggiato,¹,²,³ Monique ElMaleh,⁴ Roberto Toro,¹,² Claire S Leblond,¹,² Alexandre Mathieu,¹,² Frederique Amsellem,³ Nathalie Lemière,¹,² Alain Verloes,⁶ Marion Leboyer,⁹,¹⁰,¹¹ Christopher Gillberg,⁵,⁷ Thomas Bourgeron,¹,²,¹² and Richard Delorme¹,²,³,¹¹ *

Anna Maruani,¹,²,³
Email: maruani.anna@gmail.com

Guillaume Huguet,¹,²
Email: guillaumeaf.huguet@gmail.com

Anita Beggiato,¹,²,³
Email: anita.beggiato@gmail.com

Monique ElMaleh,⁴
Email: monique.elmaleh@rdb.aphp.fr

Roberto Toro,¹,²
Email: roberto.toro@gmail.com

Claire S Leblond,¹,²
Email: leblond.claire.s@gmail.com

Alexandre Mathieu¹,²
Email: alexandre.mathieu@pasteur.fr

Frederique Amsellem,³
Email: frederiqueamsellem@gmail.com

Nathalie Lemière,¹,²
Email: nathalie.lemiere@pasteur.fr

Alain Verloes,⁶
Email: alain.verloes@rdb.aphp.fr

Marion Leboyer,⁹,¹⁰,¹¹
Email: marion.leboyer@inserm.fr

Christopher Gillberg, 5,7

Email: christopher.gillberg@pediat.gu.se

Thomas Bourgeron, 1,2,12

Email: thomasb@pasteur.fr

Richard Delorme 1,2,3,11 *

*Corresponding author

Email: richard.delorme@rdb.aphp.fr

**Correspondence** should be addressed to: Richard Delorme, M.D., Ph.D, Human Genetics and Cognitive Functions, Institut Pasteur, 25 rue du Docteur Roux, 75015 Paris, France. Tel: + 33140613311. Fax: +33140613953. E-mail: richard.delorme@rdb.aphp.fr

**Running title:** NTM in ASD: relation to brain structures

<footnote n="1">¹ Human Genetics and Cognitive Functions, Institut Pasteur, Paris, France</footnote>

<footnote n="2">² CNRS URA 2182, Institut Pasteur, Paris, France</footnote>

<footnote n="3">³ Department of Child Psychiatry, Robert Debré Hospital, APHP, Paris, France</footnote>

<footnote n="4">⁴ Department of Radiology, Robert Debré Hospital, APHP, Paris, France</footnote>

<footnote n="5">⁵ Gillberg Neuropsychiatry Centre, Gothenburg University, Göteborg, Sweden</footnote>

<footnote n="6">⁶ Department of Human Genetics, Robert Debré Hospital, APHP, Paris, France</footnote>

<footnote n="7">⁷ Saint George's Hospital Medical School, London, United Kingdom</footnote>

<footnote n="8">⁸ Department of Clinical Sciences, Lund University, Lund, Sweden</footnote>

<footnote n="9">⁹ INSERM U955, Team 15, Faculty of Medicine, Creteil, France</footnote>

<footnote n="10">¹⁰ Department of Adult Psychiatry, Henri Mondor-Albert Chenevier Hospitals, AP-HP, Creteil, France</footnote>

<footnote n="11">¹¹ Fondation FondaMental, French National Science Foundation, Creteil, France</footnote>

<footnote n="12">¹² University Paris Diderot Paris 7, 75013 Paris, France</footnote>
ABSTRACT

Jacobsen syndrome (JS) is characterized by intellectual disability and higher risk for autism spectrum disorders (ASD). All patients with JS are carriers of contiguous *de novo* deletions of 11q24.2-25, but the causative genes remain unknown. Within the critical interval, we hypothesized that haploinsufficiency of the neuronal cell adhesion molecule Neurotrimin (NTM) might increase the risk for ASD and could affect brain structure volumes. We searched for deleterious mutations affecting *NTM* in 1256 ASD patients and 1287 controls, using SNP arrays, and by direct sequencing of 250 ASD patients and 180 controls. We compared our results to those obtained from independent cohorts of ASD patients and controls. We identified two patients with Copy Number Variants (CNV) encompassing *NTM*, one with a large *de novo* deletion, and a clinical phenotype of JS (including macrocephaly), and a second with a paternally inherited duplication, not consistent with JS. Interestingly, no similar CNVs were observed in controls. We did not observe enrichment for deleterious *NTM* mutations in our cohort. We then explored if the macrocephaly in the patient with JS was associated with a homogeneous increase of brain structures volumes using automatic segmentation. Compared to subjects without *NTM* micro-rearrangements (n=188), the patient had an increased volume of the sub-cortical structures but a decrease of the occipital gray matter. Finally our explorations could not incriminate *NTM* as a susceptibility gene for ASD, but provides new information on the impact of the 11q24.2-25 deletion on brain anatomy.

**Keywords:** autism, brain, gene, Jacobsen syndrome, macrocephaly
Introduction

Autism spectrum disorders (ASD) are characterized by impairments in reciprocal social
communication, and repetitive, stereotyped and ritualistic behaviors [Kanner, 1968].
Advances in high throughput array-based genome screening technologies allowed the
detection of copy number variants (CNVs) and single nucleotide variants (SNVs) in large
cohorts of patients [De Rubeis et al., 2013; Iossifov et al., 2014; O'Roak et al., 2014;
Pinto et al., 2014]. *De novo* CNVs or SNVs affecting genes have conferred high risk for
ASD [Iossifov et al., 2012; Pinto et al., 2014] but the majority of these de novo events are
rare or unique. Several recurrent CNVs have been associated with ASD, reaching
genome-wide significance, such as duplications at 15q11-13, or deletions/duplications at
16p11.2 and 22q11.2 [Hadley et al., 2014; Pinto et al., 2014; Sanders et al., 2011]. Whole
exome studies also revealed only a slightly elevated rate of *de novo* SNVs in probands
compared with their unaffected siblings (1.02 vs. 0.79 mutations per offspring), but
patients had two- to threefold more disruptive *de novo* mutations in comparison to their
siblings, or to a random model of mutation [De Rubeis et al., 2013; Iossifov et al., 2014].

Although the number of causative genes for ASD could be more than 250,
[Huguet et al., 2013] they affect a restricted number of biological pathways, including
chromatin remodeling, mRNA translation and synaptic functions, [De Rubeis et al., 2013;
Iossifov et al., 2014; Toro et al., 2010]. Among the synaptic genes associated with ASD,
Ig-like cell adhesion molecules such as *CNTN3* [Girirajan et al., 2011], *CNTN4*
[Fernandez et al., 2004] and *CNTNAP2* [Alarcon et al., 2008; Arking et al., 2008] have
important roles in neuronal interactions for synaptic targeting, neuronal migration, and
axon guidance. Neurotrimin (NTM), which belongs to the same molecular family, is a
glycosylphosphatidylinositol (GPI)-anchored cell adhesion protein and a member of the IgLON subfamily (containing also LAMP and OBCAM). Predominantly expressed in the brain, NTM promotes neurite outgrowth and adhesion via a homophilic mechanism [Sellar et al., 2003]. Interestingly, two genome wide association studies (GWAS) reported an association between *NTM* and cognitive function performances [Liu et al., 2007; Pan et al., 2011] and two studies have detected CNVs altering *NTM* in ASD and ID [Cooper et al., 2011; Vorstman et al., 2006].

Deletions of 11q24.2-25 and of the telomeric region of 11q are associated with Jacobsen syndrome (JS), a rare genetic condition sometimes associated with ASD, occurring in 1/100000 birth and considered as a continuous gene disease with highly variable clinical presentations [Cooper et al., 2011; Grossfeld et al., 2004; Penny et al., 1995]. The most common clinical features include mild to severe intellectual disability (ID) associated with autistic symptoms, pre- and postnatal physical growth delay, skull and facial dimorphism (specifically macrocephaly) (for review [Mattina et al., 2009]). The attempts to correlate the clinical findings to the extent of the deletion led to the identification of candidate regions within 11q24.2-25 for several abnormalities such as the Paris-Trousseau thrombocytopenia [Grossfeld et al., 2004]. Difficulties in finding the gene(s) associated with cognitive impairment in patients with JS is due to the relatively high number of compelling candidate genes located at 11q24.2-25 such as *NRGN* and *KIRREL3* involved in synaptic plasticity [Guerin et al., 2012; Zhong et al., 2009], *ARHGAP32*, *NTM*, *OPCML* involved in axon guidance and outgrowth [Akshoomoff et al., 2014; Iossifov et al., 2014; McNamee et al., 2002] or *BSX* involved in brain specific transcriptional activity [McArthur and Ohtoshi, 2007]. Critical regions for ASD and ID were suggested, mainly based on the report of small deletions identified in patients
(Figure 1) [Coldren et al., 2009; Penny et al., 1995]. A recent publication suggested a minimal region containing four genes including *KCJN1*, *KCJN5*, *TP53AIP1* and *ARHGAP32* [Akshoomoff et al., 2014]. However, no gene was formally identified as causing the cognitive phenotype of patients with JS.

Here, we first screened for 11q24.2-25 CNVs in 1256 independent patients with ASD, and detected one *de novo* deletion and one inherited duplication, both encompassing exons of *NTM* and *SNX19*. Hypothesizing that haploinsufficiency of *NTM* might increase the risk for ASD, we sequenced all coding exons of this gene in 250 patients with ASD and ID, and 180 controls evaluated for the absence of personal and familial history of psychiatric disorders. In addition, to better understand the macrocephaly frequently reported in patients with JS, we quantified the cortical and subcortical brain volumes using magnetic resonance imaging (MRI).

# METHODS

## Patients and controls

A sample of 1256 patients with ASD from the Paris Autism Research International Sibpair Study (PARIS; N=260) and from the Autism Genome Project (AGP; N=996), meeting stringent quality control (QC) criteria, were included in the study (Table S1 Supplemental material). Information concerning the phenotypic assessment of patients enrolled was described previously [Leblond et al., 2014; Pinto et al., 2010]. To summarize, we used the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) for clinical evaluation and diagnosis. In addition, 1287 control subjects, from the Autism Genome Project were also used to compare our findings [Pinto et al, 2010]. The subjects were from the SAGE cohort
(n=1261) [Bierut et al., 2010] and from HapMap (n= 26) [International HapMap, 2003]
(Table S1 Supplemental material).

In parallel to the clinical and genetic exploration, some subjects enrolled in the PARIS
cohort were systematically scanned using 3DT1 MRI: 188 subjects including 149 patients
with ASD and 39 control subjects were included in the present study (for additional
information, see Table S2 Supplemental material).

Ethics Statement

This study was approved by the local Institutional Review Board (IRB) and written
inform consents were obtained from all participants of the study. The local IRB are the
Hôpital Pitié-Salpêtrière Ethics committee (Paris, France) and the Sahlgrenska Academy
Ethics committee (Gothenburg, Sweden). Written informed consent was obtained from
all participating subjects. If the proband was under 18 years old, the proband's consent
and written parental consent were obtained.

CNV detection and validation for the PARIS cohort

DNA was extracted from blood leukocytes or B lymphoblastoid cell lines. CNV was
detected with the Illumina Human 1M and 1M-Duo BeadChip technologies, which
interrogate 1 million SNPs distributed over the human genome. Both Illumina
technologies are highly similar. Genotyping was performed at the Centre National de
Génotypage (CNG), and at the Institut Pasteur. Only samples that met stringent quality
control (QC) criteria were included: call rate ≥99%; standard deviation of the log R ratio
(LRR) ≤0.35 and of the B allele frequency (BAF) ≤0.13. We used two CNV calling
algorithms, QuantiSNP and PennCNV, and one CNV viewer, Snip Peep
(http://snippeep.sourceforge.net/). Similarly to Pinto et al. [2010], CNVs were selected by the number of consecutive probes ≥5, their size ≥1 kb and the confidence score log Bayes factor ≥15. The CNVs were validated by visual inspection of the Log R ratio and B allele frequency values in order to obtain high-confidence calls. To homogenize our CNV detection techniques with those of Pinto et al. [2010], we excluded CNVs if they had a size under 30 kb, if they were within the centromeric region or in extreme GC content regions (>70%). PennCNV was then used to confirm inheritance status of the resulting CNV calls. CNVs were validated by qPCR analysis using the Universal Probe Library (UPL) system from Roche. UPL probes were labeled with 6-FAMTM fluorescein (www.exiqon.com) and the fluorescence was read with the Applied Biosystems 7500 Real-Time PCR System. Each assay was conducted in four replicates for target region probe-set and control region probe-set. Assuming that there were two copies of DNA in the control region, the relative levels of region dosage were determined using the comparative CT method. The relative copy number for each target region was calculated as 2<sup>-DDCT</sup> with confidence interval as 2<sup>(DDCT±SD)</sup>. UPL probes and primers were designed using the Universal Probe Library Assay Design Center from Roche Applied Science.

## DNA Sequencing

The genomic structure of *NTM* was obtained from http://genome.ucsc.edu/ (hg18). Among the two main isoforms referenced (NM_001144059, NM_001048209), we sequenced all exons corresponding to the longest isoform (NM_001048209) in 250 subjects with ASD from the PARIS cohort (From European descent) and 180 Caucasian controls (From European descent) evaluated for the absence of personal and familial history of psychiatric disorders by direct assessment using the Diagnostic Interview for
Genetics Study Interview (DIGS) and the Familial Interview for Genetics Study (FIGS)
(For additional details about clinical assessment of these patients and controls, see
[Leblond et al., 2012]. Primers spanning all seven intron-exon boundaries of *NTM* were
designed and used to amplify the regions of interest. Amplicons were produced from
genomic DNA, and sequencing was performed using ABI 3730 DNA sequencer
(Applied Biosystems, Foster City, CA). Sequence comparison to reference sequence was
performed using GenalysCarbon 2.8.2b (http://software.cng.fr/).

## Protein Function Prediction

Several *in silico* applications were used to predict the functional effect of the amino acid
substitutions, including SIFT (http://sift.jcvi.org/www/SIFT_seq_submit2.html),
Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/), and SNAP
(http://rostlab.org/services/snap/).

## Intracranial and brain volume estimations based on magnetic resonance imaging

For subjects included in this cohort, MRI data were collected using the following
parameters: spoiled gradient recalled echo (SPGR), 1mm isotropic, repetition time
(TR)=25ms, echo time (TE)=6ms, flip angle=30°. To estimate the intracranial volume,
the different datasets were first reoriented to correspond with the orientation of the
MNI152 atlas. The brain was removed from the skull using AFNI tools [Cox, 1996], and
linearly normalized to the (skull-less) MNI152 atlas using FSL tools [Jenkinson et al.,
2002; Smith et al., 2004]. We then used the affine matrix of this transformation to
initialize the linear normalization of the reoriented datasets (with skull) to the MNI152
atlas (with skull). We used the inverse of the determinant of the affine matrix produced
by this transformation as an estimation of the intracranial volume, as in Buckner et al
[2004][Buckner et al., 2004]. All steps of the process were visually inspected for
accuracy using in-house software. In the cases where the skull stripping was
inappropriate (the most frequent type of failure of the automatic segmentation procedure),
we manually corrected the brain extraction using in-house software, and relaunched the
processing pipeline. Also, the estimation of brain volume was obtained by segmenting
automatically the grey and white matter. We then labeled the frontal, parietal, occipital,
temporal lobes and subcortical structures by non-linearly warping the individual datasets
into an atlas, using FSL tools [Smith et al., 2004; Zhang et al., 2001] and our own
software [Toro et al., 2009]. Intracranial and brain volume values were converted into Z-
scores. We used an open online tool to visually control the accuracy of the segmentations
(http://siphonophore.org/qccc). The subjects for whom the segmentation not fulfilled the
quality criteria were excluded.

RESULTS

A de novo deletion and a rare inherited duplication at 11q24-25 encompassing NTM

Among the 1256 independent patients with ASD genotyped using Illumina SNP arrays,
we detected one large *de novo* heterozygous deletion within 11q24.2-25 in one boy with
autism and moderate intellectual disability (see clinical data section for details / Family
I). The CNV size is about 5.4Gb (chr11: 126,633,940 to 132,060,375; hg18). Among the
20 genes within the deletion (Figure 2), three are mainly expressed in the brain:
*ARHGAP32, NTM and OPCML*. Haplotype analysis revealed that the deletion originated
on the paternal chromosome (data not shown). No additional rare CNVs, inherited or *de
novo*, encompassing a gene expressed in the brain was detected in the patient.
We also detected a 938 kb duplication located within the same region (11q24.3-q25; chr11: 130,144,889 to 131,083,311; hg18) in an independent patient with ASD. The micro-rearrangement affects two genes: *SNX19* and the first exon of *NTM*, a gene that is mainly expressed in the brain. The duplication was inherited from the father and shared by the affected sister (Figure 2). Both CNVs were validated by quantitative PCR (qPCR).

We then compared our results to the CNVs detected in the 1287 independent controls from the AGP [Pinto et al., 2010]. We found only one CNV (a duplication) affecting NTM (B209941_1007852571; chr11: 130,064,162 to 131,099,383; hg 18), which was very similar to the one observed in family II (Figure 2).

We then compared our results to those obtained in different databases [Database of Genomic Variants (DGV; http://projects.tcag.ca/variation/), Decipher (http://decipher.sanger.ac.uk)] (Figure 2). No similar heterozygous CNVs (sharing at least 80% overlap with these CNVs) were previously referenced in DGV. In Decipher, several subjects presenting with a development delay (no additional clinical information was provided) carried a large deletion or duplication within 11q24.2-q25, which encompassed *NTM* (Figure 2). Three distinct CNVs affecting only *NTM* were reported: a duplication of the first exon (chr11: 130209116-13077274) in one subject (Id: 250240, clinical information not known), a deletion of the third exon (chr11: 131406957-131574563) in one subject (Id: 250733 showing global development delay), and a deletion of the third and fourth exons (chr11: 131506987-131598665) in four individuals (A in Figure 2; Id: 1617, 2049, 2050, 2054). For these subjects, clinical information was as follow: 2049 and 2050 showed ID; 1617 had microcephaly, prominent ears, delayed language development and clinodactyly.
## Deleterious mutations in *NTM* coding sequence

All *NTM* exons were directly sequenced in a subset of patients with ASD and ID (n=250) from the PARIS study and in controls (n=180). Two heterozygous non-synonymous mutations, both in exon 8, were identified: p.V327I, observed in 11/250 patients (4.4%) and 5/180 controls (2.8%) (Fisher's exact test, Odd ratio=1.6, *p*=0.4), and p.S335L, observed in 1/250 patients (0.4%) and 1/180 controls (0.6%) (Fisher's exact test, Odd ratio=0.7, *p*=1). No additional mutations were found on the remaining allele of *NTM* in the affected probands of families I and II. *In silico*, the mutations p.V327I and p.S335L were predicted to be benign whatever the model of prediction used (Polyphen-2/Prediction: benign; PSIC score difference: 0.056 for p.V327I and 0.002 for p.S335L - SNAP/Prediction: neutral; reliability index: 6 for p.V327I and 0 for p.S335L; expected accuracy 92% for p.V327I and 53% for p.S335L - SIFT/prediction: tolerated; SIFT median sequence conservation score: 3.11 for p.V327I and 3.05 for p.S335L). Clinically, the 11 patients carrying the p.V327I mutation were not different from the whole cohort of patients in terms of severity of ID or autistic symptoms, based on ADI-R sub-scores and cognitive assessment measure (data not shown). Concerning the patient carrying p.S335L, he displayed a non-verbal autism with a severe ID. Clinical examination was in the normal range, but with minor signs of dimorphism including hypertelorism and divergent strabismus. The mutation was inherited from the mother, which displayed no personal or familial history of psychiatric illness. We then compared our results to those reported previously in two independent cohorts, which were whole-exome sequenced. We collected the data from 767 independent probants with ASD and 573 unaffected siblings from the Simons Simplex Collection (SSC) [Iossifov et al., 2012; O'Roak et al., 2012; Sanders et al., 2012] and 4477 controls from the Exome Variant Server
(evs.gs.washington.edu). Based on the NM_001144058 isoform, we observed 9 non-synonymous variants (7 within the patients, 2 in the siblings) in the SSC (Table1), but no stop-gain, in-del or *de novo* mutations (no variation has been found only in patients). Variants were predicted as benign (Table 1). The variants identified in our cohort of patients, p.V327I and p.S335L, were also observed in SSC cohorts of patients and controls and were not significantly associated with ASD (respectively : p= 0,43 et p=1; odds ratio=0,82, [0.5295; 1.2528] et 0,70 [0.0769; 3.1108]). These results were also confirmed in the recent studies from the SSC (over 3800 ASD and 9000 controls) that did not find any stop-gain, in-del or *de novo* mutations within ASD patients. Latest studies from the SSC did not find transmitted missense variants, this can been explained by new pipeline and filtered MAF (minor allele frequency ≤ 0.001) used [Iossifov et al., 2014; O'Roak et al., 2014]

**Clinical characteristics of the patients with CNVs at 11q24-25**

*Family I.* The patient carrying the large 11q24 deletion was a 21-year-old male, second child of non-consanguineous healthy parents. The father, of North African origin, had no personal medical or psychiatric history, but reported a paternal cousin with symptoms of ID and ASD, and a paternal uncle with brief recurrent delirium episodes. The maternal personal and family history revealed no prior instances of mental or neurological disorders. The clinical profile of the proband's older sister was normal with no specific medical history.

The patient was born following an unremarkable pregnancy. The delivery was preterm (35 weeks of gestation) for unknown reason. A cesarean section was performed in emergency after painful and inefficient labor due to a foetomaternal
disproportion. All birth measurements were over the 97th percentile (the head circumference of the newborn was 37 cm, the birth weight was 3.440 kg and the height was 52 cm). Fetal resuscitation was performed initially (with APGAR scores at 2 and 9 at 1 and 10 minutes respectively), but the patient was not transferred to an intensive care unit. Although early neonatal development was considered as normal by pediatricians (motor skills were in the normal range at 6 months and walking was acquired at 14 months), the mother was concerned with sleep difficulties and the appearance of body rocking at the age of 8 months. Verbal language was delayed with first words emerging at 3 years of age and sentence constructions only at 5 years. Initially echolalic, the language was considered functional at 7 years. At that time, the cognitive assessment, using the Wechsler Intelligence Scale for Children, objectified the intellectual disability, with an intellectual quotient estimated fewer than 50. Clinically, the patient had clear abnormalities of social interactions. He also had various stereotypies and self-injurious behaviors associated with major impulsivity. The clinical diagnosis of autism (in accordance with DSM-IVTR criteria) was confirmed by standard assessment using the Autism Diagnostic Interview-Revised and the Autism Diagnosis Observation Scale. Clinical examination showed various signs of dimorphism similar to those reported in JS, i.e., low-set ears, large nasal bridge, slight nose protrusion, high forehead, short neck, macrocephaly (+4 SD) and short stature (-0.5 SD). Neurological examination was in the normal range. Also, results from routine biological screens (including a complete blood count), electrocardiogram and ultrasound explorations were all in the normal ranges.

**Family II.** The patient carrying the duplication was a 15-year-old male, born at term after an uneventful twin pregnancy and delivery (38th week) from non-consanguineous
Caucasian parents. The maternal personal and family history revealed no prior instances
of mental or neurological disorders, except for a putative post-partum depression after
her twin pregnancy for which she did not receive any treatment. Her clinical exam
produced normal results. The father reported a general anxiety disorder that emerged
during childhood alongside a diagnosis of attention deficit / hyperactive disorder
(ADHD). At the time of assessment, only the anxiety disorder persisted. His clinical
exam was normal, except the presence of a unilateral left thumb macrodactyly. The older
sister, aged 17 years at the time of the assessment, did not report any psychiatric
disorder, but had experienced an idiopathic pubertal delay during childhood. Her clinical
exam was normal except for a mild strabismus. Birth weight of the patient was 3170 g
(25th percentile), length 50 cm (49th percentile), and occipito-frontal head circumference
(OFC) 36 cm (53rd percentile). APGAR scores were 10 at 1 and 5 min after birth.
Despite normal development during infancy, including walking at 15 months, the parents
reported a pronounced intolerance to frustrations and noises at age 2. They also noted a
paucity of interactions accompanied by stereotyped body movements. The patient
exhibited severely delayed speech, with first words at 4 years of age. Referred to a
psychiatric unit at the age of 5 years, he was diagnosed clinically with autism, based on
DSM-IV and ICD-10 criteria. He also met criteria for autism according to the ADI-R and
the Autism Diagnostic Observation Scale (ADOS). Assessment of his cognitive
performance with the Raven's Colored Progressive Matrices test indicated that his
intelligence quotient (IQ) was below the first percentile for his age. When examined at
the age of 15, the patient's height, weight and OFC were all in the normal range, but he
presented with minor dimorphic features (not typical of JS) including a long and narrow
face, arachnoid hand fingers and a supernumerary nipple. The neurological exam was
normal. His expressive language was limited to restricted and stereotyped sentences. His
dizygotic twin sister who shared the *NTM* duplication also exhibited a severe intellectual
disability with autistic features, but also displayed a severe psychomotor delay and
microcephaly. These symptoms were quite different from those reported in her twin
brother, but concordant with Rett syndrome, which led to a screening of the *MECP2*
gene when she was 4. A *de novo* deletion (c.1126del50) in exon 3 resulting in the
truncation of the MECP2-protein was found. None of the other members of the family
carried this *MECP2* deletion.

Macrocephaly and exploration of brain structures volume in a patient with JS

Results from brain MRI obtained in the proband from Family I were compared to similar
data collected in 188 individuals including 149 subjects with ASD and 39 controls from
our cohort Surprisingly, the macrocephaly observed in our patient (>2SD) was not
associated with an increase in brain volume. Indeed, patient's total brain volume was less
than 1SD from the mean (Figure 3). Thus, the macrocephaly was explained by the
increased volume of the skull and not by the brain itself. In parallel, automatic
segmentation of brain structures suggested that grey and white matter volumes of the
frontal, parietal and temporal lobes were in the normal range when compared to subjects
from the cohort (Figure 3). However, right and left occipital grey matter was
significantly decreased (<2SD and approximately 2SD from the mean, respectively). In
addition, when exploring the volume of the subcortical structures, we observed an
increased volume of left thalamus (approximately 2SD), left and right putamen (both
>2SD), left and right pallidum approximately 2SD and >2SD, respectively), and a slight
increase of the left hippocampus (>1SD).
DISCUSSION

In the literature, several minimal regions and genes were proposed to delineate the
location of the gene(s) that could increase the risk for ASD and ID in patients with JS:
*BSX* and *NRGN* have been described at 11q24.2 [Coldren et al., 2009]; *KIRREL3* at
11q24.2 [Guerin et al., 2012], *ARHGAP32* at 11q24.3 [Akshoomoff et al., 2014] and few
others [Bernaciak et al., 2008; Ji et al., 2010; Penny et al., 1995]. A recent morbid map of
the genome based on the investigation of 29,085 patients with developmental disorders
and 19,584 controls, showed that all genes within the JS region are strongly associated
with developmental disorders (10<sup>-5</sup> < P < 10<sup>-10</sup>) [Coe et al., 2014].

In the present study, we completed our initial finding of a 11q24.2-25 *de novo* deletion in a patient with ASD by a screening for new CNVs in that region. The identification of a duplication of the first exon of *NTM* that could disrupt the gene prompted us to search for mutations in this gene. *NTM* seemed to be a compelling candidate gene with a strong association with neurodevelopmental disorders (P=1.13.10<sup>-9</sup>) based on the morbid map of Coe et al. [2014]. In addition, *NTM* belongs to the IgG-like cell adhesion molecules and several members of this family of genes such as Contactins were previously shown to be associated with ASD [State and Levitt, 2011]. Finally, *NTM* is predominantly expressed in the brain and promotes neurite outgrowth [Gil et al., 1998].

Direct sequencing of *NTM* did not provide evidence for its involvement in
ASD and ID. Two heterozygous non-synonymous mutations (p.V327I and p.S335L),
both considered as benign by predictive models, were detected at the same frequency in
patients and controls in our study and in larger independent cohorts [Iossifov et al., 2012;
Sanders et al., 2012]. Based on the whole exome sequence data from patients with ASD, no association was observed between *NTM* and ASD. Our study indicates that *NTM* mutations do not play a major role in the susceptibility to ASD, but cannot formally exclude that haploinsufficiency of *NTM* could be a risk factor for ASD in patients with JS. In addition to the structural similarities with other genes related to ASD such as *CNTN3* [Girirajan et al., 2011] and *CNTN4* [Fernandez et al., 2004]. NTM participates in the control of the axonal pathfinding and synaptogenesis, but also in the proliferation of glial cells [Hashimoto et al., 2009; Sugimoto et al., 2012]. Overexpression of NTM in rat cortical astrocytes, results in an inhibition of cell proliferation and an increase of the cell size [Sugimoto et al., 2012]. Thus, haploinsufficiency of *NTM* reported in Family I, and in patients with JS, might impact the development of cortical astrocytes. Increased in astrocyte proliferation and decreased in the astrocyte size could participate in the white matters lesions reported on patients with JS [Mattina et al., 2009]. These lesions are mainly reported in the cerebellum and in the neocortex, both regions in which *NTM* is strongly expressed all along the life (http://hbatlas.org/). Interestingly, mutations in *HEPACAM*, a member of IgG-like cell adhesion molecules, affect the astrocyte-astrocyte junctions and cause macrocephaly and ID (with or without ASD), or benign familial macrocephaly [Mattina et al., 2009].

The exploration of brain structures using MRI in the proband from Family I has
revealed that the macrocephaly was not the consequence of increased brain volume, but
of increase circumference of the skull. On average, 20% of patients with JS [Tyson et al.,
2008] display macrocephaly, but no case report has mentioned structural changes in the
skull of the patients. Previous studies have rather highlighted the white matter lesions,
thought to be due to delayed myelinisation [Tyson et al., 2008]. In caudal regions
(reported with a high level of *NTM* mRNA in Allen Human Brain Atlas data), we observed a bilateral decrease in grey matter volume, with a maximum in the occipital region (approximately -2SD). Such an inverse correlation was also observed for subcortical structures, with an increased volume (approximately +2SD) of the left thalamus, left and right putamen, left and right pallidum, regions for which the level of *NTM* mRNA was reported low. Although these results were obtained in a single patient, they provide an interesting hypothesis on the mechanism of macrocephaly in patients with JS. Region where NTM needs to be at a high level would be more sensitive to haploinsufficiency. The major decrease in volume observed in the occipital region and the increased volume of left thalamus, the putamen and the pallidum could be explained by aberrant growth rates in regions implicated in repetitive and stereotyped behaviors and in contrast, by growth rates deceleration in cortical regions reported in young adults with ASD [Zielinski et al., 2014].

The clinical impact of the duplication reported in Family II is difficult to ascertain. The paternally transmitted rearrangement was shared by the proband and his affected sister but not by the healthy sister. The father had a lifetime diagnosis of general anxiety disorder and ADHD. He was also evaluated for autistic symptoms but he scored in the normal range on the Social Responsiveness Scale [Constantino and Todd, 2005]. A paternal cousin was also diagnosed with ID, but unfortunately, we were unable to directly assess him and his DNA was not available. Interestingly, *SNX19* is located in the duplication and might be involved in the thumb macrodactyly observed in the father. *SNX19* codes for a chondrogenic factor, and a gain of function resulting from the duplication might affect the differentiation of chondrogenic cells and chondrocyte hypertrophy [Kan et al., 2009]. Indeed, mutations of family members of SNX were
associated with skeletal dysplasia or growth retardation in humans and mice [Carlton et al., 2005; Vervoort et al., 2002].

The identification of the gene(s) involved in the cognitive phenotype of patients with JS warrants more genetic and functional studies. *OPCML* is a tail-to-head tandem duplication of *NTM* and is very similar at the protein level (>75% identity). *OPCML* codes for the Opioid binding Protein/Cell adhesion Molecule-Like and is highly specifically expressed in the brain. It might also have an accessory role in opioid receptor function. In the rat, the orthologous protein binds opioid alkaloids in the presence of acidic lipids and exhibits selectivity for mu ligands. Another gene located in the deleted region, *ARHGAP32* is highly expressed during neuronal development and plays an important role in postsynaptic NMDA signaling and neurite outgrowth [Hayashi et al., 2007]. In mice, *ARHGAP32* is expressed in the brain, involved in early brain development, including extension of axons and dendrites, and postnatal remodeling and fine-tuning of neural circuits [Nasu-Nishimura et al., 2006]. *ARHGAP32* has been recently reported to be deleted in a few patients and proposed as a good candidate gene for ASD traits in JS [Akshoomoff et al., 2014]. The *KIRREL3* gene has also been proposed as a candidate gene for autism [Guerin et al., 2012], but this gene was not included in the CNVs reported here.

## CONCLUSION

In conclusion, our study provides the screening of *NTM* mutations in ASD and the first report that the macrocephaly observed in patients with JS is associated to increased skull
size and abnormal volumes of subcortical structures. More studies linking rare and common genetic variants to brain anatomy will be helpful to better define the heterogeneity of ASD and to understand the complex mechanisms leading to neurodevelopmental disorders in humans.

## Abbreviations

ADHD, Attention Deficit / Hyperactive Disorder; ADI-R, Autism Diagnostic Interview-Revised; Acc, Nucleus accumbens; ADOS, Autism Diagnostic Observation Schedule; ADOS-G, Autism Diagnostic Observation Schedule-Generic; AGP, Autism Genome Project Consortium; Amy, Amygdala; ARHGAP32, RHO GTPase activating protein 32; ASD, Autism Spectrum Disorder; BSX, Brain-Specific Homebox; Ca, Caudate nucleus; CGH, comparative genomic hybridization; CNTN, Contactin; CNV, copy number variation; DIGS, Diagnostic Interview for Genetics Study Interview; DISCO, Diagnostic Interview for Social and Communication Disorders; FIGS, Familial Interview for Genetics Study; EEG, electroencephalography; GPI, glycosylphosphatidylinositol; GWAS, genome-wide association study; Hip, Hippocampus; ID, intellectual disability; IgLON, subgroup of the immunoglobulin superfamily cell adhesion molecules; IQ, intelligence quotient; JS, Jacobsen syndrome; KIRREL3, Kin Of Irre-Like 3; LAMP, lysosomal-associated membrane protein; MAF, minor allele frequency; MRI, magnetic resonance imaging; mRNA, messenger ribonucleic acid; NRGN, Neurogranin; NTM, Neurotrimin; OPCML, Opioid-Binding Cell Adhesion Molecule; Pa, Pallidum; Pu, Putamen; OFC, occipito-frontal head circumference; QC, quality control; SNP, single-nucleotide polymorphism; SNV, single nucleotide variation; SNX19, Sorting Nexin 19; SPGR, spoiled gradient recalled echo; SRS, Social Responsiveness Scale; SSC, Simons
Simplex Collection; Th, Thalamus; TP53AIP1, Tumor Protein P53 Regulated Apoptosis Inducing Protein 1.

**Competing interest**

The authors declare that they have no competing interests.

**Author's contributions**

AM: conception and design; data collection; psychiatric evaluation, analysis, manuscript writing; AB, CSL, GH, ME, NL and RT: extraction data, analysis; FA, ME & AV: data collection, analysis. ML, CG and TB are the main investigators with RD the corresponding authors that drafted the manuscript. All authors red and approved the final manuscript.

**Acknowledgements**

We thank the platform for Ressources Biologiques, Groupe Hospitalier Albert-Chenevier-Henri Mondor, APHP, Crident and the Centre d'Investigation Clinique of the Robert Debré Hospital for collecting and processing the biological samples used in this study. This work was supported by the Institut Pasteur, University Paris Diderot, CNRS, Fondation Orange, ANR (ANR-08-MNPS-037-01 - SynGen), Neuron-ERANET (EUHF-AUTISM), Fondation FondaMental, Fondation Recherche Médicale, and Fondation Bettencourt-Schueller. Written informed consent was obtained from the participant for publication of their individual details and accompanying images in this manuscript. The consent form is held by the authors' institution and is available for review by the editor-in-Chef.
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<figure><img src="image_2.png" /></figure>

**Figure 1.** Location of the critical regions related to cognitive impairment in the Jacobsen syndrome region. Several critical regions could be causative for cognitive impairments in the Jacobsen syndrome region. These regions were delineated by the minimum overlap of 11q24.2-q25 micro rearrangements reported in studies with more than 10 patients with ASD and/or ID. Locations of both CNVs reported in this study were also mentioned. Deletions are in red and duplications in blue. In parallel, genes located in the region were added, based on Refseq information (https://genome.ucsc.edu/). Genes expressed in the brain are in black and the others in grey.
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**Figure 2.** Rearrangements of 11q24.2-q25 were identified with the Human 1M-Duo SNP array from Illumina in both probands of family I and II. In Family I, the *de novo* deletion spans 5.4 Mb, covers 20 genes among which only two expressed in the brain, i.e., *NTM* and *RCIS*. In Family II, the duplication spans 938 kb involves two genes (*SNX19* and the first exon of *NTM*), is shared with the affected siblings and transmitted by the father. This latter had a lifetime diagnosis of attention deficit/hyperactive disorder and general anxiety disorder, and also one of his paternal cousins had an ID. The upper plot shows Log R Ratio (LRR) (in red) and B allele frequency (BAF) (in green). QuantiSNP score is represented with a blue line and indicates the copy number (CN). No similar heterozygous copy number variations (sharing at least 80% overlap with one of these rearrangements) were previously referenced in the Database of Genomic Variants (DGV; http://projects.tcag.ca/variation/). At the opposite, in Decipher (http://decipher.sanger.ac.uk), several subjects with a development delay, carrying a large deletion or duplication within 11q24.2-q25, which encompassed *NTM*, are referenced. Also, three distinct CNVs affecting only *NTM* are reported: a duplication of the first exon, a deletion of the third exon, and a deletion of the third and the fourth exons. Red bars are deletions, and blue bars are duplications. A: Four patients (1617,2050, 2054,2049) with deletion (affecting NTM) and duplication of the region, B: Four patients (1969,1967,1970,2056) with duplications.
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**Figure 3.** Schematic representation of cortical and subcortical structure volumes of a patient with a 11q24-25 de novo deletion compared to 254 subjects with ASD. **A:** Estimation of brain cortical volumes: whole brain volume (BV) and cortical volumes were obtained by automatic segmentation. Reported brain volumes are the sum of the grey (g) and white matter (w) volumes for each of the four Lobes [frontal (F), parietal (P), occipital (O), temporal (T)]. We converted intracranial and brain volume values into Z-scores. Black points represent the volumes observed in the subject compared to the cohort of 254 independent patients with ASD. **B:** Estimation of subcortical structures: Caudate nucleus (Ca), Nucleus accumbens (Acc), Putamen (Pu), Pallidum (Pa), Amygdala (Amy), Hippocampus (Hip), and Thalamus (Th). **C:** Legend used in this figure [−2 SD (blue), near the average of the cohorts (green), +2 SD (red)]. **D:** Cross sectional picture of brain.