THE GENOTYPE AND PHENOTYPE CORRELATIONS IN AUTISM SPECTRUM DISORDERS

Background of the study:
Autism spectrum disorders (ASD) form a heterogeneous group of
neurodevelopmental disorders characterized by abnormal social interaction,
abnormal communication and stereotype behavior. Autism could be seen as the
core disease of the ASD and is characterized by a wide range of symptoms in
those three fields, with an onset before four years of age. ASD includes
autism, Asperger syndrome and Pervasive Developmental Disorder, not otherwise
specified (PDD-NOS). Prevalence of autism syndrome and autism related disorders
is estimated to be 1:300. The disorder is remarkably common in boys (with a
male to female ratio of 4:1.) and most patients need continued intensive
clinical care (60%).
The causes of ASD are highly genetic and most research findings to date are
based on studies of patients with autism and their relatives: The prevalence of
autism is increased in relatives when compared to the general population (2-8%
in siblings; 60-91% in monozygotic twins). Despite this large genetic
contribution, finding the causes for ASD has proven challenging. This is
largely due to genetic heterogeneity. This means that different genetic
abnormalities may increase the risk for ASD.
The genetic data suggest that three basic genetic mechanisms are involved in
autism. First, relatively rare single gene disorders (e.g. MECP2 gene in Rett
Syndrome) may increase the risk for ASD. Second chromosomal abnormalities may
be related to ASD (Freitag CM, 2007). Third, additive effects of several common
gene variants may be involved in ASD. When one also considers the possible
effects through changes in gene expression levels, the diversity in underlying
mechanisms in ASD is even larger.
Chromosomal abnormalities that co-occur with ASD have been described in
multiple cases (see figure-1). With the recent advances of DNA-chip technology
submicroscopic chromosomal abnormalities (as small as only one or two genes)
can be identified. Cytogenetic analysis has shown a relatively high occurrence
of chromosomal abnormalities (5%) in ASD patients. The submicroscopic genomic
deletions and duplications are often referred to as copy number variants
(CNVs). CNVs can be recurrent, inherited events, or arisen de novo on paternal
or maternal chromosomes. CNVs are micro deletions or micro duplications of one
of the alleles of a chromosome.
The interpretation of copy number changes is complicated by the frequent
occurrence of CNVs in both patients and the normal population.
which makes parental studies essential in interpreting subtelomeric copy number
changes. To be able to find the relationship between genetic etiologies and
phenotypic traits in ASD, we need to investigate CNVs in probands with ASD and
their parents. These detection methods have demonstrated that submicroscopic
losses and gains of DNA are causally related to ASD in at least 10-20%
(Marshall, 2008; Sebat, 2007; Autism Genome Project Consortium, 2007).
Subtelomere FISH is recommended for the investigation of children with
unexplained mental retardation (MR) and/or developmental delay (DD) with or
without dysmorphic features. Another part of this study is to analyze the
clinical utility of micro-array-based comparative genomic hybridization
(array-CGH) in the routine diagnostic process for ASD. An array-CGH in clinical
practice maximizes the identification of clinically significant chromosome
abnormalities, especially subtelomeric rearrangements (CNVs) and can influence
clinical diagnostic strategies (Ballif et al., 2007). An array-CGH may help in
determining the etiology of a subtype of a disorder and in that way prevent
further elaborate diagnostic medical interventions. Also, it leads to more
knowledge on which complications (long and short-term) can be expected and lead
to prevention or amelioration of these complications. Another important asset
is the possibility of genetic counselling for the family members. As the
usefulness of an array-CGH is clear in children with developmental delay, the
utility of the technique in the routine diagnostic process for probands with
ASD and their needs further exploration.
It is important to note that these rates will be even higher with the expected
advances in technology. Probably, the screening for this type of small
chromosomal abnormalities will become part of diagnostics in the future.
Especially in relation to the CNV studies, it is important to mention the
importance of gene expression studies briefly. To be pathogenic, a (rare) CNV
must change the coding sequence of a gene (haplo-insufficiency). For example,
in some cases a micro deletion leads to reduced gene expression, but not
always. Consequently, study of gene-expression profiles with well-described
CNVs will be of great value for the study of the pathogenesis of ASD. While
this is a major discovery, the genetic explanation (i.e. causality) of these
findings remains ambiguous. For example, the assumption that gene-expression
profiles of brain tissue can also be observed in peripheral blood. So far,
genome wide gene expression (GWGE) with ASD patients has only been reported in
a few studies. But the sample sizes did not allow drawing definite conclusions.
Though linkage- and association studies have led to potential risk genes for
ASD (see table-1, 2, 3) results didn*t show a homogeneous picture. This may be
due to several confounders such as small sample sizes, but possibly also
because individual ASD genes are of small effect. In addition, in only a
minority of the studies replication in an independent sample has been
performed. More powerful research designs such as joint analysis has only been
performed in our own association study, while genome-wide association data is
not yet available.
In summary, genetic studies with ASD patients gradually leads to insight into
the meaning of various genetic mechanisms involved in autism.
The phenotype (including a wide range of symptoms and possibly several
subgroups) as defined on the basis of a DSM-IV classification (American
Psychiatric Association, 2000b) will be too limited to relate the phenotypic
traits with the heterogeneity of the genotype. Genetic studies that stratified
their cases into more homogeneous subgroups have been proven to be more
successful. Phenotypic features that were found to be promising in genetic
studies were: language related difficulties, repetitive or rigid behaviours and
head circumference. Therefore phenotypic information is needed to characterize
individuals with ASD and their relatives, to be able to investigate the
relationship between genetic etiologies and phenotypic traits.

Objective of the study:
The main objective of this study is to investigate the influence of different
genetic mechanisms (risk genes, CNVs and gene expression profiles) in ASD and
to study the relation between the genetic heterogeneity and specific clinical
phenotypical traits as described in this disorder.

Study design:
All subjects will be at least 4 years of age. For basic ASD evaluation the
parents of the subjects will be asked to participate in a structured interview
about the (early) development of the subject, and the subjects are asked to
participate in a standardized observation to confirm the clinical diagnosis.
For more detailed assessment of phenotypical characteristics, parents (or
subjects if appropriate) are asked to fill out some questionnaires and to
participate in a short neuropsychological assessment to measure cognitive
flexibility and attention to detailed information. Neuropsychological tasks
provide a more objective way to measure rigidity than self-report with a
questionnaire alone and cognitive functioning might be more closely related to
brain development under genetic influences than behavior in daily life
situations. Lastly, subjects will be asked to provide a sample for genetic
analysis, either by blood or saliva. Information from medical records will be
obtained and head circumference and body length will be measured.

Nature and extent of the burden and risks associated with participation,
benefit and group relatedness:
There are no known risks associated with any of the proposed methodologies, and
we believe the impact on subjects will be negligible. Vena puncture can be a
burden, but can be replaced by the use of the saliva. Research into the genetic
basis of neurobiological deficits in ASD will improve our insight into the
pathophysiology of these disorders. Studies that elucidate the neurobiology of
ASD will ultimately facilitate future design of new and effective ways to treat
this disorder.
The evaluation of the utility of the array-CGH in ASD might lead to improvement
of genetic counselling to the families of probands with ASD. It can reduce
unnecessary medical procedures to unravel an etiology of ASD and lead to
prevention strategies for complications for individual probands with ASD.
In concurrence with this research project, a routine medical evaluation will be
performed, which is a standard procedure in our diagnostic process for ASD
patients. All children are screened in collaboration with our consulting
clinical geneticist on whether a more elaborate genetic work-up is necessary.
If so, the patient will be referred to the diagnostic department of Medical
Genetics in our hospital. The parents of these patients will receive the
results of this diagnostic procedure and will receive genetic counselling.
Because neurocognitive testresults will be collected for research purposes
only, parents en patients can*t be informed about the data in a diagnostic
framework. If they do have any questions, they can contact one of the
associated psychologist.
If a result of DNA-analysis in the research project proves to have relevance
for the future health of the patient, whom has not been referred to a
diagnostic genetic setting, we will refer the patient in collaboration with our
consulting clinical geneticist