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Association of the 4 integrin subunit gene (ITGA4) with autism.

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Association of the a4 Integrin Subunit Gene (ITGA4)
With Autism
Catarina Correia,1,2 Ana M. Coutinho,1 Joana Almeida,3 Raquel Lontro,3 Cristina Lobo,3
Teresa S. Miguel,4 Madalena Martins,2 Louise Gallagher,5,6 Judith Conroy,5 Michael Gill,5,6
Guiomar Oliveira,3 and Astrid M. Vicente1,2*
Instituto Gulbenkian de Ci^encia, Oeiras, Portugal
Instituto Nacional de Saúde Dr. Ricardo Jorge, Lisboa, Portugal
Centro de Desenvolvimento da Criança, Hospital Pediatrico de Coimbra, Coimbra, Portugal
Direcç~ao Regional de Educaç~ao da Regi~ao Centro, Coimbra, Portugal
Department of Genetics, Smurfit Institute, Trinity College, Dublin, Ireland
Department of Psychiatry, Trinity College, Dublin, Ireland
Received 27 March 2008; Accepted 14 January 2009
In the present work, we provide further evidence for the involvement of the integrin alpha-4 precursor gene (ITGA4) in the
etiology of autism, by replicating previous findings of a genetic
association with autism in various independent populations.
The ITGA4 gene maps to the autism linkage region on 2q3133 and is therefore a plausible positional candidate. We
tested eight single nucleotide polymorphisms (SNPs) in the
ITGA4 gene region for association with autism in a sample
of 164 nuclear families. Evidence for association was found
for the rs155100 marker (P ¼ 0.019) and for a number of specific
marker haplotypes containing this SNP (0.00053 < P < 0.022).
a4 integrins are known to play a key role in neuroinflammatory
processes, which are hypothesized to contribute to autism. In
this study, an association was found between the ITGA4
rs1449263 marker and levels of a serum autoantibody directed
to brain tissue, which was previously shown to be significantly
more frequent in autistic patients than in age-matched
controls in our population. This result suggests that the ITGA4
gene could be involved in a neuroimmune process thought
to occur in autistic patients and, together with previous
findings, offers a new perspective on the role of integrins in
the etiology of autism to which little attention has been paid so
far. 2009 Wiley-Liss, Inc.
Key words: autism; ITGA4 gene; neuroimmune process
A strong genetic component has been postulated for autism,
a behavioral syndrome characterized by deficits in social interaction, impaired communication, and restricted and stereotyped
behaviors [Folstein and Rosen-Sheidley, 2001]. While multiple
genes are likely involved, the results from several genome scans
have provided convincing evidence for the presence of an autism
susceptibility loci on chromosome 2q31-q33 [Buxbaum et al., 2001;
IMGSAC, 2001; Shao et al., 2002; Rabionet et al., 2004; Romano
et al., 2005; Lauritsen et al., 2006; Spence et al., 2006].
2009 Wiley-Liss, Inc.
How to Cite this Article:
Correia C, Coutinho AM, Almeida J, Lontro
R, Lobo C, Miguel TS, Martins M, Gallagher L,
Conroy J, Gill M, Oliveira G, Vicente AM.
2009. Association of the a4 Integrin Subunit
Gene (ITGA4) With Autism.
Am J Med Genet Part B 150B:1147–1151.
One possible candidate in this region is the integrin alpha 4 gene
(ITGA4), which spreads over 79.7 kb and encodes the integrin
alpha-4 precursor. Several independent studies have identified this
gene as a potential autism susceptibility locus. Faham et al. [2005]
carried out a case/control study, applying multiplexed variation
screening (MVS) to scan the genes mapping under the chromosome
2q31-33 linkage peak defined by Buxbaum et al. [2001], using
probands from the Autism Genetic Resource Exchange (AGRE)
families that were part of the original linkage scan. The results
provided evidence for association between autism and one single
nucleotide polymorphism (SNP) variant in exon 22 of the ITGA4
gene. Conroy et al. [2008] followed up a previous finding of an
Additional Supporting Information may be found in the online version of
this article.
Grant sponsor: Fundaç~ao Calouste Gulbenkian; Grant sponsor: Fundaç~ao
para a Ci^encia e Tecnologia; Grant number: POCTI/39636/ESP/2001.
*Correspondence to:
A. M. Vicente, Instituto Gulbenkian de Ci^encia, Rua da Quinta Grande, 6,
2781 Oeiras, Portugal; Instituto Nacional de Saúde Dr. Ricardo Jorge, Av.
Padre Cruz, 1649-016 Lisboa, Portugal.
Published online 3 March 2009 in Wiley InterScience
DOI 10.1002/ajmg.b.30940
interstitial deletion on chromosome 2q, within the region showing
evidence for linkage [Gallagher et al., 2003], and found a significant
association with several SNPs and haplotypes within the ITGA4
gene region in two independent populations. Significant association with autism is also reported by Ramoz et al. [2008], who
performed a dense marker scan across candidate genes mapping
across the 2q24-q33 linkage region in AGRE families, including
several markers within ITGA4 and additional genes in this region.
Integrins are membrane spanning, non-covalently linked ab
heterodimers that act as cell–cell and cell–extracellular matrix
(ECM) adhesion receptors throughout the body. The a4 integrin
chain dimerizes with either the b1 chain or the b7 chain. The a4b1
integrin, also known as very late antigen4 (VLA4), is the most
important member of the beta 1 integrin subfamily of integrins,
which are expressed in a variety of different cell types, including
neurons, glial cells, meningeal cells, and endothelial cells, subject to
regional and developmental regulation [Milner and Campbell,
2002]. The activation of integrins by their extracellular ligands
results in a number of changes of integrin properties, such as spatial
localization, internalization, ligand affinity, intracellular association with signaling proteins, interaction with the cytoskeleton and
transcriptional modulation. In the central nervous system (CNS),
integrins mediate adhesive and migratory events in processes as
diverse as synaptogenesis, activation of microglia, and stabilization
of the endothelium and the blood–brain barrier [Milner and
Campbell, 2002], many of which may be disrupted in autism
[Folstein and Rosen-Sheidley, 2001].
Integrins are also vital in situations of CNS inflammation, which
is thought to occur in autistic individuals. We have previously
reported the widespread occurrence of serum autoantibody reactivities to brain tissue in autistic patients [Silva et al., 2004], while
autoantibodies to specific brain proteins, such as myelin basic
protein (MBP), have been found in sera from children with
autism [Singh et al., 1993, 1998; Connolly et al., 1999]. There is
also evidence for an active ongoing chronic neuroinflammatory
process in multiple areas of the CNS of autistic individuals, with
marked activation of microglia and astroglia and increased levels of
proinflammatory and anti-inflammatory cytokines [Vargas et al.,
2005]. In vitro experiments show that microglial activation is
accompanied by increased expression of a4b1 integrin, which is
stimulated by inflammatory cytokines [Hailer et al., 1996]. a4
integrin expressed on the surface of lymphocytes binds to their
respective endothelial counter-receptor, VCAM1, playing a fundamental role in their adhesion to the vascular endothelium and
migration into the CNS. On the other hand, the use of a4b1
antagonists has been shown to reduce inflammatory cellular infiltration in various tissues, haltering the progression of brain inflammatory diseases such as multiple sclerosis (MS) [Von Andrian
and Engelhardt, 2003].
Given its chromosomal location within a candidate region for
autism and its role in the CNS, ITGA4 is a strong candidate gene for
autism susceptibility. We further investigated the involvement of
ITGA4 in autism by carrying out a replication study in a population
sample of 164 nuclear families with one affected individual. Patients
were diagnosed as previously described [Coutinho et al., 2004], and
only idiopathic subjects with Developmental Quotient above 50
were included. The Ethical Committee at the HP approved the
collection of data and biological specimens from patients for
research purposes, and all participants signed an informed consent.
Following on the previous association study in an independent
population of Irish trios [Conroy et al., 2008], which guided the
selection of markers to replicate identified associations and cover
genetic variability throughout the gene, we tested eight SNPs at the
ITGA4 gene: rs1449263 upstream of the gene, rs3770136 and
rs1449260 in intron 2, rs155100 in intron 9, rs3770116 in intron
15, rs3770112 in intron 17, rs2305581 in intron 20, and rs3770105 in
intron 22.
All SNPs were genotyped using the competitive allele specific
PCR system (KASPar). Markers were tested for Hardy–Weinberg
equilibrium and call rates were above 90% (Supplementary Table I).
TABLE I. Association Analysis of ITGA4 Individual Markers With Autism
Not transmitted
FIG. 1. Haploview-generated LD map of the eight SNPs within ITGA4 gene in the Portuguese population. The numbers within the boxes indicate the D0
statistic values between corresponding two SNPs. Black shading indicates strong LD (no number means a score of 1), gray shading indicates
uninformative, and white shading indicates strong evidence for recombination. Two-, three-, and four-marker haplotypes significantly associated
with autism. Horizontal bars represent significant over transmitted (black lines) and under transmitted ITGA4 marker haplotypes (gray lines).
The extended transmission disequilibrium test (ETDT) was used to
examine the association of individual markers with autism [Sham
and Curtis, 1995]. Haplotypes were analyzed using TRANSMIT
[Clayton and Jones, 1999]. Pair-wise linkage disequilibrium
between markers was calculated using the Haploview program.
Other statistical analyses were performed using the SPSS package.
All results are presented uncorrected for multiple testing.
Association results for all markers are shown in Table I. A
significant transmission disequilibrium of alleles at the rs155100
marker was found (c2 ¼ 5.5059; P ¼ 0.019), with preferential
transmission of allele A. Evidence of association with autism
was also found for multiple marker haplotypes involving this
SNP (Fig. 1, Supplementary Table II). Intermarker linkage disequilibrium (LD) was calculated and we found that the associated
marker rs155100 is located in a region with low levels of LD between
two regions with high levels of LD (Fig. 1). The fact that only the
rs155100 marker was associated with autism may therefore be
explained by the LD structure of this gene in our population.
Considering that all risk haplotypes identified include the A allele
of marker rs155100, the association of these haplotypes is therefore
likely driven by this marker. Although SNP rs155100, located in
intron 9, per se is not likely to have a functional consequence, it may
be in LD with a nearby functional variant. Notably, exon 9 encodes a
FG-GAP domain important in ligand binding (http://pfam.
Our results replicate previous studies reporting the association of
the ITGA4 gene with autism in various population groups [Faham
et al., 2005; Conroy et al., 2008; Ramoz et al., 2008], thus providing
additional evidence for association of the ITGA4 gene with autism
in an independent cohort. Different SNPs were, however, associated in the different studies, including our own. For instance, in the
study by Conroy et al. SNP and haplotype associations were
identified in the Irish population that were not replicated in
the AGRE and/or Vanderbilt samples, although in some cases the
combined sample provided evidence of association. On the other
hand, specific SNPs were associated in the Vanderbilt but not in the
Irish or AGRE populations. Faham et al. only found an associated
SNP in exon 22, while a number of different markers in introns
13 and 15–28 were significantly associated in the study by Ramoz
et al., even though these two studies may possibly have an important
overlap in terms of probands tested, as both used AGRE families,
and thus would be expected to identify associations in the same gene
regions. The observation of association with different SNPs in
different populations is not unexpected, and may have several
explanations. Many of the tested SNPs are not likely to be functional
polymorphisms, but may be in LD with a nearby functional variant;
it is therefore very plausible that variable underlying patterns of LD
lead to different SNPs being associated with autism in populations
with different origins and likely variable ethnic constitution. Other
factors may also contribute to the discrepancies in associated
markers between populations. For instance, the SNPs tested in the
different populations did not necessarily overlap. In our study, only
one marker significantly associated in the work by Ramoz et al.
[2008] was tested, whereas the associated SNP in our population,
rs155100, was only tested in the study of Conroy et al. Four markers
were common between the Ramoz et al. study and the Irish
population, of which only one was nominally associated in both
studies. Study design also led to discrepancies: while the Ramoz
et al. and Faham et al. studies possibly shared a proportion of the
sample, they have a very different design, one searching for sequence
variants in exons that might be associated with autism and the other
testing linkage and association with non-functional SNPs. The
discrepancies among populations regarding the associated SNPs
may therefore be explained by different patterns of LD in the
populations tested and/or by variable study designs with little SNP
We have not attempted any correction for multiple testing of
either SNPs or haplotypes, as there is no consensus regarding the
most appropriate method for this purpose. The application of the
most common correction methods, such as the Bonferroni correction, may result in an excess of type II errors, especially in relatively
small samples, and therefore the replication, in several independent
populations, of a genetic association is considered the best evidence
of a true association. The present study is a replication of a
previously reported association with this gene in multiple independent populations. It thus reinforces the suggestion of an involvement of the ITGA4 gene in autism susceptibility, indicating
that this region should be further explored.
One of the described pathologic functions of a4 integrins in
humans is the recruitment of circulating activated T-cells, monocytes, and macrophages to the CNS [Von Andrian and Engelhardt,
2003]. Besides mediating leukocyte adhesion, a4b1 has been implicated in costimulatory signals for T-cell proliferation [Nojima
et al., 1990; Sato et al., 1995] and in the differentiation of T cells and
B cells through their interaction with fibronectin [Williams et al.,
1991; Salomon et al., 1994]. In agreement with the hypothesis of a
chronic neuroinflammatory process in autism, we have previously
shown the widespread occurrence of autoantibodies to brain tissue
in autistic individuals, suggestive of an immune dysregulation in
autism [Silva et al., 2004]. More specifically, we defined patterns of
autoantibody repertoires directed to brain tissue that were characteristic of subsets of autistic patients, and identified one specific
autoantibody, to an as yet unidentified brain antigen, which
strongly discriminated between patients and controls sera
(P ¼ 0.00046, Mann–Whitney U-test). Autoantibody reactivities
were assessed by a quantitative immunoblotting technique defining
a quantitative population distribution of optical densities for each
specific autoantibody reactivity. Given the role of a4 integrins in
neuroimmune processes, we tested the association of ITGA4 with
the occurrence and strength of this specific autoantibody reactivity
in our patients, using a non-parametric analysis of variance
(Kruskal–Wallis test). We found a significant association of the
ITGA4 promoter marker rs1449263 with the optical density distri-
bution of this autoantibody reactivity (c2 ¼ 9.58 (2 df); P ¼ 0.008).
Interestingly, this marker is associated with susceptibility to MS
[O’Doherty et al., 2007], with the MS risk allele (rs1449263 C) also
more frequent in patients with higher levels of this autoantibody
reactivity in our population of autistic individuals. However, this
marker was not associated with autism and was not in LD with the
associated marker. While we cannot fully explain this inconsistency,
we believe it may mostly be related to methods of analysis and
population issues, possibly reflecting a variation in statistical power
to detect association, rather than diverse genetic effects. In fact,
association with autism was tested using a family-based approach,
examining allelic transmissions, while association with autoantibodies reactivities was tested using a non-parametric analysis of
variance, which tests the effect of genotypes rather than alleles in the
patient population. In addition, there is a small percentage of the
population sample (8.6%) that is not overlapping in the two
analyses. The upregulation of integrin a4, among other inflammation molecules, in conjunction with a well-documented autoantigen upregulation, represents a signature of enhanced immune cell
activation and costimulation in MS, while a4 integrin antagonists
are used for MS treatment because they prevent the migration of
autoimmune cells to the CNS [Iglesias et al., 2004]. Together with
other observations in autism and MS, our results lead us to
hypothesize that a chronic neuroinflammatory process occurs in
autism involving both cell-mediated and humoral autoimmune
responses. This process requires the recruitment of inflammatory
cells to the brain, which is known to be influenced by variants of the
ITGA4 gene, while the association of specific autoreactivities with
an ITGA4 marker can be explained by the parallel occurrence of the
humoral and cellular processes.
In summary, the present study provides supporting evidence for
an association between the ITGA4 gene and autism and, taken
together with previous work, suggests a plausible role for this
integrin in a neuroinflammatory process occurring in some autistic
This work was supported by the Fundaç~ao Calouste Gulbenkian
and by the Fundaç~ao para a Ci^encia e Tecnologia (POCTI/39636/
ESP/2001). C. Correia and A.M. Coutinho are supported by fellowships from the Fundaç~ao para a Ci^encia e Tecnologia (SFRH/BD/
16907/2004 and SFRH/BD/3145/2000). The genotyping was performed by KBiosciences.
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