Master thesis as part of the master Experimental and Clinical Neuroscience
The role of genes implicated in Autism Spectrum Disorders
Max Koppers
Abstract
Autism spectrum disorders are pervasive developmental disorders that are considered to be
highly genetic. ASD show a high a clinical heterogeneity which may be explained by the high
genetic heterogeneity. Many linkage regions, genes, chromosomal abnormalities and copy
number variations have been linked to ASD. In this thesis and overview of the genetics of
ASD is given and it was investigated by reviewing relevant literature whether certain ASD
associated genes could explain ASD phenotypes. Several genes seem to play an important
role in certain phenotypes (CNTNAP2, SHANK3, PTEN) while others do not clearly show any
correlation (GRIK2, NGLN3, NRXN1). Some genes seem to be related to several phenotypes
(EN2, GABRB3, OXTR) and animal models of these genes could be used to study ASD in more
detail. However, the discovery of more ASD associated genes is needed as well as more
research into the biological function/interactions of ASD associated genes is needed to
clarify whether there is a clear correlation between genes and phenotypes in ASD.
Introduction
Autism spectrum disorders (ASD) are pervasive developmental disorders that are
characterised by severe impairment in socialisation and communication, and by repetitive or
unusual behaviours. Most researchers include autistic disorder, Asperger’s syndrome (AS),
and pervasive developmental disorders not otherwise specified (PDD-NOS) in the autism
spectrum disorders (Levy et al. 2009). The prevalence of autistic disorder seems to have
increased over time. In the 1960s and 1970s it was estimated that ~5 per 10,000 children
had autistic disorder and this increased in the 1980s to ~10 per 10,000 children. Most recent
reports estimate the prevalence of autistic disorder between 10 and 20 per 10,000 children
(Newschaffer et al. 2007). The prevalence of ASD has been more consistent over time and is
about 60 per 10,000 children. The male-female ratio is typically approximately 4:1
(Newschaffer et al. 2007).
Autism spectrum disorders show a high clinical heterogeneity. The three core symptoms of
ASD, with an onset of before 3 years of age, affect socialisation, communication, and
restricted, stereotyped and repetitive patterns of behaviour (Table 1).
Table 1. Core symptoms of ASD (DSM-IV-TR)
DSM-IV-TR criteria for autism spectrum disorders. Core symptoms of ASD can be divided into the domains of
socialisation, communication and restricted, stereotyped, and repetitive patterns of behaviour.
Besides these core symptoms co-morbid disorders are very common in children with ASD
(Matson and Nebel-Schwalm 2006). These co-morbid disorders might have a greater effect
on function and outcome than the core symptoms. Intellectual disability (ID) is very common
in children with autistic disorder and PDD-NOS and symptoms, particularly language delays,
stereotypy and self-injuries increase with the severity of intellectual disability (Matson and
Nebel-Schwalm 2006). Other behavioural and developmental co-morbid disorders include
attention deficit-hyperactivity disorder, depression, anxiety, psychosis, obsessive compulsive
disorder, sleep disruption and sensory changes (Matson and Nebel-Schwalm 2006, Hughes
2009). Also gastro-oesophageal reflux, food selectivity, and neurological disorders such as
tics and seizures and epilepsy are common in children with ASD (Levy et al. 2009, Hughes
2009).
The neurobiology of ASD has been studied by examination of brain growth,
neuropathological studies, imaging studies, electrophysological, and neurochemical studies.
Several theories about the underlying neurobiology have been posed such as pragmatic
language impairment, difficulties in intersubjectivity (‘Theory of mind’), and in executive
function, weak central coherence, difficulty with integration of information into meaningful
wholes, and underconnectivity (Levy et al. 2009, Hughes 2009).
Brain growth has been found to be consistently altered in ASD patients. Neuroimaging
studies have shown overgrowth in ASD patients as compared to controls in cortical white
matter and abnormal patterns of growth in the frontal lobe, temporal lobes, and limbic
structures such as the amygdala (Levy et al. 2009). Macrocephaly is observed in about 20%
of ASD cases (Lainhart et al. 2006).
Several brain regions are implicated in ASD by neuropathological findings studies. One of the
most consistent finding is the observation of errors in neuronal migration in the frontal and
temporal lobes (Abrahams and Geschwind 2010). Examination of cortical minicolumns lead
to the observation that the spacing between of these minicolumns is reduced in the frontal
and temporal cortex of individuals with an autism spectrum condition (Abrahams and
Geschwind 2010).
The amygdala, which is involved in the modulation of social behaviour and emotions, has
also been implicated in ASD. A pathological study in an individual with autism and seizures
showed that neurons in the amygdala were abnormally small and showed elevated packing
density (Abrahams and Geschwind 2010). Another study in seizure-free autistic patients
found abnormalities with fewer neurons in the amygdala of cases compared with controls
(Schumann and Amarall 2006). The effects were most prominent in the lateral nucleus of the
amygdala is interesting because a reduced neuronal number in this area has also been
observed in schizophrenia patients (Abrahams and Geschwind 2010). The cerebellum is also
consistently affected in ASD patients. A decrease in the number of Purkinje cells in the
cerebellum has been observed in autopsy samples from ASD patients in several studies
(Palmen et al. 2004). Because neurodegenerative signs are mostly absent from these
samples, it is suggested that these defects are developmental.
Neuroimaging studies have also implicated the cerebellum in ASD. These imaging studies
have consistently demonstrated posterior cerebellar hypoplasia. Besides coordinating and
controlling motor function, tasks associated with cerebellar activation include attention,
executive control, language, working memory, learning, pain, and emotion (Strick et al.
2009).
Functional MRI (fMRI) studies have shown lowered functional connectivity relating to
language, working memory, problem solving, and social cognition (Levy et al. 2009). A well
replicated finding in fMRI studies is the hypoactivation of the fusiform face area, which is
associated with deficits in perception of people compared with objects (Levy et al. 2009).
Other fMRI studies have suggested impaired mirror neuron activity in the inferior frontal
gyrus. The mirror neuron system is active during the observation, imitation, and
understanding of the intentions of other people’s actions. When acting together with the
limbic system it is thought to be involved in the understanding of emotions (Minshew and
Williams 2007).
Furthermore diffusion tensor imaging studies have shown disruption of white matter in
brain regions associated with social functioning (Levy et al. 2009).
Neurochemical studies have implicated the serotonergic, dopaminergic, cholinergic, and
glutaminergic systems as well as oxytocin and amino acid neurotransmitters in the
development of ASD (Levy et al. 2009).
Autism spectrum disorders are considered to be ‘highly’ genetic disorders. Twin studies on
autistic disorder showed that the concordance rates for monozygotic is between 70 and
90%. For dizygotic twins this is between 0 and 30% (Freitag 2007). There have been no twin
studies performed on AS or PDD-NOS. The sibling recurrence of autistic disorder was
estimated at 2-3% (Folstein and Rosen-Sheidley 2001). Based on these concordance rates
and sibling recurrence the heritability estimate is more than 90%. Another indication that
ASD have a strong genetic component comes from the presence of mild autistic traits in first
degree relatives of ASD patients (Piven et al. 1997).
Genetics of ASD
ASD in genetic disorders
As mentioned, ASD are considered to be highly genetic disorders. A large number of
chromosomes and genetic loci have been implicated in ASD which indicates that it is caused
by complex multigenic interactions (Freitag et al. 2010, Persico and Bourgeron 2006).
Insights in candidate genes and possible molecular pathways involved in ASD have been
obtained by looking at genetic disorders that are associated with ASD. This group of ASD
cases is often called ‘syndromic’ ASD while ASD cases with an unknown genetic cause are
labeled as ‘idiopathic’ or ‘non-syndromic’ ASD (Persico and Bourgeron 2006). Around 10-15%
of the cases with ASD have an identified genetic disorder (cytogenic abnormalities and single
gene disorders). A list of genetic disorders of which association to ASD has been suggested is
shown in Table 2. In this section several genetic disorders and chromosomal abnormalities
associated with ASD will be discussed.
Table 2. Genetic syndromes associated with ASD (Moss and Howlin 2009, Caglayan 2010,
Abrahams and Geschwind 2008)
Genetic syndromes associated with ASD. The genetic syndromes that will be discussed in this section are
highlighted in bold.
Fragile X syndrome
Between 3-5% of the ASD have fragile X syndrome (FXS) (Freitag et al. 2010). Between 25
and 33% of FXS patients meet the diagnostic criteria for autism. In addition, of the male
children with FXS around 90% show one or more features of autism such as perseverative
speech, social avoidance and stereotypic behaviour (Roberts et al. 2007). Fragile X syndrome
is the main cause of inherited mental retardation (D’Hulst and Kooy 2009). Macroorchidism,
large ears, a prominent jaw and high-pitched speech are characteristics of this disorder. The
syndrome is caused by an expansion of a CGG repeat (>200 repeats) in the 5’ untranslated
region of the fragile X mental retardation 1 (FMR1) gene that is located on chromosome
Xq27.3. This expansion results in hypermethylation of the CpG island in the promoter region
which causes transcriptional silencing of FMR1 (D’Hulst and Kooy 2009). FMRP, the protein
encoded by FMR1 is a RNA binding protein that is most abundantly expressed in the brain.
The protein is thought to play an important role in synaptic plasticity through the regulation
of messenger RNA (mRNA) translation and transport (Jin et al. 2004).
Rett syndrome
Rett syndrome (RS) is a X-linked dominantly inherited neurological disorder that
predominantly affects females. According to the DSM-IV-TR criteria Rett syndrome is now
classified as a pervasive developmental disorder and it is not included in the autism
spectrum disorders although some authors do classify Rett syndrome as an ASD. Patients
with classic RS seem to develop normally until 6-18 months of age. They then gradually show
a loss of speech and motor skills and develop microchepaly, seizures, ataxia, and autistic
features such as social withdrawal, lack of eye-to-eye contact, and unresponsiveness to
social cues (Caglayan 2010). Estimate rates of ASD in Rett syndrome 25% to 40% and up to
97% in cases of the preserved speech variant of RS (Moss and Howlin 2009).
In most cases RS is caused by mutations in the gene located on chromosome Xq28 encoding
the methyl CpG binding protein 2 (MeCP2) (Amir et al. 1999). Mutation type and pattern of X
chromosome inactivation (in females) create a wide range of phenotypes (Zoghbi 2003).
MeCP2 regulates chromatin structure and can repress gene expression dependent on
methylation (Fuks et al. 2003). Dysfunction of MeCP2 might thus result in disruption of the
normal developmental program of gene silencing. MeCP2 is most abundantly expressed in
the brain in neurons but not in glia. The levels of expression increase in cortical neurons
during development. This pattern of expression suggests that MeCP2 modulates neuronal
plasticity and maturation (Zoghbi 2003).
Tuberous sclerosis complex
Tuberous sclerosis complex (TSC) is present in about 1% of ASD cases (Freitag et al. 2010). It
is a multisystem disorder that is characterized by hamartomas in several organs, including in
the brain (Curatolo et al. 2010). TSC is caused by different mutations in either the tuberous
sclerosis 1 (TSC1) gene on chromosome 9q34 or the tuberous sclerosis 2 (TSC2) gene on
chromosome 16p13. The TSC1 gene and TSC2 gene code for hamartin and tuberin
respectively. These two form a complex that inhibits the mammalian target of rapamycin
(mTOR) pathway that controls translation, proliferation and cell growth. Dysregulation of
this pathway due to the mutations results in the production of cortical tubers and cortical
disorganization (Curatolo et al. 2010). Patients with mutations in TSC2 are more likely to
develop ASD than those with TSC1 mutations (Lewis et al. 2004).
Phenylketonuria
Less than 1% of ASD cases is caused by phenylketonuria (PKU) (Freitag et al. 2010). PKU is a
genetic disorder characterized by a deficiency in the enzyme phenylalanine hydroxylase
(PAH). Over 200 mutations have been identified that affect the functioning of the enzyme.
This results in the inability to metabolize phenylalanine. Toxic levels of PAH can be produced
as a result of high levels of protein in the diet. This toxicity may cause reduction of myelin,
neuronal loss and a decreased level of neurotransmitter density, damaging the brain (Baieli
et al. 2003). This will result in seizures and intellectual disability. The association of PKU and
ASD has been inconsistent, most likely due to the increased availability of early detection
methods and treatment. When treatment is started early a normal neurodevelopment
occurs, preventing damage to the brain. The role of PAH in developing ASD symptoms might
help to understand the underlying causal factors of ASD (Moss and Howlin 2009).
Angelman/ Prader-Willi Syndrome
Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are caused by anomalies of the
chromosomal regions 15q11-q13. Anomalies on the maternal chromosome cause AS
whereas anomalies on the paternal chromosome cause PWS. Together with inherited
duplications of chromosomal region 15q11-q13, which are discussed below, these
syndromes account for approximately 1-2% of ASD cases (Abrahams and Geschwind 2008).
Prevalence rates of ASD in AS patients range from 50 to 81% (Moss and Howlin 2009).
Angelman syndrome is characterized by moderate to severe intellectual disability,
microcephaly, seizures, no language development, excessive laughter and motor
stereotypies. Several genetic mechanisms are known to cause AS. Around 70% of AS cases
are due to maternal deletions. In 2-3% of cases an uniparental disomy of the chromosomal
region with lack of the maternal copy is the cause and approximately 2-3% have imprinting
defects. Between 22% and 25% of AS patients have mutations in the ubiquitin protein ligase
E3A (UBE3A) gene region or in the imprinting centre of region 15q11-q13 (Kishino et al.
1997, Moss and Howlin 2009). The UBE3A enzyme is part of the ubiquitin protein
degradation system and is maternally expressed in the brain. It directs ubiquitin molecules
to specific substrates.
Prader-Willi syndrome is characterized by moderate intellectual disability, hypotonia, growth
retardation, obsessive-compulsive behaviours, and hyperphagia. Genetic mechanisms that
cause PWS are interstitial deletions of region 15q11-q13 (70-80%), maternal uniparental
disomy with lack of paternal copy (20-30%), and mutations in the imprinting centre (1-2%)
(Freitag 2007). Single genes responsible for PWS have not been identified yet. ASD features
are more likely to develop in PWS patients with maternal uniparental disomy (Hogart et al.
2010).
Timothy Syndrome
Timothy syndrome is a multisystem disorder characterized by cardiac arrhythmias,
congenital heart disease, webbing of fingers and toes, immune deficiency, cognitive
abnormalities and autism (Splawski et al. 2004). Between 60% and 80% of Timothy
Syndrome patients have an ASD (Abrahams and Geschwind 2008). It is caused by mutations
in the calcium channel voltage-dependent L type alpha 1C subunit gene (CACNA1C) (Splawski
et al. 2004). In the brain, this calcium channel mediates the increase of cytosolic Ca 2+ and
may control the number of excitatory synapses (Persico and Bourgeron 2006). Altered Ca2+
signalling due to mutations could results in altered synaptogenesis.
Other chromosomal abnormalities in ASD
Besides Prader-Willi and Angelman Syndrome, several chromosomal abnormalities exist in
ASD cases. A rate of between 3-5% of cytogenic abnormalities has been estimated in ASD
(Freitag et al. 2010). Many rearrangements can occur in the chromosome region 15q11-q13
due to the presence of repeated DNA elements. Deletions (AS and PWS), translocations,
inversions, and supernumerary isodincentric marker chromosomes formed by inverted
duplications are the most common rearrangements. Interstitial duplications and triplications
can occur but are less frequent (Battaglia et al. 2008). The most common chromosomal
abnormality that is present in approximately 1% of the ASD patients are inverted
duplications (inv dup (15) or idic (15) syndrome) in chromosomal region 15q11-q13. This
syndrome is characterized by muscle hypotonia, developmental delay and intellectual
disability, epilepsy, and autism or autistic behaviour (Battaglia et al. 2008). Several studies
have shown a strong association between autism and the idic (15) syndrome (Rineer et al.
1998, Hogart et al. 2010). Patients with maternally derived interstitial duplications and
triplications of chromosome 15 often present with a neurodevelopmental disorder including
ASD (Hogart et al. 2010).
Deletions of chromosome 2q37, chromosome 7q11, chromosome 16p11, chromosome
22q11 and 22q13.3 have also been associated with ASD (Freitag 2007, Abrahams and
Geschwind 2008). Deletions of chromosome 2q37 have been identified in over 70 autistic
individuals (Wassink et al. 2005, Abrahams and Geschwind 2008). It is not clear which gene
or genes in this region are contributing but the involvement of the centaurin gamma 2
(CENTG2), a GTP-ase activating protein involved in membrane trafficking and cytoskeleton
dynamics, has been implicated (Wassink et al. 2005).
Deletions and duplications of chromosome 16p11 have been seen in around 1% of ASD
patients (Abrahams and Geschwind 2008, Weiss et al. 2008).
Two regions on chromosome 22 (22q11.2 and 22q13.3) have been associated with ASD.
Velocardiofacial syndrome (VCF) is a syndrome with a microdeletion on chromosome
22q11.2. Anomalies in patients with VCF include cardiac malformations, vascular anomalies
and cleft palate (Kates et al. 2007). Also developmental problems, speech and language
delays, anxiety, and social withdrawal are seen in these patients. At least 20% of children
with VCF are diagnosed with autism (Kates et al. 2007). Phelan-McDermid syndrome or
22q13.3 deletion syndrome is a microdeletion syndrome characterized by hypotonia,
developmental delay, delayed speech, and minor dysmorphic features. Autistic-like
behaviour including poor eye contact and stereotypic movements are seen in these patients
(Manning et al. 2004).
Genomic syndromes in ASD as well as chromosomal abnormalities have provided valuable
insights in candidate genes and possible molecular pathways involved in ASD. In the next
section candidate genes for ASD that were identified partly because of these syndromes as
well as linkage regions for ASD will be discussed.
Linkage and genome-wide association studies
Numerous linkage studies have been performed in ASD. Nearly every chromosome has been
implicated through linkage studies, but not all linkage regions have been replicated
(Abrahams and Geschwind 2008). Linkage has been found in at least two independent
studies for chromosome regions 2q21-33, 3q25-27, 3p25, 4q32, 6q14-21, 7q22, 7q31-36,
11p12-13, and 17q11-21 (Freitag et al. 2010). However, probably due to the heterogeneity
of ASD, these linkage studies did not find any strong association. A meta-analysis did confirm
the region 7q22-32, and showed suggestive evidence for linkage to 10p12-q11.1 and
17p11.2-q12 (Trikalinos et al. 2006).
Similar to linkage studies, genome wide association studies (GWAS) are considered to be non
hypothesis driven studies. GWAS are designed to detect common causal variants, which
might be difficult because a common disease – common variant genetic model might not be
true for most ASD cases (Freitag et al. 2010). To date, three GWAS have been performed in
ASD (Wang K et al. 2009, Weiss et al. 2009, Ma et al. 2009). Common genetic variants were
identified and replicated on 5p14.1 (Wang K et al. 2009, Ma et al. 2009) and 5p15 (Weiss et
al. 2009). The 5p14.1 region contains highly conserved genomic elements which suggest a
potential regulatory function. The linkage disequilibrium block associated with ASD is located
in an intergenic region between the cadherin 9 (CDH9) and 10 (CDH10) genes (Wang K et al.
2009). These genes will be discussed in the candidate gene section below. One single
nucleotide polymorphism (SNP) on region 5p15 was significantly associated with autism
(Weiss et al. 2009). This SNP is located between the genes TAS2R1, a gene encoding a bitter
taste receptor, and semaphoring 5A (SEMA5A) which is involved in axon guidance during
neural development. SEMA5A expression was reduced in the occipital lobe cortex of brains
of autism patients (Weiss et al. 2009).
One GWAS was performed on autistic-like traits in the general population (Ronald et al.
2010). One SNP located at 2p21, a region not previously linked to autism, was associated
with social autistic-like traits, but only nominally significant (Ronald et al. 2010).
Copy-number variation studies
Both de novo and inherited copy number variation (CNV) are emerging as important causes
of ASD, either as rare variants that modulate disease risk or as syndromes linked to the ASD
as described above. A CNV is currently defined as a genomic segment longer than 1 kb with a
variable copy number compared to a reference genome (Freitag et al. 2010). It can be a
deletion, duplication, insertion or a complex multi-site variant that disrupt a variable number
of genes, which may then result in different gene products or changes in expression (Freitag
et al. 2010). Recently, whole-genome copy number variation studies reported the presence
of de novo CNVs in 7-10% of sporadic ASD cased, in 2-3% of multiplex ASD families and in
only 1% of control families (Sebat et al. 2007, Marshall et al. 2008). Since these findings,
several other studies have implicated CNVs in ASD (Autism Genome Project Consortium
2007, Christian et al. 2008, Kumar et al. 2008, Weiss et al. 2008, Bucan et al. 2009, Cho et al.
2009, Glessner et al. 2009, Gregory et al. 2009, Fernandez et al. 2010). One of the most
replicated findings of these CNV studies is a 555 kb deletion on chromosomal region 16p11
that is present in ~1% of ASD patients (Marshall et al. 2008, Kumar et al. 2008, Weiss et al.
2008, Fernandez et al. 2010). This deletion is observed in controls, however it is enriched a
100-fold in ASD patients (Weiss et al. 2008).
Besides this region several other genes have been implicated trough these CNV studies.
These genes will be mentioned in the next section. It is important that future research
determines how a CNV affects gene function or expression.
Candidate genes and their role in ASD
Numerous candidate gene association and resequencing studies have been performed to
elucidate variants associated with ASD. These studies, together with genes identified in
syndromic ASD, linkage studies and genome-wide association studies, have identified a large
number of candidate genes associated with ASD. Insight into the molecular mechanisms
involved in ASD can be obtained from looking at the biological function of these candidate
genes. As a result from these genetic findings several molecular pathways are thought to be
involved in ASD. Up to date, mechanisms or pathways that seem to be involved include
synaptic function, cell-cell interaction, and excitatory and inhibitory neurotransmission. In
this section several candidate genes, their functions, and the molecular pathways in which
they are involved will be discussed (Freitag et al. 2010). Several studies indicate that specific
traits or phenotypes are highly heritable, suggesting that the different phenotypic
components of ASD are under different genetic influences (Ronald et al. 2006, Happé et al.
2006). Therefore, in this section the question if certain genes can explain certain ASD
phenotypes will be discussed by looking at the expression and/or function of these genes,
animal models, and phenotypes of patients carrying gene mutations.
Neuroligins and neurexins
One of the first genes in which causal mutations were identified in idiopathic or nonsyndromic ASD patients are the genes neuroligin 3 (NLGN3) and neuroligin 4X (NLGN4X)
(Jamain et al. 2003). In one Swedish family a mutation in NLGN3 was identified in two
affected brothers, one with typical autism and one with Asperger’s syndrome. In another
Swedish family a frameshift mutation in NLGN4X resulting in a premature stop was identified
in two affected brothers, also one with typical autism and one with Asperger’s syndrome
(Jamain et al. 2003). These two genes lie on chromosome Xq13 and Xp22.3 respectively.
Since this finding several other groups have identified coding mutations in NLGN4X in ASD
and mental retardation patients (Laumonnier et al. 2004, Yan et al. 2005, Zhang C et al.
2009, Pampanos et al. 2009). Also a de novo mutation in the promoter region of NLGN4X
was identified in a patient with autism and non-syndromic mental retardation (Daoud et al.
2009). Besides these mutations several deletions in the NLGN4X locus were observed in
autistic patients (Chocholska et al. 2006, Macarov et al. 2007, Lawson-Yuen et al. 2008,
Marshall et al. 2008). Also, suggestive association with ASD for a SNP close to NGLN4X was
found in a large GWAS (Wang K et al. 2009). However there are some reports in which no
mutations were found in ASD patients in these two genes (Vincent et al. 2004, Talebizadeh
et al. 2004, Gauthier et al. 2005, Ylisaukko-oja et al. 2005, Blasi et al. 2006a). These
contradicting findings imply that the NLGN4X and to a lesser extent NLGN3 mutations are a
responsible for a small fraction (<1 %) of ASD cases. Recently, duplications of the NLGN1
gene were identified in autism patients in a genome wide copy number variation study
(Glessner et al. 2009). Also modest association between a SNP in NGLN1 and ASD has been
found (Ylisaukko-oja et al. 2005).
Although the role of neuroligin mutations or copy number variations is not inconclusive,
their function and interaction with other genes implicated in ASD (e.g. NRXN1, SHANK3,
CNTNAP2; discussed below) have provided insights into the role of synaptic function in ASD.
Humans express five neuroligin genes (NLGN1, NLGN2, and NLGN3, NLGN4X and NLGN4Y)
that are widely expressed in brain. Neuroligins are cell adhesion molecules that are localized
postsynaptically in glutameric (NLGN1, NLGN3, NLGN4X, and NLGN4Y) and/or in gammaaminobutyric acid-ergic (NLGN2, NLGN3) synapses (Lintas and Persico 2009, Südhof 2008).
They are thought to play a role in synapse function and maturation. Neuroligins form a
complex by binding to the presynaptic receptor protein neurexin 1β (NRXN1β) to exert their
synaptic functions (Südhof 2008).
Two NLGN3 mouse models have been studied for an implication in ASD (Tabuchi et al. 2007,
Chadman et al. 2008, Radyushkin et al. 2009). A mouse with the R451C mutation in NLGN3,
which was identified in a Swedish family (Jamain et al. 2003), was created (Tabuchi et al.
2007, Chadman et al. 2008). These mutant mice had a 90% reduction of NLGN3 expression
and showed impaired social interactions but enhanced spatial learning abilities. These
behavioural changes were accompanied by an increase in inhibitory synaptic transmission
(Tabuchi et al. 2007). However, in another study using the same R451C knockin mice no
autistic-like phenotypes was observed (Chadman et al. 2008). Since the R451C knockin mice
only had a 90% reduction of NLGN3 expression Radyushkin et al. created a NLGN3 knockout
mice. These knockout mice showed impaired vocal communication and altered social
memory, characterized by a deficit in recognition that is potentially related to an olfactory
deficiency. A NLGN4 knockout mouse was also investigated for behavioural similarities to
ASD (Jamain et al. 2008). These knockout mice exhibited deficits in reciprocal social
interactions and vocal communication. Thus both NLGN3 and NLGN4 knockout mice show
deficits in vocal communication, suggesting a role for these genes in impaired vocal
communication as seen in ASD patients.
However, based on the broad spectrum of phenotypes of ASD patients with NLGN3 or
NLGN4X mutations it is not likely that mutations in these genes underlie a certain
phenotype.
Interestingly, several mutations and genomic rearrangements in the neuroligin binding
partner neurexin 1 gene (NRXN1) have been found in autism patients. A mutational
screening of NRXN1β in 264 ASD patients was conducted and two mutations were identified
in a total of 4 patients (Feng et al. 2006). The mutations were not present in 729 controls but
they were detected in first-degree relatives who displayed heterogeneous phenotypes
ranging from hyperactivity, depression, and learning problems to apparently normal
behaviour (Feng et al. 2006). After this, several other studies have identified mutations and
genomic rearrangements (deletions and disruptions) in NRXN1 (α and β) ASD patients
(Autism Genome Project Consortium 2007, Bucan et al. 2008, Kim HG et al. 2008, Marshall et
al. 2008, Yan et al. 2008, Glessner et al. 2009, Ching et al. 2010, Wiśniowiecka-Kowalnik et al.
2010). However some of these mutations were also present in controls. Furthermore NRXN1
was found to be associated with ASD in one of the large GWAS (Wang K et al. 2009).
Humans express three neurexin genes (NRXN1, NRXN2, and NRXN3) that are located at
chromosome loci 2q32, 11q13, and 14q24.3-q31.1 respectively. These genes have two
independent promoters, which determine two mRNA classes: long mRNAs, encoding for αneurexins and short mRNAs, encoding for β-neurexins (Südhof 2008). As mentioned above
neurexins are presynaptic receptors for neuroligins with which they form complexes. The
first evidence of the role of neuroligins in synapse function came from experiments that
demonstrated that expression of neuroligin 1 and 2 in a non-neuronal cell induced cocultured neurons to form presynaptic specializations onto the non-neuronal cell (Scheiffele
et al. 2000). A similar experiments showed that β-neurexin expressed in non-neuronal cells
induced postsynaptic specializations in co-cultured neurons (Nam and Chen 2005). Also,
overexpression of neuroligins in transfected neurons caused an increase in synapses (Chih et
al. 2004). These studies indicated that these two proteins may induce synapse formation.
However, studies with knockout mice showed that neuroligins and neurexins are essential
for synaptic function and maturation, not formation (Südhof 2008). The knockout mice
studies are more reliable since the assays using cultured neurons do not directly measure
synapse induction (Südhof 2008). The neuroligin/neurexin complex thus plays a role in
synapse formation and maturation, and not in the initial formation of synaptic junctions.
Mice with a neurexin1-α deletion displayed repetitive grooming behaviour, suggesting that
NRXN1 might be involved in developing repetitive behaviour as seen in ASD patients
(Etherton et al. 2009). The heterogeneous phenotypes observed in patients harbouring
NRXN1 mutations however indicate that it is unlikely that NRXN1 underlies a specific
phenotype in human ASD.
Contactin associated protein-like 2
In addition to NRXN1, another gene from the neurexin family has been implicated in ASD.
Mutations, disruptions and deletions have been identified in the contactin associated
protein-like 2 (CNTNAP2) gene in ASD patients. The first evidence for the contribution of
CNTNAP2 in autism came from the finding of a homozygous mutation in patients with
cortical dysplasia. All patients with the mutations had seizures and language regression and
two thirds of these patients qualified for ASD according to the DSM-IV criteria (Strauss et al.
2006). A similar case was found with a single base pair deletion in CNTNAP2 (Jackman et al.
2009). Further evidence came when two independent studies showed an association
between ASD and common CNTNAP2 alleles (Alarcón et al. 2008, Arking et al. 2008).
Association between a common CNTNAP2 variant and ASD in the Chinese Han population
was also found (Li X et al. 2010). Additionally, structural variation and mutations in the
CNTNAP2 gene were identified in ASD patients (Rossi et al. 2008, Bakkaloglu et al. 2008,
Poot et al. 2010). Suggestive evidence for association with ASD was found for a SNP close to
CNTNAP2 in a large genome-wide association study (Wang K et al. 2009).
Recently, it was shown control subjects that are homozygotes for a risk allele for autism in
CNTNAP2 showed significant reductions in grey and white matter volume. Also reductions in
fractional anisotropy in the cerebellum, fusiform gyrus, occipital and frontal cortices, regions
implicated in ASD, were seen in these homozygotes (Tan et al. 2010).
CNTNAP2 is a very large gene that is located in chromosomal region 7q35-36.1, a region that
has been implicated multiple times in ASD by linkage studies as mentioned above (Freitag et
al. 2010). It is a transmembrane protein with multiple protein interaction motifs: epidermal
growth factor repeats, laminin globular domains, an F5/8-type C domain and a putative PDZ
binding site. CNTNAPs can form complexes with contactins (CNTN) that are thought to
mediate neuron-glial interactions, neuronal migration and dendritic orientation.
CNTNAP2 was shown to help create functional subdomains on myelinated axons by
associating with Shaker-type voltage activated potassium channels in the juxtaparanodal
regions (Poliak et al. 2003). The role of CNTNAP2 in brain development is less well known. It
was shown by in situ hybdrization in the human fetal brain that CNTNAP2 expression is
restricted to frontotemporal-subcortical circuits that are critical for executive function
(Alarcón et al. 2008). This circuitry is thought to subserve joint attention, a precursor to
verbal communication, which is disrupted in ASD (Alarcón et al. 2008). Together with the
finding that patients with cortical dysplasia that have homozygous mutations in CNTNAP2 all
showed language regression an two thirds of them qualified for ASD, this expression data
suggests a role for CNTNAP2 in language impairment in ASD patients (Strauss et al. 2006,
Alarcón et al. 2008). An association between CNTNAP2 and a measure of language delay (age
of first word spoken) in autism families further supports this possible role for CNTNAP2
(Alarcón et al. 2008).
Another line of evidence supporting this comes from a study investigating the transcription
factor FOXP2 (Vernes et al. 2008). FOXP2 is involved in speech and language function and
mutations in this gene cause a monogenic speech and language disorder. In this study it was
found that FOXP2 can bind to and significantly reduce CNTNAP2 messenger RNA (mRNA)
levels. Furthermore, they observed an association between CNTNAP2 SNPs and nonsenseword repetition in children without an ASD (Vernes et al. 2008). Interestingly, these
associated SNPs are in the same region of CNTNAP2 that was associated with language delay
(Alarcón et al. 2008).
Together, these data suggest that CNTNAP2 variants represent susceptibility factors for
language-related deficits in ASD.
SH3 and multiple ankyrin repeat domains 3
Additional evidence for a role for synaptic function in the pathogenesis of ASD arose when
mutations and deletions in the SH3 and multiple ankyrin repeat domains 3 (SHANK3) were
identified in ASD (Durand et al. 2007, Moessner et al 2007, Sebat et al. 2007, Marshall et al.
2008, Gauthier et al. 2009). The first evidence of involvement of SHANK3 in neuropsychiatric
disorders emerged when patients with 22q13.3 deletion syndrome (or Phelan-McDermid
syndrome) were found to have a disruptions or deletions of the SHANK3 gene (Bonaglia et
al. 2001, Sykes et al. 2009). As mentioned above, the phenotype of patients with the
22q13.3 deletion syndrome includes autistic-like behaviour with speech and language
disability (Manning et al. 2004). Because of this Durand et al. analyzed the SHANK3 gene in
ASD patients and found three families with disruptions or deletions of SHANK3. In the first
family, the autism patient carrying a deletion of SHANK3, had absent language and moderate
mental retardation. In the second family, the two brothers with autism carrying a
heterozygous insertion in exon 21 both had severely impaired speech and severe mental
retardation. In the third family, a 22q deletion was identified in a girl with autism and severe
language delay. The brother of this girl with partial 22q13 trisomy had Asperger syndrome
with early language development and fluent speech (Durand et al. 2007). In another study
deletions and a non-synonymous mutation were identified in four ASD families. The affected
individuals showed non-verbal communication and social deficits (Moessner et al. 2007).
One autism patient with a deletion on 22q13.33 also showed nonverbal communication
(Marshall et al. 2008). Gauthier et al. (2009) identified two putative causative mutations in
ASD patients. A missense mutation was identified in a PDD-NOS patient with a language
deficiency. Also a splice variant was identified in an autism patients with normal speech
(Gauthier et al. 2009). Two studies in ASD patients did not identify SNP associations, CNVs or
mutations in SHANK3 (Sykes et al. 2009, Qin et al. 2009). In patients with schizoaffective
disorder two mutations in the SHANK3 gene were also identified (Gauthier et al. 2010). In
one family three individuals with schizoaffective disorder carried a nonsense mutation.
These individuals did not display any autistic features. The other mutation was identified in a
woman with schizoaffective disorder that had speech impairment (Gauthier et al. 2010).
SHANK3 is located on the telomeric terminal of chromosome 22q13.3, spans 58 kb of
genomic region, and contains 24 exons, 7 of which are alternatively spliced. SHANK3 is
expressed in the brain in the cortex, hippocampus and in the granule cell layer of the
cerebellum (Sheng et al. 2000). SHANK3 is an intracellular scaffolding protein found in the
postsynaptic density complex of excitatory synapses directly opposite to the presynaptic
active zone. SHANK3 can bind to neuroligins (Meyer et al. 2004).
Phenotypic data from patients harbouring mutations and deletions in SHANK3 gene, patients
with partial 22q13 trisomy, and patients with 22q13.3 deletion syndrome suggest that fine
tuning of SHANK3 gene dosage may be crucial for language development in humans.
Cadherins 9 and 10
As mentioned above the genes cadherin 9 and cadherin 10 (CDH9 and CDH10) located on
chromosome 5p14.1 were associated with ASD in a genome wide association study (Wang K
et al. 2009). In addition to NRXN1 and CNTNAP2, cadherin 9 and cadherin 10 are also cell
adhesion molecules. CDH9 and CHD 10 are type II classical cadherins from the cadherin
superfamily. These integral membrane proteins mediate calcium-dependent cell-cell
junctions in the nervous system and are involved in the generation of synaptic complexity in
the developing brain (Redies 2000). The expression pattern of CDH9 and CDH10 in the
human fetal brain was also investigated (Wang K et al. 2009).
The expression of CDH9 showed only low uniform levels of expression and thus was not
informative. However the expression pattern of CDH10 was enriched in the frontal cortex,
similar to the pattern of CNTNAP2 expression (Alarcón et al. 2008, Wang K et al. 2009).
Interestingly, besides several other cell-cell adhesion genes implicated in ASD that are
mentioned above (NGLN3, NGLN4X, NRXN1, CNTNAP2, CNTN4), protocadherin 10 (PCDH10)
was also implicated in ASD (Morrow et al. 2008). A large deletion in the PCHD10 gene was
found in an ASD patient. PCDH10 encodes a cadherin superfamily protein which is expressed
predominantly in the developing nervous system. The protein is essential for normal axon
outgrowth in the forebrain (Uemura et al. 2007).
A recent study investigated whether there is an association between the SNP (rs4307059)
associated with ASD that was identified in a large GWAS study and ASD phenotypes
(Pourcain et al. 2010, Wang K et al. 2009). They showed that an increased load of rs4307059
risk allele is associated with stereotyped conversation and decreased ability to understand
pragmatic aspects of communication. Also they showed evidence for a joint association
between rs4307059 and the phenotypic spectrum of traits. The results of this study support
the hypothesis that rs4307059 variability is related to an underlying quantitative trait locus
for ASD in the general population (Pourcain et al. 2010).
Phosphatase and tensin homologue
The phosphatase and tensin homologue (PTEN) gene is a tumour suppressor gene that is
localized on chromosome 10q23. PTEN functions as a dual-specificity phosphatise active in
various pathways involved in cellular growth. Heterozygous mutations have been identified
in families with several disorders, including Cowden syndrome, Bannayan-Riley-Ruvalcaba
syndrome, and Proteus syndrome. These syndromes are often characterized by
hamartomas. Furthermore, a patient with Cowden syndrome due to a PTEN mutation was
found to exhibit autistic behaviour and mental retardation (Goffin et al. 2001). Since this
finding several patients with Cowden syndrome were found to have autistic features and
macrocephaly (Lintas and Persico 2009). For this reason PTEN was screened in individuals
with an ASD and macrocephaly without any family history of Cowden syndrome, and three
mutations were identified in PTEN (Butler et al. 2005). Subsequently, several other
mutations were identified in patients with ASD and macrocephaly (Buxbaum et al. 2007,
Herman et al. 2007, Varga et al. 2009, Orrico et al. 2009, McBride et al. 2010). Macrocephaly
is a feature found in around 20% of ASD cases (Lintas and Persico 2009). This overgrowth
typically develops over the first few years of life (Lintas and Persico 2009). Based on the
observation of PTEN mutations in ASD patients with macrocephaly, genetic screening for
PTEN mutations of autism patients with macrocephaly is recommended (Lintas and Persico
2009).
In the central nervous system, inactivation of PTEN resulted in an increased dendritic spine
density and enlarged calibre of neuronal projections. Enlarged and abnormal synaptic
structures were observed in the cerebral cortex and cerebellum as well as myelination
defects (Fraser et al. 2008). This suggests that PTEN is involved in the development of
neuronal and synaptic structures and in synaptic function.
Oxytocin receptor
A number of studies have found association of the oxytocin receptor gene (OXTR) with ASD
(Wu et al. 2005a, Jacob et al. 2007, Lerer et al. 2008, Yrigollen et al. 2008, Liu et al. 2010,
Wermter et al. 2010). Two studies did however not find association between OXTR and ASD
(Kelemenova et al. 2010, Tansey et al. 2010). One study identified a genomic deletion of
OXTR in an autism case. This study also suggested that aberrant DNA methylation of OXTR
may contribute to ASD (Gregory et al. 2009).
The OXTR gene is located on a chromosomal region for which linkage with ASD was found in
several studies, region 3p25 (Freitag et al. 2010). OXTR is a G-protein coupled receptor for
the neuropeptide oxytocin. Oxytocin is suggested to be involved in the regulation of a
variety of social behaviours, social cognition, social memory formation and intelligence
(Landgraf and Neumann 2004). Several studies in humans further implicate oxytocin and
OXTR in ASD. A reduction of plasma oxytocin levels have been observed in autistic children
(Modahl et al. 1998). Also a reduction in OXTR mRNA expression has been observed in the
temporal cortex of autism patients (Gregory et al. 2009). Furthermore, oxytocin
administration in ASD patients led to improvement of symptoms such as repetitive
behaviour and social cognition (Hollander et al. 2007). Association of OXTR and social
cognition has been found in patients with attention deficit hyperactivity disorder (Park et al.
2010). In the association study by Lerer at al. association was also found between OXTR and
IQ (Lerer et al. 2008). Also association of OXTR with affect, emotional loneliness and IQ was
found in normal subjects, further supporting a role for OXTR in social behaviour and
intelligence (Lucht et al. 2009).
As mentioned above amygdala activity has been proposed to be one of the disrupted
processes underlying autism. The OXTR is highly concentrated in the amygdala and oxytocin
has shown binding in this region (Park et al. 2010). Intranasal application oxytocin has been
shown to reduce amygdala activation and reduced coupling of the amygdala to brainstem
regions. (Kirsch et al. 2005) Also intranasal oxytocin reduced right-sided amygdala response
to fearful, angry and happy faces (Domes et al. 2007). These imaging study data show that
the amygdala is modulated by oxytocin. It has been suggested that oxytocin reduces the
activation of the amygdala, inhibiting social anxiety, which indicates a neural mechanisms for
the effects of oxytocin in social cognition in humans (Domes et al. 2007). Reduced oxytocin
function in the amygdala may contribute to the social deficits seen in autistic patients.
Further evidence for a role of oxytocin and its receptor was obtained from mouse studies.
Mice deficient of oxytocin failed to develop social memory while wild-type mice showed
intact social memory. Treatment with oxytocin in these mice rescued social memory
(Ferguson et al. 2000). Mice deficient of the oxytocin receptor display pervasive social
deficits, namely fewer vocalization in response to social isolation in infant mice and deficits
in social discrimination and elevated aggressive behaviour in adult mice (Takayanagi et al.
2005).
Combined, all data suggest that oxytocin and the oxytocin receptor play an important role in
deficiencies in social behaviour, social memory and social cognition that are characteristic in
autism.
Glutamate receptor ionotropic kinase 2
The glutamate receptor ionotropic kainite 2 (GRIK2) gene, also called glutamate receptor 6
(GluR6) is located on chromosome 6q21, which is a region for which linkage with ASD was
found in several studies (Freitag et al. 2010). Several studies found association for the GRIK2
gene with ASD (Jamain et al. 2002, Shuang et al. 2004, Kim SA et al. 2007, Holt et al. 2010).
Also, suggestive evidence for association with ASD was found for a SNP close to GRIK2 in a
large GWAS (Wang K et al. 2009). One study performed on an Indian population did not find
significant association between GRIK2 and autism (Dutta et al. 2007). GRIK2 encodes the
glutamate receptor 6, which is expressed during brain development. Glutamate and its
receptors are involved in cognitive functions such as memory and learning as well as in stress
response and anxiety. It has been proposed that autism could be a hypoglutamergic disorder
based on similarities between symptoms produced by glutamate antagonists in healthy
persons and autistic patients (Jamain et al. 2002). Furthermore, disturbed glutamate
concentrations in the plasma have been seen in autistic patients (Jamain et al. 2002).
Another line of evidence for the role of glutamate in autism comes from animal models.
Animal models of hypoglutamatergia show autistic features such as defective habituation,
restricted behavioural repertoire and the inability to change behavioural program (Jamain et
al. 2002). Two studies examined the behavioural displays of GRIK2 knockout mice (Ko et al.
2005, Shaltiel et al. 2008). In the first study it was shown that mice lacking GRIK2 showed a
significant reduction in fear memory. Also, synaptic potentiation in the lateral amygdala was
significantly reduced in these mice (Ko et al. 2002). The second study observed that GRIK2
knockout mice exhibited less anxious or more risk-taking type behaviour and less despairtype manifestations. The mice also had more aggressive displays (Shaltiel et al. 2008).
Based on the function of GRIK2 and data from animal models no clear genotype to
phenotype relation can be made for this gene in ASD.
Engrailed homeobox 2
The engrailed homeobox 2 (EN2) gene is located on chromosome 7q36, a region for which
several studies have found linkage with ASD (Freitag et al. 2010). Besides lying in this linkage
region, several lines of evidence implicate EN2 as a candidate gene for ASD.
Association between several different EN2 SNPs and ASD has been found in several studies
(Petit et al. 1995, Gharani et al. 2004, Benayed et al. 2005, Brune et al. 2008, Wang L et al.
2008, Yang et al. 2008, Sen et al. 2010, Yang et al. 2010). One study did not find association
between autism and EN2 (Zhong H et al. 2003).
EN2 plays an important role in regionalization, patterning and neuronal differentiation of the
midbrain and hindbrain. EN2 is predominantly expressed in the developing cerebellum and
ventral midbrain. In the adult brain EN2 is also expressed in the hippocampus and the
cerebral cortex (Tripathi et al. 2009).
Another line of evidence comes from mouse studies. EN2 knockout mice display cerebellar
hypoplasia, a reduced number of Purkinje cells in the cerebellum, and defects in the
anteroposterior pattern of cerebellar foliation (Cheh et al. 2006). Also altered anatomical
structure of the amygdale has been reported in these knockout mice (Kuemerle et al. 2007).
Interestingly, the EN2 knockout mice also displayed deficits in social behaviour as well as
deficits in spatial learning and memory tasks (Morris water maze and object recognition
test). Also deficits in specific motor tasks were observed (Cheh et al. 2006). EN2 knockout
mice also showed increased susceptibility to kainic acid induced seizures (Tripathi et al.
2009). These data show that EN2 knockout mice display neurochemical and behavioural
features that are similar to those seen in ASD patients and can be used to study at least
some of the pathological aspects of ASD.
Gamma-aminobutyric acid A receptor beta 3
As mentioned above, duplications of region 15q11-13 are present in around 1% of autism
patients. Several genes in this region have been investigated for their role in ASD, all with
inconclusive results. The gamma-aminobutyric acid A receptor beta 3 (GABRB3) gene will be
discussed here. Two other genes in this region will be discussed in the ‘other candidate
genes’ section below.
Association between the markers or SNPS at the GABRB3 gene and ASD was found in several
studies (Cook et al 1998., Menold et al. 2001, Buxbaum et al. 2002, McCauley et al. 2004a,
Curran et al. 2005, Kim SA et al. 2006). However several studies could not replicate this
finding (Maestrini et al. 1999, Salmon et al. 1999, Martin et al. 2000, Tochigi et al. 2007).
One study found an association between a rare signal peptide variant (P11S) of GABRB3 and
autism when transmitted maternally (Delahanty et al. 2009). Association between GABRB3
and childhood absence epilepsy has also been found (Urak et al. 2006).
Another line of evidence for the involvement of GABRB3 in autism is the observation that
GABRB3 expression is reduced in the brains of autism patients (Fatemi et al. 2009).
Expression was significantly reduced in the parietal cortex, superior frontal cortex, and in the
cerebellum (Fatemi et al. 2009).
The GABRB3 gene encode a member of the ligand-gated ionic channel family. The protein is
one of many distinct subunits of a multisubunit chloride channel that serves as a receptor for
gamma-aminobutyric acid (GABA), which is the major inhibitory transmitter in the nervous
system. GABRB3 is the major has a major role in the development of the central nervous
system (Delahanty et al. 2009). During development GABAA receptors, of which GABRB3 is
one, play role in proliferation, migration, and differentiation of precursor cells that organize
the development of the embryonic brain. The consequence of disruption of the GABRB3
gene in mice has been studied (Homanics et al. 1997, Delorey et al. 1998, Delorey et al.
2008). GABRB3 deficient mice display occasional epileptic seizures and hypersensitive
behaviour (Homanics et al. 1997, DeLorey et al. 1998). Furthermore, they were found to
display phenotypic similarities to Angelman syndrome and autism, such as poor learning and
memory, poor motor coordination, hyperactivity, and repetitive and stereotypical behaviour
(DeLorey et al. 1998).
Another study showed that GABRB3 knockout mice display more ASD features such as
deficits in social behaviour, deficits in exploratory behaviour. These mice also had hypoplasia
of cerebellar vermal lobules, a feature seen in ASD patients (DeLorey et al. 2008).
Thus, the observation that GABRB3 knockout mice display several behavioural and
anatomical features of ASD, most notably deficits in social behaviour as well as repetitive,
stereotypic behaviour, indicates that these mice could be a good animal model for autism.
Other candidate genes
Besides the candidate genes discussed above, numerous other genes involving several
molecular pathways have been implicated in ASD. In this section these genes will be
discussed shortly.
On chromosome 1q42, duplications and deletions have been seen in the disrupted in
schizophrenia 1 (DISC1) gene in ASD patients (Kilpinen et al. 2008, Williams et al. 2009,
Crepel et al. 2010). This gene has been associated with other psychiatric disorders such as
schizophrenia, bipolar disorder, and major depression. DISC1 has been implicated to play a
role in several biological processes, such as neurite outgrowth, neuronal migration,
synaptogenesis, glutamergic transmission and cAMP signalling (Kilpinen et al. 2008). This
indicates that DISC1 plays an important role in neurodevelopment.
The solute carrier family 25 (mitochondrial carrier, Aralar) member 12 (SLC25A12) gene has
also been associated with autism (Ramoz et al. 2004, Segurado et al. 2005, Turunen et al.
2008). Several studies could however not replicate this association (Blasi et al. 2006b,
Correia et al. 2006, Rabionet et al. 2006, Chien et al. 2010). One study found an association
between autism related routines and rituals and an SLC25A12 variant (Silverman et al. 2008).
Interestingly, expression of SLC25A12 has been found to be upregulated in the prefrontal
cortex of autistic patients (Lepagnol-Bestel et al. 2008).
The gene is located on chromosome 2q24, a region for which linkage to ASD was previously
found (Freitag et al. 2010). The gene encodes the calcium dependent mitochondrial
aspartate/glumate carrier (AGC1), which is involved in neurite outgrowth and is expressed in
the developing brain and in skeletal muscle. SLC25A12 knockout mice showed that it is
involved in brain growth, myelin formation, and neurofilaments (Sakurai et al. 2010).
It was found that disruptions in one of the genes encoding a binding partner of CNTNAPs,
the contactin 4 (CNTN4) gene were present in pedigrees with autism (Roohi et al. 2009). A
disruption was also found in a patient with 3p deletion syndrome that is characterized by
developmental delay and that was also diagnosed with an ASD (Fernandez et al. 2004 and
2008). Also, deletions and duplications in CNTN4 were found to be associated with ASD in a
genome-wide CNV study (Glessner et al. 2009). The CNTN4 gene is located on chromosome
3p26.3 which lies in a region for which linkage with ASD has been found in several studies
(Freitag et al. 2010).
Deletions in CNTN3 were also found to be associated with ASD and CNVs in CNTN5 and
CNTN6 were found in genotypic data of autism families (Morrow et al. 2008, Burbach and
van der Zwaag 2009). Contactin 4 is highly expressed in the brain, particularly in the
cerebellum, thalamus, amygdala, and the cerebral cortex (Fernandez et al. 2004). It is an
axon associated cell adhesion molecule which are believed to play crucial roles in axonal
elongation and fasciculation, and the formation, maintenance and plasticity of some
synaptic connections (Roohi et al. 2009). Knockouts of homologous neuronal adhesion
molecules in mouse resulted in mutants that demonstrated morphological, neurological, and
behavioural abnormalities (Fernandez et al. 2004).
As mentioned above, in several studies including a meta-analysis, linkage has been found
with ASD on chromosome 7q22-32 (Freitag et al. 2010). The reelin (RELN) gene lies in this
chromosomal region and 5’UTR CGG repeat alleles were first associated with autism (Persico
et al. 2001). However several studies have produced conflicting results (Zhang H et al. 2002,
Bonora et al. 2003, Devlin et al. 2004, Skaar et al. 2005, Li H et al. 2008, Kelemenova et al.
2010). The association of RELN SNPs with ASD has also been investigated but this has been
inconclusive so far (Li J et al. 2004, Dutta et al. 2008, He et al. 2010, Holt et al. 2010). RELN
has also been associated with other psychiatric disorders such as schizophrenia and mood
disorders (Fatemi et al. 2005). The RELN gene encodes for reelin, which is an extracellular
matrix, glycoprotein, that acts as the signalling protein in neuron migration, lamination and
connection during embryonic brain development. Interestingly, brain alterations seen in
autism patients show overlap with developmental alterations in the brains of reeler mice,
which lack RELN expression (Persico et al. 2001). Furthermore, reelin protein levels were
found to be reduced in post-mortem tissue of autistic patients in the superior frontal cortex,
parietal cortex and cerebellum (Fatemi et al. 2005). Reelin mRNA levels were also reduced in
the superior frontal cortex and cerebellum of autistic patients (Fatemi et al. 2005). Also,
reelin levels were found to be reduced in plasma of autism patients (Fatemi et al. 2002).
Another gene in the linkage region 7q22-32 is the met proto-oncogene (MET), on 7q31. This
gene has been associated with ASD in several studies (Campbell et al. 2006, Campbell et al.
2008, Sousa et al. 2009, Jackson et al. 2009). MET encodes for a transmembrane tyrosine
kinase receptor of the hepatocyte growth factor/scatter factor. It was first identified as a
proto-oncogene and is overexpressed and deregulated in diverse human tumours. Besides
this, MET signalling is involved in regulation of the immune system, embryogenesis, and in
peripheral organ development and repair. MET is also expressed in the developing nervous
system, and has been implicated in neuronal development of the cerebral cortex and the
cerebellum. Impaired MET signalling results in impaired interneuron migration and
disruption of neuronal growth in the cortex. It also leads to a decreased proliferation of
granule cells which reduces the size of the cerebellum. These features have been observed
in the brain of autistic patients. Furthermore, analysis of post-mortem tissue of the temporal
lobe of ASD patients showed a decreased MET protein level compared to controls. Also
mRNA expression for proteins involved in regulating MET signalling were increased in ASD
brains (Campbell et al. 2007).
Recently, MET was also found to be associated with schizophrenia and general cognitive
ability (Burdick et al. 2010). The role of MET in neurocognitive function might point to a
mechanism trough which MET variation influences risk for both autism and schizophrenia,
since neurocognitive impairment is common in both diseases (Burdick et al. 2010). MET
variation might not influence risk specifically to autism or schizophrenia but to cognitive
impairment.
As mentioned above several genes in the 15q11-13 region have been investigated for their
role in ASD. GABRB3 was discussed above, the other two genes in this region will be
discussed here. One of these two genes is the UBE3A gene. Mutations in this gene are the
causal factor for between 22-25% of patients with Angelman syndrome. Association for
UBE3A was found in families with autism (Nurmi et al. 2001, Guffanti et al. 2010). This
finding could however not be replicated (Nurmi et al. 2003). In two genome-wide copy
number variation studies, CNVs in UBE3A were identified in ASD cases and not in controls
(Bucan et al. 2008, Glessner et al. 2009).
The third gene in the 15q11-13 region is the ATPase, class V, type 10A (ATP10A or ATP10C)
gene. ATP10C encodes an aminophospholipid translocase, and may be involved in central
nervous system signaling. Significant association between ATP10C and ASD was found in
three independent studies (Nurmi et al. 2003, Kato et al. 2008, Guffanti et al. 2010).
However one study did not find this association (Kim et al. 2002a).
Psychomotor regression, an autistic trait, was significantly associated with an intergenic
region between UBE3A and ATP10C (Guffanti et al. 2010).
The solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (SLC6A4 or
5-HTT) gene has been extensively investigated for its role in ASD. The gene is located on
chromosomal region 17q11, which has been implicated in ASD through linkage studies
(Freitag et al. 2010). There are several reasons why SLC6A4 has been studied for its role in
ASD. One reason is the involvement of the neurotransmitter serotonin in the development
of the central nervous system and in social behaviour, aggression, anxiety, and other
psychological processes (Huang and Santangelo 2008). The SLC6A4 gene encodes a
transporter that modulates serotoninergic neurotransmission by reuptake of serotonin in
the synaptic cleft. Another reason for its involvement is the finding of elevated blood
serotonin levels in autistic children. Also, serotonin reuptake inhibitors, drugs that target the
transporter, are effective in treating some autism symptoms (Huang and Santangelo 2008).
Over 20 polymorphisms have been identified in SLC6A4. Association between SLC6A4 and
ASD have looked mostly at two polymorphisms in the SLC6A4 gene. The first polymorphism
is a deletion/insertion in the regulatory region, the 5-HTT gene-linked polymorphic region (5HTTLPR). This polymorphism has two common allelic forms, the long and the short variant.
The second polymorphism is a variable number of tandem repeats in the second intron,
which as three common alleles corresponding to the number of repeat unit (9, 10, and 12). A
large number of studies have examined the association between autism and the
transmission of the alleles of the two polymorphisms as well as several other SNPs (Klauck et
al. 1997, Cook et al. 1997, Maestrini et al. 1999, Zhong N et al. 1999, Persico et al. 2000,
Yirmiya et al. 2001, Betancur et al. 2002, Kim et al. 2002b, Conroy et al. 2004, McCauley et
al. 2004b, Devlin et al. 2005, Mulder et al. 2005, Sutcliffe et al. 2005, Wu et al. 2005b, Ramoz
et al. 2006, Guerini et al. 2006, Cho et al. 2007, Guhathakurta et al. 2008, Longo et al. 2009,
Ma et al. 2010). Results from these association studies are inconclusive, although more
studies report preferential transmission for the short 5-HTTLPR allele and the 12 repeat
allele to individuals with autism. The 5-HTTLPR short allele was found to decrease gene
expression and serotonin uptake activity (Huang and Santangelo 2008). A meta-analysis
done in 2008 did not find a significant overall association between the SLC6A4
polymorphisms and autism. They did find a significant transmission of the 5-HTTLPR short
allele in the US samples, but not in European and Asian samples (Huang and Santangelo
2008). It has been suggested that gene-gene and gene-environment interaction (e.g. with
ITGB3) and family history might provide explanations for inconsistent association (Ma et al.
2010). Also the possibility that multiple rare alleles in SLC6A4 play a role in the susceptibility
to ASD has to be considered. One study screened all coding exons of SLC6A4 and identified
several novel coding variants. There was however no association between these variants and
ASD (Sakurai et al. 2008). A study investigating the effect of the 5-HTTLPR polymorphism on
cortical gray matter showed that the short allele was related to cortical gray matter
overgrowth in autistic children (Wassink et al. 2007).
Conclusion/ Discussion
A large amount of genes and chromosomal regions have been implicated in ASD. The clinical
heterogeneity of ASD could possibly be explained by this genetic heterogeneity. Therefore in
this thesis the relationship between causal gene mutations or chromosomal abnormalities
and phenotypes of ASD was investigated. This relationship was investigated in several ways
by reviewing relevant literature. The biological function and expression and animal models
of the genes implicated in ASD, as well as the phenotypes of ASD patients with gene
mutations or abnormalities could help elucidate whether certain genes can explain certain
phenotypes in ASD. This knowledge could possibly also be used for genetic screening in the
clinical practice (Lintas and Persico 2009).
Based on the information presented above several genes seem the show a relationship to
certain ASD phenotypes. Both CNTNAP2 and SHANK3 seem to play an important role in
normal language development and thus mutations or abnormal expression of these genes
could underlie the language impairment seen in these ASD patients.
The PTEN gene is strongly correlated to macrocephaly based among other things on the
finding of PTEN mutations in ASD patients with macrocephaly. This shows that genetics
might underlie morphological phenotypes besides behavioral phenotypes.
The SNP that was recently identified to be associated with ASD in a large GWAS, located
between the genes CDH9 and CDH10 may play a role in stereotyped conversation and
understanding pragmatic aspects of communication (Wang K et al. 2009, Pourcain et al.
2010). However since this SNP was just recently discovered, further research is necessary to
investigate the role of CHD9 and CHD10 in ASD.
Some genes seem to play a role in several characteristics as opposed to just one ASD
phenotype or characteristic. Several lines of evidence suggest that the oxytocin receptor
plays an important role in deficiencies in social behavior, social cognition and social memory.
Based on data from animal models, EN2 is thought to play a role in deficits in social behavior
as well as deficits in spatial learning and memory tasks. Furthermore they display
neurochemical features seen in ASD patients. GABRB3 animal models show behavioral and
anatomical features of ASD such as deficits in social behavior and repetitive, stereotypic
behavior. The GABRB3 deficient mice model seems to be a strong candidate for an ASD
mouse model based on this information.
The genes for NGLN3, NGLN4X, NRXN1, and GRIK2 do not seem to show any clear
correlation to any single phenotype or to a specific subset of phenotypes.
As mentioned, animal models using these genes could be used to study ASD. Either animal
models for just one phenotype (SHANK3, CNTNAP2) or for several phenotypes (OXTR, EN2,
GABRB3) seen in ASD can be made using these genes.
However based on the data gathered in this thesis it seems to be difficult to draw clear-cut
correlations between genotype and phenotype. Most genes seem to play a role in the
development of several characteristics of ASD. It could also be that patients have mutations
in several different genes or have additional chromosomal abnormalities besides a gene
mutation.
Research into the function of the proteins involved and their interaction with other proteins
is very important and might provide better insights into how a certain gene causes a certain
phenotype. It might also help find out whether mutations in several genes are necessary to
develop all the characteristics of ASD or whether some single genes can cause all these
characteristics.
To better understand the genetics of specific phenotypes, more phenotype directed
research is also a possibility. ASD patient groups divided on the base of their phenotypes
could help provide better information on the genotype-phenotype relation. This could also
be employed using next generation sequencing.
The development of next generation sequencing is thought to accelerate the understanding
of genetics and the discovery of disease-related genes. In the form of whole genome
sequencing and/or exome sequencing, next generation sequencing might provide valuable
information into the genetics of ASD. Patient groups or families showing the same specific
ASD phenotypes could be screened using this technique to try and elucidate the genetics
behind these phenotypes. This in turn might provide valuable information into the
treatment of specific ASD phenotypes.
Based on the information found in this thesis one can conclude that for most ASD associated
genes, no clear genotype-phenotype relation can be drawn. This can be explained in part
because for several genes their function in brain and their interaction with other proteins is
not well known yet.
The understanding of the genotype-phenotype relation in ASD is growing as more and more
genes are being discovered. Next generation sequencing is likely to provide even more ASD
associated genes. Subsequently, research into the function and interaction of ASD associated
proteins will be very important.
Acknowledgements
Thanks to Martien Kas, Ph.D. for his help and corrections with writing this thesis.
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