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Genetic, transcriptional and biochemical studies of the mitochondrial aspartate/glutamate…
Italian probands only were assessed also using the Griffiths Mental Developmental
Scales (27) and the Autism Behavior Checklist (28) ; patients from Arizona
were also administered the Leiter International Performance Scale (29) . The
Autism Diagnostic Observation Schedule – G (30) and the Autism Diagnostic
Interview (31) , which represent the most up-to-date approach to the clinical
characterization of autistic disorder, have been translated and published in
Italian only very few months ago, so data collection using these scales is still in
early progress.
All parents gave written informed consent for themselves and for their
children. The consent form was approved by the IRB of UCBM. Blood is drawn
from all patients and family members, DNA is extracted from leukocytes and
is used for ongoing genetic studies. In reference to the biochemical and morphological
characterization of our patients, since serotonin blood levels, urinary
peptide excretion rates, and cranial circumference have been consistently
found altered in subgroups of autistic patients (22-26) , we have also collected
platelet-rich plasma and urines from the majority of our families, and now
have serotonin blood levels and urinary peptide excretion rates from well over
300 first-degree relatives and 150 autistic patients. Cranial circumference is
also available for 212 patients and 54 unaffected siblings. To our knowledge,
no other consortium has collected to this date such a large dataset of biochemical
and morphological parameters. We will continue to measure serotonin
blood levels in platelet-rich plasma (PRP) by HPLC, and urinary peptide excretion
rates on the first morning urine samples of all family members again by
HPLC, as described (9,26) . Finally, frontal-occipital head circumference is measured
using a non-stretchable measuring tape and macrocephaly defined as a
head circumference > 97 th percentile (26) .
WORK PROGRAM
We plan to proceed using genetic and biochemical approaches in parallel.
Genetic and gene expression studies – Initially, genomic DNA and total RNA
will be extracted from post-mortem tissue using standard techniques. Total RNA
quality will be verified using an Agilent Bioanalyzer. Total RNA of good quality will
be retrotranscribed, and cDNA will be sequenced using the CEQ TM sequencing protocol
(Beckman-Coulter). Potential polymorphisms or mutations detected by DNA
sequencing will be confirmed by restriction analysis. cDNA will also be used to
assess gene expression using real-time PCR (Applied Biosystems).
In second phase, we shall also seek to replicate in our sample of families
the positive linkage/association findings recently reported by two independent
groups. We shall genotype the two single nucleotide polymorphisms (SNPs)
located in the third and sixteenth introns, i.e. rs2056202 (I3–21A/G) and
rs2292813 (I16+70A/G), respectively, previously shown to be associated with
autism (2) . Genotyping will be performed according to standard PCR- and
restriction digest-based methods and by a primer extension-based, allelespecific
fluorescent detection method (i.e., the Acycloprime Primer Extension
kit.and the Victor2 multilabel counter by Perkin-Elmer). Genetic data will be
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