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Deregulation of EIF4E: a novel mechanism for
autismM Neves-Pereira, B Müller, D Massie, J HG Williams, P CM O'Brien, A Hughes,
S-B Shen, D St Clair and Z Miedzybrodzka
J. Med. Genet. published online 25 Jun 2009;
doi:10.1136/jmg.2009.066852

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JMG Online First, published on June 25, 2009 as 10.1136/jmg.2009.066852

Deregulation of EIF4E: a novel mechanism for autism
M Neves-Pereira1, B Müller2, D Massie3, J H G Williams1, P C M O’Brien4, A Hughes1, S-B
Shen2, D St Clair1 & Z Miedzybrodzka1,3.
1School of Medicine, and 2School of Medical Sciences, University of Aberdeen, Foresterhill,

Aberdeen, Scotland AB25 2ZD. 3Department of Medical Genetics, NHS Grampian, Foresterhill,
Aberdeen, Scotland AB25 2ZD. 4Molecular Cytogenetics Group, Department of Veterinary
Medicine, University of Cambridge, Madingley Road, Cambridge, CB3 0ES, UK.
Correspondence should be addressed to D. St Clair (d.stclair@abdn.ac.uk) and Z.
Miedzybrodzka (zosia@abdn.ac.uk).
Professor David St. Clair
University of Aberdeen
Institute of Medical Sciences
Foresterhill, Aberdeen
Scotland, UK.
Tel: +44 (0)1224 557950
Fax: 01224 557400

Dr Zosia Miedzybrodzka
University of Aberdeen
Department of Genetics
Polwarth Building
Foresterhill, ABERDEEN AB25 2ZD
Telephone : + 44 1224 559399
Fax : + 44 1224 555766
Email Zosia@abdn.ac.uk

Keywords: mTOR, translation, synaptic plasticity, autism, ASD
Word count: 3,689

Copyright Article author (or their employer) 2009. Produced by BMJ Publishing Group Ltd under licence.

Downloaded from jmg.bmj.com on 14 July 2009

ABSTRACT
Autism is a common childhood onset neurodevelopmental disorder, characterized by severe andsustained impairment of social interaction and social communication, as well as a markedly
restricted repertoire of activities and interests. Its aetiology is multifactorial with a strong genetic
basis. EIF4E is the rate-limiting component of eukaryotic translation initiation, and plays a keyrole in learning and memory through its control of translation within the synapse. EIF4E
mediated translation is the final common process modulated by the mTOR, PTEN and Fragile X
mental retardation protein (FMRP) pathways which are implicated in autism. Linkage of autism
to the EIF4E region on chromosome 4q has been found in genome wide linkage studies.
Here we present evidence that directly implicates EIF4E in autism. In a boy with classic autism
we observed a de novo chromosome translocation between 4q and 5q and mapped the breakpointsite to within a proposed alternative transcript of EIF4E. We then screened for mutations 120
autism families. We found two unrelated families where in each case both autistic siblings andone of the parents harboured the same single nucleotide insertion at position -25 in the basal
element of the EIF4E promoter. Electrophoretic mobility shift assays and reporter gene studies
show that this mutation enhances binding of a nuclear factor and EIF4E promoter activity. Theseobservations implicate EIF4E, and more specifically control of EIF4E activity, directly in
autism. Our findings raise the exciting possibility that pharmacological manipulation of EIF4E
may provide therapeutic benefit for those with autism caused by disturbance of the converging
pathways controlling EIF4E activity.

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INTRODUCTION
Autism (OMIM 209850) is a common childhood onset neurodevelopmental disorder,
characterized by severe and sustained impairment of social interaction and social communicative
abilities, as well as a markedly restricted repertoire of activities and interests. Althoughmultifactorial in origin, autism has a strong genetic basis. For autistic disorder, monozygotic twin
concordance is 60%, and when a broader spectrum of autistic features is considered,concordance rates approach 90%, in contrast to 0% and 10% for dizygotic twins [1]. Autism isclinically heterogeneous with up to 10% of cases associated with well defined neurologicaldisorders such as tuberous sclerosis and fragile X syndrome [1]. Genome wide linkage studies
yielded several linkage peaks including 2q, 5p, 7q, 11p 17q, and, of interest here, 4q [2-5]. Rare
mutations have been found at several loci including the neuroligin, neurexin and SHANK3 genes[6-8]. Microscopic chromosomal rearrangements are seen in 3-6% of autism [9], submicroscopic
copy number variations (CNVs) are found in at least 10% of sporadic, but less than 2% infamilial cases, and many individual loci remain to be identified [10;11].
EIF4E activity is the rate-limiting component of eukaryotic translation initiation, which directs
ribosomes to the mRNA 5’ cap structure for initiation of protein synthesis. In brain, EIF4E
activity is fundamental to the regulation of lasting alterations in synaptic strength or plasticity,and of long-term potentiation (LTP): these are important in learning and memory [12;13].
Increased activity in these systems can lead to repetitive, perseverative behaviour patterns [12].
EIF4E is highly conserved across species and no germline mutations have been reported to date.
Synaptic translation is the final step in pathways already implicated in autism. Deregulation of
PTEN/PI3K and tuberous sclerosis pathways, which converge on mammalian target ofDownloaded from jmg.bmj.com on 14 July 2009

rapamycin (mTOR), an upstream regulator of EIF4E, have been implicated in conditions comorbid with autism [14]. Likewise, in Fragile X syndrome, absence of FMRP upregulates
synaptic translation through failure of recruitment of CYFIP1, a newly recognised EIF4E
binding protein [15;16]. Local protein synthesis is required for synaptic plasticity, a mechanismthat underpins learning and memory [17-21].
Regulation of EIF4E activity is known to play a key role in learning and memory through its
control of translation within the synapse [12;13]. Genome wide linkage studies in autism patients
have shown linkage to the region containing the EIF4E locus on chromosome 4q [2;4]. Here we
present evidence from three independent families that strongly implicate EIF4E in autism.

METHODS
Subjects. The child with the translocation was assessed using an informal clinical interview and
the Autism Diagnostic Observation Schedule (ADOS; module 1 of generic version) at 4 yrs 9months, and the Autism Diagnostic Interview Revised (ADI-R) at 6 yrs 7 months [22;23].
Blood samples were obtained from the translocation carrier and his parents with informed
consent. The study protocol was approved by the Grampian Local Research Ethics Committee. A
lymphocyte cell line was derived from the translocation carrier by transformation with EBV(European Collection of Cell Cultures, Health Protection Agency Culture Collections, Salisbury
UK).
Cases of non-syndromic autism were obtained from the Autism Genetic Research Exchange
collection (http://www.agre.org/). Cases were selected on the basis of availability of DNA fromboth parents and two siblings with a definite diagnosis of autism (120 multiplex families).

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One thousand and twenty control DNA samples from healthy individuals with no evidence ofneuropsychiatric disorder were screened for the mutations found in autism cases. Putative
mutations identified in autism cases were screened for in family members.
Cytogenetic analysis Conventional cytogenetic analysis was performed on GTG banded
metaphase nuclei at the 550 band level. Nuclei were obtained from a lithium-heparinised
peripheral blood sample following standard cytogenetic culture and harvest protocols [24].
Fluorescent in-situ hybridization (FISH) was performed with commercial unique sequence
telomere-specific probes (Vysis), bacterial artificial chromosomes (BACs), and fosmid clones
from the regions flanking the cytogenetic breakpoints. The BACs and fosmids were selectedusing the University of California at Santa Cruz Genome Bioinformatics Browser(http://genome.ucsc.edu/) and obtained from BACPAC Resources (Children's Hospital Oakland
Research Institute). Genomic DNA was labelled by direct incorporation of fluorochromes bynick translation (Vysis nick translation kit). BACs were hybridized for 24 hours, followed by 2
min washes in 0.4 x SSC/0.1% IPEGAL CA-630 (Sigma) at 72°C visualized at 100x
magnification (Zeiss Neofluar objective) using an epifluorescence microscope (Zeiss Axiscop)
and an Applied Imaging analysis system using the MacProbe version 4.3 software.
Chromosome flow-sorting Derivative chromosomes were separated from their homologues by
dual laser flow sorting at the Molecular Cytogenetics Group, University of Cambridge.
Generation of chromosome-specific paint probes followed previously described methods [25;26].
Detailed analysis of the breakpoint regions DNA from der (4) and der (5) was amplified using
the GenomePlex® Single Cell Whole Genome Amplification Kit (WGA4) from Sigma-Aldrich.
PCR primer pairs from across the region of interest were used to amplify DNA of derivativeDownloaded from jmg.bmj.com on 14 July 2009chromosomes. Primer pairs were selected to walk across both breakpoints to characterize thetranslocation by direct sequencing. Copy-number variation analysis on the Affymetrix humanGene-Chip 10K array was used to exclude other cryptic rearrangements.
Mutation analysis Direct sequencing was used to examine the coding regions and the promoterof the EIF4E gene. PCR products were purified by a Y-100 column (Fisher Scientific) and direct
sequencing was performed using the Big Dye Terminator v. 3.1 Cycle Sequencing Kit (Applied
Biosystems). Sequencing reactions were analysed using an ABI 3100 Genetic Analyzer andresults were analyzed using the programs SEQUENCHER 3.1.1 (Gene Codes Corp.). Putative
mutations were validated by sequencing DNA from the affected sibling (and both parents)
showing a variation, and 56 controls. Variants found in affected sibs but not controls wereadditionally screened for in 1020 anonymous control samples using denaturing high performanceliquid chromatography (dHPLC) on a Transgenomic WAVE apparatus, with a positive control
on each run.
Binding reactions and EMSA Binding reactions with 80 fmol 32P 5’ end-labelled doublestranded oligonucleotides were performed in 16 mM Hepes-KOH (pH 8), 16 % glycerol, 80 mM
KCl, 0.16 mM EDTA, 0.8 mM DTT and 10 mg/ml HeLa cell nuclear extract (Abcam). After 30
min incubation on ice, reactions were analysed by 5 % polyacrylamide gel electrophoresis, andvisualised using autoradiography or a Fuji Phosphoimager with AIDA software for quantitation.
Double-stranded DNA molecules used were the wild type genomic sequence 5’TTTCCTCTTACCCCCCCTTCTGGAGCGGTT (C7-4EBE) and the derivative C8-4EBE withan additional C added to the C7 stretch element. Where indicated, 200-fold or 500-fold excess ofcold double-stranded competitor DNA was added.

Downloaded from jmg.bmj.com on 14 July 2009Luciferase assays A 410 base pair EIF4E promoter fragment spanning the region from a PstI
site up to the major transcription initiation site (Position +1 in Fig. 3a) was amplified using the
Roche Expand High Fidelity PCR kit, with oligonucleotides to create a C7-4EBE and a C8-4EBE
version. PCR fragments were inserted into pGEM-T easy (Promega) and verified by sequencing.
The primers contained KpnI and HindIII restriction sites that were used to insert the promoter
fragments into the firefly luciferase reporter vector pGL3 basic (Promega). Near confluent HeLa
cells grown in 24 well plates were transfected with 0.7 µg pGL3 based reporter, and with 100 ng
pRL-Tk (Promega) expressing Renilla luciferase. Cells were lysed 24 h - 48 h after transfection
and luciferase activities were determined using a dual luciferase assay (Promega). Firefly
luciferase activity was standardised with respect to Renilla luciferase. Transfections were done intriplicates and each transfection was measured three times. Shown are the average and standard
deviation calculated from the three parallel transfections.

RESULTS
Routine cytogenetic screening identified a de novo balanced 46,XY,t(4;5)(q23;q31.3)
translocation in a boy with classic autism (fig 1a). There is no family history suggestive ofautistic traits, he had no dysmorphic features, other than a double hair whorl on the crown, andno malformations. On the Autism Diagnostic Observation Schedule- Generic (Module) the childscored 21 points on algorithm items and on the Autism Diagnostic Interview Revised (2000)[22;23] he scored at ceiling level for most algorithm items with a total of 28 points in the domainof reciprocal social interaction, 14 points for non-verbal communication and 8 points for
repetitive behaviours. Thus, he demonstrated a typical and severe autistic phenotype and on allDownloaded from jmg.bmj.com on 14 July 2009

measures, DSMIV criteria for the diagnosis of Autistic disorder were fulfilled. Development wasapparently normal up until the age of two. This was followed by a period of severe regression
characterized by loss of speech, social interaction and communication skills, and thedevelopment of stereotyped and repetitive patterns of behaviour. At the age of 6.5 years his
speech consisted of little other than stereotyped vocalizations but did not display co-morbid
global developmental delay.
Fluorescent in situ hybridization (FISH) analysis with bacterial artificial chromosomes (BACs)
and fosmid clones localized the sites of the breakpoints in immortalised lymphocytes. BAC clone
RP11-911N10 spans the breakpoint on chromosome 4 (fig 1b, fig 1c), and fosmids mapped thebreakpoint on chromosome 5 to a 47.6 kb interval (142,854,992-142,902,586) (data not shown).
Fine mapping was performed by PCR using DNA amplified from flow sorted derivative
chromosomes [25;26]. The breakpoint was further characterized by Sanger sequencing [27] (fig1d). Chromosome translocation occurred by breakage and re-ligation of chromosomes 4 and 5
and involved the addition of few (1-4) residues at the breakpoint. Copy-number variation
analysis using the GeneChip Human Mapping 10K 2.0 Array (Affymetrix) excluded othercryptic rearrangements (data not shown).
The breakpoint on chromosome four is located in a region linked to autism [2] and maps 56 kb
downstream of the EIF4E reference sequence. The breakpoint on chromosome five is not in alinked region, the nearest gene being NR3C1. Families with heterozygous mutation of NR3C1
are reported with hypertension, hypokalaemia and female masculinisation, but not autisticfeatures [28]. Our subject by contrast is normotensive with a normal urinary screen forcatecholamines. Thus our finding supports previous data implicating the chromosome 4q region
in autism.

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To further explore a role of EIF4E in autism, we screened for mutations in 120 multiplex
families with two autistic siblings obtained from the Autism Genetic Research Exchange
collection (AGRE). In two independent families, direct sequencing revealed a heterozygous
single base C insertion in the EIF4E promoter region in the proband. In both of these families,
the variant was also present in the second autistic sibling and the father. The variant was notfound in 1020 anonymous control samples. In fig 2, the chromatograms from the analysis of one
family are shown, clearly revealing the presence of the insertion in the father and the two
affected offspring. Inheritance of the mutation is identical for the second family (data notshown). AGRE SNP data from Affymetrix 10k 2.0 arrays confirmed that the two families areunrelated. This sequence variant is not present in 2040 control chromosomes. It is located in aregion previously identified as the EIF4E basal promoter element (4EBE) that binds hnRNPKand contains a stretch of 7 C nucleotides (C7-4EBE) [29;30] (fig 2b). The C insertion extends it
to an 8 C nucleotide run (C8-4EBE). A number of rare base changes were also present in theautism families (see Table S1).
To determine whether this mutation changes the properties of the EIF4E promoter we performed
binding studies with short double-stranded DNA fragments derived from 4EBE, using HeLa cell
nuclear extract as source of binding factors. Electrophoretic mobility shift assays (EMSA)
demonstrate that the C8-4EBE DNA sequence variant has an increased affinity for an abundantnuclear protein, probably hnRNPK [30]. This is revealed in binding experiments where excess
unlabeled C8-4EBE DNA competes more efficiently with binding of the nuclear factor to 32Plabelled C7-4EBE DNA then an identical excess of unlabeled C7-4EBE DNA (fig 3a).
Promoter activity assays were performed using a 0.4 kb EIF4E promoter fragment with either a
C7- or a C8-4EBE upstream of the firefly luciferase reporter gene, resulting in a construct whereDownloaded from jmg.bmj.com on 14 July 2009

luciferase expression is exclusively controlled by one or the other of these elements. Bothvariants were transfected into HeLa cells and showed a significant increase in expression
compared to the promoterless control construct. Importantly, the single C insertion (C8-4EBE)
resulted in a significant two-fold increase in EIF4E promoter activity compared to the wild type(C7-4EBE) promoter (fig 3b). Thus this mutation causes de-regulation of EIF4E expression.
All four of the affected children with this promoter variant scored highly within all domains of
the ADI-R interview [22] and had marked language delay. One showed regression. The ADI-R is
not designed to provide an index of severity but scores indicate that all subjects had a high levelof symptoms.

DISCUSSION
Our findings implicate the EIF4E gene in the pathogenesis of autism, and provide a strong casefor further study of EIF4E and related pathways in autism. Our evidence is twofold. First, thechromosomal translocation in the boy with autism implicates the region containing EIF4E inautism. Second, we observed a heterozygous C insertion in the EIF4E promoter in two further
unrelated families. In both families, this mutation changes a basal promoter element of EIF4E,
shown to be the binding site of hnRNPK [30]. Importantly, the mutation leads to a higher affinity
for its binding protein, and causes a two-fold increase in promoter activity.
Genomic data indicates that the EIF4E gene has at least two different transcription start sites.
The transcript ENST00000280892, a member sequences of the human EIF4E consensus CDS set
CCDS34031, is transcribed from the proximal promoter that contains 4EBE (fig 2B) [29;31],

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whereas transcription of NM_001968.3 is initiated about 1.5 kb further upstream. Our data
implicate the C insertion in the development of autism and suggest increased activity of theproximal promoter EIF4E promoter as a mechanism.
In both families, the C insertion is shared by a reportedly unaffected parent and two children
diagnosed with autism. Variable penetrance and expression is frequently seen in families with
autism. Likewise, in tuberous sclerosis, a disorder caused by TSC1 and TSC2 mutations
associated with aberrant mTOR signaling, variation in severity of phenotype is common even
within families. Furthermore, these parents may have mild behavioral features that are notcatalogued with the AGRE collection. Of the five cases of autism we describe with deregulationof EIF4E, all had a high level of symptoms, and two of five exhibited regression. In contrast to
findings at other autism loci, none of the affected cases with mutations in this study had either
associated mental retardation or epilepsy.
The translocation may have its effect in two ways. The effect of downstream brain specificregulatory elements may be disrupted by the translocation. The translocation also disrupts analternative EIF4E transcript. The GENSCAN algorithm [32] predicts an EIF4E transcript
NT_016354.401 that encodes a larger protein encompassing the EIF4E reference sequence
including the cap-binding domain required for the role of EIF4E in translation initiation.
NT_016354.401 has three additional downstream exons, and two of these exons map
downstream of the breakpoint (fig 4). Hybridisation to probes 2778989 and 2778996 on the
Affymetrix Human Exon 1.0 ST array indicates that the two proximal downstream exons of
NT_016354.401, which flank the breakpoint, are expressed at elevated levels in the cerebellum.
Our own data revealed widespread expression of the terminal exon in the amygdala,hippocampus and cerebral cortex, regions of the brain implicated in autism (not shown).

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Disruption of NT_016354.401 by the translocation would most likely result in the degradation of
the mRNA by a mechanism related to nonsense-mediated mRNA decay, and cause
downregulation of EIF4E activity.
Clearly, further investigation of function is limited by the unavailability of brain material fromthe affected individuals. EIF4E levels in transformed lymphocytes derived from the translocationcase and the children carrying the C insertion were similar to levels in control transformed
lymphocyte cell lines (not shown). Expression of the four genes flanking the breakpoint (see fig1) was similar in the lymphocyte cell line from the translocation case and in control cells (notshown). We reviewed the available genomic data across the EIF4E locus for evidence ofalternative isoforms. Our analysis of EST data revealed that there is little alternative splicing,with some transcripts containing a cryptic second exon. Differences between EIF4E transcripts
are concentrated at the 5’ end and 3’ ends. Differences at the 5’ end reflect at least in part thedifferent promoter usage. In addition, a significant proportion of mRNAs lack the first exon.
Differences at the 3’ end are mostly due to mRNAs ending prematurely and lacking the terminalexons. Many of these differences may be artificially caused by the methods used for analysis.
However it is possible that there might be several EIF4E transcripts, some of which may be brain
specific. Future investigations will focus on identifying such transcripts and on the effects ofderegulating EIF4E expression on synapse function in appropriate models.
A number of independent lines of evidence support a role of EIF4E in autism. First, it is locatedin a linkage hot spot implicated through linkage studies [2]. Second, EIF4E activity is regulatedby the highly conserved PTEN/PI3K and tuberous sclerosis pathways. These pathways converge
on mammalian target of rapamycin (mTOR), an upstream regulator of EIF4E. In tuberous
sclerosis complex (TSC), where 25 - 50% have autistic features, mutations in TSC1 and TSC2,

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remove inhibition of mTOR and increase EIF4E activity [13;33]. Similarly, individuals with
germline mutations in PTEN often have associated autistic features [14]. Mice with knockouts of
the intracellular receptor mediator of rapamycin activity, Fkbp12, display repetitive and otherbehavioural features like those found in autism [34]. Deregulation of these signaling pathways
can result in abnormalities of brain growth and synaptic plasticity in a manner analogous to
Fragile X syndrome, a learning disability disorder with prominent autistic features, where
inactivation of FMRP causes upregulation of synaptic translation mediated through CYFIP1, a
recently recognised EIF4E binding protein [16]. Deregulation of glutamate signaling is also seen
in both Fragile X and TSC. Cap-dependent translation is active during mGluR-LTD, and both
MEK-ERK and PI3K-mTOR signaling pathways regulate EIF4E activity [35]. Indeed, on thebasis of the association of mutations in FMRP, TSC1/2 and PTEN with autism, Kelleher et al.
(2008) [36] hypothesized that “defects in translational repression may represent a possible
mechanism leading to autistic phenotypes”. Finally, mutation in another sub-unit of thetranslation

initiation

complex,

EIF2B,

causesthepediatric

neurologicaldisorder,leukoencephalopathy with vanishing white matter [37]. We previously reported decreased whitematter and increased grey matter in autism [38]. Thus variation in EIF4E or interacting proteins
is consistent with the white matter variation observed in autism.
Regulation of synaptic plasticity by EIF4E is highly complex and probably governed by as yet
unknown additional EIF4E binding proteins [12]. In oncogenesis, increased EIF4E activity
results in specific upregulation of translation of particular mRNAs that are normally inefficiently
translated [13]. Subtle deregulation, either up or down, of synaptic EIF4E through modificationof translation of specific brain transcripts may significantly impact on delicate processes such as
synaptic consolidation. Penetrance and expressivity of such genetic variants will depend upon

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genetic background and environmental factors, possibly at specific stages in development. This
could account for our finding of reportedly asymptomatic carriers of the insertion mutation
among the parents of the affected cases. Larger studies are now required to determine theprevalence and penetrance of EIF4E mutations.
EIF4E then is the endpoint of a number of pathways already implicated in autism. Our work
showing direct involvement of EIF4E therefore provides key additional support for Kelleher’s
protein synthesis hypothesis for autism [36]]. The drug rapamycin suppresses EIF4E expression
through its effects on mTOR. Exciting preliminary observational data from trials of rapamycin in
TSC, suggest that rapamycin may not only control tumour growth, but may also improve
behaviour [39]. Our findings raise the exciting possibility that in cases of autism caused byderegulation of translation, pharmacological manipulation EIF4E expression, either directly or,for example, through manipulation of mTOR signaling, could provide therapeutic benefit.

ACKNOWLEDGEMENTS
We thank Helen Murdoch for putting us in contact with the translocation case; Elaine Durward,
Ben Milner, Ronggai Li, Guoqing Liu and Lynne Gray for technical assistance; Caroline Clark
and Christine Bell for technical advice and Dee Rasalam for advice on diagnostic criteria for
autism; the Medical Genetics and Tau Rx groups for sharing physical resources; and Irwin
McLean and Ian Booth for invaluable comments. We gratefully acknowledge the family of ourtranslocation case, participating AGRE families and the Autism Genetic Resource Exchange(AGRE) Consortium. The Autism Genetic Resource Exchange is a program of Cure Autism
Now and is supported, in part, by grant MH64547 from the National Institute of Mental Health toDownloaded from jmg.bmj.com on 14 July 2009

Daniel H. Geschwind (PI). Flow sorting was made possible by a Wellcome Trust grant to
Malcolm Ferguson-Smith for the Cambridge Resource Centre for Comparative Genomics. This
project was funded by the Scottish Government Chief Scientist Office, NHS Grampian
Endowments and an anonymous grant to the University of Aberdeen Development Trust.

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Downloaded from jmg.bmj.com on 14 July 2009

FIGURE LEGENDS
Figure 1: A chromosomal translocation in a case of classical autism. (a) GTG banding ofchromosomes 4 and 5 and der (4) and der (5). (b) FISH analysis of breakpoint. FISH analysis
with bacmids and fosmids was performed as described. RP11-911N10 maps to base pairs
99887465-100079064 on chromosome 4, RP11-359H9 and RP11-659D8 map to base pairs
146356329-14651515 and 142011154-142179956 on chromosome 5, respectively. RP11911N10 labels both derivative chromosomes and thus spans the breakpoint. RP11-359H9 andRP11-659D8 (indicated by an arrow and a diamond) flank the chromosome 5 breakpoint. (c)Schematic representation of the breakpoint regions of chromosomes 4 and 5, and derivative
chromosomes der (4) and der (5). The breakpoints are indicated with arrows. Representation was
prepared using the UCSC Genome Bioinformatics Browser. Not all forms of the reference genes
are shown, nor are all exons. Chromosome coordinates are based on the human genome build
36.2. (d) Breakpoint sequences. Shown are chromatograms of the breakpoint sequence on der (4)and der (5) obtained by direct sequencing.

Indicated are the sequences derived form

chromosomes 4 and 5, respectively. In the sequence alignment, chromosome 4 sequences are in
red letters and chromosome 5 sequences are in blue capital letters. Residues in black capitalletters at the break point are not derived from either chromosome 4 or chromosome 5. Note thatan AC dinucleotide at the breakpoint in chromosome 5 has been duplicated and occurs in der (4)and der (5).
Figure 2: An inherited C insertion in the EIF4E promoter element 4EBE in families withchildren with autism (a) Insertions of a C in the EIF4E-4EBE promoter element were detectedby sequencing in two independent families of the AGRE cohort (C8-4EBE). Pedigree andsequencing traces are shown for one family only as the inheritance pattern and sequence are theDownloaded from jmg.bmj.com on 14 July 2009

same in both families. (b) EIF4E promoter region showing transcription initiation sites (capitalletter), the start of the EIF4E coding sequence (boxed) and the 4EBE promoter element (4EBE,
underlined) with a stretch of 7 nucleotides (C7-4EBE) [29]. The major transcription initiation site
is indicated with +1 [31].
Figure 3. The C insertion into the 4EBE promoter element increases binding of a nuclear
factor and EIF4E promoter activity (a) Effect of the C insertion on binding of nuclear factors.
Binding reactions with wild type C7-4EBE element or the derivative C8-4EBE and HeLa nuclear
were done and analyzed by EMSA followed by visualisation using autoradiography or a Fuji
Phosphoimager as described. Where indicated a 200-fold or 500-fold excess of unlabelledcompetitor DNA was included in the binding reaction. The histogram summarizes the proportionof 32-P labelled DNA in complexes. The proportion of complexed DNA in reaction withoutcompetitor was defined as 100 %. Shown are data from two independent experiments (in black
and white, respectively). (b) C insertion increases EIF4E promoter activity. Dual luciferase
assays were done by transfecting HeLa cells with pGL3 basic (no promoter) or pGL3 with the
0.4 kb EIF4E promoter fragments containing either the C7-4EBE or the C8-4EBE. The major
EIF4E transcription initiation site (Position +1 in fig 2b) and two natural myc binding sites areincluded in both constructs. Plasmid pRL-Tk was co-transfected and Renilla luciferase activity
was used as control. Transfections were done as described and shown are relative firefly
luciferase units 24 h after transfection, standardized with respect to Renilla luciferase expression.
This data is representative of three independent experiments.
Figure 4: EIF4E transcripts (a) Schematic representation of EIF4E transcripts. Shown are theintron/exon structures of EIF4E transcript ENST00000280892 (top) and of the predicted
alternative transcript (NT_016354.401; bottom) (2 additional 5’ exons have been omitted forDownloaded from jmg.bmj.com on 14 July 2009

simplicity). Shared exon sequences are shown as black boxes; unique exons are shown in white.
Additional downstream exons of the alternative transcript are also shown as white boxes. The
translocation breakpoint disrupts the alternative transcript between two of these exons. Note that
intron and exon sequences are not represented to scale.

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exclusive license (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing GroupLtd and its Licensees to permit this article (if accepted) to be published in Journal of Medical Genetics and any other
BMJPGLproducts

toexploitallsubsidiary(http://jmg.bmj.com/misc/ifora/licenceform.shtml).

rights,

as

set

out

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our

licenceTable S1: Sequence variations identified by direct sequencing in autism families.
NM_001968*/
NT_016354.401

Observationsin 120 cases

Confirmation

Locationon chr 4

SNP id(dbSNP)

5’UTR/exon 2

1

HapMap100070056

rs17028283-/3’exon2

4

Seen in 2 / 100
controls

100011889

rs7674319

non-synonymous in
NT_016354.401
non-synonymous in
NT_016354.401* EIF4E mRNA reference sequence.

1