The Italian autism network (ITAN): a resource for molecular genetics and biomarker investigations.

BACKGROUND: A substantial genetic component accounts for Autism Spectrum Disorders (ASD) aetiology, with some rare and common genetic risk factors recently identified. Large collections of DNAs from thoroughly characterized ASD families are an essential step to confirm genetic risk factors, identify new variants and investigate genotype-phenotype correlations. The Italian Autism Network aimed at constituting a clinical database and a biorepository of samples derived from ASD subjects and first-degree relatives extensively and consistently characterized by child psychiatry centers in Italy. METHODS: The study was approved by the ethical committee of the University of Verona, the coordinating site, and by the local ethical committees of each recruiting site. Certified staff was specifically trained at each site for the overall study conduct, for clinical protocol administration and handling of biological material. A centralized database was developed to collect clinical assessment and medical records from each recruiting site. Children were eligible for recruitment based on the following inclusion criteria: age 4-18 years, at least one parent or legal guardian giving voluntary written consent, meeting DSM-IV criteria for Autistic Disorder or Asperger's Disorder or Pervasive Developmental Disorder NOS. Affected individuals were assessed by full psychiatric, neurological and physical examination, evaluation with ADI-R and ADOS scales, cognitive assessment with Wechsler Intelligence Scale for Children or Preschool and Primary, Leiter International Performance Scale or Griffiths Mental Developmental Scale. Additional evaluations included language assessment, the Krug Asperger's Disorder Index, and instrumental examination such as EEG and structural MRI. DNA, RNA and plasma were collected from eligible individuals and relatives. A central laboratory was established to host the biorepository, perform DNA and RNA extraction and lymphocytes immortalisation. DISCUSSION: The study has led to an extensive collection of biological samples associated with standardised clinical assessments from a network of expert clinicians and psychologists. Eighteen sites have received ADI/ADOS training, thirteen of which have been actively recruiting. The clinical database currently includes information on 812 individuals from 249 families, and the biorepository has samples for 98% of the subjects. This effort has generated a highly valuable resource for conducting clinical and genetic research of ASD, amenable to further expansion.

Elevated fetal steroidogenic activity in autism.

Autism affects males more than females, giving rise to the idea that the influence of steroid hormones on early fetal brain development may be one important early biological risk factor. Utilizing the Danish Historic Birth Cohort and Danish Psychiatric Central Register, we identified all amniotic fluid samples of males born between 1993 and 1999 who later received ICD-10 (International Classification of Diseases, 10th Revision) diagnoses of autism, Asperger syndrome or PDD-NOS (pervasive developmental disorder not otherwise specified) (n=128) compared with matched typically developing controls. Concentration levels of Δ4 sex steroids (progesterone, 17α-hydroxy-progesterone, androstenedione and testosterone) and cortisol were measured with liquid chromatography tandem mass spectrometry. All hormones were positively associated with each other and principal component analysis confirmed that one generalized latent steroidogenic factor was driving much of the variation in the data. The autism group showed elevations across all hormones on this latent generalized steroidogenic factor (Cohen's d=0.37, P=0.0009) and this elevation was uniform across ICD-10 diagnostic label. These results provide the first direct evidence of elevated fetal steroidogenic activity in autism. Such elevations may be important as epigenetic fetal programming mechanisms and may interact with other important pathophysiological factors in autism.

Molecular sex differences in human serum.

BACKGROUND: Sex is an important factor in the prevalence, incidence, progression, and response to treatment of many medical conditions, including autoimmune and cardiovascular diseases and psychiatric conditions. Identification of molecular differences between typical males and females can provide a valuable basis for exploring conditions differentially affected by sex. METHODOLOGY/PRINCIPAL FINDINGS: Using multiplexed immunoassays, we analyzed 174 serum molecules in 9 independent cohorts of typical individuals, comprising 196 males and 196 females. Sex differences in analyte levels were quantified using a meta-analysis approach and put into biological context using k-means to generate clusters of analytes with distinct biological functions. Natural sex differences were established in these analyte groups and these were applied to illustrate sexually dimorphic analyte expression in a cohort of 22 males and 22 females with Asperger syndrome. Reproducible sex differences were found in the levels of 77 analytes in serum of typical controls, and these comprised clusters of molecules enriched with distinct biological functions. Analytes involved in fatty acid oxidation/hormone regulation, immune cell growth and activation, and cell death were found at higher levels in females, and analytes involved in immune cell chemotaxis and other indistinct functions were higher in males. Comparison of these naturally occurring sex differences against a cohort of people with Asperger syndrome indicated that a cluster of analytes that had functions related to fatty acid oxidation/hormone regulation was associated with sex and the occurrence of this condition. CONCLUSIONS/SIGNIFICANCE: Sex-specific molecular differences were detected in serum of typical controls and these were reproducible across independent cohorts. This study extends current knowledge of sex differences in biological functions involved in metabolism and immune function. Deviations from typical sex differences were found in a cluster of molecules in Asperger syndrome. These findings illustrate the importance of investigating the influence of sex on medical conditions.

Identification of a biological signature for schizophrenia in serum.

Biomarkers are now used in many areas of medicine but are still lacking for psychiatric conditions such as schizophrenia (SCZ). We have used a multiplex molecular profiling approach to measure serum concentrations of 181 proteins and small molecules in 250 first and recent onset SCZ, 35 major depressive disorder (MDD), 32 euthymic bipolar disorder (BPD), 45 Asperger syndrome and 280 control subjects. Preliminary analysis resulted in identification of a signature comprised of 34 analytes in a cohort of closely matched SCZ (n=71) and control (n=59) subjects. Partial least squares discriminant analysis using this signature gave a separation of 60-75% of SCZ subjects from controls across five independent cohorts. The same analysis also gave a separation of ~50% of MDD patients and 10-20% of BPD and Asperger syndrome subjects from controls. These results demonstrate for the first time that a biological signature for SCZ can be identified in blood serum. This study lays the groundwork for development of a diagnostic test that can be used as an aid for distinguishing SCZ subjects from healthy controls and from those affected by related psychiatric illnesses with overlapping symptoms.

Sex-specific serum biomarker patterns in adults with Asperger's syndrome.

Autism spectrum conditions have been hypothesized to be an exaggeration of normal male low-empathizing and high-systemizing behaviors. We tested this hypothesis at the molecular level by performing comprehensive multi-analyte profiling of blood serum from adult subjects with Asperger's syndrome (AS) compared with controls. This led to identification of distinct sex-specific biomarker fingerprints for male and female subjects. Males with AS showed altered levels of 24 biomarkers including increased levels of cytokines and other inflammatory molecules. Multivariate statistical classification of males using this panel of 24 biomarkers revealed a marked separation between AS and controls with a sensitivity of 0.86 and specificity of 0.88. Testing this same panel in females did not result in a separation between the AS and control groups. In contrast, AS females showed altered levels of 17 biomarkers including growth factors and hormones such as androgens, growth hormone and insulin-related molecules. Classification of females using this biomarker panel resulted in a separation between AS and controls with sensitivities and specificities of 0.96 and 0.83, respectively, and testing this same panel in the male group did not result in a separation between the AS and control groups. The finding of elevated testosterone in AS females confirmed predictions from the 'extreme male brain' and androgen theories of autism spectrum conditions. We conclude that to understand the etiology and development of autism spectrum conditions, stratification by sex is essential.

[Catecholamines and their metabolites in children with Asperger and Kanner syndromes].

Children with Asperger and Kanner syndromes in the stable state demonstrate similar decrease in plasma norepinephrine. In the aggravated state, these changes become more expressed and are characterized by a decrease in plasma tyrosine, norepinephrine, normetanephrine and by an increase in dopamine and homovanylic acid and a decrease in excretion of norepinephrine and an increase in excretion of homovanylic acid, epinephrine and MHPG. Only in children with Kanner syndrome in the aggravated state plasma MHPG increases, excretion of tyrosine decreases and excretion of normetanephrine increases. The observed imbalance in dopamine and epinephrine/norepinephrine systems justifies combined analysis of changes in catecholamines and their metabolites levels as the most informative approach in the study of the effect of autistic disorders.

Immune allergic response in Asperger syndrome.

Asperger's syndrome is a subgroup of autism characterized by social deficits without language delay, and high cognitive performance. The biological nature of autism is still unknown but there are controversial evidence associating an immune imbalance and autism. Clinical findings, including atopic family history, serum IgE levels as well as cutaneous tests showed that incidence of atopy was higher in the Asperger group compared to the healthy controls. These findings suggest that atopy is frequent in this subgroup of autism implying that allergic inflammation might be an important feature in Asperger syndrome.

The plasma zinc/serum copper ratio as a biomarker in children with autism spectrum disorders.

The frequency of zinc deficiency, copper toxicity and low zinc/copper in children with autism spectrum disorders (ASDs) may indicate decrement in metallothionein system functioning. A retrospective review of plasma zinc, serum copper and zinc/copper was performed on data from 230 children with autistic disorder, pervasive developmental disorder-NOS and Asperger's syndrome. The entire cohort's mean zinc level was 77.2 microg dl(-1), mean copper level was 131.5 microg dl(-1), and mean Zn/Cu was 0.608, which was below the 0.7 cut-off of the lowest 2.5% of healthy children. The plasma zinc/serum copper ratio may be a biomarker of heavy metal, particularly mercury, toxicity in children with ASDs.

Motion and form coherence detection in autistic spectrum disorder: Relationship to motor control and 2:4 digit ratio.

Children with autistic spectrum disorder and controls performed tasks of coherent motion and form detection, and motor control. Additionally, the ratio of the 2nd and 4th digits of these children, which is thought to be an indicator of foetal testosterone, was measured. Children in the experimental group were impaired at tasks of motor control, and had lower 2D:4D than controls. There were no group differences in motion or form detection. However a sub-group of children with autism were selectively impaired at motion detection. There were significant relationships between motion coherence detection and motor control in both groups of children, and also between motion detection, fine motor control and 2D:4D in the group of children with autistic spectrum disorder.

Higher plasma ACTH levels in adults with Asperger syndrome.

OBJECTIVE: The aim of this preliminary study was to characterize the levels of plasma adrenocorticotropic hormone (ACTH) and cortisol in adult patients with Asperger syndrome (AS). METHODS: Twenty medication-free individuals with high-functioning AS were recruited from a clinic specialized in autism spectrum disorders. Ten age-matched healthy persons (hospital staff or students) with no neuropsychiatric disorders served as controls. Blood samples for the assessment were collected at 8:00 a.m. RESULTS: The patients with AS had significantly higher plasma-ACTH values than did the healthy controls. Plasma-cortisol levels were similar in both groups. CONCLUSION: Increased plasma-ACTH levels are associated with AS. Future studies are needed to clarify whether this finding is a biological consequence of chronic anxiety and elevated stress, or a sign of facilitated response to an acute novel stressor.

Increased repetitive behaviours and prolactin responsivity to oral m-chlorophenylpiperazine in adults with autism spectrum disorders.

Autism is a neurodevelopmental disorder characterized by dysfunction in three primary behavioural domains: repetitive behaviours, social deficits, and language abnormalities. There is evidence that abnormalities exist in the serotonin (5-HT) system in autism spectrum patients. Furthermore, 5-HT is known to play a role in repetitive and social behaviours. This study examined the effect of m-chlorophenylpiperazine (m-CPP) on repetitive behaviours and prolactin response in 11 adults with autism or Aspergers disorder and 8 age- and gender-matched healthy controls via randomized double-blind, m-CPP and placebo challenges. The primary outcome measure was an instrument rating six repetitive behaviours: need to know, repeating, ordering, need to tell/ask, self-injury, and touching. Patients with autism spectrum disorders showed a significant increase in repetitive behaviours at end-point following oral m-CPP in comparison to placebo. Additionally subjects with autism spectrum disorders showed a significantly increased prolactin response to m-CPP compared to normal controls, with neither group responding to placebo. This study provides further evidence for altered 5-HT sensitivity in individuals with autism spectrum disorders, as well as a possible relationship between repetitive behaviours in autism spectrum disorders and abnormalities in the 5-HT system.

Red blood cell fatty acid compositions in a patient with autistic spectrum disorder: a characteristic abnormality in neurodevelopmental disorders?

The fatty acid compositions of red blood cell (RBC) phospholipids from a patient with autistic spectrum disorder (ASD) had reduced percentages of highly unsaturated fatty acids (HUFA) compared to control samples. The percentage of HUFA in the RBC from the autistic patient was dramatically reduced (up to 70%) when the sample was stored for 6 weeks at -20 degrees C. However, only minor HUFA reductions were recorded in control samples stored similarly, or when the autistic sample was stored at -80 degrees C. A similar instability in RBC HUFA compositions upon storage at -20 degrees C has been recorded in schizophrenic patients. In a number of other neurodevelopmental conditions, including attention deficit hyperactivity disorder (ADHD) and dyslexia, reduced concentrations of RBC HUFA have been recorded. The extent and nature of these aberrations require further assessment to determine a possible common biochemical origin of neurodevelopmental disorders in general. To facilitate this, a large scale assessment of RBC fatty acid compositions in patients with ASD, and related disorders, should be performed as a matter of urgency. Supplementing cells in culture with the tryptophan metabolite indole acrylic acid (IAA) affected the levels of cellular HUFA and prostaglandin production. Indole acroyl glycine (IAG), a metabolite of IAA excreted in urine, is found in high concentrations in patients with neurodevelopmental disorders including ASD, ADHD, dyslexia, Asperger's syndrome and obsessive compulsive disorder.